脂联素对LX-2细胞COL-Ⅰ表达的调节及其机制

Adiponectin down-regulates COL-I expression in human hepatic stellate cells

目的:研究全长脂联素真核表达质粒对体外培养人类肝星状细胞株(LX-2)细胞,Ⅰ型胶原(collagenⅠ,COL-Ⅰ)基因及蛋白的影响,探讨全长脂联素对细胞凋亡的作用及其调节COL-Ⅰ表达的作用机制.方法:实验分为对照组、空质粒组、全长脂联素真核表达质粒组,各组转染48h后Real-timePCR方法检测COL-ⅠmRNA的表达,ELISA法检测COL-Ⅰ蛋白水平的表达,MTT检测细胞存活率变化,Annexin V-FITC检测细胞凋亡率变化,Western blot检测细胞凋亡蛋白caspase-3的变化.结果:与空质粒组及对照组相比,全长脂联素真核表达质粒组COL-ⅠmRNA及蛋白的表达下降(179.00ng/L±16.83ng/Lvs532.30ng/L±27.52ng/L,570.00ng/L±16.12ng/L,均P<0.01),细胞存活率下降(65.70%±1.56%vs93.15%±1.90%,95.82%±2.52%,均P<0.01),凋亡率增加(14.70%±2.34%vs1.60%±0.23%,1.80%±0.15%,均P<0.01),caspase-3活性蛋白的表达增加(0.62±0.09vs0.21±0.04,0.22±0.07,均P<0.01).结论:脂联素可下调LX-2细胞COL-Ⅰ表达而抑制肝纤维化,其机制可能是通过抑制LX-2细胞增殖及增加caspase-3蛋白的途径促进其凋亡.

AIM：To observe the effect of transfection of full-length adiponectin cDNA on the expression of collagen I（COL-I）in human hepatic stellate cells（LX-2）and to explore possible mechanisms involved.METHODS：LX-2 cells were transfected with the empty plasmid（P7）or the recombinant plasmid carrying the full-length adiponectin cDNA.Forty-eight hours later,the expression of COL-I mRNA and protein was detected by real-time PCR and ELISA;cell survival was detected by MTT assay;apoptosis was detected by annexin V-FITC staining;and the changes in caspase-3 protein expression were detected by Western blot.RESULTS：Compared to cells transfected with the empty plasmid and untransfected cells,the expression of COL-I mRNA and protein（179.00ng/L±16.83 ng/L vs 532.30 ng/L±27.52 ng/L,570.00 ng/L±16.12 ng/L,both P0.01）were significantly down-regulated,cell viability decreased（65.70%±1.56%vs 93.15%±1.90%,95.82%±2.52%,both P0.01）,apoptosis rate increased（14.70%±2.34%vs 1.60%±0.23%,1.80% ±0.15%,both P0.01）,and the expression of caspase-3 was up-regulated（0.62±0.09 vs 0.21 ±0.04,0.22±0.07,both P0.01）in cells transfected with the recombinant plasmid.CONCLUSION：Adiponectin can down-regulate COL-I expression,inhibit cell proliferation,in-duce caspase-3-mediated apoptosis,and thus restrain liver fibrosis.