小鼠B7-1cDNA克隆、测序、表达及其抗瘤效应的初步探讨

小鼠B7-1cDNA克隆、测序、表达及其抗瘤效应的初步探讨 Cloning, Expression, and Antitumor Effect of Mouse Costimulatory Molecule B7 1 刘然义 LIU Ran yi 1 first-author 屈伸 QU Shen 1 尤颖健 YOU Ying jian 1 邓耀祖 DENG Yao zu 1 同济医科大学基础医学部 同济医科大学基础医学部 Base Courses Department of Medicine, Tongji Medical University Base Courses Department of Medicine, Tongji Medical University 用反转录ＰＣＲ的方法，从ＢＡＬＢ／ｃ小鼠脾细胞中扩增出Ｂ７－１ｃＤＮＡ后，插入ｐｃＤＮＡ３质粒中构建成小鼠Ｂ７－１ｃＤＮＡ的真核表达载体ｐＣＤ－ｍＢ７．１，经酶切鉴定和序列分析证实此表达载体中插入的Ｂ７－１ｃＤＮＡ的序列与文献报道一致．通过脂质体介导将ｐＣＤ－ｍＢ７．１导入Ｂ７－１的小鼠黑色素瘤细胞系Ｂ１６（Ｆ０）中，经ＲＴ－ＰＣＲ和ＲＮＡ斑点杂交初步证实Ｂ７－１在肿瘤细胞中获得了稳定有效的表达．同源小鼠脾淋巴细胞与肿瘤细胞混合培养后采用ＬＤＨ释放改良法测定淋巴细胞特异杀伤活性，结果显示，与野生型和模拟转染的Ｂ１６细胞相比，Ｂ７－１基因转染的Ｂ１６细胞能较有效诱导淋巴细胞产生针对野生型Ｂ１６细胞的特异杀伤活性（ｐ＜０．０２）．这说明，将Ｂ７－１基因导入肿瘤细胞表达能提高其免疫原性，诱导有效的抗肿瘤免疫反应 Recent studies suggested that expression of B7 1 in tumor cells is effective at inducing antitumor immune responses. In this research, mouse B7 1 cDNA was amplified by RT PCR from spleen cells of BALB/c mice, and inserted into a eukaryotic expression vector, pcDNA3. Then the inserter was sequenced, the results were corresponed to the data from Freeman et al. The recombinant, named pCD mB7.1, was transfected into B7 1 negative melanoma B16（F0） cells by lipofectin mediated gene transfer. The mouse B7 1 expression in modified B16 cells （B16 mB7.1） were detected within at least six months by RT PCR and RNA molecular hybrid consequently. Non adherent splenocytes from non immunized C57BL/6 mice were incubated with non replicating B16 wt, B16 neo as control, or B16 mB7.1 respectively. Then the lymphocytes were tested for cytotoxic activity against B16 wt cells. The results suggested that the costimulation signal of B7 1 is required for the activation of T lymphocytes, the expression of B7 1 by tumor cells is effective at inducing antitumor immune responses. B7－1 基因表达 反转录PCR RNA分子杂交 基因治疗 B7 1 RT PCR gene expression RNA molecular hybrid gene therapy R392.11 国家教委基金 1999-02-01 2021-04-01 2