膀胱癌EJ细胞的CD44异常糖基化及其单抗KMP1的生物学功能研究

研究EJ细胞单抗KMP1的结合抗原CD44的变化和KMP1对EJ细胞生物学功能的影响。方法:采用人膀胱癌EJ细胞株免疫BALB/c小鼠,获得单抗KMP1,免疫荧光检测KMP1的亲和性、结合部位及效价。流式细胞检测KMP1对膀胱癌细胞的特异性。免疫组化检测对膀胱癌组织的特异性。亲和层析分离纯化特异性抗原,检测抗原的氨基酸序列。糖基转化酶抑制剂和过碘酸氧化后分析抗原决定簇的理化性质改变。软琼脂培养克隆形成实验和细胞划痕实验检测KMP1对EJ细胞株的增殖和迁移功能的影响。结果:获得的单抗KMP1是IgG1,能与EJ、BIU-87、T24膀胱癌细胞和膀胱癌组织特异性结合,而与Lovo、HeLa、K562、HepG2、Jurkat、293、HCV29、人的红细胞和白细胞无结合性,也不能结合正常膀胱黏膜。其结合EJ细胞的抗原是异常糖基化的CD44,糖基转化酶抑制剂BG作用EJ细胞7d,KMP1对EJ细胞结合性降低,过碘酸氧化后Western blot显示KMP1对CD44结合性降低,软琼脂培养克隆形成实验和细胞划痕实验结果示KMP1还能减弱EJ细胞的增殖和迁移功能。结论:膀胱癌的高复发性、易转移可能与出现异常糖基化的CD44高表达具有一定的相关性,KMP1可结合异常糖基化的CD44,并抑制EJ细胞的增殖和迁移。      

人膀胱癌非经典型多药耐受性研究

目的：为了建立人膀胱癌耐阿霉素细胞系并研究它们的生物学特性及药物耐受性的机理。方法：采用人膀胱癌细胞系EJ,经递增阿霉素剂量的方法,历时1年,建立一株耐药亚株EJ／DOR,对其生物学特性及耐药机理进行了研究,并应用反转录PCR检测了MDR1、MRP和DNA TopoⅡ基因的表达。结果：EJ／DOR对阿霉素的相对耐受度较亲本细胞提高了14．3倍;对蒽环类、长春花属生物碱及DNA TopoⅡ靶制剂足叶乙甙有明显的交叉耐药性,但对顺铂、丝裂霉素无明显的交叉耐受性;耐药细胞对柔红霉素的细胞内聚集量显著减少;EJ／DOR并不表达MDR1基因,而MRP基因过表达,但细胞内DNA TopoⅡ基因表达低于亲本细胞。结论：细胞内DNA TopoⅡ基因表达下降及MRP基因过表达是EJ／DOR表现为多药耐受性亚型的主要原因,这种非P－gp介导的非经典型多药耐受性细胞为寻求包括阿霉素在内的化疗方案提供了良好的实验模型。     

CD44+CD24-/low乳腺癌干细胞分选鉴定及其多药耐药性研究

目的:观察MACS免疫磁珠法分选CD44+CD24-/low乳腺癌干细胞活性,并检测其与多药耐药的关系。方法:运用MACS免疫磁珠法从多药耐药乳腺癌细胞株MCF-7/ADR中分选CD44+CD24-/low乳腺癌干细胞,流式细胞术测定分选前后CD44+CD24-/low细胞比例,微球体培养法检测分选细胞自我更新能力,流式检测CD44+CD24-/low细胞表面P-糖蛋白(P-gp)表达水平,Real-time PCR检测多药耐药相关基因MDR1表达水平。结果:MACS免疫磁珠法分选后,CD44+CD24-/low细胞比例为93.85%,其成球能力明显强于non-CD44+CD24-/low细胞亚群。MCF-7/ADR细胞株和CD44+CD24-/low乳腺癌干细胞P-gp表达强度分别为101 177.10±2 171.86和114 906.70±2 560.19,P<0.05。CD44+CD24-/low乳腺癌干细胞MDR1基因表达水平为MCF-7/ADR细胞株的(1.07±0.02)倍,P<0.05。结论:经MACS免疫磁珠法分选所得CD44+CD24-/low细胞亚群有更强的自我更新能力,高表达P-gp蛋白和MDR1基因可能是引起乳腺癌多药耐药的重要原因。     

曲古霉素A对人膀胱癌细胞EJ增殖的影响及其作用机制

目的 探讨曲古霉素A(TSA)对人膀胱癌细胞EJ增殖及WNT抑制因子-1(WIF-1)mRNA表达的影响,并分析其可能的作用机制.方法 采用四甲基偶氮唑蓝(MTT)比色法检测不同浓度的TSA对人膀胱癌细胞EJ增殖的影响;采用流式细胞术检测经TSA处理后人膀胱癌细胞EJ的细胞周期分布情况;采用实时逆转录聚合酶链反应(RT-PCR)检测用药前后人膀胱癌细胞EJ中WIF-1 mRNA的表达情况;采用染色质免疫沉淀技术分析用药前后WIF-1基因启动子区组蛋白H3第9位赖氨酸(H3-K9)的乙酰化状态.结果 MTT比色法检测结果显示,处理72 h后,0.1、0.2、0.4、0.8和1.6μmol/L人膀胱癌细胞EJ的增殖抑制率均高于空白对照组(P﹤0.05).流式细胞仪检测结果显示,人膀胱癌细胞EJ经0.2、0.4和0.8μmol/L浓度的TSA处理72 h后,G0/G1期、G2/M期细胞的数量随着TSA浓度的升高而逐渐增加,S期细胞的数量随着TSA浓度的升高而逐渐减少.RT-PCR检测结果显示,与空白对照组相比,不同浓度(0.2、0.4和0.8μmol/L)的TSA处理24、48、72 h后,人膀胱癌细胞EJ中WIF-1 mRNA的相对表达量均升高(P﹤0.05).琼脂糖凝胶电泳结果显示,电泳条带符合预期的片段长度,经TSA处理后,人膀胱癌细胞EJ中扩增出目的片段,且随着TSA浓度的增加,目的片段的产物增多.结论 TSA对人膀胱癌细胞EJ的增殖具有抑制作用,其作用机制可能涉及细胞周期阻滞、WIF-1基因启动子区组蛋白H3-K9乙酰化水平升高及该基因的表达水平升高.     

5-氮-2′-脱氧胞苷诱导膀胱癌EJ细胞凋亡研究

[目的]探讨5-氮-2′-脱氧胞苷（5-Aza-CdR）诱导膀胱癌EJ细胞凋亡机制。[方法]以不同浓度的5-Aza-CdR作用于膀胱癌EJ细胞，以四甲基偶氮唑蓝（MTT）法检测细胞的增殖程度，原位凋亡细胞检测技术（TUNEL）和流式细胞术（FCM）检测5-Aza-CdR对膀胱癌EJ细胞凋亡和细胞周期的影响。[结果]EJ细胞加入不同浓度的5-Aza-CdR（0.625，1.25，2.50，5.00mg/ml），与对照组相比各组EJ细胞增长速度减慢。与对照组相比，不同浓度的各组5-Aza-CdR（0.625，1.25，2.50，5.00mg/ml）可以显著降低EJ细胞的增殖比和增高EJ细胞的凋亡指数（P〈0.05），且与5-Aza-CdR呈浓度依赖关系（P〈0.05）；不同浓度的5-Aza-CdR作用EJ细胞后G0/G1期细胞明显增加（P〈0.05）。[结论]5-氮-2′-脱氧胞苷诱导膀胱癌EJ细胞凋亡并使EJ细胞阻滞于G0/G1期，从而抑制膀胱癌EJ细胞的增殖。     

抗CD3或B7.1与CD28共刺激PBLs膜表面分子表达与肝癌细胞凋亡的相关性

为探讨腺病毒载体介导的外源性Rb基因导入对膀胱癌细胞生长的抑制作用,为膀胱癌的基因治疗提供实验依据,构建了Rb基因的复制缺陷型重组腺病毒载体.体外转染人膀胱癌细胞株EJ.应用免疫组化法、免疫印迹及聚合酶链反应技术检测外源性Rb基因腺病毒载体的转染效率及Rb基因表达效果;用流式细胞仪分析细胞周期;以细胞计数及同位素掺入技术观察外源性Rb基因对EJ细胞生长的抑制效果.结果显示腺病毒载体可有效地将外源基因导入EJ细胞.Rb基因重组腺病毒载体转染细胞后,胞内DNA合成减少,细胞生长受到抑制.流式细胞仪分析显示68%的EJ细胞生长停滞在GO/GI期.结果提示,外源性Rb基因重组腺病毒载体转染膀胱癌细胞可有效抑制细胞生长     

5-氮-2''''-脱氧胞苷诱导膀胱癌EJ细胞凋亡研究

[目的]探讨5-氮-2'-脱氧胞苷(5-Aza-CdR)诱导膀胱癌EJ细胞凋亡机制.[方法]以不同浓度的5-Aza-CdR作用于膀胱癌FJ细胞,以四甲基偶氮唑蓝(MTT)法检测细胞的增殖程度,原位凋亡细胞检测技术(TUNEL)和流式细胞术(FCM)检测5-Aza-CdR对膀胱癌EJ细胞凋亡和细胞周期的影响.[结果]EJ细胞加入不同浓度的5-Aza-CdR(0.625,1.25,2.50,5.00 mg/ml),与对照组相比各组FJ细胞增长速度减慢.与对照组相比,不同浓度的各组5-Aza-CdR(0.625,1.25,2.50,5.00 mg/ml)可以显著降低EJ细胞的增殖比和增高EJ细胞的凋亡指数(P＜0.05),且与5-Aza-CdR呈浓度依赖关系(P＜0.05);不同浓度的5-Aza-CdR作用FJ细胞后G0/G1期细胞明显增加(P＜0.05).[结论]5-氮-2'-脱氧胞苷诱导膀胱癌FJ细胞凋亡并使EJ细胞阻滞于G0/G1期,从而抑制膀胱癌FJ细胞的增殖.     

人膀胱癌非经典型多药耐受性研究

目的：为了建立人膀胱癌耐阿霉素细胞系并研究它们的生物学特性及药物耐受性的机理。方法：采用人膀胱癌细胞系EJ,经递增阿霉素剂量的方法,历时1年,建立一株耐药亚株EJ／DOR,对其生物学特性及耐药机理进行了研究,并应用反转录PCR检测了MDR1、MRP和DNA TopoⅡ基因的表达。结果：EJ／DOR对阿霉素的相对耐受度较亲本细胞提高了14.3倍;对蒽环类、长春花属生物碱及DNA TopoⅡ靶刺剂足叶乙甙有明显的交叉耐药性,但对顺铂、丝裂霉素无明显的交叉耐受性;耐药细胞对柔红霉素的细胞内聚集量显著减少;EJ／DOR并不表达MDR1基因,而MRP基因过表达,但细胞内DNA TopoⅡ基因表达低于亲本细胞。结论：细胞内DNA TopoⅡ基因表达下降及MRP基因过表达是EJ／DOR表现为多药耐受性亚型的主要原因,这种非P-gp介导的非经典型多药耐受性细胞为寻求包括阿霉素在内的化疗方案提供了良好的实验模型。     

LncRNA RP11-79H23.3在膀胱癌细胞中的作用及其发生、发展的研究

背景与目的：虽然许多长链非编码RNA(long non-coding RNA，lncRNA)的异常表达与膀胱癌的发生有密切关系，但对于lncRNA RP11-79H23.3暂未见报道。该研究旨在探讨lncRNA RP11-79H23.3在膀胱癌EJ细胞中的作用及其发生、发展的机制。方法：采用微阵列方法对4对膀胱癌患者的癌和癌旁组织进行组学分析，随后用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction，RTFQ-PCR)检测膀胱癌组织、癌旁组织及正常人膀胱上皮细胞sv-HUC-1、膀胱癌EJ细胞中RP11-79H23.3的表达。通过转染pIRES2-RP11-79H23.3上调该基因后，采用细胞计数试剂盒(cell counting kit-8，CCK-8)和EdU的方法检测EJ细胞的增殖活性，通过Transwell小室和平板划痕实验分别检测EJ细胞的侵袭和迁移能力，应用流式细胞术、Hoechst33342及Tunel检测细胞凋亡，应用细胞免疫荧光检测PTEN在膀胱癌细胞中的定位，采用鬼笔环肽染色观察细胞骨架形成，应用蛋白［质］印迹法(Western blot)分析过表达RP11-79H23.3后EJ细胞中PI3K/AKT信号通路中相关蛋白的表达情况。结果：LncRNA RP11-79H23.3在膀胱癌组织和膀胱癌EJ细胞中表达下调(P ＜0.001，P＜0.01)，pIRES2-RP11-79H23.3转染EJ细胞结果显示，RP11-79H23.3的表达量较转染前显著增加(P＜0.01)。上调RP11-79H23.3的表达可诱导膀胱癌EJ细胞的凋亡，相反，转染pIRES2-EGFP可促进EJ细胞的增殖、侵袭和迁移能力，同时，Western blot结果显示，转染pIRES2-RP11-79H23.3后可上调PTEN在EJ细胞中的表达，下调p-PI3K、p-AKT及p-Gsk3β蛋白的表达(P＜0.05)。结论：LncRNA RP11-79H23.3在膀胱癌组织和膀胱癌EJ细胞中低表达(P＜0.001，P＜0.01)，并且过表达RP11-79H23.3会降低膀胱癌细胞增殖、侵袭和迁移能力，其作用机制可能与PI3K/AKT信号通路有关。提示lncRNA RP11-79H23.3在膀胱癌恶性肿瘤中发挥重要的作用，可能会成为治疗膀胱癌的药物作用靶点。     

模拟胃癌外周血微转移免疫磁珠检测法的建立

目的:探讨建立胃癌外周血微转移癌细胞的免疫磁珠检测法及其意义。方法:体外培养胃癌SGC-7901细胞,将胃癌细胞与PBMC及全血细胞混合,细胞混合液与单克隆抗体(CA199、CEA单抗)在4℃下作用90分钟,然后与免疫磁珠(M-450)作用60分钟后,在磁性细胞分离器中分离,涂片,HE染色,计算玫瑰花环形成率,并进行灵敏度的检测。结果:免疫磁珠技术敏感性高,当PBMC与SGC-7901细胞比为5×105:1时可检测到恶性细胞。1 ml外周血中有20个肿瘤细胞即可被检测出来。结论:免疫磁珠技术是一种简便、快速、敏感性强且临床实用价值高的检测技术,可提高胃癌转移的早期诊断率。     

白血病细胞来源的树突状细胞在治疗白血病中的应用

目的:探讨免疫毒素BDI-1-PEA特异杀伤膀胱癌细胞的体外及体内研究.方法:应用MTT法检测不同浓度的BDI-1-PEA,BDI-1和PEA的混合物(BDI-1 PEA),PEA对体外癌细胞的杀伤能力.接种于裸鼠背部皮下的癌细胞长至0.3cm大小实体瘤时,于尾静脉隔日分别注入BDI-1-PEA、BDI-I、正常小鼠IgG共6次,观察不同的药物对癌的抑制作用.结果:免疫毒素BDI-1-PEA在体外抗膀胱癌细胞系E-J的作用明显优于PEA及BDI-1与PEA的混合物,在荷瘤裸鼠体内明显优于BDI-1及IgG,与对非靶细胞的细胞毒性作用相比较差异非常显著.结论:免疫毒素BDI-1-PEA是一种很有潜力的抗癌药物,为进一步临床研究奠定基础.     

免疫毒素抗膀胱癌的实验与初步临床应用

目的:探讨抗人膀胱癌单克隆抗体绿脓杆菌外毒素(BDI-1-PEA)对膀胱癌细胞的特异杀伤作用。方法:应用四甲基偶氮唑盐(MTT)法检测10-10～10-7mol/L浓度下的BDI-1-PEA、抗人膀胱癌单克隆抗体(BDI-1)、BDI-1和绿脓杆菌外毒素(PEA)的混合物(PEA BDI-1)对体外癌细胞的杀伤能力。接种于裸鼠背部皮下的癌细胞长至直径0.3cm大小实体瘤时,随机分三组,每组10只裸鼠,于尾静脉隔日分别注入100倍半致死剂量浓度(1×10-7mol/L)的BDI-1-PEA100!l及相同剂量的BDI-1、正常小鼠IgG共6次,观察不同的药物对癌的抑制作用。24例膀胱移行细胞癌术后患者应用100倍半细胞致死剂量浓度(1×10-7mol/L)50mlBDI-1-PEA进行了膀胱灌注治疗,每周2次,共8次。每3个月复查膀胱镜或B超。结果:BDI-1-PEA抗膀胱癌细胞系E-J的作用明显优于单抗及毒素与单抗的混合物(P<0.01),与对非靶细胞肾肿瘤细胞系786-0的细胞毒性作用相比较差异非常显著。24例膀胱移行细胞癌术后患者,用药后随访7～22个月,平均15个月,2例患者复发。结论:BDI-1-PEA对膀胱癌细胞具有特异杀伤作用,对预防膀胱癌术后复发有较好疗效。     

Expression of Inhibitor of Apoptosis Gene Family Members in Bladder Cancer Tissues and the 5637 Tumor Cell Line

Background: Apoptosis is suppressed in cancer tissues and tumor cell lines because anti-apoptosis genes are over-expressed. The inhibitor of apoptosis proteins (IAP) gene family contributes to control of apoptosis. The expression profile of eight genes of the IAP family in biopsies from patients with a history of bladder cancer and normal bladder tissues, as well as a bladder tumor cell line (5637), was assessed in the present study. Methods: Cancer tissue samples were obtained at surgery and the 5637 tumor cell line was cultured in RPMI1640 medium. Beyond tumor margins were selected as normal tissue. Expressional profile of interested genes was obtained by using specific primers and the real-time PCR method. Results: The results showed that expression of seven of the studied genes was up-regulated in cancer tissues and the cell line whereas BIRC4 (XIAP) was down-regulated in both. Conclusions: The results showed that these genes were expressed to a greater extent in cancer tissue and cancer cells than in normal tissues. The data suggested that over-expression of anti-apoptotic genes such as IAP family members, can trigger cells to escape from apoptosis.     

Construction and expression of a human-mouse chimeric antibody against human bladder cancer

Human bladder cancer is one of the most common malignant diseases in urogenital system. Traditional therapies are far from successful, especially in advanced cases[1,2]. Targeted therapy with specific antibody is considered a promising strategy for the treatment of diseases as carcinoma, immune disorders and infectious diseases, and so on[3-5]. BDI-1 is an anti-human bladder cancer monoclonal antibody produced through hybridoma technique. It has undergone a series of trials with good results…     

miR-623在膀胱癌中的表达及靶向Fascin1抑制膀胱癌细胞迁移和侵袭

目的:探讨miR-623在膀胱癌中的表达及通过靶向Fascin1对膀胱癌细胞迁移和侵袭能力的影响。方法:采用实时荧光定量PCR检测正常膀胱上皮组织、膀胱癌组织、正常永生化膀胱上皮细胞系(SV-HUC-1)和膀胱癌细胞系（T24、UMUC3）中miR-623的表达量;采用脂质体瞬时转染miR-623 mimics,划痕实验和Transwell侵袭实验检测miR-623过表达后膀胱癌细胞迁移、侵袭能力的改变;生物信息学预测miR-623的作用靶蛋白。miR-623过表达后Western blot及双荧光素酶报告基因检测其靶点的表达及结合情况;使用Fascin1特异性siRNA观察膀胱癌细胞迁移和侵袭能力的变化,并同时转染miR-623 inhibitor进行恢复实验。结果:miR-623在膀胱癌中的表达水平显著低于在正常膀胱组织中的表达（P＜0.05）,在膀胱癌细胞系（T24、UMUC3）中的表达水平显著低于正常膀胱细胞系(SV-HUC-1)（P＜0.05）;miR-623过表达显著抑制T24和UMUC3细胞的迁移和侵袭能力。生物信息学预测Fascin1为miR-623的靶基因,在T24和UMUC3细胞中过表达miR-623,能够显著降低Fascin1的蛋白水平;荧光素酶报告基因分析结果证实miR-623作用于Fascin1的3'-UTR。下调Fascin1表达能够抑制膀胱癌T24和UMUC3细胞的迁移和侵袭能力,同时抑制miR-623的表达能够提高细胞的迁移和侵袭能力。结论:miR-623在膀胱癌中表达水平降低,是一个抑癌因子;并可能通过靶向Fascin1调节膀胱癌的侵袭和转移能力。     

Cloning and analysis of human UroplakinII promoter and its application for gene therapy in bladder cancer

The differential expression of the desired gene product in the target tissue is central for gene therapy. One approach is to use a tissue-specific promoter to drive therapeutic gene expression. UroplakinII (UPII) is a urothelium-specific membrane protein. To investigate the feasibility of targeting gene therapy for bladder cancer, a DNA fragment of 2542-bp upstream of the UPII gene was amplified by PCR and linked to a promoterless firefly luciferase reporter gene. The transient transfection showed that the DNA fragment resulted in preferential expression in bladder carcinoma cells, with negligible expression in nonurothelium cells. Furthermore, the DNA segment located between -2545 and -1608 decided the tissue-specificity of the UPII promoter, the segment located between -328 and -4 being the core promoter of UPII. We generated two recombinant adenoviruses under the control of the UPII promoter: Ad-hUPII-GFP, carrying green fluorescence protein (GFP), and Ad-hUPII-TNF, carrying the tumor necrosis factor alpha (TNFalpha). ELISA revealed that the secretion of TNFalpha by Ad-hUPII-TNF-infected bladder cancer cells was significantly higher than Ad-hUPII-TNF-infected nonurothelium cells. The conditioned medium from Ad-hUPII-TNF-infected bladder cancer cells apparently inhibited the proliferation of L929 cells, a TNFalpha-sensitive cell line, comparing to Ad-hUPII-TNF-infected nonurothelium cells. Intravesical inoculation with Ad-hUPII-TNF inhibited tumor growth in the orthotopic human bladder cancer model. The sustained high level of TNFalpha in urine was identified with ELISA. Taken together, these data suggest that most of the cis elements that confer the bladder-specificity and differentiation-dependent expression of the human UPII gene reside in the 2542-bp sequence, and TNFalpha driven by the human UPII (hUPII) promoter is effective in the specific inhibition of bladder cancer growth both in vivo and in vitro. These results may yield a new therapeutic approach for bladder cancer and provide information on the molecular regulation of urothelial growth, differentiation, and disease.     

瞬时受体电位阳离子通道蛋白在膀胱癌细胞系中的差异表达及其意义

目的:研究瞬时受体电位阳离子通道蛋白(TRPV1)在三种膀胱癌细胞系(RT4、5637和T24)中的差异表达情况,并探讨其意义.方法:Real-time PCR和Western blot 实验分别检测TRPV1 mRNA和蛋白在三种膀胱癌细胞系中的表达情况;MTT 细胞增殖实验检测TRPV1特异性激动剂-辣椒素对三种肿瘤细胞增殖能力的影响,并计算其半数抑制浓度(IC50).结果:Real-time PCR 实验显示TRPV1 mRNA 在膀胱癌细胞系中差异性表达.RT4细胞相对表达量最高,为41.56±4.64;5637表达量次之,为8.81±0.62;T24细胞最少,为1.00±0.063,统计学分析表明三种细胞系表达TRPV1 mRNA具有统计学差异(P<0.01).TRPV1蛋白在膀胱癌细胞系中的表达与mRNA一致.MTT实验表明辣椒素能抑制TRPV1阳性肿瘤细胞的增殖,膀胱癌细胞系对辣椒素作用的敏感性依赖于TRPV1的表达程度.结论:TRPV1在膀胱癌细胞系中存在表达,膀胱癌细胞系对辣椒素的敏感性依赖于TRPV1的表达程度,TRPV1可能是膀胱癌潜在的治疗靶点.     

Periostin is down-regulated in high grade human bladder cancers and suppresses in vitro cell invasiveness and in vivo metastasis of cancer cells

We have previously reported that expression of periostin mRNA is markedly reduced in a variety of human cancer cell lines, suggesting that downregulation of periostin mRNA expression is correlated with the development of human cancers. In our study, to clarify the role of the periostin in human bladder carcinogenesis, we examined the expression of periostin mRNA in normal bladder tissues, bladder cancer tissues and bladder cancer cell lines by Northern blot analysis and RT-PCR analysis. Although the expression of periostin mRNA was detected in 100% (5/5) of normal bladder tissues, it was not detected in 3 human bladder cancer cell lines examined. It was also detected in 81.8% (9/11) of grade 1, 40.0% (4/10) of grade 2 and 33.3% (4/12) of grade 3 bladder cancer tissues, indicating that downregulation of periostin mRNA is significantly related to higher grade bladder cancer (p<0.05). To assess the tumor suppressor function of periostin, we investigated the ability of periostin gene to suppress malignant phenotypes of a bladder cancer cell line, SBT31A. Ectopic expression of periostin gene by a retrovirus vector suppressed in vitro cell invasiveness of the bladder cancer cells without affecting cell proliferation and tumor growth in nude mice. Periostin also suppressed in vivo lung metastasis of the mouse melanoma cell line, B16-F10. Mutational analysis revealed that the C-terminal region of periostin was sufficient to suppress cell invasiveness and metastasis of the cancer cells. Periostin may play a role as a suppressor of invasion and metastasis in the progression of human bladder cancers.     

人类DBC2基因重组腺病毒的构建及其在膀胱癌中的表达(英文)

Objective: The aim of the study was to construct recombinant type 5 adenovirus expressing the human DBC2(deleted in breast cancer 2) gene for in vitro and in vivo assay in human bladder cancer research. Methods: The human DBC2gene was first subcloned into a shuttle plasmid pAdTrack-CMV. After recombining with pAdEasy-1 vector in BJ5183 cells, thenew recombinant vector pAdEasy-DBC2-CMV was transfected into HEK-293 cells to produce adenovirus. The human bladdercancer cell line T24 was infected with DBC2-containing adenovirus particles. Both RNA and protein were collected from cellsharvested at 72 h after infection. Real time quantitative PCR (qPCR) and Western blot were used to examine mRNA and proteinlevels. Fluorescence microscopy was utilized to observe the expression of reporter green fluorescence protein. Results:Electrophoresis showed there was a 2.2 kb size band produced from high fidelity PCR. Pac I digest of the final producedrecombinant vector yielded band sizes of approximately 30 kb and 4.5 kb. After virus infection with the pAdEasy-DBC2-CMVvector, the T24 cell line was observed to highly express green fluorescence protein under a fluorescence microscope. qPCRand Western blot assay identified that the DBC2 gene was overexpressed at both the mRNA and protein levels in virustransfected cells. Conclusion: By using the pAdEasy adenovirus system, we successfully constructed an adenovirus thatcould highly overexpress the tumor suppressor DBC2 gene in a bladder cancer cell line. This viral construct would be widelyused for our further research in gene functional assays and gene therapy in bladder cancer.