中华急诊医学杂志
中華急診醫學雜誌
중화급진의학잡지
CHINESE JOURNAL OF EMERGENCY MEDICINE
2010年
6期
615-620
,共6页
郎明健%闵新文%李健%郭敏%杨汉东
郎明健%閔新文%李健%郭敏%楊漢東
랑명건%민신문%리건%곽민%양한동
结缔组织生长因子%肾脏纤维化%RNA干扰%细胞外基质%纤维连接蛋白%质粒%胶原%转染
結締組織生長因子%腎髒纖維化%RNA榦擾%細胞外基質%纖維連接蛋白%質粒%膠原%轉染
결체조직생장인자%신장섬유화%RNA간우%세포외기질%섬유련접단백%질립%효원%전염
Connective tissue growth factor%Renal fibrosis%RNA interference%Extracellular matrix%Fi-bronectin%Collagen%Plasmid%Transfection
目的 研究RNA干扰靶向抑制结缔组织生长因子(CTGF)对高血压大鼠肾脏纤维化指标的影响.方法 本研究于2006-2008年在武汉协和医院心血管病研究所完成,实验以自发性高血压大鼠(SHR)为动物模型,随机(随机数字法)分为未干预的SHR组(n=10)和RNA干扰组(RNAi组,n=10),并设置WKY大鼠(n=8)为正常对照组.构建并筛选出靶向大鼠CTGF的RNAi质粒重组体,通过升主动脉钳夹冠脉灌注法联合尾静脉注射将质粒转染至大鼠体内,RNA干扰结束后,获取肾脏标本,RT-PCR和Western blotting检测肾脏组织CTGF及纤维连接蛋白(FN)的mRNA和蛋白表达,免疫组化技术分析CTGF及FN在肾脏的空间表达.0.1%天狼星红-饱和苦味酸胶原染色分析肾组织胶原类型及代谢水平(以胶原容积分数CVF表示),比色法测定肾脏组织羟脯氨酸含量.所有数据以(x-±s)表示,多个均数的组间比较采用单因素方差分析,以P<0.05为有统计学意义.结果 RNA干扰使高血压大鼠肾脏组织CTGF的mRNA和蛋白表达分别下调66%和62%(P<0.01),FN mRNA和蛋白的表达也相应降低达56%和51%(P<0.01);免疫组化显示了同样的抑制效应,而且观察到CTGF及FN不同的空间表达,CTGF在肾实质和间质中均可表达,而FN主要在肾脏间质表达.天狼星红染色显示RNA干扰组胶原沉积减少,偏光显微镜下进一步观察到RNA 干扰主要抑制了I型胶原的合成;肾脏组织羟脯氨酸含量在RNA干扰组显著减少[SHR组:(0.596±0.067)μg/mg,RNAi组:(0.368±0.084)μg/mg,P<0.01].结论 C-CTGF靶向性RNA干扰显著下调肾组织中CTGF的表达并相应抑制细胞外基质在肾间质的沉积,且这种效应独立于降压之外.提示CTGF在肾脏纤维化发生及进展中起着关键作用.
目的 研究RNA榦擾靶嚮抑製結締組織生長因子(CTGF)對高血壓大鼠腎髒纖維化指標的影響.方法 本研究于2006-2008年在武漢協和醫院心血管病研究所完成,實驗以自髮性高血壓大鼠(SHR)為動物模型,隨機(隨機數字法)分為未榦預的SHR組(n=10)和RNA榦擾組(RNAi組,n=10),併設置WKY大鼠(n=8)為正常對照組.構建併篩選齣靶嚮大鼠CTGF的RNAi質粒重組體,通過升主動脈鉗夾冠脈灌註法聯閤尾靜脈註射將質粒轉染至大鼠體內,RNA榦擾結束後,穫取腎髒標本,RT-PCR和Western blotting檢測腎髒組織CTGF及纖維連接蛋白(FN)的mRNA和蛋白錶達,免疫組化技術分析CTGF及FN在腎髒的空間錶達.0.1%天狼星紅-飽和苦味痠膠原染色分析腎組織膠原類型及代謝水平(以膠原容積分數CVF錶示),比色法測定腎髒組織羥脯氨痠含量.所有數據以(x-±s)錶示,多箇均數的組間比較採用單因素方差分析,以P<0.05為有統計學意義.結果 RNA榦擾使高血壓大鼠腎髒組織CTGF的mRNA和蛋白錶達分彆下調66%和62%(P<0.01),FN mRNA和蛋白的錶達也相應降低達56%和51%(P<0.01);免疫組化顯示瞭同樣的抑製效應,而且觀察到CTGF及FN不同的空間錶達,CTGF在腎實質和間質中均可錶達,而FN主要在腎髒間質錶達.天狼星紅染色顯示RNA榦擾組膠原沉積減少,偏光顯微鏡下進一步觀察到RNA 榦擾主要抑製瞭I型膠原的閤成;腎髒組織羥脯氨痠含量在RNA榦擾組顯著減少[SHR組:(0.596±0.067)μg/mg,RNAi組:(0.368±0.084)μg/mg,P<0.01].結論 C-CTGF靶嚮性RNA榦擾顯著下調腎組織中CTGF的錶達併相應抑製細胞外基質在腎間質的沉積,且這種效應獨立于降壓之外.提示CTGF在腎髒纖維化髮生及進展中起著關鍵作用.
목적 연구RNA간우파향억제결체조직생장인자(CTGF)대고혈압대서신장섬유화지표적영향.방법 본연구우2006-2008년재무한협화의원심혈관병연구소완성,실험이자발성고혈압대서(SHR)위동물모형,수궤(수궤수자법)분위미간예적SHR조(n=10)화RNA간우조(RNAi조,n=10),병설치WKY대서(n=8)위정상대조조.구건병사선출파향대서CTGF적RNAi질립중조체,통과승주동맥겸협관맥관주법연합미정맥주사장질립전염지대서체내,RNA간우결속후,획취신장표본,RT-PCR화Western blotting검측신장조직CTGF급섬유련접단백(FN)적mRNA화단백표체,면역조화기술분석CTGF급FN재신장적공간표체.0.1%천랑성홍-포화고미산효원염색분석신조직효원류형급대사수평(이효원용적분수CVF표시),비색법측정신장조직간포안산함량.소유수거이(x-±s)표시,다개균수적조간비교채용단인소방차분석,이P<0.05위유통계학의의.결과 RNA간우사고혈압대서신장조직CTGF적mRNA화단백표체분별하조66%화62%(P<0.01),FN mRNA화단백적표체야상응강저체56%화51%(P<0.01);면역조화현시료동양적억제효응,이차관찰도CTGF급FN불동적공간표체,CTGF재신실질화간질중균가표체,이FN주요재신장간질표체.천랑성홍염색현시RNA간우조효원침적감소,편광현미경하진일보관찰도RNA 간우주요억제료I형효원적합성;신장조직간포안산함량재RNA간우조현저감소[SHR조:(0.596±0.067)μg/mg,RNAi조:(0.368±0.084)μg/mg,P<0.01].결론 C-CTGF파향성RNA간우현저하조신조직중CTGF적표체병상응억제세포외기질재신간질적침적,차저충효응독립우강압지외.제시CTGF재신장섬유화발생급진전중기착관건작용.
Objective To investigate the impact on renal fibrosis by inhibition of connective tissue growth factor( CTGF) by RNA interference in spontaneous hypertension rat( SHR) . Method Twenty SHR were randomly (random number) divided into SHR group ( n = 10) and RNAi group ( n = 10), eight Wistar-Kyoto rats were set as control. At the end of RNA interference procedure, all the rats were sacrificed and the kidneys were harvested. The mRNA and plasmosin of CTGF and fibronectin(FN) of renal tissue were extracted and measured by RT-PCR and Western Blotting. And the localization of CTGF and FN were analyzed with immunohistochernistry technique. The collagen deposition(shown as collagen volume traction, CVF) were evaluated with 0.1% sirius-picric staining, and the hydroxyproline of myocardium were detected by colorimetry. Results The mRNA and protein expression of CTGF decreased 66% and 62% in RNAi group (P < 0.01). The mRNA and protein expression of FN decreased 56% and 51% in RNAi group.The same inhibition effect was observed by hislological analysis. Immuno-histochemistry showed that CTGF localized both in renal parenchyma and renal interstitium, whereas FN majorly expressed in renal interstitium. Observation with light microscope showed that collagen deposition(CVF)decreased sharply in RNAi group versus SHR group. And the same effect was viewed in hydroxypnoline assay[SHR group: (0.596 ± 0.067) μg/mg, RNAi group: (0.368±0.084) μg/mg, P < 0.01 ] .Further study by polarized microscope displayed that RNA interference mainly suppressed type I collagen synthesis. Conclusions Targeted inhibition of CTGF by RNA interference leads significant decrease of extracellular matrix deposition in kidney. And the anti-fibrotic effect independent of lower the blood pressure. This study indicated CTGF take a key role in the development and progress of renal fibrosis.
