白细胞介素12基因转染抑制B16细胞成瘤性及转移性的研究

Interleukin12 gene transfection into murine B16 melanoma cells suppresses tumorigenicity and decreases metastatic potential

目的探讨白细胞介素１２（ＩＬ１２）基因转移对弱免疫原性的小鼠Ｂ１６黑色素瘤细胞生长与转移的影响。方法构建小鼠ＩＬ１２两个亚单位ｐ４０、ｐ３５ｃＤＮＡ表达载体；经脂质体ＬｉｐｏｆｅｃｔＡＭＩＮ介导，将两真核表达载体同时导入Ｂ１６细胞；应用逆转录聚合酶链反应（ＲＴＰＣＲ）检测ｐ４０、ｐ３５ｍＲＮＡ表达；用生物学活性检测法（致分裂激活的活化的小鼠脾细胞增殖）检测ＩＬ１２分泌；经皮下或静脉接种肿瘤细胞，检测其体内成瘤性及转移性。结果ＩＬ１２基因转染的Ｂ１６细胞（Ｂ１６ＴＩＬ１２）能够表达ｐ４０及ｐ３５ｍＲＮＡ，分泌有生物活性的ＩＬ１２；Ｂ１６ＴＩＬ１２细胞体内成瘤性降低，成瘤延迟，肿瘤生长慢，荷瘤小鼠存活期延长（Ｐ＜００１）；肺转移率降低，肺内转移灶明显减少（Ｐ＜００１）。

Objective To study the tumorigenicity and metastasis of poorly immunogenic murine B16 melanoma cells transfected with murine interleukin12 (IL12) gene. Methods Two recombinant vectors containing the full length cDNA of p40 or p35 subunit of IL12 were constructed. They were cotransfected into B16 melanoma cells by LipofectAMIN method. The expressions of p40 and p35 mRNAs were analyzed by means of reverse transcription polymerase chain reaction (RTPCR). The secretion of bioactive IL12 was detected by bioassay (measuring the proliferative response of PMAactivated murine splenocytes). To determine the effects of IL12 secreted by genetically engineered B16 cells (B16TIL12) on tumorigenesis and metastasis, the cells were inoculated s.c. or i.v. into C57BL/6 mice. Results B16TIL12 cells expressed both p40 and p35 mRNAs, also produced bioactive IL12. Only 70% of the mice injected with B16TIL12 cells developed palpable tumors. The emergence of palpable tumors in the mice inoculated with B16TIL12 cells was significantly delayed (P<0.01), the growth rate of the tumors was obviously decreased (P<0.01), and the survival time of tumorbearing mice was prolonged substantially (P<0.01), compared with the control B16 or B16Tneo cells. The incidence of experimental pulmonary metastasis of B16TIL12 cells was inhibited, and the number of pulmonary metastases was reduced markedly, in contrast to the i.v. inoculation of B16 cells (P<0.01). Conclusion Transfection of IL12 gene into B16 cells results in the inhibition of tumorigenesis and in the suppression of experimental pulmonary metastasis.