干预“石门”穴对着床期小鼠子宫内膜容受性影响的实验研究

【作者】 李惠；

【导师】 张鸥；

【作者基本信息】 辽宁中医药大学 ， 针灸推拿学， 2009， 硕士

【摘要】 目的:通过观察干预“石门”穴对着床期小鼠子宫内膜胞饮突、白血病抑制因子(LIF)表达及血清中孕酮(P)含量的影响,初步探讨干预“石门”穴对小鼠胚胎着床的影响及其作用机制、可能途径和相关靶点,为针灸疗法用于抗着床提供科学依据。材料与方法:将昆明种雌性小鼠60只按体重分层随机分为空白对照组(A组)、电针“石门”组(B组)、手针“石门”组(C组)、埋线“石门”组(D组)、西药对照组(E组)、非穴电针组(F组),每组10只,本实验以妊娠第一天(Pd1)小鼠为研究对象。B、C、D组选取“石门”穴进行电针、手针、埋线刺激;E组皮下注射米非司酮;F组在非穴位点进行电针刺激。疗程:B、C、F组自Pd1上午8:00开始针刺,一天一次,连续5天;D组Pd1上午8:00施治一次;E组于Pd4上午9:00施治一次。以上各组小鼠均在Pd5上午9:30末次干预后处死,迅速剖腹取出子宫,用扫描电镜观察小鼠子宫内膜表面胞饮突的表达;光镜下观察小鼠子宫内膜形态变化(HE染色);用免疫组化SABC法检测小鼠子宫内膜LIF的表达;用放射免疫法检测小鼠血清中P的含量。结果:1.各组小鼠Pd5子宫内膜形态变化:A组小鼠腺腔内充盈着丰富的被染成淡红色的分泌物,腺腔扩大呈锯齿状,间质内血窦多。F组子宫内膜发育与A组相一致。B、C、D、E组小鼠子宫内膜比A组发育滞后。2.各组小鼠Pd5子宫内膜表面胞饮突的表达:A组小鼠子宫内膜表面均匀分布着丰富的胞饮突,大小一致,胞饮突之间界限清楚,此即发育成熟的胞饮突;F组亦可见发育成熟的胞饮突;B、C、D组小鼠子宫内膜表面膜状突起明显低于A组,且大小不均,边界欠清,表面覆盖为微绒毛,子宫内膜表面胞饮突呈局灶性表达,且与胚胎发育不同步,胞饮突从形态上看较A组发育受到抑制,从发生时相上看延迟于A组;E组小鼠子宫内膜表面无胞饮突,可见呈多边形的上皮细胞,上皮细胞顶端膜覆以细的微绒毛,细胞间界限清楚,表现为增生早期的子宫内膜变化,其发育亦明显延后于A组。3.各组小鼠Pd5血清中P含量结果和变化:B、C、D、E组小鼠血清中P含量与A组相比均有不同程度下降,比较差异显著(P＜0.05),有统计学意义。4.各组小鼠Pd5子宫内膜LIF的表达:各组小鼠LIF表达部位均位于子宫内膜腔上皮和腺上皮细胞的胞浆;A、F组小鼠子宫内膜LIF的表达强度以“++”为主;B、C、D、E组小鼠子宫内膜LIF的表达强度以“+”为主。图像分析结果显示B、C、D、E组小鼠子宫内膜LIF的表达与A组比较差异显著(P＜0.05),有统计学意义。结论:1.干预“石门”穴在整体水平上对着床期小鼠有抗着床作用,进一步证实石门穴具有这一特异性;2.干预“石门”穴影响小鼠子宫内膜容受性建立和着床窗开放,使子宫内膜和胚胎发育不同步,从而使得胚胎不能正常植入着床;3.在抗着床过程中,干预“石门”穴的作用模式可能为“一点触发,多点调控,整合调节”,这种作用模式来源于孕酮的后续连锁反应;4.电针、手针、埋线三种方法干预“石门”穴都能影响胚胎着床,其中以电针作用最明显,都是通过调节孕酮来实现的。更多还原

【Abstract】 PurposeTo object the effects of intervening at "Shimen" on the expression of pinopodes and LIF on the mouse endometrium and progesterone during implantation period,so as to study the effect of intervening at "Shimen" on the implantation of mouse embryos and action mechanism and pathway and relative target and explain action character of acupuncture.Material and methodsSixty female mice were randomly divided into blank control group(group A,n=10),electro-acupuncture "Shimen" group(group B,n=10),hand-acupuncture "Shimen" group(group C,n=10),catgut embedding "Shimen" group(group D,n=10), positive medicine group(group E,n=10),and non-acupoint group(group F,n=10). Pregnance day 1 was determined on the vaginal plug day."Shimen" were treated with electroacupuncture and acupuncture;Catgut embedding group choose "Shimen";On Pd4,group E were injected subcutaneously with mifepristone diluted in propylene glycol;Group F were treated with electroacupuncture.Course of treatment:group B,C and D treated once on the first day,being treated 5 times insuccession;Group D treated one on first day;Group E infused with medicine on the fourth day.The animals used as experiment of every the above-mentioned groups put to death at 9:30 a.m on Pd5 after the end treatment.Progesterone was determined by radioimmunoassay;The morphologic structure of endometrium was observed by light microscope and scanning electron microscope;The expression of LIF was detected by immunohistochemistry techniques.Results1.The development of endometrium in group B,C,D and E was inhibited.2.The expression of pinopodes in mouse endometrium.In group A,day 5 of vaginal Plug,the endometrial surface is covered with fully developed pinopodes which are larger,completely smooth with well defined border of the cells,resembling a mushroom;group F is covered with fully developed pinopodes,too;In group B and C and D,day 5 of vaginal plug,the endometrial surface is covered with small projections without distinguished borders of cells.The endometrial surface expresses developing pinopodes locally which are asynchronous;In group E,day 5 of vaginal plug,the endometrial surface is covered with hexagonal and flat appeal cells without any pinopodes formation.Compared with group A,the development of pinopodes lagged behind obviously and was repressed.3.The resuIts and enchangings of the levels of P in the serums of different group mouses.After treating,there are various decreases of comparing treat group(B,C,D and E) to group A(P＜0.05).4.The expression of LIF in mouse endometrium of different group.Immunostaining in mouse endometrial epithelial cell and glandular epithelial cell is observed on the implantation day with different level in different group.The results of Semi-quantitative assay show:compared with group A,the level of LIF is significantly decreased in groups of B,C,Dand E(P＜0.05).Conclusion1.Interventing at "Shimen" could inhibit blastocyst implantation in vivo. Specificity of acupoint was further confirmed;2.Intervening at "Shimen" is not beneficial to establish endometrial respectively and to open the implantation window,thus affecting the establishment of embryo implantation;3.In the circumstance of anti-implantation,intervention Shimen possesses the effect character of ’key point trigger-multidrop control-integrate regulation’ This kind of character maybe derives from chain reaction after P is decreased; 4.Different needling methods have different effects on anti-implantation.Their mechanism may be by way of progesterone.更多还原