人工抗凋亡促进胚胎发育的研究

发布时间：2018-06-20 19:06

本文选题：颗粒细胞 + 凋亡　；参考：《南方医科大学》2007年博士论文

【摘要】： 卵细胞和胚胎的衰老和死亡是制约体外受精-胚胎移植(in vitro fertilization embryo transfer，IVF-ET)等辅助生殖技术(assisted reproduetive techniques，ART)成功率提高的一个关键因素。而细胞凋亡是卵细胞和胚胎死亡的主要方式，也是导致卵细胞受精障碍和胚胎发育停滞、着床失败的主要原因。可以设想，若能抑制卵细胞和胚胎的凋亡，将显著提高胚胎的质量及辅助生殖的成功率。导致细胞凋亡的原因很多，抗凋亡分子和促凋亡分子在调节卵细胞和胚胎细胞凋亡方面起着极为重要的作用，它们之间的平衡决定着细胞的存活和死亡。其中，Bcl-2家族起着重要的作用，Bax、Bak是其亚家族中的成员，具有促凋亡作用。本研究从一个全新的角度，首次提出通过抗凋亡人工干预的方法来促进胚胎发育。我们以人颗粒细胞和小鼠早期胚胎为研究对象，采用基因转染法，在人颗粒细胞和小鼠胚胎内转入Bax、Bak等凋亡分子的小分子干扰RNA(small interfering RNA，siRNA)，通过干扰Bax等凋亡分子mRNA表达从而提升Bcl-2等抗凋亡分子的含量比例等方法分析体外人工抗凋亡干预措施对早期胚胎发育的影响，寻找一条提高卵子和胚胎质量的新途径，也为提高胚胎干细胞培养中的囊胚形成率奠定一定的基础，从而为最终将抗凋亡干预应用于临床不孕症辅助治疗提供理论和实验依据。第一部分干扰Bax-Bak基因表达目的通过转染Bax、Bak小分子干扰RNA(siRNA)，，干扰Bax、Bak等凋亡分子的功能，研究其对人颗粒细胞凋亡的影响，为提高卵子、胚胎质量提供新的思路及理论依据。方法 1、用Westernblot检测Bax、Bak-siRNA； 2、将Bax、Bak-siRNA分别或同时转染入人颗粒细胞，观察基本形态，用流式细胞仪检测颗粒细胞的凋亡。 3、实验设置空白对照组、阳性对照组、阴性对照组、Bax干扰组、Bak干扰组、gax-Bak干扰组。结果 1、esternblot检测：转染了Bax-siRNA和Bak-siRNA后的条带减弱，说明细胞内Bax、Bak含量降低。 2、基本形态观察： 1)空白对照组、Bak干扰组、Bak+Bax干扰组细胞形态正常，多为多边形和梭形，以贴壁为主。 2)Bax干扰组细胞形态基本正常，胞质中有少数黑点，多为多边形和梭形，以贴壁为主。 3)阳性对照组、阴性对照组细胞变圆，收缩，细胞间距大，胞质中有较多黑点。 3、流式细胞仪检测结果： 1)Bak干扰组的凋亡指数为3.44％，BaxBak联合干扰组的凋亡指数为3.97％，明显低于阴性对照。 2)Bax干扰组的凋亡指数为19.98％，与阴性对照组相似。结论 1、干扰Bak等凋亡分子的表达，可以抑制颗粒细胞的凋亡。 2、Bak干扰组以及BaxBak联合干扰组抗颗粒凋亡效果显著。 3、Bax干扰组抑制颗粒细胞凋亡的作用不明显。第二部分人工干预小鼠胚胎细胞凋亡目的通过转染Bax-siRNA和Bak-siRNA，研究其对鼠胚凋亡的影响，寻找干预胚胎的凋亡、促进胚胎发育的新途径。方法 1、激光打孔转染FAM标记的阴性siRNA，荧光检测转染方法是否可行。 2、通过激光打孔分别以及同时将Bax、Bak-siRNA转入小鼠胚胎，进行普通形态、卵裂观察；并计算囊胚形成率。 3、通过激光打孔分别以及同时将Bax、Bak-siRNA转染入小鼠胚胎，Hoechst33342和PI荧光染色检测凋亡，并计数凋亡细胞。 4、免疫荧光检测Bak、Bax的表达，并检测其光密度与阴性对照比较。 5、分别用荧光检测Bak及Bax干扰组中caspase-3前体的表达。结果 1、转染FAM标记的阴性siRNA的胚胎有荧光，证明转染方法有效。 2、囊胚形成率：空白对照组81.0％，阴性对照组69.5％，Bak干扰组92.5％，Bax干扰组74.0％，联合干扰组89.5％。其中，Bak干扰组、联合干扰组与阴性对照有显著差异，p＜0.001；Bax干扰组与阴性对照无显著差异，p=0.318；阴性对照与空白对照有差异，p=0.011。 3、Hoechst33342和PI染色检测细胞凋亡：空白对照组26.0％，阴性对照组33.0％，Bak干扰组17.6％，Bax干扰组30.5％，联合干扰组19.1％；与阴性对照组比较，Bak干扰组和联合干扰组均有显著差异，p＜0.001；但Bax干扰组无显著差异，p=0.470。 4、荧光检测Bak、Bax的表达：Bax干扰组及其阴性对照组光密度分别为25.39±4.73、52.89±3.91，前者显著减弱p＜0.001；Bak干扰组及其阴性对照组光密度分别为：17.10±3.79、35.26±4.88，前者显著减弱p＜0.001；联合干扰组及其阴性对照组光密度分别为：TRITC标记组：19.68±4.86、35.26±4.88；前者显著减弱p＜0.001；FITC标记组：32.78±3.73、52.89±3.91，均为前者显著减弱p＜0.001。 5、荧光检测caspase-3前体的表达。 Bak干扰组及其阴性对照中caspase-3光密度分别为：65.68±4.79、30.24±3.40，p＜0.001，有显著差异；Bax干扰组及其阴性对照中caspase-3光密度分别为：31.48±2.65、30.24±3.40，p=0.318，无显著差异。结论 1．首次在小鼠胚胎上应用激光打孔后转染的实验方法。 2．在转染Bak／Bax-SiRNA后，Bak和Bax的表达减弱。 3．在Bak干扰和联合干扰组中，鼠胚的凋亡细胞数明显减少，囊胚的形成率明显增加；而在Bax干扰组中，胚胎的凋亡细胞数、囊胚形成率与阴性对照相比无明显改变；说明Bak干扰以及Bak-Bax联合干扰对于抑制鼠胚的凋亡是有效的。 4．鼠胚中caspase-3前体的表达在Bak干扰组中增强；在Bax干扰组中无明显变化。第三部分鼠胚移植与染色体检测目的 1、初步研究鼠胚在进行抗凋亡处理后是否能发育至成熟个体。 2、初步观察出生小鼠是否有生理缺陷及染色体异常。方法 1、挑选Bak／Bax干扰组及联合干扰组中发育较好的囊胚各10个移植入小鼠子宫；观察出生小鼠的基本情况。 2、出生小鼠染色体的检查。结果 1、Bak干扰组共出生6只小鼠，Bax干扰组和联合干扰组均出生5只小鼠，出生小鼠均无明显畸形和行为异常。 2、出生小鼠染色体无明显异常。结论 1、经Bak、Bax抗凋亡处理的鼠胚可以正常发育至成熟个体。 2、抗凋亡处理对出生小鼠无明显影响。 3、抗凋亡处理对出生小鼠染色体无明显影响。
[Abstract]:The aging and death of eggs and embryos is a key factor that restricts the success rate of the assisted reproductive technology (in vitro fertilization embryo transfer, IVF-ET), such as assisted reproduetive techniques (ART), and the apoptosis is the main way of egg and embryo death, and also the fertilization of egg cells. The main reasons for the stagnation of the barrier and embryo development and the failure of the implantation of the embryo can be conceived that, if the apoptosis of the eggs and embryos can be suppressed, the quality of the embryos and the success rate of assisted reproduction will be significantly increased. There are many reasons for the apoptosis, and the anti apoptotic molecules and the apoptotic molecules are very important in regulating the apoptosis of the egg cells and embryo cells. The balance between them determines the survival and death of cells. Among them, the Bcl-2 family plays an important role, Bax, Bak is a member of its subfamily, and has the role of promoting apoptosis. From a new point of view, the first proposed method of anti apoptosis artificial intervention to promote the development of embryos. We use human granulosa cells and mice. Early embryos as the research object, using gene transfection, the small molecules of apoptotic molecules such as Bax (small interfering RNA, siRNA) were transferred into RNA (small interfering RNA, siRNA) in human granulosa cells and mouse embryos, and the anti apoptosis intervention in vitro was analyzed by interfering mRNA expression of Bax and so on to enhance the content ratio of anti apoptotic molecules such as Bcl-2 and so on. The effect of measures on the development of early embryo, looking for a new way to improve the quality of egg and embryo, also lay a foundation for improving the rate of blastocyst formation in the culture of embryonic stem cells, so as to provide theoretical and experimental basis for the application of anti apoptosis intervention to the auxiliary treatment of clinical infertility.
The first part interferes with the expression of Bax-Bak gene
objective
By transfection of Bax, Bak small interfering RNA (siRNA), Bax interference, apoptosis Bak and other functions, to study its effect on the apoptosis of human granulosa cells, in order to improve the egg, to provide new way of embryo quality.
Method
1, Bax and Bak-siRNA were detected by Westernblot.
2, Bax and Bak-siRNA were transfected into human granulosa cells at the same time or at the same time. The basic morphology was observed and the apoptosis of granulosa cells was detected by flow cytometry.
3, the experiment was set up in blank control group, positive control group, negative control group, Bax interference group, Bak interference group and gax-Bak interference group.
Result
1, esternblot detection:
After transfection of Bax-siRNA and Bak-siRNA, the bands decreased, indicating that Bax and Bak contents in cells decreased.
2, basic morphological observation:
1) in the blank control group, the Bak interference group and the Bak+Bax interference group had normal morphology, mostly polygonal and fusiform, and mainly adhered to the wall.
2) the morphology of Bax interference group was basically normal, and there were a few black spots in the cytoplasm, mostly polygons and fusiform, mainly adhered to the wall.
3) in the positive control group, the cells in the negative control group became round and contracted, the cell spacing was large, and there were more black spots in the cytoplasm.
3, flow cytometry results:
1) the apoptotic index of the Bak interference group was 3.44%, and the BaxBak combined interference group had an apoptotic index of 3.97%, which was significantly lower than that of the negative control group.
2) the apoptotic index of the Bax interference group was 19.98%, which was similar to that of the negative control group.
conclusion
1, interfering with the expression of apoptotic molecules such as Bak can inhibit the apoptosis of granulosa cells.
2, Bak interference group and BaxBak combined interference group had significant effect on particle apoptosis.
3, inhibition of granulosa cell apoptosis by Bax interference group was not obvious.
The second part of artificial intervention in mouse embryonic cell apoptosis
objective
The effects of Bax-siRNA and Bak-siRNA on apoptosis of mouse embryos were studied.
Method
1, laser scanning was used to transfect FAM labeled negative siRNA, and fluorescence detection of transfection method was feasible.
2, through laser drilling, Bax and Bak-siRNA were transferred into mouse embryos at the same time, and the normal morphology and cleavage were observed, and the rate of blastocyst formation was calculated.
3, Bax and Bak-siRNA were transfected into mouse embryos by laser drilling respectively. Hoechst33342 and PI were used to detect apoptosis and apoptosis cells were counted.
4, the expression of Bak and Bax was detected by immunofluorescence and the optical density was compared with that of negative control.
5, the expression of Caspase-3 precursor in Bak and Bax interference groups was detected by fluorescence.
Result
1, transfected FAM labeled negative siRNA embryos showed fluorescence, indicating that the transfection method was effective.
2, the rate of blastocyst formation: blank control group 81%, negative control group 69.5%, Bak interference group 92.5%, Bax interference group 74%, combined interference group 89.5%., Bak interference group, combined interference group and negative control has significant difference, P < 0.001; Bax interference group and negative control no significant difference, p=0.318; negative control and blank control is different, p= 0.011.
3, Hoechst33342 and PI staining detected cell apoptosis: blank control group 26%, negative control group 33%, Bak interference group 17.6%, Bax interference group 30.5%, combined interference group 19.1%, compared with negative control group, Bak interference group and combined interference group were significantly different, P < 0.001; Bax interference group had no significant difference, p=0.470.
4, the expression of fluorescence detection Bak, Bax: the light density of Bax interference group and negative control group was 25.39 + 4.73,52.89 + 3.91 respectively, the former significantly weakened P < 0.001; the light density of Bak interference group and negative control group was 17.10 + 3.79,35.26 + 4.88, the former significantly weakened P < 0.001; the light density of the combined interference group and the negative control group was respectively. : TRITC tag group: 19.68 + 4.86,35.26 + 4.88; the former decreased P < 0.001; FITC tag group: 32.78 + 3.73,52.89 + 3.91, the former was significantly reduced P < 0.001.
5, the expression of Caspase-3 precursor was detected by fluorescence.
The Bak interference group and the negative control in the caspase-3 optical density was 65.68 + 4.79,30.24 + 3.40, P < 0.001, there are significant differences; the Bax interference group and the negative control in the caspase-3 optical density was 31.48 + 2.65,30.24 + 3.40, p=0.318, no significant difference.
conclusion
1. it is the first time to apply laser drilling to transfect mouse embryos.
2. after transfection of Bak / Bax-SiRNA, the expression of Bak and Bax decreased.
3. in Bak interference and combined interference group, the number of apoptotic cells in mouse embryos decreased significantly, and the rate of blastocyst formation was obviously increased. In the Bax interference group, the number of apoptotic cells, the rate of blastocyst formation and the negative ratio were not significantly changed, indicating that Bak interference and Bak-Bax interference were effective in inhibiting the apoptosis of rat embryos.
4. the expression of Caspase-3 precursor in mouse embryos increased in the Bak interference group, but there was no significant change in the Bax interference group.
The third part of mouse embryo transfer and chromosome detection
objective
1, we can preliminarily study whether mouse embryos can develop to mature individuals after anti apoptotic treatment.
2, we initially observed whether the birth mice had physiological defects and chromosomal abnormalities.
Method
1, in the Bak / Bax interference group and the combined interference group, 10 blastocysts with better blastocyst were transplanted into the mouse uterus, and the basic conditions of the mice were observed.
2, the examination of the chromosomes of the born mice.
Result
1, 6 mice were born in the Bak interference group, 5 mice were born in the Bax interference group and the combined interference group.
2, there were no obvious abnormalities in the chromosomes of the mice born.
conclusion
1, the anti apoptotic mouse embryos developed by Bak and Bax can normally develop to mature individuals.
2, the anti apoptosis treatment had no obvious effect on the birth mice.
3, the anti apoptotic treatment had no obvious effect on the chromosomes of the born mice.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R321-33

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