Lat1在妊娠小鼠子宮內(nèi)膜蛻膜化進(jìn)程的功能研究
發(fā)布時(shí)間：2018-07-26 16:27
【摘要】：背景:L-型氨基酸轉(zhuǎn)運(yùn)載體1(lat1)主要負(fù)責(zé)轉(zhuǎn)運(yùn)大的、中性氨基酸,并選擇性的表達(dá)在人胎兒的肝臟、胎盤(pán)、大腦等組織中。胚胎植入和蛻膜化對(duì)成功的妊娠至關(guān)重要。小鼠子宮內(nèi)膜蛻膜化發(fā)生在妊娠D4晚上22:00-24:00的囊胚著床后,圍繞植入胚胎周圍的基質(zhì)細(xì)胞發(fā)生的廣泛的增殖和分化,這一過(guò)程被稱為蛻膜化。母體蛻膜細(xì)胞在母-胎對(duì)話中扮演著多功能作用,如:母-胎免疫耐受和胎盤(pán)發(fā)育等。而研究表明氨基酸在胚胎植入前/后的胚胎和胎盤(pán)發(fā)育中扮演著重要的作用,并且成功的胚胎植入和胎盤(pán)形成需要激活的氨基酸轉(zhuǎn)運(yùn)系統(tǒng),除此之外,在胎盤(pán)形成進(jìn)程中l(wèi)at1在滋養(yǎng)層巨細(xì)胞的侵襲表型中扮演著一定的作用,然而,lat1在胚胎植入時(shí)蛻膜化進(jìn)程的母-胎對(duì)話中與卵巢激素之間的相互作用還有待證明。目的:以妊娠D4-8小鼠為研究對(duì)象,從離體、體內(nèi)兩方面研究lat1在小鼠子宮內(nèi)膜蛻膜化過(guò)程中所扮演的作用。方法:1.RT-PCR、免疫組織化學(xué)、Western Blot方法檢測(cè)lat1在妊娠D4-8小鼠子宮中的表達(dá)。2.體外水平建立小鼠子宮內(nèi)膜基質(zhì)細(xì)胞誘導(dǎo)蛻膜化模型,轉(zhuǎn)染lat1過(guò)表達(dá)質(zhì)粒或干擾質(zhì)粒,Western Blot方法檢測(cè)誘導(dǎo)72h時(shí)蛻膜化基質(zhì)細(xì)胞中prl蛋白的表達(dá)變化;亮氨酸轉(zhuǎn)運(yùn)競(jìng)爭(zhēng)性抑制劑BCH(濃度分別為0μM、0.05μM、0.1μM、0.5μM、2μM和4μm)干預(yù)后觀察誘導(dǎo)72h的蛻膜化基質(zhì)細(xì)胞形態(tài)變化,同時(shí)westernblot方法檢測(cè)細(xì)胞prl蛋白的表達(dá)。3.妊娠d4子宮角注射bch(濃度分別為50μg/ml、5μg/ml),對(duì)照分為溶劑1mol/l氨水處理組及空白對(duì)照組,于妊娠d8時(shí),采用he染色法檢測(cè)其對(duì)子宮蛻膜化的形態(tài)學(xué)影響。4.妊娠d4子宮角注射bch(濃度分別為50μg/ml、5μg/ml),對(duì)照分為溶劑1mol/l氨水處理組及空白對(duì)照組,采用免疫組化檢測(cè)其對(duì)妊娠d8子宮植入位點(diǎn)prl表達(dá)區(qū)域及相對(duì)含量的變化;收集d8子宮植入位點(diǎn)組織,westernblot方法檢測(cè)其對(duì)prl蛋白表達(dá)的變化。結(jié)果:1.d4時(shí),lat1主要分布在子宮腔上皮、腺上皮和內(nèi)膜基質(zhì)細(xì)胞的胞質(zhì)中,d5開(kāi)始,lat1在蛻膜基質(zhì)細(xì)胞胞質(zhì)中的表達(dá)水平增加,d6時(shí),lat1主要表達(dá)在初級(jí)蛻膜帶的蛻膜細(xì)胞和胚胎中,d7和d8時(shí),lat1主要定位在次級(jí)蛻膜帶的蛻膜細(xì)胞的胞質(zhì)和胚胎中。lat1mrna和蛋白表達(dá)于妊娠小鼠d4-d8子宮,且在小鼠子宮非植入位點(diǎn)(iis)的表達(dá)水平低于植入位點(diǎn);與d4相比,妊娠d5-d8植入位點(diǎn)處lat1mrna和蛋白表達(dá)水平呈上升趨勢(shì),在d8達(dá)到峰值(p0.05),而非植入位點(diǎn)表達(dá)無(wú)差異。2.westernblot結(jié)果顯示:體外誘導(dǎo)蛻膜化進(jìn)程中轉(zhuǎn)染lat1過(guò)表達(dá)質(zhì)粒后,能促進(jìn)lat1蛋白在蛻膜化基質(zhì)細(xì)胞中表達(dá),同時(shí)能顯著上調(diào)prl蛋白在誘導(dǎo)72h時(shí)蛻膜化基質(zhì)細(xì)胞中的表達(dá)水平;轉(zhuǎn)染篩選后的lat1-sirna1有效干擾質(zhì)粒后,能顯著抑制prl蛋白在誘導(dǎo)72h時(shí)蛻膜化基質(zhì)細(xì)胞中的表達(dá)水平(p0.05)。3.bch處理后,誘導(dǎo)72h時(shí)基質(zhì)細(xì)胞蛻膜化多核化比例降低;westernblot結(jié)果顯示:bch能顯著降低lat1蛋白在誘導(dǎo)72h時(shí)蛻膜化基質(zhì)細(xì)胞中的表達(dá)含量,同時(shí)prl的表達(dá)水平也出現(xiàn)下調(diào)趨勢(shì),并與BCH濃度呈劑量依賴關(guān)系,4μM BCH能顯著抑制prl在蛻膜化基質(zhì)細(xì)胞中的表達(dá)(P0.05)。4.HE染色后顯示,與空白組比較50μg/ml和5μg/ml BCH處理組的妊娠D8子宮植入位點(diǎn)的蛻膜區(qū)域減小(P0.05),而氨水處理組無(wú)明顯變化,但各組之間不同功能蛻膜區(qū)所占比例和胚胎植入數(shù)量無(wú)顯著差異。5.與空白對(duì)照組相比,BCH能降低妊娠D8子宮植入位點(diǎn)prl陽(yáng)性表達(dá)細(xì)胞的積分光密度(P0.05),同時(shí)Western Blot結(jié)果顯示,BCH能有效降低妊娠D8子宮植入位點(diǎn)lat1的表達(dá),prl蛋白在妊娠D8子宮植入位點(diǎn)的表達(dá)水平受到抑制(P0.05),而氨水處理組無(wú)明顯差異。結(jié)論:Lat1在離體與在體水平均可通過(guò)調(diào)控prl表達(dá)水平促進(jìn)小鼠子宮內(nèi)膜蛻膜化進(jìn)程。
[Abstract]:Background: L- type amino acid transporter 1 (LAT1) is mainly responsible for transporting large, neutral amino acids and selectively expressing in human fetal liver, placenta, brain and other tissues. Embryo implantation and decidua are essential for successful pregnancy. Endometrial decidua occurs around the implantation of the blastocyst at 22:00-24:00 in the D4 night of pregnancy and surrounds the implantation. The extensive proliferation and differentiation of stromal cells around the embryo is known as decidua. Maternal decidual cells play a multi-functional role in the mother fetal dialogue, such as maternal fetal immune tolerance and placental development. In addition, LAT1 plays a role in the invasion phenotype of trophoblast giant cells in the process of placental formation. However, the interaction of LAT1 with ovarian hormones in the mother fetal dialogue during the process of deciduating during embryo implantation remains to be proved. The role of LAT1 in endometriosis of endometrium in mice was studied from two aspects in vitro and in vivo. Methods: 1.RT-PCR, immunohistochemistry, Western Blot method was used to detect the expression of LAT1 in the uterus of pregnant D4-8 mice to establish the induced deciduization of endometrial stromal cells in mouse endometrium in vitro. Model, transfection of LAT1 overexpression plasmid or interference plasmid, Western Blot method was used to detect the expression of PRL protein in the deciduating matrix cells induced by 72h, and the leucine transporter competitive inhibitor BCH (concentration of 0 mu M, 0.05 mu M, 0.1 u M, 0.5 mu M, 2 mu M and 4 Mu m) observed the morphological changes of the deciduating matrix cells The lot method was used to detect the expression of cell PRL protein in.3. pregnancy D4 uteri injection BCH (the concentration was 50 mu g/ml, 5 g/ml respectively), the control was divided into the solvent 1mol/l ammonia water treatment group and the blank control group. At the pregnancy D8, the HE staining method was used to detect the morphological effects of the uterus decidua on the.4. pregnancy induced pregnancy uteri angle injection (50 mu, 5 mu respectively). The control group was divided into the solvent 1mol/l ammonia treatment group and the blank control group. The changes in the PRL expression area and relative content of the pregnancy D8 implantation site were detected by immunohistochemistry. The D8 uterus implantation site tissue was collected and the Westernblot method was used to detect the changes in the expression of PRL protein. In 1.d4, LAT1 was mainly distributed on the uterine cavity epithelium and on the gland. In the cytoplasm of skin and endometrial stromal cells, D5 begins, and the expression level of LAT1 in the cytoplasm of decidual stromal cells increases. When D6, LAT1 is mainly expressed in decidual cells and embryos of the primary decidua. When D7 and D8, LAT1 mainly locates in the cytoplasm of the decidual cells in the secondary decidual zone and in the embryo,.Lat1mrna and protein are expressed in the pregnant mice d4-d8. The expression level of the non implantation site (IIS) of the uterus was lower than that of the implantation site. Compared with D4, the expression level of lat1mrna and protein at the site of d5-d8 implantation at pregnancy was on the rise, at the peak of d8 (P0.05), but the non difference.2.westernblot results of non implantation site expression showed that the transfection of LAT1 overexpression plasmid in the process of induced deciduate in vitro After that, it can promote the expression of LAT1 protein in the deciduated stroma cells and up regulate the expression level of PRL protein in the deciduating matrix cells when inducing 72h. After the transfected lat1-sirna1 effectively interferes with the plasmid, it can significantly inhibit the expression level of PRL protein in the deciduating matrix cells (P0.05).3.bch treated with the induced 72h. The deciduation ratio of matrix cell deciduation decreased in 72h, and the results of Westernblot showed that BCH could significantly reduce the expression of LAT1 protein in the deciduating matrix cells induced by 72h, and the expression level of PRL also decreased, and was dose-dependent with the concentration of BCH. The 4 u M BCH could significantly inhibit the PRL in the deciduating matrix cells. After P0.05.4.HE staining, the decidua region of the pregnancy D8 uterus implantation site decreased (P0.05) compared with the blank group in 50 u g/ml and 5 g/ml BCH treatment group, but there was no significant change in the ammonia treatment group, but there was no significant difference in the proportion of the functional decidua and the number of embryo implantation among the groups. The BCH decreased in.5. and in the blank control group. The integral light density (P0.05) of PRL positive cells expressed in the pregnancy D8 implantation site, and Western Blot results showed that BCH could effectively reduce the expression of LAT1 in pregnancy D8 implantation site, and the expression level of PRL protein in the pregnant D8 uterus implantation site was inhibited (P0.05), but there was no significant difference in the ammonia treatment group. Conclusion: Lat1 is in vitro and in vivo. All levels can promote the deciduating process of mouse endometrium by regulating the expression level of PRL.
【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】：2017
【分類號(hào)】：R714.2
本文編號(hào)：2146595
[Abstract]:Background: L- type amino acid transporter 1 (LAT1) is mainly responsible for transporting large, neutral amino acids and selectively expressing in human fetal liver, placenta, brain and other tissues. Embryo implantation and decidua are essential for successful pregnancy. Endometrial decidua occurs around the implantation of the blastocyst at 22:00-24:00 in the D4 night of pregnancy and surrounds the implantation. The extensive proliferation and differentiation of stromal cells around the embryo is known as decidua. Maternal decidual cells play a multi-functional role in the mother fetal dialogue, such as maternal fetal immune tolerance and placental development. In addition, LAT1 plays a role in the invasion phenotype of trophoblast giant cells in the process of placental formation. However, the interaction of LAT1 with ovarian hormones in the mother fetal dialogue during the process of deciduating during embryo implantation remains to be proved. The role of LAT1 in endometriosis of endometrium in mice was studied from two aspects in vitro and in vivo. Methods: 1.RT-PCR, immunohistochemistry, Western Blot method was used to detect the expression of LAT1 in the uterus of pregnant D4-8 mice to establish the induced deciduization of endometrial stromal cells in mouse endometrium in vitro. Model, transfection of LAT1 overexpression plasmid or interference plasmid, Western Blot method was used to detect the expression of PRL protein in the deciduating matrix cells induced by 72h, and the leucine transporter competitive inhibitor BCH (concentration of 0 mu M, 0.05 mu M, 0.1 u M, 0.5 mu M, 2 mu M and 4 Mu m) observed the morphological changes of the deciduating matrix cells The lot method was used to detect the expression of cell PRL protein in.3. pregnancy D4 uteri injection BCH (the concentration was 50 mu g/ml, 5 g/ml respectively), the control was divided into the solvent 1mol/l ammonia water treatment group and the blank control group. At the pregnancy D8, the HE staining method was used to detect the morphological effects of the uterus decidua on the.4. pregnancy induced pregnancy uteri angle injection (50 mu, 5 mu respectively). The control group was divided into the solvent 1mol/l ammonia treatment group and the blank control group. The changes in the PRL expression area and relative content of the pregnancy D8 implantation site were detected by immunohistochemistry. The D8 uterus implantation site tissue was collected and the Westernblot method was used to detect the changes in the expression of PRL protein. In 1.d4, LAT1 was mainly distributed on the uterine cavity epithelium and on the gland. In the cytoplasm of skin and endometrial stromal cells, D5 begins, and the expression level of LAT1 in the cytoplasm of decidual stromal cells increases. When D6, LAT1 is mainly expressed in decidual cells and embryos of the primary decidua. When D7 and D8, LAT1 mainly locates in the cytoplasm of the decidual cells in the secondary decidual zone and in the embryo,.Lat1mrna and protein are expressed in the pregnant mice d4-d8. The expression level of the non implantation site (IIS) of the uterus was lower than that of the implantation site. Compared with D4, the expression level of lat1mrna and protein at the site of d5-d8 implantation at pregnancy was on the rise, at the peak of d8 (P0.05), but the non difference.2.westernblot results of non implantation site expression showed that the transfection of LAT1 overexpression plasmid in the process of induced deciduate in vitro After that, it can promote the expression of LAT1 protein in the deciduated stroma cells and up regulate the expression level of PRL protein in the deciduating matrix cells when inducing 72h. After the transfected lat1-sirna1 effectively interferes with the plasmid, it can significantly inhibit the expression level of PRL protein in the deciduating matrix cells (P0.05).3.bch treated with the induced 72h. The deciduation ratio of matrix cell deciduation decreased in 72h, and the results of Westernblot showed that BCH could significantly reduce the expression of LAT1 protein in the deciduating matrix cells induced by 72h, and the expression level of PRL also decreased, and was dose-dependent with the concentration of BCH. The 4 u M BCH could significantly inhibit the PRL in the deciduating matrix cells. After P0.05.4.HE staining, the decidua region of the pregnancy D8 uterus implantation site decreased (P0.05) compared with the blank group in 50 u g/ml and 5 g/ml BCH treatment group, but there was no significant change in the ammonia treatment group, but there was no significant difference in the proportion of the functional decidua and the number of embryo implantation among the groups. The BCH decreased in.5. and in the blank control group. The integral light density (P0.05) of PRL positive cells expressed in the pregnancy D8 implantation site, and Western Blot results showed that BCH could effectively reduce the expression of LAT1 in pregnancy D8 implantation site, and the expression level of PRL protein in the pregnant D8 uterus implantation site was inhibited (P0.05), but there was no significant difference in the ammonia treatment group. Conclusion: Lat1 is in vitro and in vivo. All levels can promote the deciduating process of mouse endometrium by regulating the expression level of PRL.
【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】：2017
【分類號(hào)】：R714.2
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相關(guān)期刊論文 前2條
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