采用sPD-1协同4-1 BBL进行肿瘤免疫基因治疗的研究  

The Immunogene Therapy with Soluble PD-1 and 4-1BBL for Hepato-ceUuar Carcinoma

作　　者：耿婷[1] 张桂梅[1] 黄波[1] 肖晗[1] 冯作化[1] 

机构地区：[1]华中科技大学同济医学院基础医学院生物化学与分子生物学系,武汉市430030

出　　处：《医学分子生物学杂志》2007年第3期209-214,共6页Journal of Medical Molecular Biology

基　　金：国家自然科学基金(No.30471587);国家重点基础研究发展计划(973计划)(No.2002CB513100)~~

摘　　要：目的探讨采用sPD-1协同4-1BBL进行肿瘤免疫基因治疗的效果及相关的免疫学机制。方法以不同剂量的H22肝癌细胞接种于BALB/c小鼠右后腿肌肉内,建立小鼠肿瘤模型;采用可溶性PD-1 (sPD-1)和4-1BBL真核表达质粒体内转染进行基因治疗;观察接种不同剂量肿瘤细胞、不同治疗时间小鼠的成瘤率及肿瘤治疗效果;RT-PCR检测肿瘤微环境中免疫调控相关基因的表达;组织切片检测肿瘤细胞浸润肌肉组织的组织学变化;流式细胞仪检测脾脏细胞毒性T细胞(CTLs)的杀瘤效率。结果转染4- 1BBL/sPD-1基因治疗后,接种低剂量(1×10~4个/ml)H22肿瘤细胞的小鼠肿瘤生长完全受到抑制;接种高剂量(1×10~5个/ml)H22肿瘤细胞的小鼠肿瘤也受到显著抑制。通过延长基因治疗,荷瘤小鼠的成瘤率随着治疗时间的延长逐渐递减,至8周时成瘤率为0;基因治疗不仅促进IFN-γ和IL-2基因表达上调,而且也使TGF-β、IL-10的表达下调;瘤组织中CD8^+ T淋巴细胞数量增多和脾淋巴细胞的杀瘤效率显著增加。结论利用体内存在的少量肿瘤可作为抗原刺激淋巴细胞的激活;基因治疗适用于对手术、化疗、放疗后体内残存的少量肿增细胞的清除;当体内存在大量肿瘤细胞时,适当延长基因治疗时间可获得较好的治疗效果。Objective The purpose of this study is to investigate the synergistic effects of soluble PD-1 used in combination with 4-1BBL in the treatment of hepatocelluar carcinoma and the possible mechanism. Methods BALB/c mice were inoculated i.m. to establish different hepatoma models. Soluble PD-1 （ sPD-1 ） and 4-1BBL were used for in vivo transfection for gene therapy. The tumor-generating rate and treatment effect at different dosese and time points were observed. RT-PCR was used for the detection of immunoegulation-related genes in tumorous environment; the histological changes in muscle tissues were observed by pathological section; the tumor-killing efficiency of CTLs was detected by flow cytometry. Results After transfection with 4-1BBL/ SPD-1, the tumors inoculated with low-dose （ 1 × 10^4 ） H22 tumor cells was completely inhibited in mice and the tumors inoculated with high-dose （ 1 × 10^5 ） H22 tumor cells were also significantly inhibited. By prolonged gene therapy, the tumor-generating rate in tumor-loaded mice was lowered progressively with the treatment time and the rate was 0 at the 8th week. Gene therapy not only can up-regulate the expression of IFN-γ/and IL-2 genes but also down-regulate the expression of TGF-β and IL-IO. The number of CD8^＋ T cells in tumor tissues and the tumor-killing rate of lymphocytes in spleen were significantly increased. Conclusion The small amount of remnant tumor cells can be used as antigens to generate tumor-specific T cells. Gene therapy is indicated for the removal of the few tumor cells left in body after operative, chemotherapeutic and irradiation treatment. When large amount of tumor cells are involved, prolonged gene therapy can achieve better therapeutic results.

关 键 词：肿瘤坏死因子4-1 BBL SPD-1 共刺激分子 基因治疗 肝癌 

分 类 号：R349.83[医药卫生—基础医学]