TNF—α基因转染对胃癌细胞系的生长抑制效应

本工作以逆转录病毒为载体将TNF-α基因导入胃癌细胞系MKN28和BGC823,经筛选获阳性克隆MKN28-TNF和BCC823-TNF.对TNF基因在肿瘤细胞中的表达及其对肿瘤细胞的作用进行了探讨.证明外源TNF基因在肿瘤细胞中获得表达;肿瘤细胞生长速度明显减慢,生长抑制率分别达71%和 66%.HTdR掺入率在TNF基因转染细胞明显降低.转染后的肿瘤细胞丧失了裸鼠体内成瘤能力.实验结果表明TNF基因导入胃癌细胞后,细胞生长及致癌能力受到明显抑制,为TNF最终能用于临床胃癌基因治疗提供了有意义的资料.     

TNF-α对人肺癌细胞系的生长抑制作用和对细胞周期的影响

为探讨TNF-α对肺癌细胞的毒性作用及其作用机制,本研究利用MTT法检测了TNF-α对人肺癌细胞系A549的细胞毒作用.TNF-α对A549细胞的生长抑制作用有时相(24、48、72、96小时)及剂量依赖关系.采用流式细胞分析仪观察了TNF-α对A549细胞周期的影响,结果发现,TNF-α与A549细胞作用24小时后,G2 M,S期的比例由22.05%下降为14.00%,G0 G1期的比例由77.95%增加到86.00%.推测TNA-α对A549细胞的作用是通过仰制细胞DNA的合成及有丝分裂,促使细胞由G2 M、S期转至G0 G1期.     

膜结合型TNF-α基因在人胃癌细胞中的表达及其对肿瘤细胞生长的影响

TNF-α是一种具有抗肿瘤作用的细胞因子．它有二种形式，一种是26kD膜结合型，另一种是17kD分泌型，二者在体外均有细胞毒活性。我们利用逆转录病毒载体LXSN构建了插入突变的膜结合型TNF-α基因的重组逆转录病毒载体。用Lipofectin将逆转录病毒载体及重组逆转录病毒载体引入包装细胞CRIP，     

重组TNF-α慢病毒感染的脐血间质干细胞对胃癌移植瘤生长的抑制作用

目的:以脐血间质干细胞(umbilical cord blood mesenchyme stem cell,UCBMSC)作为肿瘤坏死因子-α(tumor necro-sis factor-alpha,TNF-α)载体,研究重组TNF-α慢病毒Lv-TNF-α感染的UCBMSC对胃癌移植瘤生长的抑制作用。方法:人胃癌细胞SGC-7901注射到裸鼠腹股沟皮下建立胃癌移植瘤裸鼠模型。将表达TNF-α的重组慢病毒Lv-TNF-α和对照慢病毒Lv-EGFP分别感染UCBMSC后获得Lv-TNF-α感染的UCBMSC(UCBMSC-TNF-α)以及Lv-EGFP感染的UCBMSC(UCBMSC-EGFP)。荷瘤裸鼠随机分为3组:分别注射UCBMSC-TNF-α、UCBMSC-EGFP及生理盐水(NaCl),观察注射后瘤体生长情况,RT-PCR和ELISA方法分别测定各组胃癌组织中TNF-αmRNA和蛋白的表达;H-E染色观察瘤体的坏死情况。结果:成功建立SGC-7901胃癌细胞裸鼠移植瘤模型。治疗后三组荷瘤裸鼠肿瘤体积分别为(0.51±0.27)、(0.64±0.36)和(1.21±0.80)cm3,UCBMSC-TNF-α组肿瘤体积最小(F=3.88,P<0.05);RT-PCR法检测结果显示,3组荷瘤裸鼠肿瘤组织TNF-αmRNA分别为(1.92±0.12)、(1.21±0.26)、(0.81±0.22),UCBMSC-TNF-α组TNF-αmRNA表达量最大(F=54.82,P<0.01);ELISA法检测结果显示,3组荷瘤裸鼠肿瘤组织TNF-α蛋白表达分别为(148.29±3.76)、(78.22±6.24)、(42.80±3.02)pg/ml,MSC-TNF-α组表达量最大(F=694.54,P<0.01);H-E染色病理切片显示,UCBMSC-TNF-α治疗组肿瘤坏死面积最大。结论:TNF-α转基因UCBMSC可通过旁分泌TNF-α抑制胃癌的生长。     

IL—4基因转染人胃癌细胞的实验研究

目的：评价ＩＬ－４基因转染人胃癌细胞株分泌ＩＬ－４的可行性。方法：用脂质体转染法将ＩＬ－４基因转入ＳＧＣ－７９０１人胃癌细胞株，并检测细胞分泌的ＩＬ－４和对ＬＡＫ细胞杀伤肿瘤细胞作用的影响。结果：ＩＬ－４基因可以转染入人胃癌细胞，并表达有活性ＩＬ－４，基因转染可以明显增强ＩＬ－２激活的ＬＡＫ细胞的肿瘤杀伤作用，用大剂量的γ射线照射后，转染细胞仍可以持续分泌ＩＬ－４３～４周期。结论：ＩＬ－４基因转染不改变胃癌细胞的体外生长特性，但可以明显增强抗肿瘤免疫反应     

p16基因转染对肺腺癌细胞系Anip973和AGZY83—a的生长抑制作用

目的 探讨肿瘤抑制基因p16对肺腺癌细胞生长的抑制作用。方法 应用FuGene转染方法将p16基因的表达质粒转入一对分别具高、低转移能力的肺腺癌细胞系Anip973和AGZY83－a中。对p16蛋白过表达的细胞系进行了细胞生长曲线、克隆形成率、原位末端标记分析和流式细胞术分析。结果 p16基因的过表达只能使AGZY83－a和Anip973的G1期细胞比例提高,但细胞生长曲线、克隆形成率均未发生改变,原位末端标记分析也未发现凋亡信号。结论 本实验结果提示p16基因在AGZY83－a和Anip973中的过表达不能抑制它们的生长。     

野生基因p53联合TNF-α对Hep3B凋亡作用的研究

  目的 研究补充外源性的野生型p53（wt-p53）基因联合应用小剂量TNF-α对人肝癌Hep3B细胞的凋亡作用。方法 采用lipofectAMINE 和Plus介导的脂质体转染技术将人wt-p53质粒pCEP4wtp53和空载体pCEP4导入人肝癌细胞系Hep3B内, 于转染p53 基因12 h后，在培养基中加入肿瘤坏死因子-α（20 μg／ml）。免疫荧光法检测p53蛋白定位表达。DNA片断分析法、原位末端标记（TUNEL）法观察细胞的凋亡情况。结果 TNF-α可导致细胞凋亡，与阴性对照组比较差异有统计学意义（P＜0.05）；wt-p53组及wt-p53+TNF-α组的Hep3B细胞内有p53表达，并且能促进凋亡；TNF-α与wt-p53二者联用凋亡作用更加显著。结论 wt-p53与小剂量TNF-α联用对Hep3B肿瘤细胞有协同促进凋亡的作用。     

野生型p16基因对胃癌细胞生长的抑制作用

目的　探讨p16基因对胃癌细胞生长的抑制作用 ,为胃癌发生的分子基础和基因治疗提供理论依据。方法　采用亚克隆技术将p16克隆入真核表达载体pCI中 ,用Lipofectamine ,将p16基因转染入胃癌细胞株 ,观察p16基因的表达对胃癌细胞株生长的影响。结果　p16基因克隆入真核表达载体 ,转染胃癌细胞后 ,可抑制胃癌细胞的生长。经Dotblot和Westernblot杂交证实 ,在mRNA和蛋白质水平均有外源性p16基因的表达。结论　野生型p16基因导入p16基因功能缺失的细胞 ,能恢复其抑制癌细胞生长的功能。     

野生型p16基因对胃癌细胞生长的抑制作用

目的 探讨p16基因对胃癌细胞生长的抑制作用。为胃癌发生的分子基础和基因治疗提供理论依据。方法 采用亚克隆技术将p16克隆入真核表达载体pCI中，用Lipofectamine，将p16基因转染入胃癌细胞株，观察p16基因的表达对胃癌细胞株生长的影响。结果 p16基因克隆入真核表达载体。转染胃癌细胞后，可抑制胃癌细胞的生长。经Dot blot和Westernblot杂交证实。在mRNA和蛋白质水平均有外源性p16基因的表达。结论 野生型p16基因导入p16基因功能缺失的细胞，能恢复其抑制癌细胞生长的功能。     

RIP3及TNF-α在胃癌组织中的表达及临床意义

目的:探讨RIP3、TNF-α在胃癌组织中的表达及临床意义.方法:收集55例胃癌组织及相应癌旁正常组织,采用实时荧光定量PCR检测RIP3 mRNA及TNF-α mRNA的表达,并分析其与临床病理特征的关系及两者相关性;运用免疫组织化学方法检测RIP3及TNF-α的表达,并分析其与临床病理特征的关系及两者相关性.结果:胃癌组织中RIP3 mRNA表达水平明显低于癌旁正常组织的表达,差异有统计学意义(P<0.05);TNF-α mRNA的表达水平明显高于对照组,差异有统计学意义(P<0.05).两指标的mRNA的表达情况均与患者的淋巴结转移、TNM分期、浸润深度有关(P<0.05),与患者性别、年龄无关(P>0.05).进一步分析RIP3 mRNA及TNF-α mRNA在胃癌中表达呈负相关(r=-0.492,P<0.05).免疫组织化学法显示RIP3在胃癌组织中表达阳性率(34.5%)明显低于对照组(67.3%);TNF-α在胃癌组织中表达阳性率(61.8%)明显高于对照组(34.5%),差异具有统计学意义(P<0.05),并且随着淋巴结转移越多、TNM分期越高、浸润越深,RIP3的表达水平逐渐降低,但TNF-α与之相反,两者均与性别、年龄无关.RIP3与TNF-α呈负相关(r=-0.489,P<0.05).结论:胃癌中可能存在RIP3/TNF-α信号通路上调,RIP3参与细胞程序化坏死可能是导致胃癌发生、发展的一个重要机制.     

人胃癌细胞系SGC-7901放射敏感性及异质性的研究

 目的: 研究人胃癌细胞系SGC-7901及其克隆亚系的放射敏感性。方法: SGC-7901细胞株及其两个克隆亚系（大集落、小集落）的细胞用8MeV直线加速器电子束以不同剂量照射, 分别经48小时、72小时培养及集落培养后, 计数并得出各组存活曲线。结果: 1.两个克隆亚系之间的D₀值有显著差异(P<0.05)大集落亚系与SGC母系之间在集落形成实验中, 其D₀值有差异（P<0.05）。结论: SGC-7901细胞系及其克隆亚系对放射的敏感性存在着异质性。     

人突变K-ras基因修饰lewis肺癌细胞株的建立及其生物学特性

目的:探讨以K-ras基因为靶点,建立人K-ras(12位密码子点突变)基因修饰的lewis肺癌细胞株3LL-pcDNA3-K-ras/V12及其生物学特性.方法:应用脂质体法将人K-ras(12位密码子点突变)基因cDNA导入lewis肺癌细胞株3LL,应用流式细胞仪检测p21/V12蛋白的表达,同时检测3LL细胞株转基因前后生长、克隆形成率和体内成瘤的变化.结果:成功的将人K-ras(12位密码子点突变)基因cDNA导入lewis肺癌细胞株3LL,p21/V12蛋白在3LL-pcDNA3-K-ras/V12中稳定表达,转基因细胞株的生长曲线和克隆形成率与原代细胞株3LL无显著差异;但转基因细胞株在C57BL/6小鼠体内的成瘤性显著高于原代细胞株3LL.结论:成功的建立了导入K-ras(12位密码子点突变)的lewis肺癌细胞株3LL-pcDNA3-K-ras/V12.     

Effect of First Line Gastric Cancer Chemotherapy Regime on the AGS Cell Line - MTT Assay Results

Background: Combination chemotherapy regimes are common treatments for cancer. The aim of this study was to evaluation the effect of individual chemotherapeutic agents in comparison with a first line chemotherapy regime treatment in the AGS gastric cancer cell line by MTT assay. Materials and Methods: In this experimental study, AGS cells were grown in RPMI-1640 supplemented with 10% fetal calf serum and 100 IU/ml penicillin, and 10 μg/ml streptomycinin, under a humidified condition at 37°C with 5% CO2. All cells were washed with PBS and detached with trypsin, centrifuged and 8000 cells re-plated on to 96- well plates. LD50 doses of Epirubicin, Cisplatin and 5-fluorouracil were added to each well in mono or triple therapy. Anti-proliferative activities were determined by MTT assay after 24, 48 or 72 h. Results: Results of MTT assays showed that there were no significant differences among 3 drugs in monotherapy (p=0.088), but there was significant difference between combination therapy with epirubicin (P=0.031) and 5FU (p=0.013) on cell survival at 24 h. After 48 and 72 hours, cell viability showed significant differences between the 3 drugs (p=0.048 and P=0.000 for 48 and 72 h, respectively) and there was significant difference between combination therapy with epirubicin (P=0.035 and P=0.002 for 48 and 72 h, respectively). Conclusions: The results showed no significant differences between these chemotherapy drugs each given alone, but combination therapy with 3 drugs had significant effects on cell viability in comparison with epirubicin alone.     

Third- and Late Line Treatments of Metastatic Gastric Cancer: Still More to Be Done

In recent years, advances of anticancer and supportive therapies have determined a gradual improvement in survival rates and patients’ general conditions in metastatic gastric cancer (mGC), allowing them to receive further treatments. The choice of treatment is driven by performance status, age, stage of disease, number of metastatic sites and time from the first to third line of treatment. Targets such as microsatellite instability, PD-L1 expression, and HER2 overexpression or amplification may be addressed to personalise treatment and prolong survival. Despite a growing number of third line options that have provided clinicians with greater opportunities to customise treatments, up to date few agents have been demonstrated as effective after two standard lines for mGC; for these reasons, chemotherapy, immunotherapy, and targeted therapy were all widely investigated in both phase II and phase III studies. Overall, TAS-102, apatinib, regorafenib, nilotinib, trastuzumab, and pembrolizumab were demonstrated to be valid options in the third line scenario for mGC patient refractory to at least two lines of therapy. A multimodal approach based on chemotherapy, immunotherapy, targeted agents, a personalised nutritional programme as well as the research of new predictive biomarkers may pave the way to new strategies to identify the best treatment for each patient.     

mrp反义RNA逆转胃癌细胞系SGC7901对VCR的耐受性

目的：探讨以多药耐药相关蛋白（ＭＲＰ）作为靶分子进行胃癌多药耐药基因治疗的可行性。方法：采用已构建成功的ｍｒｐ反义ＲＮＡ真核载体ｐｃＤＮＡ－Ａｍｒｐ转染胃癌细胞系ＳＧＣ７９０１,用Ｇ４１８抗性筛选出稳定细胞克隆,命名为Ｍ－ＳＧＣ７９０１;用＾３２Ｐ标记的寡核苷酸探针打点杂交检测Ｍ－ＳＧＣ７９０１细胞中ｍｒｐ ｍＲＮＡ表达的变化;从细胞生长曲线中,观察Ｍ－ＳＧＣ７９０１细胞生长速度的变化;经０．００     

Expression of Inhibitor of Apoptosis Gene Family Members in Bladder Cancer Tissues and the 5637 Tumor Cell Line

Background: Apoptosis is suppressed in cancer tissues and tumor cell lines because anti-apoptosis genes are over-expressed. The inhibitor of apoptosis proteins (IAP) gene family contributes to control of apoptosis. The expression profile of eight genes of the IAP family in biopsies from patients with a history of bladder cancer and normal bladder tissues, as well as a bladder tumor cell line (5637), was assessed in the present study. Methods: Cancer tissue samples were obtained at surgery and the 5637 tumor cell line was cultured in RPMI1640 medium. Beyond tumor margins were selected as normal tissue. Expressional profile of interested genes was obtained by using specific primers and the real-time PCR method. Results: The results showed that expression of seven of the studied genes was up-regulated in cancer tissues and the cell line whereas BIRC4 (XIAP) was down-regulated in both. Conclusions: The results showed that these genes were expressed to a greater extent in cancer tissue and cancer cells than in normal tissues. The data suggested that over-expression of anti-apoptotic genes such as IAP family members, can trigger cells to escape from apoptosis.     

Expression of Fas Antigen and Its Mediation of Apoptosis in Human Gastric Cancer Cell Lines

Fas, a member of the tumor necrosis factor receptor/nerve growth factor receptor family, induces apoptosis by crosslinking with Fas ligand or anti-Fas antibody in a variety of cultured cells. We examined the expression of Fas antigen and its mediation of apoptosis in six human gastric carcinoma cell lines. Flow cytometric analysis and western blotting revealed relatively high expression of Fas antigen in MKN-74 (wild-type p53 gene) and MKN-45 (wild-type), followed by MKN-1 (mutated), MKN-7 (mutated) and KATO-III (deleted). MKN-28 (mutated) showed minimal expression of the antigen. The expression was apparently enhanced by interferon-γ, except for MKN-1 and MKN-28. Anti-Fas antibody (100 ng/ml) induced nuclear fragmentation characteristic of apoptosis. Apoptosis occurred in a delayed fashion and the apoptotic index at 72 h was approximately 60% in MKN-74, 35% in MKN-45, and 20% in MKN-1 and KATO-III. A DNA ladder was noted in MKN-74 at 72 h. Expression levels of P53 and P21^Wafl did not change for up to 48 h in MKN-74. The biological effects did not correlate with endogenous Bcl-2 expression. These results indicated that a) Fas antigen is variably expressed in human cultured gastric carcinoma cells, b) the protein transduces an apoptotic signal which leads to delayed cell death, and c) susceptibility to the antibody correlates well with the expression level of Fas antigen.     

人胃癌细胞系(BGC-823)的细胞遗传学研究

本研究以人胃癌(BGC-823)细胞系为材料,自建系后40～60代进行了细胞遗传学研究。先后共计数600个分裂中期细胞的染色体数,并对其中的30个分裂相作了胰酶消化和G-分带。核型分析结果表明:染色体众数为亚三倍体,发现了规律性出现的标记染色体和Ⅰ号染色体长臂的部分缺失,部分核型出现双微小体。带型分析表明。Ⅰ号标记染色体为染色体11,12间的易位,t(11,12)(11pter→11q23::12q13→12qter).12号染色体的断裂点和染色体的脆性部位位于同一染色体区带(12q13)。这将有助于阐明此系的癌基因ras与标记染色体的关系,进而了解癌变过程中癌基因的作用。     

Down-regulation and It's Molecular Biology of Nm-23H1 Gene Transfection on PKC Signal Path-way in Human High-metastatic Large Cell Lung Cancer Cell Line L9981

Backgroud and Objective At present, lung cancer has become the most frenquent malignant tumor in China and in the world which its mortality and morbidity is increasing fastest. It