人膀胱移行细胞癌各种透明质酸合成酶基因亚型的表达及意义

目的:研究各种透明质酸合成酶亚型(HASs)mRNA在人膀胱移行细胞癌中的表达及其潜在的临床价值.方法:运用半定量RT-PCR方法,对145例不同恶性度的人膀胱移行细胞癌、22例正常人膀胱黏膜组织标本和4种人BTCC细胞系的HAS亚型mRNA的表达情况进行研究.结果:PCR 23循环后,正常膀胱黏膜组织中7例扩增出微弱的HASl mRNA条带(28%,7/25),其余无任何条带扩增出来;所有膀胱移行细胞癌组织标本和细胞系有一种以上的HAS亚型mRNA被扩增出来.不同膀胱癌组织、细胞系表达的HASs mRNA的种类及表达程度各不相同,但随着肿瘤恶性程度的增高,其表达的各种HASs mRNA的种类趋向相同.每个膀胱癌组织、细胞系均有一种HASs mRNA优势表达,但其类型及优势表达程度互不相同.结论:人膀胱移行细胞癌出现了HAS亚型mRNA的异常高表达,可能与肿瘤的发生、发展有密切关系,是BTCC生物治疗潜在的靶点;同时进一步证实了尿透明质酸作为膀胱癌的"标志物"的可靠性.     

PREPARATION AND IDENTIFICATION OF MONOCLONAL ANTIBODIES AGAINST THE EXTRACELLULAR DOMAIN OF INTEGRIN α6 SUBUNIT-THE SPECIFIC LAMININ RECEPTOR

Objective: To prepare monoclonal antibody (McAb) against the Integrin α6 extracellular domain and identify its biological activities. Methods: Fusion-protein of integrin α6 extracellular domain (GST-IAGED) was expressed in E.coli. JM109 and used for immunizing BALB/C mice. The spleen cells from immunized mice were fused with SP2/0 cells and selectively cultured with HAT medium. ELISA and immunocytochemistry staining were used to select hybridomas. Results: One strain of hybridoma cells that secreted specific monoclonal antibody against integrin α6 extracellular domain was indentified. The immunoglobulin subclass of the McAb was IgG1. Conclusion: The McAb against the extracellular domain of integrin α6 was successfully prepared by using GST-IA6ED fusion protein expressed by E.Coli. And the McAb had positive reaction with human hepatocarcinoma cells-BEL-7402.     

二甲基甲酰胺所致肝细胞毒性及谷胱甘肽含量的变化

以ＤＭＦ与大鼠肝细胞体外孵育，观察ＤＭＦ所致细胞毒性及对肝细胞内ＧＳＨ含量的影响。发现ＤＭＦ在短时间内即可使ＧＳＨ明显下降。细胞死亡率也随着孵育时间和剂量的增加而增加，两者呈负相关。ＤＴＴ对ＤＭＦ所致的细胞毒性有明显的拮抗作用。     

PREPARATION AND IDENTIFICATION OF MONOCLONAL ANTIBODIES AGAINST THE EXTRACELLULAR DOMAIN OF INTEGRIN a6 SUBUNIT-THE SPECIFIC LAMININ RECEPTOR

Integrin family belongs to the adhesive molecules of the cell surface. Integrins are transmembrane glycoproteins. Their receptor function requires the noncovalent linkage of an a and a b subunits. Integrin a6 can bind with either b1 or b4, and functions as a laminin receptor[1]. The heterodimers?extracellular domain mediates the recognization and combination of the cell-cell and cell-extracellular matrix. The cytoplasmic domain interacts with cytoskeletal proteins and other signal transductio…     

CD2相关蛋白和肾质在多房囊性肾细胞癌中的表达

目的 探讨CD2相关蛋白(CD2AP)及肾质(nephrin)在成人多房囊性肾细胞癌中的表达及意义.方法 收集多房囊性肾细胞癌标本28例、肾透明细胞癌标本32例和单纯性肾囊肿标本13例,采用免疫组化方法 检测CD2AP在上述组织中的表达.从上述3组组织蜡块中每组各随机抽取5例共15例,重新切片,用CD2AP和肾质,做免疫组化染色对照实验.结果 在多房囊性肾细胞癌的肿瘤细胞中CD2AP表达较强,尤其在囊壁的内层上皮表达强度明显增加.CD2AP在肾透明细胞癌中有弱表达,在单纯性肾囊肿中没有表达.肾质和CD2AP在3种组织的对照实验结果 中染色强度和部位完全一致.结论 CD2AP在多房囊性肾细胞癌中的表达具有特异性,其表达与肾透明细胞癌和单纯性肾囊肿有显著差异,对于多房囊性肾细胞癌的鉴别诊断有重要意义.CD2AP和肾质在多房囊性肾细胞癌中分布一致的特点,以及上述两种蛋白的足细胞特异性对于多房囊性肾细胞癌的形成原因具有重要提示.     

PREPARATION AND IDENTIFICATION OF MONOCLONAL ANTIBODIES AGAINST THE EXTRACELLULAR DOMAIN OF INTEGRIN α6 SUBUNIT-THE SPECIFIC LAMININ RECEPTOR

Objective: To prepare monoclonal antibody (McAb) against the Integrin α6 extracellular domain and identify its biological activities. Methods: Fusion-protein of integrin α6 extracellular domain (GST-IAGED) was expressed in E.coli. JM109 and used for immunizing BALB/C mice. The spleen cells from immunized mice were fused with SP2/0 cells and selectively cultured with HAT medium. ELISA and immunocytochemistry staining were used to select hybridomas. Results: One strain of hybridoma cells that secreted specific monoclonal antibody against integrin α6 extracellular domain was indentified. The immunoglobulin subclass of the McAb was IgG1. Conclusion: The McAb against the extracellular domain of integrin α6 was successfully prepared by using GST-IA6ED fusion protein expressed by E.Coli. And the McAb had positive reaction with human hepatocarcinoma cells-BEL-7402.     

NSBP1基因干扰RNA慢病毒载体的构建及效应检测

目的 构建和鉴定NSBP1基因RNA干扰慢病毒表达载体。 方法 针对NSBP1 mRNA设计了3条siRNA,并构建pGCSIL-GFP-NSBP1慢病毒质粒,测序鉴定。用pGCSIL-GFP-NSBP1 、pHelper1.0和 pHelper2.0质粒共转染293T细胞包装产生慢病毒,测定病毒滴度。将慢病毒转染前列腺癌DU145细胞,Western-blot检测NSBP1表达, MTT法检测细胞生长活性,用转染细胞种植裸鼠成瘤,观察抑瘤效果。结果 PCR和测序证实,成功构建LV-shNSBP1的慢病毒载体,病毒滴度达2×108TU/ml。转染细胞中NSBP1蛋白表达显著降低,且MTT检测细胞生长活性明显减慢,转染细胞在裸鼠体内成瘤率没有影响,但对肿瘤的生长有明显抑制作用。结论 人NSBP1基因RNA干扰慢病毒载体的成功构建,且NSBP1对前列腺癌激素非依赖DU145细胞的生长具有重要作用。     

层粘连蛋白特异性受体--整合素α6亚单位胞外区单克隆抗体的制备及鉴定

目的：制备针对整合素α6胞外区的单克隆抗体并鉴定其生物学特异性。方法：利用整合素α6 cDNA构建可表达整合素α6胞外区的原核表达载体，在大肠杆菌中表达GST－整合素α6胞外区融合蛋白。融合蛋白经凝胶分离纯化后免疫Balb/C小鼠，取其脾细胞，用传统杂交瘤技术与小鼠骨髓瘤细胞融合及次黄嘌呤，氨基喋呤与胸苷选择性培养。单克隆杂交瘤上清经ELISA双筛后，分析其亚类及进行免疫细胞化学鉴定。结果：经ELISA双筛，获得了一株能稳定分泌对整合素α6胞外区的单克隆抗体的杂交瘤细胞株，其分泌抗体亚类为IgG1。免疫细胞化学显示此单抗在人肝癌细胞系BEL－7402呈强阳性反应。结论：通过原核表达产物制备并纯化了针对整合素α6胞外区的单克隆抗体，对分离纯化α6亚单位、研究其生物学作用及鉴定正常和疾病状态下是否表达整合素α6及其水平具有重要意义。     

临床有效剂量的洛伐他汀对前列腺癌PC3细胞的影响

目的：研究临床有效剂量的洛伐他汀对前列腺癌PC3细胞的影响。方法：将PC3细胞分为空白对照组、二甲基亚砜（dimethyl sulfoxide, DMSO)对照组、洛伐他汀处理组、洛伐他汀与甲羟戊酸（mevalonic acid）同时处理组,处理时间点分别为24、48和72 h。运用MTT方法检测细胞活力,［³H］掺入法和细胞计数法检测细胞增殖改变,Western blot检测凋亡关键分子casepase3、casepase7、多聚ADP-核糖聚合酶［poly(ADP-ribose)polymerase,PARP］裂解蛋白（cleaved PARP, cPARP）等。结果：临床有效剂量2 μmol/L洛伐他汀作用PC3细胞48 h后细胞增殖率比DMSO对照组降低了39.29%［（63.69%±3.69%）vs（102.98%±6.84%）,P=0.000］,72 h后降低了44.24%［（52.79%±9.88%）vs (97.03%±0.87%), P=0.048］; 2 μmol/L洛伐他汀作用PC3细胞72 h后,数量显著减少［(4.86×10⁵ ± 0.10×10⁵) vs (9.66×10⁵ ± 0.10×10⁵), P=0.000］; PC3细胞活力在2 μmol/L洛伐他汀作用48和72 h后分别降低了50.12% ［(56.52%±6.40%) vs (106.64%±5.27%),P=0.000］和60.05%［(41.99%±11.64% ) vs (102.94%±8.49%),P=0.000］,此外,2 μmol/L洛伐他汀还诱导凋亡关键分子caspase7活化及其受体PARP蛋白裂解。结论：临床有效剂量的洛伐他汀可抑制前列腺癌细胞PC3的增殖并诱导细胞凋亡。