Maspin在早孕小鼠子宫内膜的表达规律及功能研究

发布时间：2018-05-15 15:04

本文选题：Maspin + 胚泡着床　；参考：《重庆医科大学》2012年硕士论文

【摘要】：背景胚泡着床是处于活化状态的胚泡与处于容受态的子宫内膜相互作用并进入子宫内膜的复杂过程。成功的胚泡着床是人类和其他哺乳类动物妊娠的标志。研究发现，受孕母体的子宫内膜只能在一个极短的时期内适应胚泡植入，将内膜处于接受状态的时期称为“着床窗口期”[1]，进入子宫腔的胚泡在“着床窗口期”之前或之后都不能粘附和植入子宫内膜。当子宫内膜处于“着床窗口期”，同时受精卵经过一系列生长发育、卵裂，胚泡形成和脱透明带，最终具有侵入能力时，胚泡才能成功着床[2-4]。胚泡着床一般分为三个阶段，即定位、黏附、侵入，此过程涉及到众多调控因子参与的错综复杂的信号传导通路[2,5]，目前的研究仍为完全明确其重要的分子调控机制。1892年，有学者提出了肿瘤胚胎性起源的概念[6]，肿瘤生长、侵袭、转移的生物学行为与胚泡在子宫内膜的着床过程非常相似。因此，对胚泡着床分子机制的研究，我们可以从研究癌症相关基因作为切入点。Maspin基因是近年来发现的一种抑癌基因，它可以通过促进肿瘤细胞的凋亡，增加细胞之间以及细胞与胞外基质间的粘附来抑制肿瘤的生长和转移[7-8]。胚胎滋养层细胞在子宫内膜的侵入与肿瘤浸润十分相似，但肿瘤的浸润是没有节制地无限扩张，而胚胎植入是生理性的节制性入侵，Dokras等[9]的研究发现，Maspin在人类胎盘滋养层细胞中有表达。因此我们推测，Maspin基因可能通过调节胚泡与子宫内膜之间的黏附、胚胎滋养细胞的侵入和诱导子宫内膜细胞的凋亡来调控胚泡着床。目的检测未孕和早孕小鼠子宫内膜Maspin mRNA和蛋白的表达规律以及初步研究在胚泡植入过程中，Maspin基因对胚泡着床率的影响，为进一步明确胚泡着床过程中的重要分子机制提供理论和实验依据。方法 1. Maspin mRNA和蛋白表达规律的实验研究在无菌条件下，取未孕和早孕1-7天小鼠的子宫内膜，共8个组，每个实验，每组20只小鼠。荧光定量PCR技术定量检测未孕和早孕小鼠子宫内膜Maspin mRNA的表达规律；免疫组织化学技术（SP法）定位检测Maspin蛋白在未孕及早孕小鼠子宫内膜的阳性表达；Western blotting技术定量检测未孕和早孕小鼠子宫内膜Maspin蛋白的表达规律。 2.在体功能实验研究Maspin基因对胚泡着床率的影响将妊娠第4天的40只小鼠随机平均地分为实验组和对照组，每组20只，进行在体的功能实验。于孕4天上午8AM-9AM进行小鼠子宫角注射，实验组小鼠一侧子宫角注射Maspin抗体，另一侧注射兔源免疫球蛋白G，，对照组小鼠一侧子宫角注射兔源免疫球蛋白G，另一侧不做任何处理。于妊娠第8天上午8AM-9AM观察胚泡着床数的变化，分析Maspin基因对胚泡着床率的影响。结果 1.荧光定量聚合酶链反应(FQ-PCR): Maspin mRNA在未孕和早孕1-7天小鼠的子宫内膜均有表达，未孕组的表达量明显低于早孕组(P0.05)，并且随着妊娠天数的增加，表达量逐渐增加，妊娠第5天达高峰（p0.05）。 2.免疫组织化学（SP法）：Maspin蛋白在未孕和早孕1-7天小鼠子宫内膜组织中均有表达。子宫内膜中Maspin蛋白的棕黄色阳性表达主要分布于腔上皮和腺上皮细胞的胞浆中，在基质细胞的胞浆中也有少量表达。未孕组Maspin蛋白的棕黄色阳性着色明显弱于早孕各组(P0.05)，孕1-4天棕黄色阳性表达逐渐增强，在孕5天棕黄色阳性着色最强(P0.05)，孕6-7天又逐渐减弱。 3. Western blotting试验: Maspin蛋白在未孕和早孕1-7天子宫内膜均有表达，早孕组表达量明显高于未孕组(P0.05)。Maspin蛋白表达的整体趋势是：随着妊娠天数的增加，表达量逐渐增加，于妊娠第5天表达量最高，孕6-7天又逐渐降低，结果与FQ-PCR检测MaspinmRNA的表达规律一致(P0.05)。 4.在体功能实验：实验组在子宫角注射Maspin抗体后胚泡着床数较阴性对照组明显减少，差异有显著性(P0.05)。结论 Maspin mRNA和蛋白的表达随着妊娠天数的增加，逐渐增强，于孕5天即“着床窗口期”达到最高峰，随后逐渐降低。在体功能实验显示，小鼠胚泡植入前的子宫角注射Maspin多克隆抗体，从而阻断了Maspin在胚泡植入时的作用，妊娠第8天发现小鼠胚泡着床数明显下降。由此，我们推测Maspin可能参与了促进子宫内膜细胞的凋亡和调节胚泡与子宫内膜细胞黏附和侵入的调控。
[Abstract]:background
The implantation of the blastocyst is a complex process of interaction between the activated blastocyst and the receptive endometrium and into the endometrium. The successful implantation of the blastocyst is a sign of the pregnancy of the human and other mammals. The study found that the endometrium of the pregnant woman can only adapt to the implantation of the blastocyst in a very short period of time and the endometrium The period of acceptance is called the "implantation window period" [1]. The blastocyst entering the uterine cavity cannot adhere to and implant the endometrium before or after the "bed window period". When the endometrium is in the "implantation window period", the fertilized egg passes through a series of growth, cleavage, blastocyst formation and deuterohyaline zone, eventually having an invading energy. When the blastocyst is successful, the [2-4]. blastocyst implantation is usually divided into three stages, namely, location, adhesion and invasion. This process involves a complex signal transduction pathway, [2,5], which is involved in many regulatory factors. The current research is still completely clear about the important molecular regulatory mechanism of.1892 years, and some scholars have proposed the possibility of the embryonic origin of the tumor. [6], the biological behavior of tumor growth, invasion and metastasis is very similar to the process of the implantation of the blastocyst in the endometrium. Therefore, the study of the molecular mechanism of the blastocyst implantation is a tumor suppressor gene found in recent years from the study of the cancer related genes as the entry point of the.Maspin gene, which can promote the apoptosis of tumor cells, Increasing the adhesion between cells and between the cells and the extracellular matrix to inhibit the growth and metastasis of the tumor, the invasion of [7-8]. embryo trophoblast cells in the endometrium is very similar to the invasion of the tumor, but the invasion of the tumor is unrestricted, and the implantation of the embryo is a physiological, joint invasion, and the study of the [9], such as Dokras, has found that Maspin It is expressed in human placental trophoblast cells. Therefore, we speculate that the Maspin gene may regulate the implantation of the blastocyst by regulating the adhesion between the blastocyst and the endometrium, the invasion of the embryonic trophoblastic cells and the induction of apoptosis in the endometrium cells.
objective
The expression of Maspin mRNA and protein in the endometrium of unpregnant and early pregnant mice and the preliminary study on the effect of Maspin gene on the implantation rate of the blastocyst in the process of blastocyst implantation provide theoretical and experimental basis for further clarifying the important molecular mechanisms of the blastocyst implantation.
Method
Experimental study on the expression of 1. Maspin mRNA and protein
Under the aseptic condition, the endometrium of the uterus of the mice of 1-7 days of unpregnant and early pregnancy was taken, 8 groups, each experiment, 20 mice in each group. The expression of Maspin mRNA in the endometrium of the unpregnant and early pregnant mice was quantified by fluorescence quantitative PCR technique; the immuno histochemical technique (SP method) was used to detect the positive of the Maspin protein in the endometrium of the unpregnant and early pregnant mice Western Blotting Technology was used to detect the expression of Maspin protein in endometrium of unpregnant and early pregnant mice.
2. in vivo functional experiments, the effect of Maspin gene on blastocyst implantation rate.
The 40 mice of fourth days of pregnancy were randomly divided into experimental group and control group, with 20 mice in each group. The mice were injected into the uterus at 8AM-9AM 4 morning. The mice in the experimental group were injected with Maspin antibody on the corner of uterus, the other side of the rabbit source immunized egg white G was injected, and the rabbit angle of one side of the uterus of the control group was injected into the rabbit source immunity. The globulin G and the other side did not do any treatment. The changes of blastocyst implantation number were observed at eighth 8AM-9AM on the first day of pregnancy, and the effect of Maspin gene on blastocyst implantation rate was analyzed.
Result
1. fluorescence quantitative polymerase chain reaction (FQ-PCR): Maspin mRNA was expressed in the endometrium of mice at 1-7 days of unpregnancy and early pregnancy. The expression of the unpregnant group was significantly lower than that of the early pregnancy group (P0.05), and with the increase of the number of gestation days, the expression increased gradually, and the fifth day of pregnancy reached the high peak (P0.05).
2. the immunohistochemical (SP):Maspin protein was expressed in the endometrium of the mouse endometrium on the 1-7 day of pregnancy and early pregnancy. The brown yellow positive expression of Maspin protein in the endometrium was mainly distributed in the cytoplasm of the cavities and glandular epithelial cells, and a small amount of expression in the cytoplasm of the stromal cells. The brown and yellow positive of the Maspin protein in the unpregnant group was positive. The coloring was significantly weaker than that of the early pregnancy (P0.05). The positive expression of brown yellow in the 1-4 day of pregnancy increased gradually, and the positive staining of brown yellow was the strongest on the 5 day of pregnancy (P0.05), and gradually weakened on the 6-7 day of pregnancy.
3. Western blotting test: the expression of Maspin protein in the endometrium on the 1-7 day of unpregnancy and early pregnancy, the expression of the early pregnancy group was significantly higher than that of the unpregnant group (P0.05), the overall trend of the expression of.Maspin protein was: with the increase of the number of days of pregnancy, the expression increased gradually, the amount of the expression was highest at fifth days of pregnancy, and the 6-7 days of pregnancy gradually decreased, and the result was detected with FQ-PCR examination. The expression of MaspinmRNA was consistent (P0.05).
4. in vivo function experiment: the number of blastocyst implantation in the experimental group was significantly lower than that in the negative control group after injection of Maspin antibody at the uterine horn (P0.05).
conclusion
The expression of Maspin mRNA and protein gradually increased with the increase of the number of gestation days. The "implantation window period" reached its peak at the 5 day of pregnancy, and then gradually decreased. In the body function experiment, the Maspin polyclonal antibody was injected into the horn of the uterus before the implantation of the blastocyst in mice, which blocked the effect of Maspin on the implantation of the blastocyst, and found the mice for eighth days of pregnancy. The number of blastocyst implantation decreased significantly. Thus, we speculate that Maspin may be involved in the regulation of the apoptosis of endometrium cells and the regulation of the adhesion and invasion of the blastocyst and endometrium cells.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R321

【参考文献】