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目的观察转染人Endostatin(hEndostatin)基因对胰腺癌细胞株SW1990增殖、凋亡及血管内皮生长因子(VEGF)表达的影响。方法携带hEndostatin基因的复制缺陷型重组腺病毒载体体外转染SW1990细胞,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,实时荧光定量PCR和ELISA分析检测hEndostatin和VEGF的表达。建立裸鼠胰腺癌模型,随机分为3组(n=8),瘤体内分别注射磷酸盐缓冲液(PBS组)、报告基因LacZ重组腺病毒(Ad-LacZ组)、hEndostatin重组腺病毒(Ad-hEnd组)100μl,隔日1次,共4次。4周后免疫组织化学染色检测肿瘤组织中hEn- dostatin、VEGF、增殖性细胞核抗原(PCNA)的表达,TUNEL法检测肿瘤细胞凋亡。结果体外转染hEndostatin基因不影响SW1990细胞的增殖和凋亡(P>0.05),但下调VEGF的表达(P<0.01),下调作用第5天最大,在mRNA和蛋白水平的抑制率分别为84.67%和81.41%。体内转染4周后,Ad- hEnd组VEGF、PCNA阳性表达率分别为(36.3±7.1)%、(38.2±3.9)%,显著低于Ad-LacZ组的(81.2±6.6)%、(93.2±4.9)%和PBS组的(78.4±6.2)%、(90.1±5.7)%(P<0.01);肿瘤细胞凋亡率为(31.2±5.4)%,显著高于Ad-LacZ组的(9.4±4.9)%和PBS组的(8.5±3.7)%(P<0.01)。结论hEndostatin基因转染抑制SW1990细胞的体内增殖并促进凋亡;下调SW1990细胞内源性VEGF的表达可能是抗肿瘤的作用机制之一。
Objective To observe the effect of hEndostatin gene on the proliferation, apoptosis and the expression of vascular endothelial growth factor (VEGF) in pancreatic cancer cell line SW1990. Methods The recombinant adenovirus carrying hEndostatin gene was transfected into SW1990 cells in vitro. The proliferation of SW1990 cells was detected by MTT assay. The apoptosis of cells was detected by flow cytometry. The expression of hEndostatin and VEGF was detected by real-time fluorescence quantitative PCR and ELISA. The model of pancreatic cancer in nude mice was established and randomly divided into 3 groups (n = 8). The mice were inoculated with phosphate buffered saline (PBS), LacZ recombinant adenovirus (Ad-LacZ), hEndostatin recombinant adenovirus -hEnd group) 100 μl every other day for 4 times. After 4 weeks, the expression of hEn-dostatin, VEGF and proliferating cell nuclear antigen (PCNA) in tumor tissues were detected by immunohistochemical staining. TUNEL method was used to detect the apoptosis of tumor cells. Results In vitro, hEndostatin gene transfection did not affect the proliferation and apoptosis of SW1990 cells (P> 0.05), but down-regulated the expression of VEGF (P <0.01). The down-regulation effect of hEndostatin gene was the largest at day 5 with the inhibition rates at mRNA and protein levels of 84.67 % And 81.41%. After 4 weeks of transfection, the positive rates of VEGF and PCNA in Ad-hEnd group were (36.3 ± 7.1)% and (38.2 ± 3.9)%, respectively, which were significantly lower than those in Ad-LacZ group (81.2 ± 6.6% and 93.2 ± 4.9% in PBS group and 78.4 ± 6.2% and 90.1 ± 5.7% in PBS group (P <0.01). The apoptosis rate of tumor cells was (31.2 ± 5.4)%, which was significantly higher than that of Ad-LacZ group ± 4.9% vs 8.5 ± 3.7% in PBS group (P <0.01). Conclusion Transfection of hEndostatin gene can inhibit the proliferation and induce apoptosis of SW1990 cells in vitro. Down-regulation of endogenous VEGF expression in SW1990 cells may be one of the mechanisms of anti-tumor effect.