腺病毒介导的HSV-tk基因治疗人脑胶质瘤SHG_(44)的研究

Adenovirus mediated HSV tk gene therapy for experimental human gliomas

目的 探讨带有ＨＳＶｔｋ 基因的重组腺病毒（Ａｄ．ｔｋ） 结合ＧＣＶ 对国人脑胶质瘤ＳＨＧ４４ 的治疗作用。方法 Ｘｇａｌ 染色比较带有报告基因ＬａｃＺ的重组腺病毒（Ａｄ．ＬａｃＺ） 转染ＳＨＧ４４ 细胞和大鼠脑胶质瘤Ｃ６ 细胞的效率，ＭＴＴ法比较Ａｄ．ｔｋ／ＧＣＶ 系统对ＳＨＧ４４ 和Ｃ６ 细胞的敏感性，ＤＮＡ片段分析细胞杀伤机制；在活体用Ａｄ．ｔｋ／ＧＣＶ 治疗ＳＨＧ４４ 移植瘤。结果 ＭＯＩ为５０ 时，Ａｄ．ＬａｃＺ感染ＳＨＧ４４ 细胞的效率近１００％ ，较Ｃ６ 细胞高；ＭＯＩ为１００ 时，Ａｄ．ｔｋ 感染的ＳＨＧ４４ 细胞对ＧＣＶ 的敏感性较Ｃ６ 细胞高，其半致死剂量（ＩＣ５０） 分别为０ ．１２ μｍｏｌ／Ｌ 和０ ．２６ μｍｏｌ／Ｌ，有凋亡机制参与其细胞杀伤；５ ×１０８ＰＦＵ 的Ａｄ．ｔｋ 肿瘤原位注射结合ＧＣＶ治疗，有效抑制ＳＨＧ４４ 移植瘤生长，肿瘤组织坏死，出血伴有纤维组织增生；与Ａｄ ．ｔｋ／ＰＢＳ组、Ａｄ．ＬａｃＺ／ＧＣＶ 组、ＰＢＳ组相比，肿瘤抑制效率分别为９２．５ ％ 、９２．６％ 、８９ ．５％ （ Ｐ＜０ ．０１） 。结论 Ａｄ．ｔｋ／ＧＣＶ系统对人脑胶质瘤细胞的感染效率和敏感性较高，治疗国人脑胶质瘤效果显著。

Objective Adenovirus mediated gene therapy using HSV tk and ganciclovir(GCV) treatment is examined for efficacy as the treatment for Chinese human gliomas SHG 44 . Methods The efficiency of Ad.LacZ infecting SHG 44 cells and rat C 6 glioma cells was studied by using X gal staining, the sensitivity of these two kinds of cells to Ad.tk/GCV system was studied by the MTT assay, the cells treated with Ad.tk/GCV analyzed by the method of DNA fragmentation, Subcutaneous SHG 44 gliomas were treated with Ad.tk/GCV system in vivo. Results The SHG 44 cells infected in vitro with Ad.LacZ at the multiplicity of infection (MOI) of 50 showed higher transduction efficacy than C 6 cells did, nearly all SHG 44 cells expressed β galactosidase. The SHG 44 cells infected with Ad.tk at MOI of 100 had higher sensitivity to GCV than C 6 cells with IC 50 values of 0.12 μmol/L and 0.26 μmol/L. Apoptosis was observed in the cells treated with Ad.tk/GCV. A target therapeutic Ad.tk dose of 5×10 8 PFU combined with 40mg·kg -1 ·d -1 GCV intraperitoneally resulted in tumor regression as compared with control animals (P<0.01). The absense of tumors cells and extensive necrosis were noted in the treated animals. Conclusion The human glioma cells are more sensitive and higher transduction to the treatment of Ad.tk/GCV system, and it has the potential efficacy in the treatment of human gliomas.