体外诱导人胚胎生殖细胞向心肌细胞分化的实验研究
发布时间：2018-11-19 16:46
【摘要】： 目的:1.探讨维甲酸(RA)、参麦、抗坏血酸作为诱导剂诱导人胚胎生殖细胞(EG细胞)体外分化为心肌细胞的可行性;2.观察人胚胎生殖细胞经三种诱导剂诱导不同时间后的分化状态;3.寻求能使人胚胎生殖细胞体外分化为心肌细胞的更安全可靠且高效的诱导剂。 方法:取5-10周流产人胚胎生殖腺嵴和背侧肠系膜,进行组织块培养。以源于自身胚胎组织的成纤维细胞作为饲养层,在培养液中添加白血病抑制因子(LIF),观察人EG细胞生长及集落形成情况。将培养得到的细胞消化成单细胞悬液,差速贴壁1h去除成纤维细胞(MEF),细胞计数后以1×105/ml的初始密度接种于100mm的培养皿进行悬浮培养,培养液中去除了LIF。培养5天后,小心挑出类胚体(EBs)接种于24孔板中,然后分别添加含不同浓度RA、参麦、抗坏血酸的诱导液进行诱导分化培养,观察细胞的分化状态。取诱导不同时间(1周、2周、3周、4周)后的细胞做免疫细胞化学染色,鉴定诱导后细胞心肌特异转录因子GATA-4和心肌肌钙蛋白T(cTnT)的表达情况。用统计学方法计算人EG细胞经三种诱导剂诱导后的心肌样细胞转化率,从而找出各种诱导剂的最佳诱导浓度,并比较三种诱导剂的诱导效果。 结果:经RA、参麦、抗坏血酸三种诱导剂诱导1周后,EBs周边长出的细胞呈梭形或不规则形;诱导2周,细胞之间的突起相互连接;诱导3周,细胞之间的连接更加紧密,可见几个细胞连接形成闰盘样结构;诱导4周,细胞呈条索状排列,走向趋于一致。诱导1周后细胞即出现GATA-4弱表达,核呈棕黄色,数量较少;诱导2周,阳性细胞数量增多且颜色加深,核椭圆形,染成棕色;诱导3周GATA-4的表达达高峰,核长椭圆形染色为棕褐色;诱导4周,阳性细胞数量较3周时减少且颜色变淡。诱导2周cTnT开始有弱表达,胞浆染成浅棕色;诱导3周,cTnT的表达增强,胞浆内出现横纹样阳性结构,但主要分布于核周区,染色为棕色;诱导4周,胞浆阳性反应产物明显增强且分布广泛,染色为棕褐色。对照组GATA-4和cTnT阴性表达。RA诱导人EG细胞向心肌细胞分化的最佳诱导浓度为10-8mol/l或10-9mol/l,诱导效率最高达(81.7±0.41)%;参麦的最佳诱导浓度为1g/l,诱导效率最高达(57.0±3.25)%;抗坏血酸的最佳诱导浓度为0.1mg/ml,诱导分化率最高达(85.2±1.79)%。 结论:RA、参麦和抗坏血酸三种诱导剂均能诱导人EG细胞向心肌细胞分化,诱导后的细胞GATA-4和cTnT阳性表达。抗坏血酸和传统的诱导剂RA一样,对人EG细胞向心肌细胞分化具有很强的诱导作用,而且是一种更安全高效的诱导剂。参麦虽然诱导效率低一点,但与RA相比较,更安全可靠,所以也是一种比较好的诱导剂。
[Abstract]:Objective: 1. To explore the feasibility of inducing human embryonic germ cells (EG cells) to differentiate into cardiomyocytes by (RA), and ascorbic acid as inducers in vitro. 2. To observe the differentiation state of human embryonic germ cells induced by three inducers for different time. To seek a safer and more effective inducer to differentiate human embryonic germ cells into cardiomyocytes in vitro. Methods: gonadal ridge and dorsal mesentery were collected from 5-10 weeks aborted human embryo and cultured with tissue mass. The growth and colony formation of human EG cells were observed by adding leukemia inhibitory factor (LIF),) into the culture medium with fibroblasts derived from their own embryonic tissues as the feeder layer. The cultured cells were digested into single cell suspensions, the fibroblast (MEF), cells were removed from the fibroblasts for 1 h, and then the cells were seeded into the culture dish of 100mm at the initial density of 1 脳 105/ml. The LIF. was removed from the culture medium. After 5 days of culture, the embryoid (EBs) was carefully selected and inoculated into the 24-well plate, and then the differentiation of the cells was observed by adding different concentrations of RA, and ascorbic acid respectively. The expression of cardiomyocyte specific transcription factor (GATA-4) and cardiac troponin (T (cTnT) were detected by immunocytochemical staining at different time (1 week, 2 weeks, 3 weeks and 4 weeks) after induction. The myocardial cell transformation rate of human EG cells induced by three inducers was calculated by statistical method, and the optimal concentration of the inducers was found out, and the induction effects of the three inducers were compared. Results: after 1 week of induction by RA, and ascorbic acid, the cells around EBs were fusiform or irregular, and the processes were connected with each other after 2 weeks of induction. After 3 weeks of induction, the connection between the cells became more close, and several cells formed intercalated disk-like structure, and at 4 weeks, the cells were arranged in stripe shape and tended to be in the same direction. After 1 week of induction, the cells showed weak expression of GATA-4, the nucleus was brownish yellow, and the number of the positive cells increased at 2 weeks, and the nucleus was oval and stained brown. After 3 weeks of induction, the expression of GATA-4 reached its peak and the nucleus was stained brown with long ellipse, and at 4 weeks, the number of positive cells decreased and the color became lighter than that at week 3. After 2 weeks of induction, the expression of cTnT began to be weakly expressed, and the cytoplasm was stained with light brown color. After 3 weeks of induction, the expression of cTnT increased, and the striated positive structure appeared in the cytoplasm, but mainly distributed in the perinuclear area and stained brown. After 4 weeks of induction, the cytoplasmic positive reaction products were significantly increased and widely distributed and stained brown. The negative expression of GATA-4 and cTnT in the control group showed that the best concentration of RA induced human EG cells to differentiate into cardiomyocytes was 10-8mol/l or 10-9 mol / L, and the highest induction efficiency was (81.7 卤0.41)%. The best inductive concentration of Shenmai was 1 g / l, the highest induction efficiency was (57.0 卤3.25)%, and the optimum concentration of ascorbic acid was 0.1 mg / ml, and the highest induced differentiation rate was (85.2 卤1.79)%. Conclusion: both RA, and ascorbic acid can induce human EG cells to differentiate into cardiomyocytes and induce the positive expression of GATA-4 and cTnT. Ascorbic acid, like the traditional inducer RA, can induce the differentiation of human EG cells into cardiomyocytes, and it is a safer and more efficient inducer. Although the induction efficiency of Shenmai is lower, it is more safe and reliable than RA, so it is also a good inducer.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
[Abstract]:Objective: 1. To explore the feasibility of inducing human embryonic germ cells (EG cells) to differentiate into cardiomyocytes by (RA), and ascorbic acid as inducers in vitro. 2. To observe the differentiation state of human embryonic germ cells induced by three inducers for different time. To seek a safer and more effective inducer to differentiate human embryonic germ cells into cardiomyocytes in vitro. Methods: gonadal ridge and dorsal mesentery were collected from 5-10 weeks aborted human embryo and cultured with tissue mass. The growth and colony formation of human EG cells were observed by adding leukemia inhibitory factor (LIF),) into the culture medium with fibroblasts derived from their own embryonic tissues as the feeder layer. The cultured cells were digested into single cell suspensions, the fibroblast (MEF), cells were removed from the fibroblasts for 1 h, and then the cells were seeded into the culture dish of 100mm at the initial density of 1 脳 105/ml. The LIF. was removed from the culture medium. After 5 days of culture, the embryoid (EBs) was carefully selected and inoculated into the 24-well plate, and then the differentiation of the cells was observed by adding different concentrations of RA, and ascorbic acid respectively. The expression of cardiomyocyte specific transcription factor (GATA-4) and cardiac troponin (T (cTnT) were detected by immunocytochemical staining at different time (1 week, 2 weeks, 3 weeks and 4 weeks) after induction. The myocardial cell transformation rate of human EG cells induced by three inducers was calculated by statistical method, and the optimal concentration of the inducers was found out, and the induction effects of the three inducers were compared. Results: after 1 week of induction by RA, and ascorbic acid, the cells around EBs were fusiform or irregular, and the processes were connected with each other after 2 weeks of induction. After 3 weeks of induction, the connection between the cells became more close, and several cells formed intercalated disk-like structure, and at 4 weeks, the cells were arranged in stripe shape and tended to be in the same direction. After 1 week of induction, the cells showed weak expression of GATA-4, the nucleus was brownish yellow, and the number of the positive cells increased at 2 weeks, and the nucleus was oval and stained brown. After 3 weeks of induction, the expression of GATA-4 reached its peak and the nucleus was stained brown with long ellipse, and at 4 weeks, the number of positive cells decreased and the color became lighter than that at week 3. After 2 weeks of induction, the expression of cTnT began to be weakly expressed, and the cytoplasm was stained with light brown color. After 3 weeks of induction, the expression of cTnT increased, and the striated positive structure appeared in the cytoplasm, but mainly distributed in the perinuclear area and stained brown. After 4 weeks of induction, the cytoplasmic positive reaction products were significantly increased and widely distributed and stained brown. The negative expression of GATA-4 and cTnT in the control group showed that the best concentration of RA induced human EG cells to differentiate into cardiomyocytes was 10-8mol/l or 10-9 mol / L, and the highest induction efficiency was (81.7 卤0.41)%. The best inductive concentration of Shenmai was 1 g / l, the highest induction efficiency was (57.0 卤3.25)%, and the optimum concentration of ascorbic acid was 0.1 mg / ml, and the highest induced differentiation rate was (85.2 卤1.79)%. Conclusion: both RA, and ascorbic acid can induce human EG cells to differentiate into cardiomyocytes and induce the positive expression of GATA-4 and cTnT. Ascorbic acid, like the traditional inducer RA, can induce the differentiation of human EG cells into cardiomyocytes, and it is a safer and more efficient inducer. Although the induction efficiency of Shenmai is lower, it is more safe and reliable than RA, so it is also a good inducer.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
【参考文献】
相关期刊论文 前10条
1 刘晓萍,高英茂,邴鲁军;定向诱导小鼠原始生殖细胞向肝细胞分化的研究[J];解剖学报;2005年06期
2 陈永珍;陈谦;李芳;朱e，
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