【作者】 段恒；

【导师】 陆华；

【作者基本信息】 成都中医药大学 ， 中医妇科学， 2010， 博士

【摘要】 目的探讨含补肾中药鼠血清对小鼠卵细胞体外成熟的影响及其机制。方法未成熟卵细胞来自孕马血清促性腺激素(PMSG)刺激后的90只ICR小鼠的卵巢。将收集的小鼠卵丘细胞-卵母细胞复合体(COCs)245个随机分为含补肾中药鼠血清1#、2#、3#组、空白鼠血清组、空白组,分别置入添加了2%含补肾中药鼠血清1#、2#、3#、2%空白鼠血清及未添加鼠血清的M199培养液中,采用微滴法培养24h,体视显微镜和倒置显微镜观察并计算小鼠卵细胞体外第一极体(PB1)排出率即成熟率。将COCs约238个随机分为含药鼠血清1#组、卵泡刺激素(FSH)组、空白鼠血清组、空白组,分别置入添加了2%含药鼠血清1#、100IU/L FSH、2%空白鼠血清及未添加鼠血清的M199培养液中,显微镜观察并计算小鼠卵细胞体外成熟率、受精率及卵裂率,实验重复3次。将COCs随机分为含药鼠血清1#组、卵泡刺激素(FSH)组、空白鼠血清组、空白组,免疫荧光标记法结合反射荧光显微镜观察细胞骨架指标(纺锤体、染色体),及成熟促进因子(MPF)调节亚基周期蛋白B(CyclinB)的表达；荧光检测仪定量分析Cyclin B1的表达强度。将COCs随机分为含药鼠血清1#组、空白鼠血清组,半定量RT-PCR检测卯母细胞MPF调节亚基Cyclin Bl mRNA的表达。采用酶联免疫法测定含药鼠血清1#、2#、3#与空白鼠血清FSH和雌二醇(E2)水平。结果1.含药鼠血清1#、2#组均较空白组卵细胞成熟率明显增高(42.31%、36.17% vs 16.67%,分别P<0.01、P<0.05);含药鼠血清1#组较空白鼠血清组卵细胞成熟率明显增高(42.31% vs 22.92%,P<0.05)；含药鼠血清1#组较空白组生发泡破裂和第一极体(GVBD+PBl)出现率明显增高(84.62% vs 66.67%,P<0.05)；含药鼠血清各组之间GVBD+PBl和成熟率比较,无显著性差异。2.FSH组、含药鼠血清1#组均较空白组卵细胞成熟率明显增高(33.69%、40.98% vs 17.71%,P<0.01)：FSH组、含药鼠血清1#组均较空白鼠血清组卵细胞成熟率明显增高(33.69%、40.98% vs 22.81%,分别P<0.05、P<0.01)；FSH组、含药鼠血清1#组均较空白组GVBD+PBl明显增高(80.75%、83.61% vs 64.00%,P<0.01)：FSH组、含药鼠血清1#组均较空白鼠血清组GVBD+PBl明显增高(80.75%、83.61% vs 71.35%,分别P<0.05、P<0.01);FSH组与含药鼠血清1#组GVBD+PBl和成熟率比较,无显著性差异;各组之间受精率及卵裂率总体上无明显差异。3次重复实验分次结果显示基本一致。FSH组、含药鼠血清1#组均较空白组卵细胞成熟率明显增高,分别P<0.05、P<0.05或P<0.01；含药鼠血清1#组均较空白血清组卵细胞成熟率明显增高,P<0.05;FSH组、含药鼠血清1#组均较空白组GVBD+PBl明显增高,分别P<0.05、P<0.05或P<0.01；FSH与含药鼠血清1#组GVBD+PBl和成熟率比较,无显著性差异。3.FSH、含药鼠血清1#组CyclinBl的荧光强度较空白组、空白鼠血清组明显增强(15069.00±3908.88、17936.50±4717.42 vs 5029.50±1656.61、6625.33±1777.41,P<0.01);FSH与含药鼠血清1#组比较,无显著性差异。含药鼠血清1#组Cyclin Bl mRNA的表达(Cyclin Bl/GAPDH=2.78)较空白鼠血清组(Cyclin Bl/GAPDH=2)增强。空白组、空白鼠血清组、FSH组、含药鼠血清1#组卵子骨架各组之间比较,无显著性差异。含药鼠血清1#、2#、3#组血清FSH和E2水平均较空白鼠血清组高,无显著性差异。结论含补肾中药鼠血.清1#体外可以促进小鼠未成熟卵细胞生长发育,主要是促进卵细胞核成熟,并且不增加成熟卵细胞细胞骨架的异常几率。其机制与增强卵细胞成熟促进因子调节亚基Cvclin Bl的表达有关。更多还原

【Abstract】 Objective To study the effect of traditional chinese medicine for reinforcing the kidney on mouse oocyte in vitro maturation.Method Inmature oocytes were retreived from 90 ICR murine ovaries stimulated by pregnant mare serum gonadotropin (PMSG).245 mouse cumulus oocyte complexes (COCs) were collected and randomly divided into five groups, rat serum containing traditional chinese medicine 1#,2#,3# for reinforcing the kidney, blank serum(rat serum without traditional chinese medicine) and blank group, and microdrop matured in M199 media for 24 h with 2% rat serum containing traditional chinese medicine 1#,2#,3# for reinforcing the kidney,2% blank serum and medium without rat serum, separately. Oocyte maturation rate were counted. About 238 mouse COCs were collected and randomly divided into four groups, medicated serum 1#, follicle-stimulating hormone (FSH), blank serum and blank group, and matured in media with 2% medicated serum 1#,100IU/L FSH,2% blank serum and medium without rat serum, separately. Oocyte maturation rate, fertilization rate and cleavage rate were counted. The experiment was repeated 3 times. The mouse COCs were collected and randomly divided into four groups, medicated serum 1#, follicle-stimulating hormone (FSH), blank serum and blank group. The expression of oocyte cytoskeleton (spindle and chromosome) and regulatory subunit Cyclin B of mature promoting factor (MPF) were detected by immunofluorescence and observed by reflection fluorescence microscope. And the expression intensity of Cyclin B1 was analyzed by fluorescence testing instrument. The mouse COCs were collected and randomly divided into two groups, medicated serum 1# and blank serum group. Semi-quantity RT-PCR method was used to observe the expression of Cyclin B1 mRNA in mouse oocytes. FSH and E2 in rat blood were determinated by enzyme immunoassay (EIA).Results1. The oocyte maturation rate in the medicated serum 1#,2# groups were higher than that in the blank group (42.31%、36.17% vs 16.67%, P<0.01 or P<0.05). The oocyte maturation rate in the medicated serum 1# group was higher than that in the blank serum group (42.31% vs 22.92%, P<0.05). germinal vesicle breakdown and the first polar body (GVBD+PB1) in the medicated serum 1# group was higher than that in the blank group (84.62% vs 66.67%, P<0.05). There was no significant difference between the medicated serum 1#,2#,3# groups.2. The oocyte maturation rate in the FSH group and the medicated serum 1# group were higher than that in the blank group (33.69%,40.98% vs 17.71%, P<0.01), The oocyte maturation rate in the FSH group and the medicated serum 1# group were higher than that in the blank serum group (33.69%、40.98% vs 22.81%, P <0.05 or P<0.01). GVBD+PB1 in the FSH group and the medicated serum 1# group were higher than that in the blank group (80.75%、83.61% vs 64.00%, P<0.01). GVBD+PB1 in the FSH group and the medicated serum 1# group were higher than that in the blank serum group (80.75%、83.61% vs 71.35%, P<0.05 or P<0.01) There was no significant difference between the FSH group and the medicated serum 1# group.The experiment was repeated threel times to get the basically consistent outcome. The oocyte maturation rate in the FSH group and the medicated serum 1# group were higher than that in the blank group, respectively, P<0.05, P<0.05 or P<0.01. The oocyte maturation rate in the FSH group and the medicated serum 1" group were higher than that in the blank serum group, P<0.05. GVBD+PB1 in the FSH group and the medicated serum 1# group were higher than that in the blank group, respectively, P<0.05、P<0.05 or P<0.01. There was no significant difference between the FSH group and the medicated serum 1# group.3. Fluorescence intensity of Cyclin B1 in the FSH group and the medicated serum 1# group were higher than that in the blank group and the blank serum group (15069.00±3908.88,17936.50±4717.42 vs 5029.50±1656.61、6625.33±1777.41, P<0.01).There was no significant difference between the FSH group and the medicated serum 1" group. the expression of Cyclin B1 mRNA in the medicated serum 1# group (Cyclin B1/GAPDH=2.78) were higher than that in the blank serum group (Cyclin B1/GAPDH=2) There was no significant, difference of oocyte cytoskeleton between medicated serum 1", follicle-stimulating hormone (FSH), blank serum and blank group. The levels of FSH and E2 in the medicated serum 1#,2#,3# groups were higher than that in the blank serum group, but there was no significant difference between the four groups.Conclusion Rat serum containing traditional chinese medicine for reinforcing the kidney 1# has the effect of improving the mouse oocyte development in vitro. The medicated serum 1# has no adverse effect on the cytoskeleton configuration of the mature oocytes. The effect of medicated serum 1" for improving the mouse oocyte in vitro maturation may be ralated to the effect on increasing the expression of maturation promoting factor regulatory subunit Cyclin B1.更多还原