基质细胞衍生因子-1及其受体CXCR4对膀胱癌细胞侵袭能力及腔内种植的影响  

Effect of chemokine stromal cell-derived factor-1 and its receptor CXCR4 on invasion capacity and intraluminal implantation of human bladder cancer cells

作　　者：杨德林[1] 霍倩[1] 王乙水[1] 杨旭升[1] 王凯[1] 王剑松[1] 徐鸿毅[1] 王海峰 

机构地区：[1]昆明医科大学第二附属医院泌尿外科(云南省泌尿外科研究所),昆明医学博士650101

出　　处：《医学研究生学报》2014年第10期1028-1032,共5页Journal of Medical Postgraduates

基　　金：云南省科技计划项目(2007C0016R;2008ZC135M)

摘　　要：目的复发和转移是膀胱癌治疗失败的主要原因。文中利用RNA干扰技术使趋化性细胞因子受体CXCR4基因沉默,探讨其沉默对膀胱癌细胞侵袭能力及腔内种植的影响。方法设计合成CXCR4的特异性短发卡状RNA(short hairpin,shRNA),筛选出能够稳定抑制CXCR4表达的细胞,并分为空白对照组、阴性对照质粒组、pshRNA-CXCR4-1实验组,pshRNA-CXCR4-2实验组,应用RT-PCR和免疫荧光分别检测CXCR4 mRNA和蛋白表达水平,应用Boyden小室模型检测体外侵袭能力的变化。将20只裸小鼠随机数表法分为实验裸小鼠和对照裸小鼠,各10只。向实验裸小鼠膀胱内注入100μL ShRNA-EJ-M3,向对照裸小鼠膀胱内注入100μL EJ-M3细胞,检测shRNA-CXCR4对膀胱癌细胞腔内种植能力的影响。结果稳定转染后的pshRNA-CXCR4-1实验组CXCR4 mRNA表达(62.05±1.35)较空白对照组(174.38±1.96)和阴性对照组(166.27±1.82)明显下降(P<0.05),pshRNA-CXCR4-2实验组CXCR4 mRNA表达水平(182.58±4.2)与空白对照组和阴性对照组比较,差异无统计学意义(P>0.05);免疫荧光实验中pshRNA-CXCR4-1实验组红色免疫荧光细胞数(32.24±2.23)较空白对照组(89.61±4.47)和阴性对照组(92.45±3.68)降低(P<0.05);pshRNA-CXCR4-2实验组红色免疫荧光细胞数(76.87±5.11)与空白对照组和阴性对照组比较,差异无统计学意义(P>0.05)。体外趋化侵袭实验结果显示,pshRNACXCR4-1实验组穿膜细胞数(39.67±8.45)明显少于空白对照组(135.33±9.28)及阴性对照组(123.63±6.36),差异有统计学意义(P<0.05)。实验裸小鼠腔内种植能力与对照裸小鼠比较,差异有统计学意义(10%vs 70%,P<0.01)。结论以CXCR4为靶向的特异性shRNA能有效抑制膀胱癌细胞基因CXCR4的表达,显著降低膀胱癌细胞体外侵袭能力及腔内种植能力,SDF-1/CXCR4生物轴有望成为预防和治疗膀胱癌侵袭和转移的重要靶点之一。Objective Bladder cancer , which has a high rate of recurrence and invasion , is the most common genitourinary cancer.The article was to study the effect of specific chemokine receptor CXCR 4 on invasion capacity and intraluminal implantation of human bladder cancer cells . Methods A CXCR4 specific recombinant plasmid vector （short hairpin, shRNA） was constructed to select those cells which could inhibit the expression of CXCR 4, and these cells were divided into blank control group , negative control plasmid group and recombinant plasmid group （pshRNA-CXCR4-1, pshRNA-CXCR4-2）.RT-PCR and immunofluorescence technique were used to detect the mRNA and protein expression of CXCR 4 respectively .Invasion capability in vitro of the cells was evaluated by Boyden chamber .20 nude mice were randomly divided into experimental group and control group （ n=10 ） .The experimental group was established by injection of 100μL shRNA-EJ-M3 into the bladder , while the control group was established by injection of 100μL EJ-M3, aiming to detect the effect of shRNA-CXCR4 on intraluminal implantation of human bladder cancer cells . Results The CXCR4 mRNA expression of the pshRNA-CXCR4-1 group （62.05 ± 1.35） was significantly lower than that of blank control group （174.38 ±1.96, P〈0.05）.In immunofluores-cence experiment, the red cell amount of the pshRNA-CXCR4-1 group（32.24 ±2.23） was lower than that of the blank control group （89.61 ±4.47,P0.05）.The Boyden chamber experiment showed that the number of penetrating cells of the pshRNA -CXCR4-1 group （39.67 ±8.45） was significantly lower than that of the blank control group （135.33 ±9.28, P〈0.05） and that of the negative control plasmid group（123.63 ±6.36, P〈0.05）.As to the intraluminal implanting capability, the difference between the ex-perimental group and the control group of statistical significance （10%vs 70%,P〈0.01）. Conclusion CXCR4 shRNA can inhibit the expression of CXCR4 and significantly decrease the invasion capacity

关 键 词：CXCR4基因 短发卡状RNA 膀胱癌 侵袭 

分 类 号：R737.14[医药卫生—肿瘤]