Survivin shRNA—APC双基因共表达稳转株对HT-29结肠癌细胞裸鼠皮下移植瘤组织新生血管形成的影响

Effects of survivin shRNA-APC double gene co-expression stably transfected cell lines on the angiogenesis of HT-29 colon cancer cells subcutaneous xenografts in nude mice

目的探讨Survivin shRNA-APC双基因共表达稳转株对HT-29结肠癌细胞裸鼠皮下移植瘤组织细胞中血管内皮生长因子（vascular endothelial growth factor，VEGF）、环氧合酶2（cyclooxygenase-2，COX-2）表达情况及新生血管形成的影响。方法采用随机数字表法将40只裸鼠分成5组，分别为阴性对照组、空载组、Survivin shRNA组、APC组、Survivin shRNA-APC双基因组。阴性对照组：接种HT-29细胞，空载组：接种空载稳转株，Survivin shRNA组：Survivin shRNA稳转株，APC组：接种APC稳转株，Survivin shRNA-APC双基因组：接种Survivin shRNA-APC双基因组共表达稳转株。将各组稳转株及HT-29结肠癌细胞进行培养，磷酸盐缓冲液（phosphate buffer saline，PBS）重悬使细胞密度达到2×107/ml，每只裸鼠右前腋下分别接种相对应的细胞悬液，构建裸鼠皮下移植瘤模型。待接种6周后统一处死所有裸鼠，剥离移植瘤，分别应用Real time PCR及免疫组化方法检测各组移植瘤组织细胞中VEGF、COX-2mRNA及蛋白表达水平，选取CD34抗体标记血管内皮细胞，应用免疫组化方法测定移植瘤组织微血管密度（MVD）。结果每只裸鼠皮下均形成肿瘤。VEGF及COX-2 mRNA表达相对含量Survivin shRNA组[（50.84±3.64）%、（50.11±3.91）%]、APC组[（74.28±6.87）%、（72.39±6.55）%]、Survivin shRNA-APC双基因组[（21.78±4.00）%、（20.74±5.12）%]较空载组[（100.00±0.00）%、（100.00±0.00）%]、阴性对照组[（98.22±0.38）%、（97.61±0.77）%]明显降低，差异均有统计学意义（P均〈0.05）；且Survivin shRNA-APC双基因组中VEGF、COX-2 mRNA表达相对含量较Survivin shRNA组、APC组亦明显降低，差异有统计学意义（P均〈0.05）。VEGF、COX-2蛋白表达量Survivin shRNA组（5.15±1.02、5.26±0.91）、APC组（4.96±1.12、4.93±1.18）、Survivin shRNA-APC双基因组（1.86±0.84，1.80±0.81）较阴性对照组（8.95±0.55、8.77±0.60）和空载组（9.17±0.49、9.01±0.80）明显降低，差异均有统计学意义（P均〈0.05）；且Survivin shRNA-APC双基因组中VEGF、COX-2蛋白表达量较Survivin shRNA组、APC组亦明显降低，差异均有统计学意义（P均〈0.05）。Survivin shRNA组（12.14±3.45）、APC组（11.39±2.94）、Survivin shRNA-APC双基因组（3.96±2.20）MVD值较阴性对照组（25.09±5.59）和空载组（27.87±7.36）明显降低，差异均有统计学意义（P均〈0.05）；Survivin shRNA-APC双基因组MVD值较Survivin shRNA组、APC组亦显著降低，差异均有统计学意义（P均〈0.05）。结论Survivin shRNA-APC双基因共表达稳转株能明显下调移植瘤组织细胞中VEGF、COX-2 mRNA及蛋白表达水平，进而有效抑制移植瘤组织新生血管形成，并且其抑制效果较Survivin shRNA及APC单基因稳转株更为显著。

ObjectiveTo investigate the effects of survivin shRNA-APC double gene co-expression stably transfected cell lines on the VEGF、COX-2 expressions and angiogenesis of subcutaneous exnotransplanted tumor tissues cell of HT-29 colon cancer in nude mice.MethodsForty nude mice were randomly divided into five groups, the negative control group, empty vector group, Survivin shRNA group, APC group, double-gene group.The stably transfected cell lines and HT-29 colon cancer cells were cultured, PBS suspension resulted in cell density of 2×107/ml, injected with respective stably transfected cell lines to establish an SXT model.All the mice were sacrificed after six weeks in order to separate the subcutaneous tumor, the expressions of the VEGF, COX-2mRNA and protein were detected by Real time PCR and immunohistochemistry, CD34 antibody was used to mark the vascular endothelial cells, and the MVD values were detected by immunohistochemistry.ResultsTumors were formed in the nude mice of each group.The expressions of VEGF, COX-2 mRNA in Survivin shRNA group （（50.84±3.64）%, （50.11±3.91）%）, APC group（（74.28±6.87）%, （72.39±6.55）%）and Survivin shRNA-APC double-gene group （（21.78±4.00）%, （20.74±5.12）%）were significantly lower than those in the empty vector groups（（100.00±0.00）%, （100.00±0.00）%） or negative control group （（98.22±0.38）%, （97.61 ＋ 0.77））, the differences were statistically significant（P〈0.05）; the expressions of VEGF, COX-2 mRNA in Survivin shRNA-APC double-gene group were significantly lower than those in APC groups, Survivin shRNA group, the differences were statistically significant（P〈0.05）. The expressions of VEGF, COX-2 protein in Survivin shRNA group（5.15±1.02, 5.26±0.91）, APC group（4.96±1.12, 4.93±1.18）, and Survivin shRNA-APC double-gene groups（1.81±0.84, 1.80±0.81）were significantli lower than those in the negative control group （8.95±0.55, 8.77±0.60） and empty vector group（9.17±0.49, 9.01±0.80）, the differences were statistically significant（P〈0.05）, the expressions of VEGF, COX-2 protein in the Survivin shRNA-APC double-gene group were significantly lower than those than in APC group, Survivin shRNA group（P〈0.05）; the expressions of MVD in APC group（12.14±3.45）, Survivin shRNA group（11.39±2.94）and Survivin shRNA-APC double-gene group（3.96±2.20）were lower than those in the negative control group（25.09±5.59）and empty vector group（27.87±7.36）, the differences were statistically significant（P〈0.05）, the MVD in the Survivin shRNA-APC double-gene group was even lower than that in APC group, Survivin shRNA group, the differences were statistically significant （P〈0.05）.ConclusionSurvivin shRNA-APC double gene co-expression stably transfected cell lines can significantly reduce the expression of the VEGF, COX-2 mRNA and protein and then inhibit the angiogenesis of transplanted tumor tissue, and its inhibitory effect is more effective than that og Survivin shRNA and APC single gene stable strain.