CAJ | 학술논문

目的探讨转化生长因子β_1(TGF-β_1)的RNA干扰质粒(shRNA-TGF-β_1)对大鼠移植肾细胞外基质生成的影响.方法预先构建shRNA-TGF-β_1.取SD大鼠肾脏,置于4℃肝素生理盐水中以强化缺血再灌注损伤,用于移植.切除Wistar大鼠左肾后移植入供肾,术中采用以流体力学为基础的肾脏基因转染技术进行质粒转染.实验分为4组.T组为质粒组,受者注射shRNA-TGF-β_1质粒;H组为空质粒组,受者注射空质粒;Y组为单纯移植组,受者仅行肾移植,不注射任何质粒;J组为假手术组,只打开腹腔切除左肾,不进行肾移植.移植术后1、2和3个月时,切取各组受者的移植肾,检测TGF-β_1、Ⅰ型胶原及其mRNA的表达,检测Ⅰ型胶原组织定位,观察移植肾细胞外基质的沉积.结果 J组大鼠肾脏组织中仅有少量的TGF-β_1 mRNA表达.H组及Y组TGF-β_1 mRNA的表达较高.术后1个月时,T组TGF-β_1 mRNA的表达显著低于其他各组,随着时间的延长,其表达有所升高,但仍显著低于H组及Y组.各组TGF-β_1的表达与TGF-β_1 mRNA的表达有相同的变化趋势.T组Ⅰ型胶原mRNA的表达低于H组和Y组.各组Ⅰ型胶原的表达与其mRNA的表达有相同的变化趋势,Ⅰ型胶原主要位于H组和Y组的皮质小管区及髓质间质区,肾小球部位相对较少.T组移植肾纤维化程度低于H组和Y组.结论转染shRNA-TGF-β_1能抑制移植肾TGF-β_1的表达,减少细胞外基质的生成,在一定程度上预防移植肾纤维化.
목적 탐토전화생장인자β_1(TGF-β_1)적RNA간우질립(shRNA-TGF-β_1)대대서이식신세포외기질생성적영향.방법 예선구건shRNA-TGF-β_1.취SD대서신장,치우4℃간소생리염수중이강화결혈재관주손상,용우이식.절제Wistar대서좌신후이식입공신,술중채용이류체역학위기출적신장기인전염기술진행질립전염.실험분위4조.T조위질립조,수자주사shRNA-TGF-β_1질립;H조위공질립조,수자주사공질립;Y조위단순이식조,수자부행신이식,불주사임하질립;J조위가수술조,지타개복강절제좌신,불진행신이식.이식술후1、2화3개월시,절취각조수자적이식신,검측TGF-β_1、Ⅰ형효원급기mRNA적표체,검측Ⅰ형효원조직정위,관찰이식신세포외기질적침적.결과 J조대서신장조직중부유소량적TGF-β_1 mRNA표체.H조급Y조TGF-β_1 mRNA적표체교고.술후1개월시,T조TGF-β_1 mRNA적표체현저저우기타각조,수착시간적연장,기표체유소승고,단잉현저저우H조급Y조.각조TGF-β_1적표체여TGF-β_1 mRNA적표체유상동적변화추세.T조Ⅰ형효원mRNA적표체저우H조화Y조.각조Ⅰ형효원적표체여기mRNA적표체유상동적변화추세,Ⅰ형효원주요위우H조화Y조적피질소관구급수질간질구,신소구부위상대교소.T조이식신섬유화정도저우H조화Y조.결론 전염shRNA-TGF-β_1능억제이식신TGF-β_1적표체,감소세포외기질적생성,재일정정도상예방이식신섬유화.
Objective To investigate the effects of shRNA-TGF-β_1 plasmid on extracellular matrix synthesis of rat renal allograft.Methods shRNA-TGF-β_1 was constructed.The donor's kidney resected from SD rats was put in 4℃ heparin saline in order to get enhanced ischemiareperfusion injury.The donor's kidney was transplanted to the Wistar rat after removing its left kidney,and transfected with the plasmid using renal gene transfection technology based on the hydromechanics.The recipients were divided into four groups:group T(plasmid group)injected with shRNA-TGF-β_1;group H(vacant plasmid group)injected with vacant plasmid;group Y(simply transplantation group) injected with no plasmid;group J (sham-operated group) only removing the right kidney with no transplantation.Transplanted kidneys and blood samples were collected at the first,second and third month after transplantation.The expression levels of TGF-β_1 and type Ⅰ collagen mRNA and protein were detected by RT-PCR and Western blot respectively.Immunohistochemical staining was used to assess the tissue location of typeⅠcollagen,and the fibrosis degree was assessed by Masson staining.Results A small amount of TGF-β_1 mRNA was detected in group J,but great amount in groups H and Y.At the first month,TGF-β_1 mRNA expression in group T was significantly lower than in other groups.Although its expression was increased with the time,but still significantly lower than in groups H and Y.Type Ⅰ collagen mRNA expression in group T was lower than in groups H and Y.The protein expression of type Ⅰ collagen was similar to its mRNA expression in all groups, mainly located at cortical tubules and medulla mesenchyme,and less at glomerulus in groups H and Y.The fibrosis in group T was significantly milder than in groups H and Y.Conclusion The shRNA-TGF-β_1 plasmid could inhibit the expression of TGF-β_1 and reduce the synthesis of extracellular matrix,which could prevent fibrosis of renal allograft to varying degrees.