表达RANTES基因的腺病毒载体构建及抗肿瘤作用研究

Construction of adenovirus vector expressing RANTES gene and its anti-cancer effect

目的:构建表达活化T细胞表达和分泌的调节因子(regulatedonactivationnormalTexpressedandsecreted,RANTES)基因的腺病毒载体并研究其抗肿瘤作用机制和效果。方法:RT-PCR法克隆人和鼠源性(regulatedonacti-vationnormalTexpressedandsecreted,RAN-TES)基因,采用AD-easyXLAdenovirusVectorSystem试剂盒分别构建表达上述基因的腺病毒载体。通过趋化试验验证表达RANTES基因的腺病毒载体转染细胞后所具有的趋化肿瘤免疫细胞的作用。应用于体外及小鼠体内试验,包括成瘤试验及抑瘤试验,通过观察免疫效应细胞对小鼠体内肿瘤的抑制作用来评价其在肿瘤治疗方面的作用。结果:克隆了人和鼠源性RANTES基因编码序列并成功地构建腺病毒表达载体;功能试验表明由腺病毒表达的RANTES具有趋化肿瘤免疫细胞的作用。体内试验表明该病毒载体可抑制肿瘤细胞成瘤作用,应用于荷瘤小鼠可抑制肿瘤的发展,延长荷瘤小鼠的生存时间。结论:表达RANTES基因的腺病毒载体可显著地趋化免疫细胞到肿瘤局部杀伤肿瘤细胞,抑制肿瘤细胞成瘤作用并延长荷瘤小鼠的生存时间,为肿瘤的生物学治疗提供了新的研究方向。

OBJECTIVE： To construct the adenovirus vectors expressing the human and murine origin RANTES genes, and analyze the mechanisms and evaluate the anti-tumor effects of RANTES gene that mediated by adenovirus vector. METHODS： The human and mouse origin RANTES gene was cloned by RT-PCR method and the adenovirus vector was constructed by using the AD-easy XL Adenovirus Vector System. Chemotactic test was used to analyze the functions of the adenovirus vectors expressing RANTES gene. Tumor formation and tumor suppressing tests were used to analyze the anti-cancer functions of the adenovirus vector in vivo. RESULTS： RANTES gene was cloned by RT-PCR pathway from lymph tissue such as tonsil and spleen, and constructed into adenovirus vector by using AdEasy XL Adenoviral Vector System kid. Western blot showed that the eukaryotic cell infected by the vectors expressed RANTES. Functional tests showed that RANTES mediated by adenovirus vector mimic ked the natural factor＇s chemotactic activity in vivo or in vitro. The vectors expressing RANTES inhibited the tumor formation and tumor genesis. Treatment of the mice bearing H22 tumor cells using RANTES gene mediated by adenovirus vector gained encouraging outcome that the volume and increasing rate of ascites, as well as the survival time were distinctively different from the controls. CONCLUSIONS.. RANTES gene can improve the anti-cancer immune response and inhibit the progression of the cancer. It gives a new way to treat the cancer.