成熟心肌细胞对早期胚胎或胚胎干细胞定向分化诱导作用的研究

发布时间：2018-04-25 04:34

本文选题：胚胎干细胞 + 心肌细胞　；参考：《重庆医科大学》2008年硕士论文

【摘要】： 目的拟证实成熟心肌细胞对早期胚胎或胚胎干细胞(ESCs)定向分化存在特殊的诱导作用,为建立一套简单实用的胚胎干细胞移植技术提供理论依据。方法分离培养SD大鼠乳鼠心肌细胞,以DAPI(4’6-联眯-2-苯基吲哚)染核作为细胞标记。分别应用自然合笼及超排卵技术获取昆明小鼠的早期胚胎(8～16细胞期胚)与心肌细胞及相关心肌微环境共培养;取昆明小鼠(3.5～4天)的囊胚培养后分离出ESCs,以1到2代的小鼠ESCs细胞团与心肌细胞及心肌成纤维细胞共培养。录像动态观察早期胚胎或ESCs向心肌细胞分化的情况;分别于共培养后3、7、14天行肌钙蛋白T(cTnT)、α-肌动蛋白(α-Actinin)免疫荧光染色检测。结果(1)早期胚胎与心肌细胞或心肌成纤维细胞共培养时,可继续发育分化,所分化出的细胞无cTnT表达。(2)1代或2代的ESCs细胞和/或细胞团与心肌细胞共培养约7天左右出现节律搏动的心肌样细胞;观察至14天时,搏动频率约为30～50次/min。最好实验批次,可计数到1/4以上的ESCs细胞和/或ESCs细胞团出现节律性搏动;ESCs细胞和/或细胞团与心肌细胞共培养并添加0.6%的DMSO时,节律搏动的心肌样细胞分化率未见显著提高。(3)心肌细胞诱导组,记录已搏动和未搏动ESCs诱导分化后的心肌样细胞cTnT、α-Actin蛋白荧光染色阳性率为27%;ESCs与心肌成纤维细胞共培养后的成纤维细胞诱导组,分化的细胞cTnT、α-Actin蛋白荧光染色均阴性;0.6%DMSO诱导组中约3%的分化细胞cTnT、α-Actin蛋白荧光染色阳性;DMSO联合心肌细胞诱导组,其结果与心肌细胞诱导组相似。结论(1)心肌细胞和心肌成纤维细胞所提供的微环境有利于早期胚胎发育分化,分化出的细胞可以继续发育增殖。但是,不能诱导出cTnT阳性表达的心肌样细胞,考虑主要是滋养层细胞的分化影响了细胞间的直接接触,导致诱导失败。(2)未经建系操作的ESCs可被诱导分化为节律搏动的心肌样细胞;成熟心肌细胞与ESCs共培养状态下,成熟心肌细胞可诱导ESCs向心肌细胞分化,这种微环境下ESCs向心肌细胞定向分化率显著高于自然分化组,即成熟心肌细胞是诱导ESCs细胞定向分化中较强的诱导因素。
[Abstract]:Objective to prove that mature cardiomyocytes can induce the directional differentiation of early embryonic or embryonic stem cells (ESCs) and provide a theoretical basis for the establishment of a set of simple and practical techniques for embryonic stem cell transplantation. Methods the neonatal rat cardiomyocytes were isolated and cultured, and the nuclei of DAPI4 (6-diphenylin-2-phenylindole) were used as cell markers. Natural cage and superovulation were used to obtain the embryo of Kunming mouse. The blastocysts of Kunming mice were cultured for 4 days. The blastocysts were isolated and co-cultured with cardiomyocytes and cardiac fibroblasts in 1 to 2 passages of mouse ESCs cells. The differentiation of early embryos or ESCs into cardiomyocytes was observed dynamically by video recording, and the expression of troponin TnTnT, 伪 -actin (伪 -actin) was detected by immunofluorescence staining on the 7th and 14th days after co-culture. Results 1) when the early embryo was co-cultured with cardiomyocytes or myocardial fibroblasts, it could continue to develop and differentiate. There was no cTnT expression in the differentiated cells. The ESCs cells and / or cell clusters of the first or second passage were co-cultured with cardiomyocytes for about 7 days, and the beating frequency was about 30 ~ 50 beats / min at the 14th day. The best experiment batch can be counted when more than 1 / 4 of ESCs cells and / or ESCs cell clusters have rhythmic pulsating ESCs cells and / or cell clusters co-cultured with cardiomyocytes and added 0.6% DMSO. The differentiation rate of cardiomyoid cells in rhythmic pulsatile group was not significantly increased. The positive rate of cTnT, 伪 -actin protein fluorescence staining in cardiac myoid cells induced by pulsatile and non-pulsatile ESCs was 27% in the fibroblast induction group after co-culture with myocardial fibroblast. CTnT, 伪 -Actin protein fluorescent staining of differentiated cells was negative. About 3% of differentiated cells in DMSO group were positive for cTnT, 伪 -Actin protein fluorescence staining and combined with cardiomyocyte induction. The results were similar to those in cardiomyocyte induction group. Conclusion 1) the microenvironment provided by cardiac myocytes and myocardial fibroblasts is conducive to early embryonic development and differentiation, and the differentiated cells can continue to develop and proliferate. However, cardiomyoid cells with cTnT positive expression could not be induced. It was considered that the differentiation of trophoblast cells affected the direct contact between cells. ESCs can be induced to differentiate into rhythmically beating cardiomyocytes, and mature cardiomyocytes can induce ESCs to differentiate into cardiomyocytes when co-cultured with ESCs. In this microenvironment, the differentiation rate of ESCs into cardiomyocytes was significantly higher than that of natural differentiation group, that is, mature cardiomyocytes were the strong inducing factors in inducing the directional differentiation of ESCs cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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