导入外源肝细胞生长因子基因对肝细胞增殖的影响  

Proliferation of hepatocytes after delivery of exogenous hepatocyte growth factor gene

作　　者：林勇[1] 谢渭芬[1] 陈伟忠[1] 张新 曾欣[1] 陈岳祥[1] 杨秀疆[1] 张忠兵[1] 

机构地区：[1]第二军医大学长征医院消化内科,上海200003 [2]上海市国家人类基因组南方研究中心

出　　处：《中华肝脏病杂志》2003年第6期331-333,共3页Chinese Journal of Hepatology

基　　金：国家自然科学基金(30200122)

摘　　要：目的 利用重组腺病毒载体将外源人肝细胞生长因子(HGF)基因导入原代培养的大鼠肝细胞, 观察HGF表达对肝细胞增殖特性的影响。方法 同源重组构建表达HGF的复制缺陷型重组腺病毒AdHGF,用 其感染原代培养的肝细胞。逆转录聚合酶链反应检测肝细胞HGF和c—met(HGF受体)mRNA的表达;酶联免 疫吸附实验测定培养上清液中HGF水平。MTS测定感染前后细胞增殖情况;流式细胞仪测定细胞周期的变化; 细胞免疫荧光法检测HGF基因导入后增殖细胞核抗原(PCNA)表达。结果 同源重组获得约4×10^(10)efu/ml 滴度的AdHGF。AdHGF感染原代培养肝细胞后,肝细胞HGF和c-met mRNA表达均明显上调;细胞上清液 中HGF分泌水平显著增加,达到(5 939.00±414.39)pg/ml(F=13.661,P<0.01)。细胞增殖能力增强(F ≥15.158,P<0.01),细胞周期由G_0/G_1期向S期转化(X^2=41.616,P<0.01);细胞免疫荧光法提示HGF 基因导入后PCNA指数显著提高(F=42.122,P<0.01)。结论 通过重组腺病毒载体将外源HGF基因 导入肝细胞后可维持HGF高效表达并能促进肝细胞增殖,是基因修饰供体肝细胞、增强肝细胞移植治疗效 果的有效方法。Objective To explore the proliferation of primary cultured rats hepatocytes after delivery of exogenous hepatocyte growth factor (HGF) gene which was inserted into the genome of replication-deficient recombi-nant adenovirus vector. Methods The recombinant adenovirus-AdHGF which could express HGF was generated by homologous recombination. After the HGF gene was delivered into the hepatocytes, the expression of both HGF and c-met/HGF receptor mRNA in the cells was detected by RT-PCR and the level of HGF in the culture supernatant was also assayed by ELISA. On the other hand, cell proliferation was compared between before and after delivery of the HGF gene by MTS assay and the percentages of cell cycles were analyzed by flow cytometry. In addition, the expression of proliferating cell nuclear antigen (PCNA) was determined by immunocytofluorescent stain. Results 4 x 1010 efu/ml liter of AdHGF was obtained after recombination, RT-PCR indicated that the expression of HGF mRNA in hepatocytes increased on the third day after infected by the viruses and c-met/HGF receptor mRNA was also up-regulated. The HGF level in the culture supernatant assayed by ELISA was (5 939.0±414.39) pg/ml, which was much higher than that in the control (208.1 pg/ml ± 37.20 pg/ml, F = 13.661, P < 0.01). In addition, the proliferation of hepatocytes infected with AdHGF increased significantly according to MTS method (F > 15.158, P < 0.01) and more hepatocytes in G0/G1 stages changed into S stage ( x2= 41.616, P < 0.01), accordingly, PCNA index increased from 6.42 ± 1.88 to 14.56 ± 2.85 (F = 42.122, P < 0.01). Conclusions The results show that HGF gene delivered into hepatocytes by AdHGF can be expressed with high efficiency in the cells, which can stimulate hepatocytes proliferation. It may be an effective tool for hepatocyte transplantation by gene modified donor hepatocytes.

关 键 词：外源肝细胞生长因子 基因 肝细胞增殖 影响 肝移植 

分 类 号：R657.3[医药卫生—外科学]