论文部分内容阅读
目的: 构建人类CD40膜蛋白siRNA的真核表达载体, 观察其对CA46细胞上CD40表达、细胞增殖能力和凋亡的影响。方法: 合成两条编码发夹siRNA序列的单链DNA,并将其克隆到pSilenCircle载体中, 构建含目的基因片段的重组质粒siCD40 /pSilenCircle。以同样的方法, 分别构建相对应的编码反义RNA及无关基因的重组质粒antiCD40 /pSilenCir cle和siFly/pSilenCircle。以上述重组质粒分别瞬时转染CA46细胞后, 用流式细胞仪检测CA46细胞上CD40的表达和细胞凋亡情况, 用MTS法(改良的MTT法)测定细胞的增殖能力。结果: ①成功地构建了两个CD40发夹siRNA的真核表达载体siCD40 /pSilenCircle、两个相对应的反义RNA真核表达载体antiCD40 /pSilenCircle和无关基因重组质粒siFly/pSilenCir cle。②与siFly/pSilenCircle转染组相比较, siCD40 /pSilenCir cle转染组和antiCD40 /pSilenCircle转染组CA46细胞上CD40的表达均明显减少, 但细胞的增殖能力和凋亡未发现明显变化。结论: 构建的两个CD40发夹siRNA的真核表达载体siCD40 /pSilenCircle, 可有效地抑制CA46细胞上CD40分子的表达, 但不影响细胞的增殖和凋亡。RNA干扰技术可望作为一种有效地调控基因功能的方法。
OBJECTIVE: To construct eukaryotic expression vector of human CD40 membrane protein siRNA and observe its effect on CD40 expression, cell proliferation and apoptosis in CA46 cells. METHODS: Two single-stranded DNA encoding hairpin siRNA sequences were synthesized and cloned into pSilenCircle vector to construct recombinant plasmid siCD40 / pSilenCircle containing the target gene fragment. In the same way, the corresponding recombinant anti-CD40 / pSilenCir cle and siFly / pSilenCircle were constructed respectively encoding antisense RNA and unrelated genes. After transient transfection of CA46 cells with the above recombinant plasmids respectively, the expression and apoptosis of CD40 on CA46 cells were detected by flow cytometry. The proliferation of cells was measured by MTS method (modified MTT method). RESULTS: Two eukaryotic expression vectors, siCD40 / pSilenCircle, were constructed successfully. Two antisense RNA eukaryotic expression vectors antiCD40 / pSilenCircle and unrelated recombinant plasmid siFly / pSilenCircle were successfully constructed. ② Compared with the siFly / pSilenCircle transfection group, the expression of CD40 on CA46 cells in siCD40 / pSilenCir cle transfection group and antiCD40 / pSilenCircle transfection group were significantly reduced, but no significant changes in cell proliferation and apoptosis. CONCLUSION: The eukaryotic expression vector siCD40 / pSilenCircle of two CD40 hairpin siRNAs can effectively inhibit the expression of CD40 on CA46 cells but does not affect cell proliferation and apoptosis. RNA interference technology is expected as a way to effectively regulate the function of genes.