中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2012年
8期
576-580
,共5页
蒋玉凤%孙华丽%张建军%黄飞%刘加群
蔣玉鳳%孫華麗%張建軍%黃飛%劉加群
장옥봉%손화려%장건군%황비%류가군
肝硬化%转染%短发夹RNA%基质金属蛋白酶组织抑制因子-1%结缔组织生长因子
肝硬化%轉染%短髮夾RNA%基質金屬蛋白酶組織抑製因子-1%結締組織生長因子
간경화%전염%단발협RNA%기질금속단백매조직억제인자-1%결체조직생장인자
Liver cirrhosis%Transfection%Short hairpin RNA%Tissue inhibitor of metalloproteinase-1%Connective tissue growth factor
目的 研究结缔组织生长因子(CTGF)和基质金属蛋白酶组织抑制因子-1(TIMP-1)的短发夹RNA(shRNA)表达质粒分别及共转染肝星状细胞(HSC)对CTGF、TIMP-1、I型前胶原(PCI)mRNA表达及细胞外基质(ECM)分泌的影响. 方法 筛选并成功构建靶向大鼠CTGF和TIMP-1有效RNA干扰靶位的shRNA表达质粒,分别及共转染转化生长因子β1刺激活化的大鼠HSC-T6细胞,荧光定量PCR检测各组细胞中CTGF、TIMP-1、PCI mRNA的表达;放射免疫法分析细胞上清液中Ⅲ型前胶原(PCⅢ)、透明质酸(HA)和层黏连蛋白(LN)的含量.组间比较采取方差分析;多组间两两比较采用SNK-q检验. 结果 CTGFshRNA转染和双质粒共转染HSC-T6的CTGFmRNA相对表达量分别为0.59±0.03、0.62±0.01,与空白对照组、CTGFshRNA转染组的1和1.00±0.07比较,CTGF mRNA表达量均明显下降,F=66.515,P值均＜0.05,差异有统计学意义;而TIMP-1 shRNA组和双质粒共转染组的TIMP-1 mRNA相对表达量分别为0.66±0.04、0.68±0.03,与空白对照组、CTGFshRNA转染组的1和1.05±0.03比较,TIMP-1 mRNA表达量均明显下降,F=83.835,P值均＜0.05,差异有统计学意义;CTGFshRNA转染、TIMP-1shRNA转染和共转染组的PCI rnRNA相对表达量分别为0.55±0.02、0.57±0.02和0.41±0.01与空白对照组的1比较,PC I mRNA表达量均明显下降,F=709.905,P值均＜0.05,差异有统计学意义;双质粒联合转染对CTGF、TIMP-1、PC I的mRNA表达量抑制作用优于单质粒转染.CTGF shRNA转染、CIGF shRNA转染和双质粒共转染组的PCⅢ含量分别为(78.02±6.50) ng/ml、(79.03±5.47) ng/ml、(53.91±4.01)ng/ml,与空白对照组的(113.79±15.88)比较,PCⅢ含量均明显下降,P值均＜0.05,差异有统计学意义; CTGF shRNA转染、CTGF shRNA转染和双质粒共转染组的HA含量分别为(127.36±8.33)ng/ml、(116.55±3.37) ng/ml、(82.84±9.03) ng/ml,与空白对照组的(163.10±7.01)ng/ml比较,HA含量均明显下降,P值均＜0.05,差异有统计学意义CTGFshRNA转染、CTGF shRNA转染和双质粒共转染组的LN含量分别为(55.41±9.37)ng/ml、(49.77±6.70) ng/ml、(30.72±1.22) ng/ml,与空白对照组的(83.99±4.67) ng/ml比较,LN含量均明显下降,P值均＜0.05,差异有统计学意义.CTGF shRNA与TIMP-1 shRNA双质粒联合转染对PCⅢ、HA、HA分泌的抑制作用优于单质粒转染.结论 CTGF shRNA.TIMP-1 shRNA可明显抑制HSC的CTGF、TIMP-1、PCI基因表达及ECM的分泌,且shRNA联合干扰效果更显著,有望成为抗肝纤维化基因治疗的有效方法.
目的 研究結締組織生長因子(CTGF)和基質金屬蛋白酶組織抑製因子-1(TIMP-1)的短髮夾RNA(shRNA)錶達質粒分彆及共轉染肝星狀細胞(HSC)對CTGF、TIMP-1、I型前膠原(PCI)mRNA錶達及細胞外基質(ECM)分泌的影響. 方法 篩選併成功構建靶嚮大鼠CTGF和TIMP-1有效RNA榦擾靶位的shRNA錶達質粒,分彆及共轉染轉化生長因子β1刺激活化的大鼠HSC-T6細胞,熒光定量PCR檢測各組細胞中CTGF、TIMP-1、PCI mRNA的錶達;放射免疫法分析細胞上清液中Ⅲ型前膠原(PCⅢ)、透明質痠(HA)和層黏連蛋白(LN)的含量.組間比較採取方差分析;多組間兩兩比較採用SNK-q檢驗. 結果 CTGFshRNA轉染和雙質粒共轉染HSC-T6的CTGFmRNA相對錶達量分彆為0.59±0.03、0.62±0.01,與空白對照組、CTGFshRNA轉染組的1和1.00±0.07比較,CTGF mRNA錶達量均明顯下降,F=66.515,P值均＜0.05,差異有統計學意義;而TIMP-1 shRNA組和雙質粒共轉染組的TIMP-1 mRNA相對錶達量分彆為0.66±0.04、0.68±0.03,與空白對照組、CTGFshRNA轉染組的1和1.05±0.03比較,TIMP-1 mRNA錶達量均明顯下降,F=83.835,P值均＜0.05,差異有統計學意義;CTGFshRNA轉染、TIMP-1shRNA轉染和共轉染組的PCI rnRNA相對錶達量分彆為0.55±0.02、0.57±0.02和0.41±0.01與空白對照組的1比較,PC I mRNA錶達量均明顯下降,F=709.905,P值均＜0.05,差異有統計學意義;雙質粒聯閤轉染對CTGF、TIMP-1、PC I的mRNA錶達量抑製作用優于單質粒轉染.CTGF shRNA轉染、CIGF shRNA轉染和雙質粒共轉染組的PCⅢ含量分彆為(78.02±6.50) ng/ml、(79.03±5.47) ng/ml、(53.91±4.01)ng/ml,與空白對照組的(113.79±15.88)比較,PCⅢ含量均明顯下降,P值均＜0.05,差異有統計學意義; CTGF shRNA轉染、CTGF shRNA轉染和雙質粒共轉染組的HA含量分彆為(127.36±8.33)ng/ml、(116.55±3.37) ng/ml、(82.84±9.03) ng/ml,與空白對照組的(163.10±7.01)ng/ml比較,HA含量均明顯下降,P值均＜0.05,差異有統計學意義CTGFshRNA轉染、CTGF shRNA轉染和雙質粒共轉染組的LN含量分彆為(55.41±9.37)ng/ml、(49.77±6.70) ng/ml、(30.72±1.22) ng/ml,與空白對照組的(83.99±4.67) ng/ml比較,LN含量均明顯下降,P值均＜0.05,差異有統計學意義.CTGF shRNA與TIMP-1 shRNA雙質粒聯閤轉染對PCⅢ、HA、HA分泌的抑製作用優于單質粒轉染.結論 CTGF shRNA.TIMP-1 shRNA可明顯抑製HSC的CTGF、TIMP-1、PCI基因錶達及ECM的分泌,且shRNA聯閤榦擾效果更顯著,有望成為抗肝纖維化基因治療的有效方法.
목적 연구결체조직생장인자(CTGF)화기질금속단백매조직억제인자-1(TIMP-1)적단발협RNA(shRNA)표체질립분별급공전염간성상세포(HSC)대CTGF、TIMP-1、I형전효원(PCI)mRNA표체급세포외기질(ECM)분비적영향. 방법 사선병성공구건파향대서CTGF화TIMP-1유효RNA간우파위적shRNA표체질립,분별급공전염전화생장인자β1자격활화적대서HSC-T6세포,형광정량PCR검측각조세포중CTGF、TIMP-1、PCI mRNA적표체;방사면역법분석세포상청액중Ⅲ형전효원(PCⅢ)、투명질산(HA)화층점련단백(LN)적함량.조간비교채취방차분석;다조간량량비교채용SNK-q검험. 결과 CTGFshRNA전염화쌍질립공전염HSC-T6적CTGFmRNA상대표체량분별위0.59±0.03、0.62±0.01,여공백대조조、CTGFshRNA전염조적1화1.00±0.07비교,CTGF mRNA표체량균명현하강,F=66.515,P치균＜0.05,차이유통계학의의;이TIMP-1 shRNA조화쌍질립공전염조적TIMP-1 mRNA상대표체량분별위0.66±0.04、0.68±0.03,여공백대조조、CTGFshRNA전염조적1화1.05±0.03비교,TIMP-1 mRNA표체량균명현하강,F=83.835,P치균＜0.05,차이유통계학의의;CTGFshRNA전염、TIMP-1shRNA전염화공전염조적PCI rnRNA상대표체량분별위0.55±0.02、0.57±0.02화0.41±0.01여공백대조조적1비교,PC I mRNA표체량균명현하강,F=709.905,P치균＜0.05,차이유통계학의의;쌍질립연합전염대CTGF、TIMP-1、PC I적mRNA표체량억제작용우우단질립전염.CTGF shRNA전염、CIGF shRNA전염화쌍질립공전염조적PCⅢ함량분별위(78.02±6.50) ng/ml、(79.03±5.47) ng/ml、(53.91±4.01)ng/ml,여공백대조조적(113.79±15.88)비교,PCⅢ함량균명현하강,P치균＜0.05,차이유통계학의의; CTGF shRNA전염、CTGF shRNA전염화쌍질립공전염조적HA함량분별위(127.36±8.33)ng/ml、(116.55±3.37) ng/ml、(82.84±9.03) ng/ml,여공백대조조적(163.10±7.01)ng/ml비교,HA함량균명현하강,P치균＜0.05,차이유통계학의의CTGFshRNA전염、CTGF shRNA전염화쌍질립공전염조적LN함량분별위(55.41±9.37)ng/ml、(49.77±6.70) ng/ml、(30.72±1.22) ng/ml,여공백대조조적(83.99±4.67) ng/ml비교,LN함량균명현하강,P치균＜0.05,차이유통계학의의.CTGF shRNA여TIMP-1 shRNA쌍질립연합전염대PCⅢ、HA、HA분비적억제작용우우단질립전염.결론 CTGF shRNA.TIMP-1 shRNA가명현억제HSC적CTGF、TIMP-1、PCI기인표체급ECM적분비,차shRNA연합간우효과경현저,유망성위항간섬유화기인치료적유효방법.
Objective To investigate the effect of short hairpin RNA (shRNA)-mediated silencing of CTGF and TIMP-1 in hepatic stellate cells (HSCs) on mRNA expression of TIMP-1,CTGF,and procollagen type-I (PC I),as well as secretion of extracellular matrix (ECM) proteins.Methods Two recombinant expression plasmids harboring shRNAs against CTGF and TIMP-1 (psiRNA-GFP-CTGF and psiRNA-GFP-TIMP-1) were transfected alone or together into TGFβ1-activated HSC-T6 cells.The mRNA expression levels of CTGF,TIMP-1,and PC I were detected by fluorescence quantitative PCR (FQ-PCR).The concentrations of secreted PC type-Ⅲ,hyaluronate (HA),and laminin (LN) were measured by radioimmonoassay (RIA) of culture supernatants.Results FQ-PCR analysis showed that CTGFshRNA and TIMP-(l)shRNA specifically inhibited the expression of CTGF,TIMP-1,and PC I mRNA in activated HSC-T6 cells.The concentrations of secreted PC Ⅲ,HA,and LN were decreased significantly in HSC-T6 cells with shRNA-silenced CTGF or TIMP-1 (P＜0.01 orP＜0.05).Moreover,HSC-T6 cells with shRNA-silenced CTGF and TIMP- 1 showed a more robust decrease in synthesis of PC Ⅲ,HA and LN (all,P＜0.01),as well as in mRNA expression of PC I (P＜0.05).Conclusion CTGFshRNA and TIMP-1 shRNA effectively inhibit expression of the respective target genes,as well as of PC I,and decrease secretion of ECM components from HSC-T6 cells.Silencing of both CTGF and TIMP-1 produces more robust effects than either in isolation.These data suggest that CTGF and TIMP-1 may be effective targets of shRNA-based gene therapy to treat liver fibrosis.
