血管内皮生长因子对诱导多能干细胞向心肌细胞分化的影响

发布时间：2018-06-24 21:14

本文选题：血管内皮生长因子 + 小鼠诱导多能干细胞　；参考：《重庆医科大学》2012年硕士论文

【摘要】：目的观察血管内皮生长因子（VEGF）对小鼠诱导多能干细胞（miPSc）向心肌细胞分化的影响。方法在白血病抑制因子(LIF)和成纤维细胞存在的条件下培养小鼠iPSc，从小鼠iPSc的镜下形态特征，干细胞基因Oct-4的表达及iPSc的碱性磷酸酶染色方面鉴定iPSc。并采用悬滴培养法启动iPSc分化，分别以10ng/ml、20ng/ml、50ng/ml的VEGF作为诱导剂对小鼠iPSc诱导分化，以自然分化作为阴性对照组，以加入1%二甲亚砜（DMSO）诱导剂作为阳性对照组。每天在倒置显微镜下观察细胞生长情况，记录各组跳动的拟胚体（EBs）出现的具体时间及每天跳动的拟胚体的数目，进一步计算心肌细胞分化率。PT-PCR检测分化过程中心肌发育基因α-MHC和β-MHC mRNA的表达,细胞免疫荧光检测iPSc分化细胞cTnT的表达。结果1.在LIF和成纤维细胞存在的条件下，iPSc呈集落样或巢状生长且不断增殖；荧光显微镜下小鼠iPSc呈绿色荧光（Oct-4启动），碱性磷酸酶染色提示iPSc细胞团着紫黑色。 2.各分化组均可在光镜下见自发搏动的EBs，与自然分化组相比，三个浓度组的VEGF均可提高iPSc的心肌细胞分化率(P0.05)；VEGF浓度为20ng/ml时，，iPS细胞的心肌细胞分化率最高，为53.17%±3.63%（P0.05），与二甲亚砜组的心肌细胞分化率（57.07%±3.22%）相比无统计学差异（P0.05）。 3.各分化组分化而来的细胞均表达调控心肌发育的基因α-MHC和β-MHC;VEGF可上调α-MHC和β-MHC的表达，VEGF浓度为20ng/ml时,其上调作用最为明显（P0.05）。 4.各分化组分化而来的心肌细胞可自发搏动，同时表达心肌特异蛋白cTnT。结论1.在LIF和成纤维细胞的环境中，小鼠iPSc保持未分化状态，去除这两者后，小鼠iPSc可以通过形成拟胚体开始分化。 2.一定浓度的外源性的VEGF可以促进诱导多能干细胞向心肌细胞的分化。
[Abstract]:Objective to investigate the effect of vascular endothelial growth factor (VEGF) on the differentiation of mouse pluripotent stem cells (miPSc) into cardiomyocytes. Methods iPScwas cultured in the presence of leukemia suppressor (LIF) and fibroblasts. The morphological characteristics of mouse iPSC, the expression of stem cell gene Oct-4 and the alkaline phosphatase staining of iPSc were identified. The differentiation of iPSC was induced by 10 ng / ml 20 ng / ml VEGF as inducer, natural differentiation as negative control group and 1% dimethyl sulfoxide (DMSO) inducer as positive control group. The cell growth was observed under inverted microscope every day. The specific time of emergence of the beating embryoid body (EBs) and the number of the beating embryoid bodies in each group were recorded. The expression of 伪 -MHC and 尾 -MHC mRNA and cTnT of iPSc differentiated cells were detected by PT-PCR and immunofluorescence. Result 1. In the presence of LIF and fibroblasts, iPSc grew in the shape of colony or nesting and continued to proliferate, while the mouse iPSc showed green fluorescence (Oct-4 priming) under fluorescence microscope, and alkaline phosphatase staining showed that iPSc cells were purple and black. The spontaneous pulsatile EBs were observed in each differentiation group under light microscope. Compared with the natural differentiation group, the differentiation rate of cardiac myocytes in the three concentration groups (P0.05) was higher than that in the natural differentiation group (P0.05) when the concentration of VEGF was 20ng/ml, the differentiation rate of cardiac myocytes was the highest. Compared with dimethyl sulfoxide group (57.07% 卤3.22%), the differentiation rate of cardiomyocytes was 53.17% 卤3.63% (P0.05), and there was no significant difference between dimethyl sulfoxide group and dimethyl sulfoxide group (P0.05). All the differentiated cells expressed 伪 -MHC and 尾 -MHC genes that regulated myocardial development. When the expression of 伪 -MHC and 尾 -MHC was 20ng/ml, the up-regulation effect was the most obvious (P0.05). The cardiac myocytes differentiated from each differentiation group could spontaneously pulsatile and express the myocardial specific protein cTnT at the same time. Conclusion 1. In the environment of LIF and fibroblast, mouse iPSc remained undifferentiated, and the mouse iPSc could begin to differentiate by forming embryoid body. 2. Exogenous VEGF at a certain concentration can promote the differentiation of pluripotent stem cells into cardiomyocytes.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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