ABCE1基因沉默对人膀胱癌T24细胞增殖和迁移能力的影响

目的:本实验通过沉默ABCE1基因在人膀胱癌T24细胞中的表达,探讨ABCE1基因对细胞的生长、细胞周期及迁移等方面的作用。方法:设计及合成ABCE1的siRNA序列,LipofectamineTM2000转染T24细胞。采用RT-PCR和Western blot法检测基因沉默后ABCE1 mRNA和蛋白的表达情况,采用流式细胞术检测T24细胞周期。并通过CCK-8增殖实验、划痕愈合实验和细胞侵袭实验评价沉默ABCE1基因对T24细胞增殖、迁移以及侵袭能力的影响。结果:沉默ABCE1基因48 h后,T24细胞ABCE1 mRNA及蛋白表达明显降低,差异有统计学意义。T24的细胞周期被阻滞于G0/G1期,且S期的细胞数减少,差异有统计学意义。与对照组和空白组相比,CCK-8增殖实验显示,实验组T24细胞的增殖受到明显抑制。划痕愈合实验显示,实验组迁移能力显著下降,差异有统计学意义。Transwell小室法示,实验组T24细胞的侵袭能力显著下降,差异有统计学意义。结论:特异性干扰ABCE1基因表达可抑制人膀胱癌T24细胞的迁移能力,并抑制肿瘤细胞增殖,因此,ABCE1的siRNA序列可能成为治疗膀胱癌的有效靶点。     

膀胱癌T24顺铂耐药细胞系的建立及生物学特性

目的 采用浓度梯度法诱导建立人膀胱癌顺铂耐药细胞系(T24/DDP),并观察其生物学特性.方法 利用人膀胱癌细胞株T24,采用顺铂(DDP)作为诱导剂在体外建立人膀胱癌顺铂耐药细胞模型T24/DDP(0.6μg/mL).通过倒置相差显微镜下观察T24、T24/DDP细胞形态,MTT法检测细胞多药耐药性,生长曲线检测细胞的增殖能力,流式细胞术测细胞的周期,qRT-PCR检测耐药基因MRP(muhidrug resistance-associated protein)的表达.结果 经过顺铂14个月的诱导,成功建立了T24/DDP.倒置相差显微镜下观察与T24细胞相比较,T24/DDP排列不规则,形态出现多形性,出现巨细胞;与T24相比较,T24/DDP对顺铂、阿霉素、长春新碱、甲氨蝶呤均产生不同程度的交叉耐药,比T24细胞倍增时间明显延长,细胞周期中G1期细胞增多,G2、S期细胞减少.qRT-PCR结果显示,耐药细胞MRP基因的表达明显高于亲本细胞.结论 人膀胱癌耐顺铂细胞株T24/DDP具有多药耐药性,可以作为研究膀胱癌细胞多药耐药的良好实验模型.     

免疫球蛋白G在膀胱癌细胞中的表达及其抗体诱导肿瘤细胞凋亡的研究

目的： 研究免疫球蛋白G在膀胱癌细胞中的表达以及其抗体诱导肿瘤细胞凋亡的效应。方法： 免疫组织化学法检测56例临床膀胱癌组织和11例正常膀胱黏膜中以及T24和BIU-87细胞中IgG蛋白表达；对体外培养的膀胱癌细胞系T24和BIU-87采用Western blotting、ELISA法和流式细胞术（FCM）检测IgG蛋白的表达，原位杂交法和RT-PCR检测IgG1 mRNA的表达，MTT法检测羊抗人IgG抗体对肿瘤细胞的生长抑制的影响，FCM检测羊抗人IgG抗体诱导肿瘤细胞凋亡效应。结果： 免疫组化显示临床膀胱癌组织中IgG表达率为91.1％（51/56），正常膀胱移行上皮细胞IgG的表达为45.4％（5/11）；T24和BIU-87细胞浆中IgG明显阳性表达。RT-PCR和原位杂交显示IgG mRNA在T24和BIU-87细胞中明显表达。Western blotting显示T24和BIU-87细胞中均有IgG表达。FCM则表明IgG蛋白主要在细胞质内表达，细胞膜上则小部分表达。ELISA法未能在T24和BIU-87细胞培养液的上清液中检测到IgG。MTT显示羊IgG和羊抗人IgG抗体在25 mg/L时对T24和BIU-87细胞的生长抑制率分别为(4.73±3.73)%、（24.98±3.81）% 和（5.36±1.53）%、（22.70±3.72）%，(P<0.05)。FCM 显示羊IgG和羊抗人IgG抗体在25 mg/L时对T24和BIU-87细胞的凋亡率分别为2.3%、20.7％和1.3%、15.3％。结论:膀胱癌细胞能够表达IgG，但其为非分泌型IgG。IgG的抗体体外对膀胱肿瘤细胞的生长具有一定抑制作用，且具有促进肿瘤细胞凋亡的效应，这可能对膀胱肿瘤的治疗提供一个新的靶点。     

青蒿琥酯对膀胱癌T-24细胞生长的影响

目的：探讨青蒿琥酯(artesunate,Art)对人膀胱癌T-24细胞的抑制作用及机制。方法用不同浓度的Art干预人膀胱癌T-24细胞,采用MTT法检测用药后细胞增殖的改变;划痕实验检测鱼藤素对细胞迁移能力的影响;流式细胞术(FCM)检测细胞周期的改变和凋亡率。结果与对照组比较,Art可显著抑制人膀胱癌T-24细胞株增殖(<0.01);划痕实验结果显示,鱼藤素可降低T-24细胞的迁移能力;FCM结果显示Art可使人膀胱癌T-24细胞周期阻滞在G0/G1期,并可诱导T-24细胞的凋亡(<0.01)。结论 Art可显著抑制T-24细胞的增殖以及降低其迁移能力,其机制可能与Art使T-24细胞生长周期阻滞在G0/G1期并诱导其凋亡相关。     

顺铂和阿霉素对K562细胞周期及凋亡影响的研究

目的：观察化疗药物顺铂（DDP）和阿霉素（ADM）对细胞周期及凋亡的影响。方法： 以不同剂量的药物处理细胞，用流式细胞仪检测处理后不同时间细胞周期变化。结果： 10 μmol/L的DDP作用36 h及1.36 μmol/L的ADM作用48 h,K562细胞以S期阻滞最明显。20 μmol/L的DDP作用24 h出现G1期阻滞，0.17 μmol/L的ADM作用下以G2/M期阻滞为主。在10 μmol/L的DDP 和1.36 μmol/L的ADM作用下，随作用时间延长，阻滞的细胞释放，部分进入凋亡导致凋亡增加。结论： 不同剂量的DDP或ADM作用不同时间导致不同的细胞周期检测点激活，从而使细胞周期变化呈现不同的规律。     

蛋白酶体抑制剂MG132对NK/T淋巴瘤细胞增殖、凋亡和细胞周期的影响

目的： 探讨蛋白酶体抑制剂MG132（Z-leu-leu-leu-CHO,三肽基乙醛）对NK/T淋巴瘤细胞的增殖和细胞周期分布的影响,研究蛋白酶体抑制剂MG132治疗NK/T细胞淋巴瘤的潜在应用价值。方法: 用蛋白酶体抑制剂MG132处理细胞,噻唑蓝［ 3（4,5二甲基噻唑2）2,5二苯基四氮唑溴盐,3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide,MTT］法检测细胞增殖抑制率,倒置显微镜观察细胞形态改变,流式细胞仪检测细胞周期分布和凋亡。结果: 1 μmol/L MG132作用24 h,HANK1细胞增殖抑制率为（57.72±7.44）%,而0.1 μmol/L MG132作用24 h,增殖抑制率仅为（3.98±0.07）%;MG132剂量大,增殖抑制率高;1 μmol/L MG132作用24 h后,HANK1细胞G1和G2期细胞分别为（72.33±3.44）%和（12.86±1.29）%,对照组为（63.63±2.29）%和（7.94±1.91）%,用药组G1和G2期细胞明显高于对照组（P<0.01,P<0.05）; 早期凋亡细胞和晚期凋亡细胞分别为（33.57±2.10）%和（16.66±0.47）%,而对照组仅为（7.18±0.82）%和（3.81±1.06）%,与对照组相比,凋亡率明显增高 (P<0.01,P<0.01)。结论: MG132抑制NK/T细胞淋巴瘤细胞增殖,使细胞周期G1和G2期阻滞,促进细胞凋亡,并存在剂量和时间依赖关系,蛋白酶体抑制剂MG132很可能成为治疗进展期NK/T细胞淋巴瘤的药物。     

膀胱癌术后沙培林膀胱灌注的临床疗效观察

目的观察沙培林膀胱灌注预防膀胱癌术后复发的疗效及并发症.方法 42例膀胱癌术后患者应用沙培林定期行膀胱内灌注,并随访8～24个月,了解灌注后肿瘤复发情况及并发症.结果肿瘤复发率为14.3%(6/42),无肿瘤复发率为85.7%(36/42),并发症发生率为11.9%(5/42).结论沙培林膀胱灌注预防膀胱癌术后复发的有效率高,不良反应小,患者耐受性好,值得临床推广应用.     

5-氮-2′-脱氧胞苷逆转膀胱癌细胞hepaCAM基因的表达及对其生长的抑制

目的 研究5-氮-2-脱氧胞苷(5-aza-CdR)逆转hepaCAM表达及抑制膀胱癌细胞的生长.方法 MTT法检测细胞增殖;流式细胞术检测细胞凋亡;RT-PCR检测hepaCAM mRNA的表达.结果 在T24细胞中,3.0和10.0 μmol/L5-aza-CdR药物浓度的抑制率明显高于0.3和1.0 μmol/L 5-aza-CdR(P＜0.05),在BIU-87细胞中,5.0 μmol/L5-aza-CdR药物浓度的抑制率明显高于0.1和0.5μmol/L(P＜0.05),并且药物处理细胞72 h的抑制率明显高于24和48 h(P＜0.05);5-aza-CdR处理细胞后,T24和BIU-87细胞凋亡数明显增加(P＜0.05);并可逆转hepaCAMmRNA表达.结论 5-aza-CdR可使hepaCAM基因重新表达,并通过诱导凋亡而抑制膀胱癌细胞的生长.     

重组人白细胞介素-24及其联合顺铂对卵巢癌细胞体外生长的影响

目的探讨重组人白细胞介素24(rhIL-24)对SKOV3卵巢癌细胞株、卵巢癌顺铂(DDP)耐药SKOV3/DDP细胞株的抑制效应,并观察rhIL-24对卵巢癌SKOV3和SKOV3/DDP细胞凋亡和细胞周期的影响。方法用rhIL-24、DDP及rhIL-24联合DDP作用于SKOV3、SKOV3/DDP细胞,以CCK-8法检测细胞增殖、用流式细胞仪检测细胞凋亡及周期的变化。结果 rhIL-24对体外培养的卵巢癌SKOV3和SKOV3/DDP细胞的生长有明显抑制作用,rhIL-24联合DDP对SKOV3/DDP细胞株抑制率为30.7%,与单用DDP组抑制率6.5%相比有显著性差异,流式细胞仪检测rhIL-24、DDP和rhIL-24联合DDP各组凋亡率分别为14.95%、12.99%和16.32%,与阴性对照组凋亡率1.32%比较具有显著性差异;细胞周期检测中,SKOV3细胞周期变化显示rhIL-24组G2、S期增多,联合用药组出现S期增多;SKOV3/DDP细胞中,rhIL-24组处理出现G2期增多,联合用药组出现G1期增多。结论 rhIL-24对卵巢癌细胞DDP敏感和耐药株都具有抑制作用,联合用药可增强DDP的效应;rhIL-24对卵巢癌细胞的抑制作用通过诱导细胞凋亡实现,rhIL-24处理细胞后周期变化出现G2期阻滞,联合用药组出现G1期阻滞。     

SEA诱导人膀胱癌BIU-87细胞凋亡机制的研究

目的：为了观察葡萄球菌肠毒素A(SEA)对人膀胱癌BIU-87细胞株诱导凋亡机制。方法：应用MTT比色法和流式细胞术检测SEA对BIU-87细胞增殖抑制率和对细胞周期的影响。结果：SEA对BIU-87细胞株有明显的抑制作用，且呈剂量依赖性。流式细胞术分析，SEA能阻止G1期细胞向S期的进程，G1期细胞堆积。结论：SEA能诱导BIU-87细胞凋亡，具有细胞毒性效应，可用于膀胱内灌注，从而抑制膀胱癌的浸润转移。     

Non-viral tumor necrosis factor-alpha gene transfer decreases the incidence of orthotopic bladder tumors

Our aim was to evaluate the feasibility and efficacy of tumor necrosis factor-alpha gene therapy in preventing bladder tumor recurrence using an orthotopic model of bladder cancer. We transiently transfected a murine bladder cancer cell line MB49 with pBud-TNF-alpha using a transfection system consisting of the cationic liposome N-(1-(2,3-dioleoyloxyl)propyl)-N,N,N-trimethylammoniummethyl sulfate (DOTAP) plus methyl-beta-cyclodextrin solubilized cholesterol (MBC). MB49 cells produced 893.7+/-24.0 pg/ml of TNF-alpha 2 days after transfection. Cell growth was inhibited, apoptosis was induced and MHC class I, B7.1 and Fas expression on the MB49 cells were increased. In vivo, an orthotopic murine bladder cancer model was established by intravesical instillation of bladder cancer cells after transurethral cauterization of the mouse bladder mucosa. TNF-alpha gene transfer was initiated 2 days after the tumor inoculation, when the tumor burden was small, and given twice per week for 3 weeks. RT-PCR showed TNF-alpha mRNA was observed to increase after the first instillation and then return to basal level 1 month after the sixth instillation. Histology revealed that TNF-alpha gene transfer decreases the bladder tumor incidence from 75% for the control group to 25% for the treated group. Increased level of T lymphocytes and NK cells was found in the TNF-alpha transfected bladders. In situ cytokine gene transfer provides significant protection against tumor growth. This approach may be useful to reduce the incidence of a subsequent tumor after endoscopic resection when used for prophylaxis.     

Correlation of ANXA1 expression with drug resistance and relapse in bladder cancer

Objective: To investigate the expression of annexin a1 (ANXA1) in adriamycin-resistant human bladder cancer cell line (pumc-91/ADM) compared with the parental cell line (pumc-91) and its relevance to the drug resistance of bladder cancer, as well as explore the relevance of ANXA1 in recurrent bladder cancer tissues as pertinent to relapse. Methods: qRT-PCR and Western blot were implemented to research the level of ANXA1 in two cell lines (pumc-91/ADM and pumc-91). Immunohistochemistry was applied to explore ANXA1 expression in bladder cancer tissues of different intervals of relapse. The association of ANXA1 with clinicopathological parameters was analyzed. Results: The expression of ANXA1 was downregulated in drug-resistant cell line pumc-91/ADM compared to pumc-91. The bladder cancer tissues recurring two years later exhibited higher ANXA1 levels. ANXA1 expression level was positively correlated with T stage, while it was not connected with histological grade strongly. The expression level and influencing factors of ANXA1 in recurrent tissues of bladder cancer were clarified for the first time. Conclusion: ANXA1 may become a promising marker to predict the recurrence and drug resistance of bladder cancer and provide guidance for surveillance.     

黄芩素和U0126 体外协同抗人膀胱癌细胞的作用及机制研究

目的：探讨黄芩素和U0126体外协同抗人膀胱癌T-24细胞的作用及机制研究。方法：用黄芩素、U0126及二者联合处理T-24细胞,流式细胞术检测细胞周期、细胞凋亡;显微镜下计数细胞数量变化;TUNEL法检测细胞凋亡指数;实时定量PCR和Western blot分别检测T-24 细胞胞外信号调节激酶1/2(ERK1/2) 、CyclinD1、GSK-3β和AKT ＲNA水平、蛋白水平,分析黄芩素与U0126对膀胱癌细胞凋亡和增殖的影响。结果：T-24细胞经不同浓度黄芩素处理后, 细胞凋亡率显著增加;T-24细胞经黄芩素处理24 h,G0/G1 期细胞比例明显增多,S 期细胞比例明显下降,细胞数减少,采用黄芩素联合U0126 处理T-24细胞24 h,S 期细胞比例显著降低;黄芩素或U0126单独处理T-24细胞引起明显细胞凋亡,二者联合处理凋亡更明显;利用两种药物单独或联合作用T-24细胞24 h,GSK-3β、ERK1/2、AKT磷酸化水平显著降低, ERK1/2、CyclinD1的 mRNA表达减少,联合处理作用更显著。结论：黄芩素和U0126 均可诱导T-24细胞凋亡,增加G0/G1 期细胞比例,降低S 期细胞比例,联合应用效果更明显。     

The effects of intravesical instillation of Thio-Tepa on the recurrence rate of bladder tumors

Seventy-eight patients were treated with intravesical instillation of Thio-Tepa in an attempt to prevent postoperative recurrences of bladder tumors. Fifty-six patients who were given no preventive treatment against recurrences were taken as the control group. The patients in this series presented at the Okayama University Hospital between 1961 and 1976 and only the first recurrence after the primary operation was taken into consideration. There was no significant difference in the recurrence rates of the control and instillation groups.     

异丹叶大黄素下调膀胱癌细胞的细胞周期蛋白D1表达并诱导G0/G1细胞周期阻滞

 目的：探索异丹叶大黄素（ISO）抑制膀胱癌细胞侵袭、转移和增殖活性的机制。方法：以ISO作用于人源性膀胱癌UMUC3细胞后,倒置相差显微镜下观察并以ATP生物荧光法检测癌细胞增殖活性;以RT-PCR和Western blotting法检测细胞周期蛋白D1表达;以流式细胞术检测细胞周期的变化;细胞划痕法检测细胞迁移活性。结果：20 μmol/L浓度以上ISO可显著抑制UMUC3细胞的增殖,其IC50为(22.5±2.8) μmol/L。同时 UMUC3细胞的细胞周期蛋白D1 mRNA和蛋白水平均显著降低。以低浓度5 μmol/L ISO预处理UMUC3细胞后,与对照组（47.33%）进行比较,可分别在12 h（58.82％）和24 h（63.94％）显著诱导细胞G0/G1期阻滞（P<0.01）。细胞划痕法检测证实5 μmol/L ISO可显著抑制膀胱癌细胞的迁移活性。结论：ISO可抑制膀胱癌细胞增殖,下调细胞周期蛋白D1表达,诱导G0/G1细胞周期阻滞,抑制细胞的迁移能力。     

Knockdown of lncRNA SNHG7 inhibited cell proliferation and migration in bladder cancer through activating Wnt/β-catenin pathway

It is identified that long non-coding RNAs (lncRNAs) play important roles in tumorigenesis. LncRNA SNHG7 has been found to be an oncogene in varieties of tumors including bladder cancer. However, its potential regulatory mechanism in bladder cancer still remains unknown. In this study, we discovered that the expression levels of SNHG7 were significantly increased in bladder cancer tissues and cell lines. Patients with high expression level of SNHG7 suffered from poor prognosis. Additionally, knockdown of SNHG7 induced declined cell viability, proliferation as well as G0/G1 cell cycle arrest. Furthermore, we found that cell migratory ability was markedly reduced after silencing SNHG7. Next, we verified that knockdown of SNHG7 reduced the protein level of β-catenin and thus decreased the level of its downstream targets including c-myc, cyclin D1 and E-cadherin, implying that SNHG7 might impact bladder cancer via Wnt/β-catenin pathway. Subsequently, the rescue assays performed in SNHG7 silenced T24 cells by using activator of Wnt/β-catenin signaling elucidated that re-activation of this pathway partly restored the inhibitory effects of SNHG7 suppression on biological behaviors of T24 cells. Collectively, SNHG7 elicited carcinogenic functions in bladder cancer partially via activating Wnt/β-catenin signaling pathway, suggesting a potential target for the treatment and prognosis of bladder cancer.     

Protective effects of N-acetylcysteine against cadmium-induced damage in cultured rat normal liver cells

In this study, the protective effects of N-acetylcysteine (NAC), a precursor of reduced glutathione, were studied by measuring the viability, the levels of antioxidant enzymes, and by analyzing the cell cycle in cadmium (Cd)-treated rat liver cells. The cells were treated with 150 μM CdCl2 alone or co-treated with 150 μM CdCl2 and 5 mM NAC (2 h pre-, simultaneous or 2 h post-treatment) for 24 h. The viability of the cells treated with 150 μM CdCl2 alone decreased to 40.1%, while that of the cells co-treated with 5 mM NAC (pre-, simultaneous and post-treatment) significantly increased to 83.7, 86.2 and 83.7%, respectively in comparison to the control cells (100%). The catalase enzyme level decreased to undetectable level in the cells treated with CdCl2 alone, while it significantly increased in the co-treated cells (pre-, simultaneous and post-treatment) to 40.1, 34.3 and 13.2%, respectively. In the cells treated with CdCl2 alone, the glutathione peroxidase enzyme level decreased to 78.3%, while it increased in the co-treated cells (pre-, simultaneous, and post-treatment) to 84.5, 83.3 and 87.9%, respectively. The glutathione reductase enzyme level decreased to 56.1% in the cells treated with cadmium alone, but significantly increased in the cells co treated with NAC (pre-, simultaneous and post-treatment) to 79.5, 78.5 and 78.2%, respectively. Cd caused cell cycle arrest at the S and G2/M phases. The co-treatment with NAC inhibited cell cycle arrest by shifting the cells to the G1 phase. These results clearly show the protective effects of NAC against Cd-induced damage in rat liver cells.     

吡柔比星膀胱灌注预防浅表性膀胱癌术后复发的疗效观察

目的探讨吡柔比星（THP）膀胱内灌注预防浅表性膀胱癌术后复发的效果。方法患者随机分成两组,一组患者膀胱灌注卡介苗（BCG）（40例）,另一组膀胱灌注THP（40例）,随访6～24个月,观察两组复发情况及不良反应。结果BCG组和THP组2年复发率分别为12．5％（5／40）、14．0％（6／43）;两组间比较差异无统计学意义（x^2=0．038,P〉0．05）,而THP组不良反应发生率明显低于BCG组（x^2=9．565,P〈0．01）。结论膀胱灌注THP预防浅表性膀胱癌术后复发的疗效确切,不良反应小,安全性好。     

卡介苗素膀胱灌注预防膀胱癌术后复发的临床研究

目的探讨卡介苗素膀胱内灌注预防膀胱癌术后复发的疗效、安全性．方法47例膀胱癌术后患者分2组。分别应用卡介苗素和卡介苗定期行膀胱内灌注，随访10～32个月，了解灌注后肿瘤复发情况及并发症。并于灌注前、后检测2组尿液中IL－2、IL－6、IL－8的变化情况．结果卡介苗膀胱灌注组肿瘤复发4例（17．4％）。副反应发生18例（78．3％），卡介苗素膀胱灌注组肿瘤复发5例（20．8％）。副反应发生率11例（45．8％），2组尿液中IL－2、IL－6、IL－8值灌注后高于灌注前（P〈0．05）．两组肿瘤复发率、IL-2、IL-6、IL-8值灌注前后比较无显著差异，副反应卡介苗组明显高于卡介苗素组（P〈0．05）．结论卡介苗素膀胱灌注预防膀胱癌术后复发的有效率与卡介苗相同，但不良反应明显减少。患者耐受性好，因此卡介苗素可成为膀胱浅表移行上皮细胞癌临床治疗和预防复发的一种有效药物．     

Anterior urethral recurrence of superficial bladder cancer: its clinical significance

The aim of this study was to reveal the clinical features of anterior urethral recurrence in patients with superficial bladder cancer, and to determine the appropriate treatment. Three hundred and three patients with superficial bladder cancer, who were newly diagnosed and initially treated conservatively in our hospital between 1965 and 1990, were followed for at least 5 years and their clinical outcomes were analyzed. Clinical factors, including anterior urethral recurrence, were evaluated statistically regarding tumor progression. Eight patients (2.6%) had anterior urethral recurrence following superficial bladder cancer. Twenty-four patients (7.9%) had tumor progression and 149 (49.2%) had tumor recurrence. In a multivariate analysis using a logistic model, anterior urethral recurrence was the most important factor, followed by histological grade. Four of 5 patients who were treated for anterior urethral recurrent tumors by transurethral resection showed progression and died of the cancer within one year. Two of the remaining three patients who underwent radical cysto-urethrectomy at the time of anterior urethral recurrence survived. Anterior urethral recurrence following superficial bladder cancer is a predictor for rapid subsequent malignant progression. Once there is anterior urethral recurrence, radical intensive therapy, including radical cysto-urethrectomy, should be carried out immediately.