人脐带血间充质干细胞移植治疗大鼠急性心肌梗死的实验研究

发布时间：2018-01-07 13:05

本文关键词：人脐带血间充质干细胞移植治疗大鼠急性心肌梗死的实验研究　出处：《广州医学院》2010年硕士论文　论文类型：学位论文

【摘要】：目的: 1.研究脐带血来源的间充质干细胞体外分离、纯化及培养的条件,以建立稳定的体外培养体系,满足实验和临床的需要。 2.探讨脐血间充质干细胞移植到心肌梗死大鼠体内,观察其是否可以存活及对大鼠心功能的影响。方法: 1.无菌条件下采取健康育龄产妇正常分娩胎儿的脐带血42份,所有样本均于采集8h内分离。 2.脐带血42份随机分成3组: FBS(胎牛血清)未包被组,FBS包被组,Mesencult培养基组,每组12份。FBS未包被组和FBS包被组均使用含10%FBS的LG-DMEM培养基,Mesencult培养基组使用培养干细胞专用的Mesencult培养基,与其添加物配合使用。3组均用Ficoll淋巴细胞分离液,密度梯度法分离脐血单个核细胞(MNC),将单个核细胞按密度1 X 108/ml接种于T25培养瓶中 3.培养瓶置37℃饱和湿度含5%CO2孵育箱培养,72h后全量换液,去除未贴壁细胞,以后均7天换液一次。待细胞长到80%铺满瓶底时结束原代培养,按1:1进行消化传代。观察不同培养条件对脐血MSCs生长的影响。 4.流式细胞术分析P2代细胞表面CD29、CD34、CD45、CD105标志。 5.将36只SD大鼠随机分成MSCs移植组、假手术组和心肌梗死组各12只。结扎左冠状动脉前降支制备大鼠心肌梗死模型, 1周后,经尾静脉注射带DAPI标记的脐血MSCs。 6.4周后测定大鼠血流动力学,并行免疫组织化学检测移植细胞存活与分化情况及检测梗死组织中FactorⅧ表达来比较三组微血管密度。结果: 1.实验观察显示:经典的MSCs培养方法,用未经胎牛血清包被培养瓶培养的MNC很少出现贴壁,且大多为形态多样的混杂细胞,无明显的细胞集落形成。在少数标本中约2周后细胞可达到融合,主要表现为大的扁圆形细胞、破骨细胞和梭形成纤维样的MSCs,尽管这些混杂细胞原代培养也可以达到融合,但其扩增能力明显受到限制,且传代未达2代。而脐血单个核细胞在合适的培养基中,以较高的密度种植于经胎牛血清包被的培养瓶内,原代培养产生的贴壁细胞以间充质干细胞为优势细胞,混杂有少量破骨样细胞。而在Mesencult培养基下培养,经传代后这些贴壁细胞可以被纯化为生长良好的间充质干细胞。其能保持良好的扩增能力和均质性。42份脐血中单个核细胞原代培养,其中仅8份传代培养成功,而其中Mesencult条件培养基有5份培养成功,明显高于经典培养方法。原代和P2代脐血MSCs的生长特性进行观察比较,原代MSCs在接种后的2-8d为生长的潜伏期,10-14d以后细胞进入对数生长期,3-4周左右逐渐进入平台期,P2代脐血MSCs扩增速度明显快于原代培养,2-4d倍增1次,5-10d后细胞进入到对数生长期,10-14d进入平台期。 2.通过流式细胞仪检测P2代的脐血MSCs结果显示,P2代MSCs极弱表达CD34、CD45造血细胞标志,稳定地高表达CD29、CD105间充质细胞相关的表面抗原。这与骨髓MSCs的表面抗原标志相一致。表明脐血MSCs是存在于脐血中区别于造血细胞的一群处于未分化状态的非定向干/祖细胞。 3.移植后4周,经血流动力学检测,与心梗组比较,MSCs移植组左室心功能明显改善(P0.05),移植组心肌组织中可以观察到DAPI标记细胞存在,但标记细胞并未表达Troponin-T及connexin43,免疫组化染色检测示MSCs移植组心肌微血管密度(MVD)明显高于心梗组和假手术组。结论 1.脐血单个核细胞以高密度(1X108cells/ml )接种在Mesencult培养基中、FBS包被培养瓶等条件下,可以在体外成功的培养出较纯化的脐血MSCs,其培养成功率较高。脐血MSCs的免疫表型符合间充质干细胞特征。 2.经尾静脉移植的脐血MSCs能存活在心肌梗死大鼠心肌组织中。 3.脐血MSCs移植能促进心梗大鼠心功能恢复,并刺激梗死部位血管生成。
[Abstract]:Objective:
1., we studied the isolation, purification and culture conditions of umbilical cord blood derived mesenchymal stem cells in vitro, so as to establish a stable in vitro culture system to meet experimental and clinical needs.
2. the umbilical cord blood mesenchymal stem cells were transplanted into the rats with myocardial infarction to observe whether it could survive and influence the cardiac function of rats.
Method:
1. under aseptic conditions, 42 umbilical cord blood samples from healthy childbearing age parturients were taken from normal childbirth, and all the samples were isolated in 8h.
2. 42 copies of umbilical cord blood were randomly divided into 3 groups: FBS (fetal bovine serum) non coated group, FBS group, Mesencult medium group, each group of 12.FBS coated group and FBS group were coated with 10%FBS LG-DMEM medium, Mesencult medium was used for stem cell culture special Mesencult medium and additives are used Ficoll with lymphocyte separation liquid using.3 group, mononuclear cells separated from umbilical cord blood by density gradient method (MNC), the mononuclear cells by density of 1 X 108/ml were seeded in T25 culture flask
3. of the culture flask containing 5%CO2 37 DEG C saturated humidity incubator culture, after 72h was all changed, the nonadherent cells were removed after 7 days, all was changed once. When the cells grow to 80% covered the bottom of the bottle at the end of primary culture, digestion and passage by 1:1. To observe the effects of different culture conditions on the growth of umbilical cord blood MSCs.
4. flow cytometry was used to analyze the CD29, CD34, CD45, and CD105 markers on the surface of P2 cells.
5. 36 SD rats were randomly divided into MSCs transplantation group, sham operation group and myocardial infarction group, 12 rats. The left anterior descending branch of left coronary artery was ligated to prepare rat myocardial infarction model. After 1 weeks, umbilical cord blood with DAPI labeled MSCs. was injected through tail vein.
Determination of hemodynamics in rats after 6.4 weeks, compared with three groups of parallel to microvessel density Factor VIII expression survival chemical detection and differentiation of transplanted cells and immunohistochemical detection in myocardial tissue.
Result:
1.: experimental observation shows that the culture method of the classic MSCs, without fetal bovine serum coated culture flask MNC rarely adherent, and mostly mixed cell morphological diversity, no obvious cell colony formation. In a few specimens in about 2 weeks after cell fusion can be achieved, the main performance for large the flat and round cells, osteoclasts and spindleformation fiber like MSCs, although these hybrid cells in primary culture can also achieve the fusion, but its ability was significantly restricted, and the passage is not up to the 2 generation. The umbilical cord blood mononuclear cells in a suitable medium to high density planting in the fetal bovine serum clear coated culture flask, primary culture of adherent cells to mesenchymal stem cells as the dominant cells, a small amount of osteoclast like cells mixed with. Cultured in the Mesencult culture medium, then the adherent cells can be purified for good growth of mesenchymal The stem cells can maintain good amplification ability and heterogeneity of.42 in umbilical cord blood mononuclear cells were cultured, which only 8 copies were cultured successfully, and the Mesencult conditioned medium 5 successfully cultured, was significantly higher than that of classical methods. The growth characteristics of cultured primary and P2 cells of umbilical cord blood MSCs were compared between the original. Generation of MSCs in 2-8d after inoculation for growth after incubation, 10-14d cells in logarithmic growth phase, 3-4 weeks gradually into the platform, the P2 generation of umbilical cord blood MSCs amplification significantly faster than the primary culture, 2-4d doubled 1 times, after 5-10d cells into the number of students for a long period of time, 10-14d into the platform.
2. by flow cytometry P2 generation of umbilical cord blood MSCs results showed that P2 MSCs very weak expression of CD34, CD45 hematopoietic cell markers, stable and high expression of CD29, CD105 mesenchymal cell associated surface antigens. This is consistent with the surface antigen of bone marrow MSCs sign. That is present in the umbilical cord blood MSCs iswho in a group of hematopoietic cells in non directional undifferentiated stem / progenitor cells.
4 3. weeks after transplantation, the hemodynamics, compared with myocardial infarction group, MSCs transplantation group significantly improved cardiac function of left ventricular (P0.05), transplantation group, myocardial tissue can be observed in DAPI labeled cells, but not the expression of Troponin-T and connexin43 labeled cells, immunohistochemical staining showed MSCs group myocardial microvascular density (MVD) was significantly higher than that in MI group and sham operation group.
conclusion
1. human umbilical cord blood mononuclear cells at high density (1X108cells/ml) were inoculated in Mesencult medium, FBS coated bottles and other conditions, can be successfully cultured with purified MSCs in umbilical cord blood in vitro, the success rate is high. The culture of umbilical cord blood MSCs immune phenotype of mesenchymal stem cells.
2. the umbilical cord blood MSCs transplanted through the tail vein can survive in the myocardium of rats with myocardial infarction.
3. MSCs transplantation in umbilical cord blood can promote the recovery of cardiac function in myocardial infarction rats and stimulate angiogenesis in the infarct site.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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