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前列腺癌PSA特异性树突状细胞瘤苗的构建及其体外免疫活性的观察
徐丹枫，高轶，刘玉杉，崔心刚，车建平，阴雷，邢继章，姚亚成，任吉忠，闵志廉
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(第二军医大学长征医院泌尿外科，全军泌尿外科中心，上海 200003)

摘要:

目的:构建前列腺癌PSA特异性树突状细胞（PSADC）瘤苗，并观察其体外免疫活性，为后续研究奠定基础。方法:分离骨髓前体细胞，大量制备骨髓DCs（bone marrow derived DC,BMDC）；分别以PSA、癌细胞裂解产物（lysate，Lys）、无关蛋白卵清蛋白（Ova）冲击DC制备PSADC、LysDC、OvaDC瘤苗。ELISA法检测PSADC瘤苗培养上清中细胞因子（IL12 p70和IL1β）水平的变化；观察PSADC瘤苗刺激抗原特异性T细胞增殖活性和诱导抗原特异性CTL杀伤活性，并与LysDC、OvaDC瘤苗及未加入抗原冲击的DC（NonDC）组作比较。结果:成功获得成熟DC，纯度可以达到>95%。ELISA法检测结果表明，PSADC、LysDC和OvaDC组培养上清中IL12 p70和IL1β水平均较NonDC组明显升高（P<0.05）。混合淋巴细胞培养（MLR）显示：PSADC、LysDC组DCs刺激CD4+T细胞增殖的能力明显优于OvaDC、NonDC组（P<0.01）；PSADC、LysDC组培养上清中IL2、IFNγ水平明显高于后两者（P<0.01），而IL10和IL4水平下调(P<0.05)。PSADC、LysDC组可产生针对PSA和肿瘤裂解物的DTH反应，而对无关抗原无效(P<0.05)。与OVADC、NonDC组相比，LysDC、PSADC组体外诱导的CTL细胞对LNCaP细胞的杀伤活性较强 (P<0.05)，且具有抗原特异性。结论:利用PSA蛋白冲击DC可成功制备PSA特异性DC瘤苗，该瘤苗体外具有较强的免疫活性，能有效杀伤LNCaP细胞。

关键词: 树突细胞前列腺特异抗原前列腺癌癌症疫苗细胞毒性T淋巴细胞

DOI：10.3724/SP.J.1008.2009.0265

投稿时间：2007-12-14修订日期：2008-09-16

基金项目:上海市科委基金(044119615).

Construction of PSA specific dendritic cell vaccine and its in vitro immune activity

XU Danfeng*, GAO Yi, LIU Yushan*, CUI Xingang, CHE Jianping, YIN Lei, XING Jizhang, YAO Yacheng, REN Jizhong, MIN Zhilian

(Department of Urology, Urology Center of PLA, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China)

Abstract:

Objective:To construct of PSAspecific dendritic cell (DC) vaccine and to observe its in vitro antitumour activity, so as to pave a way for future study. Methods: Bone marrow precursors were isolated and bone marrow derived DCs were prepared. Mature DCs were pulsed by PSA, Lysate of cancer cells, OVA and PBS to yield PSADC, Lys DC, OvaDC, and NonDC, respectively. After primed by antigen, the changes of IL12 p70 and IL1β in the supernatant of dendritic cells were assessed by ELISA. The antigenspecific proliferation and cytotoxicity activity of T cellprimed by PSApulsed DCs were observed and the results were compared with those by Lys, Ova and PBSpulsed DCs. Results: Mature DCs were successfully derived from bone marrow cells with a purity higher than 95%. ELISA assay showed PSADC, LysDC and OvaDC group secreted high levels of IL12 p70 and IL1β than NonDC group (P＜0.05). In addition, PSADCs and LysDCs had significantly stronger ability to stimulate the proliferation of CD4+T cells in 3day classic mixed lymphocyte reaction (MLR) compared with OvaDCs and NonDCs (P＜0.01). Higher levels of IFNγ and IL2 were detected in PSADCs and LysDCs groups compared with the other two groups (P＜0.01), whereas the levels of IL10 and IL4 were lower than the other two groups (P＜0.05). Moreover, PSADCs and Lys DCs enhanced DTH responses of C57BL/6 mice after antigen immunization; the third antigen and control did not show the enhancement effect (P＜0.05). To observe the in vitro antiPSA CTL reactions induced by PSADCs and LysDCs, the LNCaP cell line (PSA specific) was used as syngeneic target and the E.G7 cell line (H2b) was used as Ovaspecific target cells. Compared with OvaDCs and NonDCs, CTL cells induced by PSADCs, LysDCs had significantly enhanced antigenspecific CTL activity to LNCaP cells (P＜0.05).Conclusion: DCbased PSAepitope vaccine can be prepared by pulsing DCs with PSA protein; the prepared vaccine has strong in vitro immune activity and can kill LNCaP cells.

Key words: dendritic cells prostate specific antigen prostatic neoplasms cancer vaccines cytotoxic T lymphocytes