负载人HUVECs抗原的DC疫苗诱导小鼠对宫颈癌U14的特异性免疫反应

Study on the specific immunity of dendritic cell vaccine loading human umbilical vein endothelial cell antigen against U14 cervical cancer

目的:探讨负载人脐静脉内皮细胞(HUVEC)抗原的DC疫苗诱导小鼠对宫颈癌U14的特异性免疫反应。方法:原代培养和鉴定人HUVECs,制备抗原,负载树突状细胞(DC),免疫小鼠,免疫结束后1周小鼠皮下注射小鼠宫颈癌U14细胞,观察肿瘤生长大小、检测免疫后小鼠脾细胞体外CTL效应、表面CD3+CD8+百分率和血清抗体滴度,通过细胞免疫组化和Western blot血清特异性反应分析。结果:经免疫组化鉴定成功培养出纯度高的HUVECs,抗原负载DC并注射BALB/c小鼠后,PBS组肿瘤生长最快,DC组和HUVEC-DC组肿瘤也有生长,但生长较慢,在2周后HUVEC-DC组肿瘤消失,3周后DC组肿瘤消失。小鼠脾细胞体外CTL结果显示HUVEC-DC组有明显的特异性杀伤HUVEC的作用。CD3+CD8+百分比提示HUVEC-DC组细胞毒性T细胞比例较其他两组高。小鼠血清抗体滴度示有抗体产生,免疫组化检测示组与HUVEC有特异性抗原抗体反应,Western blot显示在相对分子质量(Mr)130000和220000处有特异性条带。结论:负载人HUVECs抗原的DC疫苗和DC疫苗对小鼠宫颈癌U14有抑制作用,HUVEC-DC疫苗诱导了小鼠的细胞和体液免疫,并诱导了针对HUVECs细胞中的某些抗原的应答,这些抗原可能是整合素αν和VEGF-R2。

AIM：To explore the specific immunity of dendritic cell（DC）vaccine loading human umbilical vein endothelial cell（HUVEC）antigen.against U14 cervical cancer of mice.METHODS：Primary HUVECs were cultured,identified and made into antigen.The BALB/c mice were immuned with DCs loading HUVEC antigen 4 times a week.One week after last vaccination,U14 cervical cancer cells were injected subcutaneously into the mice.The tumor size,CTL response of spleen lymphocytes in vitro,the percentage of CD3＋CD8＋ surface markers of spleen lymphocytes,and the titer of serum antibody were detected.The specific immunity was examined by immunocytochemistry and Western blot.RESULTS：HUVECs with high purity were successfully cultured and by identified by immunocytochemistry and RT-PCR.After the vaccine was injected into mice,the tumors of mice in PBS group grew faster than the those in other groups.The tumors of mice in HUVEC-DC groups grew slowly and disappeared after 2 weeks and The tumors of mice in DC group disappeared after 3 weeks.The CTL response of spleen lymphocytes in vitro showed that HUVEC-DC-T cells could kill HUVEC cells.The percentage of CD3＋CD8＋ surface markers of spleen lymphocytes in HUVEC-DC group was higher than that in other groups.The titer of serum antibody was 1∶800.immunocytochemistry analysis indicated HUVEC-DC group had specific antigen-antibody reaction to HUVECs through and Western blot analysis revealed there were specific bands at 130 KDa and 220 KDa.CONCLUSION：HUVEC-DC vaccine and DC vaccine can inhibit the tumor growth of U14 cervical cancer of mice.The special cellular and humoral immunity are induced by HUVEC-DC vaccine.Furthermore,some antigens in HUVECs maybe have special immune reaction with integrin α ν and VEGF-R2.