骨髓间充质干细胞对小鼠Heps肝癌移植瘤组织作用的研究

Study on murine Heps hepatoma tissue after mesenchymal stem cells inoculation

目的观察骨髓间充质干细胞（MSC）接种于小鼠Heps肝癌移植瘤后局部免疫效应及肿瘤细胞促血管生成因子表达的变化，探讨MSC用于肝癌治疗的可能性及安全性。方法体外贴壁培养法制备昆明小鼠骨髓间充质干细胞，流式细胞仪鉴定MSC表型。4，6-二脒基-2-苯基吲哚（DAPI）对MSC进行标记备用。随机取54只8同龄雄性昆明小鼠建立Heps肝癌移植瘤模型。移植瘤长径达0．5～0．8cm时，随机分为MSC组、DAPI组、生理盐水（NS）对照组。MSC组及DAPI组分别向小鼠Heps肝癌移植瘤内注射2×10^6 MSC及DAPI标记的MSC，观察荷瘤小鼠生存期。利用免疫组化法检测肿瘤组织中CD4^＋、CD8^＋T及血管内皮生长因子（VEGF）的表达，并行常规组织学检查。结果MSC组平均生存期为45（95％C，33～56）d，NS对照组平均生存期为33（95％C1：28—37）d，两组比较差异有统计学意义（P〈0．05）。免疫组化检测发现接种MSC早期肿瘤组织CD4^＋、CD8^＋T细胞下降明显，接种MSC晚期肿瘤组织VEGF表达明显下调，组织坏死严重，荷瘤小鼠生存期延长。结论MSC可在原发性肝癌Heps荷瘤小鼠肿瘤组织内定植，MSC接种后早期降低CD4^＋T、CD8^＋T表达．抑制荷瘤小鼠T细胞免疫，促进肿瘤的生长；晚期下调VEGF表达，诱发肿瘤组织大片坏死，延长荷瘤小鼠的生存期。

Objective To observe the local immue response and changes of angiogenic factors of tumor cells in Hepsbearing mice after mesenchymal stem cell （ MSC ） are administrated. And to explore the feasibility and safety of MSC for liver tumors therapy. Methods MSC were obtained through adherent culture method. Phenotypes of MSC were analyzed by flow cytometry. MSC were labeled with DAPI in vitro. 54 Mice of 8 weeks of age with subcutaneously transplanted liver carcinomas were developed randomly. When the maximal diameters of the tumor reached 0. 5 ～0. 8cm,they were divided into three groups randomly：MSC group, DAPI group and NS control group. 2 × 10^6 MSC and MSC marked by DAPI were administrated into the mice right rear back tumor tissue. The survival time of the tumor-bearing mice was recorded and the mean survival time was calculated. Immunohistochemical staining was performed to count CD4 ＋T cells and CD8 ＋T cells in the local tumor,as well as to examine the expression of vascular endothelial growth factor （VEGF） in tumor cells. Results In the MSC group ,the mean survival time was 45 d （95% CI：33 ～ 56 d）, in the NS control group, the mean survival time was 33 d （95%（CI： 28 -37 d）. There was a statistical significance in the difference between them （ P 〈 0.05）. lmmunohistochemical staining results showed as follow ： the number of CD4^＋ T cells and CD8^＋ T cells in the MSC group decreased significantly in comparison with the NS control group at early stage. The expression of VEGF also decreased obviously in comparison with the NS control group and induced tumor cells necrosis at late stage. The survival time of MSC group was prolonged. Conclusion MSC can engraft in Heps-bearing tumor tissue, and inhibit T lymphocyte cellular immunity at early stage. It can reduce the number of CD4^＋T cells and CD8^＋ T cells and promote tumor growth. MSC can down regulate VEGF expression and induce tumor cells necrosis at late stage. By this way, it can prolong the survival time of Heps-bearing mice.