【作者】 刘晓会；

【导师】 吴珊；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 目的:观察TGF-β1在诱导大鼠肾小管上皮细胞系(NRK-52E)的上皮-间充质转分化过程中,对细胞基质金属蛋白酶2(MMP-2)和基质金属蛋白酶9(MMP-9)的影响,探讨MMP-2和MMP-9与肾小管上皮细胞转分化的相关性。方法:选择E钙粘附蛋白为上皮细胞标志物,α平滑肌动蛋白(α-SMA)为间叶组织标志物,以低血清培养的NRK-52E做为对照组,低血清培养的NRK-52E细胞经TGF-β1(10ng/ml)诱导72小时为实验组,分别以免疫细胞化学染色和RT-PCR方法检测NRK-52E的转分化状态, RT-PCR法观察NRK-52E转分化后的细胞MMP-2与MMP-9的表达。结果:RT-PCR和免疫细胞化学染色结果均显示,NRK-52E细胞经TGF-β1体外诱导72小时后,其上皮细胞标志物E钙粘附蛋白的mRNA和蛋白的表达与相应的对照组相比明显减弱,同时其间叶组织标志物α-SMA mRNA和蛋白表达与相应的对照组相比均增强,表明培养的NRK-52E经TGF-β1体外诱导72小时发生了上皮-间充质细胞的转分化;此时,转分化细胞的MMP-2和MMP-9 mRNA表达均明显高于对照组细胞,说明TGF-β1诱导NRK-52E发生转分化后可使细胞的MMP-2与MMP-9表达增多。结论:TGF-β1成功的诱导大鼠肾小管上皮细胞(NRK-52E)发生转分化,使细胞MMP-2与MMP-9mRNA表达增多。更多还原

【Abstract】 Renal interstitial fibrosis is the final stage of many kinds of renal diseases[1]. Renal interstitial fibrosis is characteristic of the accumulation of excessive extra cellular matrix and the destruction of renal tissue induced by the development of kinds of renal diseases. Now, it is accepted that the excessive extra cellular matrix is secreted by the Myofibroblast[4], the precise resource of the Myofibroblast has been always considered activation of interstitial fibroblasts. But recently it was suggested that there might be other sources in addition to activation of interstitial fibroblasts. Some scientists have proposed that epithelial to mesenchymal transition is a major resource of the Myofibroblast,and renal tubular epithelial to mesenchymal transition plays an important role in renal interstitial fibrosis. So more and more scientists pay close attention to its role in the renal fibrosis. The so-called renal tubular epithelial cells to mesenchymal cell transdifferentiation usually pointed tubular epithelial cells lost E-cadherin, a marker of endothelial cells and acquired a phenotype of mesenchymal cells, a-SMA[26]. EMT of renal tubular cells can be affected by many factors, among these factors, TGF-β1[27] was proved to be an important cytokine which can induce the EMT of renal tubular cells. So TGF-β1 is selected to induce renal tubular epitheial cell to mesenchymal transition.For the past few years, researches about the EMT of renal tubular cells emerged. It was noted that the integrity of tubular basement membrane plays an important role in renal tubular epithelial cell transdifferentiation. Tubular basement membrane is mainly composed of typeⅣcollagen, fibronectin and laminin and other extracellular matrix. While matrix metalloproteinases (MMPs) can digest the basement membrane. Specifically, MMP-2 and MMP-9 can digest collagenⅣ[32], which is the major component of glomerular basement membrane and renal tubular basement membrane. So more and more scientists pay close attention to its role in the EMT of renal tubular cells.Purpose: In this study, renal tubular epithelial cell line (NRK-52E) be selected as the object, TGF-β1 to be selected as induction factor, we designed the experiment on the basis of above to observe the influence of TGF-β1 on MMP-2 and MMP-9, when it inducing the EMT of rat renal tubular cell line (NRK-52E). Detect the relationship between MMP-2, MMP-9 and the EMT of renal tubular cells.Method: Rat renal tubular cell line (NRK-52E) was cultured in vivo. Performed Immunocytochemistry and RT-PCR to detect and clarify the EMT of NRK-52E cells 72 hours after been stimulated by TGF-β1(10ng/ml), selected E-cadherin as a marker of endothelial cells,α- SMA as a phenotype of mesenchymal cells. Then we observed the expression of MMP-2 and MMP-9 in the transformed NRK-52E cells. The NRK-52E cells cultured in the low- blood serum media was used as control.Result: Growth of the normal cultured renal tubular epithelial cell (NRK-52E) showed a confluent monolayer of cells with a classical epithelial polygon cobblestone-like morphology.NRK-52E were cultured for 72 hours in the presence or absence of TGF-β1,we observed that that in the experimental group and control group has an obvious change in cell morphology. Growth of the renal tubular epithelial cell of control group always showed a confluent monolayer of cells with a classical epithelial polygon cobblestone-like morphology, whether , growth of the renal tubular epithelial cell of experimental group showed scattered singly, showed morphological changes typical of EMT, from epithelial cobblestone to fibroblastic spindle-shaped morphology. Rat renal tubular cell line (NRK-52E) was cultured in vivo, Performed Immunocytochemistry to detect and clarify the EMT of NRK-52E cells 72 hours after been stimulated by TGF-β1(10ng/ml), selected as a marker of endothelial cells,α- SMA as a phenotype of mesenchymal cells. Result showed that the expression of E-cadherin were stronger in the cell membrane of control group than experimental group,but, the expression ofα- SMA were less in the cell cytoplasm of control group than experimental group. Immunocytochemical results suggest that cultured NRK-52E cells in the induction of TGF-β1 has undergone epithelial - mesenchymal cell transdifferentiation. To further verify the immunocytochemistry result, RT-PCR to detect and clarify the EMT of NRK-52E cells 72 hours after been stimulated by TGF-β1(10ng/ml), selected E-cadherin as a marker of endothelial cells,α- SMA as a phenotype of mesenchymal cells.With the mRNA expression of GAPDH as an internal of RT-PCR, to RT-PCR detect and clarify the EMT of NRK-52E cells 72 hours after been stimulated by TGF-β1(10ng/ml), selected E-cadherin as a marker of endothelial cells,α- SMA as a phenotype of mesenchymal cells. Result showed that the mRNA expression of GAPDH were similarly, the mRNA expression of E-cadherin were stronger in the cell membrane of control group than experimental group, but, the mRNA expression ofα- SMA were less in the cell cytoplasm of control group than experimental group. It further verify the immunocytochemistry result. Result testified that TGF-β1 has successfully induced renal tubular epitheial cell to mesenchymal transition.In the same time,we observed the mRNA expression of MMP-2 and MMP-9 in the transformed NRK-52E cells. With the mRNA expression of GAPDH as an internal of RT-PCR,and the mRNA expression of GAPDH were similarly, result showed that the mRNA expression of MMP-2 were less in the cell cytoplasm of control group than experimental group, the mRNA expression of MMP-9 were less in the cell cytoplasm of control group than experimental group, then ,it further supported Yang and his co-worker′s conclusion that transformed HK-2 cells secrete a lot of MMP-2 and MMP-9 in the earlier process of renal tubular cells transit into the mesenchymal cells, then they raised that transformed NRK-52E cells secreted large quantities of MMP-2, MMP-9 were associated with tubular basement membrane disintegration of the basement membrane, then reinforcement of the capability of migration and invasion in the cells. The cells migrate to the interstitial area, secrete extra cellular matrix and promote the development of fibrosis.Conclusion: TGF-β1 can induce rat renal tubular cells develop EMT, and the expression of MMP-2 and MMP-9 were enhanced in the transformed cells.更多还原