TRPC6在足细胞损伤中的作用研究
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摘要
第一部分
足细胞损伤时TRPC6表达的变化及TRPC6高表达对裂孔隔膜蛋白及细胞骨架的影响
目的：观察PAN作用下体外培养的小鼠足细胞TRPC6分布以及表达的变化，进一步研究TRPC6高表达对足细胞裂孔隔膜分子Nephrin及细胞骨架成分α-actinin-4和α-tubulin的影响。
方法：在33℃许可条件下培养的小鼠足细胞传代后转入37℃非许可条件下继续培养14天，以不同浓度(10μg／ml，30μg/ml，50μg／ml，70μg／ml)、不同时间(6小时，12小时，24小时，48小时)PAN处理足细胞，分别应用免疫荧光技术观察TRPC6分布的变化；用逆转录-聚合酶链反应(reverse transcription-polymerase chainreaction，RT-PCR)及Western印迹方法分别检测TRPC6 mRNA及蛋白水平的变化。用脂质体法将小鼠TRPC6真核表达载体pEGFP-N_1-mTRPC6转染足细胞，48小时后用荧光显微镜观察EGFP的表达；48小时后Western印迹方法检测转染后TRPC6蛋白表达的变化；应用免疫荧光技术观察转染后Nephrin、α-actinin-4、α-tubulin分布的变化；用RT-PCR及Western印迹方法分别检测Nephrin、α-actinin-4、α-tubulin mRNA及蛋白水平的变化。
结果：成熟足细胞TRPC6主要分布于细胞核周围和细胞膜，PAN作用24小时后足细胞上TRPC6分布出现变化，细胞膜上分布明显增强，48小时后变化更加明显。PAN作用于足细胞后，TRPC6 mRNA及蛋白表达水平明显升高，并呈现剂量依赖性和时间依赖性，在30μg／ml PAN作用下，TRPC6的表达即明显升高(P＜0．05)，当更高浓度PAN作用时，TRPC6的增高更加明显(P＜0．01)；应用50μg／ml PAN时，在12小时TRPC6 mRNA及蛋白表达开始上调(P＜0．05)并随作用时间的延长逐渐增高。pEGFP-N_1-mTRPC6转染足细胞后约35％细胞出现绿色荧光；足细胞TRPC6蛋白表达水平明显增高(P＜0．01)。转染TRPC6以后Nephrin、α-actinin-4分布均出现变化；Nephrin在mRNA及蛋白水平分别下降42%及26%左右(P＜0．05)；α-actinin-4在mRNA及蛋白水平分别下降22%及25%左右(P＜0．05)；TRPC6高表达对α-tubulinmRNA及蛋白水平无影响，但可影响其分布。
结论：在PAN作用于足细胞导致其损伤后，TRPC6分布出现变化并且其表达增高；TRPC6高表达干扰了足细胞裂孔隔膜分子Nephrin以及细胞骨架蛋白α-actinin-4、α-tubulin的正常定位及功能。
第二部分
TRPC6在AngⅡ诱导的小鼠足细胞凋亡过程中的作用
目的：研究TRPC6高表达对AngⅡ诱导的小鼠足细胞凋亡的影响并初步探讨其作用机制。
方法：在33℃许可条件下培养的小鼠足细胞传代后转入37℃非许可条件下继续培养14天，四甲基偶氮唑盐法(MTT法)检测不同浓度AngⅡ对足细胞活性的影响；用脂质体法将针对小鼠TRPC6的基因真核表达载体pEGFP-N_1-mTRPC6转染体外培养的永生化小鼠足细胞系，同时转染pEGFP-N_1空载体作为对照，48小时后用倒置荧光显微镜观察EGFP的表达，Western印迹方法检测转染后TRPC6蛋白表达的变化；应用AngⅡ(10~(-6)mol／L)处理足细胞，Fluo-3AM结合激光共聚焦显微镜观察转染TRPC6基因后足细胞胞浆内钙离子浓度的变化；将细胞分组，分别采用低剂量(10~(-10)mol／L)及高剂量(10~(-6)mol／L)AngⅡ刺激细胞，24小时后用逆转录-聚合酶链反应(RTPCR)及Western印迹方法分别检测Bax、Bcl-2mRNA及蛋白水平的变化；流式细胞仪及Hoechst33258染色法检测足细胞凋亡。
结果：低剂量AngⅡ(10~(-10)mol／L)作用24小时，对足细胞活力无影响；高剂量AngⅡ(10~(-6)mol／L)作用24小时后足细胞活力下降约50％；pEGFP-N_1-mTRPC6转染足细胞后约35％细胞出现绿色荧光，足细胞TRPC6蛋白表达水平明显增高(P＜0．01)；转染TRPC6以后可以明显促进AngⅡ诱导的足细胞钙离子内流(P＜0．01)；低剂量AngⅡ(10~(-10)mol／L)作用24小时后，与对照组相比，Bax、Bcl-2mRNA及蛋白的表达无明显变化；转入TRPC6基因后，Bax mRNA及蛋白表达水平明显增高(P＜0．01)而Bcl-2 mRNA及蛋白表达水平明显下调(P＜0．05)；高剂量AngⅡ(10~(-6)mol／L)作用24小时可上调Bax的表达而下调Bcl-2的表达(P＜0．01)，转染TRPC6基因则Bax的表达进一步升高而Bcl-2的表达进一步下降(P＜0．01，P＜0．05)；在低剂量AngⅡ(10~(-10)mol／L)作用下，足细胞凋亡率为(2．50±0．72)％，转染TRPC6以后凋亡率为(4．33±0．45)％(P＜0．05)；在高剂量AngⅡ(10~(-6)mol／L)作用下，足细胞凋亡率为(15．46±1．40)％，转染TRPC6以后凋亡率为(18．33±0．87)％(P＜0．01)，转染pEGFP-N_1空载体对凋亡率则无影响。
结论：TRPC6在AngⅡ诱导的足细胞凋亡中发挥重要作用；TRPC6可能通过增加钙离子内流，进而启动其下游的凋亡调节成分参与凋亡过程。
第三部分
TRPC6 shRNA真核表达质粒的构建及其对足细胞损伤的影响
目的：构建针对TRPC6基因的短发夹结构RNA(shRNA)真核表达质粒，观察基因敲低TRPC6对PAN诱导的小鼠足细胞损伤的影响。
方法：设计两条针对TRPC6mRNA不同靶序列的干扰序列，将其插入真核表达质粒pGCsi-U6-Neo-GFP-shRNA，构建针对TRPC6基因的shRNA表达载体pGCsi-TRPC6A和pGCsi-TRPC6B，经酶切和DNA测序证实；选择不与任何基因同源的序列NC作为阴性对照；用脂质体法将构建成功的3种重组质粒转染体外培养的永生化小鼠足细胞系。24及48小时后用荧光显微镜观察GFP的表达；48小时后RT-PCR法和Western印迹法检测TRPC6的干扰效率。将细胞分为4组：正常对照组、PAN干预组、PAN干预+转染pGCsi-TRPC6B组以及PAN干预+转染pGCsi-NC组。PAN作用48小时后应用Western印迹法检测Nephrin、CD2AP表达的变化，应用RT-PCR及Western印迹法分别在mRNA及蛋白水平检测Bax、Bcl-2表达的变化；应用流式细胞术检测各组细胞的凋亡率。
结果：应用质粒提取试剂盒抽提重组质粒pGCsi-TRPC6A及pGCsi-TRPC6B后进行测序分析，基因测序结果证明插入到pGCsi-U6-Neo-GFP-shRNA的序列与设计的序列完全一致，符合设计要求；小鼠足细胞转染shRNA24小时后，GFP的表达率为45％左右；pGCsi-TRPC6A及pGCsi-TRPC6B均下调了TRPC6 mRNA及蛋白的表达(P＜0．05)，但pGCsi-TRPC6B作用较pGCsi-TRPC6A更强，因此后续实验采用pGCsi-TRPC6B；PAN作用48小时后Nephrin及CD2AP表达明显下调，PAN干预+转染pGCsi-TRPC6B组Nephrin及CD2AP下降不明显(P＜0．05)，PAN作用48小时后在mRNA及蛋白水平均上调了Bax的表达而下调了Bcl-2的表达；转染TRPC6shRNA质粒后可明显逆转上述改变(P＜0．05)；基因敲低TRPC6可以明显降低PAN诱导的足细胞凋亡率(P＜0．05)。
结论：成功构建了针对TRPC6基因的shRNA真核表达质粒，转染足细胞后能发挥确切的RNA干扰作用使TRPC6基因沉默；基因敲低TRPC6能有效保护PAN诱导的足细胞损伤。
PartⅠ
Effects of puromycin aminonucleoside on the expression of TRPC6of murine podocytes and the significance of it
Objective:To investigate the effect of PAN on the expression of TRPC6 of murinepodocytes and to further study the effect of overexpression of TRPC6 on Nephrin,α-actinin-4 andα-tubulin in murine podocytes in vitro.
Methods:Conditionally immortalized murine podocyte cell line was cultured underpermissive condition at 33℃.After passage the cells were shifted to non-permissivecondition at 37℃and cultrued for 14 days.PAN was added to the medium at theconcentration of 30μg/ml.Immunofluorescent assay was used to observe the distribution ofTRPC6.The expression of TRPC6 mRNA was assessed by RT-PCR and the expression ofTRPC6 protein was measured by Western-blot at 6,12,24 and 48 hours.Mouse TRPC6cDNA eukaryotic expression vector pEGFP-N_1-mTRPC6 was transfected to podocytes byliposome.The fluorescent microscopy was used to examine the expression of EGFP after48 hours.The change of TRPC6 protein expression was observed by Western-blot after 48hours.Immunofluorescent assay was used to observe the distribution of Nephrin, α-actinin-4 andα-tubulin.RT-PCR and Western-blot were used to measure the expressionof mRNA and protein of Nephrin,α-actinin-4 andα-tubulin,respectively.
Results:TRPC6 was expressed in podocytes,expecialy in the perinuclear and the cellmembrane.The expression of membrane TRPC6 increased after exposure to PAN for 24h.After treated with 50μg/ml PAN for 48h,more significant changes were observed.Podocytes treated with different concentrations of PAN resulted in the stimulation ofTRPC6 in a dose-and time-dependent manner.The addition of PAN to the media ofcultured mouse podocytes effectively stimulated the mRNA and protein production ofTRPC6.A noticeable increase in TRPC6 was detected at 30μg/ml PAN.A higherconcentration,50μg/ml,of PAN stimulated PAN production even more.At a fixed dose of50μg/ml PAN,the stimulation of TRPC6 production was evident in 12h.Additional time ofexposure to PAN appear to increase the quantity of TRPC6 mRNA and protein in the mousepodocytes further.About 35% of the cells expressed EGFP.A up-regulation of proteinexpression of TRPC6 was detected in podocytes when transfected with pEGFP-N_1-mTRPC6 (P＜0.01).The distribution changes of Nephrin andα-actinin-4 were found ascompared to control groups.The reductions of the mRNA and the protein expression inNephrin were apparently detected by about 42% and 26%,respectively.The mRNA andprotein expression levels ofα-actinin-4 decreased by about 22% and 25%,respectively.(P＜0.05).The overexpression of TRPC6 had no effect on the expression ofα-tubulin butchanged the distribution of it.
Conclusion:The distribution of TRPC6 was changed after exposure toPAN.Exposureto PAN resulted in the stimulation of TRPC6 both in mRNA and protein level.Theoverexpression of TRPC6 interfere with the normal distribution and function of Nephrin,α-actinin-4 andα-tubulin.
PartⅡEffect of overexpression of TRPC6 on angiotensinⅡ-inducedapoptosis of mouse podocytes
Objective:To study the effect of overexpression of TRPC6 on AngⅡ-inducedapoptosis of mouse podocytes in vitro and to further explore the possible mechanisms.
Methods:Conditionally immortalized murine podocyte cell line was cultured underpermissive condition at 33℃.After passage the cells were shifted to non-permissivecondition at 37℃and cultrued for 14 days.Podocytes were treated by differentconcentrations of AngⅡand the cell viability was dected by methyl thiazolyl tetrazoliummethod(MTT).Mouse TRPC6 cDNA eukaryotic expression vector pEGFP-N_1-mTRPC6was transfeced to podocytes by liposome.The fluorescent microscopy was used to examinethe expression of EGFP after 48 hours.The change of TRPC6 protein expression wasobserved by Western-blot.The podocyte intracellular calcium concentration was measuredwith laser-scanning confocal microscope.Podocytes were divided into groups and differentconcentrations of AngⅡwere used.The expression of Bax and Bcl-2 mRNA was assessedby RT-PCR and the expression of Bax and Bcl-2 protein was measured by Western-blot.The apoptotic ratio of podocytes was monitored by flow cytometry and Hoechst33258staining.
Results:The cell viability han no change after treated with low-dose (10~(-10)mol/L) ofAngⅡbut decreased 50% by using high-dose (10~(-6)mol/L)of AngⅡfor 24 hours.About35% of the cells expressed EGFP.A up-regulation of protein expression of TRPC6 wasdetected in podocytes when transfected with pEGFP-N_1-mTRPC6(P＜0.01).Theoverexpression of TRPC6 promoted the AngⅡ-induced influx of extracellular calcium andelevated the expression of Bax but decreased the expression of Bcl-2(P＜0.01,P＜0.05).The apoptotic ratio of podocyte was (2.50±0.72)% on the effect of low-doseAngⅡ(10~(-10)mol/L),it was increased to (4.33±0.45)% when transfected with pEGFP-N_1 -mTRPC6(P＜0.05).Transfection with pEGFP-N_1-mTRPC6 increased apoptosis ratefrom (15.46±1.40)% to (18.33±0.87)% (P＜0.01) on the high-dose (10~(-6)mol/L) of AngⅡ.
Conclusion:TRPC6 plays an important role in the AngⅡ-induced apoptosis ofpodocytes.This effect can be attributed to its ability of promoting the influx of extracellularcalcium and initiating the apoptosis cascade.
PartⅢ
Effect of Down-regulation of TRPC6 on the Mouse Podocyte injuryinduced by Puromycin Aminonucleoside
Objective:To construct eukaryotic expression vectors carrying the small hairpinRNA(shRNA) for TRPC6 mRNA and observe the effect of knocking-down TRPC6 onPAN-induced injury of mouse podocytes.
Methods:Two DNA sequences containing small hairpin structure targeting TRPC6were designed and synthesized and were then inserted into the plasmid pGC containinggreen fluorescence protein (GFP) to form plasmid pGCsi-TRPC6A and pGCsi-TRPC6B.The inserted sequences were verified by enzyme digestion and DNA sequeneing.Plasmidexpressing irrelevant shRNA was used as negative control named PGCsi-NC.The plasmidswere transfeced to conditionally immortalized murine podocyte cell line by liposome.Thefluorescent microscopy was used to examine the expression of GFP after 24 and 48 hours.The changes of TRPC6 mRNA and protein expression were observed by RT-PCR andWestern-blot after 48 hours.Cultured podocytes were divided into four groups:controlgroup;PAN treatment group;PAN treatment+shRNA transfection group and PANtreatment+negative control group.The expression of Nephrin and CD2AP protein wasmeasured by Western-blot.The expression of Bax and Bcl-2 mRNA was assessed byRT-PCR and the expression of Bax and Bcl-2 protein was measured by Western-blot.The apoptotic ratio ofpodocytes was monitored by flow cytometry.
Results:The sequence of recombinant plasmids pGCsi-TRPC6A and pGCsi-TRPC6Bwas confirmed by further sequencing after extracted using plasmid extraction kit.Theresults showed that the inserted sequence of the pGCsi-U6-Neo-GFP-shRNA was the sameas the designed sequence.About 45% of the cells expressed GFP.A down-regulation ofmRNA and protein expression of TRPC6 was detected in mouse podocytes whentransfected with pGCsi-TRPC6A and pGCsi-TRPC6B(P＜0.05).Plasmid pGCsi-TRPC6Bwas used for subsequent study because of its higher efficiency of knocking-down theTRPC6 gene compared with plasmid pGCsi-TRPC6A.Compaired with the control group,dreased protein of Nephrin and CD2AP was detected in the cultured podocytes treated withPAN for 48 hours (P＜0.05).This effect was interfered by transfecting the plasmidpGCsi-TRPC6B into the podocytes(P＜0.05).The expression of Bax increased and theexpression of Bcl-2 decreased at protein and mRNA levels after treated with PAN for 48hours but knocking-down TRPC6 could prevent these changes.Knocking-down TRPC6could effectively decreased the PAN-induced apoptosis of podocytes.
Conclusion:The eukaryotic expression vectors carrying the small hairpin RNA(shRNA) for TRPC6 mRNA was successfully constructed.The reeombinant plasmid couldexpress stably and eode small interfering RNA in cells to make target genes silence.Knocking-down TRPC6 gene could effectively prevent the podocytes from injury inducedby PAN.
足细胞损伤时TRPC6表达的变化及TRPC6高表达对裂孔隔膜蛋白及细胞骨架的影响
目的：观察PAN作用下体外培养的小鼠足细胞TRPC6分布以及表达的变化，进一步研究TRPC6高表达对足细胞裂孔隔膜分子Nephrin及细胞骨架成分α-actinin-4和α-tubulin的影响。
方法：在33℃许可条件下培养的小鼠足细胞传代后转入37℃非许可条件下继续培养14天，以不同浓度(10μg／ml，30μg/ml，50μg／ml，70μg／ml)、不同时间(6小时，12小时，24小时，48小时)PAN处理足细胞，分别应用免疫荧光技术观察TRPC6分布的变化；用逆转录-聚合酶链反应(reverse transcription-polymerase chainreaction，RT-PCR)及Western印迹方法分别检测TRPC6 mRNA及蛋白水平的变化。用脂质体法将小鼠TRPC6真核表达载体pEGFP-N_1-mTRPC6转染足细胞，48小时后用荧光显微镜观察EGFP的表达；48小时后Western印迹方法检测转染后TRPC6蛋白表达的变化；应用免疫荧光技术观察转染后Nephrin、α-actinin-4、α-tubulin分布的变化；用RT-PCR及Western印迹方法分别检测Nephrin、α-actinin-4、α-tubulin mRNA及蛋白水平的变化。
结果：成熟足细胞TRPC6主要分布于细胞核周围和细胞膜，PAN作用24小时后足细胞上TRPC6分布出现变化，细胞膜上分布明显增强，48小时后变化更加明显。PAN作用于足细胞后，TRPC6 mRNA及蛋白表达水平明显升高，并呈现剂量依赖性和时间依赖性，在30μg／ml PAN作用下，TRPC6的表达即明显升高(P＜0．05)，当更高浓度PAN作用时，TRPC6的增高更加明显(P＜0．01)；应用50μg／ml PAN时，在12小时TRPC6 mRNA及蛋白表达开始上调(P＜0．05)并随作用时间的延长逐渐增高。pEGFP-N_1-mTRPC6转染足细胞后约35％细胞出现绿色荧光；足细胞TRPC6蛋白表达水平明显增高(P＜0．01)。转染TRPC6以后Nephrin、α-actinin-4分布均出现变化；Nephrin在mRNA及蛋白水平分别下降42%及26%左右(P＜0．05)；α-actinin-4在mRNA及蛋白水平分别下降22%及25%左右(P＜0．05)；TRPC6高表达对α-tubulinmRNA及蛋白水平无影响，但可影响其分布。
结论：在PAN作用于足细胞导致其损伤后，TRPC6分布出现变化并且其表达增高；TRPC6高表达干扰了足细胞裂孔隔膜分子Nephrin以及细胞骨架蛋白α-actinin-4、α-tubulin的正常定位及功能。
第二部分
TRPC6在AngⅡ诱导的小鼠足细胞凋亡过程中的作用
目的：研究TRPC6高表达对AngⅡ诱导的小鼠足细胞凋亡的影响并初步探讨其作用机制。
方法：在33℃许可条件下培养的小鼠足细胞传代后转入37℃非许可条件下继续培养14天，四甲基偶氮唑盐法(MTT法)检测不同浓度AngⅡ对足细胞活性的影响；用脂质体法将针对小鼠TRPC6的基因真核表达载体pEGFP-N_1-mTRPC6转染体外培养的永生化小鼠足细胞系，同时转染pEGFP-N_1空载体作为对照，48小时后用倒置荧光显微镜观察EGFP的表达，Western印迹方法检测转染后TRPC6蛋白表达的变化；应用AngⅡ(10~(-6)mol／L)处理足细胞，Fluo-3AM结合激光共聚焦显微镜观察转染TRPC6基因后足细胞胞浆内钙离子浓度的变化；将细胞分组，分别采用低剂量(10~(-10)mol／L)及高剂量(10~(-6)mol／L)AngⅡ刺激细胞，24小时后用逆转录-聚合酶链反应(RTPCR)及Western印迹方法分别检测Bax、Bcl-2mRNA及蛋白水平的变化；流式细胞仪及Hoechst33258染色法检测足细胞凋亡。
结果：低剂量AngⅡ(10~(-10)mol／L)作用24小时，对足细胞活力无影响；高剂量AngⅡ(10~(-6)mol／L)作用24小时后足细胞活力下降约50％；pEGFP-N_1-mTRPC6转染足细胞后约35％细胞出现绿色荧光，足细胞TRPC6蛋白表达水平明显增高(P＜0．01)；转染TRPC6以后可以明显促进AngⅡ诱导的足细胞钙离子内流(P＜0．01)；低剂量AngⅡ(10~(-10)mol／L)作用24小时后，与对照组相比，Bax、Bcl-2mRNA及蛋白的表达无明显变化；转入TRPC6基因后，Bax mRNA及蛋白表达水平明显增高(P＜0．01)而Bcl-2 mRNA及蛋白表达水平明显下调(P＜0．05)；高剂量AngⅡ(10~(-6)mol／L)作用24小时可上调Bax的表达而下调Bcl-2的表达(P＜0．01)，转染TRPC6基因则Bax的表达进一步升高而Bcl-2的表达进一步下降(P＜0．01，P＜0．05)；在低剂量AngⅡ(10~(-10)mol／L)作用下，足细胞凋亡率为(2．50±0．72)％，转染TRPC6以后凋亡率为(4．33±0．45)％(P＜0．05)；在高剂量AngⅡ(10~(-6)mol／L)作用下，足细胞凋亡率为(15．46±1．40)％，转染TRPC6以后凋亡率为(18．33±0．87)％(P＜0．01)，转染pEGFP-N_1空载体对凋亡率则无影响。
结论：TRPC6在AngⅡ诱导的足细胞凋亡中发挥重要作用；TRPC6可能通过增加钙离子内流，进而启动其下游的凋亡调节成分参与凋亡过程。
第三部分
TRPC6 shRNA真核表达质粒的构建及其对足细胞损伤的影响
目的：构建针对TRPC6基因的短发夹结构RNA(shRNA)真核表达质粒，观察基因敲低TRPC6对PAN诱导的小鼠足细胞损伤的影响。
方法：设计两条针对TRPC6mRNA不同靶序列的干扰序列，将其插入真核表达质粒pGCsi-U6-Neo-GFP-shRNA，构建针对TRPC6基因的shRNA表达载体pGCsi-TRPC6A和pGCsi-TRPC6B，经酶切和DNA测序证实；选择不与任何基因同源的序列NC作为阴性对照；用脂质体法将构建成功的3种重组质粒转染体外培养的永生化小鼠足细胞系。24及48小时后用荧光显微镜观察GFP的表达；48小时后RT-PCR法和Western印迹法检测TRPC6的干扰效率。将细胞分为4组：正常对照组、PAN干预组、PAN干预+转染pGCsi-TRPC6B组以及PAN干预+转染pGCsi-NC组。PAN作用48小时后应用Western印迹法检测Nephrin、CD2AP表达的变化，应用RT-PCR及Western印迹法分别在mRNA及蛋白水平检测Bax、Bcl-2表达的变化；应用流式细胞术检测各组细胞的凋亡率。
结果：应用质粒提取试剂盒抽提重组质粒pGCsi-TRPC6A及pGCsi-TRPC6B后进行测序分析，基因测序结果证明插入到pGCsi-U6-Neo-GFP-shRNA的序列与设计的序列完全一致，符合设计要求；小鼠足细胞转染shRNA24小时后，GFP的表达率为45％左右；pGCsi-TRPC6A及pGCsi-TRPC6B均下调了TRPC6 mRNA及蛋白的表达(P＜0．05)，但pGCsi-TRPC6B作用较pGCsi-TRPC6A更强，因此后续实验采用pGCsi-TRPC6B；PAN作用48小时后Nephrin及CD2AP表达明显下调，PAN干预+转染pGCsi-TRPC6B组Nephrin及CD2AP下降不明显(P＜0．05)，PAN作用48小时后在mRNA及蛋白水平均上调了Bax的表达而下调了Bcl-2的表达；转染TRPC6shRNA质粒后可明显逆转上述改变(P＜0．05)；基因敲低TRPC6可以明显降低PAN诱导的足细胞凋亡率(P＜0．05)。
结论：成功构建了针对TRPC6基因的shRNA真核表达质粒，转染足细胞后能发挥确切的RNA干扰作用使TRPC6基因沉默；基因敲低TRPC6能有效保护PAN诱导的足细胞损伤。
PartⅠ
Effects of puromycin aminonucleoside on the expression of TRPC6of murine podocytes and the significance of it
Objective:To investigate the effect of PAN on the expression of TRPC6 of murinepodocytes and to further study the effect of overexpression of TRPC6 on Nephrin,α-actinin-4 andα-tubulin in murine podocytes in vitro.
Methods:Conditionally immortalized murine podocyte cell line was cultured underpermissive condition at 33℃.After passage the cells were shifted to non-permissivecondition at 37℃and cultrued for 14 days.PAN was added to the medium at theconcentration of 30μg/ml.Immunofluorescent assay was used to observe the distribution ofTRPC6.The expression of TRPC6 mRNA was assessed by RT-PCR and the expression ofTRPC6 protein was measured by Western-blot at 6,12,24 and 48 hours.Mouse TRPC6cDNA eukaryotic expression vector pEGFP-N_1-mTRPC6 was transfected to podocytes byliposome.The fluorescent microscopy was used to examine the expression of EGFP after48 hours.The change of TRPC6 protein expression was observed by Western-blot after 48hours.Immunofluorescent assay was used to observe the distribution of Nephrin, α-actinin-4 andα-tubulin.RT-PCR and Western-blot were used to measure the expressionof mRNA and protein of Nephrin,α-actinin-4 andα-tubulin,respectively.
Results:TRPC6 was expressed in podocytes,expecialy in the perinuclear and the cellmembrane.The expression of membrane TRPC6 increased after exposure to PAN for 24h.After treated with 50μg/ml PAN for 48h,more significant changes were observed.Podocytes treated with different concentrations of PAN resulted in the stimulation ofTRPC6 in a dose-and time-dependent manner.The addition of PAN to the media ofcultured mouse podocytes effectively stimulated the mRNA and protein production ofTRPC6.A noticeable increase in TRPC6 was detected at 30μg/ml PAN.A higherconcentration,50μg/ml,of PAN stimulated PAN production even more.At a fixed dose of50μg/ml PAN,the stimulation of TRPC6 production was evident in 12h.Additional time ofexposure to PAN appear to increase the quantity of TRPC6 mRNA and protein in the mousepodocytes further.About 35% of the cells expressed EGFP.A up-regulation of proteinexpression of TRPC6 was detected in podocytes when transfected with pEGFP-N_1-mTRPC6 (P＜0.01).The distribution changes of Nephrin andα-actinin-4 were found ascompared to control groups.The reductions of the mRNA and the protein expression inNephrin were apparently detected by about 42% and 26%,respectively.The mRNA andprotein expression levels ofα-actinin-4 decreased by about 22% and 25%,respectively.(P＜0.05).The overexpression of TRPC6 had no effect on the expression ofα-tubulin butchanged the distribution of it.
Conclusion:The distribution of TRPC6 was changed after exposure toPAN.Exposureto PAN resulted in the stimulation of TRPC6 both in mRNA and protein level.Theoverexpression of TRPC6 interfere with the normal distribution and function of Nephrin,α-actinin-4 andα-tubulin.
PartⅡEffect of overexpression of TRPC6 on angiotensinⅡ-inducedapoptosis of mouse podocytes
Objective:To study the effect of overexpression of TRPC6 on AngⅡ-inducedapoptosis of mouse podocytes in vitro and to further explore the possible mechanisms.
Methods:Conditionally immortalized murine podocyte cell line was cultured underpermissive condition at 33℃.After passage the cells were shifted to non-permissivecondition at 37℃and cultrued for 14 days.Podocytes were treated by differentconcentrations of AngⅡand the cell viability was dected by methyl thiazolyl tetrazoliummethod(MTT).Mouse TRPC6 cDNA eukaryotic expression vector pEGFP-N_1-mTRPC6was transfeced to podocytes by liposome.The fluorescent microscopy was used to examinethe expression of EGFP after 48 hours.The change of TRPC6 protein expression wasobserved by Western-blot.The podocyte intracellular calcium concentration was measuredwith laser-scanning confocal microscope.Podocytes were divided into groups and differentconcentrations of AngⅡwere used.The expression of Bax and Bcl-2 mRNA was assessedby RT-PCR and the expression of Bax and Bcl-2 protein was measured by Western-blot.The apoptotic ratio of podocytes was monitored by flow cytometry and Hoechst33258staining.
Results:The cell viability han no change after treated with low-dose (10~(-10)mol/L) ofAngⅡbut decreased 50% by using high-dose (10~(-6)mol/L)of AngⅡfor 24 hours.About35% of the cells expressed EGFP.A up-regulation of protein expression of TRPC6 wasdetected in podocytes when transfected with pEGFP-N_1-mTRPC6(P＜0.01).Theoverexpression of TRPC6 promoted the AngⅡ-induced influx of extracellular calcium andelevated the expression of Bax but decreased the expression of Bcl-2(P＜0.01,P＜0.05).The apoptotic ratio of podocyte was (2.50±0.72)% on the effect of low-doseAngⅡ(10~(-10)mol/L),it was increased to (4.33±0.45)% when transfected with pEGFP-N_1 -mTRPC6(P＜0.05).Transfection with pEGFP-N_1-mTRPC6 increased apoptosis ratefrom (15.46±1.40)% to (18.33±0.87)% (P＜0.01) on the high-dose (10~(-6)mol/L) of AngⅡ.
Conclusion:TRPC6 plays an important role in the AngⅡ-induced apoptosis ofpodocytes.This effect can be attributed to its ability of promoting the influx of extracellularcalcium and initiating the apoptosis cascade.
PartⅢ
Effect of Down-regulation of TRPC6 on the Mouse Podocyte injuryinduced by Puromycin Aminonucleoside
Objective:To construct eukaryotic expression vectors carrying the small hairpinRNA(shRNA) for TRPC6 mRNA and observe the effect of knocking-down TRPC6 onPAN-induced injury of mouse podocytes.
Methods:Two DNA sequences containing small hairpin structure targeting TRPC6were designed and synthesized and were then inserted into the plasmid pGC containinggreen fluorescence protein (GFP) to form plasmid pGCsi-TRPC6A and pGCsi-TRPC6B.The inserted sequences were verified by enzyme digestion and DNA sequeneing.Plasmidexpressing irrelevant shRNA was used as negative control named PGCsi-NC.The plasmidswere transfeced to conditionally immortalized murine podocyte cell line by liposome.Thefluorescent microscopy was used to examine the expression of GFP after 24 and 48 hours.The changes of TRPC6 mRNA and protein expression were observed by RT-PCR andWestern-blot after 48 hours.Cultured podocytes were divided into four groups:controlgroup;PAN treatment group;PAN treatment+shRNA transfection group and PANtreatment+negative control group.The expression of Nephrin and CD2AP protein wasmeasured by Western-blot.The expression of Bax and Bcl-2 mRNA was assessed byRT-PCR and the expression of Bax and Bcl-2 protein was measured by Western-blot.The apoptotic ratio ofpodocytes was monitored by flow cytometry.
Results:The sequence of recombinant plasmids pGCsi-TRPC6A and pGCsi-TRPC6Bwas confirmed by further sequencing after extracted using plasmid extraction kit.Theresults showed that the inserted sequence of the pGCsi-U6-Neo-GFP-shRNA was the sameas the designed sequence.About 45% of the cells expressed GFP.A down-regulation ofmRNA and protein expression of TRPC6 was detected in mouse podocytes whentransfected with pGCsi-TRPC6A and pGCsi-TRPC6B(P＜0.05).Plasmid pGCsi-TRPC6Bwas used for subsequent study because of its higher efficiency of knocking-down theTRPC6 gene compared with plasmid pGCsi-TRPC6A.Compaired with the control group,dreased protein of Nephrin and CD2AP was detected in the cultured podocytes treated withPAN for 48 hours (P＜0.05).This effect was interfered by transfecting the plasmidpGCsi-TRPC6B into the podocytes(P＜0.05).The expression of Bax increased and theexpression of Bcl-2 decreased at protein and mRNA levels after treated with PAN for 48hours but knocking-down TRPC6 could prevent these changes.Knocking-down TRPC6could effectively decreased the PAN-induced apoptosis of podocytes.
Conclusion:The eukaryotic expression vectors carrying the small hairpin RNA(shRNA) for TRPC6 mRNA was successfully constructed.The reeombinant plasmid couldexpress stably and eode small interfering RNA in cells to make target genes silence.Knocking-down TRPC6 gene could effectively prevent the podocytes from injury inducedby PAN.
引文
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