LMP1-CTAR3对鼻咽癌干细胞SP18生长的影响

背景：课题组前期构建了CTAR3缺失突变型LMP1(LMP1△232-351)表达载体,初步了解该活性区在LMP1发挥作用中扮演十分重要的角色。 目的：观察LMP1-CTAR3对鼻咽癌干细胞SP18生长的影响。 方法：采用逆病毒感染方式,建立稳定表达LMP1及CTAR3缺失突变型LMP1(LMP1△232-351)的鼻咽癌干细胞SP18细胞系,通过细胞生长曲线、软琼脂集落形成实验和平皿克隆形成实验观察LMP1△232-351对鼻咽癌干细胞SP18生长的影响,然后选用流式细胞术检测细胞周期,计算细胞增殖指数。另外,利用Western-blot检测细胞中JAK3蛋白的磷酸化水平。 结果与结论：①与SP18-LMP1细胞比较,SP18-LMP1△232-351细胞生长速度减慢,增殖指数降低(P < 0.01)。②在SP18-  LMP1△232-351细胞中JAK3的磷酸化水平低于SP18- LMP1细胞。结果说明LMP1-CTAR3可能通过影响SP18细胞中JAK3的磷酸化,下调细胞的增殖指数,降低促细胞增殖的能力。     

siRNA干扰Msi1对人胃癌SGC-7901细胞的影响

目的探讨siRNA沉默Msi1基因表达对人胃癌SGC-7901细胞增殖、凋亡、侵袭与转移的影响。方法针对Msi1 mRNA序列设计合成siRNA,转染SGC-7901细胞。RT-PCR法检测Msi1基因表达;Westernblot检测survivin、caspase3及MMP-9表达;MTT法检测SGC-7901细胞增殖;流式细胞术检测细胞凋亡情况。结果Msi1 siRNA能有效抑制胃癌SGC-7901细胞中Msi1的表达,与空白对照组比较,Msi1 siRNA组SGC-7901细胞生长、增殖速度减缓(P<0.05);survivin、MMP9蛋白表达水平显著下调(P<0.05),而caspase3蛋白表达水平上调(P<0.05);Msi1 siRNA组凋亡率增高。结论沉默Msi1对人胃癌SGC-7901细胞增殖、凋亡、侵袭与转移有负性调节作用。     

角蛋白19表达与鼻咽癌分化转移的关系

目的 研究角蛋白19(Keratin 19)表达与鼻咽癌分化和转移的关系.方法 免疫组织化学检测62例鼻咽癌、17例淋巴结转移鼻咽癌和16例慢性鼻炎标本中角蛋白19的表达水平,并分析其表达水平与鼻咽癌临床病理特征的关系;采用 Western Blot检测不同分化程度和不同转移潜能NPC细胞系中角蛋白19的表达.结果 鼻咽癌患者中角蛋白19阳性率为74.2%(46/62);淋巴结转移鼻咽癌中阳性率为88.2%(15/17);对照组正常鼻咽上皮中阳性率为12.5%(2/16);角蛋白19在转移癌中表达高于鼻咽癌,而在鼻咽癌中又显著高于正常对照组,两者间差异有显著性(P＜0.05).角蛋白19表达水平与鼻咽癌的组织学类型、肿瘤原发灶大小、淋巴结转移和远处转移有相关性(P＜0.05),而与临床分期、年龄和性别无相关性.结论 角蛋白19表达与鼻咽癌的分化和转移有关,可作为鼻咽癌分化、转移的分子标志物.     

人胃癌细胞高转移亚系的初步筛选及母亚两系生物学特性的比较

目的 筛选高转移胃癌细胞亚系并与其母系进行生物学特性的比较.方法 利用transwell小室筛选高转移力胃癌细胞亚系,通过HE染色比较母亚两系细胞形态改变;流式细胞仪检测母亚两系细胞周期;免疫细胞化学法检测两系细胞中MMP-9蛋白表达;transwell侵袭小室模型比较母亚两系的迁移能力.结果 利用transwell小室从母系MKN-28中成功筛选出高转移力胃癌细胞亚系MKN-28S, 母亚两系细胞形态上无明显差异,亚系增殖指数(PI)高于母系,且亚系中 MMP-9蛋白表达同母系相比显著增高,显微镜下细胞计数示亚系迁移至膜背面的数量较母系明显增多 (P＜0.05),细胞运动能力明显增强.结论 MKN-28S细胞较MKN-28细胞具有更强增殖能力和更高迁移力.     

人胃癌高低侵袭转移潜能细胞系对化疗药物敏感性的比较研究

目的探讨肿瘤细胞侵袭转移能力的改变对肿瘤细胞化疗敏感性的影响。方法Westem blotting法比较筛选出的人胃癌细胞MKN-28S（亚系）与MKN-28（母系）中E-Cadherin、MMP-2蛋白表达；transwell体外侵袭实验比较两系侵袭潜能。采用MTT法比较两系对阿霉素（ADM）、丝裂霉素（MMC）、5-氟尿嘧啶（5-Fu）、紫杉醇（PTX）4种化疗药物的敏感性。结果筛选出亚系的E-Cadherin蛋白表达与母系相比显著减弱、而MMP-2蛋白表达则显著增强；显微镜下细胞计数示亚系侵袭至基质胶膜背面的数量较母系明显增多（P〈0．05）；MTr示两系对5-Fu、ADM、MMC的敏感性差异有显著性（P〈0．05），而两系对PTX的敏感性差异无显著性（P〉0．05）。结论肿瘤细胞侵袭转移能力的改变可以影响肿瘤细胞对化疗药物的敏感性，同一遗传背景下的高侵袭转移亚系较母系耐药性更强。     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

Preparation,purification and characterization of the polyclonal antibody against α1-AT

Objective To prepare the polyclonal antibody against α1-antitrypsin(α1-AT)by immunizing rabbits with α1-AT,and to purify and characterize the antibodies produced from rabbits in order to establish the production process of the polyclonal antibody against α1-AT.Methods The healthy rabbits were routinely immunized with α1-AT to prepare polyclonal antibody against α1-AT.The serum antibody titers were determined by ELISA.The rabbits with qualified antibody titers were killed to collect blood.The antisera,obtained by centrifugalizing the whole blood,were purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography.The purified polyclonai antibodies were analyzed by 15%SDS-PAGE,and antibody titers weIe measured by ELISA.Results ELISA assay showed that the titers of antisera were over 105.The polyclonal antibodies,purified by caprylic acid-ammonium sulfate precipitation and protein A affinity chromatography,had good purity and immunological activity.Conclusion The production and purification processes of polyclonal antibody against α1-AT are successfully established.     

外源性p16基因对人原发性肝癌细胞生物学行为的影响及其机制

目的:探讨导入外源性p16cDNA真核表达载体对人原发性肝癌细胞系SMMC- 772 1生物学行为的影响及其分子机制。方法:构建外源性p16基因真核表达载体并转染SMMC- 772 1细胞,经G4 18抗性筛选和扩增,得到稳定的表达株,并经RT -PCR及免疫细胞化学鉴定。通过生长曲线,流式细胞术,免疫细胞化学及Western -Blot等实验方法,对转基因后肿瘤细胞生物学行为及细胞周期调控因子CDK4 ,CyclinD1及pRb进行观察。结果:转染外源性p16基因的SMMC- 772 1细胞有外源性p16基因的整合及表达,细胞生长速度明显减慢,倍增时间明显延长,G1期细胞明显多于转基因前(P <0 .0 5 ) ;免疫细胞化学显示外源性p16表达可下调CDK4及cylinD1的表达(P <0 .0 5 ) ,Western -Blot提示转染外源性p16基因后细胞中磷酸化pRb表达明显降低。结论:外源性p16基因导入人肝癌细胞株SMMC -772 1中可稳定表达并使细胞停滞在G1期,抑制细胞生长,其机制可能为下调CDK4、cyclinD1的表达和抑制pRb磷酸化有关。