血管内皮生长因子及其受体双靶向阻断对膀胱癌细胞生长及血管生成的抑制作用

Inhibition of bladder cancer cell growth and angiogenesis by co-blockage of vascular endothelial growth factor and its receptor kdr

目的探讨血管内皮生长因子（VEGF）及其受体（KDR）双靶向阻断对人膀胱癌T24细胞和裸鼠膀胱癌移植瘤生长的抑制作用。方法构建VEGF siRNA和可溶性KDR（sKDR）表达质粒的共转染细胞系，采用二苯基溴化四氮唑蓝（MTT）法和流式细胞仪测定T24细胞的增殖和凋亡，采用免疫组化法检测裸鼠移植瘤组织中VEGF的表达、瘤间质微血管密度（MVD）和细胞DNA拓扑异构酶（Topo）Ⅱα的表达，采用原位末端标记（TUNEL）法检测裸鼠移植瘤中肿瘤细胞的凋亡。结果MTT法检测结果显示，VEGF siRNA、sKDR和联合应用组细胞的生存率分别为56．3％±8．3％、42．6％±13．8％和32．5％±4．3％，均明显低于阴性对照组（97．3％±11．6％，P〈0．0001）。流式细胞仪分析显示，VEGF siRNA、sKDR和联合应用组在G0期前均出现亚二倍体凋亡峰，凋亡率分别为5．1％±0．9％、4．2％±0．5％和8．8％±0．7％，均高于阴性对照组（0．9％±0．4％，P〈0．05），而且联合应用组的凋亡率还明显高于VEGF siRNA和sKDR组（P〈0．01）。体内实验结果显示，VEGF siRNA、sKDR和联合应用组的肿瘤生长均受到不同程度的抑制，联合应用组从16d开始肿瘤体积即明显小于阴性对照组（P〈0．05），28d起肿瘤生长几乎处于停滞状态。免疫组化分析显示，联合应用组肿瘤组织中VEGF的表达水平为54．37±5．28，显著低于阴性对照组（141．66±8．59，P〈0．0001）；瘤间质MVD仅为8．22±3．79，明显低于阴性对照组（61．76±5．28，P〈0．0001）和sKDR组（19．46±4．16，P=0．0089）；瘤细胞增殖指数为1．5％±0．7％，显著低于阴性对照组（11．8％±5．2％，P〈0．0001）；而凋亡率达到67．2％±8．5％，明显高于阴性对照组（8．7％±2．7％，P〈0．0001）、VEGF siRNA组（54．3％±4．8％，P=0．0492）和sKDR组（52．3％±6．4％，P=0．0293）。结论VEGF siRNA与sKDR单独应用均可不同程度地抑制肿瘤细胞增殖并诱导细胞凋亡，但二者联合应用时靶向双位点的治疗效果更为显著。

Objective To study the effect of co-blockage of vascular endothelial growth factor （VEGF） and its receptor （KDR） on growth of bladder carcinoma T24 cells and nude mice xenograft. Methods T24 cell line co-transfected with VEGF siRNA and sKDR expression plasmids was developed and its proliferation was assayed by MTT and apoptosis by FCM. The nude mice model bearing bladder carcinoma xenograft was established. The tumor cell VEGF expression, stroma microvessel density （MVD） and tumor cell topoisomerase Ⅱ alpha （Topo Ⅱα） expression were detected by immunohistochemistry. Cell apoptosis was estimated by TUNEL assay. Results MTT assay showed that cell proliferation in VEGF siRNA, sKDR and combination groups was 56.3% ± 8.3%, 42.6% ± 13.8% and 32.5% ± 4. 3%, respectively, significantly lower than that in the scramble control （97.3% ± 11.6%, P 〈 0. 0001 ）. FCM showed there were sub-diplod apoptotic peaks before G1 phase in VEGF siRNA, sKDR and combination groups, and apoptosis ratio was 5.1% ± 0.9%, 4.2% ± 0. 5% and 8.8% ± 0.7%, respectively, all of which were higher than that in the scramble control （0.9% ± 0.4%, P 〈 0.05 ）, and the combination group had even more higher apoptosis than the two singlely treated groups （P 〈 0.01 ）. In vivo test showed that tumor growth was inhibited in VEGF siRNA, sKDR and combination groups, and from day 16 the tumor volume in combination group was significantly smaller than that in scramble control （P 〈0.05） , and from day 28 the tumor almost lost the ability to further growth. Immunohistoehemistry revealed VEGF expression in combination group was 54.37 ±5.28, significantly lower than that in the scramble control （ 141.66 ± 8.59, P 〈 0.0001 ）. MVD number was only 8.22 ±3.79, much less compared with that in the scramble control （61.76 ±5.28, P〈0. 0001 ） or sKDR group （ 19. 46 ± 4. 16, P = 0. 0089）. Tumor cell proliferation index in the combination group （1.5% ±0.7% ） was significantly decreased compared with that in the scramble control （ 11.8% ± 5.2%, P 〈 0. 0001 ）, and apoptosis index （67.2% ± 8.5% ） was much higher than that in the scramble control （ 8.7% ± 2.7%, P 〈 0. 0001 ）, VEGF siRNA group （ 54. 3% ± 4.8%, P = 0. 0492 ） or sKDR group （52.3% ±6.4%, P = 0. 0293）. Conclusion VEGF siRNA or sKDR alone can inhibit tumor cell proliferation and induce cell apoptosis, but co-blockage of VEGF and KDR by their combination shows more significant therapeutic efficacy.