垂体腺瘤双重靶向性基因治疗的实验研究

目的构建并实验评价转染与转录双重靶向性基因治疗系统对垂体腺瘤的治疗作用。方法构建GE7基因导入系统介导的生长激素启动子调控的基因治疗系统,通过对垂体生长激素腺瘤GH3细胞的体外实验,以及垂体腺瘤裸鼠模型的体内实验,观察此系统用于垂体腺瘤基因治疗的可能性和靶向性。结果成功构建双重靶向性基因治疗系统;基因转染后,GH3细胞可检测到HSVTK蛋白,对照细胞为阴性;在此基础上予以GCV(4mg/L),GH3细胞存活率平均为106%,相同条件下,U2OS、HO8910PM的存活率分别为829%和935%(P<001);裸鼠皮下种植肿瘤经基因治疗后,治疗组肿瘤体积明显缩小(168mm3±17mm3),各对照组肿瘤体积均有不同程度的增大(P<001);治疗组裸鼠的生存期也较对照组明显延长(平均123d和40d,P<001)。结论GE7转染的生长激素启动子调控的基因治疗有望成为垂体腺瘤的靶向性治疗策略。

Experimental study of dually targeting gene therapy system for pituitary adenomas

OBJECTIVE: To construct a dually targeting gene therapy system for pituitary adenomas and investigate its effect. METHODS: Promoter hGHp containing human growth hormone gene was obtained from human genome and cloned into the plasmid pcDNA3.1/His A with the promoter cut to construct the recombinant plasmid pcDNA3.1/His A-hGHp. HSV-TK gene was obtained from the plasmid pcDNA3.1/His A-TK and integrated into the plasmid pcDNA3.1/His A-hGHp to construct the recombinant plasmid pcDNA3.1/His A-hGHp-TK. A GE7 gene delivery system-mediated human growth hormone promoter controlled gene therapy system was constructed by adding the mixture of GE7-polylysine and HA20-polylysine into the DNA solution. Human growth hormone-secreting pituitary adenoma cells of the GH3 line, human myeloma cells of the U-2OS line, and human oophoroma cells of the HO8910PM line were cultured and transfected with PBS or GE7 packaged pcDNA3.1/HisA-TK or pcDNA3.1/HisA-hGHp-TK. Western blotting was used to examine the expression of PBS or GE7 packaged pcDNA3.1/HisA-TK or pcDNA3.1/HisA-hGHp-TK protein, MTT method was used to detect the cell survival rate. Another GH3, U-2OS, and HO8910PM cells were cultured and transfected with PBS or GE7 packaged pcDNA3.1/HisA-TK or pcDNA3.1/HisA-hGHp-TK and then ganciclovir (GCV) was added. MTT method was used to examine the cell survival rates. GH3 cells were injected subcutaneously into the right axilla of 200 SD nude rats loaded with human pituitary adenoma. Three weeks after the rats were randomly divided into 5 equal groups: PBS group in which PBS was injected into the tumor and GCV was injected peritoneally; GE7 group in which GE7-polylysine and HA20-polylysine were injected into the tumor and GCV was injected peritoneally; without TK group in which GE7-packaged pcDNA3.1/His A-hGHp was injected into the tumor and GCV was injected peritoneally; without GCV group in which GE7-packaged pcDNA3.1/His A-hGHp-TK was injected into the tumor and PBS was injected peritoneally; and treatment group in which GE7-packaged pcDNA3.1/His A-hGHp-TK was injected into the tumor and GCV was injected peritoneally. Peritoneal injection lasted 21 days for all groups. On the days 3, 7, 14, and 21 eight rats from each group were killed to measure the volume of tumor. The survival rate of the rest 8 rats was observed. RESULTS: A dually targeting gene therapy system for pituitary adenoma was composed successfully. HSV-TK protein was expressed in the GH3 cells but not in the U-2OS and HO8910PM cells after transfection of GE7-packaged pcDNA3.1/HisA-hGHp-TK; and was expressed in the GH3 and HO8910PM cells but not in the U-2OS cells after transfection of GE7-packaged pcDNA3.1/HisA-TK. Transfection of GE7-packaged pcDNA3.1/HisA-hGHp-TK and addition of GCV significantly decreased the survival rate of the GH3 cells, but did not influence the survival rates of the U-2OS and HO8910PM cells. Transfection of GE7-packaged pcDNA3.1/HisA-TK and addition of GCV significantly decreased the survival rate of GH3 and HO8910PM cells but did not influence the survival of the U-2OS and HO8910PM cells. When the GH3 cells were transfected with GE7-packaged pcDNA3.1/HisA-hGHp-TK with the addition of GCV of the concentration of 4 mg/L the survival rate decreased to 10%, when the GCV concentration was raised to 8 mg/L the survival rate of the GH3 cells was < 5%. Three days after the beginning of treatment the tumor volume of different groups of rats increased at different degrees and the tumor was smallest in the treatment group in comparison with the other groups (all P < 0.05). Seven days after the beginning of treatment the tumor volume of the treatment group significantly decreased and the tumors of the other groups still increased (all P < 0.001). The survival time of the treatment group was over 120 days, significantly longer than those of the other groups (all about 40 days). CONCLUSION: GE7 system-mediated hGHp controlled gene therapy system is hopeful to be the targeted therapeutic strategy for pituitary adenomas.