TβR-Ⅱ-RANTES融合基因重组腺病毒的抗肿瘤作用

Construction of recombinant adenovirus vector expressing extracellular domain of TβR-Ⅱ-RANTES fusion gene and its anti-tumor effects

目的构建表达融合基因TGF-βⅡ型受体(TβR-Ⅱ)胞外区及活化T细胞表达和分泌的调节因子(RANTES)重组腺病毒载体,并观察其抗肿瘤作用。方法RT-PCR扩增小鼠TβR-Ⅱ胞外区和RANTES基因,重叠PCR扩增融合基因TβR-Ⅱ胞外区-RANTES。采用adMax adenovjrus vector creation试剂盒构建表达融合基因重组腺病毒。体外感染小鼠肺腺痛LA795细胞系,绘制细胞生长曲线。采用Western blot法检测感染后细胞融合基因的表达;酶联免疫吸附试验(FLISA)法检测其培养上清的蛋白含量;Annexin V-FITC法检测感染后细胞的凋亡;并观察感染后细胞培养上清对小鼠脾细胞的趋化作用。将感染后的细胞(1×105个)接种于T739小鼠,观察成瘤时间和生存时间;1×1010pfu的重组腺病毒局部注射荷瘤小鼠,观察肿瘤的大小变化,并统计肿瘤的重量和计算抑瘤率。结果经测序证实,RT-PCR正确扩增小鼠TβR-Ⅱ胞外区和RANTFS基因,重叠PCR正确扩增融合基因TβR-Ⅱ胞外区-RANTES。重组质粒pDC316-融合基因经酶切鉴定正确,与pJM17双质粒共转染获得表达融合基因重组腺病毒,病毒滴度为8×1010pfu/ml。Western blot结果表明,感染后的LA795细胞有融合蛋白的特异性条带出现;培养上清中,游离TGF-β1的水平显菩减低,而RANTES的水平显著升高。感染融合基因重组腺病毒的细胞生长速度明显减低,凋亡率为16.9%;培养上清可趋化小鼠脾细胞,与对照组之间差异均有统计学意义。感染融合基因重组腺病毒组与对照组相比,体内成瘤时间和生存时间明显延长;局部注射融合基因重组腺病毒可显著抑制肿瘤的生长,抑瘤率为37.6%。结论成功构建表达融合基因TβR-Ⅱ胞外区-RANTES重组腺病毒可有效结合TGF-β1,显著逆转TGF-β介导的免疫抑制状态,高水平表达RANTES强化肿瘤局部的免疫功能,具有显著的抗肿瘤作用。

Objective To construct a recombinant adenovirus vector expressing TβR-Ⅱ extracellular domain-RANTES fusion gene and evaluate its anti-tumor effects. Methods Mouse origin TβR- Ⅱ extracellular domain and RANTES gene were amplified by RT-PCR. The TβR- Ⅱ extracellular domain- RANTES fusion gene was amplified by overlapping PCR method. TβR-Ⅱ extracellular domain-RANTES fusion gene was cloned into pDC316 vector. The recombinant adenovirus vector expressing the fusion gene was constructed by adMax adenovirus vector creation system. Recombinant adenovirus vector expressing the fusion gene was transfected into LA795 cells. The expression of recombinant adenovirus was checked by Westen blot. The levels of TGF-β1, RANTES in supematant were checked by ELISA. The transfected cells were counted and growth curve was obtained, Apoptosis of transfected cells was detected by Annexin V FITC method. The chemotactic activity of supernatant of transfected cells to splenic lymphocytes was assayed. Transfected cells （ 1 ×10^5 ） were inoculated into T739 mice and to observe the tumor growth and survival time. Ad-TβR- Ⅱ extracellular domain, Ad-RANTES and Ad-TβR- Ⅱ extracellular domain-RANTES fusion gene（1 × 10^10 pfu）were injected into the tumor in T739 mice. The tumor size and tumor weight were recorded and tumor growth inhibition rate was counted and statistically analyzed. Results TβR-Ⅱ extracellular domain and RANTES gene were amplified by RT-PCR and TβR-Ⅱ extracellular domain-RANTES fusion gene amplified by overlapping PCR, were identified by DNA sequence analysis. Restriction enzyme digestion analysis showed that the recombinant vector was constructed correctly. The recombinant adenovirus vector expressing the fusion gene was constructed successfully using the AdMax Adenovirus Vector Creation System. Its titer was 8 × 10^10 pfu/ml. Ad-TβR-Ⅱ extracellular domain-RANTES fusion gene was transfected into LA795 cells and had specific protein fragment proved by Western Blot. The concentration of TGF-β1 was decreased and RANTES was increased in supernatant of transfected cells. The growth curve showed that recombinant adenovirus vector expressing the fusion gene could delay tumor development and induce apoptosis, with an apoptosis rate in vitro of 16.9%. The supernant of infected cells showed chemotactic activity to splenic lymphocytes. Tumor growth and survival time were prolonged significantly in group tranfected with recombinant adenovirus vector expressing the fusion gene, and tumor growth was effectively inhibited after injecting recombinant adenovirus vector expressing the fusion gene, with a tumor growth inhibition rate of 37.6%. Conclusion A recombinant adenovirus vector expressing TβR- Ⅱ extracellular domain-RANTES fusion gene has been constructed successfully. The recombinant adenovirus vector can bind TGF-β1 effectively, counteract immune suppression mediated by TGF-β, enhance immune function, induce significant antitumor immune respone, inhibit tumor growth, and prolong the survival time of tumor-bearing mice.