APJ基因在人胚胎干细胞定向心肌细胞分化过程中表达特征的实验研究

发布时间：2018-03-07 18:53

本文选题：人胚胎干细胞　切入点：定向心肌分化　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：第一部分化学成分明确的无饲养层培养基定向诱导人胚胎干细胞分化为心肌细胞的实验研究研究目的以E8培养基培养人胚胎干细胞(human embryonic stem cell,ESCs),探索化学成分明确的CDM3诱导分化培养基在人ESCs定向心肌细胞分化中的作用及最佳细胞接种密度。研究方法人ESCs细胞复苏后传代3次,接种至铺有生长因子减量的Matrigel胶上,待细胞汇合度达90%左右时,换用CDM3诱导分化培养基,诱导分化的d0-d2加入6umol/L CHIR99021,d3-d4加入2umol/L Wnt-59,之后单用CDM3培养基,直至开始出现跳动的心肌细胞,诱导分化过程中观察细胞形态及跳动心肌细胞数量的变化。再分别以0.5×10~4、1×10~4、1.5×10~4、2×10~4个/cm~2的接种密度分别进行诱导分化实验,观察不同接种密度ESCs定向心肌细胞诱导分化的效率。研究结果诱导d7开始出现跳动的细胞,跳动细胞数量d7-d16逐渐增加,d16达高峰。细胞免疫荧光结果显示:跳动细胞心肌特异性标志物a-Actinin、cTNNT2表达阳性,并可见明显的肌节及润盘结构;ESCs接种密度为0.5×10~4个/cm~2时不能成功诱导为心肌细胞,接种密度为2×10~4个/cm2的诱导效率较密度为1×10~4、1.5×10~4个/cm~2者低。结论CDM3诱导分化培养基可以成功诱导人ESCs定向分化为心肌细胞,1-1.5×10~4个/cm~2为诱导分化的最佳接种密度。第二部分人胚胎干细胞定向心肌分化过程中APJ表达特征的研究研究目的人ESCs定向心肌分化过程中,以中胚层阶段、心肌细胞起始分化阶段、心肌细胞后分化阶段为时间节点,探索APJ的表达特征。研究方法经单层培养后,应用化学成分明确的诱导分化培养基定向诱导人ESCs向心肌细胞分化,于中胚层阶段(d2)、心肌细胞起始分化阶段(d3)、心肌细胞后分化阶段(d7)分别进行细胞免疫荧光检测,共聚焦显微镜观察Brachyury T、mesp1、Nkx2.5与APJ的蛋白表达情况,实时荧光定量PCR(Real-Time PCR)法检测四种标志物m RNA的表达水平。研究结果诱导d7开始出现跳动的细胞,跳动细胞数量d7-d16逐渐增加,d16达高峰。细胞免疫荧光结果显示:APJ在d2、d3、d7三个阶段分别与阶段特异性标志物Brachyury T、mesp1、Nkx2.5共表达。APJ m RNA在三个阶段中呈持续性表达,中胚层起始时表达最高,中胚层后表达逐渐降低。结论APJ与代表人ESCs心肌分化过程中阶段特异性标志物Brachyury T、mesp1、Nkx2.5共表达,证实APJ在人心肌细胞发育的整个过程中持续性表达。
[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.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R542.22

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