Lat1在妊娠小鼠子宫内膜蜕膜化进程的功能研究
发布时间：2018-07-26 16:27
【摘要】：背景:L-型氨基酸转运载体1(lat1)主要负责转运大的、中性氨基酸,并选择性的表达在人胎儿的肝脏、胎盘、大脑等组织中。胚胎植入和蜕膜化对成功的妊娠至关重要。小鼠子宫内膜蜕膜化发生在妊娠D4晚上22:00-24:00的囊胚着床后,围绕植入胚胎周围的基质细胞发生的广泛的增殖和分化,这一过程被称为蜕膜化。母体蜕膜细胞在母-胎对话中扮演着多功能作用,如:母-胎免疫耐受和胎盘发育等。而研究表明氨基酸在胚胎植入前/后的胚胎和胎盘发育中扮演着重要的作用,并且成功的胚胎植入和胎盘形成需要激活的氨基酸转运系统,除此之外,在胎盘形成进程中lat1在滋养层巨细胞的侵袭表型中扮演着一定的作用,然而,lat1在胚胎植入时蜕膜化进程的母-胎对话中与卵巢激素之间的相互作用还有待证明。目的:以妊娠D4-8小鼠为研究对象,从离体、体内两方面研究lat1在小鼠子宫内膜蜕膜化过程中所扮演的作用。方法:1.RT-PCR、免疫组织化学、Western Blot方法检测lat1在妊娠D4-8小鼠子宫中的表达。2.体外水平建立小鼠子宫内膜基质细胞诱导蜕膜化模型,转染lat1过表达质粒或干扰质粒,Western Blot方法检测诱导72h时蜕膜化基质细胞中prl蛋白的表达变化;亮氨酸转运竞争性抑制剂BCH(浓度分别为0μM、0.05μM、0.1μM、0.5μM、2μM和4μm)干预后观察诱导72h的蜕膜化基质细胞形态变化,同时westernblot方法检测细胞prl蛋白的表达。3.妊娠d4子宫角注射bch(浓度分别为50μg/ml、5μg/ml),对照分为溶剂1mol/l氨水处理组及空白对照组,于妊娠d8时,采用he染色法检测其对子宫蜕膜化的形态学影响。4.妊娠d4子宫角注射bch(浓度分别为50μg/ml、5μg/ml),对照分为溶剂1mol/l氨水处理组及空白对照组,采用免疫组化检测其对妊娠d8子宫植入位点prl表达区域及相对含量的变化;收集d8子宫植入位点组织,westernblot方法检测其对prl蛋白表达的变化。结果:1.d4时,lat1主要分布在子宫腔上皮、腺上皮和内膜基质细胞的胞质中,d5开始,lat1在蜕膜基质细胞胞质中的表达水平增加,d6时,lat1主要表达在初级蜕膜带的蜕膜细胞和胚胎中,d7和d8时,lat1主要定位在次级蜕膜带的蜕膜细胞的胞质和胚胎中。lat1mrna和蛋白表达于妊娠小鼠d4-d8子宫,且在小鼠子宫非植入位点(iis)的表达水平低于植入位点;与d4相比,妊娠d5-d8植入位点处lat1mrna和蛋白表达水平呈上升趋势,在d8达到峰值(p0.05),而非植入位点表达无差异。2.westernblot结果显示:体外诱导蜕膜化进程中转染lat1过表达质粒后,能促进lat1蛋白在蜕膜化基质细胞中表达,同时能显著上调prl蛋白在诱导72h时蜕膜化基质细胞中的表达水平;转染筛选后的lat1-sirna1有效干扰质粒后,能显著抑制prl蛋白在诱导72h时蜕膜化基质细胞中的表达水平(p0.05)。3.bch处理后,诱导72h时基质细胞蜕膜化多核化比例降低;westernblot结果显示:bch能显著降低lat1蛋白在诱导72h时蜕膜化基质细胞中的表达含量,同时prl的表达水平也出现下调趋势,并与BCH浓度呈剂量依赖关系,4μM BCH能显著抑制prl在蜕膜化基质细胞中的表达(P0.05)。4.HE染色后显示,与空白组比较50μg/ml和5μg/ml BCH处理组的妊娠D8子宫植入位点的蜕膜区域减小(P0.05),而氨水处理组无明显变化,但各组之间不同功能蜕膜区所占比例和胚胎植入数量无显著差异。5.与空白对照组相比,BCH能降低妊娠D8子宫植入位点prl阳性表达细胞的积分光密度(P0.05),同时Western Blot结果显示,BCH能有效降低妊娠D8子宫植入位点lat1的表达,prl蛋白在妊娠D8子宫植入位点的表达水平受到抑制(P0.05),而氨水处理组无明显差异。结论:Lat1在离体与在体水平均可通过调控prl表达水平促进小鼠子宫内膜蜕膜化进程。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.2
本文编号：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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.2
【参考文献】
相关期刊论文 前2条
1 张晋平;罗文萍;张倩;彭洪英;谭冬梅;王应雄;谭毅;;肿瘤坏死因子相关的凋亡诱导配体(TRAIL)基因抑制小鼠子宫基质细胞的蜕膜化[J];生物化学与生物物理进展;2010年03期
2 张婧婧;侯丽辉;丛晶;李威;匡洪影;;子宫内膜蜕膜化的分子细胞学机制[J];医学研究杂志;2009年10期
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