稳定表达HBx的人肝星形细胞系的建立及其生物学特性初探

目的建立稳定表达HBVX蛋白（HBx）的LX-2细胞系，并初步研究HBx对于肝星形细胞（HSC）的增殖及其I型胶原蛋白表达的影响。方法构建包含HBx基因的重组慢病毒载体LV5-X，并转染LX-2细胞，同时用对照质粒转染LX-2细胞，经嘌呤霉素筛选后获得HBx稳定表达的LX-2-X和对照LX-2-C细胞系。荧光显微镜下观察感染效率，Western印迹检测细胞株中HBx的表达。在LX-2细胞系的生物学特性探索方面，应用CCK_8法检测细胞的增殖；Real—timePCR检测细胞中I型胶原蛋白mRNA的表达；ELISA法愉测细胞培养上清中I型胶原蛋白的表达。结果成功构建重组慢病载体LV5-X，感染LX-2细胞后建立细胞系LX-2-X和LX-2-C，嘌呤霉素筛选3d后，荧光显微镜显示近100％细胞发出绿色荧光，Western印迹证实LX-2-X细胞稳定表达HBx。培养72h和96h后LX-2-C细胞增殖明显高于LX-2和LX-2-C细胞组（P〈0．05）；PCR证实LX-2X细胞I型胶原蛋白mRNA合成高于LX-2和LX-2-C细胞组（P〈0．05）；ELISA结果显示LX一2一X细胞I型胶原蛋白表达高于LX-2和LX-2-C细胞组（P〈0．05）。结论成功构建了稳定表达HBx的LX-2细胞系；HBx能够促进HSC的增殖，并增加I型胶原蛋白的表达。

Establishment of hepatic stellate cell line with stable expression of HBx protein and its preliminary characterization

Objective To establish a LX-2 cell line with stable expression of HBV X protein （HBx） and preliminary study the effect of HBx to hepatic stellate cell （HSC） proliferation and collagen I expression. Methods A recombinant lentivirus vector LV5-X and a vehicle control plasmid were constructed, both of which subsequently transfeeted into LX-2 cells. Then a stable HBx-expressing LX-2-X cell line and a control LX-2-C cell line were established after puromysin selection. The transfection rate was determined by fluorescene microscope and HBx expression was detected by westeru blot. For the preliminary characterization of the LX-2 cell line, the cell proliferation was detected by CCK-8, the mRNA expression of collagen I was qualified by Real-time PCR and the collagen I secretion was measured by ELISA. Results The recombinant lentivirus LV5-X was successfully constructed and then infected LX-2 cell to establish the cell lines named LX-2-X and LX-2-C. After selection of puromysin for 3 d, approximately 100% of the LX-2-X cells expressed green fluorescent protein（GFP） and western blot showed that LX-2-X cell line expressed HBx stably. LX-2-X cells showed the higher proliferation compared to LX-2 and LX-2-C cells after 72 h and 96 h （P〈0.05）. The collagen I expression of LX-2-X cells at both mRNA and protein level were higher than LX-2 and LX-2-C cells （P〈0.05）. Conclusions The LX-2 cell line named LX- 2-X which can express HBx stably is successfully established. HBx could promote the proliferation of HSC and inerease the expression of collagen 1.