本文選題：顆粒細胞 + 凋亡��； 參考：《南方醫(yī)科大學》2007年博士論文

【摘要】： 卵細胞和胚胎的衰老和死亡是制約體外受精-胚胎移植(in vitro fertilization embryo transfer，IVF-ET)等輔助生殖技術(assisted reproduetive techniques，ART)成功率提高的一個關鍵因素。而細胞凋亡是卵細胞和胚胎死亡的主要方式，也是導致卵細胞受精障礙和胚胎發(fā)育停滯、著床失敗的主要原因�？梢栽O想，若能抑制卵細胞和胚胎的凋亡，將顯著提高胚胎的質量及輔助生殖的成功率。導致細胞凋亡的原因很多，抗凋亡分子和促凋亡分子在調節(jié)卵細胞和胚胎細胞凋亡方面起著極為重要的作用，它們之間的平衡決定著細胞的存活和死亡。其中，Bcl-2家族起著重要的作用，Bax、Bak是其亞家族中的成員，具有促凋亡作用。本研究從一個全新的角度，首次提出通過抗凋亡人工干預的方法來促進胚胎發(fā)育。我們以人顆粒細胞和小鼠早期胚胎為研究對象，采用基因轉染法，在人顆粒細胞和小鼠胚胎內轉入Bax、Bak等凋亡分子的小分子干擾RNA(small interfering RNA，siRNA)，通過干擾Bax等凋亡分子mRNA表達從而提升Bcl-2等抗凋亡分子的含量比例等方法分析體外人工抗凋亡干預措施對早期胚胎發(fā)育的影響，尋找一條提高卵子和胚胎質量的新途徑，也為提高胚胎干細胞培養(yǎng)中的囊胚形成率奠定一定的基礎，從而為最終將抗凋亡干預應用于臨床不孕癥輔助治療提供理論和實驗依據。 第一部分干擾Bax-Bak基因表達 目的 通過轉染Bax、Bak小分子干擾RNA(siRNA)，，干擾Bax、Bak等凋亡分子的功能，研究其對人顆粒細胞凋亡的影響，為提高卵子、胚胎質量提供新的思路及理論依據。 方法 1、用Westernblot檢測Bax、Bak-siRNA； 2、將Bax、Bak-siRNA分別或同時轉染入人顆粒細胞，觀察基本形態(tài)，用流式細胞儀檢測顆粒細胞的凋亡。 3、實驗設置空白對照組、陽性對照組、陰性對照組、Bax干擾組、Bak干擾組、gax-Bak干擾組。 結果 1、esternblot檢測： 轉染了Bax-siRNA和Bak-siRNA后的條帶減弱，說明細胞內Bax、Bak含量降低。 2、基本形態(tài)觀察： 1)空白對照組、Bak干擾組、Bak+Bax干擾組細胞形態(tài)正常，多為多邊形和梭形，以貼壁為主。 2)Bax干擾組細胞形態(tài)基本正常，胞質中有少數黑點，多為多邊形和梭形，以貼壁為主。 3)陽性對照組、陰性對照組細胞變圓，收縮，細胞間距大，胞質中有較多黑點。 3、流式細胞儀檢測結果： 1)Bak干擾組的凋亡指數為3.44％，BaxBak聯合干擾組的凋亡指數為3.97％，明顯低于陰性對照。 2)Bax干擾組的凋亡指數為19.98％，與陰性對照組相似。 結論 1、干擾Bak等凋亡分子的表達，可以抑制顆粒細胞的凋亡。 2、Bak干擾組以及BaxBak聯合干擾組抗顆粒凋亡效果顯著。 3、Bax干擾組抑制顆粒細胞凋亡的作用不明顯。 第二部分人工干預小鼠胚胎細胞凋亡 目的 通過轉染Bax-siRNA和Bak-siRNA，研究其對鼠胚凋亡的影響，尋找干預胚胎的凋亡、促進胚胎發(fā)育的新途徑。 方法 1、激光打孔轉染FAM標記的陰性siRNA，熒光檢測轉染方法是否可行。 2、通過激光打孔分別以及同時將Bax、Bak-siRNA轉入小鼠胚胎，進行普通形態(tài)、卵裂觀察；并計算囊胚形成率。 3、通過激光打孔分別以及同時將Bax、Bak-siRNA轉染入小鼠胚胎，Hoechst33342和PI熒光染色檢測凋亡，并計數凋亡細胞。 4、免疫熒光檢測Bak、Bax的表達，并檢測其光密度與陰性對照比較。 5、分別用熒光檢測Bak及Bax干擾組中caspase-3前體的表達。 結果 1、轉染FAM標記的陰性siRNA的胚胎有熒光，證明轉染方法有效。 2、囊胚形成率：空白對照組81.0％，陰性對照組69.5％，Bak干擾組92.5％，Bax干擾組74.0％，聯合干擾組89.5％。其中，Bak干擾組、聯合干擾組與陰性對照有顯著差異，p＜0.001；Bax干擾組與陰性對照無顯著差異，p=0.318；陰性對照與空白對照有差異，p=0.011。 3、Hoechst33342和PI染色檢測細胞凋亡：空白對照組26.0％，陰性對照組33.0％，Bak干擾組17.6％，Bax干擾組30.5％，聯合干擾組19.1％；與陰性對照組比較，Bak干擾組和聯合干擾組均有顯著差異，p＜0.001；但Bax干擾組無顯著差異，p=0.470。 4、熒光檢測Bak、Bax的表達：Bax干擾組及其陰性對照組光密度分別為25.39±4.73、52.89±3.91，前者顯著減弱p＜0.001；Bak干擾組及其陰性對照組光密度分別為：17.10±3.79、35.26±4.88，前者顯著減弱p＜0.001；聯合干擾組及其陰性對照組光密度分別為：TRITC標記組：19.68±4.86、35.26±4.88；前者顯著減弱p＜0.001；FITC標記組：32.78±3.73、52.89±3.91，均為前者顯著減弱p＜0.001。 5、熒光檢測caspase-3前體的表達。 Bak干擾組及其陰性對照中caspase-3光密度分別為：65.68±4.79、30.24±3.40，p＜0.001，有顯著差異；Bax干擾組及其陰性對照中caspase-3光密度分別為：31.48±2.65、30.24±3.40，p=0.318，無顯著差異。 結論 1．首次在小鼠胚胎上應用激光打孔后轉染的實驗方法。 2．在轉染Bak／Bax-SiRNA后，Bak和Bax的表達減弱。 3．在Bak干擾和聯合干擾組中，鼠胚的凋亡細胞數明顯減少，囊胚的形成率明顯增加；而在Bax干擾組中，胚胎的凋亡細胞數、囊胚形成率與陰性對照相比無明顯改變；說明Bak干擾以及Bak-Bax聯合干擾對于抑制鼠胚的凋亡是有效的。 4．鼠胚中caspase-3前體的表達在Bak干擾組中增強；在Bax干擾組中無明顯變化。 第三部分鼠胚移植與染色體檢測 目的 1、初步研究鼠胚在進行抗凋亡處理后是否能發(fā)育至成熟個體。 2、初步觀察出生小鼠是否有生理缺陷及染色體異常。 方法 1、挑選Bak／Bax干擾組及聯合干擾組中發(fā)育較好的囊胚各10個移植入小鼠子宮；觀察出生小鼠的基本情況。 2、出生小鼠染色體的檢查。 結果 1、Bak干擾組共出生6只小鼠，Bax干擾組和聯合干擾組均出生5只小鼠，出生小鼠均無明顯畸形和行為異常。 2、出生小鼠染色體無明顯異常。 結論 1、經Bak、Bax抗凋亡處理的鼠胚可以正常發(fā)育至成熟個體。 2、抗凋亡處理對出生小鼠無明顯影響。 3、抗凋亡處理對出生小鼠染色體無明顯影響。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R321-33

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