CAJ | 학술논문

目的探讨转化生长因子β1(TGF-β1)的RNA干扰质粒shRNA-TGF-β1对大鼠移植肾Smads信号通路的影响.方法以SD大鼠和Wistar大鼠分别作为供、受者,建立大鼠左肾原位移植及移植肾纤维化加快模型.将受者分为3组,质粒组受者肾移植后转染shRNA-TGF-β1质粒,空质粒组受者肾移植后转染空质粒,单纯移植组受者仅行肾移植,术后不转染任何质粒.另取健康Wistar大鼠作为假手术对照组,仅切除其左肾,不进行肾移植和转染任何质粒.质粒转染采用以流体力学为基础的肾脏基因转染技术.移植后1、2和3个月,采用酶联免疫吸附试验检测各组大鼠血清肌酐(SCr)和尿素氮(BUN)的水平,采用逆转录聚合酶链反应法和蛋白质印迹法分别检测各组大鼠移植肾组织中TGF-β1、磷酸化-Smad3(p-Smad3)和p-Smad7的mRNA和蛋白的表达.结果术后各检测时点,质粒组SCr和BUN水平显著高于假手术组(P＜0.01或P＜0.05),但明显低于空质粒组和单纯移植组(P＜0.05或P＜0.01);质粒组TGF-β1的表达显著高于假手术组(P＜0.01或P＜0.05),但显著低于空质粒组和单纯移植组(P＜0.05或P＜0.01);质粒组p-Smad3的表达显著高于假手术组(P＜0.01或P＜0.05),但明显低于空质粒组和单纯移植组(P＜0.01或P＜0.05);质粒组p-Smad7表达显著低于假手术组(P＜0.01或P＜0.05),但明显高于空质粒组和单纯移植组(P＜0.01或P＜0.05).结论质粒shRNA-TGF-β1能够显著改善大鼠的移植肾功能,其机制可能是shRNA-TGF-β1可以明显下调信号蛋白p-Smad3的表达,上调p-Smad7的表达,抑制TGF-β1的促纤维化作用,延缓移植肾的纤维化.
목적 탐토전화생장인자β1(TGF-β1)적RNA간우질립shRNA-TGF-β1대대서이식신Smads신호통로적영향.방법 이SD대서화Wistar대서분별작위공、수자,건립대서좌신원위이식급이식신섬유화가쾌모형.장수자분위3조,질립조수자신이식후전염shRNA-TGF-β1질립,공질립조수자신이식후전염공질립,단순이식조수자부행신이식,술후불전염임하질립.령취건강Wistar대서작위가수술대조조,부절제기좌신,불진행신이식화전염임하질립.질립전염채용이류체역학위기출적신장기인전염기술.이식후1、2화3개월,채용매련면역흡부시험검측각조대서혈청기항(SCr)화뇨소담(BUN)적수평,채용역전록취합매련반응법화단백질인적법분별검측각조대서이식신조직중TGF-β1、린산화-Smad3(p-Smad3)화p-Smad7적mRNA화단백적표체.결과 술후각검측시점,질립조SCr화BUN수평현저고우가수술조(P＜0.01혹P＜0.05),단명현저우공질립조화단순이식조(P＜0.05혹P＜0.01);질립조TGF-β1적표체현저고우가수술조(P＜0.01혹P＜0.05),단현저저우공질립조화단순이식조(P＜0.05혹P＜0.01);질립조p-Smad3적표체현저고우가수술조(P＜0.01혹P＜0.05),단명현저우공질립조화단순이식조(P＜0.01혹P＜0.05);질립조p-Smad7표체현저저우가수술조(P＜0.01혹P＜0.05),단명현고우공질립조화단순이식조(P＜0.01혹P＜0.05).결론 질립shRNA-TGF-β1능구현저개선대서적이식신공능,기궤제가능시shRNA-TGF-β1가이명현하조신호단백p-Smad3적표체,상조p-Smad7적표체,억제TGF-β1적촉섬유화작용,연완이식신적섬유화.
Objective To evaluate the effects of shRNA-TGF-β1 plasmid on Smads signal transduction of rat renal allograft.Methods A Sprague-Dawley to Wistar rat orthotopic transplant kidney-sclerosis accelerated model was constructed and transfected with short hairpin RNA-TGF-β1 based on the hydromechanics.The recipients were divided into three groups:group T(plasmid group)injected with shRNA-TGF-β1;group H(vacant plasmid group)injected with vacant plasmid;group Y(simply transplantation group)injected with no plasmid.In group J(sham-operated group)only right kidney was removed with no transplantation as control group.Transplanted kidneys and blood samples were collected at the first,second and third month after transplantation.The blood urea nitrogen(BUN)and serum Cr were tested by enzyme-linked immunoadsordent assay.The gene transcriptional level of TGF-β1 and Smad3/7 was detected by RT-PCR,and the protein variations of TGF-β1 and phosphorylated Smad3/7 were examined by Western blotting.Results At each test time point,the BUN and serum Cr were significantly higher in the plasmid group than in the sham-operated group(P<0.05 or P<0.01),but obviously lower than in the vacant plasmid group and simply transplantation group(P<0.05 or P<0.01).The expression of TGF-β1 as well as phosphorylated Smad3 was significantly higher in the plasmid group than in the sham-operated group(P<0.05 or P<0.01),but obviously lower than in the vacant plasmid group and simply transplantation group(P<0.05 or P<0.01).However,the expression of phosphorylated Smad7 was significantly lower in the plasmid group than in the sham-operated group(P<0.05 or P<0.01),but obviously higher than in the vacant plasmid group and simply transplantation group(P<0.05 or P<0.01).Conclusion Short hairpin RNA-TGF-β1 plasmid could significantly improve the renal function of rat renal allografts probably by downregulating phosphorylated Smad3 and upregulating phosphorylated Smad7,leading to the inhibition of TGF-beta 1 promoting fibrosis role and delay of the allograft fibrosis.