本文選題：人胚胎干細(xì)胞　切入點(diǎn)：定向心肌分化　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：第一部分化學(xué)成分明確的無(wú)飼養(yǎng)層培養(yǎng)基定向誘導(dǎo)人胚胎干細(xì)胞分化為心肌細(xì)胞的實(shí)驗(yàn)研究研究目的以E8培養(yǎng)基培養(yǎng)人胚胎干細(xì)胞(human embryonic stem cell,ESCs),探索化學(xué)成分明確的CDM3誘導(dǎo)分化培養(yǎng)基在人ESCs定向心肌細(xì)胞分化中的作用及最佳細(xì)胞接種密度。研究方法人ESCs細(xì)胞復(fù)蘇后傳代3次,接種至鋪有生長(zhǎng)因子減量的Matrigel膠上,待細(xì)胞匯合度達(dá)90%左右時(shí),換用CDM3誘導(dǎo)分化培養(yǎng)基,誘導(dǎo)分化的d0-d2加入6umol/L CHIR99021,d3-d4加入2umol/L Wnt-59,之后單用CDM3培養(yǎng)基,直至開(kāi)始出現(xiàn)跳動(dòng)的心肌細(xì)胞,誘導(dǎo)分化過(guò)程中觀察細(xì)胞形態(tài)及跳動(dòng)心肌細(xì)胞數(shù)量的變化。再分別以0.5×10~4、1×10~4、1.5×10~4、2×10~4個(gè)/cm~2的接種密度分別進(jìn)行誘導(dǎo)分化實(shí)驗(yàn),觀察不同接種密度ESCs定向心肌細(xì)胞誘導(dǎo)分化的效率。研究結(jié)果誘導(dǎo)d7開(kāi)始出現(xiàn)跳動(dòng)的細(xì)胞,跳動(dòng)細(xì)胞數(shù)量d7-d16逐漸增加,d16達(dá)高峰。細(xì)胞免疫熒光結(jié)果顯示:跳動(dòng)細(xì)胞心肌特異性標(biāo)志物a-Actinin、cTNNT2表達(dá)陽(yáng)性,并可見(jiàn)明顯的肌節(jié)及潤(rùn)盤(pán)結(jié)構(gòu);ESCs接種密度為0.5×10~4個(gè)/cm~2時(shí)不能成功誘導(dǎo)為心肌細(xì)胞,接種密度為2×10~4個(gè)/cm2的誘導(dǎo)效率較密度為1×10~4、1.5×10~4個(gè)/cm~2者低。結(jié)論CDM3誘導(dǎo)分化培養(yǎng)基可以成功誘導(dǎo)人ESCs定向分化為心肌細(xì)胞,1-1.5×10~4個(gè)/cm~2為誘導(dǎo)分化的最佳接種密度。第二部分人胚胎干細(xì)胞定向心肌分化過(guò)程中APJ表達(dá)特征的研究研究目的人ESCs定向心肌分化過(guò)程中,以中胚層階段、心肌細(xì)胞起始分化階段、心肌細(xì)胞后分化階段為時(shí)間節(jié)點(diǎn),探索APJ的表達(dá)特征。研究方法經(jīng)單層培養(yǎng)后,應(yīng)用化學(xué)成分明確的誘導(dǎo)分化培養(yǎng)基定向誘導(dǎo)人ESCs向心肌細(xì)胞分化,于中胚層階段(d2)、心肌細(xì)胞起始分化階段(d3)、心肌細(xì)胞后分化階段(d7)分別進(jìn)行細(xì)胞免疫熒光檢測(cè),共聚焦顯微鏡觀察Brachyury T、mesp1、Nkx2.5與APJ的蛋白表達(dá)情況,實(shí)時(shí)熒光定量PCR(Real-Time PCR)法檢測(cè)四種標(biāo)志物m RNA的表達(dá)水平。研究結(jié)果誘導(dǎo)d7開(kāi)始出現(xiàn)跳動(dòng)的細(xì)胞,跳動(dòng)細(xì)胞數(shù)量d7-d16逐漸增加,d16達(dá)高峰。細(xì)胞免疫熒光結(jié)果顯示:APJ在d2、d3、d7三個(gè)階段分別與階段特異性標(biāo)志物Brachyury T、mesp1、Nkx2.5共表達(dá)。APJ m RNA在三個(gè)階段中呈持續(xù)性表達(dá),中胚層起始時(shí)表達(dá)最高,中胚層后表達(dá)逐漸降低。結(jié)論APJ與代表人ESCs心肌分化過(guò)程中階段特異性標(biāo)志物Brachyury T、mesp1、Nkx2.5共表達(dá),證實(shí)APJ在人心肌細(xì)胞發(fā)育的整個(gè)過(guò)程中持續(xù)性表達(dá)。
[Abstract]:The first part of a chemically defined without feeder layer of human embryonic stem cells by differentiation of human embryonic stem cells for the purpose of experimental research on myocardial cells by E8 medium (human embryonic stem cell training, ESCs), to explore the culture medium in ESCs differentiate myocardial cells differentiation induced by chemical composition to clarify the role of CDM3 and the optimal cell density. The method of ESCs cell recovery after 3 generations, with a growth factor reduction inoculated to Matrigel gel, when cell confluence in about 90%, for medium differentiation induced by CDM3 induced differentiation of d0-d2 into 6umol/L CHIR99021, d3-d4 joined 2umol/L Wnt-59, after cultured with CDM3 alone the base, until began beating cardiomyocytes, observe the changes of cell morphology and beating myocardial cells induced differentiation. Then in 0.5 * 10~4,1 * 10~4,1.5 * 10~4,2 * 10~ Inoculation density of 4 /cm~2 were induced differentiation experiments, observe the efficiency of directional myocardial cells of different cell density ESCs differentiation induced by D7. The results began beating beating cells, the number of d7-d16 cells increased gradually, reached the peak at D16. Immunofluorescence results showed that myocardial beating cell specific markers a-Actinin, cTNNT2 expression positive, and the visible sarcomere and run disk structure; ESCs inoculation density of 0.5 * 10~4 /cm~2 can be successfully induced into cardiomyocytes, inoculation density of 2 * 10~4 /cm2 induction efficiency compared with the density of 1 * 10~ 4,1.5 * 10~4 /cm~2 low. Medium can induce ESCs differentiate into success conclusion myocardial cell differentiation induced by CDM3, 1-1.5 * 10~4 /cm~2 for differentiation of inoculation density. The research purpose of expression characteristics of APJ cells in the cardiac differentiation of human embryonic stem part second The ESCs orientation in the process of myocardial differentiation, in mesoderm stage, initial stage of differentiation of myocardial cells, myocardial cells after differentiation stage as the time node, to explore the expression characteristics of APJ. The method of the monolayer culture after the application of a chemically defined culture medium induced directional differentiation of ESCs into cardiomyocytes, from the mesoderm stage (D2), myocardial cell initial differentiation stage (D3), myocardial cell differentiation stage (D7) cells were detected by immunofluorescence confocal microscopy, Brachyury T, mesp1, Nkx2.5 and APJ protein expression situation, real-time fluorescence quantitative PCR (Real-Time PCR) was used to detect expression levels of four markers in M RNA results D7 induced began beating cells, beating the number of d7-d16 cells increased gradually, reached the peak at D16. Immunofluorescence results showed that APJ in D2, D3, D7 three stages respectively and stage specific markers Br Achyury T, mesp1.APJ, m RNA was sustained in three stages the expression of co expression of Nkx2.5, at the start of the highest expression of mesoderm mesoderm, expression gradually decreased. Conclusion the cardiac differentiation of APJ and ESCs in the representative stage specific markers Brachyury T, mesp1 Nkx2.5, co expression, APJ expression in the last confirmed the process of myocardial cell development.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R542.22

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