CCR1基因过表达对骨髓间充质干细胞靶向肝癌迁移能力的影响

Effect of CCRI gene overexpression on the migration of bone marrow- derived mesenchymal stem cells towards hepatocellular carcinoma

目的研究过表达人的CCR1（hCCR1）受体对人的骨髓间充质干细胞（hMSCs）靶向肝癌迁移能力的影响，探讨MSCs作为细胞载体在肝癌基因治疗中的应用前景。方法构建慢病毒表达质粒pLV—hCCR1，经293T细胞包装，收集病毒上清液，感染hMSCs。收集各组细胞，通过RT—PCR、Western blot分析各组细胞hCCR1mRNA及蛋白表达情况，并通过Transwell迁移实验研究hCCR1过表达对hMSCs体外迁移能力的影响。建立肝癌裸鼠原位模型，将荷瘤裸鼠随机分为2组，每组8只；通过尾静脉注射将hMSCs输入到荷瘤裸鼠体内，研究hCCR1过表达对hMSCs体内靶向肝癌迁移能力的影响。两组数据之间的比较采用独立样本t检验，多组间均数比较采用单因素方差分析。结果限制性内切酶酶切及测序表明，成功构建hCCR1慢病毒表达载体。以293T包装的重组慢病毒能够高效感染hMSCs，hMSCs感染重组慢病毒后可高效表达hCCR1。Transwell体外迁移实验表明，hCCR1基因转染的hMSCs向肝癌细胞条件培养基的趋向迁移能力明显增强[（134．8±15．7）／LPF对比（83．5±10．9）／LPF，t=10．40，P〈0．O0]。体内迁移实验显示，hMSCs经尾静脉注射后14d，到达小鼠肝癌部位的hMSCs—hCCR1显著多于hMSCs-RFP[（86．7±14．1）／HPF对比（54．5土9．6）／HPF，t=-7．32，P〈0．01]。结论CCR1基因过表达可显著增强骨髓MSCs靶向肝癌迁移的能力，提示开发迁移能力更强的细胞载体及MSCs的临床应用具有重要的意义。

Objective To evaluate the effect of human CCR1 （hCCR1） gene overexpression on the migration of human bone marrow-derived mesenchymal stem cells （hMSCs） towards hepatocellular carcinoma （HCC）, and to examine the application prospects of MSCs as gene delivery vectors in the treatment of HCC. Methods The hCCR1 gene was subcloned into a lentiviral vector to generate the recombinant plasmid pLV- hCCR1. The pLV-hCCR1 plasmid and two other packaging plasmids were co-transfected into 293T cells using calcium phosphate, and the virus-containing supematant was collected, hMSCs were then infected with the recombinant lentivirus, and the expression of hCCR1 mRNA and protein was analyzed by RT-PCR and Western blot, respectively. The effect of CCR1 gene overexpression on the in vitro migration of hMSCs was examined using the Transwell migration assay. Orthotopic nude mice models of HCC were established using the MHCC- 97H-GFP cell line, and the mice were divided into two groups （n = 8 per group）, hMSCs were then intravenously injected via the tail vein into the tumor-beating nude mice to examine the effect of hCCR1 overexpression on the in vivo migration of hMSCs towards HCC. Unpaired Student＇s t-test was used for two-group comparisons, and one-way ANOVA was used for multi-group comparisons. Results Restriction enzyme digestion and DNA sequencing demonstrated that the recombinant plasmid pLV-hCCR1 was constructed successfully. The LV-hCCR1 lentivirus packaged by 293T cells has high infection efficiency in hMSCs, and hCCR1 was overexpressed in hMSCs after LV-hCCR1 infection. Transwell migration assay showed that hCCR1transfected hMSCs had significantly enhanced migration towards HCC cell line-derived condition medium （CM） compared with the control RFP-hMSCs [（134.8±15.7）/LPF vs （83.5±10.9）/LPF, t = 10.40,P 〈 0.01]. In vivo migration experiment also demonstrated that there was significantly higher number of hCCRI-hMSCs localized within the MHCC-97H-GFP xenografts than hMSCs-RFP on day 14 following intravenous injection ofhMSCs in mice [（86.7±14.1）/HPF vs （54.5±9.6）/HPF, t = -7.32, P 〈 0.01]. Conclusion Overexpression of CCR1 gene can significantly enhance the migration capacity of hMSCs towards HCC cells in vitro and in vivo. This study provides evidence for potential clinical application of MSCs as more effective delivery vehicles in cancer gene therapy.