骆驼蓬脂转移蛋白基因真核表达载体的构建及其体内外抗瘤效应的研究  

Construction of the Eukaryotic Expression Vector of PhLTP Gene and Its Primary Antitumor Effects in vitro and in vivo

作　　者：赵晶[1] 吕慧[1] 贾依娜古丽.朱玛拜 孙素荣[1] 

机构地区：[1]新疆大学生命科学与技术学院新疆生物资源基因工程重点实验室,乌鲁木齐830046

出　　处：《中国生物工程杂志》2014年第8期7-13,共7页China Biotechnology

基　　金：新疆维吾尔自治区科技支疆项目(20091129);新疆生物资源基因工程重点实验室开放基金项目(XJDX0201-2010-06)资助项目

摘　　要：目的:构建骆驼蓬脂转移蛋白(lipid transfer protein from Peganum harmala,PhLTP)基因真核表达质粒,并探讨其对黑色素瘤B16细胞在体内外的抗肿瘤作用。方法:将PhLTP基因亚克隆至pcDNA3.1上,获得重组质粒pcDNA3.1-PhLTP;用脂质体转染法将重组质粒及空载体外转染B16细胞,MTT检测其对B16细胞生长的影响。建立B16荷瘤小鼠模型,设重组质粒(pcDNA3.1-PhLTP)、空载(pcDNA3.1)、生理盐水和阳性药物(CTX)组,分别处理小鼠后测量各组肿瘤体积并称瘤重,计算抑瘤率。光镜观察鼠脾、肝等组织变化;免疫组织化学法检测各瘤体中PhLTP、血管内皮生长因子(VEGF)及碱性成纤维细胞生长因子(bFGF)的表达。结果:pcDNA3.1-PhLTP转染B16细胞72 h后,细胞增殖能力明显受到抑制(P<0.01)。注射pcDNA3.1-PhLTP组的小鼠肿瘤生长速度明显减慢,肿瘤体积小于空载和生理盐水组(P<0.05)。显微镜下可见重组质粒组肿瘤细胞有不同程度的点、片状坏死,而肝、肺等无明显病理损伤。重组质粒组肿瘤组织中有PhLTP蛋白的表达,且VEGF和bFGF的阳性表达指数都低于空载和生理盐水组(P<0.01)。结论:成功构建了重组表达质粒pcDNA3.1-PhLTP,体内外实验结果显示其能有效地抑制B16细胞的生长,预示了该重组质粒在治疗黑色素瘤中的潜在应用价值。Objective： To construct an eukaryotic expressing plasmid of lipid transfer protein from Peganum harmala,and investigate its inhibitory effects on the growth of murine melanoma（ B16） cells in vitro and in vivo.Methods： The eukaryotic expressing plasmid of PhLTP（ pcDNA3. 1-PhLTP） was constructed by DNA recombination technique. The recombination plasmid and empty vector were transfected into B16 cells,respectively. The effect of pcDNA3. 1-PhLTP on the growth of B16 cells was measured by MTT assay. B16 cells were inoculated subcutaneously of C57 BL /6J mice,which randomly divided into 4 groups. Tumor volume and weigh were measured,and the tumor inhibition rates were calculated. The change of the mouse spleen and liver were observed under microscope. The expressions of PhLTP,vascular endothelial growth factor（ VEGF） and basic fibroblast growth factor（ bFGF） in tumor tissues were detected by immunohistochemistry. Results： The proliferation of B16 cells were inhibited after transfection with pcDNA3. 1-PhLTP for 72 h（ P〈0. 01）. Growth rate of tumor in mice model was also significantly inhibited in pcDNA3. 1-PhLTP therapy group as compared with the control group and empty vector group（ P〈0. 05）. The tumor tissue cells had different levels of necrosis in recombinant plasmid group. The liver,lungs and other organs in mice haven＇t significant pathological damage.The PhLTP could be expressed in recombinant plasmid group. And the positive expression indexes of VEGF and bFGF in recombinant plasmid group were significantly lower than ones of VEGF and bFGF in control group and empty vector group（ P〈0. 01）. Conclusion： The recombinant eukaryotic expression vector pcDNA3. 1-PhLTP is constructed successfully,and pcDNA3. 1-PhLTP can inhibit the growth of B16 cells in vitro and in vivo. It is suggestted that the recombinant plasmid pcDNA3. 1-PhLTP may have potential applications in the treatment of melanoma.

关 键 词：骆驼蓬脂转移蛋白基因 转染 生长抑制 鼠黑色素瘤B16 血管生成 

分 类 号：Q255[生物学—细胞生物学] R730.54[医药卫生—肿瘤]