补肾活血方强化骨髓干细胞原位移植治疗急性心肌梗死的研究
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摘要
急性心肌梗死是冠心病中最危重的临床类型,目前临床上应用的药物治疗与手术治疗均无法使梗死的心肌再生,由于干细胞有可能通过参与坏死心肌组织修复而显著改善心肌梗死患者的远期预后,因此,如何通过干细胞移植促使心肌细胞再生成为当前研究的热点。
骨髓干细胞诱导分化的心肌样细胞能与宿主心肌细胞间形成有效的电-机械耦联,加上采集及自我扩增容易,无免疫排斥反应,无伦理道德问题,成为治疗心肌梗死较为理想的细胞来源。骨髓干细胞原位移植法(骨髓干细胞动员)简单无创,不需外科手术介入,不受细胞来源、免疫排斥反应等条件的限制,更易于为患者接受,更有可能抓住有限治疗时机,其临床应用前景可能更好。目前常用的骨髓动员剂为粒细胞集落刺激因子,大量动物试验及小样本临床研究证实了粒细胞集落刺激因子的有效性及安全性,但仍存在较多不良反应。祖国医学认为:“肾主骨生髓”,既往大量证据也证实补肾活血中药具有促进骨髓干细胞分化增殖、改善骨髓微环境等作用,结合中医药对缺血性心脏病的良好疗效,且中药具有临床应用安全和长期有效的特点,如能将中药用于骨髓干细胞动员以治疗心肌梗死,将有可能克服粒细胞集落刺激因子诸多不足之处,无疑将会成为传统中医药研究及发展的又一亮点,并为研究心肌缺血性疾病的防治提供新的思路,具有重要的临床意义和广阔的应用前景。
本研究围绕骨髓干细胞动员这一中心,应用流式细胞技术、病理组织学、免疫组织化学等多种技术比较不同浓度补肾活血方与粒细胞集落刺激因子对急性心肌梗死大鼠骨髓干细胞动员的效果,并在此基础上,进一步观察了补肾活血方对急性心肌梗死患者外周血CD34~+细胞百分比、心肌梗死面积、左室结构与心功能等指标的作用,探讨了补肾活血方可能通过动员骨髓干细胞的途径而发挥了对修复坏死心肌组织的作用。
1补肾活血中药动员急性心肌梗死大鼠骨髓干细胞的研究
方法:通过结扎大鼠左冠状动脉前降支造成心肌梗死模型后,随机分为空白组、基因重组人粒细胞集落刺激因子(CSF)组、补肾活血方(简称BS)低剂量组与BS高剂量组。分别于造模后1天、5天和14天后采用流式细胞仪检测外周血CD34~+细胞,HE染色观察病理变化和免疫组织化学检测心肌CD34~+细胞,比较不同组间血管密度。
结果:急性心梗1天后测各组间的CD34~+细胞比例无明显差异。5天后BS高剂量组与CSF组外周血CD34~+细胞测值明显升高,与空白组比较存在显著性差异;BS高剂量组心梗5天后测值虽略低于CSF组测值,但两组间比较无显著性差异;造模5天后BS低剂量组较空白组亦明显升高。2周后空白组、CSF组及BS低剂量组基本恢复至动员前水平,BS高剂量组测值虽有下降,但仍维持在较高水平,与其余3组相比具有显著性差异。心梗5天后和14天后CSF组与BS低剂量组、BS高剂量组心肌梗死区均可见大量CD34~+单个核细胞浸润,梗死灶周围可见CD34~+心肌细胞样细胞生长,且血管密度明显高于空白组。空白组造模后5天于梗死灶可见少数新生血管及较多纤维母细胞增生,梗死灶的周围可见少数CD34~+单个核细胞生长;造模后14天,可见较大片心肌疤痕组织,呈散在片状分布,心肌组织排列有序的基本结构被破坏,瘢痕化。
结论:急性心肌梗死后可出现骨髓干细胞的自发动员现象。不同剂量的补肾活血方有可能促进骨髓干细胞进入外周血液,致外周血CD34~+细胞数量增加,且高剂量组补肾活血方的动员效果明显强于低剂量组。动员的外周血CD34~+细胞有可能流入心肌梗死的部位,进而分化为心肌细胞与血管内皮细胞等。与G-CSF相比较,高剂量补肾活血方疗效确定,且动员作用更为持久。
2补肾活血方对急性心肌梗死患者的治疗作用
方法:将20例急性心肌梗死患者随机分为补肾活血组和对照组,每组各10例,治疗参照2001年《中华心血管病杂志》编委会制定的急性心肌梗死治疗指南中的方法,均常规给予硝酸甘油、抗凝剂、血小板抑制剂及ACEI类等药物,有心衰、心律失常等并发症者对症治疗,其中补肾活血组每日加服补肾活血方。分别在治疗前和治疗后对各组患者经流式细胞仪测定外周血中CD34~+细胞百分比,通过QRS记分系统测定AMI组患者心肌梗死面积,并经彩色多普勒超声测定左室结构和功能。
结果:治疗后补肾活血组和对照组外周血CD34~+细胞百分比均呈逐步升高,其中补肾活血组CD34~+细胞百分比较对照组明显升高,于第5天达高峰。而CSA组不同时间点外周血CD34~+细胞百分比测值无明显变化。至治疗后第14天时,对照组测值已明显降低,而补肾活血组测值仍维持于较高水平。补肾活血治疗4周后,补肾活血组QRS记分值明显减低,梗死面积明显缩小,与对照组治疗后28天比较,存在显著性差异。补肾活血组与对照组治疗后7天时LVEF、LVEDd及LVEDs的基线值均无明显差异,经治疗28天后,补肾活血组LVEF显著改善,LVEDd及LVEDs明显减低,而对照组无明显变化。
结论:本研究进一步证实了心梗后存在自发的骨髓干细胞动员现象。补肾活血方有可能通过动员骨髓造血干细胞,达到提高外周血骨髓干细胞水平的作用,从而能防止心肌梗死患者急性期防止梗死灶的延展、缩小梗死面积,对心肌梗死后心功能的恢复有显著的保护作用。
通过上述试验我们得出以下结论:
本研究根据祖国医学对“肾主骨生髓”、精血同源、心肾相关等理论,通过动物实验及临床观察发现,心梗后存在自发的骨髓干细胞动员现象,同时,补肾活血方有可能通过动员骨髓干细胞向心肌损伤部位迁移,并使其在心脏微环境中分化为心肌细胞和血管内皮细胞,从而起到改善梗死后心肌组织的血供、促进心肌细胞再生的作用。本研究利用中医药的整体治疗优势,从肾精、血脉立论,为补肾活血治法提供新的现代医学依据,在理论与临床实践中均具有重要价值,可望为中医药治疗急性心肌梗死提供新的理论基础,开辟新的治疗途径。其确切机理有待进一步探讨。
Acute myocardial infarction(AMI)is the most common cause of morbidity and mortality in coronary heart diseases.Current clinical therapy to minimize the devastating effects of myocardial infarction range from administration of drugs,acute percutaneous coronary intervention to coronary artery bypass grafting are usually not sufficient to prevent left ventricular remodeling and subsequent development of heart failure as they fall short of actual replacement of necrotic cardiac myocytes.By the time of end-stage heart failure,the effect of drug treatment is limited,and cardiac transplantation is the only viable alternative.However,this strategy is costly and severely limited by the availability of donor hearts.In addition,the use of immunosuppressive agents may diminish the patient' s quality of life.The recent progress in the area of stem cell research has led to the suggestion that stem cells might potentially be used to regenerate cardiomyocytes and improve impaired heart function.Current insights into stem cell plasticity have opened up new perspectives for regenerating the infarcted heart.
There are several different types of stem cells containing embryonic stem cells(ESCs),skeletal muscle satellite cells and bone marrow-derived stem cells(BMSCs)being investigated for their capacity to proliferate and differentiate into cardiomyocytes.Embryonic stem cells are highly proliferative and totipotent,but they have some drawbacks,such as ethical issue,immunosuppressive therapy,the potential to form teratomas,etc. Skeletal muscle satellite cells and BMSCs,although limited in their differentiation capacity,provide a safer and ethically acceptable alternative to ESCs.For skeletal myoblasts,the question is that the cells differentiated from skeletal muscle satellite cell are different from cardiomyocytes because of the lack of the intercalated disks which are essential for mechanical activity in the myocardium.The bone marrow contains several reconstructing stem cell types,with overlapping phenotypes, including haematopoietic stem cells(HSCs),endothelial progenitor cells (EPCs),mesenchymal stem cells(MSCs),etc.Recent data suggest that BMSCs are the ideal sources because the cardiomyocyte-like cells induced from BMSCs might be able to form effective mechanoelectric coupling with host cardiomyocyte.Bone marrow stem cells can be harvested as autologous cells, avoiding the use of immunosuppressive drugs.Besides,BMSCs can be collected and expanded easily from adults without moral principles.The BMSCs mobilization is an attractive administration candidate because the treatment is noninvasive so that it is more convenient and acceptable for patients.There is increasing evidence that stem cell mobilization homing to the infarcted myocardium and differentiation into cardiac myocytes is a naturally occurring process.However,the amount of mobilized stem cells is too low to be sufficient to ameliorate the remodelling process after ischemia,preventing preexisting cardiomyocytes from undergoing apoptosis and improving heart function. Pharmacological mobilization of bone marrow stem cells is one of the strategies for cardiac repair.Granulocyte colony-stimulating factor(G-CSF)is an effective cytokine capable of mobilizing hematopoietic stems and progenitor cells from the bone marrow to the peripheral circulation.The previous studies demonstrated that G-CSF exert favorable effects on cardiac remodeling is enhancement of endogenous cardiac repair mechanisms that include both bone marrow stem cells mobilization,engraftment,and differentiation as well as proliferation of cardiomyocytes.Its efficacy and safety have been proved by many animal experiments and some small-sample clinic trials and G-CSF treatment was well tolerated despite of some adverse reaction,such as excessive instent restenosis,embolism,blood coagulation,ostalgia,fever, etc.It is also doubtful whether G-CSF therapy will benefit elderly patients because the increased age is associated with reduced angiogenic capacity and diminished cell cycling of cardiac stem cells.
In the theories of traditional Chinese Medicine(TCM),kidney is in charge of the condition of the bone and marrow,heart and kidney is closely-related。If we tonify the kidney,the function of bone and marrow will be strengthened. Plenty of evidences showed that Chinese medicines of invigorating kidney and promoting blood circulation can facilitate the differentiation of bone marrow stem cells and improve the microenviroment of bone marrow.Besides,the Chinese medicines are effective and safe in treating the ischemic heart diseases.If the Chinese herbs could be used for the mobilization of BMSC to cure the AMI, the disadvantages of G-CSF may be overcome and it may offer a new therapeutic strategy for ischemic heart diseases.So,in this present study,we hypothesized that bone marrow stem cells could be mobilized by Bu Shen Huo Xue Formula,which is composed of invigorating kidney and promoting blood circulation Chinese medicines,and the mobilized stem cells would colonize and differentiate at the site of myocardial injury,limit the size of damage incurred by the infarction,and thereby improve myocardial function.
The present study compared the BMSCs mobilization effects of the different-doses of Bu Shen Huo Xue.Formula and G-CSF on AMI in experimental rats with flow cytometer,pathohistology and immunohistochemical staining technique.On the base of the animal experiment,the further clinic study was carried out to observe the contribution of Bu Shen Huo Xue Formula to the percentages of CD34 positive cells expression in peripheral blood of AMI patients,the area of myocardial infarction,the left ventricular structure and ejection function.We explored that Bu Shen Huo Xue Formula might improve cardiac performance by increasing the number of bone marrow stem cell in the peripheral circulation.
PartⅠStudy of Bu Shen Huo Xue Formula on the mobilization of bone marrow stem cells of Acute Myocardial Infarction experimental Rat
Methods AMI was induced in rat by permanent ligation of the left descending coronary artery.The rats were divided into four groups at random:CSF group, BS low-dose group,BS high-dose group and control group.Three hours after left anterior coronary artery ligation,the rats in CSF group were assigned to receive a first subcutaneous injection of recombinant human granulocyte colony-stimulating factor 10μg/kg of body weight followed by daily injections for 5 consecutive days.The rats in BS low-dose group and BS high-dose group took the different concentration of Bu Shen Huo Xue Formula separately daily for 5 consecutive days.Control group received equal volumes of isotonic saline. The percentages of CD34 positive cells expression in peripheral blood were measured with flow cytometer on day 1,day 5 and day 14 after model established separately.The histological changes of morphological characteristics of myocardium were observed with the haematine-eosin staining on day 1,day 5 and day 14 after MI.Immunohistochemistry for CD34 positive cells were performed in the tissues of myocardial infarcted area and marginal area.
Results(1)The percentages of CD34 positive cells expression in peripheral blood of all groups had no difference on day lafter MI.On day 5 after MI, the values of CD34positive cells expression in BS low-dose group,BS high-dose group and CSF group increased significantly from(0.53±0.15),(0.55±0.19)% and(0.59±0.14)%at baseline to(1.77±0.46),(2.49±0.62)%and (3.31±0.87)%(p＜0.01 versus control).The values of BS high-dose group and CSF group had no significantly difference(p＞0.05).On day 14 after models established the percentages of CD34 positive cells expression in G-CSF groups, BS low-dose group,and control group showed a similar decline,except the value of BS high-dose group keep in high level(p＜0.05 versus other three groups). (2)It was observed in histological changes that there were CD34 positive infiltrative monocytes in the infarcted area and CD34 positive myocardium-like cells in the marginal area on day 5 and day 14 after mobilization in the CSF group,BS high-dose group and BS low-dose group separately.The vascular densities of above three groups were significantly greater(p＜0.01 versus control).In control group,it is showed that there were a few newborn vessels and more fibroblasts on day 5 after model established.A few CD34 positive infiltrative monocytes were observed in the marginal infractedarea.On day 14 after MI,there were larger myocardial scar tissues distributed in patch. The well-arranged basic structure of myocardial tissues was destroyed and the formation of scars could be observed.
Conclusion In this study,we induced MI in rats via ligation of the LAD and applied rhG-CSF and different-doses Bu Shen Huo Xue Formula three hours after MI daily for 5 consecutive days.Our main findings were as follows:(1)AMI is followed by enhanced spontaneous mobilization of BMSCs;(2)Bu Shen Huo Xue Formula in different doses might both have mobilization effects on bone marrow stem cells,which could increase the percentages of CD34 positive cells expression in peripheral blood after acute myocardial infarction.It was shown that Bu Shen Huo Xue Formula in high dose application increased peripheral blood stem cells after MI better than that in low dose did.The mobilized bone marrow stem cells may migrate to the site of myocardial infarction and differentiated into myocardium-like cells and vascular endothelials which might lead to neovascularisation and resulted in the reduced scar extension; (3)High-dose Bu Shen Huo Xue Formula has more continuous mobilization effect when compared to G-CSF.
PartⅡProtective effect of Bu Shen Huo Xue Formula on acute myocardial infarction patients
Methods Twenty AMI patients were randomly assigned to receive treatment with Bu Shen Huo Xue Formula(BS group)and to receive placebo(control group). Two groups patients had comparable demographic and clinical and infracted-related characteristics.The standard care included the glyceryltrinitrate,anti-coagulation drugs,platelet aggregation inhibitors and angiotensin converting enzyme inhibitor,etc.Symptomatic treatments were practiced when the complications such as heart failure and arrhythmia occurred. Chinese medicines of invigorating kidney and promoting blood circulation(Bu Shen Huo Xue Formula)were administered daily after MI in BS group.The percentages of CD34 positive cells expression in peripheral blood were measured by flow cytometry on day 3,5,7,14 after treatment.The myocardial infarction sizes were estimated by QRS Scoring System before treatment and on day 7,28 after treatment.The left ventricular structure and function were determined with color doppler ultrasonography on day 7,28 after treatment.
Results(1)The percentages of CD34 positive cells expression in peripheral blood in all AMI patients increased gradually after treatment and peaked on day 5(p＜0.01 versus baseline).BS group had significantly higher mean level of CD34 positive cells expression(p＜0.05 versus control).The CD34 positive cells expression of control group decreased obviously on day 14 while BS group keep in high level(p＜0.05 versus control).(2)According to QRS Scoring System, the score on day 28 after treatment in BS group decreased from 5.59±0.65 to 3.79±0.46(p＜0.05 versus baseline),and the left ventricular infarct size on day 28 after treatment reduced significantly from 37.98±3.45%to 28.00±2.26%(p＜0.01 versus baseline),while the score and infarct size in control group patients showed no significant decrease(p＞0.05).(3)The baseline values of left ventricular ejection fraction(LVEF),left ventricular end-diastole dimension(LVEDd)and left ventricular end-systole dimension (LVEDs)had no differences between BS group and control group.But on day 28 after treatment,the LVEF in BS group improved markedly from 52.67±4.69%to 60.11±4.57(p＜0.01 versus baseline)and LVEDd and LVEDs lowered obviously from 61.11±5.90 mm,40.67±5.72 mm to 54.11±2.62 and 34.67±4.12(p＜0.01 and p＜0.05 versus baseline),while in control group,LVEDd and LVEDs on day 28 after treatment had no significantly differences respectively(p＞0.05 versus baseline).
Conclusion In this study,we examined the effect of Bu Shen Huo Xue Formula administration on AMI patients' peripheral blood stem cells expressions, infarction areas,cardiac structures and ejection functions at the base of animal experiment in different time set after treatment.Our main findings were as follows:(1)the spontaneous mobilization of BMSCs after AMI was proven to be existent;(2)our results showed beneficial effects of Bu Shen Huo Xue Formula treatment after MI on infarction areas,cardiac structure and function in a follow up of 4 weeks.The beneficial outcome on cardiac structure and function may related to the increasing number of bone marrow stem cells in the peripheral circulation because of bone marrow stem cells mobilization through which may attenuate left ventricular dilation,decrease infarction size and reversed a decline in cardiac function.
In conclusion,this present investigation suggests that there may be a naturally occurring but inefficient repair process that attempts to regenerate myocardial tissue in response to a myocardial infarction.Furthermore,our results offered a discovery that Bu Shen Huo Xue Formula might have a mobilization effect on bone marrow stem cells.The mobilized bone marrow stem cells may migrate to the site of myocardial infarction and differentiated into cardiomyocytes and vascular endothelial so that myocardial perfusion could be improved and cardiomyocytes be promoted to regenerate which would be beneficial for left-ventricular remodeling after myocardial infarction.
骨髓干细胞诱导分化的心肌样细胞能与宿主心肌细胞间形成有效的电-机械耦联,加上采集及自我扩增容易,无免疫排斥反应,无伦理道德问题,成为治疗心肌梗死较为理想的细胞来源。骨髓干细胞原位移植法(骨髓干细胞动员)简单无创,不需外科手术介入,不受细胞来源、免疫排斥反应等条件的限制,更易于为患者接受,更有可能抓住有限治疗时机,其临床应用前景可能更好。目前常用的骨髓动员剂为粒细胞集落刺激因子,大量动物试验及小样本临床研究证实了粒细胞集落刺激因子的有效性及安全性,但仍存在较多不良反应。祖国医学认为:“肾主骨生髓”,既往大量证据也证实补肾活血中药具有促进骨髓干细胞分化增殖、改善骨髓微环境等作用,结合中医药对缺血性心脏病的良好疗效,且中药具有临床应用安全和长期有效的特点,如能将中药用于骨髓干细胞动员以治疗心肌梗死,将有可能克服粒细胞集落刺激因子诸多不足之处,无疑将会成为传统中医药研究及发展的又一亮点,并为研究心肌缺血性疾病的防治提供新的思路,具有重要的临床意义和广阔的应用前景。
本研究围绕骨髓干细胞动员这一中心,应用流式细胞技术、病理组织学、免疫组织化学等多种技术比较不同浓度补肾活血方与粒细胞集落刺激因子对急性心肌梗死大鼠骨髓干细胞动员的效果,并在此基础上,进一步观察了补肾活血方对急性心肌梗死患者外周血CD34~+细胞百分比、心肌梗死面积、左室结构与心功能等指标的作用,探讨了补肾活血方可能通过动员骨髓干细胞的途径而发挥了对修复坏死心肌组织的作用。
1补肾活血中药动员急性心肌梗死大鼠骨髓干细胞的研究
方法:通过结扎大鼠左冠状动脉前降支造成心肌梗死模型后,随机分为空白组、基因重组人粒细胞集落刺激因子(CSF)组、补肾活血方(简称BS)低剂量组与BS高剂量组。分别于造模后1天、5天和14天后采用流式细胞仪检测外周血CD34~+细胞,HE染色观察病理变化和免疫组织化学检测心肌CD34~+细胞,比较不同组间血管密度。
结果:急性心梗1天后测各组间的CD34~+细胞比例无明显差异。5天后BS高剂量组与CSF组外周血CD34~+细胞测值明显升高,与空白组比较存在显著性差异;BS高剂量组心梗5天后测值虽略低于CSF组测值,但两组间比较无显著性差异;造模5天后BS低剂量组较空白组亦明显升高。2周后空白组、CSF组及BS低剂量组基本恢复至动员前水平,BS高剂量组测值虽有下降,但仍维持在较高水平,与其余3组相比具有显著性差异。心梗5天后和14天后CSF组与BS低剂量组、BS高剂量组心肌梗死区均可见大量CD34~+单个核细胞浸润,梗死灶周围可见CD34~+心肌细胞样细胞生长,且血管密度明显高于空白组。空白组造模后5天于梗死灶可见少数新生血管及较多纤维母细胞增生,梗死灶的周围可见少数CD34~+单个核细胞生长;造模后14天,可见较大片心肌疤痕组织,呈散在片状分布,心肌组织排列有序的基本结构被破坏,瘢痕化。
结论:急性心肌梗死后可出现骨髓干细胞的自发动员现象。不同剂量的补肾活血方有可能促进骨髓干细胞进入外周血液,致外周血CD34~+细胞数量增加,且高剂量组补肾活血方的动员效果明显强于低剂量组。动员的外周血CD34~+细胞有可能流入心肌梗死的部位,进而分化为心肌细胞与血管内皮细胞等。与G-CSF相比较,高剂量补肾活血方疗效确定,且动员作用更为持久。
2补肾活血方对急性心肌梗死患者的治疗作用
方法:将20例急性心肌梗死患者随机分为补肾活血组和对照组,每组各10例,治疗参照2001年《中华心血管病杂志》编委会制定的急性心肌梗死治疗指南中的方法,均常规给予硝酸甘油、抗凝剂、血小板抑制剂及ACEI类等药物,有心衰、心律失常等并发症者对症治疗,其中补肾活血组每日加服补肾活血方。分别在治疗前和治疗后对各组患者经流式细胞仪测定外周血中CD34~+细胞百分比,通过QRS记分系统测定AMI组患者心肌梗死面积,并经彩色多普勒超声测定左室结构和功能。
结果:治疗后补肾活血组和对照组外周血CD34~+细胞百分比均呈逐步升高,其中补肾活血组CD34~+细胞百分比较对照组明显升高,于第5天达高峰。而CSA组不同时间点外周血CD34~+细胞百分比测值无明显变化。至治疗后第14天时,对照组测值已明显降低,而补肾活血组测值仍维持于较高水平。补肾活血治疗4周后,补肾活血组QRS记分值明显减低,梗死面积明显缩小,与对照组治疗后28天比较,存在显著性差异。补肾活血组与对照组治疗后7天时LVEF、LVEDd及LVEDs的基线值均无明显差异,经治疗28天后,补肾活血组LVEF显著改善,LVEDd及LVEDs明显减低,而对照组无明显变化。
结论:本研究进一步证实了心梗后存在自发的骨髓干细胞动员现象。补肾活血方有可能通过动员骨髓造血干细胞,达到提高外周血骨髓干细胞水平的作用,从而能防止心肌梗死患者急性期防止梗死灶的延展、缩小梗死面积,对心肌梗死后心功能的恢复有显著的保护作用。
通过上述试验我们得出以下结论:
本研究根据祖国医学对“肾主骨生髓”、精血同源、心肾相关等理论,通过动物实验及临床观察发现,心梗后存在自发的骨髓干细胞动员现象,同时,补肾活血方有可能通过动员骨髓干细胞向心肌损伤部位迁移,并使其在心脏微环境中分化为心肌细胞和血管内皮细胞,从而起到改善梗死后心肌组织的血供、促进心肌细胞再生的作用。本研究利用中医药的整体治疗优势,从肾精、血脉立论,为补肾活血治法提供新的现代医学依据,在理论与临床实践中均具有重要价值,可望为中医药治疗急性心肌梗死提供新的理论基础,开辟新的治疗途径。其确切机理有待进一步探讨。
Acute myocardial infarction(AMI)is the most common cause of morbidity and mortality in coronary heart diseases.Current clinical therapy to minimize the devastating effects of myocardial infarction range from administration of drugs,acute percutaneous coronary intervention to coronary artery bypass grafting are usually not sufficient to prevent left ventricular remodeling and subsequent development of heart failure as they fall short of actual replacement of necrotic cardiac myocytes.By the time of end-stage heart failure,the effect of drug treatment is limited,and cardiac transplantation is the only viable alternative.However,this strategy is costly and severely limited by the availability of donor hearts.In addition,the use of immunosuppressive agents may diminish the patient' s quality of life.The recent progress in the area of stem cell research has led to the suggestion that stem cells might potentially be used to regenerate cardiomyocytes and improve impaired heart function.Current insights into stem cell plasticity have opened up new perspectives for regenerating the infarcted heart.
There are several different types of stem cells containing embryonic stem cells(ESCs),skeletal muscle satellite cells and bone marrow-derived stem cells(BMSCs)being investigated for their capacity to proliferate and differentiate into cardiomyocytes.Embryonic stem cells are highly proliferative and totipotent,but they have some drawbacks,such as ethical issue,immunosuppressive therapy,the potential to form teratomas,etc. Skeletal muscle satellite cells and BMSCs,although limited in their differentiation capacity,provide a safer and ethically acceptable alternative to ESCs.For skeletal myoblasts,the question is that the cells differentiated from skeletal muscle satellite cell are different from cardiomyocytes because of the lack of the intercalated disks which are essential for mechanical activity in the myocardium.The bone marrow contains several reconstructing stem cell types,with overlapping phenotypes, including haematopoietic stem cells(HSCs),endothelial progenitor cells (EPCs),mesenchymal stem cells(MSCs),etc.Recent data suggest that BMSCs are the ideal sources because the cardiomyocyte-like cells induced from BMSCs might be able to form effective mechanoelectric coupling with host cardiomyocyte.Bone marrow stem cells can be harvested as autologous cells, avoiding the use of immunosuppressive drugs.Besides,BMSCs can be collected and expanded easily from adults without moral principles.The BMSCs mobilization is an attractive administration candidate because the treatment is noninvasive so that it is more convenient and acceptable for patients.There is increasing evidence that stem cell mobilization homing to the infarcted myocardium and differentiation into cardiac myocytes is a naturally occurring process.However,the amount of mobilized stem cells is too low to be sufficient to ameliorate the remodelling process after ischemia,preventing preexisting cardiomyocytes from undergoing apoptosis and improving heart function. Pharmacological mobilization of bone marrow stem cells is one of the strategies for cardiac repair.Granulocyte colony-stimulating factor(G-CSF)is an effective cytokine capable of mobilizing hematopoietic stems and progenitor cells from the bone marrow to the peripheral circulation.The previous studies demonstrated that G-CSF exert favorable effects on cardiac remodeling is enhancement of endogenous cardiac repair mechanisms that include both bone marrow stem cells mobilization,engraftment,and differentiation as well as proliferation of cardiomyocytes.Its efficacy and safety have been proved by many animal experiments and some small-sample clinic trials and G-CSF treatment was well tolerated despite of some adverse reaction,such as excessive instent restenosis,embolism,blood coagulation,ostalgia,fever, etc.It is also doubtful whether G-CSF therapy will benefit elderly patients because the increased age is associated with reduced angiogenic capacity and diminished cell cycling of cardiac stem cells.
In the theories of traditional Chinese Medicine(TCM),kidney is in charge of the condition of the bone and marrow,heart and kidney is closely-related。If we tonify the kidney,the function of bone and marrow will be strengthened. Plenty of evidences showed that Chinese medicines of invigorating kidney and promoting blood circulation can facilitate the differentiation of bone marrow stem cells and improve the microenviroment of bone marrow.Besides,the Chinese medicines are effective and safe in treating the ischemic heart diseases.If the Chinese herbs could be used for the mobilization of BMSC to cure the AMI, the disadvantages of G-CSF may be overcome and it may offer a new therapeutic strategy for ischemic heart diseases.So,in this present study,we hypothesized that bone marrow stem cells could be mobilized by Bu Shen Huo Xue Formula,which is composed of invigorating kidney and promoting blood circulation Chinese medicines,and the mobilized stem cells would colonize and differentiate at the site of myocardial injury,limit the size of damage incurred by the infarction,and thereby improve myocardial function.
The present study compared the BMSCs mobilization effects of the different-doses of Bu Shen Huo Xue.Formula and G-CSF on AMI in experimental rats with flow cytometer,pathohistology and immunohistochemical staining technique.On the base of the animal experiment,the further clinic study was carried out to observe the contribution of Bu Shen Huo Xue Formula to the percentages of CD34 positive cells expression in peripheral blood of AMI patients,the area of myocardial infarction,the left ventricular structure and ejection function.We explored that Bu Shen Huo Xue Formula might improve cardiac performance by increasing the number of bone marrow stem cell in the peripheral circulation.
PartⅠStudy of Bu Shen Huo Xue Formula on the mobilization of bone marrow stem cells of Acute Myocardial Infarction experimental Rat
Methods AMI was induced in rat by permanent ligation of the left descending coronary artery.The rats were divided into four groups at random:CSF group, BS low-dose group,BS high-dose group and control group.Three hours after left anterior coronary artery ligation,the rats in CSF group were assigned to receive a first subcutaneous injection of recombinant human granulocyte colony-stimulating factor 10μg/kg of body weight followed by daily injections for 5 consecutive days.The rats in BS low-dose group and BS high-dose group took the different concentration of Bu Shen Huo Xue Formula separately daily for 5 consecutive days.Control group received equal volumes of isotonic saline. The percentages of CD34 positive cells expression in peripheral blood were measured with flow cytometer on day 1,day 5 and day 14 after model established separately.The histological changes of morphological characteristics of myocardium were observed with the haematine-eosin staining on day 1,day 5 and day 14 after MI.Immunohistochemistry for CD34 positive cells were performed in the tissues of myocardial infarcted area and marginal area.
Results(1)The percentages of CD34 positive cells expression in peripheral blood of all groups had no difference on day lafter MI.On day 5 after MI, the values of CD34positive cells expression in BS low-dose group,BS high-dose group and CSF group increased significantly from(0.53±0.15),(0.55±0.19)% and(0.59±0.14)%at baseline to(1.77±0.46),(2.49±0.62)%and (3.31±0.87)%(p＜0.01 versus control).The values of BS high-dose group and CSF group had no significantly difference(p＞0.05).On day 14 after models established the percentages of CD34 positive cells expression in G-CSF groups, BS low-dose group,and control group showed a similar decline,except the value of BS high-dose group keep in high level(p＜0.05 versus other three groups). (2)It was observed in histological changes that there were CD34 positive infiltrative monocytes in the infarcted area and CD34 positive myocardium-like cells in the marginal area on day 5 and day 14 after mobilization in the CSF group,BS high-dose group and BS low-dose group separately.The vascular densities of above three groups were significantly greater(p＜0.01 versus control).In control group,it is showed that there were a few newborn vessels and more fibroblasts on day 5 after model established.A few CD34 positive infiltrative monocytes were observed in the marginal infractedarea.On day 14 after MI,there were larger myocardial scar tissues distributed in patch. The well-arranged basic structure of myocardial tissues was destroyed and the formation of scars could be observed.
Conclusion In this study,we induced MI in rats via ligation of the LAD and applied rhG-CSF and different-doses Bu Shen Huo Xue Formula three hours after MI daily for 5 consecutive days.Our main findings were as follows:(1)AMI is followed by enhanced spontaneous mobilization of BMSCs;(2)Bu Shen Huo Xue Formula in different doses might both have mobilization effects on bone marrow stem cells,which could increase the percentages of CD34 positive cells expression in peripheral blood after acute myocardial infarction.It was shown that Bu Shen Huo Xue Formula in high dose application increased peripheral blood stem cells after MI better than that in low dose did.The mobilized bone marrow stem cells may migrate to the site of myocardial infarction and differentiated into myocardium-like cells and vascular endothelials which might lead to neovascularisation and resulted in the reduced scar extension; (3)High-dose Bu Shen Huo Xue Formula has more continuous mobilization effect when compared to G-CSF.
PartⅡProtective effect of Bu Shen Huo Xue Formula on acute myocardial infarction patients
Methods Twenty AMI patients were randomly assigned to receive treatment with Bu Shen Huo Xue Formula(BS group)and to receive placebo(control group). Two groups patients had comparable demographic and clinical and infracted-related characteristics.The standard care included the glyceryltrinitrate,anti-coagulation drugs,platelet aggregation inhibitors and angiotensin converting enzyme inhibitor,etc.Symptomatic treatments were practiced when the complications such as heart failure and arrhythmia occurred. Chinese medicines of invigorating kidney and promoting blood circulation(Bu Shen Huo Xue Formula)were administered daily after MI in BS group.The percentages of CD34 positive cells expression in peripheral blood were measured by flow cytometry on day 3,5,7,14 after treatment.The myocardial infarction sizes were estimated by QRS Scoring System before treatment and on day 7,28 after treatment.The left ventricular structure and function were determined with color doppler ultrasonography on day 7,28 after treatment.
Results(1)The percentages of CD34 positive cells expression in peripheral blood in all AMI patients increased gradually after treatment and peaked on day 5(p＜0.01 versus baseline).BS group had significantly higher mean level of CD34 positive cells expression(p＜0.05 versus control).The CD34 positive cells expression of control group decreased obviously on day 14 while BS group keep in high level(p＜0.05 versus control).(2)According to QRS Scoring System, the score on day 28 after treatment in BS group decreased from 5.59±0.65 to 3.79±0.46(p＜0.05 versus baseline),and the left ventricular infarct size on day 28 after treatment reduced significantly from 37.98±3.45%to 28.00±2.26%(p＜0.01 versus baseline),while the score and infarct size in control group patients showed no significant decrease(p＞0.05).(3)The baseline values of left ventricular ejection fraction(LVEF),left ventricular end-diastole dimension(LVEDd)and left ventricular end-systole dimension (LVEDs)had no differences between BS group and control group.But on day 28 after treatment,the LVEF in BS group improved markedly from 52.67±4.69%to 60.11±4.57(p＜0.01 versus baseline)and LVEDd and LVEDs lowered obviously from 61.11±5.90 mm,40.67±5.72 mm to 54.11±2.62 and 34.67±4.12(p＜0.01 and p＜0.05 versus baseline),while in control group,LVEDd and LVEDs on day 28 after treatment had no significantly differences respectively(p＞0.05 versus baseline).
Conclusion In this study,we examined the effect of Bu Shen Huo Xue Formula administration on AMI patients' peripheral blood stem cells expressions, infarction areas,cardiac structures and ejection functions at the base of animal experiment in different time set after treatment.Our main findings were as follows:(1)the spontaneous mobilization of BMSCs after AMI was proven to be existent;(2)our results showed beneficial effects of Bu Shen Huo Xue Formula treatment after MI on infarction areas,cardiac structure and function in a follow up of 4 weeks.The beneficial outcome on cardiac structure and function may related to the increasing number of bone marrow stem cells in the peripheral circulation because of bone marrow stem cells mobilization through which may attenuate left ventricular dilation,decrease infarction size and reversed a decline in cardiac function.
In conclusion,this present investigation suggests that there may be a naturally occurring but inefficient repair process that attempts to regenerate myocardial tissue in response to a myocardial infarction.Furthermore,our results offered a discovery that Bu Shen Huo Xue Formula might have a mobilization effect on bone marrow stem cells.The mobilized bone marrow stem cells may migrate to the site of myocardial infarction and differentiated into cardiomyocytes and vascular endothelial so that myocardial perfusion could be improved and cardiomyocytes be promoted to regenerate which would be beneficial for left-ventricular remodeling after myocardial infarction.
引文
[1]Makino S,Fuknda K,Migoshi S,et al.Cardiomyocytes can be generated from marrow stromal cells in vitro.J Clin Invest,1999;103:697-705.
[2]Tomita S,Li RK,Weisel RD,et al.Autologous transplantation of bone marrow cells improve damaged heart function.Circulation,1999;100(19supp1):247-256.
[3]Wang JS,Shum TD,Galipeau J,et al.Marrow stromal cells for cellular cardiomyoplasty:feasibility and potentiall clinical adventages.J Thorac Cardiovasc Surg,2000;120:999-1005.
[4]Goodell MA,Jackson KA,Majka SM,et al.Stem cell plasticity in muscle and bone marrow.ANN N Y Acad Sci,2001;938:208-218.
[5]Orlic D,kajstura J,Chimenti S,et al.Bone marrow cells regenerate infarcted myocardium.Nature,2001;410(6829):701-705.
[6]Jackson KA,Majka SM,Wang H,et al.Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cell.J Clin Invest,2001;107:1395-1402.
[7]Edward TH,Sui Zhang,Henry D,et al.Transdifferentiation of human peripheral blood CD34~+-enriched cell population into cardiomyocytes,endothelial cells,and smooth muscle cells in vivo.Circulation,2003;108(17):2070-2073.
[8]Askari AT,Unzek S,Popovic ZB,et al.Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy.Lancet,2003;362(9385):697-703.
[9]Kocher AA,Schuster MD,Szabolcs MJ,et al.Neovascularation of ischemic myocardium by human bone-marrow derived angioblasts prevents cardiomyocyte apoptosis,reduces remodeling and improves cardiac function.Nat Med,2001;7(4):430-436.
[10]Badorff C,Brandes RP,Popp R,et al.Transdifferentiation of blood-derived human adult endothelial progenitor cells into functionally active cardiomyocytes.Circulation,2003;107:1024-1032.
[11]Shintani S,Murohara T,Ikeda H,et al.Mobilization of endothelial progenito cells in patients with acute myocardial infarction.Circulation,2001;103(23):2776-2779.
[12]Ciulla MM,Lazzari L,Pacchiana R,et al.Homing of peripherally injected bone marrow cells in the rat after experimental myocardial injury.Haematologica,2003;88:614-621.
[13]邓争荣,杨春,马爱群,等.急性心肌梗死患者血浆VEGF、SDF-1和外周血CD34~+细胞的动态改变及其意义.南方医科大学学报,2006;26(11):1637-1640.
[14]牛丽丽,郑敏,曹丰,等.同种异体骨髓间充质干细胞在大鼠心脏的迁移及分化.中华医学杂志,2004;84(1):38-42.
[15]Hill JM,Dick AJ,Raman VK,et al.Serial cardiac magnetic resonance imaging of injected mesenchymal stem cells.Circulation,2003;108(8):1009-1014.
[16]Orlic D,Kajstura J,Chimenti S,et al.Mobilized bone marrow cells repair the infarcted heart,improving funetion and survival.Proc Natl Aead Sci.2001;98:10344-10349.
[17]Miki T,Sakamoto J,Nakano A,et al.Mobilization of bone marrow cells by G-CSF/GM-CSF improves ventricular function in the heart undergoing post-infarct remodeling.Circulation,2002;106(19 supp Ⅰ):15.
[18]Adachi Y,Imagawa J,Suzuki Y,Yogo K,Fukazawa M,Kuromaru O and Saito Y.G-CSF treatment increase side population cell infiltration after myocardial infarction in mice.J Mol Cell Cardiol,2004;36:707-710.
[19]Fukuhara S,Tomita S,Nakatani T,et al.G-CSF promotes bone marrow cells to migrate into infarcted Mice Heart,and Differentiate Into Cardiomyocytes.Cell Transplant,2004;13(7-8):741-748.
[20]Tang YL,Qian K,Zhang YC,et al.Mobilizing of haematopoietic stem cells to ischemic myocardium by plasmid mediated stromal-cell-derived factor-1alpha(SDF-1 alpha)treatment.Regul Pept.2005;125(1-3):1-8.
[21]陈运贤,欧瑞明,钟雪云,等.粒细胞集落刺激因子干预性治疗大鼠心肌梗塞的实验研究.中国病理生理杂志,2001;17(12):1153-1155.
[22]陈运贤,欧瑞明,钟雪云,等.粒细胞集落刺激因子动员骨髓干细胞治疗大鼠急性心肌梗塞.中国病理生理杂志,2002;18(1):1-3.
[23]陈运贤,欧瑞明,钟雪云,等.CD34~+细胞的心肌细胞分化潜能研究.中国病理生理杂志,2002;18(2):117-119.
[24]李连达,张荣利,张旋,等.聚乙二醇重组人粒细胞集落刺激因子对大鼠心肌梗死的影响.中国新药杂志,2003;9(12):721-724.
[25]郭军,贾光宏,林国生,等.体外模拟心肌微环境下骨髓间充质干细胞向心肌样细胞分化的实验研究.心脏杂志,2004;15(6):501-504.
[26]路璐,彭建军,胡大一,等.骨髓动员与细胞移植对直接经皮腔内冠状动脉成形术后急性心肌梗死治疗作用.中国心血管病研究杂志,2004;7(2):557-560.
[27]黄国倩,刘虹,舒先红,等.超声心动图评价粒细胞集落刺激因子动员自身骨髓干细胞对心肌梗死后左心室重构及心功能的影响.中华超声影像学杂志,2004;11(13):848-852.
[28]郭素萍,高传玉,刘煜昊.骨髓干细胞动员剂对大鼠急性心肌梗死心肌肌钙蛋白Ⅰ的影响.实用诊断与治疗杂志,2005;2(19):114-115.
[29]刘宏伟,盖鲁粤,张端珍,等.粒细胞集落刺激因子骨髓干细胞动员治疗兔心肌梗死的实验研究.军医进修学院学报,2005;26(2):132-134.
[30]毛文凯,刘志勇.自体骨髓干细胞动员治疗心肌梗死.现代医学,2005;3(33):154-157.
[31]林玲,周胜华,祁述善,等.粒细胞集落刺激因子和干细胞因子对急性心肌梗死后心室重构的影响.中国介入心脏病学杂志,2005;13(3):178-181.
[32]Ohtsuka M,Takano H,Zou Y,et al.Cytokine therapy prevents left ventricular remodeling and dysfunction after myocardial infarction through neovascularization.FASEB J,2004;18(7):851-853.
[33]Laudmesser U,Engberding N,Bahlmann FH,et al.Statin-indueed improvement of endothelial progenitor cell mobilization,myocardial neovascularization,left ventricular function,and survival after experimental myocardial infarction requires endothelial nitric oxide synthase.Circulation,2004;110(14):1933-1939.
[34]姜宇,何力鹏,王农荣,等.辛伐他汀对大鼠外周血单个核CD34~+细胞动员作用的研究.武警医学,2004;15(10):740-743.
[35]吴贤仁,杨敏,李玉光.骨髓干细胞动员对大鼠缺血心肌的治疗作用.中国免疫学杂志,2004;7(20):471-474.
[36]李宾公,曾秋棠,晏凯利.骨髓干细胞动员治疗大鼠急性心肌梗死的实验研究.中国急救医学,2005;4(25),268-271.
[37]Seiler C,Pohl T,Wustmann K,et al.Promotion of colleteral growth by granulocyte macrophage colony-stimulating factor in patients with coronary artery disease:a randomized,double-blind,placebo-controlled study.Circulation,2001;104:2012-2017.
[38]Kang HJ,Kim HS,Zhang SY,et al.Effects of intraeoronary infusion of peripheral blood stem-cells mobilised with granulocyte-colony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction:the MAGIC cell randomised clinical trial.Lancet,2004;363(9411):751-756.
[39]Kang HJ,Lee HY,Na SH,et al.Myocardial infarction:the MAGIC cell-3-DES randomized,controlled trial and remodeling in patients with acute myocardial infarction versus old cells by granulocyte colony-stimulating factor on left ventricular function differential effect of intracoronary infusion of mobilized peripheral blood stem.Circulation,2006;114:145-151.
[40]Ince H,Petzsch M,Kleine HD,et al.Prevention of left ventrieular remodeling with granulocyte colony-stimulating factor after acute myocardial infarction:final 1-year results of the Front-Integrated Revaseularization and Stem Cell Liberation in Evolving Acute Myocardial Infarction by Granulocyte Colony-Stimulating Factor(FIRSTLINE-AMI)Trial. Circulation,2005;112(9 Suppl):173-180.
[41]Kuethe F,Figulla HR,Voth M,et al.Mobilization of stem cells by granulocyte colony-stimulating factor for the regeneration of myocardial tissue after myocardial infarction.Dtsch Med Woehenschr,2004;129(9):424-428.
[42]Huseyin Ince,Michael Petzsch,Hans Dieter Kleine,et al.Prevention of left ventricular remodeling with Granulocyte Colony-Stimulating factor after acute myocardial infarction.Circulation,2005;112:I-73-I-80.
[43]Valgimigli M,Rigolin GM,Cittanti C,et al.Use of granulocyte-colony stimulating factor during acute myocardial infarction to enhance bone marrow stem cell mobilization in humans:clinical and angiographic safety profile.Eur Heart J,2005;26(18):1838-1845.
[44]Suarez de Lezo J,Torres A,Herrera I,et al.Effects of stem-cell mobilization with recombinant human granulocyte colony stimulating factor in patients with percutaneously revascularized acute anterior myocardial infarction.Rev Esp Cardiol,2005;58(3):253-261.
[45]陈运贤,欧瑞明,钟雪云,等.自体骨髓造血干细胞原位移植治疗急性心肌梗死的临床研究.中国病理生理杂志,2003;19(4):452-454.
[46]徐新,马绍椿,罗东云,等.应用G-CSF动员骨髓干细胞治疗急性心肌梗死的研究.中国分子心脏病学杂志,2003;4(3):223-225.
[47]佟铁壁.集落刺激因子动员骨髓干细胞治疗心肌梗死临床观察.辽宁医学杂志,2006;20(2):99-100.
[48]马绍椿,徐新,罗东云,等.粒细胞集落刺激因子动员骨髓干细胞治疗急性心肌梗死的观察.中西医结合心脑血管病杂志,2004;2(2):63-65.
[49]Norol F,Merlet P,Isnard R,et al.Influence of mobilized stem cells on myocardial infarct repair in a nonhuman primate model.Blood,2003;102(13):4361-4368.
[50]Ripa RS,Jφgensen E,Wang Y,et al.Stem cell mobilization induced by subcutaneous granulocyte-colony stimulating factor to improve cardiac regeneration after acute ST-elevation myocardial infarction:result of the double-blind,randomized,placebo-controlled stem cells in myocardial infarction(STEMI)trial.Circulation,2006;113(16):1983-1992.
[51]Zohlnhofer D,Ott I,Mehilli J,et al.Stem cell mobilization by granulocyte colony-stimulating factor in patients with acute myocardial infarction:a randomized controlled trial.JAMA,2006;295:1003-1010.
[52]范文茂,陆东风,张东成,等.人重组粒细胞刺激因子治疗动物模型急性心肌梗死的病理学观察.血栓与止血学,2004;10(4):172-174.
[53]庄俊合,邓志刚,陆东风,等.人重组粒细胞集落刺激因子对急性心肌梗死患者心 功能恢复的疗效观察.血栓与止血学,2005;11(3):109-111.
[54]Balsam LB,Wagers A.J,Christensen JL,et al.Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium.Nature,2004;428(6983):668-673.
[55]Murry CE,Soonpaa MH,Reinecke H,et al.Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts.Nature,2004;428(6983):664-668.
[56]Alvarez-Dolado M,Pardal R,Gareia-Verdugo JM,et al.Fusion of bone-marrow-derived cells with purkinje neurons,cardiomyocytes and hepatocytes.Nature,2003;425(10):968-973.
[57]Kronenwett R,Martin S,Hass R.The role of cytokines and adhesion molecules for mobilization of peripheral blood stem cells.Stem ceil,2000;18(5):320-330.
[58]Li J,Brown LF,Hibberd MG,et al.VEGF,FIK-1 and Flt-1 expression in rat myocardial model of angiogenesis.Am J Physiol,1996;270:H1803-H1181.
[59]Scarborough J E,Smith M L.Basic fibroblast growth factor is upregulated in hibernating myoearditun.Surg Res,2002;107(1):119-123.
[60]Levi-Sehaffer F,Garbuzenko E,Rubin A,et al.Human eosinophils regulate human lung-and skin-derived fibroblast properties in vitro:a role for transforming growth factor beta(TGF-beta).Proc Natl Aead Sci USA,1996;96(17):9660-9665.
[61]Lichterfeld M,Martin S,Burkly L,et al.Mobilization of CD34~+ haematopoietic stem cells is associated with a functional inactivation of the integrin very late antigen 4.Br J Haematol,2000;110(1):71-81.
[62]Levesque JP,Takamatsu Y,Nilsson SK,et al.Vascular cell adhesion molecule-1(CD106)is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor.Blood,2001;98(5):1289-1297.
[63]翁建宇.G-CSF对造血干细胞的动员机制及对健康供者的影响.国外医学内科学分册,2002;29(8):352-355.
[64]Orlic D.Stem cell repair in isehemic heart disease:an experimental model.Iut J Hematol,2002,7(6 Suppl 1):S144-S145.
[65]Wright DE,Bowman EP,Wagers AJ,et al.Hematopoietie stem cells are uniquely selective in their migratory response to ehemokines.J Exp Med,2002;195(9):1145-1154.
[66]Satoshi S,Toyoaki M,Hisao I,et al.Mobilization of endothelial progenitor cells in patients with acute myocardial infarction.Circulation,2001;103(12):2776-2779.
[67]Rafii S,Heissig B,Hattori K.Efficient mobilization and recruitment of marrow-derived endothelial and hematopoietic stem cells by adenoviral vectors expressing angiogenic factors.Gene Ther,2002;9(10):631-641.
[68]Watt FM,Hogan B.Out of Eden:stem cells and their niches.Science,2000;287:1427-1430.
[69]Condorelli G,Borello U,De Angelis L,et al.Cardiomyocytes induce endothelial cells to transdifferentiate into cardiac muscle:implications for myocardium regeneration.Proc Natl Acad Sci U S A.2001;98:10733-10738.
[70]Tang YL,Zhao Q,Zhang Y,et al.Autologous mesenchymal stem cell transplantation induce VEGF and neovaseularization in ischernic myocardium.Regul Peptides,2004;117:3-10.
[71]尹瑞兴,李佳荃,蔡捷,等.血管内皮生长因子对急性心肌梗死大鼠心肌细胞凋亡的影响.中华老年医学杂志,2003;22(2):98-101.
[72]张少衡,贾竹青,郭静萱,等.骨髓细胞移植上调急性缺血心肌HSP32和HSP70的表达.北京大学学报(医学报),2003;35(5):476-480.
[73]Engelhardt M,Bertz H,Wasch R,et al.Analysis of stem cell apheresis products using intermediate-dose filgraseim plus large volume apheresis for allogeneic transplantation.Ann Hematol,2001;80:201-208.
[74]Hill JM,Zalos G,Halcox J P,et al.Circulating endothelial progenitor ceils,vascular function,and cardiovascular risk.N Engl J Med,2003;348:593-600.
[75]刘煜昊,房佰俊,方五旺,等.急性心肌梗死患者骨髓间充质干细胞生物学特性改变的研究.临床心血管病杂志,2006;22(1):44-46.
[76]Shimoda K Okawura S,Inaba S,et al.Granulocyte colony-stimulating factor and platelet aggregation.Lancet,1993;338:633.
[77]北京市造血干细胞移植协作组,应用重组人粒细胞集落刺激因子的经验与教训专题讨论.白血病·淋巴瘤,2007;16(1):1-5.
[78]Hill JM,Paul J-D,Powell TM,et al.Efficacy and risk of granulccyte colony stimulating factor administration in patients with servere coronary artery disease.Circulation,2003;108(Suppl Ⅳ):478-483.
[79]Stroncek DF,Clay ME,Petzoldt ML,et al.Treatment of normal individuals with G-CSF:donor experiences and the effects on peripheral blood CD34~+ cell counts and on the collection of peripheral blood stem cells.Transfusion,1996;36:601-610.
[80]Anderlini P,Przepiorka D,Seong D,et al.Clinical toxicity and laboratory effects of granulocyte-colony-stimulating factor(filgrastim)mobilization and blood stem cell apheresis from normal donors,and analysis of charges for the procedures.Transfusion,1996;36:590-595.
[81]Stroncek DF,Clay ME,Jaszez W,et al.Collection of teo peripheral blood stem cell concentrates from healthy donors.Transfus Med,1999;9:37-50.
[82]张仲平,洪介民.黄芪多糖对体外入骨髓造血祖细胞生成的影响.中药药理与临床,2000;16(1):16-17.
[83]刘晓,武正炎,范萍.黄芪在外周血造血干细胞移植中应用的初步实验研究.镇江医学院学报,2001;11(1):15-17.
[84]王晓东,陈孟莉,殷建芬,等.鸡血藤活性成分SS8对骨髓抑制大鼠造血祖细胞增殖的作用.中国中药杂志,2003;28(2):152-154.
[85]钱煦岱,高瑞兰,马珂,等.三七皂甙对人骨髓CD34~+造血干/祖细胞增殖分化作用.中国实验血液学杂志,2003;11(2):120-123.
[86]孙汉英,房明皓,任天华,等.川芎嗪对骨髓移植小鼠骨髓造血的影响.中国中西医结合杂志,2002;22(5):365-368.
[87]刘文励,黄伟,何美冬,等.复方活血汤对辐射损伤大鼠骨髓基质细胞粘附功能的影响.中国中西医结合杂志,1997;17(6):354-355.
[88]王炳胜,刘秀芳,王涛,等.益气活血方对放射损伤骨髓基质细胞生长的影响.中华放射医学与防护杂志,2000;20(3):190-191.
[89]牛泱平,金锦梅,高瑞兰,等.当归注射液对正常人和再生障碍性贫血患者造血祖细胞的刺激增殖作用.实用中西医结合杂志,1998;11(9):772-773.
[90]卢衮伟,袁久荣,孙兆贵,等.四物汤及各单味药的含药血清对粒系-巨系造血祖细胞集落(CFU-GM)的影响.山东中医药大学学报,2000;24(5):385-386.
[91]章亭,谭允育,潘彦舒,等.四物汤对红细胞免疫及骨髓干细胞增殖能力的影响.北京中医药大学学报,2000;23(1):36-38.
[92]张荣华.补肾活血中药对免疫介导再障小鼠骨髓CD45~+细胞影响.中国病理生理杂志,2000;16(7):633-636.
[93]张新华,黄有文,王荣新,等.益髓生血灵对小鼠骨髓造血干/祖细胞的影响.浙江中医杂志,2002;37(10):448-449.
[94]陈志雄,曹克俭,薛恺睿,等.活髓片对环磷酰胺造模小鼠骨髓骨髓细胞DNA含量及细胞周期的影响.中医杂志,1999;40(11):686-687.
[95]吴志奎,姜葆华,陈双厚,等.采用细胞CT技术研究补肾生血方保护骨髓作用的分子基础.中药药理与临床,1995;(3):21-23.
[96]王树庆,李建华,张圣明,等.补肾化瘀方对再生障碍性贫血患者骨髓细胞DNA含量的影响.中国中西医结合杂志,1996;16(7):411-413.
[97]梁毅,陈志雄,丘和明,等.活髓片与四物汤对免疫介导再障小鼠骨髓基质细胞的影响比较.中国中西医结合急救杂志,2002;9(6):327-330.
[98]胡军祥,申屠垠,殷舒,等.中药对间质干细胞分化为神经细胞的影响.中草药, 2006;37(5),783-785.
[99]董晓先,刘金保,董燕湘,等.天麻诱导骨髓间质干细胞分化为神经元细胞的实验研究.中国中西医结合杂志,2004;24(1):51-54.
[100]张进,徐志伟.补肾法诱导间充质干细胞向神经方向分化研究.现代医院,2004;4(9):15-17.
[101]王勇,陆长青,王凡.黄芪诱导大鼠骨髓间充质干细胞分化为神经样细胞的研究.四川解剖学杂志,2006;14(1):5-8.
[102]刘金保,董晓先,董燕湘,等.多种中药成分诱导大鼠骨髓间质干细胞转变为神经元样细胞.中国药物与临床,2003;3(3):234-236.
[103]撒亚莲,李海标.川芎嗪诱导大鼠骨髓间质干细胞分化为神经元样细胞的研究.解剖学报,2003;34(5):514-517.
[104]肖庆忠,温冠媚,李浩威,等.麝香组分诱导成年大鼠骨髓间质干细胞体外定向分化为神经元样细胞的能力.中山医科大学学报,2002;23(6):405-408.
[105]董燕湘,董晓先,何慧华,等.大鼠骨髓间质干细胞用中药绞股蓝诱导为神经细胞的研究.中华神经科杂志,2003;36(5):355-358.
[106]张艳军,范祥,胡利民,等.不同治则中药单体对体外培养神经干细胞分化的影响.天津中医药,2004;21(2):156-157.
[107]卢昌均,陆兵勋,王立新,等.大鼠脑缺血再灌注损伤后神经干细胞的增殖分化以及通心络的干预效果.中国临床康复,2006;10(5):16-18.
[108]Liao Xin,Du Shao-hui,Chen Dong-feng.et al.Effect of niupozhibao weiwan on neural precursor cells after focal cerebral ischemic reperfusion.Chin J Clini Rehab,2004;8(28):6253-6255.
[109]刘柏炎,蔡光先,林琳,等.补阳还五汤对大鼠局灶性脑缺血后神经干细胞影响的初步研究.中国临床康复,2004;8(22):4532-4533.
[110]高唱,王景周.左归丸对体外培养新生大鼠海马神经干细胞增殖分化的影响.中国医药学报,2004;19(11):691-693.
[111]孔晓丹,曲鹏,王彦,等.三七皂甙诱导骨髓基质细胞分化为心肌样细胞的实验研究.中国心血管病研究杂志,2005;3(7):528-530.
[112]冼绍祥,杨忠奇,汪朝晖,等.黄芪甲苷体外诱导骨髓间充质干细胞分化为心肌样细胞的实验研究.广州中医药大学学报,2007;24(1):37-40.
[113]汪朝晖,冼绍祥,杨忠奇,等.人参总皂甙诱导骨髓间充质干细胞分化为心肌样细胞的实验研究.广州中医药大学学报,2006;23(2):100-103.
[114]杨忠奇,冼绍祥,汪朝晖,等.三七总皂苷对骨髓间充质干细胞分化为心肌样细胞的作用.中药新药与临床药理,2006;17(4):239-242.
[115]杨庆有,陆曙,龚少愚,等.益气温阳活血方对心肌梗死模型大鼠骨髓干细胞的 动员作用和左室重构的影响.中国中医药信息,2007;14(3):24-25.
[116]陈晓锋,唐礼江,朱敏,等.丹参酮ⅡA对外周血内皮祖细胞增殖、粘附和迁移功能的影响.中国药理学通报,2007,23(2):274-275.
[117]韩丽华,王振涛,索红亮,等.益气活血方促心梗后大鼠缺血心肌血管新生及对bFGF影响的研究.四川中医,2006;24(8):16-18.
[118]王宁元,吕传江,陈学海,等.人参皂甙Rg1诱导骨髓干细胞游走分化促进家兔心肌梗死后心肌血管内皮细胞再生的研究.中国中西医结合杂志,2005;25(10):916-919.
[119]王兴祥,尚云鹏,陈君柱,等.银杏叶提取物对外周血内皮祖细胞数量和功能的影响.药学学报,2004;39(8):56-660.
[120]张芙荣,陈君柱,朱军慧,等.葛根素对外周血内皮祖细胞数量和功能的影响.中国中药杂志,2004;29(8):771-781.
[121]刘小雨,王行宽,杨孝芳,等.从肝治心组方对急性心肌梗死大鼠心肌毛细血管密度的影响.中国中西医结合急救杂志,2004;11:17-20.
[122]张腾,曹洪欣,盛小禹,等.温心胶囊对大鼠实验性缺血心肌细胞凋亡及Bcl-2、Fas蛋白表达的影响.中国中西医结合杂志,2003;23(10):769-771.
[123]李连达,张荣利,刘成源,等.双龙方与自体骨髓单个核细胞经心导管移植对中国小型猪心肌梗死的研究.中国新药杂志,2003;12(12):999-1004.
[124]杨博华,秦建辉,朱龄群,等.自体骨髓干细胞移植合中药治疗下肢动脉缺血.浙江中西医结合杂志,2004;14(11):683-684.
[125]郑良朴,李楠,王和鸣.补骨合剂对体外培养骨髓基质细胞分化影响的观察.福建中医学院学报,2003;13(6):33-35.
[126]史风雷,李刚,王海彬,等.袁氏生脉成骨胶囊对激素诱导骨髓基质细胞成脂分化的拮抗作用.广州中医药大学学报,2003;20(4):302-304.
[127]曾意荣,樊粤光,刘红,等.补肾活血中药对大鼠骨髓间充质干细胞体外增殖的影响.中药新药与临床药理,2007;18(2):93-96.
[128]吴理达,李连达,刘红,等.自体骨髓干细胞移植与归元方联用治疗急慢性肝损伤实验研究.中国工程科学,2004;6(7):34-44.
[129]Fushs S,Baffour R,Zhou YF,et al.Transendocardial delivery of autologous bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia.J Am Coll Cardiol,2001;37:1726-1733.
[130]Kobayashi T,Hamano K,Li TS,et al.Enhancement of angiogenesis by the implantation of self bone marrow cells in a rat ischemic heart model.J Surg Res,2000;89(2):189-195.
[131]Nygren J,Jovinge S,Breitbach M,et al.Bone marrow-derived haematopoietic generate cardiomyocyte at a low frequency through cell fusion,but not transdifferentiation. Nat Med,2004;10:494-501.
[132]中华医学会心血管病学分会,中华心血管病杂志编辑委员会,中国循环杂志编辑委员会.急性心肌梗死诊断和治疗指南.中华心血管病杂志,2001;29(12):710-725.
[133]Wagner GS,Frege CJ,Palmeri ST,et al.Evaluation of a QRS scoring system for estimating infarct size:specificity and observer agreement.Circulation,1982;65:342-345.
[134]Yoshinko S,Staniloff HM,Shell WE,et al.Evaluation of a QRS scoring system on acute myocardial infarction:Relation to infarct size,early stage left vontricular eject fraction and exerxise performance.Am J cardiol,1983;52:37-40.
[135]Ideker E,Wagner GS,Ruth,et al.Evaluation of a QRS scoring for estimating myocardial infarct size 2 Correlation with quantitative anatomic findings for anterior infarct.Am J Cardiol,1982;49:I604.
[136]Seino Y,Staniloff HM,Shell WE,et al.Evaluation of a QRS scoring system in acutemyocardial infarction:relation to infarct size,early stage left Ventricular ejection fraction,and exercise performance.Am J Cardio 1,1983;52:37.
[137]刘德平,韩丽君,葛力等.QRS记分评价老年心肌梗塞患者的左室功能.中国心脏起搏与心电生理杂志,1997;11(2):74-75.
[138]王晓才,林谦,农一兵,等.138例冠心病的脏腑虚证分布与组合特点分析.中国中医基础医学杂志,2007;13(11):840-841.
[139]蒋梅光,张伯臾.以补法治疗老年冠心病的经验.上海中医药杂志,1989;(5):6-9.
[140]国家中医药管理局医政司胸痹急症协作组,东北分组.胸痹心厥(冠心病心肌梗塞)急症诊疗规范.中国中医急症,1995;4(4):183-186.
[141]张进,徐志伟,杜少辉,等.“精”学说与干细胞辨识.中医药学刊,2004;22(7):1198-1200.
[2]Tomita S,Li RK,Weisel RD,et al.Autologous transplantation of bone marrow cells improve damaged heart function.Circulation,1999;100(19supp1):247-256.
[3]Wang JS,Shum TD,Galipeau J,et al.Marrow stromal cells for cellular cardiomyoplasty:feasibility and potentiall clinical adventages.J Thorac Cardiovasc Surg,2000;120:999-1005.
[4]Goodell MA,Jackson KA,Majka SM,et al.Stem cell plasticity in muscle and bone marrow.ANN N Y Acad Sci,2001;938:208-218.
[5]Orlic D,kajstura J,Chimenti S,et al.Bone marrow cells regenerate infarcted myocardium.Nature,2001;410(6829):701-705.
[6]Jackson KA,Majka SM,Wang H,et al.Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cell.J Clin Invest,2001;107:1395-1402.
[7]Edward TH,Sui Zhang,Henry D,et al.Transdifferentiation of human peripheral blood CD34~+-enriched cell population into cardiomyocytes,endothelial cells,and smooth muscle cells in vivo.Circulation,2003;108(17):2070-2073.
[8]Askari AT,Unzek S,Popovic ZB,et al.Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy.Lancet,2003;362(9385):697-703.
[9]Kocher AA,Schuster MD,Szabolcs MJ,et al.Neovascularation of ischemic myocardium by human bone-marrow derived angioblasts prevents cardiomyocyte apoptosis,reduces remodeling and improves cardiac function.Nat Med,2001;7(4):430-436.
[10]Badorff C,Brandes RP,Popp R,et al.Transdifferentiation of blood-derived human adult endothelial progenitor cells into functionally active cardiomyocytes.Circulation,2003;107:1024-1032.
[11]Shintani S,Murohara T,Ikeda H,et al.Mobilization of endothelial progenito cells in patients with acute myocardial infarction.Circulation,2001;103(23):2776-2779.
[12]Ciulla MM,Lazzari L,Pacchiana R,et al.Homing of peripherally injected bone marrow cells in the rat after experimental myocardial injury.Haematologica,2003;88:614-621.
[13]邓争荣,杨春,马爱群,等.急性心肌梗死患者血浆VEGF、SDF-1和外周血CD34~+细胞的动态改变及其意义.南方医科大学学报,2006;26(11):1637-1640.
[14]牛丽丽,郑敏,曹丰,等.同种异体骨髓间充质干细胞在大鼠心脏的迁移及分化.中华医学杂志,2004;84(1):38-42.
[15]Hill JM,Dick AJ,Raman VK,et al.Serial cardiac magnetic resonance imaging of injected mesenchymal stem cells.Circulation,2003;108(8):1009-1014.
[16]Orlic D,Kajstura J,Chimenti S,et al.Mobilized bone marrow cells repair the infarcted heart,improving funetion and survival.Proc Natl Aead Sci.2001;98:10344-10349.
[17]Miki T,Sakamoto J,Nakano A,et al.Mobilization of bone marrow cells by G-CSF/GM-CSF improves ventricular function in the heart undergoing post-infarct remodeling.Circulation,2002;106(19 supp Ⅰ):15.
[18]Adachi Y,Imagawa J,Suzuki Y,Yogo K,Fukazawa M,Kuromaru O and Saito Y.G-CSF treatment increase side population cell infiltration after myocardial infarction in mice.J Mol Cell Cardiol,2004;36:707-710.
[19]Fukuhara S,Tomita S,Nakatani T,et al.G-CSF promotes bone marrow cells to migrate into infarcted Mice Heart,and Differentiate Into Cardiomyocytes.Cell Transplant,2004;13(7-8):741-748.
[20]Tang YL,Qian K,Zhang YC,et al.Mobilizing of haematopoietic stem cells to ischemic myocardium by plasmid mediated stromal-cell-derived factor-1alpha(SDF-1 alpha)treatment.Regul Pept.2005;125(1-3):1-8.
[21]陈运贤,欧瑞明,钟雪云,等.粒细胞集落刺激因子干预性治疗大鼠心肌梗塞的实验研究.中国病理生理杂志,2001;17(12):1153-1155.
[22]陈运贤,欧瑞明,钟雪云,等.粒细胞集落刺激因子动员骨髓干细胞治疗大鼠急性心肌梗塞.中国病理生理杂志,2002;18(1):1-3.
[23]陈运贤,欧瑞明,钟雪云,等.CD34~+细胞的心肌细胞分化潜能研究.中国病理生理杂志,2002;18(2):117-119.
[24]李连达,张荣利,张旋,等.聚乙二醇重组人粒细胞集落刺激因子对大鼠心肌梗死的影响.中国新药杂志,2003;9(12):721-724.
[25]郭军,贾光宏,林国生,等.体外模拟心肌微环境下骨髓间充质干细胞向心肌样细胞分化的实验研究.心脏杂志,2004;15(6):501-504.
[26]路璐,彭建军,胡大一,等.骨髓动员与细胞移植对直接经皮腔内冠状动脉成形术后急性心肌梗死治疗作用.中国心血管病研究杂志,2004;7(2):557-560.
[27]黄国倩,刘虹,舒先红,等.超声心动图评价粒细胞集落刺激因子动员自身骨髓干细胞对心肌梗死后左心室重构及心功能的影响.中华超声影像学杂志,2004;11(13):848-852.
[28]郭素萍,高传玉,刘煜昊.骨髓干细胞动员剂对大鼠急性心肌梗死心肌肌钙蛋白Ⅰ的影响.实用诊断与治疗杂志,2005;2(19):114-115.
[29]刘宏伟,盖鲁粤,张端珍,等.粒细胞集落刺激因子骨髓干细胞动员治疗兔心肌梗死的实验研究.军医进修学院学报,2005;26(2):132-134.
[30]毛文凯,刘志勇.自体骨髓干细胞动员治疗心肌梗死.现代医学,2005;3(33):154-157.
[31]林玲,周胜华,祁述善,等.粒细胞集落刺激因子和干细胞因子对急性心肌梗死后心室重构的影响.中国介入心脏病学杂志,2005;13(3):178-181.
[32]Ohtsuka M,Takano H,Zou Y,et al.Cytokine therapy prevents left ventricular remodeling and dysfunction after myocardial infarction through neovascularization.FASEB J,2004;18(7):851-853.
[33]Laudmesser U,Engberding N,Bahlmann FH,et al.Statin-indueed improvement of endothelial progenitor cell mobilization,myocardial neovascularization,left ventricular function,and survival after experimental myocardial infarction requires endothelial nitric oxide synthase.Circulation,2004;110(14):1933-1939.
[34]姜宇,何力鹏,王农荣,等.辛伐他汀对大鼠外周血单个核CD34~+细胞动员作用的研究.武警医学,2004;15(10):740-743.
[35]吴贤仁,杨敏,李玉光.骨髓干细胞动员对大鼠缺血心肌的治疗作用.中国免疫学杂志,2004;7(20):471-474.
[36]李宾公,曾秋棠,晏凯利.骨髓干细胞动员治疗大鼠急性心肌梗死的实验研究.中国急救医学,2005;4(25),268-271.
[37]Seiler C,Pohl T,Wustmann K,et al.Promotion of colleteral growth by granulocyte macrophage colony-stimulating factor in patients with coronary artery disease:a randomized,double-blind,placebo-controlled study.Circulation,2001;104:2012-2017.
[38]Kang HJ,Kim HS,Zhang SY,et al.Effects of intraeoronary infusion of peripheral blood stem-cells mobilised with granulocyte-colony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction:the MAGIC cell randomised clinical trial.Lancet,2004;363(9411):751-756.
[39]Kang HJ,Lee HY,Na SH,et al.Myocardial infarction:the MAGIC cell-3-DES randomized,controlled trial and remodeling in patients with acute myocardial infarction versus old cells by granulocyte colony-stimulating factor on left ventricular function differential effect of intracoronary infusion of mobilized peripheral blood stem.Circulation,2006;114:145-151.
[40]Ince H,Petzsch M,Kleine HD,et al.Prevention of left ventrieular remodeling with granulocyte colony-stimulating factor after acute myocardial infarction:final 1-year results of the Front-Integrated Revaseularization and Stem Cell Liberation in Evolving Acute Myocardial Infarction by Granulocyte Colony-Stimulating Factor(FIRSTLINE-AMI)Trial. Circulation,2005;112(9 Suppl):173-180.
[41]Kuethe F,Figulla HR,Voth M,et al.Mobilization of stem cells by granulocyte colony-stimulating factor for the regeneration of myocardial tissue after myocardial infarction.Dtsch Med Woehenschr,2004;129(9):424-428.
[42]Huseyin Ince,Michael Petzsch,Hans Dieter Kleine,et al.Prevention of left ventricular remodeling with Granulocyte Colony-Stimulating factor after acute myocardial infarction.Circulation,2005;112:I-73-I-80.
[43]Valgimigli M,Rigolin GM,Cittanti C,et al.Use of granulocyte-colony stimulating factor during acute myocardial infarction to enhance bone marrow stem cell mobilization in humans:clinical and angiographic safety profile.Eur Heart J,2005;26(18):1838-1845.
[44]Suarez de Lezo J,Torres A,Herrera I,et al.Effects of stem-cell mobilization with recombinant human granulocyte colony stimulating factor in patients with percutaneously revascularized acute anterior myocardial infarction.Rev Esp Cardiol,2005;58(3):253-261.
[45]陈运贤,欧瑞明,钟雪云,等.自体骨髓造血干细胞原位移植治疗急性心肌梗死的临床研究.中国病理生理杂志,2003;19(4):452-454.
[46]徐新,马绍椿,罗东云,等.应用G-CSF动员骨髓干细胞治疗急性心肌梗死的研究.中国分子心脏病学杂志,2003;4(3):223-225.
[47]佟铁壁.集落刺激因子动员骨髓干细胞治疗心肌梗死临床观察.辽宁医学杂志,2006;20(2):99-100.
[48]马绍椿,徐新,罗东云,等.粒细胞集落刺激因子动员骨髓干细胞治疗急性心肌梗死的观察.中西医结合心脑血管病杂志,2004;2(2):63-65.
[49]Norol F,Merlet P,Isnard R,et al.Influence of mobilized stem cells on myocardial infarct repair in a nonhuman primate model.Blood,2003;102(13):4361-4368.
[50]Ripa RS,Jφgensen E,Wang Y,et al.Stem cell mobilization induced by subcutaneous granulocyte-colony stimulating factor to improve cardiac regeneration after acute ST-elevation myocardial infarction:result of the double-blind,randomized,placebo-controlled stem cells in myocardial infarction(STEMI)trial.Circulation,2006;113(16):1983-1992.
[51]Zohlnhofer D,Ott I,Mehilli J,et al.Stem cell mobilization by granulocyte colony-stimulating factor in patients with acute myocardial infarction:a randomized controlled trial.JAMA,2006;295:1003-1010.
[52]范文茂,陆东风,张东成,等.人重组粒细胞刺激因子治疗动物模型急性心肌梗死的病理学观察.血栓与止血学,2004;10(4):172-174.
[53]庄俊合,邓志刚,陆东风,等.人重组粒细胞集落刺激因子对急性心肌梗死患者心 功能恢复的疗效观察.血栓与止血学,2005;11(3):109-111.
[54]Balsam LB,Wagers A.J,Christensen JL,et al.Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium.Nature,2004;428(6983):668-673.
[55]Murry CE,Soonpaa MH,Reinecke H,et al.Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts.Nature,2004;428(6983):664-668.
[56]Alvarez-Dolado M,Pardal R,Gareia-Verdugo JM,et al.Fusion of bone-marrow-derived cells with purkinje neurons,cardiomyocytes and hepatocytes.Nature,2003;425(10):968-973.
[57]Kronenwett R,Martin S,Hass R.The role of cytokines and adhesion molecules for mobilization of peripheral blood stem cells.Stem ceil,2000;18(5):320-330.
[58]Li J,Brown LF,Hibberd MG,et al.VEGF,FIK-1 and Flt-1 expression in rat myocardial model of angiogenesis.Am J Physiol,1996;270:H1803-H1181.
[59]Scarborough J E,Smith M L.Basic fibroblast growth factor is upregulated in hibernating myoearditun.Surg Res,2002;107(1):119-123.
[60]Levi-Sehaffer F,Garbuzenko E,Rubin A,et al.Human eosinophils regulate human lung-and skin-derived fibroblast properties in vitro:a role for transforming growth factor beta(TGF-beta).Proc Natl Aead Sci USA,1996;96(17):9660-9665.
[61]Lichterfeld M,Martin S,Burkly L,et al.Mobilization of CD34~+ haematopoietic stem cells is associated with a functional inactivation of the integrin very late antigen 4.Br J Haematol,2000;110(1):71-81.
[62]Levesque JP,Takamatsu Y,Nilsson SK,et al.Vascular cell adhesion molecule-1(CD106)is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor.Blood,2001;98(5):1289-1297.
[63]翁建宇.G-CSF对造血干细胞的动员机制及对健康供者的影响.国外医学内科学分册,2002;29(8):352-355.
[64]Orlic D.Stem cell repair in isehemic heart disease:an experimental model.Iut J Hematol,2002,7(6 Suppl 1):S144-S145.
[65]Wright DE,Bowman EP,Wagers AJ,et al.Hematopoietie stem cells are uniquely selective in their migratory response to ehemokines.J Exp Med,2002;195(9):1145-1154.
[66]Satoshi S,Toyoaki M,Hisao I,et al.Mobilization of endothelial progenitor cells in patients with acute myocardial infarction.Circulation,2001;103(12):2776-2779.
[67]Rafii S,Heissig B,Hattori K.Efficient mobilization and recruitment of marrow-derived endothelial and hematopoietic stem cells by adenoviral vectors expressing angiogenic factors.Gene Ther,2002;9(10):631-641.
[68]Watt FM,Hogan B.Out of Eden:stem cells and their niches.Science,2000;287:1427-1430.
[69]Condorelli G,Borello U,De Angelis L,et al.Cardiomyocytes induce endothelial cells to transdifferentiate into cardiac muscle:implications for myocardium regeneration.Proc Natl Acad Sci U S A.2001;98:10733-10738.
[70]Tang YL,Zhao Q,Zhang Y,et al.Autologous mesenchymal stem cell transplantation induce VEGF and neovaseularization in ischernic myocardium.Regul Peptides,2004;117:3-10.
[71]尹瑞兴,李佳荃,蔡捷,等.血管内皮生长因子对急性心肌梗死大鼠心肌细胞凋亡的影响.中华老年医学杂志,2003;22(2):98-101.
[72]张少衡,贾竹青,郭静萱,等.骨髓细胞移植上调急性缺血心肌HSP32和HSP70的表达.北京大学学报(医学报),2003;35(5):476-480.
[73]Engelhardt M,Bertz H,Wasch R,et al.Analysis of stem cell apheresis products using intermediate-dose filgraseim plus large volume apheresis for allogeneic transplantation.Ann Hematol,2001;80:201-208.
[74]Hill JM,Zalos G,Halcox J P,et al.Circulating endothelial progenitor ceils,vascular function,and cardiovascular risk.N Engl J Med,2003;348:593-600.
[75]刘煜昊,房佰俊,方五旺,等.急性心肌梗死患者骨髓间充质干细胞生物学特性改变的研究.临床心血管病杂志,2006;22(1):44-46.
[76]Shimoda K Okawura S,Inaba S,et al.Granulocyte colony-stimulating factor and platelet aggregation.Lancet,1993;338:633.
[77]北京市造血干细胞移植协作组,应用重组人粒细胞集落刺激因子的经验与教训专题讨论.白血病·淋巴瘤,2007;16(1):1-5.
[78]Hill JM,Paul J-D,Powell TM,et al.Efficacy and risk of granulccyte colony stimulating factor administration in patients with servere coronary artery disease.Circulation,2003;108(Suppl Ⅳ):478-483.
[79]Stroncek DF,Clay ME,Petzoldt ML,et al.Treatment of normal individuals with G-CSF:donor experiences and the effects on peripheral blood CD34~+ cell counts and on the collection of peripheral blood stem cells.Transfusion,1996;36:601-610.
[80]Anderlini P,Przepiorka D,Seong D,et al.Clinical toxicity and laboratory effects of granulocyte-colony-stimulating factor(filgrastim)mobilization and blood stem cell apheresis from normal donors,and analysis of charges for the procedures.Transfusion,1996;36:590-595.
[81]Stroncek DF,Clay ME,Jaszez W,et al.Collection of teo peripheral blood stem cell concentrates from healthy donors.Transfus Med,1999;9:37-50.
[82]张仲平,洪介民.黄芪多糖对体外入骨髓造血祖细胞生成的影响.中药药理与临床,2000;16(1):16-17.
[83]刘晓,武正炎,范萍.黄芪在外周血造血干细胞移植中应用的初步实验研究.镇江医学院学报,2001;11(1):15-17.
[84]王晓东,陈孟莉,殷建芬,等.鸡血藤活性成分SS8对骨髓抑制大鼠造血祖细胞增殖的作用.中国中药杂志,2003;28(2):152-154.
[85]钱煦岱,高瑞兰,马珂,等.三七皂甙对人骨髓CD34~+造血干/祖细胞增殖分化作用.中国实验血液学杂志,2003;11(2):120-123.
[86]孙汉英,房明皓,任天华,等.川芎嗪对骨髓移植小鼠骨髓造血的影响.中国中西医结合杂志,2002;22(5):365-368.
[87]刘文励,黄伟,何美冬,等.复方活血汤对辐射损伤大鼠骨髓基质细胞粘附功能的影响.中国中西医结合杂志,1997;17(6):354-355.
[88]王炳胜,刘秀芳,王涛,等.益气活血方对放射损伤骨髓基质细胞生长的影响.中华放射医学与防护杂志,2000;20(3):190-191.
[89]牛泱平,金锦梅,高瑞兰,等.当归注射液对正常人和再生障碍性贫血患者造血祖细胞的刺激增殖作用.实用中西医结合杂志,1998;11(9):772-773.
[90]卢衮伟,袁久荣,孙兆贵,等.四物汤及各单味药的含药血清对粒系-巨系造血祖细胞集落(CFU-GM)的影响.山东中医药大学学报,2000;24(5):385-386.
[91]章亭,谭允育,潘彦舒,等.四物汤对红细胞免疫及骨髓干细胞增殖能力的影响.北京中医药大学学报,2000;23(1):36-38.
[92]张荣华.补肾活血中药对免疫介导再障小鼠骨髓CD45~+细胞影响.中国病理生理杂志,2000;16(7):633-636.
[93]张新华,黄有文,王荣新,等.益髓生血灵对小鼠骨髓造血干/祖细胞的影响.浙江中医杂志,2002;37(10):448-449.
[94]陈志雄,曹克俭,薛恺睿,等.活髓片对环磷酰胺造模小鼠骨髓骨髓细胞DNA含量及细胞周期的影响.中医杂志,1999;40(11):686-687.
[95]吴志奎,姜葆华,陈双厚,等.采用细胞CT技术研究补肾生血方保护骨髓作用的分子基础.中药药理与临床,1995;(3):21-23.
[96]王树庆,李建华,张圣明,等.补肾化瘀方对再生障碍性贫血患者骨髓细胞DNA含量的影响.中国中西医结合杂志,1996;16(7):411-413.
[97]梁毅,陈志雄,丘和明,等.活髓片与四物汤对免疫介导再障小鼠骨髓基质细胞的影响比较.中国中西医结合急救杂志,2002;9(6):327-330.
[98]胡军祥,申屠垠,殷舒,等.中药对间质干细胞分化为神经细胞的影响.中草药, 2006;37(5),783-785.
[99]董晓先,刘金保,董燕湘,等.天麻诱导骨髓间质干细胞分化为神经元细胞的实验研究.中国中西医结合杂志,2004;24(1):51-54.
[100]张进,徐志伟.补肾法诱导间充质干细胞向神经方向分化研究.现代医院,2004;4(9):15-17.
[101]王勇,陆长青,王凡.黄芪诱导大鼠骨髓间充质干细胞分化为神经样细胞的研究.四川解剖学杂志,2006;14(1):5-8.
[102]刘金保,董晓先,董燕湘,等.多种中药成分诱导大鼠骨髓间质干细胞转变为神经元样细胞.中国药物与临床,2003;3(3):234-236.
[103]撒亚莲,李海标.川芎嗪诱导大鼠骨髓间质干细胞分化为神经元样细胞的研究.解剖学报,2003;34(5):514-517.
[104]肖庆忠,温冠媚,李浩威,等.麝香组分诱导成年大鼠骨髓间质干细胞体外定向分化为神经元样细胞的能力.中山医科大学学报,2002;23(6):405-408.
[105]董燕湘,董晓先,何慧华,等.大鼠骨髓间质干细胞用中药绞股蓝诱导为神经细胞的研究.中华神经科杂志,2003;36(5):355-358.
[106]张艳军,范祥,胡利民,等.不同治则中药单体对体外培养神经干细胞分化的影响.天津中医药,2004;21(2):156-157.
[107]卢昌均,陆兵勋,王立新,等.大鼠脑缺血再灌注损伤后神经干细胞的增殖分化以及通心络的干预效果.中国临床康复,2006;10(5):16-18.
[108]Liao Xin,Du Shao-hui,Chen Dong-feng.et al.Effect of niupozhibao weiwan on neural precursor cells after focal cerebral ischemic reperfusion.Chin J Clini Rehab,2004;8(28):6253-6255.
[109]刘柏炎,蔡光先,林琳,等.补阳还五汤对大鼠局灶性脑缺血后神经干细胞影响的初步研究.中国临床康复,2004;8(22):4532-4533.
[110]高唱,王景周.左归丸对体外培养新生大鼠海马神经干细胞增殖分化的影响.中国医药学报,2004;19(11):691-693.
[111]孔晓丹,曲鹏,王彦,等.三七皂甙诱导骨髓基质细胞分化为心肌样细胞的实验研究.中国心血管病研究杂志,2005;3(7):528-530.
[112]冼绍祥,杨忠奇,汪朝晖,等.黄芪甲苷体外诱导骨髓间充质干细胞分化为心肌样细胞的实验研究.广州中医药大学学报,2007;24(1):37-40.
[113]汪朝晖,冼绍祥,杨忠奇,等.人参总皂甙诱导骨髓间充质干细胞分化为心肌样细胞的实验研究.广州中医药大学学报,2006;23(2):100-103.
[114]杨忠奇,冼绍祥,汪朝晖,等.三七总皂苷对骨髓间充质干细胞分化为心肌样细胞的作用.中药新药与临床药理,2006;17(4):239-242.
[115]杨庆有,陆曙,龚少愚,等.益气温阳活血方对心肌梗死模型大鼠骨髓干细胞的 动员作用和左室重构的影响.中国中医药信息,2007;14(3):24-25.
[116]陈晓锋,唐礼江,朱敏,等.丹参酮ⅡA对外周血内皮祖细胞增殖、粘附和迁移功能的影响.中国药理学通报,2007,23(2):274-275.
[117]韩丽华,王振涛,索红亮,等.益气活血方促心梗后大鼠缺血心肌血管新生及对bFGF影响的研究.四川中医,2006;24(8):16-18.
[118]王宁元,吕传江,陈学海,等.人参皂甙Rg1诱导骨髓干细胞游走分化促进家兔心肌梗死后心肌血管内皮细胞再生的研究.中国中西医结合杂志,2005;25(10):916-919.
[119]王兴祥,尚云鹏,陈君柱,等.银杏叶提取物对外周血内皮祖细胞数量和功能的影响.药学学报,2004;39(8):56-660.
[120]张芙荣,陈君柱,朱军慧,等.葛根素对外周血内皮祖细胞数量和功能的影响.中国中药杂志,2004;29(8):771-781.
[121]刘小雨,王行宽,杨孝芳,等.从肝治心组方对急性心肌梗死大鼠心肌毛细血管密度的影响.中国中西医结合急救杂志,2004;11:17-20.
[122]张腾,曹洪欣,盛小禹,等.温心胶囊对大鼠实验性缺血心肌细胞凋亡及Bcl-2、Fas蛋白表达的影响.中国中西医结合杂志,2003;23(10):769-771.
[123]李连达,张荣利,刘成源,等.双龙方与自体骨髓单个核细胞经心导管移植对中国小型猪心肌梗死的研究.中国新药杂志,2003;12(12):999-1004.
[124]杨博华,秦建辉,朱龄群,等.自体骨髓干细胞移植合中药治疗下肢动脉缺血.浙江中西医结合杂志,2004;14(11):683-684.
[125]郑良朴,李楠,王和鸣.补骨合剂对体外培养骨髓基质细胞分化影响的观察.福建中医学院学报,2003;13(6):33-35.
[126]史风雷,李刚,王海彬,等.袁氏生脉成骨胶囊对激素诱导骨髓基质细胞成脂分化的拮抗作用.广州中医药大学学报,2003;20(4):302-304.
[127]曾意荣,樊粤光,刘红,等.补肾活血中药对大鼠骨髓间充质干细胞体外增殖的影响.中药新药与临床药理,2007;18(2):93-96.
[128]吴理达,李连达,刘红,等.自体骨髓干细胞移植与归元方联用治疗急慢性肝损伤实验研究.中国工程科学,2004;6(7):34-44.
[129]Fushs S,Baffour R,Zhou YF,et al.Transendocardial delivery of autologous bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia.J Am Coll Cardiol,2001;37:1726-1733.
[130]Kobayashi T,Hamano K,Li TS,et al.Enhancement of angiogenesis by the implantation of self bone marrow cells in a rat ischemic heart model.J Surg Res,2000;89(2):189-195.
[131]Nygren J,Jovinge S,Breitbach M,et al.Bone marrow-derived haematopoietic generate cardiomyocyte at a low frequency through cell fusion,but not transdifferentiation. Nat Med,2004;10:494-501.
[132]中华医学会心血管病学分会,中华心血管病杂志编辑委员会,中国循环杂志编辑委员会.急性心肌梗死诊断和治疗指南.中华心血管病杂志,2001;29(12):710-725.
[133]Wagner GS,Frege CJ,Palmeri ST,et al.Evaluation of a QRS scoring system for estimating infarct size:specificity and observer agreement.Circulation,1982;65:342-345.
[134]Yoshinko S,Staniloff HM,Shell WE,et al.Evaluation of a QRS scoring system on acute myocardial infarction:Relation to infarct size,early stage left vontricular eject fraction and exerxise performance.Am J cardiol,1983;52:37-40.
[135]Ideker E,Wagner GS,Ruth,et al.Evaluation of a QRS scoring for estimating myocardial infarct size 2 Correlation with quantitative anatomic findings for anterior infarct.Am J Cardiol,1982;49:I604.
[136]Seino Y,Staniloff HM,Shell WE,et al.Evaluation of a QRS scoring system in acutemyocardial infarction:relation to infarct size,early stage left Ventricular ejection fraction,and exercise performance.Am J Cardio 1,1983;52:37.
[137]刘德平,韩丽君,葛力等.QRS记分评价老年心肌梗塞患者的左室功能.中国心脏起搏与心电生理杂志,1997;11(2):74-75.
[138]王晓才,林谦,农一兵,等.138例冠心病的脏腑虚证分布与组合特点分析.中国中医基础医学杂志,2007;13(11):840-841.
[139]蒋梅光,张伯臾.以补法治疗老年冠心病的经验.上海中医药杂志,1989;(5):6-9.
[140]国家中医药管理局医政司胸痹急症协作组,东北分组.胸痹心厥(冠心病心肌梗塞)急症诊疗规范.中国中医急症,1995;4(4):183-186.
[141]张进,徐志伟,杜少辉,等.“精”学说与干细胞辨识.中医药学刊,2004;22(7):1198-1200.