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目的 :研究p16过表达对HeLa细胞带 3蛋白 (band 3)阴离子交换功能的影响。方法 :细胞免疫组织化学法检测HeLa细胞中p16和带 3蛋白的表达。采用PCR方法将p16cDNA亚克隆到pEGFP -C1上 ,经双酶切和DNA测序鉴定后将其转染HeLa细胞 ,在荧光显微镜下观察细胞转染效率。应用 6甲基 - 3-磺丙基 - 1-喹啉 -水化合物 (SPQ)荧光探针检测带 3蛋白的阴离子交换功能。结果 :在HeLa细胞中p16和带 3蛋白表达均为阳性。双酶切和DNA测序结果证明所扩增的p16cDNA序列与报道序列相同。细胞转染后在荧光显微镜下可观察到 6 0 %以上的细胞发出荧光。转染pEGFP -C1-p16后细胞的阴离子交换功能增强。结论 :p16对HeLa细胞带 3蛋白的阴离子交换功能有促进作用。
Objective: To investigate the effect of overexpression of p16 on anion exchange function of band 3 in HeLa cells. Methods: Immunohistochemistry was used to detect the expression of p16 protein and 3 protein in HeLa cells. The p16 cDNA was subcloned into pEGFP-C1 by PCR, and then transfected into HeLa cells by double enzyme digestion and DNA sequencing. The transfection efficiency was observed under a fluorescence microscope. The anion exchange function of Band 3 protein was detected using a 6-methyl-3-sulfopropyl-1-quinoline-water compound (SPQ) fluorescent probe. Results: The expression of p16 and band 3 in HeLa cells was positive. Double digestion and DNA sequencing proved that the amplified p16 cDNA sequence was the same as the reported sequence. After transfection of cells, more than 60% of the cells were observed under a fluorescence microscope to fluoresce. After transfection with pEGFP-C1-p16 cells anion exchange function enhanced. Conclusion: p16 can promote the anion exchange function of band 3 protein in HeLa cells.