补肾活血中药对膝OA患者生存质量影响及相关实验研究
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摘要
研究目的
膝骨性关节炎(OA)是中老年常见的疾病,属于中医“骨痹”范畴,其主要病机为肾虚、血瘀。以补肾活血法治疗膝骨性关节炎取得良好疗效,前期临床研究显示补肾活血中药与塞来昔布具有相似的临床疗效,无统计学差异。然而,随着医学模式的发展,单纯的着眼于改善临床症状的疗效标准已经不能满足患者的要求。因疾病所导致的生理功能、心理功能、生理角色、社会角色等生存质量方面的损害越来越受到重视。本研究拟观察补肾活血中药(关节康片)对膝骨性关节炎患者生存质量(QOL)的影响,试将生存质量评价引入中医临床试验进行推广、应用,从而改善中医药界临床疗效评定指标的构成,以更好地验证中医药的疗效。
现代医学研究表明,软骨下微骨折在膝OA发病中起着重要作用。本研究拟通过实验证实补肾中药可能通过促进BMSCs增殖并诱导其向成骨细胞分化,促进软骨下微骨折的修复,从而阻断因此引起的致炎性成份及生长因子的产生增多和炎性抑制因子的产生减少这一病理过程。探讨关节康治疗膝OA的机制。
中医认为“肾主骨生髓”,现代医学主要从骨代谢本身进行研究。研究也发现,骨髓间充质干细胞(BMSCs)特性与“肾主骨”存在相关性。基于杜仲在补肾药物中的重要作用,本研究选择关节康片中药物组成的君药-杜仲作为实验对象,观察杜仲含药血清对BMSCs增殖的影响,及诱导BMSCs向成骨细胞分化结果；从细胞学的角度阐述“肾主骨生髓”的理论基础；解释杜仲补肾作用的机理。
当前通常从基因组水平研究干细胞的生物学特性。但是,以转录水平的分析研究细胞生物学功能往往不够充分,因为细胞决定于蛋白质,正是那些参与代谢和调控的蛋白质决定细胞的存活。本研究通过蛋白质组学研究方法,建立杜仲诱导BMSCs向成骨细胞分化功能蛋白质组,分析BMSCs向成骨细胞分化的分子机制。研究方法
所有入选病例均来自广州中医药大学第一附属医院骨科门诊首诊病人,共55例,利用随机表分为关节康组与塞来昔布组。所有患者在治疗前后填写SF-36生存质量调查问卷,计算治疗前后各组得分,组内比较采用t检验,治疗前后得分差异的组间比较采用两样本t检验。
从SD大鼠双下肢股骨、胫骨分离BMSCs,通过全骨髓法进行培养、胰酶消化纯化、扩增。以第4代细胞作为实验细胞。
实验分为3组,分为空白组、地塞米松组、杜仲组。将第四代BMSCs以5×106接种于9块96孔板,共3组,每组8个复孔；2块6孔板,每板6孔,每组2个复孔。2天后换液,加入诱导液。空白组加入含10%胎牛血清(FBS)的L-DMEM培养基继续培养,地塞米松组在基础培养基中上加入诱导剂(含β甘油磷酸钠10mmol／L、地塞米松0.1μmol／L、维生素C50mg／L),杜仲组为含10%杜仲含药血清的L-DMEM培养基。分别自加诱导液第1天起,采用MTT法测量96孔板细胞数(OD值),每天1块板,连续测量9天,以每天OD值平均值描绘细胞增殖曲线；在第10天取出1块6孔板做ALP染色；第20天做6孔板茜素红染色。
收集杜仲含药血清干预前、于预后第2天、第7天、第14天的细胞,采用去污剂裂解法提取总蛋白,去除杂质,并采用bradford法测定各样品中蛋白浓度。通过双向电泳对蛋白进行分离,采用PDQuest-7.3.0胶图分析软件进行分析找出差异蛋白,并进行质谱分析、数据库查询建立功能蛋白质组。分析杜仲诱导BMSCs向成骨细胞分化分子机制。研究结果
关节康组与塞来昔布组在治疗后各维度积分都有提高,具有统计学意义。表明关节康与塞来昔布治疗膝OA均可显著提高患者生存质量。但组间从各个不同的维度却有不同的结果,两组在改善生理功能、躯体疼痛方面不具有可比性,与之前的临床疗效观察结果一致,而关节康组在改善生理职能、总体健康、活力、社会功能、情感职能及精神健康方面优于塞来昔布组。
通过全骨髓培养法从SD大鼠骨髓中分离到BMSCs,通过胰酶消化、传代可进一步纯化。杜仲含药血清组细胞OD值呈现逐渐增高的趋势,而空白组和地塞米松组无明显增高的趋势；诱导后第10天,杜仲组及地塞米松组ALP染色阳性；第13天,培养板底部可见白色钙结节；第20天,茜素红染色阳性。
经bradford法测定各组总蛋白浓度分别为0天3.24ug／ul,2天4.10ug／ul,7天4.7ug／ul,14天7.27ug／ul。说明杜仲含药血清能够促进细胞增殖。
双向电泳对蛋白进行分离,得到4张图谱,从整个图谱来分析,点与点之间区分比较明显,可以对其进行蛋白质表达的差异分析。采用PDQuest-7.3.0胶图分析软件进行软件分析,找出差异蛋白点共641个,选取其中75个点行质谱分析并进行数据库查询。其中6个点具有积极意义。分别为：
F18,基质细胞衍生因子(SDF-1),可作为检测MSCs分化程度的标志。
G21,过氧化物歧化酶(SOD),属于金属酶,是一种主要的抗氧化酶,能清除超氧化物自由基,在防御氧的毒性、抑制老年疾病以及预防衰老等方面起着重要作用；
E17,钙网织蛋白(CRT),参与钙的贮藏和信号转导；细胞黏附；基因表达的调控等；
E4,波形蛋白(vimentin),是间充质来源细胞的中间纤维蛋白,附在细胞核、内质网及线粒体的旁边或末端,因而相信波形蛋白在支撑及锚固原生质内的细胞器有着重要的角色,它亦能维持细胞的形状、细胞质的完整性及稳定细胞骨架内的相互作用；
F6,核纤层蛋白A(lamin A),与波形蛋白同属于中间纤维蛋白的一种,是所有真核细胞核结构所必需的结构蛋白,主要参与构成核的功能,在核形成过程中起着重要作用。
F3,膜联蛋白A6(annexin A6),在细胞中参与膜转运及膜表面一系列依赖于钙调蛋白的活动,包括囊泡运输、胞吐作用中的膜融合、信号传导和钙离子通道的形成、调控炎症反应、细胞分化和细胞骨架蛋白间的相互作用等。研究结论
中医治疗以辨证论治为基础,综合考虑了人体各个系统的机能状态,从多环节对人体整体机能状态进行调节,这样在治疗现代社会病因复杂的慢性身心疾病时便具有了较大的优势。将QOL评价引入中医临床试验进行推广、应用,可以改善中医药界临床疗效评定指标的构成,以更好地验证中医药的疗效,从而更好发展与宏扬祖国医学。
但目前各生存质量量表繁多,均有各自适用范围。本研究采用SF-36表进行研究,此表为普适性生存质量量表,虽然从不同的角度反应疾病对患者生存质量的影响,但对于膝骨性关节炎患者而言,部分条目不能很好反应该疾病对生存质量的影响。故研究一个膝骨性关节炎的生存质量量表是我们进一步研究的目标。
关节康可能通过促进软骨下微骨折的修复,从而阻断因此引起的致炎性成份及生长因子的产生增多和炎性抑制因子的产生减少这一病理过程,达到治疗膝OA的作用。
经活体提取骨髓能更有效地获取BMSCs,有利于BMSCs的贴壁生长、分裂增殖。全骨髓培养法简单有效,细胞的密度影响着细胞的扩增。杜仲含药血清能促进BMSCs增殖并诱导BMSCs定向成骨细胞分化,在分化过程中,细胞密度起着重要作用。BMSCs向成骨细胞分化是一个复杂的过程,实验认为补肾杜仲可能通过上调波形蛋白、核纤层蛋白A的表达促进细胞分化,下调钙网织蛋白的表达,释放细胞内钙,参与钙结节的形成,与体内骨矿化过程有关。过氧化物歧化酶、与膜联蛋白A6与细胞的活力有关,调节着细胞的分裂增殖与分化,但是否与定向成骨细胞分化有着密切关系尚有待进一步研究。
Objective
Knee osteoarthritis is a common disease in the elderly, connects with rheumatism involving bone in Chinese medicine, the main pathogenesis are renal deficiency and blood stasis. Treating knee osteoarthritis with Kidney-Supplementing Blood-Quickening Chinese medicine gained good outcome, clinical studies before showed that Traditional Chinese medicine and celecoxib had similar clinical efficacy, no significant difference. However, with the development of medical model, the efficacy standard only focusing on improving the clinical symptoms can not meet patients'requirements. Damage of physiological function, psychological function, physical role, social role, et al, involve in the quality of life (QOL) caused by illness received more and more attention. This study was to observe the effect of QOL of patients with knee osteoarthritis treated with Kidney-Supplementing Blood-Quickening Chinese medicine(Guan Jie Kang), try to introduce the QOL assessment to clinical trials of Chinese medicine and promote and apply, in order to modify composition of clinical evaluation index in Chinese medicine, and to better verify the efficacy of Chinese medicine.
Modern medical research showed that subchondral micro-fractures played an important role in the pathogenesis of knee OA. This study was to confirm that by experimental Kidney-Supplementing medicine might promote the repair of subchondral micro-fracture by promotion of proliferation of BMSCs and induction them to differentiation into osteoblasts, to block the pathological process that the increase of inflammatory components and growth factors, and induce of the inflammatory inhibitors. In order to ecplore the mechanism of treatment KOA by Guan Jie Kang.
Chinese medicine considers the kidney generating marrow and dominating bone, which was studied mainly on the bone metabolism itself by modern medical research. Studies also found that features of mesenchymal stem cells (MSCs) had relationship with "the kidney being in charge of bone". Based on the important role of Duzhong(Cortex Eucommiae) in the Chinese medicine invigorating the kidney, the study chose Duzhong (Cortex Eucommiae) which was the Sovereign drug in Guan Jie Kang tables as the research object to observe proliferation of BMSCs induced by serum containing Duzhong (Cortex Eucommiae), and differentiation results of BMSCs into osteoblasts, in order to expound the theory of "the kidney generating marrow" from cytology and to explain the mechanism of Duzhong(Cortex Eucommiae) invigorating the kidney.
Usually biological characteristics of stem cells were researched at the level of genome. However, the analysis of biological function by transcription was often not sufficient, because the cells depends on protein, it is protein involved in metabolism and regulation that decided cell survival. This study used proteomics research methods to establish functional protein of BMSCs differentiating into osteoblasts induced by Duzhong (Cortex Eucommiae), and analyze molecular mechanism of BMSCs differentiating into osteoblasts. Methods
All the cases who attending out-patient first were selected from the department of Orthopaedics of the First Affiliated Hospital of Guangzhou University of Traditional Chinese medicine, there was a total of 55 cases, all the cases were divided into Guan Jie Kang group and the celecoxib group by random table. Before and after treatment all patients completed SF-36 quality of life questionnaire to calculate scores in each group, t test was used to compare before and after treatment within group, two sample t test was used to compare score difference between the two groups.
BMSCs were isolated from the two femurs and tibias of SD rats, cultured by the whole bone marrow method in vitro, purification and amplification by trypsin digestion. The quartus generation cells were used as the experimental cells.
The experiment was divided into 3 groups named blank group, DXM group and Duzhong (Cortex Eucommiae) group. Inoculated quartus generation MSCs in nine nity-six-holes cell culture plates and two six-holes cell culture plates by 5×106 cells per milliliter, all the holes were divided into three groups, each group had eight holes in nity-six-holes cell culture plates and two holes in six-holes cell culture plates. Two days later, changed medium and add new medium. The medium of DXM group contained 10mmol/Lβ-sodium glycerophosphate, 0.lumol/L DXM,50mg/L vitamin C, that of Duzhong (Cortex Eucommiae) group contained 10% serum containing Duzhong (Cortex Eucommiae). Measured cell number by MTT of nity-six-holes plates from the second day to the eighth day, one of the six-holes plates was colored by ALP staining solution at the tenth day, the last one was colored by alizarin bordeaux at the twentieth day.
Extracted total protein of cells induced by serum containing Duzhong (Cortex Eucommiae) for 0day,2days,7days,14days, and runouted them by two-dimensional electrophoresis, searched for different protein spot by PDQuest-7.3.0 software for analyzing gel picture, to establish functional proteome by mass chromatographic analysis and data base querying, to analyze molecular mechanism of BMSCs differentiation into osteoblasts induced by Duzhong (Cortex Eucommiae).
Results
Guan Jie Kang group and celecoxib group got improvement of score of each dimension after treatment, with statistical significance. It showed that Guan Jie Kang and celecoxib could significantly improve the QOL of patients with knee OA. But there were different results between the two groups from different dimensions, the two groups could not be compared in improving role limitations due to physical health and bodily pain, which was consistent with the previous clinical observation results; but Guan Jie Kang group improve physical functioning, general health, energy, social functioning, role limitations due to emotional problems, emotional well being better than celecoxib group.
BMSCs could be isolated from the bone marrow of SD rat by whole bone marrow culture, and could be further purified by trypsin digestion and passage.
The OD value of Duzhong (Cortex Eucommiae) group rose obviously, blank group and DXM group had no obvious ascension; on the tenth day DXM group and Duzhong(Cortex Eucommiae) group were colored masculine by ALP staining solution; white calcium nodus could be seen at the undersurface of the holes 13days later, which could be colored masculine by alizarin bordeaux twenty days later.
Total protein concentration in each group were determined by the bradford method, they were 3.24μg/μL on the Oday,4.10μg/μL on the 2nd day, 4.7μg/μL on the 7th day,7.27μg/μL on the 14th day. Which descripted that serum containing Duzhong (Cortex Eucommiae) could promote cell proliferation.
We separated proteins by two-dimensional electrophoresis and gained 4 maps. Analysis of all the maps, the distinction between points was more obvious, the difference of protein expression may be analyzed. We used PDQuest-7.3.0 analysis software to analyze and identified a total of 641 protein spots,75 of witch were selected to receive mass spectrometry analysis and database query. 6 points of them were positive. They were:
F18, stromal cell-derived factor (SDF-1), can be used as a sign of detection differentiation degree of BMSCs;
G21, superoxide dismutase (SOD), was metal enzyme, was a major antioxidant enzyme, superoxide can eliminate oxygen-derived free radicals, and play an important role in the defense of oxygen toxicity, suppression of senile disease, prevention of senescence and so on;
E17, calcium reticulocyte protein (CRT), involved in calcium storage and signal transduction; cell adhesion; gene expression, etc;
E4, vimentin, vimentin was a source of intermediate filament protein existing in mesenchymal cells, attached to the nucleus, endoplasmic reticulum and the side or the end of mitochondria, so it was believed that vimentin played an important role in supporting and anchoring organelle in protoplasm, It can also maintain cell shape, integrity of cytoplasm and stability of the cytoskeleton interaction;
F6, lamin A, it and vimentin belonged to a kind of intermediate filament proteins, it was a necessary structural protein for structure of all eukaryocyte, mainly involved in constructing nuclear, and playing an important role in the processes of nuclear construction;
F3, annexin A6, involved in membrane transport and a series of calmodulin dependent activities at membrane surface in cells, including vesicle transport, membrane fusion of exocytosis, signal transduction and calcium channel formation, control inflammation, cell differentiation, and interaction between cytoskeletal proteins, etc. Conclusion
TCM treatment based on determination of treatment based in pathogenesis obtained through differentiation of symptoms and signs, it synthetically considered functional state of each demic system, and adjusted the whole functional state of human from more links, so it had.a greater advantage in the treatment of chronic physical and psychological diseases with complex etiopathogenisis in modern society. Introducing the QOL assessment to clinical trials of Chinese medicine and promoting and applying, could modify composition of clinical evaluation index in Chinese medicine, in order to verify the efficacy of Chinese medicine better.
But there were various QOL scale, each had its own scope of application. In this study, SF-36 form was used, this table was a universal QOL scale, although responsed to effect on patients'QOL of disease from different angles, but in terms of knee osteoarthritis, some items can not responsed QOL very well. Therefore, a study of a QOL scale for knee osteoarthritis is our further objective of the study.
Guan Jie Kang minht cure KOA by promoting repair of subchondral microfracture which blocked the pathological process that increase of the production of inflammatory component and growth factor and decrease of the production of inflammatory inhibitors.
It was more effectively to obtain BMSCs by extraction from bone marrow in vivo, which was benefit for growth, division and proliferation of BMSCs on the parietes. Whole bone marrow culture was simple and effective, cell density affected cell proliferation. Serum containing Duzhong (Cortex Eucommiae) could promote the proliferation of BMSCs and induced differentiation of BMSCs into osteoblasts. In the differentiation process, cell density played an important role. Differentiation of BMSCs into osteoblasts was a complex process, experiments thought that Duzhong (Cortex Eucommiae) invigorating the kidney possibly promoted cell differentiation by increasing vimentin, lamin A expression, reduced the express of calreticulin to release intracellular calcium, which involved in the formation of calcium nodules, and related to the process of bone mineralization in vivo. Superoxide dismutase and annexin A6 had relation with the vitality of the cells, regulated cell division, proliferation and differentiation; but whether they had closely relation with directing differentiation of osteoblasts remained to be further studied.
膝骨性关节炎(OA)是中老年常见的疾病,属于中医“骨痹”范畴,其主要病机为肾虚、血瘀。以补肾活血法治疗膝骨性关节炎取得良好疗效,前期临床研究显示补肾活血中药与塞来昔布具有相似的临床疗效,无统计学差异。然而,随着医学模式的发展,单纯的着眼于改善临床症状的疗效标准已经不能满足患者的要求。因疾病所导致的生理功能、心理功能、生理角色、社会角色等生存质量方面的损害越来越受到重视。本研究拟观察补肾活血中药(关节康片)对膝骨性关节炎患者生存质量(QOL)的影响,试将生存质量评价引入中医临床试验进行推广、应用,从而改善中医药界临床疗效评定指标的构成,以更好地验证中医药的疗效。
现代医学研究表明,软骨下微骨折在膝OA发病中起着重要作用。本研究拟通过实验证实补肾中药可能通过促进BMSCs增殖并诱导其向成骨细胞分化,促进软骨下微骨折的修复,从而阻断因此引起的致炎性成份及生长因子的产生增多和炎性抑制因子的产生减少这一病理过程。探讨关节康治疗膝OA的机制。
中医认为“肾主骨生髓”,现代医学主要从骨代谢本身进行研究。研究也发现,骨髓间充质干细胞(BMSCs)特性与“肾主骨”存在相关性。基于杜仲在补肾药物中的重要作用,本研究选择关节康片中药物组成的君药-杜仲作为实验对象,观察杜仲含药血清对BMSCs增殖的影响,及诱导BMSCs向成骨细胞分化结果；从细胞学的角度阐述“肾主骨生髓”的理论基础；解释杜仲补肾作用的机理。
当前通常从基因组水平研究干细胞的生物学特性。但是,以转录水平的分析研究细胞生物学功能往往不够充分,因为细胞决定于蛋白质,正是那些参与代谢和调控的蛋白质决定细胞的存活。本研究通过蛋白质组学研究方法,建立杜仲诱导BMSCs向成骨细胞分化功能蛋白质组,分析BMSCs向成骨细胞分化的分子机制。研究方法
所有入选病例均来自广州中医药大学第一附属医院骨科门诊首诊病人,共55例,利用随机表分为关节康组与塞来昔布组。所有患者在治疗前后填写SF-36生存质量调查问卷,计算治疗前后各组得分,组内比较采用t检验,治疗前后得分差异的组间比较采用两样本t检验。
从SD大鼠双下肢股骨、胫骨分离BMSCs,通过全骨髓法进行培养、胰酶消化纯化、扩增。以第4代细胞作为实验细胞。
实验分为3组,分为空白组、地塞米松组、杜仲组。将第四代BMSCs以5×106接种于9块96孔板,共3组,每组8个复孔；2块6孔板,每板6孔,每组2个复孔。2天后换液,加入诱导液。空白组加入含10%胎牛血清(FBS)的L-DMEM培养基继续培养,地塞米松组在基础培养基中上加入诱导剂(含β甘油磷酸钠10mmol／L、地塞米松0.1μmol／L、维生素C50mg／L),杜仲组为含10%杜仲含药血清的L-DMEM培养基。分别自加诱导液第1天起,采用MTT法测量96孔板细胞数(OD值),每天1块板,连续测量9天,以每天OD值平均值描绘细胞增殖曲线；在第10天取出1块6孔板做ALP染色；第20天做6孔板茜素红染色。
收集杜仲含药血清干预前、于预后第2天、第7天、第14天的细胞,采用去污剂裂解法提取总蛋白,去除杂质,并采用bradford法测定各样品中蛋白浓度。通过双向电泳对蛋白进行分离,采用PDQuest-7.3.0胶图分析软件进行分析找出差异蛋白,并进行质谱分析、数据库查询建立功能蛋白质组。分析杜仲诱导BMSCs向成骨细胞分化分子机制。研究结果
关节康组与塞来昔布组在治疗后各维度积分都有提高,具有统计学意义。表明关节康与塞来昔布治疗膝OA均可显著提高患者生存质量。但组间从各个不同的维度却有不同的结果,两组在改善生理功能、躯体疼痛方面不具有可比性,与之前的临床疗效观察结果一致,而关节康组在改善生理职能、总体健康、活力、社会功能、情感职能及精神健康方面优于塞来昔布组。
通过全骨髓培养法从SD大鼠骨髓中分离到BMSCs,通过胰酶消化、传代可进一步纯化。杜仲含药血清组细胞OD值呈现逐渐增高的趋势,而空白组和地塞米松组无明显增高的趋势；诱导后第10天,杜仲组及地塞米松组ALP染色阳性；第13天,培养板底部可见白色钙结节；第20天,茜素红染色阳性。
经bradford法测定各组总蛋白浓度分别为0天3.24ug／ul,2天4.10ug／ul,7天4.7ug／ul,14天7.27ug／ul。说明杜仲含药血清能够促进细胞增殖。
双向电泳对蛋白进行分离,得到4张图谱,从整个图谱来分析,点与点之间区分比较明显,可以对其进行蛋白质表达的差异分析。采用PDQuest-7.3.0胶图分析软件进行软件分析,找出差异蛋白点共641个,选取其中75个点行质谱分析并进行数据库查询。其中6个点具有积极意义。分别为：
F18,基质细胞衍生因子(SDF-1),可作为检测MSCs分化程度的标志。
G21,过氧化物歧化酶(SOD),属于金属酶,是一种主要的抗氧化酶,能清除超氧化物自由基,在防御氧的毒性、抑制老年疾病以及预防衰老等方面起着重要作用；
E17,钙网织蛋白(CRT),参与钙的贮藏和信号转导；细胞黏附；基因表达的调控等；
E4,波形蛋白(vimentin),是间充质来源细胞的中间纤维蛋白,附在细胞核、内质网及线粒体的旁边或末端,因而相信波形蛋白在支撑及锚固原生质内的细胞器有着重要的角色,它亦能维持细胞的形状、细胞质的完整性及稳定细胞骨架内的相互作用；
F6,核纤层蛋白A(lamin A),与波形蛋白同属于中间纤维蛋白的一种,是所有真核细胞核结构所必需的结构蛋白,主要参与构成核的功能,在核形成过程中起着重要作用。
F3,膜联蛋白A6(annexin A6),在细胞中参与膜转运及膜表面一系列依赖于钙调蛋白的活动,包括囊泡运输、胞吐作用中的膜融合、信号传导和钙离子通道的形成、调控炎症反应、细胞分化和细胞骨架蛋白间的相互作用等。研究结论
中医治疗以辨证论治为基础,综合考虑了人体各个系统的机能状态,从多环节对人体整体机能状态进行调节,这样在治疗现代社会病因复杂的慢性身心疾病时便具有了较大的优势。将QOL评价引入中医临床试验进行推广、应用,可以改善中医药界临床疗效评定指标的构成,以更好地验证中医药的疗效,从而更好发展与宏扬祖国医学。
但目前各生存质量量表繁多,均有各自适用范围。本研究采用SF-36表进行研究,此表为普适性生存质量量表,虽然从不同的角度反应疾病对患者生存质量的影响,但对于膝骨性关节炎患者而言,部分条目不能很好反应该疾病对生存质量的影响。故研究一个膝骨性关节炎的生存质量量表是我们进一步研究的目标。
关节康可能通过促进软骨下微骨折的修复,从而阻断因此引起的致炎性成份及生长因子的产生增多和炎性抑制因子的产生减少这一病理过程,达到治疗膝OA的作用。
经活体提取骨髓能更有效地获取BMSCs,有利于BMSCs的贴壁生长、分裂增殖。全骨髓培养法简单有效,细胞的密度影响着细胞的扩增。杜仲含药血清能促进BMSCs增殖并诱导BMSCs定向成骨细胞分化,在分化过程中,细胞密度起着重要作用。BMSCs向成骨细胞分化是一个复杂的过程,实验认为补肾杜仲可能通过上调波形蛋白、核纤层蛋白A的表达促进细胞分化,下调钙网织蛋白的表达,释放细胞内钙,参与钙结节的形成,与体内骨矿化过程有关。过氧化物歧化酶、与膜联蛋白A6与细胞的活力有关,调节着细胞的分裂增殖与分化,但是否与定向成骨细胞分化有着密切关系尚有待进一步研究。
Objective
Knee osteoarthritis is a common disease in the elderly, connects with rheumatism involving bone in Chinese medicine, the main pathogenesis are renal deficiency and blood stasis. Treating knee osteoarthritis with Kidney-Supplementing Blood-Quickening Chinese medicine gained good outcome, clinical studies before showed that Traditional Chinese medicine and celecoxib had similar clinical efficacy, no significant difference. However, with the development of medical model, the efficacy standard only focusing on improving the clinical symptoms can not meet patients'requirements. Damage of physiological function, psychological function, physical role, social role, et al, involve in the quality of life (QOL) caused by illness received more and more attention. This study was to observe the effect of QOL of patients with knee osteoarthritis treated with Kidney-Supplementing Blood-Quickening Chinese medicine(Guan Jie Kang), try to introduce the QOL assessment to clinical trials of Chinese medicine and promote and apply, in order to modify composition of clinical evaluation index in Chinese medicine, and to better verify the efficacy of Chinese medicine.
Modern medical research showed that subchondral micro-fractures played an important role in the pathogenesis of knee OA. This study was to confirm that by experimental Kidney-Supplementing medicine might promote the repair of subchondral micro-fracture by promotion of proliferation of BMSCs and induction them to differentiation into osteoblasts, to block the pathological process that the increase of inflammatory components and growth factors, and induce of the inflammatory inhibitors. In order to ecplore the mechanism of treatment KOA by Guan Jie Kang.
Chinese medicine considers the kidney generating marrow and dominating bone, which was studied mainly on the bone metabolism itself by modern medical research. Studies also found that features of mesenchymal stem cells (MSCs) had relationship with "the kidney being in charge of bone". Based on the important role of Duzhong(Cortex Eucommiae) in the Chinese medicine invigorating the kidney, the study chose Duzhong (Cortex Eucommiae) which was the Sovereign drug in Guan Jie Kang tables as the research object to observe proliferation of BMSCs induced by serum containing Duzhong (Cortex Eucommiae), and differentiation results of BMSCs into osteoblasts, in order to expound the theory of "the kidney generating marrow" from cytology and to explain the mechanism of Duzhong(Cortex Eucommiae) invigorating the kidney.
Usually biological characteristics of stem cells were researched at the level of genome. However, the analysis of biological function by transcription was often not sufficient, because the cells depends on protein, it is protein involved in metabolism and regulation that decided cell survival. This study used proteomics research methods to establish functional protein of BMSCs differentiating into osteoblasts induced by Duzhong (Cortex Eucommiae), and analyze molecular mechanism of BMSCs differentiating into osteoblasts. Methods
All the cases who attending out-patient first were selected from the department of Orthopaedics of the First Affiliated Hospital of Guangzhou University of Traditional Chinese medicine, there was a total of 55 cases, all the cases were divided into Guan Jie Kang group and the celecoxib group by random table. Before and after treatment all patients completed SF-36 quality of life questionnaire to calculate scores in each group, t test was used to compare before and after treatment within group, two sample t test was used to compare score difference between the two groups.
BMSCs were isolated from the two femurs and tibias of SD rats, cultured by the whole bone marrow method in vitro, purification and amplification by trypsin digestion. The quartus generation cells were used as the experimental cells.
The experiment was divided into 3 groups named blank group, DXM group and Duzhong (Cortex Eucommiae) group. Inoculated quartus generation MSCs in nine nity-six-holes cell culture plates and two six-holes cell culture plates by 5×106 cells per milliliter, all the holes were divided into three groups, each group had eight holes in nity-six-holes cell culture plates and two holes in six-holes cell culture plates. Two days later, changed medium and add new medium. The medium of DXM group contained 10mmol/Lβ-sodium glycerophosphate, 0.lumol/L DXM,50mg/L vitamin C, that of Duzhong (Cortex Eucommiae) group contained 10% serum containing Duzhong (Cortex Eucommiae). Measured cell number by MTT of nity-six-holes plates from the second day to the eighth day, one of the six-holes plates was colored by ALP staining solution at the tenth day, the last one was colored by alizarin bordeaux at the twentieth day.
Extracted total protein of cells induced by serum containing Duzhong (Cortex Eucommiae) for 0day,2days,7days,14days, and runouted them by two-dimensional electrophoresis, searched for different protein spot by PDQuest-7.3.0 software for analyzing gel picture, to establish functional proteome by mass chromatographic analysis and data base querying, to analyze molecular mechanism of BMSCs differentiation into osteoblasts induced by Duzhong (Cortex Eucommiae).
Results
Guan Jie Kang group and celecoxib group got improvement of score of each dimension after treatment, with statistical significance. It showed that Guan Jie Kang and celecoxib could significantly improve the QOL of patients with knee OA. But there were different results between the two groups from different dimensions, the two groups could not be compared in improving role limitations due to physical health and bodily pain, which was consistent with the previous clinical observation results; but Guan Jie Kang group improve physical functioning, general health, energy, social functioning, role limitations due to emotional problems, emotional well being better than celecoxib group.
BMSCs could be isolated from the bone marrow of SD rat by whole bone marrow culture, and could be further purified by trypsin digestion and passage.
The OD value of Duzhong (Cortex Eucommiae) group rose obviously, blank group and DXM group had no obvious ascension; on the tenth day DXM group and Duzhong(Cortex Eucommiae) group were colored masculine by ALP staining solution; white calcium nodus could be seen at the undersurface of the holes 13days later, which could be colored masculine by alizarin bordeaux twenty days later.
Total protein concentration in each group were determined by the bradford method, they were 3.24μg/μL on the Oday,4.10μg/μL on the 2nd day, 4.7μg/μL on the 7th day,7.27μg/μL on the 14th day. Which descripted that serum containing Duzhong (Cortex Eucommiae) could promote cell proliferation.
We separated proteins by two-dimensional electrophoresis and gained 4 maps. Analysis of all the maps, the distinction between points was more obvious, the difference of protein expression may be analyzed. We used PDQuest-7.3.0 analysis software to analyze and identified a total of 641 protein spots,75 of witch were selected to receive mass spectrometry analysis and database query. 6 points of them were positive. They were:
F18, stromal cell-derived factor (SDF-1), can be used as a sign of detection differentiation degree of BMSCs;
G21, superoxide dismutase (SOD), was metal enzyme, was a major antioxidant enzyme, superoxide can eliminate oxygen-derived free radicals, and play an important role in the defense of oxygen toxicity, suppression of senile disease, prevention of senescence and so on;
E17, calcium reticulocyte protein (CRT), involved in calcium storage and signal transduction; cell adhesion; gene expression, etc;
E4, vimentin, vimentin was a source of intermediate filament protein existing in mesenchymal cells, attached to the nucleus, endoplasmic reticulum and the side or the end of mitochondria, so it was believed that vimentin played an important role in supporting and anchoring organelle in protoplasm, It can also maintain cell shape, integrity of cytoplasm and stability of the cytoskeleton interaction;
F6, lamin A, it and vimentin belonged to a kind of intermediate filament proteins, it was a necessary structural protein for structure of all eukaryocyte, mainly involved in constructing nuclear, and playing an important role in the processes of nuclear construction;
F3, annexin A6, involved in membrane transport and a series of calmodulin dependent activities at membrane surface in cells, including vesicle transport, membrane fusion of exocytosis, signal transduction and calcium channel formation, control inflammation, cell differentiation, and interaction between cytoskeletal proteins, etc. Conclusion
TCM treatment based on determination of treatment based in pathogenesis obtained through differentiation of symptoms and signs, it synthetically considered functional state of each demic system, and adjusted the whole functional state of human from more links, so it had.a greater advantage in the treatment of chronic physical and psychological diseases with complex etiopathogenisis in modern society. Introducing the QOL assessment to clinical trials of Chinese medicine and promoting and applying, could modify composition of clinical evaluation index in Chinese medicine, in order to verify the efficacy of Chinese medicine better.
But there were various QOL scale, each had its own scope of application. In this study, SF-36 form was used, this table was a universal QOL scale, although responsed to effect on patients'QOL of disease from different angles, but in terms of knee osteoarthritis, some items can not responsed QOL very well. Therefore, a study of a QOL scale for knee osteoarthritis is our further objective of the study.
Guan Jie Kang minht cure KOA by promoting repair of subchondral microfracture which blocked the pathological process that increase of the production of inflammatory component and growth factor and decrease of the production of inflammatory inhibitors.
It was more effectively to obtain BMSCs by extraction from bone marrow in vivo, which was benefit for growth, division and proliferation of BMSCs on the parietes. Whole bone marrow culture was simple and effective, cell density affected cell proliferation. Serum containing Duzhong (Cortex Eucommiae) could promote the proliferation of BMSCs and induced differentiation of BMSCs into osteoblasts. In the differentiation process, cell density played an important role. Differentiation of BMSCs into osteoblasts was a complex process, experiments thought that Duzhong (Cortex Eucommiae) invigorating the kidney possibly promoted cell differentiation by increasing vimentin, lamin A expression, reduced the express of calreticulin to release intracellular calcium, which involved in the formation of calcium nodules, and related to the process of bone mineralization in vivo. Superoxide dismutase and annexin A6 had relation with the vitality of the cells, regulated cell division, proliferation and differentiation; but whether they had closely relation with directing differentiation of osteoblasts remained to be further studied.
引文
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