本文選題：JNKs + 植入前胚胎　； 參考：《華中科技大學(xué)》2006年博士論文

【摘要】： 第一部分:JNKs蛋白在各階段植入前小鼠胚胎中的表達(dá) 目的:了解JNKs蛋白在各階段小鼠著床前胚胎中的表達(dá)規(guī)律: 方法:實(shí)驗(yàn)動(dòng)物為昆明種小鼠,經(jīng)過藥物促排卵后,分別獲取妊娠0.5天(合子),1.5天(二細(xì)胞期),2.5天(融合期桑椹胚),3.5天(早期囊胚)的胚胎;使用JNK多克隆抗體,通過免疫熒光法結(jié)合激光共聚焦技術(shù),在蛋白水平上定性且半定量檢測個(gè)階段鼠胚內(nèi)JNKs蛋白的表達(dá),并且初步找出其中規(guī)律 結(jié)果與結(jié)論: 1.在各階段的小鼠胚胎細(xì)胞的胞漿內(nèi)均有JNKs蛋白的表達(dá); 2.隨著胚胎的發(fā)育,JNKs蛋白表達(dá)呈上升趨勢,其中在妊娠1.5天(2細(xì)胞階段)到妊娠2.5天(融合期桑椹胚階段)上升最為明顯(熒光強(qiáng)度3176.9±446 vs 1297.2±392.3,P0.05): 第二部分外界培養(yǎng)環(huán)境與小鼠早期胚胎細(xì)胞內(nèi)JNKs分子的激活 目的:探討體外培養(yǎng)環(huán)境及其內(nèi)各種刺激因素對(duì)小鼠胚胎細(xì)胞內(nèi)JNKs分子激活的影響 方法: 1.分別獲取體內(nèi)及體外的妊娠第3.5天的小鼠胚胎(早期囊胚階段),以免疫熒光和Western-Blotting的檢測方法,比較兩者細(xì)胞內(nèi)JNKs分子的激活(磷酸化)水平; 2.以免疫熒光法比較不同溫度下(35攝氏度,37攝氏度,39攝氏度)培養(yǎng)9小時(shí)后小鼠著床前胚胎(早期囊胚期)細(xì)胞內(nèi)JNKs分子的激活水平。 3.以免疫熒光法比較在3種不同培養(yǎng)液(CZB,G-1,G-2)中培養(yǎng)48小時(shí)后的小鼠胚胎細(xì)胞內(nèi)JNKs分子的激活水平; 4.以免疫熒光法比較在不同培養(yǎng)密度下(3個(gè)/25ul;15-25個(gè)/25ul與50個(gè)/ul) 培養(yǎng)48小時(shí)后小鼠胚胎細(xì)胞內(nèi)JNKs分子的激活水平。 結(jié)果: 1.體外培養(yǎng)的小鼠胚胎細(xì)胞內(nèi)JNKs分子的激活水平顯著高于體內(nèi)發(fā)育者。(免疫熒光法與Western-Bloting法比較均有統(tǒng)計(jì)學(xué)意義) 2.而在39攝氏度培養(yǎng)下9小時(shí)后的鼠胚細(xì)胞內(nèi)JNKs分子的激活水平明顯高于35與37攝氏度組(P0.05); 3.而使用CZB培養(yǎng)液培養(yǎng)的胚胎細(xì)胞內(nèi)激活的JNKs分子的熒光密度顯著高于G-2培養(yǎng)液組(P0.05) 4.而在25微升的液滴中,當(dāng)培養(yǎng)密度〉50個(gè)胚胎/液滴時(shí),細(xì)胞內(nèi)激活的JNKs分子的含量顯著增加,較另外兩組差異有統(tǒng)計(jì)學(xué)意義(P0.05) 結(jié)論: 1.體外培養(yǎng)的小鼠胚胎細(xì)胞內(nèi)JNKs分子的激活水平高于體內(nèi)發(fā)育者; 2.培養(yǎng)溫度,培養(yǎng)液的成分以及培養(yǎng)密度的變化局可以影響小鼠胚胎細(xì)胞內(nèi)JNKs分子的激活。 第三部分:JNKs分子的激活與小鼠早期胚胎的發(fā)育與凋亡 目的:探討JNKs分子的激活與小鼠胚胎細(xì)胞凋亡之間的關(guān)系: 方法: 1.比較在培養(yǎng)液內(nèi)加入不同濃度(10uM,20uM與50uM)或在不同時(shí)間內(nèi)(妊娠1.5天到2.5天與妊娠2.5天到妊娠3.5天)加入JNKs激活的特異性抑制劑SP600125對(duì)小鼠胚胎囊胚形成率的影響; 2.取妊娠3.5天的小鼠早期囊胚,以TUNEL標(biāo)記法比較培養(yǎng)液加入或不加入SP600125的情況下,熱處理(41攝氏度)6小時(shí)后小鼠囊胚細(xì)胞的凋亡比例; 3.取妊娠1.5天的小鼠胚胎,以線粒體膜電位熒光探針JC-1染色法,比較加入或不加入SP600125的情況下,熱處理(41攝氏度)1小時(shí)后胚胎細(xì)胞內(nèi)線粒體膜電位的改變; 4.同上法,比較加入或不加入SP600125的情況下,熱處理3小時(shí)后小鼠胚胎細(xì)胞內(nèi)Caspase-3蛋白表達(dá)水平的差異; 結(jié)果: 1.培養(yǎng)液中添加不同濃度SP600125后,囊胚形成率有所上升,其中當(dāng)SP600125濃度為20uM是囊胚形成率最高,達(dá)72.3%,培養(yǎng)液中在妊娠1.5-2.5天時(shí)(卵裂球融合前)添加SP600125比妊娠2.5-3.5天時(shí)(卵裂球融合后)添加SP600125的囊胚形成率更高,但以上差異均未見統(tǒng)計(jì)學(xué)意義; 2.在培養(yǎng)液中加入20uM濃度的SP600125時(shí),可顯著降低熱處理后小鼠囊胚中調(diào)亡的細(xì)胞比例(39.27±7.15 Vs 64.97±8.23,P0.05): 3.在培養(yǎng)液中加入20uM濃度的SP600125時(shí),熱處理1小時(shí)后胚胎膜電位喪失的線粒體比例顯著下降(P0.05) 4.在培養(yǎng)液中加入20uM濃度的SP600125時(shí),熱處理后3小時(shí)的胚胎細(xì)胞內(nèi)Caspase蛋白表達(dá)的水平明顯下降(P0.05) 結(jié)論: 1.JNKs分子的激活可能影響小鼠胚胎進(jìn)一步的發(fā)育潛力; 2.外界刺激引起的JNKs分子的激活可以促進(jìn)小鼠胚胎細(xì)胞的凋亡過程; 3.JNKs促進(jìn)胚胎細(xì)胞凋亡的機(jī)制可能與線粒體依賴的凋亡途徑有關(guān)。
[Abstract]:Part I: the expression of JNKs protein in pre implantation mouse embryos at different stages.
Objective: to understand the expression pattern of JNKs protein in mouse preimplantation embryos at different stages.
Methods: the experimental animals were Kunming mice. After the ovulation induction, the 0.5 days of pregnancy (zygote), 1.5 days (two cell stage), 2.5 days (fusion stage morula) and 3.5 days (early blastocyst) were obtained respectively. Using JNK polyclonal antibody, the laser confocal technique was combined with immunofluorescence, and the protein level was qualitatively and semi quantified at the level of protein. The expression of JNKs protein in mouse embryos was preliminarily identified.
Results and conclusions:
1. JNKs protein was expressed in the cytoplasm of mouse embryonic cells at all stages.
2. with the development of embryo, the expression of JNKs protein showed an upward trend, in which the 1.5 day of pregnancy (2 cell stage) to 2.5 days of pregnancy (fusion stage morula stage) was the most obvious (fluorescence intensity 3176.9 + 446).
Vs 1297.2 + 392.3, P0.05):
The second part is the activation of JNKs molecules in the environment and the early embryonic cells of mice.
Objective: To investigate the effects of various stimulating factors on the activation of JNKs in mouse embryonic cells.
Method:
1. the mouse embryos (early blastocyst stage) on day 3.5 of the body and in vitro were obtained respectively, and the activation (phosphorylation) level of JNKs molecules in the two cells was compared by immunofluorescence and Western-Blotting detection.
2. the activation level of JNKs in the mouse preimplantation embryos (early blastocyst stage) was compared by immunofluorescence at different temperatures (35 degrees Celsius, 37 degrees centigrade, 39 degrees centigrade) for 9 hours.
3. immunofluorescence method was used to compare the activation level of JNKs molecules in mouse embryo cells cultured in 3 different culture media (CZB, G-1, G-2) for 48 hours.
4. immunofluorescence method was used to compare 3 /25ul, 15-25 /25ul and 50 /ul under different culture densities.
The activation level of JNKs in mouse embryonic cells was cultured after 48 hours.
Result:
1. the activation level of JNKs in the mouse embryo cells in vitro was significantly higher than that in the body. (both the immunofluorescence method and the Western-Bloting method were statistically significant).
2., the activation level of JNKs in mouse embryo cells after 9 hours of culture at 39 degrees Celsius was significantly higher than that in 35 and 37 degrees Celsius (P0.05).
3., the fluorescence density of JNKs molecules activated by CZB medium was significantly higher than that of G-2 medium (P0.05).
4., the content of JNKs molecules activated in the cells increased significantly when the culture density was 50 embryos / droplets in the 25 micro - lift droplets, and the difference was statistically significant compared with the other two groups (P0.05).
Conclusion:
1. in vitro, the activation level of JNKs in mouse embryo cells was higher than that in vivo.
2. the changes in the culture temperature, the composition of the culture medium and the density of culture can affect the activation of JNKs molecules in mouse embryonic cells.
The third part: the activation of JNKs and the development and apoptosis of early mouse embryos.
Objective: To investigate the relationship between activation of JNKs and apoptosis of mouse embryonic cells.
Method:
1. the effects of adding different concentrations (10uM, 20uM and 50uM) in the culture medium or at different times (1.5 to 2.5 days of pregnancy and 2.5 days of pregnancy to 3.5 days of pregnancy) were compared with the specific inhibitor SP600125, which was activated by JNKs, on the rate of blastocyst formation in mouse embryos.
2. the early blastocysts of mice were taken for 3.5 days of pregnancy, and the apoptosis ratio of blastocyst cells in mice after heat treatment (41 degrees centigrade) 6 hours was compared with the TUNEL labeling method.
3. the mouse embryos of 1.5 days of pregnancy were stained with mitochondrial membrane potential fluorescence probe JC-1, and the changes of mitochondrial membrane potential in the embryonic cells after 1 hours of heat treatment (41 degrees centigrade) were compared with or without SP600125.
4. in the same way, the expression level of Caspase-3 protein in mouse embryonic cells after 3 hours of heat treatment was compared with that of SP600125.
Result:
1. after adding different concentrations of SP600125 in the culture medium, the rate of blastocyst formation was increased. When the concentration of SP600125 was 20uM, the rate of blastocyst formation was the highest, up to 72.3%. The formation rate of the blastocyst with SP600125 added to the SP600125 was higher, but the above difference was higher in the culture medium at 1.5-2.5 days of pregnancy (before blastomere fusion) (before blastomere fusion) (after blastomere fusion). The difference was not statistically significant.
2. when adding 20uM concentration of SP600125 in culture medium, the percentage of cells in the blastocyst of heat treated mice decreased significantly (39.27 + 7.15 Vs 64.97 + 8.23, P0.05).
3. when the 20uM concentration of SP600125 was added to the culture medium, after 1 hours of heat treatment, the mitochondrial proportion of the membrane potential loss decreased significantly (P0.05).
4. when the 20uM concentration of SP600125 was added to the culture medium, the expression level of Caspase protein in the embryonic cells decreased significantly after heat treatment for 3 hours (P0.05).
Conclusion:
The activation of 1.JNKs molecules may affect the further developmental potential of mouse embryos.
2. activation of JNKs molecules induced by external stimulation can promote the apoptosis process of mouse embryonic cells.
The mechanism of 3.JNKs promoting embryonic apoptosis may be related to mitochondria dependent apoptotic pathway.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R321-33

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 黃明敏;絲裂原激活蛋白激酶對(duì)金魚發(fā)育的調(diào)控作用[D];湖南師范大學(xué);2008年

相關(guān)碩士學(xué)位論文 前1條

1 張衛(wèi);自發(fā)性高血壓大鼠NHE-1基因表達(dá)與左心室肥厚相關(guān)性研究[D];南方醫(yī)科大學(xué);2007年

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