發(fā)布時間：2018-05-14 10:57

  本文選題：小鼠 + 孤雌胚胎干細胞��； 參考：《華北理工大學》2017年碩士論文

【摘要】：目的研究小鼠孤雌囊胚(實驗組)及精卵結(jié)合囊胚(對照組)A、B、C三個級別內(nèi)細胞團(ICM)形成胚胎干細胞的發(fā)育潛能,為探索孤雌胚胎發(fā)育特點、發(fā)育潛能以及孤雌胚胎干細胞后續(xù)分化能力提供實驗數(shù)據(jù)。方法應(yīng)用HMG和HCG聯(lián)合法雌鼠促排卵,實驗組的MⅡ卵化學聯(lián)合法激活形成孤雌胚胎,對照組合籠飼養(yǎng)、自然受精形成受精卵,兩組體外培養(yǎng)至囊胚階段,分別獲取A、B、C三個級別的內(nèi)細胞團,置于飼養(yǎng)層細胞進行傳代培養(yǎng)至干細胞階段,檢測不同組間胚胎干細胞標志物堿性磷酸酶和端粒酶表達水平,并應(yīng)用透射電子顯微鏡觀察各組胚胎干細胞的形態(tài)和超微結(jié)構(gòu)。結(jié)果實驗組與對照組囊胚的A、B級內(nèi)細胞團形成的胚胎干細胞標志物堿性磷酸酶表達水平相同(A級兩組68.39±7.92、65.79±7.42;B級兩組66.5±8.32、66.16±8.68),差異不顯著(P0.05);兩組囊胚的C級內(nèi)細胞團形成的胚胎干細胞堿性磷酸酶表達水平不同(C級兩組24.98±4.37、44.26±4.19),實驗組較低,差異具有統(tǒng)計學意義(P0.05)。實驗組內(nèi)比較,囊胚A級、B級內(nèi)細胞團形成的孤雌胚胎干細胞堿性磷酸酶表達水平相同(A級68.39±7.92;B級66.5±8.32),差異不顯著(P0.05),C級內(nèi)細胞團形成的孤雌胚胎干細胞堿性磷酸酶表達水平低于A級、B級(C級24.98±4.37),差異有統(tǒng)計學意義(P0.05)。實驗組與對照組囊胚的A、B級內(nèi)細胞團形成的胚胎干細胞標志物端粒酶表達水平相同(A級兩組10.18±1.52、10.00±1.24;B級兩組10.00±1.50、10.11±1.43),差異不顯著(P0.05);兩組囊胚的C級內(nèi)細胞團形成的胚胎干細胞標志物端粒酶表達水平不同(5.67±1.43、8.94±0.99),實驗組較低,差異具有統(tǒng)計學意義(P0.05);實驗組內(nèi)比較,孤雌囊胚的A級、B級內(nèi)細胞團形成的孤雌胚胎干細胞標志物端粒酶表達水平相同(A級10.18±1.52、B級10.00±1.50),差異不顯著(P0.05);孤雌囊胚C級內(nèi)細胞團形成的孤雌胚胎干細胞端粒酶表達水平低于A級與B級(C級5.67±1.43),差異有統(tǒng)計學意義(P0.05)。透射電子顯微鏡下觀察孤雌囊胚A、B級內(nèi)細胞團形成的孤雌胚胎干細胞的超微結(jié)構(gòu)完整性保持較好、形態(tài)簡單規(guī)整,細胞間隙基本不可見,皮質(zhì)區(qū)內(nèi)含有大量球狀線粒體,體積小且數(shù)量多,形態(tài)正常,胞質(zhì)內(nèi)僅可見少量空泡;孤雌囊胚C級內(nèi)細胞團形成的孤雌胚胎干細胞的超微結(jié)構(gòu)不規(guī)則,核糖體數(shù)目較少,形態(tài)也相對不規(guī)整,體積明顯腫脹變圓,凋亡情況較常見,細胞基質(zhì)增多變淺,嵴減少或消失,部分細胞胞質(zhì)內(nèi)線粒體腫脹失去正常形態(tài),部分轉(zhuǎn)變?yōu)樾螒B(tài)、大小不等的空泡。結(jié)論1.小鼠孤雌囊胚A、B級內(nèi)細胞團形成的孤雌胚胎干細胞堿性磷酸酶、端粒酶表達活性相同,C級內(nèi)細胞團形成的孤雌胚胎干細胞堿性磷酸酶、端粒酶表達活性低于A、B級內(nèi)細胞團。2.小鼠孤雌囊胚具有較好的發(fā)育潛能及后續(xù)分化能力,A級與B級內(nèi)細胞團后續(xù)發(fā)育潛能較好,尤以A級最佳。
[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.

【學位授予單位】：華北理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R714.8

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