CD40反义RNA的构建及其诱导Balm细胞凋亡

王艳华　杨贵贞

　　中国图书分类号　R392.11
　　摘　要　目的：探讨CD40反义RNA对Balm细胞的影响。方法：用RT-PCR法从人扁桃体细胞获得CD40 cDNA，双脱氧终止法测序正确后，构建编码膜内段CD40基因的反义RNA表达载体(pcDNA3-CD40)，将其导入人B细胞株Balm细胞。结果：发现转导后细胞生长代谢受抑，细胞体积缩小，有细胞碎块脱落，FACS检测出现典型凋亡峰；细胞发光减弱，发光值明显低于正常对照组及转导空载体组(P＜0.001)，与放线菌素D(ActD)、氨甲喋呤(MTX)、肿瘤坏死因子(TNF)处理组有相似的形态及发光改变。结论：提示CD40反义RNA特异性阻断了CD40正常功能的发挥，导致细胞凋亡。
　　关键词　CD40反义RNA　Balm细胞　凋亡

The construction of CD40 antisense RNA and its apoptotic effect on Balm cell

WANG Yan-Hua,YANG Gui-Zhen.
Norman Bethune University of Medical Sciences,Changchun 130021

　　Abstract Objective:In order to explore the effects of CD40 antisense RNA on Balm cell.Methods:Human CD40 cDNA was acquired by RT-PCR form human tonsil cell by CD40 specific primers. The cloned CD40 products were sequenced to confirmed the specific CD40 coding region fragments by dideoxy-mediated-chain termination method.Then CD40 cyto-plasmic region(176 bp) antisense RNA expression vector of pcDNA3-CD40 was constructed,which was transfected into B cell line-Balm cell.Results:The transfected Balm cell showed apoptotic changes such as condense of nucleus and dark staining,apoptotic peak under FACS examination compared with the Balm cell transfected with only pcDNA3 vector.The luminometer counts of those CD40 antisense RNA treated cells are much lower than those of the controls(P＜0.001).Conclusion:The results suggest that CD40 antisense RNA can inhibit normal function of CD40 and induce apoptosis of CD40 bearing cells.
　　Key words CD40 antisense RNA Balm cell　Apoptosis

　　CD40是存在于B细胞、抗原提呈细胞及某些癌变上皮细胞表面的一种Ⅰ型跨膜糖蛋白［1］。它与存在于T细胞、肥大细胞及嗜碱性粒细胞表面的CD40配体(CD40 ligand,CD40L)反应，参与双方细胞的活化及效应功能的发挥。在免疫反应、炎症及肿瘤的发生发展中发挥重要作用［2，3］。这些功能的发挥是通过CD40介导信号传递而完成，用CD40反义RNA特异性封闭CD40基因，阻断CD40的表达，中止其信号传递，在炎症与肿瘤的治疗中可能有一定意义。因此本文构建编码CD40膜内段基因的反义RNA表达载体pcDNA3-CD40，导入B细胞传代细胞系Balm细胞，观察CD40基因特异性封闭后Balm细胞的形态与功能变化。

1　材料与方法
1.1　CD40反义RNA表达载体的构建
1.1.1　CD40膜内基因片段的获得　用RT-PCR法从人扁桃体细胞中获得CD40基因片段。上游引物为5′-

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