鼠肝癌细胞的热休克蛋白诱导及其抗瘤机理研究

Induced expression of HSP70 on H22 cell and the prilimary study of the mechinasm of its anti tumor effects

目的摸索鼠肝癌Ｈ２２细胞膜热休克蛋白７０（ＨＳＰ７０）最适的诱导条件，并明确其体内外的抗瘤机理。方法用免疫荧光及ＦＣＭ法检测热休克Ｈ２２细胞膜ＨＳＰ７０蛋白的动态表达。并用ＭＭＣ灭活、热休克后的Ｈ２２细胞作抗原进行体内肿瘤免疫保护试验及体外肿瘤杀伤试验。结果小鼠肝癌Ｈ２２细胞经４２℃、４３℃热休克１２小时后，其胞膜ＨＳＰ７０蛋白表达率分别达到５９．５％，９６．８％。均较对照组显著增高（Ｐ＜０．００１）。用４３℃休克１２小时的Ｈ２２细胞免疫的Ｃ３Ｈ小鼠能抵抗同种野生型肿瘤细胞的攻击，其肿瘤发生率比野生型Ｈ２２免疫组及未免疫组均显著降低，分别为１／９、７／８和８／８。同时小鼠荷瘤生存期显著延长，中位生存期分别为＞９０天，７３．５天及３９．５天。体外杀伤试验表明，经热休克Ｈ２２诱导７天后的Ｃ３Ｈ脾淋巴细胞对热休克型Ｈ２２细胞杀伤率高达６３．３８％（４３℃，２小时）和６７．８４％（４３℃，１２小时），且这种杀伤活性可被抗鼠ＨＳＰ７０的单抗所阻断。分析效应细胞的表型发现，ＣＤ４＋、ＣＤ８＋淋巴细胞在整个体外诱导过程中并不增加，而ＴＣＲγδ＋淋巴细胞随诱导时间的延长与杀伤活性同步增加，诱导第７天为３０．４３％，第１４天?

Objective To identify the optimum conditions for inducing HSP70 and the mechinasm of the anti tumor effects of the induced HSP70 in vitro and in vivo. Methods The kinetic expression of membrane protein HSP70 of H22 cell at various tempertures and the immune protective effects of the protein in vivo and cytotoxic activity in vitro to the same type of tumor cells of C3H mice which had been immunized twice with MMC inactived,heat shocked H22 cell were analysed with immunofluorescence and FCM techniques. Results The highest expressing rate of membrane HSP70 protein on H22 cells which had been heat shocked at 42℃,43℃ for 12 hours were 59.5% and 96.8%,respectively,which were significantly higher in heat shocked H22 cell than in control cells( P <0.001).Immunized with H22 cell which had been treated at 43℃ for 12 hours,the C3H mice could resist to a great extent invasion of the same wild type tumor cells by subsequent subcutaneous inoculation.The tumorgenic rate in immunized mice with heat treated H22 cells was significantly lower than that of in immunized mice with wild type H22 cells and in RPMI 1640 control mice,which were 1/9,7/8, and 8/8, respectively.And the survival periods of heat treated H22 cell immunized mice were statistically longer.The median survival time was >90 days in heat treated group.73.5 days in wild type group and 39.5 days in RPMI 1640 control groups. In vitro, the specific cytotoxicity against heat treated H22 cells were higher than that against wild type cells.The peak level of cytotoxicity against H22 cells reached to 65.38%(2 hrs,43℃) and 67.84%(12 hrs,43℃) and could be blocked by anti HSP70 McAb.Further analysis with FCM revealed that the rate of TCRγδ + cells rose along with the increasing cytotoxic activity,which got to 30.43% at the 7th day and 45.67% at the 14th day,but no similar change could be found in CD4 +CD8 +T subset.Conclusions Proper heat shock treatment can increase the expression of HSP70 on H22 cell,thus improve its immunogenicity and such function is executed by CD4 -CD8 -TCRγδ + T lymphocyte via HSP70 molecule.