小鼠孤雌激活胚胎干细胞体外发育潜能的探讨

发布时间：2018-05-14 10:57

本文选题：小鼠 + 孤雌胚胎干细胞　；参考：《华北理工大学》2017年硕士论文

【摘要】：目的研究小鼠孤雌囊胚(实验组)及精卵结合囊胚(对照组)A、B、C三个级别内细胞团(ICM)形成胚胎干细胞的发育潜能,为探索孤雌胚胎发育特点、发育潜能以及孤雌胚胎干细胞后续分化能力提供实验数据。方法应用HMG和HCG联合法雌鼠促排卵,实验组的MⅡ卵化学联合法激活形成孤雌胚胎,对照组合笼饲养、自然受精形成受精卵,两组体外培养至囊胚阶段,分别获取A、B、C三个级别的内细胞团,置于饲养层细胞进行传代培养至干细胞阶段,检测不同组间胚胎干细胞标志物碱性磷酸酶和端粒酶表达水平,并应用透射电子显微镜观察各组胚胎干细胞的形态和超微结构。结果实验组与对照组囊胚的A、B级内细胞团形成的胚胎干细胞标志物碱性磷酸酶表达水平相同(A级两组68.39±7.92、65.79±7.42;B级两组66.5±8.32、66.16±8.68),差异不显著(P0.05);两组囊胚的C级内细胞团形成的胚胎干细胞碱性磷酸酶表达水平不同(C级两组24.98±4.37、44.26±4.19),实验组较低,差异具有统计学意义(P0.05)。实验组内比较,囊胚A级、B级内细胞团形成的孤雌胚胎干细胞碱性磷酸酶表达水平相同(A级68.39±7.92;B级66.5±8.32),差异不显著(P0.05),C级内细胞团形成的孤雌胚胎干细胞碱性磷酸酶表达水平低于A级、B级(C级24.98±4.37),差异有统计学意义(P0.05)。实验组与对照组囊胚的A、B级内细胞团形成的胚胎干细胞标志物端粒酶表达水平相同(A级两组10.18±1.52、10.00±1.24;B级两组10.00±1.50、10.11±1.43),差异不显著(P0.05);两组囊胚的C级内细胞团形成的胚胎干细胞标志物端粒酶表达水平不同(5.67±1.43、8.94±0.99),实验组较低,差异具有统计学意义(P0.05);实验组内比较,孤雌囊胚的A级、B级内细胞团形成的孤雌胚胎干细胞标志物端粒酶表达水平相同(A级10.18±1.52、B级10.00±1.50),差异不显著(P0.05);孤雌囊胚C级内细胞团形成的孤雌胚胎干细胞端粒酶表达水平低于A级与B级(C级5.67±1.43),差异有统计学意义(P0.05)。透射电子显微镜下观察孤雌囊胚A、B级内细胞团形成的孤雌胚胎干细胞的超微结构完整性保持较好、形态简单规整,细胞间隙基本不可见,皮质区内含有大量球状线粒体,体积小且数量多,形态正常,胞质内仅可见少量空泡;孤雌囊胚C级内细胞团形成的孤雌胚胎干细胞的超微结构不规则,核糖体数目较少,形态也相对不规整,体积明显肿胀变圆,凋亡情况较常见,细胞基质增多变浅,嵴减少或消失,部分细胞胞质内线粒体肿胀失去正常形态,部分转变为形态、大小不等的空泡。结论1.小鼠孤雌囊胚A、B级内细胞团形成的孤雌胚胎干细胞碱性磷酸酶、端粒酶表达活性相同,C级内细胞团形成的孤雌胚胎干细胞碱性磷酸酶、端粒酶表达活性低于A、B级内细胞团。2.小鼠孤雌囊胚具有较好的发育潜能及后续分化能力,A级与B级内细胞团后续发育潜能较好,尤以A级最佳。
[Abstract]:Objective to study the developmental potential of embryonic stem cells formed by the three levels of A, B, and C cell clusters (ICM) in mouse parthenogenetic blastocysts (experimental group) and sperm oval blastocyst (control group), and to provide experimental data for exploring the developmental characteristics of parthenogenetic embryos, developmental potential and the subsequent differentiation ability of parthenogenetic embryonic stem cells. Methods the combination of HMG and HCG was used to promote ovulation in female rats. The M II egg chemical combination method of the experimental group activates the formation of parthenogenetic embryos, and the control combination cage is raised, and the fertilized eggs are formed by natural fertilization. The two groups are cultured in vitro to the blastocyst stage, and the three levels of the inner cell groups of the three levels are obtained, respectively, and are placed in the feeder layer cells to the stem cell stage to detect the basic phosphorus of the embryonic stem cell markers in different groups. The expression level of acid enzyme and telomerase was observed and the morphology and ultrastructure of the embryonic stem cells were observed by transmission electron microscope. Results the expression level of alkaline phosphatase in the embryonic stem cell markers in the A and B level cells of the experimental group was the same as that of the group A (class two, 68.39 + 7.92,65.79 + 7.42, and class B two 66.5 + 8.32,66.16 + 8.6) 8), the difference was not significant (P0.05); the expression level of alkaline phosphatase in the embryonic stem cells of the two blastocysts was different (C class two 24.98 + 4.37,44.26 + 4.19), the experimental group was lower, and the difference was statistically significant (P0.05). The alkaline phosphatase expression of the parthenogenetic embryonic stem cells formed in the group of blastocysts and the cell group B in the experimental group was compared. The same level (a 68.39 + 7.92; B grade 66.5 + 8.32), the difference was not significant (P0.05). The expression level of alkaline phosphatase in the parthenogenetic embryonic stem cells formed in the C level was lower than that of the A-class, B grade (C class 24.98 + 4.37), and the difference was statistically significant (P0.05). The telomerase table of the embryonic stem cell markers in the experimental group and the control group blastocyst and the cell mass in the B grade The level was the same (two group 10.18 + 1.52,10.00 + 1.24; B class two 10 + 1.50,10.11 + 1.43), and the difference was not significant (P0.05). The expression level of telomerase was different (5.67 + 1.43,8.94 + 0.99) in the C stage of the two blastocysts, and the experimental group was lower, and the difference was statistically significant (P0.05); in the experimental group, the parthenogenetic group was parthenogenetic. The telomerase expression level of the parthenogenetic embryonic stem cell markers in the B stage of the blastocyst was the same (10.18 + 1.52, B 10 + 1.50), and the difference was not significant (P0.05). The telomerase expression level of the parthenogenetic cell group in parthenogenetic blastocyst C was lower than that of A and B (C 5.67 + 1.43), and the difference was statistically significant (P0.05). The ultrastructural integrity of the parthenogenetic blastocyst A was observed under the transmission electron microscope. The ultrastructural integrity of the parthenogenetic embryonic stem cells formed by the cell mass in the B level remained good, the morphology was simple and regular, the space between the cells was not visible, and the cortical area contained a large number of globular mitochondria. The size of the cells was small and the form was normal, and only a few vacuoles were visible in the cytoplasm; the parthenogenetic blastocyst was within the C level. The ultrastructure of the parthenogenetic embryonic stem cells formed by the cell mass is irregular, the number of ribosomes is less, the morphology is relatively irregular, the volume is obviously swollen and round, the apoptosis is more common, the cell matrix increases and the crest decreases or disappears. The mitochondria swollen in the cytoplasm of some cells is lost to the normal form, and some change into the shape, the size of the empty space. Conclusion 1. parthenogenetic blastocysts in mice were A, the parthenogenetic embryonic stem cells formed in the B class cell group were alkaline phosphatase, and the telomerase activity was the same. The alkaline phosphatase of the parthenogenetic embryonic stem cells formed in the C class cell group was lower than that of the A. The.2. mouse blastocyst in the B level cell group had better developmental potential and subsequent differentiation ability. The subsequent developmental potential of class A and B class cell clusters is better, especially for A-class.

【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.8

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