miR-375过表达引起胰腺损伤的初步研究
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摘要
microRNA (miRNA,即微小RNA),是体内22nt的一类非编码的RNA,由miRNA的前体经Dicer酶的作用加工而来。在细胞的生长、增殖、代谢、调亡等方面都有着重要的作用。它可以通过与特定mRNA结合,来调节特定mRNA的蛋白质翻译过程,从而调控基因表达。目前人类基因组中已确认的miRNA有上千个,可能参与30%人类蛋白质的表达调控。随着许多新的miRNA及其功能的不断发现,使得miRNA的研究倍受关注,成为生命科学最热门的研究之一。
研究有关miRNA功能的文献有很多,但大部分是在细胞水平的研究,而只在细胞水平研究miRNA功能是远远不够的,因为miRNA功能最终还得在整个机体中发挥作用。在整体水平对miRNA功能的研究,目前国内外都很少。miRNA在胰腺发育和功能的维持中起着非常重要的作用,miR-375是在胰腺中特异表达的miRNA之一,它的正常表达与否对胰腺的形态、发育和功能也至关重要。有关研究表明,miR-375除了对MTPN表达的调控外,还对AdipoR2的表达有调控作用。为了研究miR-375是如何影响胰腺发育和胰腺的功能,探讨miR-375在胰腺发育过程中的作用机理,本实验采用显微注射的方法,利用胰腺内特异表达的启动子RIP(rat insulin premptor)带上编码miR-375的基因,构建miR-375过表达的转基因小鼠模型,旨在研究由miR-375过表达而引起的MTPN、AdipoR2的变化,从而引起胰腺损伤的机理。本研究共分三部分,其主要的实验方法及实验结果如下：
第一章miR-375基因的克隆和功能的初步分析
本章构建了一个含有miR-375基因序列的质粒pAAV-MCS-miR-375,用pAAV-MCS-miR-375转染Hela细胞48小时后,RT-PCR检测发现转染质粒的Hela细胞有miR-375前体的表达。用pAAV-MCS-miR-375转染Nit-1细胞48小时后,Western Blot检测miR-375过表达对Nit-1细胞中MTPN蛋白表达的调节,结果显示,转染pAAV-MCS-miR-37548小时后的Nit-1细胞中MTPN的表达,与转染pAAV-MCS和未转染的Nit-1细胞中MTPN的表达相比有略微下调。为进一步证实miR-375对MTPN表达的作用,我们用pAAV-MCS-miR-375转染Nit-1细胞48小时后,做免疫细胞化学分析。结果发现,转染pAAV-MCS-miR-37548小时后的Nit-1细胞中MTPN蛋白表达比转染pAAV-MCS和未转染的Nit-1细胞中MTPN蛋白表达下调。本研究还测定了未转染、转染pAAV-MCS-miR-375和转染pAAV-MCS的Nit-1细胞中葡萄糖刺激的胰岛素分泌。结果发现转染pAAV-MCS-miR-37548小时后的Nit-1细胞中胰岛素的分泌比未转染和转染pAAV-MCS的Nit-1细胞中胰岛素的分泌下调。这说明我们构建的质粒不但能生成成熟的miR-375,并且能抑制目的Mytrophin基因的表达。本实验成功的克隆了miR-375基因。所构建的表达miR-375的质粒pAAV-MCS-miR-375在细胞中能够表达miR-375、能够抑制MTPN的表达使得细胞受葡萄糖刺激分泌胰岛素的能力下降。
第二章转基因小鼠的构建
本实验采用显微注射法,利用胰腺内特异表达的启动子RIP(rat insulin premptor)带上编码miR-375的基因和人生长激素基因的Poly(A),构建miR-375过表达的转基因小鼠模型。通过PCR、Slot Blot、Southern Blot鉴定阳性转基因小鼠。转基因小鼠胰腺中miR-375的表达采用RT-PCR和real-time PCR测定,转基因小鼠胰腺中myotrophin(MTPN)的表达采用real-time PCR和Western Blot测定。结果发现,转基因小鼠胰腺中成熟miR-375的表达是正常小鼠的1.15倍；转基因小鼠胰腺中myotrophin(MTPN)的表达是正常小鼠的1／7.7。转基因小鼠与正常小鼠的体重和血糖测定发现,与正常同类小鼠相比,雌性转基因鼠的体重有明显下降,雄性转基因鼠的体重无变化,转基因鼠的血糖无明显变化。糖耐量实验中雌性转基因鼠在30-60分钟段血糖恢复比正常小鼠快。转基因小鼠空腹血清胰岛素含量用Linco Research公司的RAT/MOUSE INSULIN ELISA KIT测定。结果显示,与正常同类小鼠相比,雌性转基因鼠血清胰岛素含量低34.3%、有显著差异,雄性转基因鼠的血清胰岛素含量无明显变化。在对转基因小鼠进行细胞学检测时,未发现转基因小鼠存在染色体数量或结构上的改变,而RT-PCR检测与胰腺发育相关基因时发现,雌性转基因小鼠县腺中PDX-1、INS1、ISL-1、PAX6、GLK、GLG、PP的表达上升。用胰岛素抗体和胰高血糖素抗体做免疫荧光发现,转基因鼠胰腺中胰岛素和胰高血糖素的合成不受影响。对转基因鼠胰腺做病理切片HE染色发现,在胰腺中胰岛的周围有很多空泡,出现脂肪变性,另外胰腺中的胰岛还显著萎缩。对转基因鼠肺做病理H／E染色发现,肺中有严重的支气管肺炎、支气管坏死和中性淋巴细胞浸润,在支气管中有增生的现象。
第三章非β-细胞诱导成产生胰岛素细胞的初步研究
本章构建了表达PDX-1、Insulin的质粒pAAV-MCS-PDX-1和pAAV-MCS-Insulin。用pAAV-MCS-PDX-1转入293T细胞后引起了293T细胞中与胰岛发育相关基因的表达或高表达,但是没有胰岛基因的表达。另外还用pAAV-MCS-PDX-1和pAAV-MCS-Insulin共转染293T细胞时发现PDX-1有促进Insulin表达的功能。结果提示293T细胞有被诱导成产生胰岛素细胞的可能。
MicroRNAs are 22nt short no-code RNA. It is derived from pre-miRNA by Dicer enzyme. Several hundreds of miRNAs have been identified in plants and animals that regulate diverse biological processes ranging from cell metabolism to cell differentiation and growth, apoptosis and immune responses. MiRNAs serve as regulators of gene expression by binding to complementary sites on their target transcripts, regulating the expression of 30% gene in human. With new miRNAs were found, increasing number of people pay attention to the study on the miRNAs. Therefore, the research for miRNAs has become one of the hosttest fields in life science.
There are lots of papers about function of miRNAs. But they study the function of miRNAs in the cells. There is scarcely paper about the function of miRNAs in whole body. It is inadequate that we study on the function of miRNAs only in the cell, because miRNAs act as function in the whole body. In this study we microinject the transgene containing RIP, miR-375 gene and the polyadenylation signals of the human growth hormone gene into the pronucleus of fertilized oocytes to construct transgenic mice. In order to study on the injure of pancreas induced by the overexpression of miR-375.Our study includes four parts, the main methods and results are as follows:
PartⅠCloning of the miR-375 and primary study on the function of the miR-375
We construct the plasmid of pAAV-MCS-miR-375 (mouse miR-375). The HeLa cell line was transfected with the plasmid of pAAV-MCS-miR-375. After 48 hours the HeLa cells transfected, Total RNA was extracted from the HeLa cells using TRIzol (Invitrogen). RT-PCR analysis showed the expression of miR-375 in the transfected Hela cells. We transfected the Nit-1 cells.After 48 hours the Nit-1 cells transfected,the proteins are extracted from the cells, The expression of the myotrophin (MTPN) is analyzed by western blots and Immunocytochemical analysis. The level secretion of insulin in the Nit-lcell is measured by RAT/MOUSE INSULIN ELISA kit (Linco Research). The western blots and Immunocytochemical analyses revealed the protein of MTPN expressed decreasely. The level secretion of insulin in the Nit-lcell transfected with the plasmid of pAAV-MCS-miR-375 decreased 30% campared to the transfected with the plasmid of pAAV-MCS and the untransfected cells. We successly cloned the gene of the miR-375 and construct a plasmid of pAAV-MCS-miR-375 that produce the mature miR-375, which down-regulated the expression of the MTPN protein.
Part II The study on Injure of pancreas induced by overexpression of miR-375
The transgene containing RIP, miR-375 gene and the polyadenylation signals of the human growth hormone gene was injected into the pronucleus of fertilized oocytes. Four transgenic founders were identified by PCR, Slot Blot and Southern Blot hybridization. We generated 17 transgenic mice in which the over-expression of miR-375 was under the control of a rat insulin promoter (RIP).The analysis of RT-PCR and real-time PCR of the expression of miR-375 revealed the expression of miR-375 in transgenic mice was 1.15 folds compare to the normal mice. We found the expression of the myotrophin (MTPN) in transgenic mice was 1/7.7 of the normal mice by real-time PCR and western blots analysis. The measure of the fasting serum insulin level of the transgenic mice showed the fasting serum insulin level of the female transgenic mice decreased significantly compared with the untreated control group (0.56144±0.091 vs0.8542±0.044, P<0.05), whereas the male are normal (0.8542±0.044vs0.8525±0.072). Body weight administered weekly, and the fasting blood glucose levels were measured with a portable glucose meter every two weeks. We found the female transgenic mice grown significantly slower than the untreated control group while the male grown normally and the fasting blood glucose was no significant difference among female transgenic mice, male transgenic mice and the untreated control group (2.98±0.57vs3.55±0.1721, 3.521±0.227vs3.55±0.1721). The expression of the gene relate to pancreas development showed PDX-1、INS1、ISL-1、PAX6、GLK、GLG、PP is up-regulated by RT-PCR analysis. The hematoxylin/eosin (H&E) analysis of the pancreas showed there was fatty degeneration in pancreas and the islet markedly shrinked. The hematoxylin/eosin (H&E) analysis of the lungs showed transgenic mice featured lymphocytic infiltration, bronchus inflammatory and bronchus putrescence.
PartⅢPreliminary study on In Vitro Trans-differentiation of Non-β-cell into Insulin-producing cells
We construct the plasmid of pAAV-MCS-PDX-1 and transfect 293T cell with it. After 48 hours transfected 293T cell with the plasmid of pAAV-MCS-PDX-1, RT-PCR analysis revealed that some genes relative to pancreatic development (PDX-1, ISL-land PAX6) were up-regulated; others (PAX4, Bate2 and PC2) were expressed whereas they are not expressed in untransfected 293T cells. But the expression of the insulin gene has not been detected yet. We also construct the plasmid of pAAV-MCS-Insulin, and transfected 293T cell with the the plasmid of pAAV-MCS-PDX-1 and pAAV-MCS-Insulin. The result showed PDX-1 boost up the expression of the insulin.
研究有关miRNA功能的文献有很多,但大部分是在细胞水平的研究,而只在细胞水平研究miRNA功能是远远不够的,因为miRNA功能最终还得在整个机体中发挥作用。在整体水平对miRNA功能的研究,目前国内外都很少。miRNA在胰腺发育和功能的维持中起着非常重要的作用,miR-375是在胰腺中特异表达的miRNA之一,它的正常表达与否对胰腺的形态、发育和功能也至关重要。有关研究表明,miR-375除了对MTPN表达的调控外,还对AdipoR2的表达有调控作用。为了研究miR-375是如何影响胰腺发育和胰腺的功能,探讨miR-375在胰腺发育过程中的作用机理,本实验采用显微注射的方法,利用胰腺内特异表达的启动子RIP(rat insulin premptor)带上编码miR-375的基因,构建miR-375过表达的转基因小鼠模型,旨在研究由miR-375过表达而引起的MTPN、AdipoR2的变化,从而引起胰腺损伤的机理。本研究共分三部分,其主要的实验方法及实验结果如下：
第一章miR-375基因的克隆和功能的初步分析
本章构建了一个含有miR-375基因序列的质粒pAAV-MCS-miR-375,用pAAV-MCS-miR-375转染Hela细胞48小时后,RT-PCR检测发现转染质粒的Hela细胞有miR-375前体的表达。用pAAV-MCS-miR-375转染Nit-1细胞48小时后,Western Blot检测miR-375过表达对Nit-1细胞中MTPN蛋白表达的调节,结果显示,转染pAAV-MCS-miR-37548小时后的Nit-1细胞中MTPN的表达,与转染pAAV-MCS和未转染的Nit-1细胞中MTPN的表达相比有略微下调。为进一步证实miR-375对MTPN表达的作用,我们用pAAV-MCS-miR-375转染Nit-1细胞48小时后,做免疫细胞化学分析。结果发现,转染pAAV-MCS-miR-37548小时后的Nit-1细胞中MTPN蛋白表达比转染pAAV-MCS和未转染的Nit-1细胞中MTPN蛋白表达下调。本研究还测定了未转染、转染pAAV-MCS-miR-375和转染pAAV-MCS的Nit-1细胞中葡萄糖刺激的胰岛素分泌。结果发现转染pAAV-MCS-miR-37548小时后的Nit-1细胞中胰岛素的分泌比未转染和转染pAAV-MCS的Nit-1细胞中胰岛素的分泌下调。这说明我们构建的质粒不但能生成成熟的miR-375,并且能抑制目的Mytrophin基因的表达。本实验成功的克隆了miR-375基因。所构建的表达miR-375的质粒pAAV-MCS-miR-375在细胞中能够表达miR-375、能够抑制MTPN的表达使得细胞受葡萄糖刺激分泌胰岛素的能力下降。
第二章转基因小鼠的构建
本实验采用显微注射法,利用胰腺内特异表达的启动子RIP(rat insulin premptor)带上编码miR-375的基因和人生长激素基因的Poly(A),构建miR-375过表达的转基因小鼠模型。通过PCR、Slot Blot、Southern Blot鉴定阳性转基因小鼠。转基因小鼠胰腺中miR-375的表达采用RT-PCR和real-time PCR测定,转基因小鼠胰腺中myotrophin(MTPN)的表达采用real-time PCR和Western Blot测定。结果发现,转基因小鼠胰腺中成熟miR-375的表达是正常小鼠的1.15倍；转基因小鼠胰腺中myotrophin(MTPN)的表达是正常小鼠的1／7.7。转基因小鼠与正常小鼠的体重和血糖测定发现,与正常同类小鼠相比,雌性转基因鼠的体重有明显下降,雄性转基因鼠的体重无变化,转基因鼠的血糖无明显变化。糖耐量实验中雌性转基因鼠在30-60分钟段血糖恢复比正常小鼠快。转基因小鼠空腹血清胰岛素含量用Linco Research公司的RAT/MOUSE INSULIN ELISA KIT测定。结果显示,与正常同类小鼠相比,雌性转基因鼠血清胰岛素含量低34.3%、有显著差异,雄性转基因鼠的血清胰岛素含量无明显变化。在对转基因小鼠进行细胞学检测时,未发现转基因小鼠存在染色体数量或结构上的改变,而RT-PCR检测与胰腺发育相关基因时发现,雌性转基因小鼠县腺中PDX-1、INS1、ISL-1、PAX6、GLK、GLG、PP的表达上升。用胰岛素抗体和胰高血糖素抗体做免疫荧光发现,转基因鼠胰腺中胰岛素和胰高血糖素的合成不受影响。对转基因鼠胰腺做病理切片HE染色发现,在胰腺中胰岛的周围有很多空泡,出现脂肪变性,另外胰腺中的胰岛还显著萎缩。对转基因鼠肺做病理H／E染色发现,肺中有严重的支气管肺炎、支气管坏死和中性淋巴细胞浸润,在支气管中有增生的现象。
第三章非β-细胞诱导成产生胰岛素细胞的初步研究
本章构建了表达PDX-1、Insulin的质粒pAAV-MCS-PDX-1和pAAV-MCS-Insulin。用pAAV-MCS-PDX-1转入293T细胞后引起了293T细胞中与胰岛发育相关基因的表达或高表达,但是没有胰岛基因的表达。另外还用pAAV-MCS-PDX-1和pAAV-MCS-Insulin共转染293T细胞时发现PDX-1有促进Insulin表达的功能。结果提示293T细胞有被诱导成产生胰岛素细胞的可能。
MicroRNAs are 22nt short no-code RNA. It is derived from pre-miRNA by Dicer enzyme. Several hundreds of miRNAs have been identified in plants and animals that regulate diverse biological processes ranging from cell metabolism to cell differentiation and growth, apoptosis and immune responses. MiRNAs serve as regulators of gene expression by binding to complementary sites on their target transcripts, regulating the expression of 30% gene in human. With new miRNAs were found, increasing number of people pay attention to the study on the miRNAs. Therefore, the research for miRNAs has become one of the hosttest fields in life science.
There are lots of papers about function of miRNAs. But they study the function of miRNAs in the cells. There is scarcely paper about the function of miRNAs in whole body. It is inadequate that we study on the function of miRNAs only in the cell, because miRNAs act as function in the whole body. In this study we microinject the transgene containing RIP, miR-375 gene and the polyadenylation signals of the human growth hormone gene into the pronucleus of fertilized oocytes to construct transgenic mice. In order to study on the injure of pancreas induced by the overexpression of miR-375.Our study includes four parts, the main methods and results are as follows:
PartⅠCloning of the miR-375 and primary study on the function of the miR-375
We construct the plasmid of pAAV-MCS-miR-375 (mouse miR-375). The HeLa cell line was transfected with the plasmid of pAAV-MCS-miR-375. After 48 hours the HeLa cells transfected, Total RNA was extracted from the HeLa cells using TRIzol (Invitrogen). RT-PCR analysis showed the expression of miR-375 in the transfected Hela cells. We transfected the Nit-1 cells.After 48 hours the Nit-1 cells transfected,the proteins are extracted from the cells, The expression of the myotrophin (MTPN) is analyzed by western blots and Immunocytochemical analysis. The level secretion of insulin in the Nit-lcell is measured by RAT/MOUSE INSULIN ELISA kit (Linco Research). The western blots and Immunocytochemical analyses revealed the protein of MTPN expressed decreasely. The level secretion of insulin in the Nit-lcell transfected with the plasmid of pAAV-MCS-miR-375 decreased 30% campared to the transfected with the plasmid of pAAV-MCS and the untransfected cells. We successly cloned the gene of the miR-375 and construct a plasmid of pAAV-MCS-miR-375 that produce the mature miR-375, which down-regulated the expression of the MTPN protein.
Part II The study on Injure of pancreas induced by overexpression of miR-375
The transgene containing RIP, miR-375 gene and the polyadenylation signals of the human growth hormone gene was injected into the pronucleus of fertilized oocytes. Four transgenic founders were identified by PCR, Slot Blot and Southern Blot hybridization. We generated 17 transgenic mice in which the over-expression of miR-375 was under the control of a rat insulin promoter (RIP).The analysis of RT-PCR and real-time PCR of the expression of miR-375 revealed the expression of miR-375 in transgenic mice was 1.15 folds compare to the normal mice. We found the expression of the myotrophin (MTPN) in transgenic mice was 1/7.7 of the normal mice by real-time PCR and western blots analysis. The measure of the fasting serum insulin level of the transgenic mice showed the fasting serum insulin level of the female transgenic mice decreased significantly compared with the untreated control group (0.56144±0.091 vs0.8542±0.044, P<0.05), whereas the male are normal (0.8542±0.044vs0.8525±0.072). Body weight administered weekly, and the fasting blood glucose levels were measured with a portable glucose meter every two weeks. We found the female transgenic mice grown significantly slower than the untreated control group while the male grown normally and the fasting blood glucose was no significant difference among female transgenic mice, male transgenic mice and the untreated control group (2.98±0.57vs3.55±0.1721, 3.521±0.227vs3.55±0.1721). The expression of the gene relate to pancreas development showed PDX-1、INS1、ISL-1、PAX6、GLK、GLG、PP is up-regulated by RT-PCR analysis. The hematoxylin/eosin (H&E) analysis of the pancreas showed there was fatty degeneration in pancreas and the islet markedly shrinked. The hematoxylin/eosin (H&E) analysis of the lungs showed transgenic mice featured lymphocytic infiltration, bronchus inflammatory and bronchus putrescence.
PartⅢPreliminary study on In Vitro Trans-differentiation of Non-β-cell into Insulin-producing cells
We construct the plasmid of pAAV-MCS-PDX-1 and transfect 293T cell with it. After 48 hours transfected 293T cell with the plasmid of pAAV-MCS-PDX-1, RT-PCR analysis revealed that some genes relative to pancreatic development (PDX-1, ISL-land PAX6) were up-regulated; others (PAX4, Bate2 and PC2) were expressed whereas they are not expressed in untransfected 293T cells. But the expression of the insulin gene has not been detected yet. We also construct the plasmid of pAAV-MCS-Insulin, and transfected 293T cell with the the plasmid of pAAV-MCS-PDX-1 and pAAV-MCS-Insulin. The result showed PDX-1 boost up the expression of the insulin.
引文
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