骨髓源性神经干细胞移植磁共振成像的Sinerem标记及大鼠脑缺血模型中的应用
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摘要
研究背景
我国属脑血管病高发国家，脑卒中是我国疾病死亡和导致长期残疾的三大原因之一。缺血性脑梗塞约占卒中的80％，用组织纤溶酶原激活剂进行治疗的方法能使梗塞再通，但由于溶栓的副作用和较短的治疗时间窗而使其应用受到限制，因此，只有很小部分卒中病人能得到及时的溶栓治疗。而存活的患者往往伴有严重的残疾，即使最好的康复治疗也由于不可逆的脑损伤而疗效有限。因而，脑卒中的治疗仍需要寻找更好更有效的治疗措施。
由于受“中枢神经不可再生”旧理论的束缚和影响，长期以来神经细胞损伤后结构与功能修复的研究进展一直比较缓慢。20世纪末期，干细胞(Stem cells，SCs)的发现为人们治疗许多难治性疾病提供了新的历史契机。干细胞因具有很强的自我更新能力和多种细胞分化潜能而成为研究的热点，具有非常重要的理论研究和临床应用价值。其中，神经干细胞(Neural stem cells，NSCs)的发现和证实，为治疗包括脑卒中在内的难治性中枢神经系统(Center neural system，CNS)疾病开辟了新的道路。目前，已证实的NSCs来源主要有：(1)大脑室管膜下区、海马和齿状回部位的干细胞，可在脑细胞受损伤时动员到局部，但数量与作用有限；(2)胚胎干细胞，是一类从着床前囊胚内细胞团或早期胚胎原始生殖细胞分离克隆出来的一类未分化的全能性干细胞，在适当条件下能向神经干细胞方向分化，因受到伦理道德上的约束只能应用于动物实验；(3)骨髓基质细胞(Bone marrow stromal cells，BMSCs)，可以向NSCs、神经元和神经胶质细胞方向分化，为了与来源神经组织的NSCs相区别，将此种由骨髓基质细胞诱导而来的NSCs称为骨髓基质细胞源NSCs(BMSCs-D-NSCs)。由于BMSCs容易获取、可在体外大量扩增并在一定条件下能诱导成神经干细胞，因此成为NSCs的重要来源，不仅克服了从其它部位取材的危险性和局限性，也避免了胚胎组织移植中存在的伦理和免疫排斥问题。若将外源性基因导入待移植NSCs，使其在体内有效表达，则可达到长效修复受损伤脑组织功能和治疗的目的。因此，BMSCs-D-NSCs移植是一种极具潜力的治疗缺血性脑卒中策略。
NSCs移植到宿主后组织结构的改变与功能的改善程度是我们关注的重点，而这些移植的效果与移植细胞的状况密切相关，这些状况主要是细胞的存活、生长、迁移、分化情况、与周围组织的融合情况等。因此，上述细胞在宿主内转归过程是评价移植的重要内容。以往传统的方法主要是先处死动物，然后再利用组织学或分子生物学等方来观察干细胞移植后在体内的转归和如何促进行为学上的功能改善，使研究解剖学改变与行为学的恢复之间的关系研究受到限制，这种侵袭性的分析只能提供细胞的单一的“瞬间快照”(snapshot)，不能对同一受试动物移植的干细胞及宿主进行动态连续的实时观察，而且细胞迁移路径和有些病变用组织学方法很难观察到，从而丢失许多重要的信息。同时，侵袭性的研究方法更不适用于人体，严重制约干细胞移植在临床上的研究与应用。所以，必须寻找新的手段来监测干细胞移植后在体内的情况，为解决临床应用的瓶颈提供可能。
超小超顺磁性氧化铁(ultrasmall superparamagnetic iron oxide，USPIO)是一类新型的纳米级的超顺磁性氧化铁微粒，由于其自身特殊的磁性，可在磁场作用下与周围组织进行不同的成像，如果用其标记干细胞，就可以进行影像学的无创监测。目前，还未见用来标记大鼠骨髓基质细胞移植脑卒中模型的报道。本研究课题通过磁共振(Magnetic resonance，MR)成像技术和体外细胞标记技术相结合的方法，利用USPIO Sinerem标记鼠BMSCs-D-NSCs并移植，研究Sinerem对BMSCs-D-NSCs的生物学影响，及移植缺血性脑卒中动物模型后在活体脑内的识别、存活、迁移、增殖等转归，探讨NSCs移植治疗脑缺血的机制和无创伤成像技术的适用性，为NSCs移植治疗脑卒中的临床应用与安全性奠定基础，并为干细胞治疗其它神经系统疾病提供有益的启示。
第一部分：SD大鼠骨髓基质细胞体外向神经干细胞诱导的实验研究
目的：通过细胞培养的方法对分离的大鼠骨髓基质细胞进行体外诱导，向神经干细胞方向诱导。
方法：无菌条件下获取大鼠股骨中的骨髓，用淋巴分离液经梯度离心分离骨髓基质细胞。用干细胞培养基／碱性成纤维因子／白细胞抑制因子／胎牛血清进行实验组的培养、诱导，以DMEM／F12培养基／胎牛血清进行对照组的培养，在倒置光学相差显镜下观察细胞的形态、数量等生长情况；利用计数法测定细胞生长曲线；利用免疫细胞化学方法对神经巢蛋白(Nestin)特异性分子标志物进行检测鉴定。
结果：细胞刚接种后在倒置显微镜下观察可见细胞呈悬浮状态，形态为圆形，大小均一，边缘光滑、完整。接种后24h，骨髓基质细胞开始贴壁，随时间的增加，贴壁细胞逐渐增多，大约3天后贴壁细胞数量不再增加，未贴壁细胞仍呈悬浮状。此时更换培养基后，除掉了其中的非粘附细胞，可见剩余较纯的贴壁细胞。剩余的贴壁细胞折光性强，体积较前期略增大，个别细胞出现核分裂相。5～7天时出现较多的分裂相，增殖明显，出现少许细胞克隆团，9～12天时细胞增殖旺盛，可见较多的细胞克隆团形成，围绕克隆团细胞向周围呈辐射状或旋涡状分布，大部分呈圆形或椭圆形。继续培养至18～20天时，大部分细胞形态开始发生变化，呈多边形、梭形等不规则状，从胞体发出的突起向远处伸展并可相互接触，类似于神经细胞间的联络。而在DMEM／F12培养的对照组，细胞接种后第8天左右时只有极少数量的克隆团出现，且随时间的延长数量增加不明显。
生长曲线说明，实验组细胞接种后7～9天开始进入对数生长期，至14～16天融合达80％。而对照组9～11天时进入对数生长期。
实验组细胞在接种培养第8天时检测到BMSCs-D-NSCs干细胞特异性抗原神经巢蛋白(Nestin)表达呈阳性，提示BMSCs诱导为BMSCs-D-NSCs。
结论：所用的分离骨髓基质细胞方法简单易行，在一定的条件下大鼠骨髓基质细胞可向神经干细胞方向诱导，具有很强的自我更新和多潜能分化能力，可作为干细胞移植的供体细胞。
第二部分：Sinerem纳米铁标记鼠BMSCs-D-NSCs及其生物学影响
目的：明确新型超小超顺磁性氧化铁纳米微粒(USPIO)Sinerem体外标记大鼠骨髓源神经干细胞所需的合适浓度和铁微粒在细胞中的代谢、存留情况，探讨Sinerem标记大鼠骨髓源神经干细胞的可行性，为标记细胞的磁共振成像奠定基础。
方法：无菌条件下手术取SD大鼠双侧股骨的骨髓，利用淋巴细胞分离液分离其中的骨髓基质细胞，体外培养诱导成骨髓源神经干细胞。首先USPIO Sinerem和PLL通过静电作用形成的复合物。再用不同浓度的Sinerem(0、25、50、100、200、500μg／ml)和细胞过夜进行标记，分别于标记后1、3、5、7、9、11～17d不同的时间点行MTT观察细胞增殖，并用流式细胞仪观察细胞的生长周期和凋亡情况；RT-PCR方法检测铁标志对干细胞相关基因表达的影响；利用免疫细胞化学方法分析铁标记对干细胞分化能力的影响。普鲁士蓝染色和透射电镜确定细胞内铁的摄取、定位情况及细胞的标记效率。
结果：细胞形态的观察：倒置显微镜下标记组与未标记组的细胞形态无明显区别，普鲁士蓝染色的强度伴随着培养基中Sinerem浓度的增加而升高，提示进入细胞中的铁随浓度升高而逐渐增加，对照的未标记细胞无蓝染。最佳浓度的确定：与未标记组相比，Sinerem浓度为200μg／ml或以下时，标记细胞的活力、凋亡率无明显变化(P＞0.05)，但200μ／ml已接近临界值，而浓度大于等于500μg／ml时与对照组相比有显著性差异(P＜0.05)。对基因表达的影响：在适当的浓度200μg／ml时标记细胞中的相关基因表达水平无明显改变。对细胞周期的影响：与未标记组细胞相比，流式细胞仪检测结果显示，标记组细胞G_1、S、G_2／M期的细胞所占百分比没有显著性差异，说明适当浓度时Sinerem标记对细胞周期没有明显的不良影响。对分化能力的影响：该浓度时标记细胞向脂肪细胞分化的情况与对照组相比差别不大。标记效率：经显微镜下计数观察，Sinerem标记干细胞效率为98％～100％。细胞内铁的鉴定：普鲁士蓝染色显示铁颗粒存在胞质中，电镜显示铁颗粒集中于内涵体／溶酶体中。
结论：超小超顺磁性Sinerem在合适浓度(200μg／ml)时能安全有效地体外标记骨髓源神经干细胞，且对细胞无短期或长期的毒性影响，为神经干细胞移植的无创示踪奠定了基础。
第三部分：Sinerem纳米铁标记大鼠BMSCs-D-NSCs体外磁共振成像研究
目的：应用磁共振成像技术观察Sinerem标记后的SD大鼠BMSCs-D-NSCs体外成像情况，初步评价磁共振成像体外示踪Sinerem标记干细胞的可行性，为下一步体内移植成像创造条件。
方法：分离SD大鼠骨髓基质干细胞，体外培养诱导成BMSCs-D-NSCs。以200μg／ml的浓度10~5个细胞用不同的SE序列T_1WI、T_2WI与T_3~*WI在2％的琼脂糖凝胶中模拟在体环境分别行4.7T扫描确定最佳的扫描序列。将不同浓度(0、25、50、100、200、500μg／ml)的Sinerem和干细胞(10~6个)共孵育培养过夜标记后置于2％的琼脂糖凝胶中，以SE序列T_2~*WI磁共振干细胞成像；并在磁共振下观察不同数量细胞200μg／ml Sinerem标记细胞(10~1个，10~2个，10~3个，10~4个，10~5个)的信号情况。观察细胞在浓度200μg／ml(10~6个)标记时经长期培养不同时间点时的信号变化。
结果：MR扫描结果提示序列T_2~*WI扫描的敏感性最强。不同浓度的Sinerem标记细胞的磁共振信号强度不同，随着Sinerem浓度的升高MR信号呈降低趋势，浓度为500μg／ml时信号强度最低，肉眼观察可看到浓度100μg／ml以上标记细胞的MR的信号强度明显低于对照样品，可进行区分。标记细胞数量达10~3个及以上时在磁共振下其信号变化能被观察到。10~6个标记细胞的信号随培养时间的延长逐渐增加，4w时仍能在磁共振下显示可区分的低信号。
结论：利用Sinerem标记的BMSCs-D-NSCs体外可在MR下呈现可视区别的低信号影，T_2~*WI序列是最敏感的序列；信号强度的高低可用于评估标记细胞的数量。Sinerem标记的细胞可用于磁共振下进行长期的示踪。
第四部分：Sinerem纳米铁标记大鼠BMSCs-D-NSCs移植大脑的活体MR示踪及组织学检测
目的：将USPIO标记的大鼠BMSCs-D-NSCs移植正常或缺血模型脑后，初步观察其在大脑中的存活、迁移和整合情况，确定体内MR成像示踪Sinerem标记干细胞的可行性。
方法：首先分离SD大鼠BMSCs，体外培养诱导成BMSCs-D-NSCs。将200μg／ml的Sinerem纳米铁标记BMSCs-D-NSCs后，将标记细胞(1×10~6个)用脑立体定向仪移植通过微量注射器注射到正常大鼠皮层或缺血模型大鼠纹状体。同时在正常大鼠的另一侧用未标记细胞作为对照。在不同时间点(1d、1w、2w、4w)以SE序列T_2~*WI行4.7T磁共振成像示踪，然后在不同时间点处死动物，用组织学方法观察标记细胞在脑内的转归情况。
结果：在非缺血模型：大鼠移植皮层的标记细胞在磁共振下呈T_2~*WI低信号区，而未标记细胞无信号变化，与周围脑组织不能区别；1w时低信号区未见明显位移，而至4w时检测到细胞沿胼胝体的轻度迁移。在缺血模型中：移植到纹状体部位的标记细胞磁共振扫描时呈低信号区，1w时可见低信号区开始向对侧方向迁移，随时间的增加至2w时迁移至胼胝体纤维，4w时进一步沿胼胝体向缺血侧迁移。处死动物后，组织学检测可见移植的标记细胞普鲁士染色阳性，标记细胞的分布与磁共振扫描的低信号区相一致。透射电镜下可见观察到标记细胞中存在的Sinerem铁颗粒，细胞和周围组织相互紧密接触，未见到突触样结构的形成，但从形态上可见到个别细胞胞体有向两极伸展的趋势。
结论：Sinerem标记的BMSCs-D-NSCs在动物活体内能在磁共振下显像，其在脑内存活、迁移等行为可被磁共振特异性示踪，可用于干细胞移植的无创监测。
Background
Incidence of cerebro-vascular disorders is very high, and cerebral stroke is oneof the three leading causes of mortality and morbidity in China.80% of cerebral strokeis ischemic cerebral infarction. The infarction can be recovered with the tissueplasminogen activator. However, of all patients who suffer from an ischemic stroke,only a small proportion of patients were treated with acute reperfusion therapy to haltprogression of ischemic brain damage because of side effect of reperfusion therapyand shorter time window. Moreover, frequently, these survial patients suffered fromservious morbidity and the effects of therapies are limited, even the best rehabilitationwas applied. Therefore, It is necessary to search for better therapies to cure thecerebral stroke.
The recovery progress of structure and function of damaged neural cells wasslow for a long time because an old theory that center nerve can not regrow affectedthe people. Emerge of Stem cells provided a good chance for curing many intractablediseases.SCs have become a research hot point because of its self-renewal andmulti-potential differentiation, which is important for theory research and clinicalapplications. Neural stem cells, as one kind of stem cells, has brought a new way forintractable diseases of center neural system including cerebral stroke. Nowadays, It has been demonstrated that neural stem cells exist in the following parts:(1)Stem cellsfrom the subventricular zone,the hippocampus or dentate gyrus, can be actived andmigrate to the injury site. However, their number and effect are limited.(2)Embryonicstem cells originate as inner mass cells within a blastocyst and can differentiate intoneural stem cells under appropriate conditions.But the embryonic stem cells ispresently only limited to be used in animal trial for ethical issues. (3) Bone marrowstromal cells, brifely, BMSCs, can differentiate into NSCs, neurons and glial cells. Todistinguish them from the stem cells from neural tissue, the neural stem cells inducedfrom bone stromal cells were called bone marrow stromal cells-derived-neural stemcells (BMSCs-D-NSCs). BMSCs are ease of isolation and high expansion potential invitro, and can be induced into neural stem cells.Therefore, BMSCs has been theimportant resource, which overcomes the risk of gaining material, as well as avoid theproblem of ethical issue and immunological rejection. The damaged brain tissuecould be repaired by transplanted NSCs if the exogenous gene was introducedinto them. Consequently, transplantation of BMSCs-D-NSCs is a potential strategyfor treating with the ischemical stroke.
Structure change and functional improvement of tissue after NSCstransplantation are our focus, and the outcome is closely related with NSCs status invivo including cell vability, growth, migration,differentiation and integration withsurounding tissues.Hence, It is important to evaluate the process of transplanted cellsin vivo.In the past, the animals must be firstly killed and then the histological andmolecular trials were taken to observe the events and how to promote behaviorimprovement, which restricted the research on relationship between anatomy changeand behavior recovery. The invasive analysis can only provide single snapshot andcan not have a real observation on the tansplanted stem cells and animals serially.Moreover, the migration way and some pathological process were hard to be found and much important information would lost, and the invasive way can not be appliedfor human being. All of the shortcomings above had seriously restricted the clinicalresearch and application.Therefore, it is necessory to explore new method to monitorthe stem cells in vivo and resolve the bottleneck problem.
Ultrasmall superparamagnetic iron oxide(USPIO) is a new kind ofsuperparamagnetic iron particulate with nano-scale. USPIO can visualizedistinguishing from surrounding tissue because of its special magnetic property andthe labeled stem cells should be monitored by nontraumatic methods. Up to date,there was no report about rat BMSCs transplantation to cerebral stroke. This issuemainly explored the effect of USPIO sinerem on BMSCs-D-NSCs biologicalcharacteristics and the events about recognition,survial, migration and proliferation ofthe labeled stem cells,and investigated the mechanism of brain ischemic diseasetreated by NSCs transplantation and applicability of nontraumatic imagingtechnology in order to establish a base for clinical application and safety and tosupply benefical revelation for other neural system diseases.
PartⅠ: Research on inducement of SD rat BMSCs into neural stem cells invitro
Objective: To induce the isolated rat BMSCs into neural stem cells by cellculture.
Methods: Bone marrow stem cells isolated from thigh bone marrows of SD ratsby density gradient centrifugation with lymphocyte separating medium. Then theBMSCs were cultured and induced into BMSCs-D-NSCs by NSCs culturemedium/basic fibroblast growth factor/leukemia inhibitory factor/fetal bovine serum,and the control BMSCs were cultured with DMEM/F12 medium/fetal bovine serumat the same time. The cultured cells were observed by invert phase contrast lightmicroscope to investigate their morphology and quantity.Cell growth curves were destribed by counting progress. Identification of neural stem cell was performed byspecial nestin marker immunocytochemical staining.
Results: The separated BMSCs suspeneded and their shape was round,size-equal, outline-clean and intact under inverted phase contrast microscope at thebeginning of culture.The BMSCs started attaching and more cells attached with timeextending.The cell attachment would stop after 3 days and the unattached cells stillsuspended. The purified cells were left after discarding the unattached cells and theattached cells were more dioptric, bigger and emerged nuclear splitting.There weremore splitting cells during 5days～7days and proliferation was obvious and fewcloning mass appeared. Cell growth became more strong and more cloning massappeared in the day of 9～12, which surrounded one center point with radiating orswirling shape, most of them were round or elliptical.Up to 18days～20days,morphology of most cells started changing, appearing polygon or fusiform, etc.Processus from cell body extended to far place and connected with each other, similarto contact between neural cells.However, only very few cloning mass appeared ineighth day and did not increase with time extending.
The growth curving indicated that the experimental group started logarithmicgrowth during 7～9days and merged to 80% during 14～16days.In contrast, controlgroup began logarithmic growth until 9～11days.
The experimental group could express special NSCs antigen nestin proteinwhen they were detected by eighth day.
Conclusion: The strategy of isolating BMSCs was easy and accessible, and therat BMSCs could be induced into neural stem cells with strong self-renewal andmulti- potential differentiation ability, which could be used as donor cells.
PartⅡ: Sinerem labeling rat BMSCs-D-NSCs and its effect on the cells'characteristics
Objective: To determine the optimal concentration of new type USPIO Sineremlabeling BMSCs-D-NSCs in vitro and investigate the metabolism and retaining of Feparticle, and expolre the feasibility of labeling rat BMSCs-D-NSCs by Sinerem inorder to establish base for MR imaging.
Methods: Bone marrow stem cells isolated from thigh bone marrows of SD ratsby density gradient centrifugation with lymphocyte separating medium. Then theBMSCs were cultured and induced into BMSCs-D-NSCs in vitro. Firstly, Sineremand poly-l-lysine(PLL) formed complex by static electricity.Then, theBMSCs-D-NSCs
were incubated with the comples with different Sineren concentration as 0, 25,50, 100,200and500μg/ml for one night. The proliferation of labeled cells wereobserved by MTT test at the timepoint of 1,3,5,7,9,11～17day. The growth cycle andapoptosis of labeled cells were tested by flow cytometry. The effect on related keygene expression was investigated by RT-PCR. Moreover, the effect of Fe labeling ondifferentiation ability was analized by immunocytochemistry. Prussian blue stainingand transmission electron microscope were conducted for demonstrating theuptake,location and labeling ratio of intracytoplastic nanoparticles.
Results: Morphologic difference of labeled and unlabeled BMSCs-D-NSCs wasnot significant.The density of Prussian blue staining increased with the lifting ofSinerem concentration,and the control group has no blue staining. In contrast tounlabeled group, the vitality and apoptosis ratio of labeled cells had no obviouschange when the Sinerem concentration was 200μg/ml or less than 200μg/ml(P＞0.05),but this concentration has very closely near the threshold value.It was significantwhen the Sinerem concentration was 500μg/ml or more than 500μg/ml(P＜0.05). Thegene expression level has no significant change after labeled by the appropriateconcentration(200μg/ml) contrast to the control group. The cell cycle had no significant difference between the labeled and unlabeled cells, which showed Sineremlabeling has no hurt on cell cycle. The differentiation ability of BMSCs-D-NSCs intoadipose cell has no obvious difference between two groups under the concentration of200μg/ml.The ratio of Sinerem labeling was about 98%～100% whenBMSCs-D-NSCs was labeled with 200μg/ml. Prussian blue staining showed that Feparticle lied in cytoplasm and transmission electron microscope indicated that thesenano-particles concentrated in endosomes/lysosomes.
Conclusion: BMSCs-D-NSCs labeling by Sinerem was safe under appropriateconcentration of 200μg/ml and has no poisonous for short or long time, whichestablished base for tracking NSCs in vivo.
PartⅢ: Experimental study on Sinerem labeling of rat BMSCs-D-NSCswith MRI in vitro
Objective: To observe the MRI of rat BMSCs-D-NSCs labeled by Sinerem invitro and evaluate the feasibility of MRI tracking of labeled NSCs.
Methods: The SD rat BMSCs were isolated and induced into BMSCs-D-NSCsin vitro. 10~5cells labeled with 200μg/ml Sinerem were immerged in 2% gel andscanned by different SE sequence T_1WI、T_2WI与T_2~*WI respectively to determine thebest scanning sequence with 4.7T MR system.10~6 cells labeled with differentconcentration
(0,25,50,100,200,500μg/ml) were scanned in 2% gel by T_2~*WI sequence.Different number(10~1,10~2,10~3,10~4,10~5)of BMSs-D-NSCs labeled with 200μg/mlSinerem were scanned to determine the threshold of cell quantity. In the end, thesignal change of 200μg/ml Sinerem labeled cells was observed at differenttimepoints.
Results: MR result indicated that T_2~*WI was the most sensitive sequence. TheMR signal intensity of Sinerem labeled cells with different concentration was various, which reduced progressively with the increasing of Sinerem concentration. The signalintensity was lowest when the concentration was 500μg/ml. The MR signal intensityof more than 100μg/ml Sinerem labeling cells was much lower than control sampleand could be distinguished.
The MR signal change could be observed when the cell number was and 10~3 andmore. The signal intensity of 10~6 labeled cells was increaseing with the timeextending, which could still be displayed as different low signal for 4 weeks.
Conclnsion:Sinerem labeled BMSCs-D-NSCs in vitro appeared visually lowsignal area. The best sensitive sequence was T_2~*WI. The level of signal could beapplied for evaluating cell numbers. Sinerem labeled cells could be tracked for a longtime.
PartⅣ: MR tracking of Sinerem labeled rat BMSCs-D-NSCs in brain andhistological detecting
Objective: To observe the survial, migration and integration with surroundingtissue of USPIO labeled BMSCs-D-NSCs in normal or ischemic brain, and todetermine the feasibility of MRI tracking of the labeled stem cells.
Methods: The SD rat BMSCs were isolated and induced into BMSCs-D-NSCsin vitro firstly. The BMSCs-D-NSCs were transplanted into normal cortex andstriatum of ischemic (middle cerebral artery occlusion, MCAO)rat bymicrotransplantation method after 10~6 cells were labeled with Sinerem. The unlabeledcells were transplanted into other side brain as the control group in normal rat. Thelabeled cells were scanned at different timepoints by T_2~*WI with 4.7 T MR system,and then the animals were killed to have a histological detection for the Sineremlabeled cells in vivo.
Results: Normal animal: The MR imaging of labeled BMSCs-D-NSCs in ratcortex appeared low signal area and the unlabeled cells had no significent signal change, which could not be distinguished from the surrounding tissue. The low signalarea did not migrate obviously at one week, but they had a slight migration along thecarpus callosum by 4 weeks.
Ischemical model animal: The MR imaging of transplanted BMSCs-D-NSCs instriatum appeard low signal area. The low sinai area started to migrate to otherhemisphere by one week and reached carpus callosum fiber by 2 weeks, migratingmuch far along carpus callosum fiber by 4 weeks.
The transplanted labled cells were blue with Prussian blue staining and thestaining cells distributed in the same place of low signal area. Fe particles could beobserved in endosomes/lysosomes and the labeled cells contacted closely with thesurrounding tissue by transmission electron microscope. Although we could not findthe synaptic-like structure, some cell bodies had the trendency to extend toward twopoles.
Conclusion: Sinerem labeled BMSCs-D-NSCs could display in vivo with MR,whose status of survival and migratin could be tracked specially. This technologycould monitor the labeled stem cells in vivo.
我国属脑血管病高发国家，脑卒中是我国疾病死亡和导致长期残疾的三大原因之一。缺血性脑梗塞约占卒中的80％，用组织纤溶酶原激活剂进行治疗的方法能使梗塞再通，但由于溶栓的副作用和较短的治疗时间窗而使其应用受到限制，因此，只有很小部分卒中病人能得到及时的溶栓治疗。而存活的患者往往伴有严重的残疾，即使最好的康复治疗也由于不可逆的脑损伤而疗效有限。因而，脑卒中的治疗仍需要寻找更好更有效的治疗措施。
由于受“中枢神经不可再生”旧理论的束缚和影响，长期以来神经细胞损伤后结构与功能修复的研究进展一直比较缓慢。20世纪末期，干细胞(Stem cells，SCs)的发现为人们治疗许多难治性疾病提供了新的历史契机。干细胞因具有很强的自我更新能力和多种细胞分化潜能而成为研究的热点，具有非常重要的理论研究和临床应用价值。其中，神经干细胞(Neural stem cells，NSCs)的发现和证实，为治疗包括脑卒中在内的难治性中枢神经系统(Center neural system，CNS)疾病开辟了新的道路。目前，已证实的NSCs来源主要有：(1)大脑室管膜下区、海马和齿状回部位的干细胞，可在脑细胞受损伤时动员到局部，但数量与作用有限；(2)胚胎干细胞，是一类从着床前囊胚内细胞团或早期胚胎原始生殖细胞分离克隆出来的一类未分化的全能性干细胞，在适当条件下能向神经干细胞方向分化，因受到伦理道德上的约束只能应用于动物实验；(3)骨髓基质细胞(Bone marrow stromal cells，BMSCs)，可以向NSCs、神经元和神经胶质细胞方向分化，为了与来源神经组织的NSCs相区别，将此种由骨髓基质细胞诱导而来的NSCs称为骨髓基质细胞源NSCs(BMSCs-D-NSCs)。由于BMSCs容易获取、可在体外大量扩增并在一定条件下能诱导成神经干细胞，因此成为NSCs的重要来源，不仅克服了从其它部位取材的危险性和局限性，也避免了胚胎组织移植中存在的伦理和免疫排斥问题。若将外源性基因导入待移植NSCs，使其在体内有效表达，则可达到长效修复受损伤脑组织功能和治疗的目的。因此，BMSCs-D-NSCs移植是一种极具潜力的治疗缺血性脑卒中策略。
NSCs移植到宿主后组织结构的改变与功能的改善程度是我们关注的重点，而这些移植的效果与移植细胞的状况密切相关，这些状况主要是细胞的存活、生长、迁移、分化情况、与周围组织的融合情况等。因此，上述细胞在宿主内转归过程是评价移植的重要内容。以往传统的方法主要是先处死动物，然后再利用组织学或分子生物学等方来观察干细胞移植后在体内的转归和如何促进行为学上的功能改善，使研究解剖学改变与行为学的恢复之间的关系研究受到限制，这种侵袭性的分析只能提供细胞的单一的“瞬间快照”(snapshot)，不能对同一受试动物移植的干细胞及宿主进行动态连续的实时观察，而且细胞迁移路径和有些病变用组织学方法很难观察到，从而丢失许多重要的信息。同时，侵袭性的研究方法更不适用于人体，严重制约干细胞移植在临床上的研究与应用。所以，必须寻找新的手段来监测干细胞移植后在体内的情况，为解决临床应用的瓶颈提供可能。
超小超顺磁性氧化铁(ultrasmall superparamagnetic iron oxide，USPIO)是一类新型的纳米级的超顺磁性氧化铁微粒，由于其自身特殊的磁性，可在磁场作用下与周围组织进行不同的成像，如果用其标记干细胞，就可以进行影像学的无创监测。目前，还未见用来标记大鼠骨髓基质细胞移植脑卒中模型的报道。本研究课题通过磁共振(Magnetic resonance，MR)成像技术和体外细胞标记技术相结合的方法，利用USPIO Sinerem标记鼠BMSCs-D-NSCs并移植，研究Sinerem对BMSCs-D-NSCs的生物学影响，及移植缺血性脑卒中动物模型后在活体脑内的识别、存活、迁移、增殖等转归，探讨NSCs移植治疗脑缺血的机制和无创伤成像技术的适用性，为NSCs移植治疗脑卒中的临床应用与安全性奠定基础，并为干细胞治疗其它神经系统疾病提供有益的启示。
第一部分：SD大鼠骨髓基质细胞体外向神经干细胞诱导的实验研究
目的：通过细胞培养的方法对分离的大鼠骨髓基质细胞进行体外诱导，向神经干细胞方向诱导。
方法：无菌条件下获取大鼠股骨中的骨髓，用淋巴分离液经梯度离心分离骨髓基质细胞。用干细胞培养基／碱性成纤维因子／白细胞抑制因子／胎牛血清进行实验组的培养、诱导，以DMEM／F12培养基／胎牛血清进行对照组的培养，在倒置光学相差显镜下观察细胞的形态、数量等生长情况；利用计数法测定细胞生长曲线；利用免疫细胞化学方法对神经巢蛋白(Nestin)特异性分子标志物进行检测鉴定。
结果：细胞刚接种后在倒置显微镜下观察可见细胞呈悬浮状态，形态为圆形，大小均一，边缘光滑、完整。接种后24h，骨髓基质细胞开始贴壁，随时间的增加，贴壁细胞逐渐增多，大约3天后贴壁细胞数量不再增加，未贴壁细胞仍呈悬浮状。此时更换培养基后，除掉了其中的非粘附细胞，可见剩余较纯的贴壁细胞。剩余的贴壁细胞折光性强，体积较前期略增大，个别细胞出现核分裂相。5～7天时出现较多的分裂相，增殖明显，出现少许细胞克隆团，9～12天时细胞增殖旺盛，可见较多的细胞克隆团形成，围绕克隆团细胞向周围呈辐射状或旋涡状分布，大部分呈圆形或椭圆形。继续培养至18～20天时，大部分细胞形态开始发生变化，呈多边形、梭形等不规则状，从胞体发出的突起向远处伸展并可相互接触，类似于神经细胞间的联络。而在DMEM／F12培养的对照组，细胞接种后第8天左右时只有极少数量的克隆团出现，且随时间的延长数量增加不明显。
生长曲线说明，实验组细胞接种后7～9天开始进入对数生长期，至14～16天融合达80％。而对照组9～11天时进入对数生长期。
实验组细胞在接种培养第8天时检测到BMSCs-D-NSCs干细胞特异性抗原神经巢蛋白(Nestin)表达呈阳性，提示BMSCs诱导为BMSCs-D-NSCs。
结论：所用的分离骨髓基质细胞方法简单易行，在一定的条件下大鼠骨髓基质细胞可向神经干细胞方向诱导，具有很强的自我更新和多潜能分化能力，可作为干细胞移植的供体细胞。
第二部分：Sinerem纳米铁标记鼠BMSCs-D-NSCs及其生物学影响
目的：明确新型超小超顺磁性氧化铁纳米微粒(USPIO)Sinerem体外标记大鼠骨髓源神经干细胞所需的合适浓度和铁微粒在细胞中的代谢、存留情况，探讨Sinerem标记大鼠骨髓源神经干细胞的可行性，为标记细胞的磁共振成像奠定基础。
方法：无菌条件下手术取SD大鼠双侧股骨的骨髓，利用淋巴细胞分离液分离其中的骨髓基质细胞，体外培养诱导成骨髓源神经干细胞。首先USPIO Sinerem和PLL通过静电作用形成的复合物。再用不同浓度的Sinerem(0、25、50、100、200、500μg／ml)和细胞过夜进行标记，分别于标记后1、3、5、7、9、11～17d不同的时间点行MTT观察细胞增殖，并用流式细胞仪观察细胞的生长周期和凋亡情况；RT-PCR方法检测铁标志对干细胞相关基因表达的影响；利用免疫细胞化学方法分析铁标记对干细胞分化能力的影响。普鲁士蓝染色和透射电镜确定细胞内铁的摄取、定位情况及细胞的标记效率。
结果：细胞形态的观察：倒置显微镜下标记组与未标记组的细胞形态无明显区别，普鲁士蓝染色的强度伴随着培养基中Sinerem浓度的增加而升高，提示进入细胞中的铁随浓度升高而逐渐增加，对照的未标记细胞无蓝染。最佳浓度的确定：与未标记组相比，Sinerem浓度为200μg／ml或以下时，标记细胞的活力、凋亡率无明显变化(P＞0.05)，但200μ／ml已接近临界值，而浓度大于等于500μg／ml时与对照组相比有显著性差异(P＜0.05)。对基因表达的影响：在适当的浓度200μg／ml时标记细胞中的相关基因表达水平无明显改变。对细胞周期的影响：与未标记组细胞相比，流式细胞仪检测结果显示，标记组细胞G_1、S、G_2／M期的细胞所占百分比没有显著性差异，说明适当浓度时Sinerem标记对细胞周期没有明显的不良影响。对分化能力的影响：该浓度时标记细胞向脂肪细胞分化的情况与对照组相比差别不大。标记效率：经显微镜下计数观察，Sinerem标记干细胞效率为98％～100％。细胞内铁的鉴定：普鲁士蓝染色显示铁颗粒存在胞质中，电镜显示铁颗粒集中于内涵体／溶酶体中。
结论：超小超顺磁性Sinerem在合适浓度(200μg／ml)时能安全有效地体外标记骨髓源神经干细胞，且对细胞无短期或长期的毒性影响，为神经干细胞移植的无创示踪奠定了基础。
第三部分：Sinerem纳米铁标记大鼠BMSCs-D-NSCs体外磁共振成像研究
目的：应用磁共振成像技术观察Sinerem标记后的SD大鼠BMSCs-D-NSCs体外成像情况，初步评价磁共振成像体外示踪Sinerem标记干细胞的可行性，为下一步体内移植成像创造条件。
方法：分离SD大鼠骨髓基质干细胞，体外培养诱导成BMSCs-D-NSCs。以200μg／ml的浓度10~5个细胞用不同的SE序列T_1WI、T_2WI与T_3~*WI在2％的琼脂糖凝胶中模拟在体环境分别行4.7T扫描确定最佳的扫描序列。将不同浓度(0、25、50、100、200、500μg／ml)的Sinerem和干细胞(10~6个)共孵育培养过夜标记后置于2％的琼脂糖凝胶中，以SE序列T_2~*WI磁共振干细胞成像；并在磁共振下观察不同数量细胞200μg／ml Sinerem标记细胞(10~1个，10~2个，10~3个，10~4个，10~5个)的信号情况。观察细胞在浓度200μg／ml(10~6个)标记时经长期培养不同时间点时的信号变化。
结果：MR扫描结果提示序列T_2~*WI扫描的敏感性最强。不同浓度的Sinerem标记细胞的磁共振信号强度不同，随着Sinerem浓度的升高MR信号呈降低趋势，浓度为500μg／ml时信号强度最低，肉眼观察可看到浓度100μg／ml以上标记细胞的MR的信号强度明显低于对照样品，可进行区分。标记细胞数量达10~3个及以上时在磁共振下其信号变化能被观察到。10~6个标记细胞的信号随培养时间的延长逐渐增加，4w时仍能在磁共振下显示可区分的低信号。
结论：利用Sinerem标记的BMSCs-D-NSCs体外可在MR下呈现可视区别的低信号影，T_2~*WI序列是最敏感的序列；信号强度的高低可用于评估标记细胞的数量。Sinerem标记的细胞可用于磁共振下进行长期的示踪。
第四部分：Sinerem纳米铁标记大鼠BMSCs-D-NSCs移植大脑的活体MR示踪及组织学检测
目的：将USPIO标记的大鼠BMSCs-D-NSCs移植正常或缺血模型脑后，初步观察其在大脑中的存活、迁移和整合情况，确定体内MR成像示踪Sinerem标记干细胞的可行性。
方法：首先分离SD大鼠BMSCs，体外培养诱导成BMSCs-D-NSCs。将200μg／ml的Sinerem纳米铁标记BMSCs-D-NSCs后，将标记细胞(1×10~6个)用脑立体定向仪移植通过微量注射器注射到正常大鼠皮层或缺血模型大鼠纹状体。同时在正常大鼠的另一侧用未标记细胞作为对照。在不同时间点(1d、1w、2w、4w)以SE序列T_2~*WI行4.7T磁共振成像示踪，然后在不同时间点处死动物，用组织学方法观察标记细胞在脑内的转归情况。
结果：在非缺血模型：大鼠移植皮层的标记细胞在磁共振下呈T_2~*WI低信号区，而未标记细胞无信号变化，与周围脑组织不能区别；1w时低信号区未见明显位移，而至4w时检测到细胞沿胼胝体的轻度迁移。在缺血模型中：移植到纹状体部位的标记细胞磁共振扫描时呈低信号区，1w时可见低信号区开始向对侧方向迁移，随时间的增加至2w时迁移至胼胝体纤维，4w时进一步沿胼胝体向缺血侧迁移。处死动物后，组织学检测可见移植的标记细胞普鲁士染色阳性，标记细胞的分布与磁共振扫描的低信号区相一致。透射电镜下可见观察到标记细胞中存在的Sinerem铁颗粒，细胞和周围组织相互紧密接触，未见到突触样结构的形成，但从形态上可见到个别细胞胞体有向两极伸展的趋势。
结论：Sinerem标记的BMSCs-D-NSCs在动物活体内能在磁共振下显像，其在脑内存活、迁移等行为可被磁共振特异性示踪，可用于干细胞移植的无创监测。
Background
Incidence of cerebro-vascular disorders is very high, and cerebral stroke is oneof the three leading causes of mortality and morbidity in China.80% of cerebral strokeis ischemic cerebral infarction. The infarction can be recovered with the tissueplasminogen activator. However, of all patients who suffer from an ischemic stroke,only a small proportion of patients were treated with acute reperfusion therapy to haltprogression of ischemic brain damage because of side effect of reperfusion therapyand shorter time window. Moreover, frequently, these survial patients suffered fromservious morbidity and the effects of therapies are limited, even the best rehabilitationwas applied. Therefore, It is necessary to search for better therapies to cure thecerebral stroke.
The recovery progress of structure and function of damaged neural cells wasslow for a long time because an old theory that center nerve can not regrow affectedthe people. Emerge of Stem cells provided a good chance for curing many intractablediseases.SCs have become a research hot point because of its self-renewal andmulti-potential differentiation, which is important for theory research and clinicalapplications. Neural stem cells, as one kind of stem cells, has brought a new way forintractable diseases of center neural system including cerebral stroke. Nowadays, It has been demonstrated that neural stem cells exist in the following parts:(1)Stem cellsfrom the subventricular zone,the hippocampus or dentate gyrus, can be actived andmigrate to the injury site. However, their number and effect are limited.(2)Embryonicstem cells originate as inner mass cells within a blastocyst and can differentiate intoneural stem cells under appropriate conditions.But the embryonic stem cells ispresently only limited to be used in animal trial for ethical issues. (3) Bone marrowstromal cells, brifely, BMSCs, can differentiate into NSCs, neurons and glial cells. Todistinguish them from the stem cells from neural tissue, the neural stem cells inducedfrom bone stromal cells were called bone marrow stromal cells-derived-neural stemcells (BMSCs-D-NSCs). BMSCs are ease of isolation and high expansion potential invitro, and can be induced into neural stem cells.Therefore, BMSCs has been theimportant resource, which overcomes the risk of gaining material, as well as avoid theproblem of ethical issue and immunological rejection. The damaged brain tissuecould be repaired by transplanted NSCs if the exogenous gene was introducedinto them. Consequently, transplantation of BMSCs-D-NSCs is a potential strategyfor treating with the ischemical stroke.
Structure change and functional improvement of tissue after NSCstransplantation are our focus, and the outcome is closely related with NSCs status invivo including cell vability, growth, migration,differentiation and integration withsurounding tissues.Hence, It is important to evaluate the process of transplanted cellsin vivo.In the past, the animals must be firstly killed and then the histological andmolecular trials were taken to observe the events and how to promote behaviorimprovement, which restricted the research on relationship between anatomy changeand behavior recovery. The invasive analysis can only provide single snapshot andcan not have a real observation on the tansplanted stem cells and animals serially.Moreover, the migration way and some pathological process were hard to be found and much important information would lost, and the invasive way can not be appliedfor human being. All of the shortcomings above had seriously restricted the clinicalresearch and application.Therefore, it is necessory to explore new method to monitorthe stem cells in vivo and resolve the bottleneck problem.
Ultrasmall superparamagnetic iron oxide(USPIO) is a new kind ofsuperparamagnetic iron particulate with nano-scale. USPIO can visualizedistinguishing from surrounding tissue because of its special magnetic property andthe labeled stem cells should be monitored by nontraumatic methods. Up to date,there was no report about rat BMSCs transplantation to cerebral stroke. This issuemainly explored the effect of USPIO sinerem on BMSCs-D-NSCs biologicalcharacteristics and the events about recognition,survial, migration and proliferation ofthe labeled stem cells,and investigated the mechanism of brain ischemic diseasetreated by NSCs transplantation and applicability of nontraumatic imagingtechnology in order to establish a base for clinical application and safety and tosupply benefical revelation for other neural system diseases.
PartⅠ: Research on inducement of SD rat BMSCs into neural stem cells invitro
Objective: To induce the isolated rat BMSCs into neural stem cells by cellculture.
Methods: Bone marrow stem cells isolated from thigh bone marrows of SD ratsby density gradient centrifugation with lymphocyte separating medium. Then theBMSCs were cultured and induced into BMSCs-D-NSCs by NSCs culturemedium/basic fibroblast growth factor/leukemia inhibitory factor/fetal bovine serum,and the control BMSCs were cultured with DMEM/F12 medium/fetal bovine serumat the same time. The cultured cells were observed by invert phase contrast lightmicroscope to investigate their morphology and quantity.Cell growth curves were destribed by counting progress. Identification of neural stem cell was performed byspecial nestin marker immunocytochemical staining.
Results: The separated BMSCs suspeneded and their shape was round,size-equal, outline-clean and intact under inverted phase contrast microscope at thebeginning of culture.The BMSCs started attaching and more cells attached with timeextending.The cell attachment would stop after 3 days and the unattached cells stillsuspended. The purified cells were left after discarding the unattached cells and theattached cells were more dioptric, bigger and emerged nuclear splitting.There weremore splitting cells during 5days～7days and proliferation was obvious and fewcloning mass appeared. Cell growth became more strong and more cloning massappeared in the day of 9～12, which surrounded one center point with radiating orswirling shape, most of them were round or elliptical.Up to 18days～20days,morphology of most cells started changing, appearing polygon or fusiform, etc.Processus from cell body extended to far place and connected with each other, similarto contact between neural cells.However, only very few cloning mass appeared ineighth day and did not increase with time extending.
The growth curving indicated that the experimental group started logarithmicgrowth during 7～9days and merged to 80% during 14～16days.In contrast, controlgroup began logarithmic growth until 9～11days.
The experimental group could express special NSCs antigen nestin proteinwhen they were detected by eighth day.
Conclusion: The strategy of isolating BMSCs was easy and accessible, and therat BMSCs could be induced into neural stem cells with strong self-renewal andmulti- potential differentiation ability, which could be used as donor cells.
PartⅡ: Sinerem labeling rat BMSCs-D-NSCs and its effect on the cells'characteristics
Objective: To determine the optimal concentration of new type USPIO Sineremlabeling BMSCs-D-NSCs in vitro and investigate the metabolism and retaining of Feparticle, and expolre the feasibility of labeling rat BMSCs-D-NSCs by Sinerem inorder to establish base for MR imaging.
Methods: Bone marrow stem cells isolated from thigh bone marrows of SD ratsby density gradient centrifugation with lymphocyte separating medium. Then theBMSCs were cultured and induced into BMSCs-D-NSCs in vitro. Firstly, Sineremand poly-l-lysine(PLL) formed complex by static electricity.Then, theBMSCs-D-NSCs
were incubated with the comples with different Sineren concentration as 0, 25,50, 100,200and500μg/ml for one night. The proliferation of labeled cells wereobserved by MTT test at the timepoint of 1,3,5,7,9,11～17day. The growth cycle andapoptosis of labeled cells were tested by flow cytometry. The effect on related keygene expression was investigated by RT-PCR. Moreover, the effect of Fe labeling ondifferentiation ability was analized by immunocytochemistry. Prussian blue stainingand transmission electron microscope were conducted for demonstrating theuptake,location and labeling ratio of intracytoplastic nanoparticles.
Results: Morphologic difference of labeled and unlabeled BMSCs-D-NSCs wasnot significant.The density of Prussian blue staining increased with the lifting ofSinerem concentration,and the control group has no blue staining. In contrast tounlabeled group, the vitality and apoptosis ratio of labeled cells had no obviouschange when the Sinerem concentration was 200μg/ml or less than 200μg/ml(P＞0.05),but this concentration has very closely near the threshold value.It was significantwhen the Sinerem concentration was 500μg/ml or more than 500μg/ml(P＜0.05). Thegene expression level has no significant change after labeled by the appropriateconcentration(200μg/ml) contrast to the control group. The cell cycle had no significant difference between the labeled and unlabeled cells, which showed Sineremlabeling has no hurt on cell cycle. The differentiation ability of BMSCs-D-NSCs intoadipose cell has no obvious difference between two groups under the concentration of200μg/ml.The ratio of Sinerem labeling was about 98%～100% whenBMSCs-D-NSCs was labeled with 200μg/ml. Prussian blue staining showed that Feparticle lied in cytoplasm and transmission electron microscope indicated that thesenano-particles concentrated in endosomes/lysosomes.
Conclusion: BMSCs-D-NSCs labeling by Sinerem was safe under appropriateconcentration of 200μg/ml and has no poisonous for short or long time, whichestablished base for tracking NSCs in vivo.
PartⅢ: Experimental study on Sinerem labeling of rat BMSCs-D-NSCswith MRI in vitro
Objective: To observe the MRI of rat BMSCs-D-NSCs labeled by Sinerem invitro and evaluate the feasibility of MRI tracking of labeled NSCs.
Methods: The SD rat BMSCs were isolated and induced into BMSCs-D-NSCsin vitro. 10~5cells labeled with 200μg/ml Sinerem were immerged in 2% gel andscanned by different SE sequence T_1WI、T_2WI与T_2~*WI respectively to determine thebest scanning sequence with 4.7T MR system.10~6 cells labeled with differentconcentration
(0,25,50,100,200,500μg/ml) were scanned in 2% gel by T_2~*WI sequence.Different number(10~1,10~2,10~3,10~4,10~5)of BMSs-D-NSCs labeled with 200μg/mlSinerem were scanned to determine the threshold of cell quantity. In the end, thesignal change of 200μg/ml Sinerem labeled cells was observed at differenttimepoints.
Results: MR result indicated that T_2~*WI was the most sensitive sequence. TheMR signal intensity of Sinerem labeled cells with different concentration was various, which reduced progressively with the increasing of Sinerem concentration. The signalintensity was lowest when the concentration was 500μg/ml. The MR signal intensityof more than 100μg/ml Sinerem labeling cells was much lower than control sampleand could be distinguished.
The MR signal change could be observed when the cell number was and 10~3 andmore. The signal intensity of 10~6 labeled cells was increaseing with the timeextending, which could still be displayed as different low signal for 4 weeks.
Conclnsion:Sinerem labeled BMSCs-D-NSCs in vitro appeared visually lowsignal area. The best sensitive sequence was T_2~*WI. The level of signal could beapplied for evaluating cell numbers. Sinerem labeled cells could be tracked for a longtime.
PartⅣ: MR tracking of Sinerem labeled rat BMSCs-D-NSCs in brain andhistological detecting
Objective: To observe the survial, migration and integration with surroundingtissue of USPIO labeled BMSCs-D-NSCs in normal or ischemic brain, and todetermine the feasibility of MRI tracking of the labeled stem cells.
Methods: The SD rat BMSCs were isolated and induced into BMSCs-D-NSCsin vitro firstly. The BMSCs-D-NSCs were transplanted into normal cortex andstriatum of ischemic (middle cerebral artery occlusion, MCAO)rat bymicrotransplantation method after 10~6 cells were labeled with Sinerem. The unlabeledcells were transplanted into other side brain as the control group in normal rat. Thelabeled cells were scanned at different timepoints by T_2~*WI with 4.7 T MR system,and then the animals were killed to have a histological detection for the Sineremlabeled cells in vivo.
Results: Normal animal: The MR imaging of labeled BMSCs-D-NSCs in ratcortex appeared low signal area and the unlabeled cells had no significent signal change, which could not be distinguished from the surrounding tissue. The low signalarea did not migrate obviously at one week, but they had a slight migration along thecarpus callosum by 4 weeks.
Ischemical model animal: The MR imaging of transplanted BMSCs-D-NSCs instriatum appeard low signal area. The low sinai area started to migrate to otherhemisphere by one week and reached carpus callosum fiber by 2 weeks, migratingmuch far along carpus callosum fiber by 4 weeks.
The transplanted labled cells were blue with Prussian blue staining and thestaining cells distributed in the same place of low signal area. Fe particles could beobserved in endosomes/lysosomes and the labeled cells contacted closely with thesurrounding tissue by transmission electron microscope. Although we could not findthe synaptic-like structure, some cell bodies had the trendency to extend toward twopoles.
Conclusion: Sinerem labeled BMSCs-D-NSCs could display in vivo with MR,whose status of survival and migratin could be tracked specially. This technologycould monitor the labeled stem cells in vivo.
引文
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