【作者】 彭新荣；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 发育生物学， 2008， 博士

【摘要】 为提高牛体细胞核移植和生产转基因动物的效率,本研究主要从囊胚发育率,囊胚细胞数和囊胚细胞凋亡指数三个方面研究了供体细胞转染端粒酶和癌化对体细胞核移植的影响,卵母细胞胞质成熟与重构胚胎发育的关系,激活方法与重构胚发育的关系,以及核移植胚胎体外培养与胚胎发育和冷冻的关系。试验研究结果如下。1、本研究首先用含人端粒酶催化亚基(hTERT)基因和报告基因EGFP的表达载体转染了牛耳成纤维细胞系,通过RT-PCR,端粒酶活性分析和Western杂交证明hTERT可以整合在牛耳成纤维细胞基因组中并表达端粒酶蛋白,人的端粒酶基因与牛的端粒酶相关基因可以互相协作,表达端粒酶蛋白。hTERT在牛耳成纤维细胞中的表达延长了细胞寿命,建立了一株染色体条带正常,贴壁生长的牛耳成纤维细胞系,命名为HBC3。2、HBC3的生长曲线仍保持正常细胞的生物学特性,EGF和胰岛素对HBC3仍有明显的促生长作用,添加EGF和胰岛素后细胞的倍增指数分别为5.1和4.7,其中EGF的促生长作用比胰岛素促生长的作用显著(P <0.05)。HBC3细胞系仍需依赖血清维持生长。但是,端粒酶的表达明显降低了血清引起的细胞凋亡,流式细胞仪进行细胞凋亡分析,结果表明,第64代的HBC3细胞凋亡率明显低于第12代的牛耳成纤维细胞(BFC)细胞,差异极显著(P <0.01)。3、HBC3做供体细胞的核移植重构胚可以发育到囊胚,重构胚的囊胚发育率与对照组差异不显著。但是,HBC3的重构胚囊胚细胞数显著少于用BFC构建的核移植胚胎,HBC3的囊胚细胞凋亡数和凋亡指数也明显少于BFC的重构胚,且差异显著(P <0.05)。4、致癌物DMBA与促癌物TPA联合作用BFC,得到两株寿命延长的细胞系,分别命名为ABF11和ABF12。ABF11的形态为长梭形,ABF12的形态为三角形,两株细胞系均有端粒酶活性,对ABF12进行了生物学特性分析,细胞在接种后,有较长的生长潜伏期,后进入对数生长期,但没有正常细胞的生长平台期,ABF12和BFC细胞系在分别在含血清的培养液生长9天的倍增指数分别为5.3和4.3,差异显著(P <0.05),试验结果表明ABF12细胞系已经失去了细胞接触生长抑制性。传代后ABF12和BFC细胞系在无血清的培养液里生长9天的倍增指数分别为5.0和-1.2,结果表明ABF12已经失去了对血清的生长依赖性。用软琼脂培养ABF12细胞系14d, ABF12在软琼脂内生长了172个单细胞克隆,结果表明ABF12细胞已经失去了贴壁依赖性。5、ABFE12细胞用于体细胞核移植后,能支持胚胎发育至囊胚阶段,但囊胚发育率低于对照组,差异显著(P <0.05)。通过染色分析,ABF12重构胚的囊胚细胞数与对照组差异不显著,但囊胚细胞的凋亡指数明显高于对照组,二者分别为0.124和0.081,差异显著(P <0.05)。6、牛卵母细胞成熟培养液中添加EGF显著提高了牛卵母细胞成熟率,用EGF培养的卵母细胞在做为受体胞质进行体细胞核移植后,体细胞重构胚的囊胚率和囊胚细胞数均显著高于不添加EGF的对照组(P <0.05)。凋亡染色分析表明,EGF培养的卵母细胞做受体后,重构胚胎的凋亡指数为0.062,比对照组显著降低(P <0.05)。培养液中添加EGF的卵母细胞在冷冻保存和体外受精后,卵母细胞的形态正常率、受精胚胎的卵裂率和囊胚率均显著高于对照组。牛卵母细胞成熟培养液中添加VE后,牛卵母细胞成熟率和重构胚的囊胚率与对照组相比均差异不显著。凋亡染色分析表明,用VE培养的卵母细胞做受体胞质,重构胚胎的囊胚细胞凋亡指数为0.064,与对照组相比,显著降低了(P <0.05)囊胚细胞的凋亡指数。而用VE培养的卵母细胞冷冻保存后,卵母细胞的形态正常率,体外受精胚胎的卵裂率和囊胚率均与对照组差异不显著。牛卵母细胞成熟培养液中添加OCS后,牛卵母细胞成熟率显著高于添加FBS培养组(P <0.05),用OCS成熟培养的卵母细胞做胞质受体,进行体细胞核移植后,重构胚的囊胚率显著高于添加FBS的卵母细胞组(P <0.05)。囊胚细胞染色分析表明,OCS组的囊胚细胞数显著高于FBS卵母细胞组(P <0.05),凋亡染色分析表明,OCS组的囊胚细胞凋亡指数为0.072,显著低于对照FBS组。而且,培养液中添加OCS的卵母细胞在冷冻保存和体外受精后,受精胚胎的卵裂率和囊胚率均显著高于对照FBS组(P <0.05)。7、比较了不同浓度精子提取物胞质内注射对牛核移植重构胚胎的卵裂率和囊胚率的影响,结果发现单独注射精子提取物可以很好的支持体细胞胞重构胚发育到囊胚阶段,其中注射5mg/ml精子提取物组的重构胚卵裂率和囊胚率均显著高于其他两个注射剂量组(P <0.05)。5mg/ml精子提取物组的囊胚细胞数显著高于其他试验组(P <0.05),而5mg/ml精子提取物组的凋亡指数为0.071,显著低于其他组(P <0.05)。其次,比较了精子提取物联合其他激活方法使用与单独使用精子提取物激活对核移植胚胎的影响。结果表明,单独注射精子提取物或联合其他方法一起使用对体细胞核移植胚胎的的卵裂率,囊胚率,囊胚细胞数和囊胚细胞凋亡指数没有显著的影响。8、比较了传统的电激活和化学激活方法对核移植胚胎的影响,以及联合使用两种激活方法(电化学法)对核移植胚胎的卵裂率和囊胚率的影响,结果表明,电化学法激活核移植胚的卵裂率和囊胚率均显著高于单独使用电激活或化学激活方法。其次,比较了精子提取物与电化学方法对核移植胚胎的影响。这两种激活方法的卵裂率和囊胚率差异不明显,但是精子提取物激活的重构胚的囊胚细胞数明显要高于电化学法(P <0.05),精子提取物法的囊胚凋亡指数也明显(P <0.05)低于后者。9、在牛体细胞核移植胚胎培养液中添加20ng/ml EGF,重构胚的囊胚发育率为34%,显著高于不添加EGF的对照组(P﹤0.05)。添加EGF的胚胎的囊胚细胞凋亡指数为0.062,显著低于对照组的细胞凋亡指数。对EGF组和对照组的胚胎进行冷冻保存并解冻培养,EGF组胚胎在解冻24h后的存活率显著高于对照组(P﹤0.05),在解冻48h后的存活率也显著高于对照组(P﹤0.05)。在牛体细胞核移植胚胎培养液中分别添加0.1μg/ml和1μg/ml孕酮后均显著提高了重构胚的囊胚发育率均显著高于对照组21%的囊胚率。添加0.1μg/ml和1μg/ml孕酮组的细胞凋亡指数分别为0.090和0.085,两组之间的胚胎细胞凋亡数和凋亡指数均差异不显著,与对照组相比差异也不显著。不同孕酮组胚胎在解冻24h后的存活率与对照组差异不显著。在解冻48h后的存活率与对照组差异不显著。在牛体细胞核移植胚胎培养液中添加100μg/ml抗氧化剂VE并没有对核移植胚胎的卵裂率产生不利影响。添加VE显著提高了重构胚胎的发育率,降低了囊胚的凋亡指数,对照组的细胞凋亡指数为0.090,VE组为0.037,差异极显著(P﹤0.01)。10、首先,分别在核移植胚胎的冷冻保存液中添加海藻糖和蔗糖,用海藻糖冷冻的胚胎解冻后24h和48h的存活率显著(P﹤0.05)高于添加蔗糖的试验组。其次,分别对不同发育时期的核移植重构胚胎进行冷冻保存。解冻后桑胚的24h和48h的胚胎存活率显著(P﹤0.05)低于早期囊胚组和晚期囊胚组。更多还原

【Abstract】 The aim of this study was to improve the efficiency of cloning transgene animals. We evaluated the development of reconstructed embryos by three aspects: the blastocyst rate, the blastomere number and the apoptotic index of blastomere. The relationship among telomerase activity of donor cells, maturation of oocytes, activation methods and efficiency of reconstructed embryos were studied in this paper. The results of this paper were as following.1. A vector including hTERT gene and EGFP gene was transfered into the bovine ear fibroblast cells, the hTERT expression were detected by RT-PCR and Western blotting assay. The telomerase activity assay showed hTERT was compatible with bovine telomerase-related factors required for functional telomerase activity in these cells. A prolonged life span cell line, HBC3, was established with nomal karyotype and the cell line exhibited normal anchorage-dependent growth.2. The growth curve of HBC3 was same as the normal bovine fibroblast cells (BFC) and still responsible to growth factors such as EGF and insulin. The doubling index of HBC3 was 5.1 or 4.7 when added EGF or insulin respectively. Addition of serum was necessary for maintaining the growth of HBC3 and the apoptosis assay indicated that the apoptosis of HBC3 was significantly(p<0.01)lower then the 12th BFC(bovine ear fibroblast cell).3. Reconstructed embryos used HBC3 as the donor cells can develop to blastocyst, and there was no significantly difference in blastocyst rate between HBC3 and the control group. However, the blastomere number and apoptosis index of blastocyst was significantly lower than the control group(p<0.05).4. ABF11 and ABF12 cell lines were obtained when BFC was treated with DMBA and TPA, the morphology of ABF11 was fibroblast-like, and the ABF12 was triangle-like. Telomerase activity in these cell lines was tested by telomerase activity assay. And the growth curve of cells cultured with serum indicated that the ABF12 cell line had a longer logarithmic growth phase and had lost the contact inhibition. The doubling index of ABF12 and BFC cell lines with serum were 5.3 and 4.3 respectively. The doubling index of ABF12 and BFC cultured without serum was 5.0 and -1.2 respectively. Compared with the control group, the ABF12 cell line has lost the serum dependency. The ABF12 were cultured in soft sugar for 14d, and the cell formed 172 clones in soft sugar, which indicated that the ABF12 cell line had lost the contact inhibition.5. Then ABF12 cell line was used as donor cells to reconstruct bovine embryos and blastocyst was obtained, the cleavage rate and blastocyst rate was significantly lower than the BFC group. The flurescence staining indicated that the blastomere number of ABF12 and BFC was not significantly different. However, the apoptosis index of blastocyst was significantly(p<0.05) higher than the control, and the index of ABF12 and BFC was 0.124 and 0. 81 respectively.6. The maturation culture medium of oocytes was added with EGF, and the addition of EGF significantly improved the maturation rate of bovine oocytes and the subsequent development of reconstructed embryos. The subsequent blastocyst rate of oocytes cultured with EGF was significantly higher than the control. And the blastomere number of EGF group was higher than the control. Compared with the control, the apoptosis index of EGF group (0.062) was significantly lower. The oocytes cultured with EGF were cyropreserved, and the rate of normal morphology oocyte, the cleavage rate and blastocyst rate of the IVF embryos were significantly improved. The addition of VE did not significantly improve the maturation rate of bovine oocytes and the subsequent development of reconstructed embryos. And the VE in oocyte maturation had no significantly effect on the blastomere number. The normal morphology of cryopreserved oocytes, the cleavage rate and blastocyst rate of the IVF embryos were no difference between VE group and the control group. The addition of OCS in oocytes maturation medium had same effects as EGF.7. The effect of different concentration of bovine sperm extracts (BSE) on the reconstructed embryos were compared and the results indicated that BSE was able to active the SCNT embryos and supported the reconstructed embryos develop to blastocyst, the recommended concentration of BSE was 5mg/ml, the cleavage rate and blastcyst rate were significantly higher than the two others group. And the blastomere number of the 5mg/mL group was significantly(p<0.05) higher than the control, and the blastomere apoptosis index was 0.071, which was significantly lower than the control(p<0.05). The cleavage rate, the blastocyst rate, the blastomere number and the apoptosis index of the reconstructed embryos showed no significantly difference when BSE was combined with other activation methods.8. Effects of fusion activation methods and chemical activation methods were compared, and the cleavage rate and the blastocyst rate were significantly higher when the two activation methods were used together. The cleavage rate and blastocyst rate of injecting BSE were not significantly different with the chemical combined with fusion activation method. But the blastomere number of blastocyst activated by BSE was significantly higher(p<0.05) than the control, and the apoptotic index was significantly(p<0.05) lower than the control.9. 20ng/ml EGF was added into the SCNT embryos culture medium, the blastocyst rate of the reconstructed embryos was significantly higher(p<0.05)than the control. And the blastomere number of the control group and the EGF group were different significantly(p﹤0.05).The apoptotic index of the EGF group was 0.062, which was significantly lower than the control. The cryopreserved embryos survival rate after 24h and 48h in EGF group were significantly higher than the control. Addition of 0.1μg/ml and 1μg/ml progesterone in SCNT embryos culture medium significantly improved the blaststocys rate, there was no significantly difference with the control group in blastomere number and blastomere apoptotic index. Compared with the control group, addition of 100μg/ml VE in embryo culture medium showed no difference in SCNT embryo cleavage rate, but significantly(p<0.05)improved the blastocyst rate and the blastomere number. Moreover, the addition of VE significantly(p<0.01)reduced the apoptotic blastomere number and apoptotic index.10. 0.1 mol/L trelecose and fucose were added into the cryopreservation medium respectively, the survival rate after 24h and 48h of the cryopreserved SCNT embryos were significantly higher than the control. Different development stage of SCNT embryos were cryopreserved respectively, and the survival rate after 24h and 48h of morula were significantly lower than the blastocyst groups..更多还原