本文選題：Maspin + 胚泡著床��； 參考：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：背景 胚泡著床是處于活化狀態(tài)的胚泡與處于容受態(tài)的子宮內(nèi)膜相互作用并進(jìn)入子宮內(nèi)膜的復(fù)雜過(guò)程。成功的胚泡著床是人類(lèi)和其他哺乳類(lèi)動(dòng)物妊娠的標(biāo)志。研究發(fā)現(xiàn)，受孕母體的子宮內(nèi)膜只能在一個(gè)極短的時(shí)期內(nèi)適應(yīng)胚泡植入，將內(nèi)膜處于接受狀態(tài)的時(shí)期稱(chēng)為“著床窗口期”[1]，進(jìn)入子宮腔的胚泡在“著床窗口期”之前或之后都不能粘附和植入子宮內(nèi)膜。當(dāng)子宮內(nèi)膜處于“著床窗口期”，同時(shí)受精卵經(jīng)過(guò)一系列生長(zhǎng)發(fā)育、卵裂，胚泡形成和脫透明帶，最終具有侵入能力時(shí)，胚泡才能成功著床[2-4]。胚泡著床一般分為三個(gè)階段，即定位、黏附、侵入，此過(guò)程涉及到眾多調(diào)控因子參與的錯(cuò)綜復(fù)雜的信號(hào)傳導(dǎo)通路[2,5]，目前的研究仍為完全明確其重要的分子調(diào)控機(jī)制。1892年，有學(xué)者提出了腫瘤胚胎性起源的概念[6]，腫瘤生長(zhǎng)、侵襲、轉(zhuǎn)移的生物學(xué)行為與胚泡在子宮內(nèi)膜的著床過(guò)程非常相似。因此，對(duì)胚泡著床分子機(jī)制的研究，我們可以從研究癌癥相關(guān)基因作為切入點(diǎn)。Maspin基因是近年來(lái)發(fā)現(xiàn)的一種抑癌基因，它可以通過(guò)促進(jìn)腫瘤細(xì)胞的凋亡，增加細(xì)胞之間以及細(xì)胞與胞外基質(zhì)間的粘附來(lái)抑制腫瘤的生長(zhǎng)和轉(zhuǎn)移[7-8]。胚胎滋養(yǎng)層細(xì)胞在子宮內(nèi)膜的侵入與腫瘤浸潤(rùn)十分相似，但腫瘤的浸潤(rùn)是沒(méi)有節(jié)制地?zé)o限擴(kuò)張，而胚胎植入是生理性的節(jié)制性入侵，Dokras等[9]的研究發(fā)現(xiàn)，Maspin在人類(lèi)胎盤(pán)滋養(yǎng)層細(xì)胞中有表達(dá)。因此我們推測(cè)，Maspin基因可能通過(guò)調(diào)節(jié)胚泡與子宮內(nèi)膜之間的黏附、胚胎滋養(yǎng)細(xì)胞的侵入和誘導(dǎo)子宮內(nèi)膜細(xì)胞的凋亡來(lái)調(diào)控胚泡著床。 目的 檢測(cè)未孕和早孕小鼠子宮內(nèi)膜Maspin mRNA和蛋白的表達(dá)規(guī)律以及初步研究在胚泡植入過(guò)程中，Maspin基因?qū)ε吲葜猜实挠绊�，為進(jìn)一步明確胚泡著床過(guò)程中的重要分子機(jī)制提供理論和實(shí)驗(yàn)依據(jù)。 方法 1. Maspin mRNA和蛋白表達(dá)規(guī)律的實(shí)驗(yàn)研究 在無(wú)菌條件下，取未孕和早孕1-7天小鼠的子宮內(nèi)膜，共8個(gè)組，每個(gè)實(shí)驗(yàn)，每組20只小鼠。熒光定量PCR技術(shù)定量檢測(cè)未孕和早孕小鼠子宮內(nèi)膜Maspin mRNA的表達(dá)規(guī)律；免疫組織化學(xué)技術(shù)（SP法）定位檢測(cè)Maspin蛋白在未孕及早孕小鼠子宮內(nèi)膜的陽(yáng)性表達(dá)；Western blotting技術(shù)定量檢測(cè)未孕和早孕小鼠子宮內(nèi)膜Maspin蛋白的表達(dá)規(guī)律。 2.在體功能實(shí)驗(yàn)研究Maspin基因?qū)ε吲葜猜实挠绊?將妊娠第4天的40只小鼠隨機(jī)平均地分為實(shí)驗(yàn)組和對(duì)照組，每組20只，進(jìn)行在體的功能實(shí)驗(yàn)。于孕4天上午8AM-9AM進(jìn)行小鼠子宮角注射，實(shí)驗(yàn)組小鼠一側(cè)子宮角注射Maspin抗體，另一側(cè)注射兔源免疫球蛋白G，，對(duì)照組小鼠一側(cè)子宮角注射兔源免疫球蛋白G，另一側(cè)不做任何處理。于妊娠第8天上午8AM-9AM觀察胚泡著床數(shù)的變化，分析Maspin基因?qū)ε吲葜猜实挠绊憽?結(jié)果 1.熒光定量聚合酶鏈反應(yīng)(FQ-PCR): Maspin mRNA在未孕和早孕1-7天小鼠的子宮內(nèi)膜均有表達(dá)，未孕組的表達(dá)量明顯低于早孕組(P0.05)，并且隨著妊娠天數(shù)的增加，表達(dá)量逐漸增加，妊娠第5天達(dá)高峰（p0.05）。 2.免疫組織化學(xué)（SP法）：Maspin蛋白在未孕和早孕1-7天小鼠子宮內(nèi)膜組織中均有表達(dá)。子宮內(nèi)膜中Maspin蛋白的棕黃色陽(yáng)性表達(dá)主要分布于腔上皮和腺上皮細(xì)胞的胞漿中，在基質(zhì)細(xì)胞的胞漿中也有少量表達(dá)。未孕組Maspin蛋白的棕黃色陽(yáng)性著色明顯弱于早孕各組(P0.05)，孕1-4天棕黃色陽(yáng)性表達(dá)逐漸增強(qiáng)，在孕5天棕黃色陽(yáng)性著色最強(qiáng)(P0.05)，孕6-7天又逐漸減弱。 3. Western blotting試驗(yàn): Maspin蛋白在未孕和早孕1-7天子宮內(nèi)膜均有表達(dá)，早孕組表達(dá)量明顯高于未孕組(P0.05)。Maspin蛋白表達(dá)的整體趨勢(shì)是：隨著妊娠天數(shù)的增加，表達(dá)量逐漸增加，于妊娠第5天表達(dá)量最高，孕6-7天又逐漸降低，結(jié)果與FQ-PCR檢測(cè)MaspinmRNA的表達(dá)規(guī)律一致(P0.05)。 4.在體功能實(shí)驗(yàn)：實(shí)驗(yàn)組在子宮角注射Maspin抗體后胚泡著床數(shù)較陰性對(duì)照組明顯減少，差異有顯著性(P0.05)。 結(jié)論 Maspin mRNA和蛋白的表達(dá)隨著妊娠天數(shù)的增加，逐漸增強(qiáng)，于孕5天即“著床窗口期”達(dá)到最高峰，隨后逐漸降低。在體功能實(shí)驗(yàn)顯示，小鼠胚泡植入前的子宮角注射Maspin多克隆抗體，從而阻斷了Maspin在胚泡植入時(shí)的作用，妊娠第8天發(fā)現(xiàn)小鼠胚泡著床數(shù)明顯下降。由此，我們推測(cè)Maspin可能參與了促進(jìn)子宮內(nèi)膜細(xì)胞的凋亡和調(diào)節(jié)胚泡與子宮內(nèi)膜細(xì)胞黏附和侵入的調(diào)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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R321

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王麗,周劍萍,張煒,劉銀坤;小鼠容受性子宮內(nèi)膜上皮細(xì)胞和基質(zhì)細(xì)胞的共培養(yǎng)[J];生殖與避孕;2002年05期

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