补肾法、疏肝法改善超促排卵小鼠子宫内膜容受性与调控MAPK家族信号通路的关系

发布时间：2018-05-27 05:00

本文选题：补肾法 + 疏肝法　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:本研究通过观察补肾法、疏肝法对超促排卵小鼠围着床期子宫内膜VEGFR-2及其下游血管生成MAPK家族信号通路的影响,以探讨两种方法改善子宫内膜容受性的作用机制和环节之异同。方法:将240只动情周期正常的雌性小鼠随机分为正常组、模型组、补肾组、疏肝组,每组各60只。除正常组外,各组建立控制性超促排卵小鼠模型。补肾组、疏肝组分别从造模第1天开始灌胃补肾助孕方混悬液和逍遥丸混悬液。模型组和正常组给予同等剂量蒸馏水灌胃,连续11天。正常组于动情期,模型组、补肾组、疏肝组于注射HCG日,按雌:雄=2:1比例合笼饲养。次日晨见阴栓记为孕1d,各组孕鼠分别于孕2d、3d、4d、5d、6d取材,通过放射免疫法检测小鼠血清中雌二醇(E2)、孕酮(P)含量;采用HE染色观察小鼠子宫内膜厚度及形态学变化;扫描电镜观察小鼠子宫内膜胞饮突的形态,免疫组化法检测小鼠子宫内膜雌激素受体(ER)、孕激素受体(PR)、微血管密度(MVD)、VEGF、VEGFR-2、PCNA、CyclinD1、MMP-9蛋白表达,Western Blot法检测小鼠子宫内膜ER、PR、PCNA、Cyclin D1、MMP-9、VEGF、VEGFR-2及其下游血管生成相关分子蛋白表达,实时荧光定量PCR检测VEGF、VEGFR-2、PCNA、CyclinD1、MMP-9mRNA表达;统计比较各组小鼠阴栓率、妊娠率及胚胎着床数。结果:1各组小鼠阴栓率、妊娠率及胚胎着床数与正常组比较,模型组小鼠阴栓率、妊娠率均下降,胚胎着床数减少,差异有统计学意义(P0.05);与模型组相比,补肾组、疏肝组阴栓率、妊娠率均提高(均P0.05),且胚胎着床数增加(P0.05);补肾组阴栓率、妊娠率及胚胎着床数较疏肝组增加,但两组比较无统计学意义(p0.05)。2围着床期各组小鼠雌孕激素及其受体水平比较各组小鼠随着妊娠天数的增加,血清e2、p值逐渐升高。孕第2、3、4、5、6天:与正常组比较,模型组小鼠血清e2值降低,差异有统计学意义(p0.05);与模型组比较,补肾组、疏肝组血清e2值升高(p0.05)。孕第3、5天补肾组血清e2值高于疏肝组,差异有统计学意义(p0.05),其余各天两组比较无统计学差异(p0.05)。孕第2、3、4、5、6天:与正常组比较,模型组小鼠血清p值降低,差异有统计学意义(p0.05);与模型组比较,补肾组、疏肝组血清p值升高(p0.05);补肾组与疏肝组比较无明显统计学差异(p0.05)。er、pr蛋白表达主要定位于小鼠子宫内膜腺体、间质的胞浆中。孕第2、3、4、5、6天:与正常组比较,模型组er、pr蛋白表达降低(p0.05);与模型组比较,补肾组、疏肝组er、pr蛋白表达升高(p0.05);补肾组与疏肝组之间比较表达无明显差异(p0.05)。3围着床期各组小鼠子宫内膜厚度比较各组小鼠随着妊娠天数的增加,子宫内膜逐渐增厚。与正常组比较,模型组小鼠孕第2、3、4、5、6天子宫内膜厚度均明显降低(均p0.01);与模型组比较,补肾组、疏肝组孕第2、3、4、5、6天子宫内膜厚度均显著增加(均p0.01);补肾组与疏肝组比较,差异无统计学意义(p0.05)。4围着床期各组小鼠子宫内膜形态学变化he染色显示:正常组:小鼠随着妊娠天数的增加,子宫内膜逐渐增厚;腺体数量增多,腺腔变大、弯曲,腺腔分泌物增多;间质疏松水肿,基质细胞增大,在孕第4天后宫腔逐渐闭合,基质细胞增殖分化成蜕膜细胞。模型组:与正常组比较,子宫内膜较薄且发育不良,腺体少而小,腺腔扩张不明显,腺腔分泌物较少;间质疏松水肿,相对致密,部分小鼠子宫内膜呈现腺体与间质发育不同步,间质呈分泌期改变而腺体发育仍为增生期。补肾组、疏肝组内膜发育与正常组接近,优于模型组,两组间比较无明显差异。5围着床期各组小鼠子宫内膜胞饮突的变化胞饮突主要分布于子宫内膜腺体开口处。正常组:孕第3天,部分子宫内膜细胞表面形成一些膜状突起,突起表面有微绒毛覆盖,绒毛短,稀疏,为发育中的胞饮突;孕第4天子宫内膜表面有大量均匀分布、边界清楚的膜状突起,表面光滑,无微绒毛覆盖,为完全发育的胞饮突;孕第5天胞饮突表面皱缩,凹陷,微绒毛重新出现,为退化的胞饮突。模型组:孕第3天,胞饮突凸起较正常组明显,微绒毛较短较稀疏,发育较正常组提前;孕第4天,子宫内膜胞饮突数量较正常组减少,表面皱缩,大小不一,整体发育不同步,未见完全发育的胞饮突,提示胞饮突发育提前;孕第5天胞饮突皱缩较正常组明显,微绒毛较少。补肾组、疏肝组:孕第3天,膜状突起出现,表面覆有微绒毛;孕第4天为完全发育的胞饮突,较模型组表面相对饱满、分布相对均匀;孕第5天胞饮突发育与正常组接近,为退化的胞饮突。6免疫组织化学法检测mvd在不同分组之间的比较cd34为血管密度标志物,表达于子宫内膜血管内皮细胞,用于测定mvd。孕第2、3、4、5、6天:与正常组相比,模型组mvd明显下降(p0.05);与模型组比较,补肾组、疏肝组mvd显著增加(p0.05);与正常组比较,补肾组、疏肝组mvd降低(p0.05)。孕第6天,补肾组mvd高于疏肝组,差异有统计学意义(p0.05)。7免疫组织化学法检测vegf、vegfr-2、pcna、cyclind1、mmp-9蛋白定位vegf、vegfr-2蛋白在围着床期小鼠子宫内膜的表达在空间上高度一致,主要定位在子宫内膜腔上皮、腺上皮细胞、间质细胞及血管内皮细胞的胞浆中;pcna蛋白表达主要定位在子宫内膜腔上皮、间质细胞核内,腺上皮细胞核有少量表达;cyclind1主要表达于子宫内膜腔上皮、腺上皮的细胞核中,间质细胞核内表达较弱;mmp-9在子宫内膜的腔上皮、腺上皮及间质细胞浆中均有表达。8vegf、vegfr-2、pcna、cyclind1、mmp-9蛋白及其mrna时序表达规律孕第2天vegf、vegfr-2、pcna、cyclind1、mmp-9蛋白及其mrna表达较低,之后逐渐上升,于孕第4天达到峰值,与其他时间点比较差异有统计学意义(p0.05),之后表达逐渐下降。9vegf、vegfr-2、pcna、cyclind1、mmp-9蛋白及其mrna表达结果在不同分组之间的比较孕第2、3、4、5、6天:与正常组比较,模型组小鼠子宫内膜vegf和vegfr-2蛋白及其mrna表达降低,差异有统计学意义(p0.05);与模型组相比,补肾组、疏肝组vegf、vegfr-2蛋白及其mrna表达明显升高(p0.05)。但与正常组比较,孕第2、4、5、6天,补肾组、疏肝组vegf蛋白及其mrna表达降低(p0.05),孕第3、4、5、6天补肾组、疏肝组vegfr-2蛋白及其mrna表达降低(p0.05)。孕第6天补肾组vegf、vegfr-2蛋白及其mrna表达高于疏肝组,差异有统计学意义(p0.05),其余各天补肾组与疏肝组比较无统计学差异(p0.05)。孕第2、3、4、5、6天:与正常组比较,模型组小鼠子宫内膜pcna蛋白及其mrna表达低于正常组,差异有统计学意义(p0.05);与模型组相比,补肾组、疏肝组pcna蛋白及其mrna表达明显增多(p0.05)。孕第2、3、6天,补肾组与正常组比较无统计学差异(p0.05),孕第4、5天补肾组低于正常组,差异有统计学意义(p0.05)。孕第2、3、4、5天补肾组pcna蛋白及其mrna表达均高于疏肝组(p0.05)。孕第2、3、4、5、6天:与正常组相比,模型组小鼠子宫内膜cyclind1蛋白及其mrna表达明显低于正常组,差异有统计学意义(p0.05);与模型组相比,补肾组、疏肝组cyclind1蛋白及其mrna表达明显升高(p0.05);补肾组、疏肝组低于正常组(p0.05),两组间比较无统计学差异(p0.05)。孕第2、3、4、5、6天:与正常组比较,模型组小鼠子宫内膜mmp-9蛋白及其mrna表达下降,差异有统计学意义(p0.05);与模型组相比,补肾组、疏肝组mmp-9蛋白及其mrna表达明显升高(p0.05)。孕第2、3、4、5天补肾组低于正常组(p0.05),孕第2、4天疏肝组低于正常组(p0.05),孕第2、3、4、5天疏肝组高于补肾组(p0.05)。孕第6天,补肾组、疏肝组、正常组比较无统计学差异(p0.05)。10westernblot法检测vegfr-2下游mapk家族信号通路蛋白表达孕2、3、4、5、6天:与正常组比较,模型组p-erk、p-p38、p-jnk蛋白表达降低(p0.05);与模型组比较,补肾组p-erk、p-p38、p-jnk蛋白表达升高(P0.05);疏肝组p-P38、p-JNK蛋白表达增强,差异有统计学意义(P0.05),疏肝组p-ERK表达增强,但差异无统计学意义(P0.05);疏肝组p-ERK蛋白表达较补肾组降低,差异有统计学意义(P0.05)。结论:1 VEGF参与胚泡着床且在围着床期表达具有时空特异性,可作为子宫内膜容受性评价指标。2控制性超促排卵降低围着床期小鼠血清E2、P水平,影响ER、PR表达,使子宫内膜腺体与间质发育不同步,胞饮突发育提前,下调VEGF及其受体,降低微血管密度,影响VEGFR-2下游MAPK家族3条信号通路,降低通路活性,减少磷酸化ERK、P38、JNK的表达,下调通路下游目的基因PCNA、CycinD1、MMP-9的表达,影响子宫内膜血管生成,降低子宫内膜容受性,导致妊娠率、着床率降低。3补肾法、疏肝法通过改善超促排卵小鼠血清E2、P水平及ER、PR表达,改善子宫内膜形态及胞饮突发育,上调VEGF及其受体,改善微血管密度,调控VEGFR-2下游MAPK家族3条信号通路,增加通路活性,提高磷酸化ERK、P38、JNK的表达,促进通路下游目的基因PCNA、CycinD1、MMP-9的表达,促进细胞增殖迁移,有利于子宫内膜血管生成,改善子宫内膜容受性,从而提高小鼠妊娠率和着床率。4补肾法促进磷酸化ERK表达优于疏肝法;补肾法在促进内皮细胞增殖,加速血管生成,改善子宫内膜容受性方面优于疏肝法;疏肝法在水解细胞外基质(extracellular matrix,ECM),促进细胞迁移,促进血管生成,改善子宫内膜容受性方面优于补肾法。
[Abstract]:Objective: To investigate the effect of two methods to improve the endometrial receptivity of the endometrium and the differences and similarities between the two methods to improve the endometrial receptivity of the endometrium and the MAPK family signal pathway of the endometrium in the surrounding bed of the superovulation mice. Methods: 240 female mice with normal estrus cycle were randomly divided into two groups. In the normal group, the model group, the kidney tonifying group and the Liver Soothing group, each group had 60 rats in each group. In addition to the normal group, the control superovulation mice model was established in each group. The tonifying kidney group and the liver Shugan group began to fill the kidney and the Xiaoyao suspension solution and the suspension of the Xiaoyao Pill from the model first days. The model group and the normal group were given the same dose of distilled water for 11 days. The model group, the kidney tonifying group and the liver Shugan group were fed on HCG day by female: male =2:1 ratio cage. The next morning, the female mice were pregnant with 2D, 3D, 4D, 5D, 6D, respectively. The content of estradiol (E2) and progesterone (P) in the serum of mice was detected by radioimmunoassay, and the endometrium thickness and morphological changes of mice were observed by HE staining; scan of the endometrium and morphology of mice by HE staining. The morphology of endometrium inrush in mouse endometrium was observed by electron microscopy. Immunohistochemistry was used to detect the estrogen receptor (ER), progesterone receptor (PR), microvascular density (MVD), VEGF, VEGFR-2, PCNA, CyclinD1, MMP-9 protein expression, and Western Blot method for detecting the endometrium of mice. The expression of relative molecular protein, VEGF, VEGFR-2, PCNA, CyclinD1 and MMP-9mRNA were detected by real time fluorescence quantitative PCR, and the negative emboli rate, pregnancy rate and embryo implantation number of the mice were compared. The results were as follows: 1 the rate of Yin embolism, the pregnancy rate and the number of embryo implantation were compared with those of the normal group. The difference was statistically significant (P0.05). Compared with the model group, the rate of Yin suppository in the kidney tonifying group, the Liver Soothing group increased (P0.05), and the number of embryo implantation increased (P0.05), the rate of Yin embolism in the kidney group, the pregnancy rate and the number of embryo implantation were higher than those in the Liver Soothing group, but the two groups were compared with the estradiol and the water progesterone and its receptor water in each group of.2 around the bed. As compared with the normal group, the serum E2 value of mice in the model group decreased and the difference was statistically significant (P0.05). Compared with the model group, the serum E2 value of the kidney group and the Liver Soothing group was higher (P0.05) than that in the model group (P0.05). The serum E2 value of the kidney tonifying group was higher than that of the Liver Soothing group at 3,5 day of pregnancy, and there was a difference between the group and the E2 group. Statistical significance (P0.05), the remaining days of the two groups had no statistical difference (P0.05). Gestation day 2,3,4,5,6: compared with the normal group, the serum P value of the model mice decreased, the difference was statistically significant (P0.05). Compared with the model group, the serum P value of the tonifying kidney group and the Liver Soothing group increased (P0.05), and there was no significant difference between the Bushen group and the Liver Soothing group (P0.05).Er (P0.05). The expression of PR protein was mainly located in the endometrium gland of mice and interstitial cytoplasm. Gestation day 2,3,4,5,6: compared with the normal group, the model group was Er, the expression of PR protein decreased (P0.05). Compared with the model group, the tonifying group, the Liver Soothing group Er, the PR protein expression increased (P0.05), and there was no significant difference between the tonifying group and the Liver Soothing group (P0.05).3 peri implantation period (P0.05). The endometrium thickness of the mice was compared with that of the normal group. Compared with the normal group, the endometrium thickness of the mice in the model group was significantly lower than that of the model group (all P0.01). The endometrium thickness of the tonifying group and the liver Shugan group increased significantly (all P0.01) on the day 2,3,4,5,6 of the model group (all P0.01); There was no significant difference between the kidney group and the liver dredging group (P0.05). The morphological changes of endometrium in each group of mice during the.4 implantation period he staining showed that in the normal group, the endometrium gradually thickened with the increase of the number of days of pregnancy; the number of glands increased, the gland cavity became larger, the secretion of the gland cavity increased, interstitial edema, the matrix cell enlargement, and the pregnancy. Fourth the cavity of the Thean Hou Temple was gradually closed and the matrix cells proliferated and differentiated into decidual cells. Compared with the normal group, the endometrium was thinner and dysplastic, the glands were small and small, the adeno cavity dilatated not obviously, the gland cavity was less secreted, the interstitium was loose edema and relatively compact, and the endometrium in some mice showed the dyssynchrony of glandular and interstitial development and the interstitium was divided. The endometrial development was close to the normal group, which was better than the model group. There was no significant difference between the two groups in the two groups. The changes of the endometrium drinking process in each group were mainly distributed at the opening of the endometrium. The normal group: third days of pregnancy and the surface of the endometrium cells. The formation of some membranous protuberances, the surface of the protuberance with microvilli, short villi and sparsely, for the development of the cell drink process; fourth days of pregnancy the endometrium surface has a large number of evenly distributed, clear boundary membrane, smooth surface, no microvilli cover, the complete development of the cell drink process; fifth days of pregnancy, the surface crinkle, sag, microvilli re appearance, In the model group, the model group: third days of pregnancy, the protrusion of the cytosolic protrusion was more obvious than the normal group, the microvilli was shorter and thinner and the development was earlier than the normal group; the number of endometrium inrush in the endometrium was less than the normal group at the fourth day of pregnancy. The surface of the endometrium was shrinking, the size was different, the whole development was not synchronized, and the development of the cytosolic process was not seen, suggesting that the development of the cytosolic process was ahead of time; fifth gestation was pregnant. Fifth pregnancy In the group of third days of pregnancy and the appearance of the membrane, the surface was covered with microvilli; the fourth day of pregnancy was a fully developed cytosolic process, which was relatively full and relatively uniform on the surface of the model group; the fifth day gestation was close to the normal group, which was a degraded.6 immunocytosochemistry of the degenerated cytosolic process. The comparison of MVD between different groups was detected by CD34 as a vascular density marker, expressed in endometrium vascular endothelial cells, and used to determine mvd. pregnancy 2,3,4,5,6 days: compared with the normal group, MVD was significantly decreased (P0.05). Compared with the model group, the MVD in the kidney group and the Liver Soothing group increased significantly (P0.05), and compared with the normal group, the kidney tonifying group and the Liver Soothing group MVD were compared with the normal group. Decrease (P0.05). Sixth days of pregnancy, the MVD of the tonifying group was higher than that of the Liver Soothing group. The difference was statistically significant (P0.05), the.7 immunohistochemical method was used to detect VEGF, VEGFR-2, PCNA, CyclinD1, and MMP-9 protein localization VEGF. The expression of VEGFR-2 protein in the endometrium of the mouse endometrium in the surrounding bed was consistent in space, mainly located in the endometrium epithelium, gland epithelial cells. The expression of PCNA protein is mainly located in the endometrium epithelium, interstitial nuclei, and a small amount of expression in the nucleus of the gland epithelium; CyclinD1 is mainly expressed in the endometrium epithelium, the nucleus of the gland epithelium, and the weak expression in the nucleus of the stromal cells; MMP-9 is in the cavity epithelium of the endometrium, and in the gland epithelium and between the epithelial cells. The expression of.8vegf, VEGFR-2, PCNA, CyclinD1, MMP-9 protein and mRNA was expressed in the cytoplasm of the cytoplasm at second days of VEGF, VEGFR-2, PCNA, CyclinD1, MMP-9 protein and its mRNA expression were low, then increased gradually, and reached the peak at the fourth day of pregnancy, and there was a significant difference compared with the other time points. Egfr-2, PCNA, CyclinD1, MMP-9 protein and mRNA expression results were compared between different groups on the day 2,3,4,5,6 day: compared with the normal group, the VEGF and VEGFR-2 protein and its mRNA expression in the endometrium of the model mice were reduced, and the difference was statistically significant (P0.05); the kidney group, the liver soothing group VEGF, the VEGFR-2 protein and its expression were compared with the model group. Xian Shenggao (P0.05). But compared with the normal group, the expression of VEGF protein and mRNA in the kidney group and the liver group decreased (P0.05), the VEGFR-2 protein and its mRNA expression in the liver group were decreased (P0.05). The VEGF, VEGFR-2 protein and mRNA expression of the kidney group were higher than those in the liver group at the sixth day of pregnancy, and the difference was statistically significant. There was no statistical difference between the kidney tonifying group and the liver dredging group (P0.05). 2,3,4,5,6 days of pregnancy: compared with the normal group, the expression of PCNA protein and mRNA in the endometrium of the model group was lower than that of the normal group, and the difference was statistically significant (P0.05). Compared with the model group, the expression of PCNA protein and mRNA in the tonifying group and the Shugan group increased significantly (P0.05). There was no statistical difference between the kidney tonifying group and the normal group (P0.05). The kidney tonifying group was lower than the normal group on the 4,5 day of pregnancy (P0.05). The expression of PCNA protein and mRNA in the kidney tonifying group on day 2,3,4,5 was higher than that in the Liver Soothing group (P0.05). Compared with the normal group, the difference was statistically significant (P0.05). Compared with the model group, the expression of cyclinD1 protein and its mRNA expression in the tonifying group and the Liver Soothing group increased significantly (P0.05); the kidney group and the Liver Soothing group were lower than the normal group (P0.05), and there was no statistical difference between the two groups (P0.05). Gestation 2,3,4,5,6 days: compared with the normal group, the endometrial MMP-9 protein in the model group mice was MMP-9 protein. And the expression of mRNA decreased, the difference was statistically significant (P0.05). Compared with the model group, the expression of MMP-9 protein and its mRNA in the tonifying group and the Liver Soothing group increased significantly (P0.05). The tonifying group was lower than the normal group (P0.05) on the 2,3,4,5 day of pregnancy (P0.05), and the Liver Soothing group was lower than the normal group (P0.05) on the 2,4 day of pregnancy (P0.05), and the sixth day of pregnancy was higher than that of the kidney tonifying group (P0.05). Group, Liver Soothing group, normal group had no statistical difference (P0.05).10westernblot method to detect VEGFR-2 downstream MAPK family signal pathway protein expression 2,3,4,5,6 days: compared with the normal group, the model group p-ERK, p-p38, p-JNK protein expression decreased (P0.05); compared with the model group, the kidney group p-ERK, p-p38, p-JNK protein expression increased. The expression of JNK protein was enhanced, the difference was statistically significant (P0.05), the expression of p-ERK in the Liver Soothing group was enhanced, but the difference was not statistically significant (P0.05). The expression of p-ERK protein in the Liver Soothing group was lower than that of the tonifying group (P0.05). Conclusion: 1 VEGF participates in the blastocyst implantation and has a spatio-temporal specificity in the peri bed period, which can be used as the endometrial receptivity. .2 controlled ovarian hyperstimulation decreased the serum E2 and P levels of the mice in the surrounding bed, and affected the ER, PR expression, which made the endometrial glands and interstitial development unsynchronized, the development of the cytoplasm of the cytosolic process early, down the VEGF and its receptor, reducing the microvessel density, affecting the 3 signal pathways of the VEGFR-2 downstream MAPK family, reducing the activity of the pathway and reducing the ERK, P38, J. The expression of NK, down regulation of the downstream target gene PCNA, CycinD1, MMP-9, affects the angiogenesis of the endometrium, reduces the endometrium receptivity, causes the pregnancy rate, reduces the implantation rate and reduces the.3 tonifying method. The Liver Soothing method improves the serum E2, P level and ER, PR expression in the superovulation mice, improves the morphology of endometrium and the development of the endometrium, up regulation of VEGF, and up regulation of VEGF and up regulation of VEGF. Its receptor, ameliorate microvascular density, regulate 3 signal pathways in the MAPK family of VEGFR-2 downstream, increase pathway activity, increase the expression of phosphorylated ERK, P38, JNK, promote the expression of PCNA, CycinD1, MMP-9, promote the proliferation and migration of the downstream target genes, promote the angiogenesis of endometrium, improve endometrial receptivity, and improve the pregnancy induced pregnancy induced pregnancy. The rate of pregnancy and the rate of implantation of.4 to promote the expression of phosphorylated ERK is better than that of the Liver Soothing method; the kidney tonifying method is superior to the Liver Soothing method in promoting the proliferation of endothelial cells, accelerating the angiogenesis and improving the receptivity of the endometrium; the Liver Soothing method is hydrolyzing the extracellular matrix (extracellular matrix, ECM), promoting the migration of the cells, promoting the angiogenesis and improving the receptivity of the endometrium. It is superior to the method of Tonifying the kidney.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.8

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