中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2008年
8期
578-582
,共5页
陈秀玲%刘禄成%许宗革%李哲%李然伟%高瑞娟%王颂%张明%郭航
陳秀玲%劉祿成%許宗革%李哲%李然偉%高瑞娟%王頌%張明%郭航
진수령%류록성%허종혁%리철%리연위%고서연%왕송%장명%곽항
膀胱肿瘤%血管内皮生长因子%血管内皮生长因子受体2%裸鼠
膀胱腫瘤%血管內皮生長因子%血管內皮生長因子受體2%裸鼠
방광종류%혈관내피생장인자%혈관내피생장인자수체2%라서
Bladder neoplasms%Vascular endothelial growth factor%Vascular endothelial growth factor receptor 2%Nude mice
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G.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16 d开始肿瘤体积即明显小于阴性对照组(P＜0.05),28 d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.
目的 探討血管內皮生長因子(VEGF)及其受體(KDR)雙靶嚮阻斷對人膀胱癌T24細胞和裸鼠膀胱癌移植瘤生長的抑製作用.方法 構建VEGF siRNA和可溶性KDR(sKDR)錶達質粒的共轉染細胞繫,採用二苯基溴化四氮唑藍(MTT)法和流式細胞儀測定T24細胞的增殖和凋亡,採用免疫組化法檢測裸鼠移植瘤組織中VEGF的錶達、瘤間質微血管密度(MVD)和細胞DNA拓撲異構酶(Topo)Ⅱα的錶達,採用原位末耑標記(TUNEL)法檢測裸鼠移植瘤中腫瘤細胞的凋亡.結果 MTT法檢測結果顯示,VEGF siRNA、sKDR和聯閤應用組細胞的生存率分彆為56.3%±8.3%、42.6%±13.8%和32.5%±4.3%,均明顯低于陰性對照組(97.3%±11.6%,P＜0.0001).流式細胞儀分析顯示,VEGF siRNA、sKDR和聯閤應用組在G.期前均齣現亞二倍體凋亡峰,凋亡率分彆為5.1%±0.9%、4.2%±0.5%和8.8%±0.7%,均高于陰性對照組(0.9%±0.4%,P＜0.05),而且聯閤應用組的凋亡率還明顯高于VEGF siRNA和sKDR組(P＜0.01).體內實驗結果顯示,VEGF siRNA、sKDR和聯閤應用組的腫瘤生長均受到不同程度的抑製,聯閤應用組從16 d開始腫瘤體積即明顯小于陰性對照組(P＜0.05),28 d起腫瘤生長幾乎處于停滯狀態.免疫組化分析顯示,聯閤應用組腫瘤組織中VEGF的錶達水平為54.37±5.28,顯著低于陰性對照組(141.66±8.59,P＜0.0001);瘤間質MVD僅為8.22±3.79,明顯低于陰性對照組(61.76±5.28,P＜0.0001)和sKDR組(19.46±4.16,P=0.0089);瘤細胞增殖指數為1.5%±0.7%,顯著低于陰性對照組(11.8%±5.2%,P＜0.0001);而凋亡率達到67.2%±8.5%,明顯高于陰性對照組(8.7%±2.7%,P＜0.0001)、VEGF siRNA組(54.3%±4.8%,P=0.0492)和sKDR組(52.3%±6.4%,P=0.0293).結論 VEGF siRNA與sKDR單獨應用均可不同程度地抑製腫瘤細胞增殖併誘導細胞凋亡,但二者聯閤應用時靶嚮雙位點的治療效果更為顯著.
목적 탐토혈관내피생장인자(VEGF)급기수체(KDR)쌍파향조단대인방광암T24세포화라서방광암이식류생장적억제작용.방법 구건VEGF siRNA화가용성KDR(sKDR)표체질립적공전염세포계,채용이분기추화사담서람(MTT)법화류식세포의측정T24세포적증식화조망,채용면역조화법검측라서이식류조직중VEGF적표체、류간질미혈관밀도(MVD)화세포DNA탁복이구매(Topo)Ⅱα적표체,채용원위말단표기(TUNEL)법검측라서이식류중종류세포적조망.결과 MTT법검측결과현시,VEGF siRNA、sKDR화연합응용조세포적생존솔분별위56.3%±8.3%、42.6%±13.8%화32.5%±4.3%,균명현저우음성대조조(97.3%±11.6%,P＜0.0001).류식세포의분석현시,VEGF siRNA、sKDR화연합응용조재G.기전균출현아이배체조망봉,조망솔분별위5.1%±0.9%、4.2%±0.5%화8.8%±0.7%,균고우음성대조조(0.9%±0.4%,P＜0.05),이차연합응용조적조망솔환명현고우VEGF siRNA화sKDR조(P＜0.01).체내실험결과현시,VEGF siRNA、sKDR화연합응용조적종류생장균수도불동정도적억제,연합응용조종16 d개시종류체적즉명현소우음성대조조(P＜0.05),28 d기종류생장궤호처우정체상태.면역조화분석현시,연합응용조종류조직중VEGF적표체수평위54.37±5.28,현저저우음성대조조(141.66±8.59,P＜0.0001);류간질MVD부위8.22±3.79,명현저우음성대조조(61.76±5.28,P＜0.0001)화sKDR조(19.46±4.16,P=0.0089);류세포증식지수위1.5%±0.7%,현저저우음성대조조(11.8%±5.2%,P＜0.0001);이조망솔체도67.2%±8.5%,명현고우음성대조조(8.7%±2.7%,P＜0.0001)、VEGF siRNA조(54.3%±4.8%,P=0.0492)화sKDR조(52.3%±6.4%,P=0.0293).결론 VEGF siRNA여sKDR단독응용균가불동정도지억제종류세포증식병유도세포조망,단이자연합응용시파향쌍위점적치료효과경위현저.
Objective To study the effect of co-blockage of vascular endothelial growth factor (VEGF)and its receptor(KDR)on growth of bladder carcinoma 124 cells and nude mice xenograft.Methods T24 cell line co-transfeeted with VEGF siRNA and sKDR expression plasmids was developed and its pmliferation 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 MTF assay showed that cell proliferation in VEGF siRNA.sKDR and combination groupa 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 apoptotie 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.Immunohistochemistry revealed VEGF expression in combination groupWas 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 prolireration 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 bv their combination shows more significant therapeutic efficacy.
