神经菌毛素-1促进肝星状细胞活化对人肝癌HepG2细胞增殖、侵袭能力的影响

Neuropilin-1 promotes HepG2 proliferation, invasion and migration through potentiating the activity of hepatic stellate cells

目的 观察神经菌毛素-1（NRP-1）通过促进肝星状细胞（HSC）活化增殖进而对肝癌细胞增殖、侵袭及迁移能力的影响.方法 构建NRP-1腺病毒载体pDC315-NRP-1-3 Flag,感染LX2细胞;Western blot检测NRP-1蛋白表达水平,细胞免疫化学法检测LX2中α-平滑肌肌动蛋白（α-SMA）及NRP-1表达水平;通过噻唑蓝（MTT）、Transwell间接共培养检测LX2及HepG2细胞增殖能力,Transwell实验检测HepG2细胞侵袭和迁移能力.结果 构建人NRP-1基因重组过表达腺病毒pDC315-NRP-1-3Flag,最佳感染滴度为2.0×106 PFU/ml,48 h感染效率可达90％以上,Western blot示感染后LX2细胞NRP-1表达增高,转染48 h后NRP-1表达较高水平,细胞免疫化学显示转染组LX2的α-SMA及NRP-1表达增高.MTT检测显示NRP-1过表达组LX2增殖能力强于空载体组及未转染组,差异有统计学意义（P＜0.05）.细胞共培养实验证实LX2可促进HepG2的增殖、侵袭及迁移能力,NRP-1过表达组LX2对HepG2的促进作用更为明显,差异有统计学意义（P＜0.05）.结论 人NRP-1基因重组腺病毒可稳定转染LX2细胞,NRP-1可促进LX2的活化及增殖,继而促进HepG2细胞增殖及侵袭、迁移能力.

Objective To explore the effect of neuropilin-1 （NRP-1） on tumor cells proliferation,invasion and migration through activation of hepatic stellate cells in primary liver cancer.Methods An adenovirus vector containing human NRP-1 gene （pDC315-NRP-1-3Flag） was constructed at first,LX2 cells was infected with the adenovirus vector,the expression of NRP-1 in the LX2 was observed by Western blotting,the expression of α-smooth muscle actin （α-SMA） and NRP-1 in LX2 was observed by immunohistochemical staining.LX2 and HepG2 were cocultured,the proliferatio of LX2 and HepG2 were observed by methyl thiazol tetrazolium （MTT）,the invasion and migration of HepG2 was observed by Transwell chamber.Results The adenovirus vector containing human NRP-1 gene （pDC315-NRP-1-3Flag） was constructed successfully,the best virus titer for cell infection was 2.0 × 106 PFU/ml and the infection efficiency was above 90% after 48 h.Western blotting showed the expression of NRP-1 was higher in the transfection group than in the mock-vehicle and non-transfection groups,and the expression of NRP-1 was also higher after infected for 48 h than for 24 h.Immunohistochemical results showed that the expression of α-SMA and NRP-1 were higher in the experimental group than in the mock-vehicle and non-transfection groups.MTT results showed that the proliferation of LX2 was higher in the experimental group than in the two latter groups （P 〈 0.05）,the difference was significant.Coculture experiments showed that LX2 could promote proliferation,invasion and migration of HepG2,the promoting effect in the experimental group was significant higher than in the control groups （P 〈 0.05）.Conclusion LX2 cells could be infected with constructed adenovirus vector containing human NRP-1 gene stably.NRP-1 could promote activion and proliferation of LX2,which in turn promotes proliferation,migration and invasion of HepG2 cells.