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目的构建表达程序性细胞死亡因子4(PDCD4)的慢病毒表达载体,建立能够稳定表达目的基因的前列腺癌PC3细胞亚株。研究PDCD4分子对前列腺癌细胞增殖的影响。方法利用PCR扩增PDCD4编码区片段,克隆入pENTRA-3C入门载体,利用LR clonaseⅡ重组酶将目的基因片段转接入pLenti-6.3慢病毒表达载体并包装相应的慢病毒,以表达红色荧光蛋白mCherry的pLenti-6.3-mCherry慢病毒载体包装的慢病毒为对照,感染人前列腺癌PC3细胞,通过杀稻瘟素(blasticidin)筛选出能够稳定过表达PDCD4蛋白的细胞亚株。利用Western blot法检测PDCD4蛋白的表达量,MTT法和平板克隆形成实验检测细胞增殖能力的变化。结果构建了重组慢病毒载体pLenti6.3-PDCD4。利用重组慢病毒表达系统筛选稳定过表达PDCD4蛋白的细胞亚株,并用Western blot法进行了验证。MTT法及平板克隆形成实验结果显示PDCD4高表达的PC3细胞亚株的增殖能力受到抑制。结论成功构建了表达PDCD4分子的慢病毒表达载体,筛选出稳定表达PDCD4的PC3细胞株。过表达的PD-CD4分子可以显著抑制PC3前列腺癌细胞的增殖。
Objective To construct a lentiviral vector expressing PDCD4 and establish a PC3 subtype that can stably express the target gene. To investigate the effect of PDCD4 on the proliferation of prostate cancer cells. Methods The fragment of PDCD4 coding region was amplified by PCR and cloned into the pENTRA-3C promoter vector. The target gene fragment was transferred into pLenti-6.3 lentiviral expression vector and the corresponding lentivirus was packaged by LR clonase Ⅱ recombinase to express the red fluorescent protein mCherry PLenti-6.3-mCherry lentiviral vector as a control, infecting human prostate cancer PC3 cells and screening out cell sub-strains stably overexpressing PDCD4 protein by blasticidin. Western blot was used to detect the expression of PDCD4 protein. MTT assay and plate clone formation assay were used to detect the cell proliferation. Results Recombinant lentiviral vector pLenti6.3-PDCD4 was constructed. The recombinant lentivirus expression system was used to screen the cell subline stably overexpressing PDCD4 protein and verified by Western blot. The results of MTT assay and plate clone formation assay showed that the proliferative ability of PDCD4-overexpressing PC3 cells was inhibited. Conclusion The lentiviral vector expressing PDCD4 was successfully constructed and the PC3 cell line stably expressing PDCD4 was screened out. Overexpression of PD-CD4 molecules can significantly inhibit the proliferation of PC3 prostate cancer cells.