单纯疱疹病毒胸苷激酶基因在肺癌基因治疗实验中的初步探讨

Experimental studies on human lung cancer gene therapy with HSV TK/GCV system

目的探讨单纯疱疹病毒胸苷激酶（ＨＳＶＴＫ）基因在肺腺癌基因治疗中的应用。方法对转导了ＨＳＶＴＫ基因的感染人肺腺癌细胞Ａ５４９（Ａ５４９／ＴＫ）和野生型Ａ５４９，采用噻唑蓝（ＭＴＴ）法测定不同浓度丙氧鸟苷（ＧＣＶ）作用７２小时后的细胞存活率，求得半抑制浓度（ＩＣ５０）。动物实验将９×１０６的Ａ５４９／ＴＫ或野生型Ａ５４９细胞接种于Ｂａｌｂ／ｃｎｕ／ｎｕ裸鼠的皮下，当肿瘤长至２００～３００ｍｇ时，随机分组。Ａ５４９／ＴＫ组（１组）荷瘤小鼠给予每天１００ｍｇ／ｋｇＧＣＶ腹腔注射，连续给药２周，对照组（２组）Ａ５４９／ＴＫ给予生理盐水，野生型Ａ５４９组（３组）给予等量ＧＣＶ，从给药开始之日起，每周二次测量肿瘤大小，按经验公式计算肿瘤相对重量。结果体外细胞毒实验：在２０μｍｏｌ／ＬＧＣＶ存在时，野生型Ａ５４９形态无明显改变，而实验组细胞明显死亡。ＧＣＶ对野生型Ａ５４９的ＩＣ５０为５００μｍｏｌ／Ｌ，对Ａ５４９／ＴＫ的ＩＣ５０为０５μｍｏｌ／Ｌ，二者相差１０００倍（Ｐ＜０．０１）。动物实验：腹腔注射ＧＣＶ可使Ａ５４９／ＴＫ皮下肿瘤显著缩小。从注射之日起到１７天时，肿瘤体积下降了９２％，而ＧＣＶ对野生型Ａ５４９肿瘤?

Objective Evaluate the clinical potential of HSV TK/GCV system in gene therapy of tumors by using this system to treat human lung cancer A549 cells in vitro and in vivo. Method The in vitro sensitivity levels of A549/TK (TK tranfered A549) cells and wild type A549 cells to GCV were expressed as IC 50 which was measured with MTT assay after treating both cells for 72 hrs. In vivo study, The nude mice bearing A549/TK tumor or A549 tumor were used as experimental models. All the animals were divided into 3 groups. Group 1, the animals bearing A549/TK tumor would be treated with GCV. Group 2, The animals bearing A549/TK tumor would be treated with saline. Group 3, The animals bearing A549 tumor would be treated with GCV. When the tumors reached to 200～300 mg the animals would be injected intraperitoneally with GCV solution (100 mg·kg -1 ·day -1 ) or equal volume of saline for two weeks. In this period and the following four weeks, the tumor weights were measured and calculated with the experience formula once every three or four days. Result In vitro study showed that the A549/TK cells were apparently killed in a 20 μmol/L GCV solution while the wild type A549 cells grew normally under the same condition. The IC 50 of A549/TK cells in GCV solution was 0.5 μmol/L and that of A549 cells 500 μmol/L, showing a 1 000 fold difference ( P <0.01). In vivo experiment GCV had compelled the A549/TK tumors to lead to a regression of 92%, meanwhile the A549 tumors treated with GCV increased 3.9 times in weight in the first seventeen days of experiment. Group 2 tumors A549/TK tumors which were treated with saline, grew as well as group 3 did. There was a significant difference ( P <0.01) between group 1 and other groups. Conclusion GCV could specifically and effectively kill the TK transfered A549 cells in vitro and in vivo. This result suggested that the HSV TK/GCV system may have a clinical potential for gene therapy of lung cancer.