靶向树突状细胞的肿瘤疫苗OVA慢病毒载体构建及其体外抗肿瘤效应

目的构建靶向树突状细胞(dendritic cell,DC)的肿瘤疫苗pHR-CMV-EGFP-OVA慢病毒表达载体,并观察肿瘤抗原特异性DC激活T细胞对小鼠Lewis肺癌细胞(lewis lung carcinoma cell,LLC)的抗肿瘤效应,为肺癌的临床治疗提供实验依据。方法以分子克隆方法构建携带模拟肿瘤抗原鸡卵清蛋白(ovalbumin,OVA)的慢病毒表达载体,经XhoⅠ及XbaⅠ双酶切后电泳和DNA测序验证;应用脂质体法三质粒共转染293T细胞,包装产生慢病毒,转导LLC,进行单克隆化筛选培养后,经RT-PCR、Western blot检测所获得的OVA稳定表达细胞株中OVA的表达;分离纯化小鼠骨髓DC;用慢病毒感染DC;分离纯化小鼠脾T淋巴细胞;应用MLR和MTT法检测肿瘤抗原负载的DC刺激T淋巴细胞对LLC的杀伤作用。结果 (1)构建的pHR-CMV-EGFP-OVA慢病毒表达载体经酶切后电泳和DNA测序,结果显示载体构建正确;(2)获得了纯化的小鼠骨髓来源的DC;(3)获得了携带肿瘤抗原OVA的LLC和DC;(4)负载肿瘤抗原的DC刺激T淋巴细胞能有效地杀伤负载相同肿瘤抗原的LLC。结论本研究成功地构建了携带肿瘤模拟抗原OVA的慢病毒表达载体pHR-CMV-EGFP-OVA,并成功地转导LLC与DC,获得了稳定表达OVA的肺癌细胞株和携带OVA的DC,基于肿瘤抗原靶向DC的抗肿瘤策略能有效地杀伤LLC,为靶向DC的肺癌免疫治疗提供了体外实验依据。

Construction of ientiviral vector targeting dendritic cell vaccine and its antitumor effects

Objective To construct lentiviral vector containing simulated tumor antigen ovalbumin (OVA) targeting dendritic cell(DC) vaccine and to test its antitumor effects in vitro.Methods The lentiviral expression vector pHR-CMV-EGFP-OVA was constructed by introducing DNA fragment of OVA into the XhoⅠand XbaⅠsites of pHR-CMV-EGFP vector.DNA sequencing and restriction endonucleases methods were used to test the recombinant vector.DCs were collected and isolated from the mouse bone marrow and purified by FACS.293T cells were transfected with the expression vector pHR-CMV-EGFP-OVA by liposome-medium transfection,and then Lewis lung carcinoma(LLC) cells were transfected with pHR-CMV-EGFP-OVA lentivirals.The positive clones were expanded and screened for OVA production by RT-PCR and Western blot.DCs also were transfected with pHRCMV -EGFP-OVA.T cells were collected from spleen and isolated by using Nylon wool column. Results The lentiviral vector pHR-CMV- EGFP-OVA was constructed successfully.LLC cells and DCs carrying ovalbumin were successfully obtained.Tumor-specific T cells stimulated by OVA-loaded DCs exhibited effectively cytolytic activity against LLC/OVA cells in vitro.Conclusion The lentiviral expression vector pHR-CMV-EGFP-OVA has been successfully constructed and the transfected DCs vaccine can elicit the anti-tumor immunity to OVA-loaded Lewis lung cancer cells in vitro.