摘要
背景:骨髓间充质干细胞通过再生心肌细胞在心肌损伤性疾病治疗方面突显较大潜力,但目前仍未找到一种理想的诱导方式。目的:探讨微血管内皮细胞诱导大鼠骨髓间充质干细胞向心肌细胞分化的潜能,并分析其移植的可行性。设计、时间及地点:细胞学体外观察及体内移植实验,于2008-07/2009-02在重庆市神经病学重点实验室完成。材料:清洁级二三月龄健康雄性SD大鼠16只,购自重庆医科大学实验动物中心方法:孔径0.4μm的transwell小室,置入培养板孔构成双室培养体系。取SD大鼠1只,Ficoll密度梯度+贴壁筛选法分离培养骨髓间充质干细胞,组织块法分离培养微血管内皮细胞。培养瓶中融合至50%生长良好的微血管内皮细胞,每3d更换培养液,收集更换液过滤即制成条件培养液。体外实验分为4组:直接接触组、双室培养组、条件培养液组、培养液对照组,观察各种培养条件对骨髓间充质干细胞的心肌诱导效应。取SD大鼠15只建立心肌梗死模型,冠状动脉结扎后第6天任取1只大鼠明确心肌梗死建模是否成功,其余大鼠随机分为细胞移植组8只、模型对照组6只。造模1周后,细胞移植组将在双室模型中与微血管内皮细胞共培养5d的骨髓间充质干细胞调整浓度为109L-1,通过鼠尾静脉缓慢注入1mL,模型对照组予等量DMEM,观察4周。主要观察指标:体外实验通过免疫荧光染色检测心肌标志物TnI、α-actin的表达;采用心电图、荧光镜检、苏木精-伊红染色等方式评价体内移植的安全性及可行性。结果:直接接触组、双室培养组、条件培养液组部分骨髓间充质干细胞心肌标志物TnI、α-actin阳性表达,荧光镜下胞浆发绿光(或红光),前2组诱导率基本相似(P>0.05),且均明显高于条件培养液组(P<0.05);培养液对照组细胞未向心肌样细胞分化。细胞移植后两组大鼠均存活良好,未出现死亡及恶性心律失常,4周后细胞移植组心脏冰冻切片荧光镜检显示心肌内有少量胞核蓝染的细胞,提示干细胞成功归巢,结合苏木精-伊红染色,归巢细胞位于心肌梗死灶周边区域,呈单个或小团聚集;而模型对照组心肌切片未见胞核蓝染的细胞。结论:微血管内皮细胞能够通过旁分泌途径诱导骨髓间充质干细胞向心肌样细胞分化,经微血管内皮细胞预处理过的骨髓间充质干细胞移植入大鼠体内是安全的,可成功归巢到大鼠心肌梗死灶及周边区。
BACKGROUND：Bone marrow mesenchymal stem cells（BMSCs） have an enormous potential on myocardium-injured diseases via cardiomyogenesis,however,an ideal inducing method has not been found at present. OBJECTIVE：To investigate the potential of rat microvascular endothelial cells（mECs） on inducing BMSCs differentiation into cardiomyocytes,and to analyze the feasibility of transplantation. DESIGN,TIME AND SETTING：An observational experiment based on cells and animals was conducted at the Chongqing Key Laboratory of Neurology between July 2008 and February 2009. MATERIALS：A total of 16 clear healthy male Sprague Dawley rats aged 2-3 months were obtained from the Experimental Animal Center,Chongqing Medical University. METHODS：Transwell cabin of 0.4 μm bore was placed in culture plate well to construct double-cabin culture system. BMSCs and mECs were respectively isolated and cultured from 1 Sprague Dawley rat using Ficoll density gradient ＋ adherence screening method and explant method. mECs were well cultured and reached 50% confluency. The medium was changed every 3 days. Following collection and filtration,the conditioned medium was obtained. In vitro experiment was divided into direct contact group（Ⅰ） ,bi-chamber coculture group（Ⅱ） ,conditioned medium group（Ⅲ） and medium control group（Ⅳ） ,and myocardial induction effect was observed in these conditions. A total of 15 healthy Sprague Dawley rats were committed to coronary ligation for myocardial infarction models and divided into cell implantation group（n=8） and model control group（n=6） . 1 week following model induction,BMSCs（109/L） processed with mECs for 5 days and equivalent DMEM（1 mL） were injected through tail veils,respectively in the cell implantation group and model control group,and then observed for 4 weeks. MAIN OUTCOME MEASURES：Cardiac markers as TnI and α-actin expression were tested using immunofluorescent staining in vitro. Implantation safety and feasibility were evaluated by electrocardiogram,fluorescent microscope and hematoxylin-eosin staining. RESULTS：Partial BMSCs in groupⅠ,Ⅱ and Ⅲ processed morphologic changes with TnI and α-actin positively expressed after induction. Cytoplasm emitted green（or red） under the fluoroscope. Induction rate in group Ⅰand groupⅡ respectively was similar（P 0.05） ,and significantly greater than that in group Ⅲ and group Ⅳ（P 0.05） . Cells did not differentiate into cardiomyocytes in the group Ⅳ. All experiment animals lived well with no death or malignant arrhythmia following cell transplantation. Frozen sections of the heart showed a few blue-stained cells under the fluoroscope in the cell transplantation group 4 weeks later. This indicated that stem cells were successfully homing. Using hematoxylin-eosin staining,homing cells were distributed over the infarct zone,with the presence of single or mass. In the model control group,no blue-stained cells were determined. CONCLUSION：mECs can induce BMSCs differentiation into cardiac-like myocytes through paracrine mechanisms. Implantation of BMSCs pretreated with mECs into rats is safe,and can successfully homing to the rat infarct zone or surrounding the infarct zone.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第40期7811-7816,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
