摘要
目的 探讨体外构建的反义金属蛋白酶组织抑制因子 (TIMP 1)表达质粒在大鼠肝星形细胞 (HSC)中的表达情况及对HSC活化后Ⅰ、Ⅲ型胶原量的影响。方法 双酶灌注和梯度离心法分离正常大鼠HSC ,培养 7～ 10d后活化为肌成纤维样母细胞表型。应用巢式RT PCR和基因重组技术构建能在真核细胞中表达的反义TIMP 1质粒并测序鉴定。应用脂质体介导重组质粒和 pcDNA3空质粒转染HSC ,经含 40 0 μg/mlG418的DMEM培养液筛选 3～ 4周 ,另设立空白对照组。Northernblot和Westernblot检测筛选后的HSC中TIMP 1表达情况 ;用FITC标记的Ⅰ型胶原为底物测定培养上清液内间质胶原酶活性 ;应用Westernblot方法对HSC中Ⅰ、Ⅲ型胶原进行半定量分析。结果 外源重组质粒能较大程度地抑制活化HSC中TIMP 1的表达 ,而pcDNA3空质粒和空白对照组则无类似结果。TIMP 1/GAPDH比值分别为 0 .67,2 .41和 2 .97(mRNA水平 ,P <0 .0 5)。TIMP 1/ β actin比值分别为0 .3 1,0 .98和 1.3 2 (蛋白质水平 ,P <0 .0 5)。外源重组质粒转染显著提高了间质胶原酶活性 ,酶活性分别为 0 .3 0 49,0 .14 11和 0 .1196(P <0 .0 5)。HSC中Ⅰ、Ⅲ型胶原量明显减少。Ⅰ型胶原 / β actin比值分别为 0 .63 ,1.78和 1.92 (P <0 .0 5)。Ⅲ型胶原 / β
Objective To study the expressing status of antisense tissue inhibitor of metalloproteina se-1(TIMP-1) in hepatic stellate cells (HSC) constructed in vitro, and to eval u ate the effects on the production of type Ⅰ and Ⅲ collagens secreted by activated rat HSC. Methods HSC were extracted from normal rat liver by pronase and co llagenase digestion and purified by centrifugal elutriation, and were cultured pla stic until they were activated to a myofibroblastic phenotype after 7-10 days. RT-nest-PCR and gene recombinant techniques were used to construct the rat ant isense TIMP-1 expression plasmid which can express in eukaryotic cells, and seg uenced after being counstructed. The expressing plasmid and the pcDNA3 empty pla smid were transfected into HSC by Effectene reagent separately. The cells were sel ected after growing in DMEM containing 400 μg/ml G418 for 3 to 4 weeks. Exp ression of TIMP-1 in HSC was d etermined by Northern blot and Western blot. We tested the interstitial collagen ase activity in culture media with FITC-labled type Ⅰ collagen as substrate. U ltimately, we quantified the typeⅠ and type Ⅲ collagen in HSC by Wester n blot. Results The exogenous antisense TIMP-1 recombinant plasmid could block the expression of TIMP-1 greatly, while there were not the same outcome i n pcDNA3 empty plasmid g roup and non-transfecting control group. The ratio of TIMP-1/GAPDH was 0.67, 2 .41 and 2.97 respectively at mRNA level( P <0.05); the ratio of TIMP-1/β-actin was 0.31, 0.98 and 1.32 respectively at protein leve l ( P <0.05). It may also elevate interstitial collagenase activity, the collage nase activity was 0.3049, 0.1411 and 0.1196 respectively( P <0.05), which lead s to decrease the amount of type Ⅰ, Ⅲ collagen in HSC. The ratio of collagen Ⅰ/β-actin was 0.63, 1.78 and 1.92 respectively ( P <0.05), and the ratio of collagen Ⅲ/β-actin was 0.59, 1.81 and 1.98 respectively( P < 0.05 ). Conclusions The antisense TIMP-1 recombinant plasmid could express in HSC stably,
出处
《中华消化杂志》
CAS
CSCD
北大核心
2004年第1期7-10,共4页
Chinese Journal of Digestion
基金
国家自然科学基金 ( 39970 339)
