PPARγ对胎盘滋养层细胞氨基酸转运体的调节及机制研究
发布时间：2018-08-08 13:22
【摘要】：妊娠期胎盘是联系母体和胎儿之间的枢纽，胎儿在子宫内能否正常生长取决于跨胎盘营养物质转运能否顺利进行。而胎盘的转运功能异常常会导致一系列病理性妊娠的发生，对于母体和胎儿都有着极大的危害。氨基酸是妊娠期胎儿生长必不可少的营养物质，，而胎盘细胞中氨基酸转运体的功能对于胎儿生长发育更是至关重要。很多研究表明在妊娠合并胎儿宫内发育迟缓（Intrauterine Growth Retardation，IUGR）胎盘组织中A系、L系及TAUT氨基酸转运体的活性显著降低，且A系氨基酸转运体活性同胎儿出生体重呈现正相关的关系，这提示胎盘细胞中三系氨基酸转运体的功能确实同宫内胎儿的生长发育密切相关。 PPARγ是PPARs的一个亚型，属于核受体超家族。许多研究均表明，PPARγ在人胎盘滋养层细胞的发育成熟及正常妊娠的维持过程中发挥着重要的作用，而PPARγ功能异常被认为和许多病理性妊娠都密切相关。最近，Marta Díaz等人的报道显示PPARγ受体在小于胎龄儿患者胎盘组织表明显著减少，且表达水平同胎儿出生体重、胎盘重量呈现正相关的关系。那么在人胎盘滋养层细胞中，PPARγ受体对三系氨基酸转运体各亚型的表达有无调控作用呢？为了阐明这一问题，我们进行了本课题的研究。 我们以原代培养的人胎盘滋养层细胞为研究对象，利用PPARγ受体的激动剂罗格列酮及拮抗剂GW9662进行处理，明确了PPARγ受体调控三系氨基酸转运体各亚型表达的现象；然后，本文利用siRNA干扰技术证明了罗格列酮调控氨基酸转运体表达的过程是否通过PPARγ受体及RXR实现；接下来，本文利用荧光素酶报告基因及染色质免疫共沉淀的技术探究了PPARγ调控三种氨基酸转运体表达的具体机制；最后我们从临床收集了胎盘组织样本，检测了不同出身体重胎儿对应胎盘组织中三种氨基酸转运体及PPARγ受体的表达情况。从而从细胞核组织水平探讨了PPARγ受体及氨基酸转运体在妊娠期与胎儿生长发育间可能的联系。 主要实验结果如下： 1、PPAR γ对胎盘3系氨基酸转运体各亚型表达的调控作用 采用原代培养的人胎盘滋养细胞48小时后，给予PPAR γ特异性激动剂罗格列酮处理细胞，利用realtime-PCR和Western blot方法检测细胞中ATA1、ATA2、ATA4、LAT1、LAT2、TAUT mRNA及蛋白的表达情况。结果显示，罗格列酮可时间及剂量依赖性上调LAT1、LAT2、TAUT的表达，而对ATA1、ATA2、ATA4表达无明显的调节作用。 2、PPAR γ调节胎盘LAT1、LAT2、TAUT表达的机制研究 1、分别给予滋养层细胞PPAR γ受体拮抗剂GW9662、天然配体15d-PGJ2、高选择激动剂GW1929处理细胞，结果15d-PGJ2及GW1929均可上调LAT1、LAT2、TAUT表达，GW9662可以逆转罗格列酮的上调效应；利用干扰技术敲低PPAR γ表达同样可以逆转罗格列酮的上调效应。这些结果提示罗格列酮上调LAT1、LAT2、TAUT表达是通过PPAR γ受体实现的。 2、利用干扰技术敲低RXRa表达后，罗格列酮上调LAT1、LAT2、TAUT表达的现象也被逆转了，提示罗格列酮上调LAT1、LAT2、TAUT表达的过程需要RXRa的参与。 3、利用蛋白合成抑制剂放线菌酮处理细胞，结果显示，罗格列酮上调LAT1、TAUT的过程需要合成新的蛋白，而罗格列酮上调LAT2mRNA的效应则不受CHX的影响。 4、利用SP-1抑制剂及干扰技术处理细胞，结果显示SP-1抑制剂处理或者敲低SP-1表达均可逆转罗格列酮上调LAT1、TAUT表达的现象，提示罗格列酮通过PPARγ受体上调LAT1、TAUT表达的过程需要SP1的参与。 5、利用染色质免疫共沉淀的实验证实罗格列酮可促进SP-1与LAT1、TAUT基因启动子区结合，这提示罗格列酮通过促进SP1表达及其与基因启动子区结合，进而发挥上调LAT1、TAUT表达的作用的。 3、小于胎龄儿、适于胎龄儿、大于胎龄儿胎盘中LAT1、LAT2、TAUT及PPARγ的表达 为了在组织水平验证小于胎龄儿、适于胎龄儿、大于胎龄儿胎盘组织中LAT1、LAT2、TAUT及PPAR γ的蛋白表达量是否存在差异，我们在临床随机收集了小于胎龄儿（15例）、适于胎龄儿（15例）、大于胎龄儿（14例）胎盘组织，利用Western blot方法检测LAT1、LAT2、TAUT及PPARγ的表达，然后进行了统计学分析。结果显示，与AGA及LGA组相比，SGA组LAT1、LAT2及PPARγ的蛋白表达显著降低，而TAUT则无显著差异；LAT1、LAT2、PPARγ的表达量与胎儿的出生体重均呈现正相关的关系，TAUT则无明显相关性。且LAT1、LAT2及TAUT蛋白的表达同PPARγ表达呈正相关。 结论： 罗格列酮可以上调LAT1、LAT2及TAUT表达；罗格列酮上调LAT1、LAT2及TAUT表达是通过PPAR γ实现的，且需要RXRα的参与；罗格列酮通过促进SP1表达及其与基因启动子区结合，进而发挥上调LAT1、TAUT表达的作用；罗格列酮激活PPAR γ后可直接促进LAT2基因的转录；人胎盘组织中LAT1、LAT2及PPAR γ表达降低可能是导致小于胎龄儿的原因之一。
[Abstract]:The placenta in pregnancy is the junction between the mother and the fetus. The normal growth of the fetus in the uterus depends on whether the transplacental nutrient transport can be carried out smoothly. The abnormal transfer function of the placenta often leads to a series of pathological pregnancies, which have great harm to both the mother and the fetus. The function of amino acid transporters in placental cells is essential for fetal growth and development. Many studies have shown that the activity of A, L and TAUT amino acid transporters in the placental tissue of Intrauterine Growth Retardation (IUGR) is significantly reduced, and the amino group of A is amino acid. The relationship between the activity of acid transporter and fetal birth weight has a positive correlation, which suggests that the function of the three line amino acid transporter in the placental cells is closely related to the growth and development of the intrauterine fetus.
PPAR gamma is a subtype of PPARs and belongs to the nuclear receptor superfamily. Many studies have shown that PPAR gamma plays an important role in the development of human placental trophoblast cells and the maintenance of normal pregnancy. The abnormal PPAR gamma function is considered to be closely related to many pathological gestation. Recently, the reports of Marta D AZ and others show PPAR gamma The expression of PPAR gamma receptor in human placental trophoblast cells has a positive correlation with fetal birth weight and placental weight. So, in human placental trophoblast cells, is there a regulation effect on the expression of the subtypes of three series amino acid transporters in human placental trophoblast cells? To clarify this problem, we have done this lesson The study of the problem.
We use the original cultured human placental trophoblast cells as the research object, using the PPAR gamma receptor agonist rosiglitazone and antagonist GW9662 to clarify the expression of PPAR gamma receptor in the regulation of the expression of each subtype of the three line amino acid transporter. Then, this paper uses siRNA interference technique to prove that rosiglitazone regulates the amino acid transport body surface. Whether the process is realized by PPAR gamma receptor and RXR; then, this paper uses the luciferase reporter gene and chromatin immunoprecipitation technique to explore the specific mechanism of PPAR gamma regulation of the expression of three amino acid transporters. Finally, we collected samples of placental tissue from the clinic and detected the placental group of different body weight fetus. The expression of three amino acid transporters and PPAR gamma receptors in the weave, thus the possible relationship between the PPAR gamma receptor and the amino acid transporter in pregnancy and the growth and development of the fetus was discussed from the level of nuclear tissue.
The main experimental results are as follows:
1, PPAR gamma regulates the expression of 3 subtypes of placental amino acid transporters in placenta.
After 48 hours of primary cultured human placental trophoblast, PPAR gamma specific agonist rosiglitazone was used to treat cells. The expression of ATA1, ATA2, ATA4, LAT1, LAT2, TAUT mRNA and protein in cells was detected by realtime-PCR and Western blot. The expression of ATA1, ATA2 and ATA4 was not significantly regulated.
2, the mechanism of PPAR gamma regulating the expression of LAT1, LAT2 and TAUT in placenta
1, PPAR gamma receptor antagonist GW9662 of trophoblast cells, natural ligand 15d-PGJ2 and high selective agonist GW1929 are used to process cells. Results 15d-PGJ2 and GW1929 can up-regulation the expression of LAT1, LAT2, TAUT, and GW9662 can reverse the up-regulation effect of rosiglitazone; using interference technique to knock low PPAR gamma expression can also reverse the up-regulation effect of rosiglitazone. These results suggest that rosiglitazone upregulated LAT1, LAT2 and TAUT expression through PPAR gamma receptor.
2, using interference technique to knock down RXRa expression, rosiglitazone up-regulated LAT1, LAT2, and TAUT expression also reversed, suggesting that rosiglitazone up-regulated LAT1, LAT2, TAUT expression needs RXRa participation.
3, using the protein synthesis inhibitor actinomycone treatment cells, the results showed that rosiglitazone up regulation of LAT1, TAUT process needs to synthesize new proteins, while rosiglitazone up the effect of LAT2mRNA is not affected by CHX.
4, the cells were treated with SP-1 inhibitors and interference techniques. The results showed that SP-1 inhibitor treatment or low SP-1 expression could reverse the up-regulation of rosiglitazone on LAT1, TAUT expression, suggesting that rosiglitazone up-regulated LAT1 through PPAR gamma receptor. The process of TAUT expression requires the participation of SP1.
5, using chromatin immunoprecipitation experiments confirmed that rosiglitazone can promote the binding of SP-1 and LAT1, TAUT gene promoter region, which suggests that rosiglitazone promotes the expression of LAT1 and TAUT by promoting the expression of SP1 and its binding with the promoter region of the gene.
3, small for gestational age, suitable for gestational age, LAT1, LAT2, TAUT and PPAR gamma expression in placenta of large gestational age infants.
In order to verify the difference in the protein expression of LAT1, LAT2, TAUT and PPAR gamma in placenta tissues of fetal age infants at tissue level, we collected less than gestational age infants (15 cases) at random, suitable for gestational age children (15 cases), greater than fetal age (14 cases) placenta tissue, and LAT1, LAT by Western blot method. 2, the expression of TAUT and PPAR gamma, and then the statistical analysis. The results showed that the protein expression of LAT1, LAT2 and PPAR y in SGA group was significantly lower than that of AGA and LGA group, but TAUT was not significantly different. LAT1, LAT2, PPAR gamma expression was positively related to the birth weight of the fetus. The expression of white was positively correlated with the expression of PPAR gamma.
Conclusion:
Rosiglitazone can up regulate the expression of LAT1, LAT2 and TAUT; rosiglitazone up-regulated LAT1, LAT2 and TAUT expression is realized through PPAR gamma, and requires the participation of RXR a; rosiglitazone plays the role of up regulation of LAT1, TAUT expression by promoting SP1 expression and its combination with the promoter region of the gene; rosiglitazone can directly promote the activation of PPAR gamma after activating PPAR gamma. 2 the transcription of genes, the decrease of LAT1, LAT2 and PPAR gamma expression in human placenta may be one of the causes of small for gestational age infants.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714
本文编号：2171906
[Abstract]:The placenta in pregnancy is the junction between the mother and the fetus. The normal growth of the fetus in the uterus depends on whether the transplacental nutrient transport can be carried out smoothly. The abnormal transfer function of the placenta often leads to a series of pathological pregnancies, which have great harm to both the mother and the fetus. The function of amino acid transporters in placental cells is essential for fetal growth and development. Many studies have shown that the activity of A, L and TAUT amino acid transporters in the placental tissue of Intrauterine Growth Retardation (IUGR) is significantly reduced, and the amino group of A is amino acid. The relationship between the activity of acid transporter and fetal birth weight has a positive correlation, which suggests that the function of the three line amino acid transporter in the placental cells is closely related to the growth and development of the intrauterine fetus.
PPAR gamma is a subtype of PPARs and belongs to the nuclear receptor superfamily. Many studies have shown that PPAR gamma plays an important role in the development of human placental trophoblast cells and the maintenance of normal pregnancy. The abnormal PPAR gamma function is considered to be closely related to many pathological gestation. Recently, the reports of Marta D AZ and others show PPAR gamma The expression of PPAR gamma receptor in human placental trophoblast cells has a positive correlation with fetal birth weight and placental weight. So, in human placental trophoblast cells, is there a regulation effect on the expression of the subtypes of three series amino acid transporters in human placental trophoblast cells? To clarify this problem, we have done this lesson The study of the problem.
We use the original cultured human placental trophoblast cells as the research object, using the PPAR gamma receptor agonist rosiglitazone and antagonist GW9662 to clarify the expression of PPAR gamma receptor in the regulation of the expression of each subtype of the three line amino acid transporter. Then, this paper uses siRNA interference technique to prove that rosiglitazone regulates the amino acid transport body surface. Whether the process is realized by PPAR gamma receptor and RXR; then, this paper uses the luciferase reporter gene and chromatin immunoprecipitation technique to explore the specific mechanism of PPAR gamma regulation of the expression of three amino acid transporters. Finally, we collected samples of placental tissue from the clinic and detected the placental group of different body weight fetus. The expression of three amino acid transporters and PPAR gamma receptors in the weave, thus the possible relationship between the PPAR gamma receptor and the amino acid transporter in pregnancy and the growth and development of the fetus was discussed from the level of nuclear tissue.
The main experimental results are as follows:
1, PPAR gamma regulates the expression of 3 subtypes of placental amino acid transporters in placenta.
After 48 hours of primary cultured human placental trophoblast, PPAR gamma specific agonist rosiglitazone was used to treat cells. The expression of ATA1, ATA2, ATA4, LAT1, LAT2, TAUT mRNA and protein in cells was detected by realtime-PCR and Western blot. The expression of ATA1, ATA2 and ATA4 was not significantly regulated.
2, the mechanism of PPAR gamma regulating the expression of LAT1, LAT2 and TAUT in placenta
1, PPAR gamma receptor antagonist GW9662 of trophoblast cells, natural ligand 15d-PGJ2 and high selective agonist GW1929 are used to process cells. Results 15d-PGJ2 and GW1929 can up-regulation the expression of LAT1, LAT2, TAUT, and GW9662 can reverse the up-regulation effect of rosiglitazone; using interference technique to knock low PPAR gamma expression can also reverse the up-regulation effect of rosiglitazone. These results suggest that rosiglitazone upregulated LAT1, LAT2 and TAUT expression through PPAR gamma receptor.
2, using interference technique to knock down RXRa expression, rosiglitazone up-regulated LAT1, LAT2, and TAUT expression also reversed, suggesting that rosiglitazone up-regulated LAT1, LAT2, TAUT expression needs RXRa participation.
3, using the protein synthesis inhibitor actinomycone treatment cells, the results showed that rosiglitazone up regulation of LAT1, TAUT process needs to synthesize new proteins, while rosiglitazone up the effect of LAT2mRNA is not affected by CHX.
4, the cells were treated with SP-1 inhibitors and interference techniques. The results showed that SP-1 inhibitor treatment or low SP-1 expression could reverse the up-regulation of rosiglitazone on LAT1, TAUT expression, suggesting that rosiglitazone up-regulated LAT1 through PPAR gamma receptor. The process of TAUT expression requires the participation of SP1.
5, using chromatin immunoprecipitation experiments confirmed that rosiglitazone can promote the binding of SP-1 and LAT1, TAUT gene promoter region, which suggests that rosiglitazone promotes the expression of LAT1 and TAUT by promoting the expression of SP1 and its binding with the promoter region of the gene.
3, small for gestational age, suitable for gestational age, LAT1, LAT2, TAUT and PPAR gamma expression in placenta of large gestational age infants.
In order to verify the difference in the protein expression of LAT1, LAT2, TAUT and PPAR gamma in placenta tissues of fetal age infants at tissue level, we collected less than gestational age infants (15 cases) at random, suitable for gestational age children (15 cases), greater than fetal age (14 cases) placenta tissue, and LAT1, LAT by Western blot method. 2, the expression of TAUT and PPAR gamma, and then the statistical analysis. The results showed that the protein expression of LAT1, LAT2 and PPAR y in SGA group was significantly lower than that of AGA and LGA group, but TAUT was not significantly different. LAT1, LAT2, PPAR gamma expression was positively related to the birth weight of the fetus. The expression of white was positively correlated with the expression of PPAR gamma.
Conclusion:
Rosiglitazone can up regulate the expression of LAT1, LAT2 and TAUT; rosiglitazone up-regulated LAT1, LAT2 and TAUT expression is realized through PPAR gamma, and requires the participation of RXR a; rosiglitazone plays the role of up regulation of LAT1, TAUT expression by promoting SP1 expression and its combination with the promoter region of the gene; rosiglitazone can directly promote the activation of PPAR gamma after activating PPAR gamma. 2 the transcription of genes, the decrease of LAT1, LAT2 and PPAR gamma expression in human placenta may be one of the causes of small for gestational age infants.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714
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