Construction of Smac gene-carrying and human uroplakin Ib promoter-Regulated Genetic Vector and its Expression

Objective： To construct an eukaryotic expression vector that contains Smac gene, which is regulated by human Uroplakin Ib （UpIb） promoter. Methods： For the directionality of Smac expression in the transitional cell carcinoma of bladder, internal CMV and T7 promoter sequences in eukaryotic expression vector pcDNA3.1-Smac were replaced with UpIb promoter to construct a new plasmid. The plasmid DNA was identified by gel electrophoresis after being double digested at respective sites, and then the sequence was analyzed. The expression of Smac mRNA and protein in BIU87 cell line were detected after the transfection by using the newly constructed vector. Results： The Smac gene-carrying and UpIb promoter-regulated eukaryotic expression vector pcDNA3-UpIb-promoter-Smac was successfully constructed. The expression of Smac mRNA was approximately increased by 2.1 times and the expression of Smac protein was increased in about 71% BIU87 cells. Conclusion： The new vector can be effectively expressed in bladder cancer cells and be of great significance for bladder cancer-targeted gene therapy.     

Relationship between the Expression of RASSF1A Protein and Promoter Hypermethylation of RASSF1A Gene in Bladder Tumor

To investigate the relationship between the expression of RASSF1A protein and promoter hypermethylation of RASSF1A gene, RASSF1A protein expression was measured by Western blotting in 10 specimens of normal bladder tissues and 23 specimens of bladder transitional cell carcinoma （BTCC）. The promoter methylation in BTCC and normal bladder tissues was detected by methylation-specific PCR （MSP）. The results showed that the expression level of RASSF1A protein was significantly lower in BTCC tissues than that in normal bladder tissues. However, it was not correlated with its clinical stages and pathological grades. The frequency of promoter methylation of RASSF1A gene was higher in BTCC tissues than that in normal bladder tissues. In 14 patients with the aberrant promoter methylation, 13 showed loss or low expression of RASSF 1A protein. It is concluded that RASSF1A gene promoter methylation may contribute to the low level or loss of RASSF1A protein expression, the inactivation of RASSF1A gene and the genesis of BTCC. But, it may bear no correlation with its clinical stages and pathological grades.     

Expression of Pinl and Ki67 in Cervical Cancer and Their Significance

In order to investigate the expression levels of Pinl mRNA and protein in cervical cancer and its association with Ki67 and their clinical significance, amplification of Pinl gene was examined by RT-PCR, and the expression of both Pinl and Ki67 protein was detected by immunohistochemistry in cervical cancer tissues. It was shown that the expression levels of Pinl were higher in cervical cancer than in normal cervical tissues （P〈0.05）. The expression of Pinl protein was increased progressively along with the disease process from normal cervix to CIN and to cervical cancer （P〈0.05）. No significant difference in the Pinl expression was found between disease stages （FIGO）, pathological grades or pelvic lymph node metastasis status （P〉0. 05）. The expression of Pin1 was significantly higher in adenocarcinoma than in squamous carcinoma of the uterine cervix （P〈0.05）. In cervical cancer, the overexpression of Pinl was positively correlated with that of Ki67 （P〈 0. 05）. These results suggested that the overexpression of Pinl was closely related with cancer cell proliferation or progression of cervical cancer and contributed to oncogenesis. Pinl may serve as a potential marker for cervical cancer diagnosis.     

Cyclin E and p27 kipl expressions in bladder transitional cell carcinoma and its clinical implication

Objective To investigate the expression of cyclin E and p27kipl in bladder transitional cell carcinoma and its clinical significance. Methods cyclin E and p27kipl expressions were detected in 69 specimens by SP of immunohistochemistry. The results were analyzed in relation to the clinical data. Results The positive rates of cyclin Eexpression and p27kipl expression in bladder carcinoma were 42 % and 51 %, respectiely. cyclin Eexpression was positively related to the tumor grade, the higher the tumor grade, the higher the cyclin E positive expression; but there was no association between cyclin Epositive expression and tumor clinical stage. Significant correlation was found between p27kipl positive expression and tumor grade and clinical stage, the lower the p27kipl positive expression, the highter the tumor grade and clinical stage. In relapsing bladder carcinoma,both cyclin E and p27kipl positive expression decreased significantly. There was close correlation between cyclin E expression and p27kipl exp     

CLINICOPATHOLOGICAL SIGNIFICANCE OF PTEN AND CASPASE-3 EXPRESSIONS IN BREAST CANCER

Objective To investigate the expressions of PTEN and Caspase-3 proteins in human breast carcinoma, and to evaluate their clinicopathological implications during the tumorigenesis and progression of breast cancer. Methods The expressions of PTEN and Caspase-3 proteins in 95 cases of breast cancer and 15 cases of benign breast diseases were investigated immunohistochemically. Correlations between the expression of PTEN protein, Caspase-3 protein, and clinicopathological features of breast cancers were analyzed. Results The loss expression rate of PTEN protein in tumor tissues was significantly higher than that in benign breast diseases （33.7% vs. 0, P 〈 0.01 ）. Analysis of the clinicopathological features showed that PTEN expression level was negatively correlated with TNM stage, histological grade, axillary lymph node status, recurrence, and metas- tasis （ P 〈 0. 05 ）. The positive expression level of Caspase-3 was negatively correlated with TNM stage （ P 〈 0. 01 ）, but not related with histological grade, axillary lymph node status, recurrence, or metastasis （ P 〉 0.05 ）. In addition, the expression of PTEN protein had significantly positive correlation with the expression of Caspase-3 protein in breast cancer （P 〈 0. 01 ）. Conclusion The combination detection of PTEN and Caspase-3 may serve as an important index to estimate the pathobiological behavior and prognosis of breast cancer.     

Expression of Vascular Endothelial Growth Factor and Cyclooxygenase-2 in Laryngeal Squamous Cell Carcinoma and Its significance

n order to study the expressions of vascular endothelial growth factor （VEGF） and cyclooxygenase-2 （COX-2） in human laryngeal squamous cell carcinoma （LSCC） and its significance, the expression of VEGF mRNA and COX-2 mRNA in 62 cases of LSCC and 54 adjacent noncancerous laryngeal tissues and 9 normal human laryngeal mucous tissues was detected by using techniques of semi-quantitative RT-PCR. It was found that the expression level of VEGF and COX-2 mRNA was significantly increased in LSCC as compared with that in the normal human laryngeal mucous tissues （both P〈0. 01）, and the expression level of VEGF and COX-2 mRNA were significantly increased in stage Ⅲ＋ Ⅳtissues of LSCC as compared with the stage Ⅰ ＋ Ⅱ tissues of LSCC （P 〈0.01）. There was a high positive correlation between VEGF and COX-2 expression in LSCC （r= 0. 756,P〈0.01）. These data raise the possibility that VEGF and COX-2 may play key roles in the growth, invasion and metastasis of LSCC.     

Smac和XIAP基因在肾细胞癌中的表达及意义

目的 探讨Smac和XIAP基因在肾细胞癌组织中的表达及其临床意义.方法 采用免疫组织化学法检测95例肾癌标本不同分型、分级、分期及10例正常肾组织中Smac和XIAP的表达,分析其表达与肾癌分级、分期的关系及二者表达相关性.结果 Smac在各型肾细胞癌中随分期、分级的增加表达减低,XIAP在各型肾细胞癌中随分期、分级的增加表达增高,二者的表达呈负相关.结论 Smac和XIAP表达与肾细胞癌分期、分级关系密切,共同参与了肾癌的发生发展. Abstract： Objective To study the expression and significance of Smac and XIAP in renal cell carcinoma(RCC). Methods The expressions of Smac and XIAP were detected using SP immunohistochemical technique in 95 cases of RCC and 10 cases of normal renal tissue. Results The postitive expressions of Smac were statistically significant differences in different clinical statges. The postitive expressions of XIAP were higher in RCC related to the lower histological grade. Smac protein and XIAP protein expression had negative correlation. Conclusions It indicate that Smac and XIAP may play an important role in RCC. The expression of Smac and XIAP are statistically significant associated with tumor grade and clinical stage.     

UROGENITAL SURGERY——Kidney, ureter, bladder

The detection of protein expression of clusterin and Ki-67 and the status of cell apoptosis in bladder transitional cell carcinoma; Kinetics of 5-ALA induced protoporphyrin IX in bladder cancer cell line BTU-87： an in vitro study;Mucl and muc7 gene expressions in bladder transitional cell carcinoma; The effects of protein kinase C alpha cDNA on the expression of genes of multidrug resistance in renal cell carcinoma 786- 0 cell line; Construction and sequence analysis of eukaryotic expression vector of renal cell carcinoma G250 antigen gene;Clinical study on the effect of intravesical instillation of antifibrinolytic agents with bacillus Calmette Guerin in preventing bladder cancer recurrence;Expression and significance of neuronal nitric oxide synthase in ureteropelvic junction obstruction;Comparison of intravesical bacillus Calmette- Guerin versus mitomycin C for the prevention of recurrence of superficial bladder cancer and their toxicity： meta-analysis;……     

膀胱癌中环氧化酶-2的表达与临床病理的关系

目的探讨环氧化酶-2(COX-2)表达与膀胱癌生物学行为的关系及其意义.方法采用免疫组化SABC法检测54例膀胱癌、29例癌旁组织和10例正常膀胱粘膜中的COX-2表达,结合临床病理资料进行分析.结果 COX-2在不同膀胱组织中的表达差异有显著性(P＜0.05),即膀胱癌组织>癌旁组织>正常膀胱粘膜.COX-2表达随膀胱癌病理级和临床分期的增加而增加(P＜0.05、P＜0.01),且癌组织中COX-2表达与淋巴转移有关(P＜0.05).结论 COX-2表达与膀胱癌病理分级、临床分期、淋巴转移有关,提示它可能在胱癌的发生、发展中扮演重要角色,通过抑制COX-2活性可能为膀胱癌的防治提供新途径.     

卵巢上皮性癌组织中激肽释放酶7及14的表达及临床意义

目的研究激肽释放酶（kallikrein, KLK）7及KLK14在卵巢上皮性癌组织中的表达,并探讨其临床意义。方法采用RT-PCR及Western blot技术测定KLK7及KLK14 在50例不同临床分期、不同组织分级的卵巢上皮性癌中mRNA及蛋白表达。结果KLK7在卵巢癌中表达明显增高，且低分化组织较高分化组织表达高（P＜0.01）；KLK14在卵巢癌中表达亦增高（P＜0.05），且晚期卵巢癌(Ⅲ、Ⅳ期)明显高于早期卵巢癌(Ⅰ、Ⅱ期，P＜0.01)；G3级卵巢癌高于G1、G2级卵巢癌（P＜0.05）。结论KLK7、KLK14在卵巢癌的发生及转移中有潜在促进作用。     

尿素通道蛋白B在人膀胱癌组织中的表达及其临床意义

目的：检测膀胱癌患者肿瘤组织和正常膀胱黏膜组织中尿素通道蛋白B（UT-B）的表达,阐明其在膀胱癌组织中表达的临床意义。方法：收集临床膀胱癌患者术后石蜡包埋标本52例,以正常膀胱黏膜组织15例作为对照,采用免疫组织化学方法检测UT-B蛋白阳性表达率,分析UT-B蛋白阳性表达率在膀胱癌组织和正常膀胱黏膜表达的差异以及其与患者临床病理参数的关联。结果：UT-B蛋白在膀胱癌组织中低表达,阳性表达率为44.2%;在正常膀胱黏膜组织中高表达,阳性表达率为93.3%,2组UT-B蛋白阳性表达率比较差异有统计学意义（P=0.001）。膀胱癌组织中UT-B蛋白的阳性表达率随肿瘤组织分级的增高而逐渐降低,不同分级间比较差异有统计学意义（P=0.010）。膀胱癌组织中UT-B蛋白在非肌层浸润期（Ta+T1）的阳性表达率明显高于肌层浸润期（T2+T3）,组间比较差异有统计学意义（P=0.014）。结论：UT-B蛋白的表达降低或缺失可能促进了膀胱癌的发生发展和侵袭。     

FBP1在肾透明细胞癌组织中的表达变化及意义

目的 探讨果糖1,6二磷酸酶1(FBP1)在人肾透明细胞癌(RCCC)及其癌旁组织中的表达,以及在肾癌发生、发展及预后中的作用和临床意义.方法 选取手术切除的118例RCCC患者的石蜡切片和40例RCCC患者的新鲜标本,分别采用免疫组织化学方法、Western blot和逆转录聚合酶链反应(RT-PCR)方法检测肾癌及对应癌旁组织(阴性切缘)中FBP1蛋白及mRNA的表达,分析其与患者临床病理特征及预后的相关性.结果 免疫组织化学发现78.81％ (93/118)的癌旁组织中可见FBP1蛋白呈强阳性表达,仅39.83％(47/118)的肾癌组织中FBP1蛋白表达阳性.Western blot发现肾癌组织中FBP1的表达阳性率较其相应癌旁组织明显下降(P＜0.01).RT-PCR法发现癌旁组织FBP1 mRNA表达水平亦明显高于肾癌组织(P＜0.05).FBP1低表达与临床分期、病理分级、危险系数及是否复发明显相关(P＜0.05),与年龄、性别、症状及肿瘤大小、部位、是否有坏死、是否有血管侵犯和肾上腺是否有累及无关(P＞0.05).FBP1阳性表达的肾癌患者的5年生存率高于FBP1阴性表达者(P＜0.05).结论 FBP1及其蛋白在人肾癌组织中低表达,与肾癌的发生、发展相关,可能成为肾癌预后的侯选标志物之一.     

癌超甲基化基因1蛋白和卵巢癌基因1蛋白在卵巢癌组织中的表达及意义研究

目的 研究卵巢癌组织中癌超甲基化基因1蛋白和卵巢癌基因1蛋白的表达情况及其与卵巢癌病理特点的关系,探讨其在卵巢癌中的意义.方法 选择2014-2015年在该院妇产科手术切除卵巢癌组织标本63例和正常卵巢组织标本63例作为研究对象.采用Western blot法测定卵巢癌组织和正常卵巢组织中癌超甲基化基因1蛋白和卵巢癌基因l蛋白的表达情况,并分析其与卵巢癌病理特点的关系.结果 卵巢癌组织中癌超甲基化基因1蛋白、卵巢癌基因1蛋白的表达量明显低于正常卵巢组织(P＜0.05).癌超甲基化基因1蛋白在不同卵巢癌分期、不同分化程度和不同病理类型中的表达量比较差异均无统计学意义(P＞0.05).在卵巢癌Ⅰ期中卵巢癌基因1蛋白的表达高于卵巢癌Ⅱ期和Ⅲ～Ⅳ期(P＜0.05),Ⅱ期的表达量高于Ⅲ～Ⅳ期(P＜0.05);高分化的表达量高于中分化和低分化(P＜0.05),中分化和低分化的表达量比较差异无统计学意义(P＞0.05);卵巢癌基因1蛋白在卵巢癌不同病理类型中的表达比较差异无统计学意义(P＞0.05).结论 癌超甲基化基因1蛋白失活可能只参与卵巢癌的发生,卵巢癌基因1蛋白和卵巢癌的发展有一定关系.     

生存素和Caspase-3蛋白在食管癌前病变及癌组织中的表达及意义

目的: 探讨食管癌前病变及癌组织中生存素和Caspase 3蛋白的表达及其意义。方法: 用免疫组织化学法检测63例食管癌 (食管癌组)、86例食管鳞状上皮不典型增生（包含轻度、中度、重度）患者（不典型增生组)和40例正常食管黏膜（正常黏膜组）组织中生存素和Caspase-3蛋白的表达,并计算阳性表达率。分析这两类蛋白表达与食管癌临床分期的关系。结果：在正常黏膜组、不典型增生组和食管癌组中生存素的阳性表达率逐渐升高（rs＝0.732,P＜0.01）,而Caspase 3蛋白的阳性表达率则逐渐减弱（rs＝－0.687,P＜0.05）。在食管癌组,生存素表达与淋巴结是否转移及临床分期（Ⅰ+Ⅱ期／Ⅲ+Ⅳ期）相关（P＜0.01）;Caspase-3表达与食管癌分化程度相关（P＜0.01）;生存素的阳性表达率与Caspase-3蛋白的阳性表达率呈负相关(r =－0.897,P＜0.01)。结论： 生存素和Caspase-3蛋白的异常表达可能与食管癌的发生、发展密切相关,可作为食管癌早期诊断及判断预后的肿瘤标志物。     

三重靶向介导的Smac过表达对乳腺癌MDA-MB-231细胞凋亡和周期进程的影响

目的：利用基因重组技术获得三重靶向性Smac过表达的条件复制型腺病毒,探讨其对乳腺癌MDA-MB-231细胞凋亡和周期的影响。方法：利用重组技术构建Smac过表达载体pShuttle-Egr1-Smac-HRE-hTERT-E1A-E1Bp-E1B55K,与骨架载体pAdEasy在BJ5183（AdEasy-1+）菌中进行重组,获得Smac过表达的条件复制型腺病毒CRAd.pE-Smac,感染MDA-MB-231细胞后,利用化学试剂氯化钴模拟肿瘤细胞乏氧状态,设立对照组、CARd.pE-Smac组、乏氧组和CARd.pE-Smac+乏氧组,并根据是否进行4 Gy照射,每组分为未照射组和照射组,共8个实验组。利用Western blotting法检测各组细胞Smac蛋白的表达,利用流式细胞术检测细胞凋亡率和不同周期进程细胞百分率。结果：Western blotting法检测,经条件复制型腺病毒CRAd.pE-Smac感染、乏氧和照射后,Smac蛋白表达水平增加,CARd.pE-Smac+乏氧+4Gy组Smac蛋白表达水平最高。流式细胞术检测,与对照组比较,CARd.pE-Smac组、乏氧组和CARd.pE-Smac+乏氧组细胞凋亡率均明显升高（P<0.05或P<0.01）;与相应未照射组比较,4 Gy照射后CARd.pE-Smac+4Gy组、乏氧+4Gy组和CARd.pE-Smac+乏氧+4Gy组细胞凋亡率均明显升高（P<0.05或P<0.01）,CARd.pE-Smac+乏氧+4Gy组升高最为明显,且S期和G₂/M期细胞百分率明显增加（P<0.05或P<0.01）,与诱导凋亡的结果有相似的趋势。结论：在条件复制型腺病毒CRAd.pE-Smac感染MDA-MB-231细胞、并经乏氧和辐射处理后,实现了三重靶向介导的Smac过表达,其具有促进肿瘤细胞凋亡和诱导G₂/M期细胞阻滞的作用。     

Survivin反义寡核苷酸对人卵巢癌细胞株中survivin和SMAC/DIABLO基因表达的影响

目的 观察survivin反义寡核苷酸(ASODN)对人卵巢癌细胞株SKOV3中survivin、Smac/DIABLO表达的影响,探讨survivin、Smac/DIABLO在ASODN诱导卵巢癌细胞凋亡和细胞周期改变中的作用和分子机制.方法 用脂质体(LipofectamineTM 2000)介导survivin ASODN转染人卵巢癌细胞株SKOV3,用MTT比色法检测细胞生长活性,流式细胞学技术检测细胞凋亡指数、细胞周期分布及survivin、Smac蛋白表达改变,逆转录酶链反应检测survivin、Smac/DIABLO基因表达改变.结果 同对照组比较,不同浓度ASODN作用后细胞生长明显减慢,转染后48 h IC50在600 nmol/1左右.用600 nmol/LASODN转染48h后,细胞凋亡指数(AI)明显增加,(t=6.3671,P＜0.05);更多的细胞停留在G0/G1期(t=10.3832,P＜0.01).Survivin mRNA和蛋白表达明显下调(t=3.5031,P＜0.05,t=7.8146,P＜0.01),SmacmRNA和蛋白表达上调(t=2.8011,P＜0.05,t=11.3917,P＜0.01).结论 ASODN诱导细胞凋亡,使更多的细胞停留在G0/G1期,减少进入有丝分裂细胞数的作用,是通过下调survivin基因,上调Smac基因表达来共同完成的,survivin、Smac/DIABLO基因在调节SKOV3细胞周期、凋亡的功能上密切相关,二者的相互作用是卵巢癌发生、发展的重要分子机制之一.     

G蛋白信号调节蛋白2在胰腺癌组织中的表达及临床意义

目的: 探讨G蛋白信号调节蛋白2（G-protein signaling modulator 2,GPSM2）基因在胰腺癌组织中的表达及其临床意义。方法: 选取54例胰腺癌患者临床石蜡样本制作组织芯片,利用免疫组化检测GPSM2在胰腺癌组织中的表达并分析GPSM2的表达与患者临床病理参数及预后的关系;另选取同期10例新鲜胰腺癌标本及配对的癌旁组织,采用实时RT-PCR法和蛋白质印迹法检测GPSM2 mRNA和蛋白的表达水平。结果： 胰腺癌组织GPSM2 mRNA 及蛋白的表达均明显高于配对的癌旁组织（P均<0.05）。组织芯片检测结果显示,与配对的癌旁组织比较,胰腺癌组织中GPSM2表达明显上调（P<0.05）,其高表达（40例）比例达74.1%。GPSM2的表达与患者的肿瘤T分期、TNM分期、分化程度相关：T分期越差(χ2=12.654,P＜0.01),TNM分期越高(χ2=16.610,P＜0.01),肿瘤分化程度越差(χ2=10.355,P＜0.01),其GPSM2表达水平越高。生存分析表明,GPSM2高表达患者的中位生存期明显低于低表达患者（P＜0.05）。结论： 胰腺癌组织GPSM2过表达,其表达与患者的肿瘤T分期、TNM分期、肿瘤分化程度相关,且与胰腺癌的预后有一定相关性。