本文選題：血管內(nèi)皮生長因子 + 小鼠誘導(dǎo)多能干細胞��； 參考：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的觀察血管內(nèi)皮生長因子（VEGF）對小鼠誘導(dǎo)多能干細胞（miPSc）向心肌細胞分化的影響。 方法在白血病抑制因子(LIF)和成纖維細胞存在的條件下培養(yǎng)小鼠iPSc，從小鼠iPSc的鏡下形態(tài)特征，干細胞基因Oct-4的表達及iPSc的堿性磷酸酶染色方面鑒定iPSc。并采用懸滴培養(yǎng)法啟動iPSc分化，分別以10ng/ml、20ng/ml、50ng/ml的VEGF作為誘導(dǎo)劑對小鼠iPSc誘導(dǎo)分化，以自然分化作為陰性對照組，以加入1%二甲亞砜（DMSO）誘導(dǎo)劑作為陽性對照組。每天在倒置顯微鏡下觀察細胞生長情況，記錄各組跳動的擬胚體（EBs）出現(xiàn)的具體時間及每天跳動的擬胚體的數(shù)目，進一步計算心肌細胞分化率。PT-PCR檢測分化過程中心肌發(fā)育基因α-MHC和β-MHC mRNA的表達,細胞免疫熒光檢測iPSc分化細胞cTnT的表達。 結(jié)果1.在LIF和成纖維細胞存在的條件下，iPSc呈集落樣或巢狀生長且不斷增殖；熒光顯微鏡下小鼠iPSc呈綠色熒光（Oct-4啟動），堿性磷酸酶染色提示iPSc細胞團著紫黑色。 2.各分化組均可在光鏡下見自發(fā)搏動的EBs，與自然分化組相比，三個濃度組的VEGF均可提高iPSc的心肌細胞分化率(P0.05)；VEGF濃度為20ng/ml時，，iPS細胞的心肌細胞分化率最高，為53.17%±3.63%（P0.05），與二甲亞砜組的心肌細胞分化率（57.07%±3.22%）相比無統(tǒng)計學(xué)差異（P0.05）。 3.各分化組分化而來的細胞均表達調(diào)控心肌發(fā)育的基因α-MHC和β-MHC;VEGF可上調(diào)α-MHC和β-MHC的表達，VEGF濃度為20ng/ml時,其上調(diào)作用最為明顯（P0.05）。 4.各分化組分化而來的心肌細胞可自發(fā)搏動，同時表達心肌特異蛋白cTnT。 結(jié)論1.在LIF和成纖維細胞的環(huán)境中，小鼠iPSc保持未分化狀態(tài)，去除這兩者后，小鼠iPSc可以通過形成擬胚體開始分化。 2.一定濃度的外源性的VEGF可以促進誘導(dǎo)多能干細胞向心肌細胞的分化。
[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 偽 -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% 鹵3.22%), the differentiation rate of cardiomyocytes was 53.17% 鹵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 偽 -MHC and 尾 -MHC genes that regulated myocardial development. When the expression of 偽 -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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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1 王秀麗;王常勇;虞星炬;郭希民;段翠密;趙云山;;二甲基亞砜誘導(dǎo)胚胎干細胞分化伴隨凋亡發(fā)生[J];解剖學(xué)雜志;2005年06期

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