c—Ha—ras反义寡聚脱氧核苷酸对人膀胱癌细胞生长增殖的抑…

应用人工合成的ｃ－Ｈａ－ｒａｓ反义寡聚脱氧核苷酸（ＡＳＯ－ｒ）对人膀胱癌细胞株Ｔ２４进行作用。结果表明：ＡＳＯ－ｒ能够抑制膀胱癌细胞株Ｔ２４中ｒａｓＰ２１的表达及Ｔ２４细胞的生长增殖，抑制作用大小与ＡＳＯ－ｒ的浓度有关。     

联合应用c－Ha－ras，c－myc反义寡聚脱氧核苷酸对人膀胱癌细胞生长增殖的抑制作用

作者联合应用人工合成的ｃ－Ｈａ－ｒａｓ、ｃ－ｍｙｃ反义寡聚脱氧核苷酸（ＡＳＯ－ｒ，ＡＳＯ－ｍ）对人膀胱癌细胞株进行作用，观察到ＡＳＯ－ｒ、ＡＳＯ－ｍ能明显抑制癌细胞中ｒａｓＰ２１、ｍｙｃＰ６２的表达，抑制了癌细胞ＤＮＡ合成及生长增殖，经ＡＳＯ－ｒ、ＡＳＯ－ｍ处理后癌细胞裸鼠致瘤力下降。这一结果表明：针对多个癌基因联合应用反义寡聚脱氧核苷酸，可能给膀胱癌的治疗提供新的途径，有进一步探索的价值     

联合应用c—Ha—ras,c—myc反义寡聚脱氧核苷酸对人膀胱癌细胞生长增殖…

作者联合应用人工合成的ｃ－Ｈａ－ｒａｓ，ｃ－ｍｙｃ反义寡聚脱氧核苷酸（ＡＳＯ－ｒ、ＡＳＯ－ｍ）对人膀胱癌细胞株进行作用，观察到ＡＳＯ－ｒ、ＡＳＯ－ｍ能明显抑制癌细胞中ｒａｓＰ２１、ｍｙｃＰ６２的表达，抑制了癌细胞ＤＮＡ合成及生长增殖，经ＡＳＯ－ｒ、ＡＳＯ－ｍ处理后癌细胞裸鼠致瘤力下降，这一结果表明：针对多外癌基因联合应有反义寡聚脱氧核苷酸，可能给膀胱癌的治疗提供新的途径，有进一步探索的价值。     

反义N－rasl DNA片段对人膀胱癌BIU－87细胞系作用的研究

将载有反义人Ｎ－ｒａｓｌ（ｅｘｏｎｌ）ＤＮＡ片段的重组表达质粒ｆＰＧＶ１－ＭＴ－Ｎｒａｓｌ（Ａ）导入人膀胱移行细胞癌ＢＩＵ－８７细胞系，可以导致ＢＩＵ－８７细胞恶性行为的部分改变，表现在生长速度减慢２１．７％～６５．６％，３Ｈ－ＴｄＲ掺入降低４５．８％，软琼脂克隆形成能力下降４８．３％，同时，Ｎ－ｒａｓ－ｍＲＮＡ及Ｐ２１ｒａｓ表达减少。     

反义N—rasl DNA片段对人膀胱癌BIU—87细胞系作用的研究

将载有反义人Ｎ－ｒａｓｌ（ｅｘｏｎｌ）ＤＮＡ片段的重组表达质粒ｆＰＧＶ１－ＭＴ－Ｎｒａｓｌ（Ａ）导入人膀胱移行细胞癌ＢＩＵ－８７细胞系，可以导致ＢＩＵ－８７细胞恶性行为的部分改变，表现在生长速度减慢２１．７％～６５．６％，＾３Ｈ－ＴｄＲ掺入降低４５．８％，软琼脂克隆形成能力下降４８．３％，同时，Ｎ－ｒａｓ－ｍＲＮＡ３及Ｐ２１ｒａｓ表达减少。     

野生型p53基因导入对膀胱癌细胞生长抑制和H－ras基因表达调控

通过逆转录病毒载体将外源野生型ｐ５３基因导入膀胱癌细胞ＢＩＵ－８７和ＥＪ，使细胞在体外标准培养条件下生长速率降低，丧失裸鼠致瘤性。细胞周期分析显示Ｇ０＋Ｇ１细胞比率明显增高，已证明，ＢＩＵ－８７和ＥＪ细胞都有ｒａｓ基因突变和表达扩增。进一步研究发现，转染的细胞内Ｈ－ｒａｓｍＲＮＡ表达低于非转染细胞。结果提示，增加细胞内野生型ｐ５３基因表达量能抑制突变的ｒａｓ基因表达，从而抑制膀胱癌细胞的恶性增殖。     

羟甲基戊二酰辅酶A还原酶抑制剂对多囊肾病囊肿衬里上皮细胞增殖抑制及其机制的研究

目的 探讨羟甲基戊二酷辅酶 Ａ（ＨＭＧ ＣｏＡ）还原酶抑制剂对常染色体显性遗传型多囊肾病（ＡＤＰＫＤ）囊肿衬里上皮细胞的作用及其机制。方法 噻唑蓝（ＭＴＴ）法检测细胞增殖，免疫印迹法检测细胞膜ｐ２１ｒａｓ的表达．免疫细胞化学检测细胞核ｃ－ｆｏｓ、ｃ－ｊｕｎ的表达。结果ＡＤＰＫＤ囊肿衬里上皮细胞经 ＨＭＧ ＣｏＡ还原酶抑制剂处理后，细胞增殖受到显著抑制（Ｐ＜０．０１），细胞膜 ｐ２１ｒａｓ蛋白表达下调，细胞核ｃ－ｆｏｓ、ｃ－ｊｕｎ的表达显著下降（Ｐ＜０．０１）。结论 ＨＭＣ ＣｏＡ还原酶抑制剂能抑制ＡＤＰＫＤ囊肿衬里上皮细胞的增殖，其机制可能与抑制细胞内ｒａｓ蛋白的异戊二烯化并阻断ｒｓ介导的信号转导有关。     

阴茎癌组织中K－ras基因点突变和ras基因产物P21表达的探讨

应用聚合酶链反应（ＰＣＲ）结合单链构象多态性分析（ＳＳＣＰ）及免疫组化方法研究２８例阴茎癌Ｋ－ｒａｓ基因第１２位密码子点突变和ｒａｓ基因产物Ｐ２１蛋白的表达情况。结果显示２８例阴茎癌发现３例存在Ｋ－ｒａｓ基因第１２位点突变，１２例标本有Ｐ２１蛋白阳性表达，而癌旁组织Ｐ２１表达阴性，并且Ｐ２１阳性表达率与阴茎癌分化程度和临床分期有关。提示ｒａｓ基因的激活在阴茎癌的发生中有一定的作用。首先发现了阴茎癌中Ｋ－ｒａｓ基因第１２位密码子点突变和Ｐ２１蛋白表达阳性。     

c-Ha-ras癌基因反义RNA抑制膀胱癌细胞生长及粘附的研究

目的 研究降低ｃ－Ｈａ－ｒａｓ癌基因表达对膀胱癌细胞生长和粘附的影响。方法 将反义ｃ－Ｈａ－ｒａｓ基因导入膀胱癌细胞系ＴＢＣ－１细胞株，筛选阳性细胞克隆。Ｓｏｕｔｈｅｒｎ杂交鉴定外源基因整合情况，荧光分光光度法检测ｃ－Ｈａ－ｒａｓ表达，绘制生长曲线，体外检测细胞粘附率。结果 筛选出细胞克隆ＴＢＣ－ｎｅｏ和ＴＢＣ－ａｎｔｉｒａｓ，放射自显影显示只有ＴＢＣ－ａｎｔｉｒａｓ有外源性ｃ－Ｈａ－ｒａｓ杂交带     

三氧化二砷抑制人膀胱癌细胞BIU-87体外生长的实验研究

目的：观察三氧化二砷对人膀胱癌细胞的生长抑制作用,并探讨其机制。方法：采用ＭＴＴ法检测不同浓度的Ａｓ２Ｏ３对人膀胱细胞株ＢＩＵ＿８７的生长抑制率,原位末端转移酶标记技术检测细胞凋亡,ＳＡＢＣ免疫组织化分析ＢＩＵ－８７中ｂｃｌ－２的表达。结果：Ａｓ２Ｏ３可有铲地抑制ＢＩＵ＿８７细胞生长,具有时间、浓度依赖性特点;经药物作用后,膀胱癌凋亡细胞明显增多,凋亡率随作用时间的延长而增高,ＢＩＵ－８７细胞中的     

The anti-angiogenic activity of human endostatin inhibits bladder cancer growth and its mechanism

PURPOSE: We investigated whether recombinant human endostatin can inhibit the growth of bladder cancer in an experimental model and its possible mechanism of action. MATERIALS AND METHODS: The recombinant human endostatin protein was induced and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot assays. Its biological activities and the possible mechanisms of action were studied in vitro and in vivo. RESULTS: Recombinant human endostatin inhibited the proliferation of endothelial cells (ECV304) but not bladder tumor cells (EJ). Endostatin induced the expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in bladder cancer cells. Endostatin slowed the growth of xenograft bladder tumors. Immunohistochemistry revealed that endostatin blocked angiogenesis by decreasing vascular endothelial growth factor expression and inducing apoptosis in bladder cancer cells. CONCLUSIONS: These findings demonstrate that endostatin can inhibit xenograft bladder cancer growth and this effect is likely to be mediated by regulating matrix metalloproteinases, tissue inhibitors of matrix metalloproteinases and vascular endothelial growth factor expression, and by inducing apoptosis.     

The growth inhibitory effect of p21 adenovirus on human bladder cancer cells

PURPOSE: To evaluate whether p21 (WAF-1/CIP1) should be considered a potential candidate for human bladder cancer gene therapy, we determined: (1) the basal level of p21 expression in bladder cancer cell lines, (2) the response of bladder cancer cells to increased p21 expression following p21 adenovirus infection, and (3) the mechanism of growth inhibition produced by p21 overexpression. MATERIALS AND METHODS: Five established human bladder cancer cell lines and one primary culture derived from an invasive transitional cell carcinoma were used in this study. To examine the effect of p21 protein on the growth of human bladder cancer cells, a recombinant adenovirus vector system containing p21 cDNA, under the control of cytomegalovirus promoter, was constructed. A control virus containing p21 in an antisense orientation was used to eliminate potential artifacts caused by viral toxicity. RESULTS: Human bladder cancer cell lines exhibit variable endogenous p21 levels which correlate with the in vitro growth status. Significant, but highly variable increases in the steady-state level of p21 were detected in p21 adenovirus infected cells. Human bladder cancer cell lines responded heterogeneously to p21 adenovirus infection. Growth of the WH cell line was substantially inhibited in a dose and time-course dependent fashion. The mechanism of p21 growth inhibition was found to be due to G0/G1 arrest and not the induction of apoptosis. In contrast, p21 adenovirus failed to inhibit the growth of T24 bladder cancer cells because T24 cells were resistant to viral infection. The 253J bladder cancer cells exhibited marked sensitivity to adenovirus; substantial growth inhibition was seen with both sense and antisense p21 very early in the time course of infection. CONCLUSIONS: We found significant variation in the basal level of p21 protein expression in several human bladder cancer cell lines. Increased p21 expression as a result of adenoviral infection may be a potent growth suppressor in some human bladder cancer because it elicits cell cycle arrest in G0/G1 stage, but not the induction of apoptosis. Bladder cancer cells exhibit a wide spectrum of sensitivity to adenoviral infection that may be caused by the presence of viral receptor heterogeneity. This wide spectrum of sensitivity has significant basic scientific and clinical implications and warrants further study.     

Adenovirus mediated gelsolin gene therapy for orthotopic human bladder cancer in nude mice

PURPOSE: Gelsolin is an actin regulatory protein that is undetectable or reduced in human bladder tumors compared with normal epithelial cells. Whether the over expression of gelsolin could inhibit tumor growth was investigated in an orthotopic bladder cancer nude mouse model using recombinant adenovirus encoding wild-type gelsolin (Ad-GSN). MATERIALS AND METHODS: The 2 human bladder cancer cell lines KU-7 and UMUC-2 were transduced with Ad-GSN in vitro. Flow cytometric analysis was done to examine the cell cycle after transducing the adenovirus. Cell growth was compared with control groups of these cells transduced with adenovirus containing the Escherichia coli beta-galactosidase gene Ad-betagal. In vivo KU-7 cells were introduced into the bladder of nude mice (day 0), followed by 3 injections into the urethra (days 2 to 4) with Ad-GSN or Ad-betagal (1 x 10 pfu). At 8 days after initial adenovirus exposure (day 10) each bladder was sectioned and stained, and the mass of the tumor was digitally determined. RESULTS: Bladder cancer cell growth (KU-7 and UMUC-2) was inhibited after these cells were transduced with Ad-GSN in vitro. Based on flow cytometric analysis over expression of gelsolin may cause these cells to arrest or delay at the G2/M phase of the cell cycle. In the orthotopic bladder cancer model the mass of the tumor was approximately 90% less in Ad-GSN treated animals than in controls. CONCLUSIONS: Ad-GSN provides a significant tumor suppressive effect on human bladder cancer cells in this orthotopic nude mouse model. Adenovirus mediated over expression of gelsolin may be useful therapy for human bladder cancer.     

转bak基因促膀胱癌多药耐药细胞凋亡的研究

目的　观察转入bak基因对膀胱癌多药耐药 (MDR)细胞的杀伤效果 ,探讨其可能的机制。　方法　用脂质体将bak基因导入MDR细胞 ,通过原位杂交法检测bakmRNA的表达 ,SABC免疫组化法分析bak和bcl 2的表达。采用细胞计数法检测细胞生长抑制率 ,流式细胞仪检测细胞周期的变化。荧光染色观察细胞形态。　结果　bak基因可成功转入MDR细胞 ,转染第 3天bakmR NA阳性率 64 % ,bak表达阳性率 60 % ,明显高于对照组 ;转染后细胞中bcl 2表达显著减少 ,P <0 .0 5。转染第 4天EJ/bak细胞抑制率 32 % ,显著高于对照组 ,P <0 .0 5。细胞周期分析可见凋亡峰 ,凋亡率 35 %。凋亡细胞在荧光显微镜下形成凋亡小体。　结论　转入bak基因可显著促进MDR细胞的凋亡 ,其作用机制与下调bcl 2基因的表达有关     

Changes in epidermal growth factor receptor expression in human bladder cancer cell lines following interferon-alpha treatment

PURPOSE: We examined the regulation of epidermal growth factor (EGF) receptor (EGFR) expression in human bladder cancer cell lines by interferon-alpha (IFN-alpha), the ability of IFN-alpha to inhibit cell proliferation and the sensitivity of IFN-alpha pretreated cells to EGF. MATERIALS AND METHODS: Cell proliferation was determined using crystal violet colorimetric and clonogenic assays. EGFR expression was measured by flow cytometry using specific antibody or ligand binding approaches. RESULTS: After IFN-alpha (100 IU/ml) treatment cell surface EGFR expression was upregulated in 6 of 11 and down-regulated in 2 of 11 bladder cancer cell lines. The over expression of cell surface EGFR peaked within 48 to 96 hours and increased by 35% to 241% in individual cell lines. High level cell surface EGFR correlated with intracellular EGFR expression. Cell growth inhibition by IFN-alpha coexisted with EGFR over expression in the 6 lines. IFN-alpha treated cells remained sensitive to EGF treatment. CONCLUSIONS: IFN-alpha transiently up-regulates EGFR expression and inhibits in vitro growth in some human bladder cancer cells. IFN-alpha does not prevent EGFR from binding EGF or signal transduction via the EGF-EGFR pathway. This may have clinical implications for improving treatment based on EGFR targeting in select patients with bladder cancer.     

PCNA反义cDNA基因转导抑制人膀胱癌细胞增殖的研究

目的 探讨增殖细胞核抗原（PCNA）基因反义cDNA对人膀胱癌细胞体外增殖活性的抑制作用。方法 构建PCNA反义cDNA真核表达载体并转染膀胱癌DJ细胞,通过细胞生长曲线、MTT比色分析、H^3-TdR掺入法检测癌细胞增殖活性,流式细胞仪（FCM）分析细胞周期时相变化,SABC免疫组化观察癌细胞PCNA蛋白表达水平。结果 PCNA反义cDNA导入后,膀胱癌DJ细胞生长速率显著减慢（P＜0．01）,增殖活性抑制率59．02％（P＜0．05）,DNA合成速率降低52．31％（P＜0．01）,S期细胞减少,细胞周期阻滞于G0／G1期,PCNA蛋白表达显著减弱（P＜0．05）,差别均有显著性意义。结论 PCNA反义cDNA基因转导能有效抑制膀胱癌细胞的PCNA蛋白表达及体外增殖活性,为肿瘤基因治疗提供了有效途径。     

缺氧诱导因子-1α小干扰RNA对人膀胱癌小鼠移植瘤生长的实验研究

目的 观察缺氧诱导因子-1α(HIF-1α)siRNA对人膀胱癌小鼠膀胱原位移植瘤生长的抑制作用,探讨HIF-1α作为膀胱癌基因治疗靶点的有效性.方法 把已稳定转染pGC-siHIF-1α的人膀胱癌细胞株T24接种至小鼠膀胱.通过CT观察肿瘤生长,应用免疫组化(SABC)检测肿瘤细胞HIF-1α和肿瘤间质CD34的水平,根据CD34表达情况评价肿瘤微血管密度(MVD).末端脱氧核苷酸转移酶标注法(TUNEL)测定肿瘤细胞凋亡指数;二苯基溴化四氮唑蓝(MTT)法测定肿瘤细胞增值率;流式细胞仪(FCM)测定肿瘤细胞周期分布.结果 实验组与对照组相比较,实验组成瘤率低,肿瘤生长速度缓慢(p＜0.05);HIF-1α水平下降(P＜0.01);肿瘤微血管密度减少(P＜0.01);凋亡指数升高(P＜0.01);增殖能力减弱(P＜0.01);G0/GI期细胞增多,G2/M期和S期细胞均减少(P＜0.05);与对照组相比较,组间差异均具有统计学意义.结论 以HIF-1α为靶点的siRNA有抑制H1F-1α表达,遏制膀胱癌T24细胞在体内生长的作用,HIF-1 0有可能成为临床膀胱癌基因治疗的新的有效靶点.     

The expression of epidermal growth factor receptor on human bladder cancer: potential use in radioimmunoscintigraphy

Monoclonal antibody 425, which binds to an extracellular domain of the epidermal growth factor receptor, was used to evaluate the expression of this antigen on bladder cancer cells. Epidermal growth factor receptor was found on all bladder cancer cell lines tested. Immunoperoxidase staining of fourteen invasive human bladder cancers with monoclonal antibody 425 demonstrated that ten showed strong staining, one showed weak staining and three were negative. Five noninvasive tumors were similarly examined. Four of these were negative and one showed weak staining. Biodistribution experiments with human bladder tumor xenografts in athymic nude mice using radiolabeled monoclonal antibody 425 and an isotype matched control antibody demonstrated specific tumor localization at five and seven days following antibody injection. Successful imaging of a human bladder tumor xenograft was achieved five days post antibody injection. These data confirm that epidermal growth factor receptor expression correlates with bladder cancer stage and suggests that epidermal growth factor receptor may serve as a target antigen for radioimmunoscintigraphy.     

卡介苗诱导膀胱癌细胞cyclinD1、Fas表达的实验研究

目的观察卡介苗（BCG）对体外培养膀胱癌细胞生长情况及相关基因（cyclinD1、Fas）表达的影响,并探讨其可能的作用机制。方法MTT法检测不同浓度BCG（0．15、0．3、0．6、1．2mg／mL）对人膀胱癌BIU-87细胞生长的影响。流式细胞术（FCM）检测BCG处理后膀胱癌细胞周期分布的变化及对膀胱癌细胞凋亡的影响;逆转录-聚合酶链反应（RT-PCR）检测BCG作用BIU-87细胞后cyclinD1、FasmRNA表达的变化。结杲BCG体外能明显抑制BIU-87细胞的生长,且抑制作用呈明显的剂量、时间依赖性（P〈0．01）。流式细胞术检测示处理后膀胱癌细胞阻滞于G0～G1期,RT—PCR结果示BCG处理能显著诱导FasmRNA的表达和显著抑制cyclinD1mRNA的表达（P〈0．05）。结论TSA可通过诱导细胞凋亡和使细胞阻滞于G1期而发挥体外抗膀胱癌作用,其作用机制可能涉及相关基因（eyelinD1、Fas）表达的调控。