表达CD40L卵巢癌细胞的生物学特性和抗肿瘤特性研究

Biological and Antitumor Effects of CD40L on Ovarian Cancer Cell Line OVHM

背景与目的:CD40与CD40配体(CD40L)间的结合反应可以启动体液和细胞免疫应答,在宿主抗肿瘤免疫反应中起着重要作用。然而,一些肿瘤细胞如表达CD40L的卵巢癌细胞与宿主相互作用的分子机制仍不十分清楚。本文旨在探讨转染CD40LcDNA的小鼠卵巢癌细胞株OVHM(CD40L/OVHM)的生物学特性及抗肿瘤效应。方法:用脂质体转染法将CD40LcDNA转入OVHM细胞,流式细胞技术检测CD40L/OVHM和OVHM细胞中CD40、CD40L、MHC-Ⅰ、MHC-Ⅱ、CD80、CD86的表达水平。MTT法检测CD40L/OVHM和OVHM细胞的增殖反应。将OVHM或CD40L/OVHM细胞皮下接种6～8周龄的C57BL/6N×C3H/He杂交一代(B6C3F1)小鼠和BALB/c裸鼠,观察肿瘤的生长情况。ELISA法测定皮下接种OVHM或CD40L/OVHM细胞小鼠脾细胞和经放射线照射的OVHM细胞培养上清中IFN-γ释放量。结果:与亲本OVHM细胞相比,转染CD40LcDNA的CD40L/OVHM细胞CD40L表达明显升高(P<0.05),CD40、MHC-Ⅰ、MHC-Ⅱ的表达无明显变化,共刺激分子CD80、CD86的表达明显增强(P<0.05);转染和未转染CD40L的OVHM细胞增殖无显著性差异。接种CD40L/OVHM细胞的小鼠,肿瘤的生长速度与接种OVHM小鼠相比明显减慢,且无腹腔转移结节,大部分小鼠无肿瘤细胞生长。再次于接种CD40L/OVHM细胞的小鼠对侧皮下接种OVHM细胞(2×105),则无肿瘤生长。将接种CD40L/OVHM的小鼠脾细胞和经放射线照射的OVHM细胞体外混合培养,分泌IFN-γ的水平[(1802±187.7)pg/ml]明显高于接种OVHM细胞和未接种肿瘤细胞的小鼠脾细胞所分泌的IFN-γ量[(20.3±7.9)pg/ml、(100.3±19.9)pg/ml]。结论:转染CD40L基因可提高OVHM细胞的抗原性,引起较强的特异性细胞毒T淋巴细胞抗肿瘤免疫。

BACKGROUND ＆ OBJECTIVE： Accumulative evidences have indicated the importance of CD40-CD40L receptor-ligand pair in the initiation and regulation of human immune response. Whether triggering CD40-CD40L signaling pathway could induce antitumor effects in ovarian cancer cells is still unclear. This study was to investigate the biological and antitumor effects of CD40L on ovarian cancer cell line OVHM （CD40-positive）. METHODS. CD40L cDNA was transfected into OVHM cells by lipofectamineTM 2000. Immunofluorescent technique and flow cytometry were used to evaluate the expression of CD40, CD40L, MHC- Ⅰ , MHC-Ⅱ CD80, CD86. Cell proliferation was detected by MTT assay. CD40L/OVHM and OVHM cells were inoculated hypodermically into C57BL/6N × C3H/He F1 （B6C3F1）mice and BALB/c nude mice simultaneously. Tumor volume was measured. The production of IFN-γ secreted by the spleen cells from CD40L/OVHM and OVHM cell-inoculated mice co-cultured with 20 Gy-irridiated OVHM cells were measured with enzyme-linked immunosorbent assay （ELISA）. RESULTS, Expression of CD40L in OVHM/CD40L cells was significantly higher than that in OVHM cells （P〈0.05）. Moreover, the expression of costimulatory molecules CD80 and CD86 of CD40L/OVHM was increased （P〈 0.05）. The proliferation rate of parental and CD40L-expressed OVHM cells were not different. Although, delayed tumor growth was observed when CD40L-expressing OVHM cells and OVHM cells were injected simultaneously and contralaterally, the tumor growth of OVHM and CD40L/OVHM in BALB/ c nude mice was not different. The amounts of IFN-γ secreted by the coculture of irradiated OVHM cells with spleen cells from mice injected with CD40L/OVHM were much higher than those from OVHM/parental cell inoculated and naive mice. CONCLUSION： The forced expression of CD40L cDNA in OVHM cells can increase the immunogenicity, and thus develop antitumor effects against OVHM-incubated mice. In vivo immunization of immnuocompetent mice with CD40L/OVHM improves the generation of cytotoxic lymphocytes against parent ovarian cancer cells.