【摘要】 为探讨穿孔素(PFP)、颗粒酶B(GrB)共表达对人喉癌(Hep-2)细胞生长的抑制及其诱导该细胞的凋亡作用,采用RT-PCR的方法从人的喉癌组织浸润淋巴细胞中扩增全长PFP、GrB的cDNA片段,构建共表达重组体pVAX1-PIG,并将其转染入人的Hep-2细胞株中。收集转染后的Hep-2细胞,采用软琼脂集落形成实验、MTT法、原位末端标记法(TUNEL)、流式细胞仪(FCM)检测分析各组人Hep-2细胞的生长抑制及其凋亡情况。结果显示pVAX1-PIG转染组的集落形成数目比空白对照组与pVAX1转染组明显减少(P<0.05),MTT检测结果显示对照组细胞生长速度比pVAX1-PIG转染组要快。TUNEL染色、FCM法检测均显示pVAX1-PIG转染组的Hep-2细胞大量凋亡且其凋亡率显著高于对照组(P<0.05)。因此,PFP、GrB的共表达能够抑制人Hep-2细胞的生长并且可以诱导该细胞的凋亡。更多还原

【Abstract】 To investigate the potential roles of human perforin protein(PFP) and granzyme B(GrB) on the growth and induction of apoptosis of Hep-2 cells,the full-length cDNA fragments of PFP and GrB genes of the infiltrated lymphocytes from throat cancer tissues were amplified and then subcloned into plasmid pVAX1 to construct eukaryotic expression plasmid pVAX1-PIG.The recombinant pVAX1-PIG was transfected to Hep-2 cell line by lipofectamine 2000.The inhibitory effect of co-expression on the growth of Hep-2 cells was investigated by colony formation on soft agar and thiazolyl blue MTT test and the apoptosis in Hep-2 cells was detected by flow cytometry,in situ terminal deoxynucleotide transferase and nick translation assay(TUNEL).It was demonstrated that Hep-2 cells transfected with plasmid pVAX1-PIG grew slowly in comparison with the control group(P<0 05),and also with a reduced number of colony formation on soft agar and an elevation of the apoptotic rate in case of Hep-2 cells tranfected with plasmid pVAX-PIG.It is concluded that co-expression of PFP and GrB genes can induce apoptosis and inhibit growth and proliferation of Hep-2 cells.更多还原