Adenovirus-mediated FasL gene transfer into human gastric carcinoma

AIM: To evaluate the possible value of FasL in gastric cancer gene therapy by investigating the effects of FasL expression on human gastric cancer cell line. METHODS: An adenoviral vector encoding the full-length human FasL cDNA was constructed and used to infect a human gastric cancer (SGC-7901) cell line. FasL expression was confirmed by X-gal staining, flow cytometric analysis and RT-PCR. The effect of FasL on cell proliferation was determined by clonogenic assay, cytotoxicity was detected by MTT assay, and cell viability was measured by trypan blue exclusion. The therapeutic efficiency of Ad-FasL in vivo was investigated with a xenograft tumor model in nude mice. RESULTS: SGC-7901 cells infected with Ad-FasL showed increased expression of FasL, resulting in significantly decreased cell growth and colony-forming activity when compared with control adenovirus-infected cells. The cytotoxicity of anti-Fas antibody (CH-11) in gastric cancer cells was stronger than that of ActD (91±8 vs60±5,P<0.01), and the cytotoxicity of Ad-FasL was stronger than that of CH-11 (60±5 vs50±2, P<0.05). In addition, G1-phase arrest (67.75±0.39 vs 58.03±2.16, P<0.05) and apoptosis were observed in Ad-FasL-infected SGC-7901 cells, and the growth of SGC-7901 xenografts in nude mice was retarded after intra-tumoral injection with Ad-FasL (54% vs 0%,P<0.0001). CONCLUSION: Infection of human gastric carcinoma cells with Ad-FasL induces apoptosis, indicating that this target gene might be of potential value in gene therapy for gastric cancer.

Influence of FasL gene expression on hepatic metastasis of colorectal carcinoma

BACKGROUND: FasL expression was reported to be asso- ciated with hepatic metastasis of colorectal cancer. The aim of this study was to study FasL gene expression in colorectal carcinoma and its influences on biological behavior and he- patic metastasis of colorectal carcinoma. METHODS: FasL gene expressions were examined with re- verse transcriptase-polymerase chain reaction (RT-PCR) in the primary focus of colorectal carcinoma, adjacent can- cerous mucosae, and metastasized liver focus from colorec- tal cancer. HR-8348 cells of human rectal cancer cell line were transfected with FasL cDNA. Cell growth suppression rate and response to 5-FU and carboplatin were observed and analyzed with the MTT method. RESULTS: FasL gene expression was detected in the prima- ry focus of colorectal cancer ( n = 58), adjacent cancerous mucosae ( n = 58), and metastasized hepatic tumor tissues (n =28). The positive rate of FasL expression was 24% (14/ 58), 8% (5/58), and 100% (58/58) in the primary focus, adjacent cancerous mucosae and metastasized hepatic tumor tissues respectively. FasL expression rate in the me- tastasized hepatic tumor tissues was higher than that in the primary focus (χ2 = 43.49, P<0. 01) and adjacent cance- rous mucosae (χ2=57.66, P<0.01). In a group of patients with hepatic metastasis, the FasL expression rate in primary focus was higher than that in patients without hepatic me- tastasis (χ2=3.96, P <0.05). In vitro study positive expres- sion of FasL was shown in transfected HR-8348 cells. When 5-FU or carboplatin was added, there was a significant difference in growth suppression rate between FasL positive and controlled cancer cells (t=9.02, t = 11.93, P<0.01). Under the same concentration of chemotherapeutic agents, the survival rate of FasL positive HR-8348 cells was higher than that of controlled cells. CONCLUSIONS: FasL positive cancer cells are powerfullyresistant to chemotherapeutic agents. The expression of the FasL gene in colorectal cancer cells is related to immune evasion to escape from being killed by immune cells, show- ing stronger drug-resistance, and it facilitates hepatic me- tastasis.

Anti-gastric cancer active immunity induced by FasL/B7-1 gene-modified tumor cells

AIM: To study the activation of cytotoxic T lymphocytes (CTLs) against gastric cancer cells induced by FasL/B7-1 (FB-11) gene-modified tumor cells, and to explore whether co-expression of FasL and B7-1 in SGC-7901 tumor cells could initiate synergistic antitumor effect. METHODS: FasL and B7-1 genes were transfected into human SGC-7901 gastric cancer cells with adenovirus vectors. The positive clones were selected by G418. FasL and B7-1 genes were detected by flow cytometry and RT-PCR. Abdominal infiltrating lymphocytes and sensitized spleen cells were obtained from mice that were immunized with SGC-7901/FB-11 or wild type SGC-7901 cells intraperitoneally, and cytotoxicity of these CTLs against tumor cells was determined by MTT assay. RESULTS: Flow cytometry and RT-PCR showed that FasL and B7-1 genes were highly expressed. FasL and B7-1 transfected cancer cells had a high apoptosis index. DNA laddering suggested that FasL and B7-1 genes induced gastric cancer cell apoptosis. FasL+/B7-1+SGC-7901 cells (SGC-7901/FB-11) were inoculated subcutaneously in the dorsal skin of C57BL/6 mice and then decreased their tumorigenicity greatly (z = 2.15-46.10, P＜0.01). SGC- 7901/FB-11 cell-sensitized mice obtained protective immune activity against the rechallenge of wild type SGC 7901 cells (z = 2.06-44.30, P＜0.05). The cytotoxicity of CTLs induced by SGC-7901/FB-11 cells against SGC-7901 was significantly higher than that of CTLs activated by wild-type SGC-7901 cells (84.1±2.4% vs30.5±2.3%,P＜0.05).CONCLUSION: FasL and B7-1 genes can effectively promote the activity of CTLs against gastric cancer cells. FasL/B7-1 molecules play an important role in CTL cytotoxicity.

Combination of CTLA4-FasL gene transfer and allogeneic bone marrow transplantation led to durable macrochimerism and donor-specific tolerance in mouse model

Mixed hemopoietic chimerism is capable of inducing donor specific tolerance, thus eliminating the chronic immunosuppressive therapy following organ trans- plantation. As yet no safe and effective tolerance protocol is available for clinical implementation. Here we describe an alternative nonmyeloablative based strategy of using a single injection of recombination adenovirus vector encoding CTLA4-FasL fusing gene and donor bone marrow cells to promote durable mixed macrochimerism (>20% on 140 d). Chimeras exhibited robust donor-specific tolerance, as evi- denced by acceptance of fully allogeneic skin grafts (the mean survival time (MST)>200 d) and rejection of third- party skin grafts in a normal manner (MST<10 d). In this model, the frequencies of helper T lymphocyte precursor (HTLp) and cytotoxic T lymphocyte precursor (CTLp) were greatly reduced on day 14 after transplantation, suggesting that CTLA4-FasL led to rapid systemic peripheral tolerance to facilitate the bone marrow engraftment, while both HTLp and CTLp remained at low level only in recipient mice with mixed chimerism on day 140 after transplantation, demon- strating that long-term skin grafts tolerance was associated with stable mixed chimerism, and central deletion of donor specific T cell may be the main mechanism for tolerance maintenance.

补肾、活血复方对老年大鼠T细胞凋亡相关基因Fas、FasL转录的影响

目的 :探讨补肾复方下调老年大鼠T细胞凋亡的分子机理。方法 :采用RNA酶保护技术 ,测定各组Fas、FasL基因的mRNA水平 ,并以GAPDH为内对照基因 ,对各组Fas、FasL基因的转录进行半定量分析。结果 :老年对照组Fas/GAPDH、FasL/GAPDH的积分光密度值分别为 0 81± 0 17、0 84± 0 12 ;年轻对照组分别为 0 36± 0 16、0 2 6± 0 12。两组比较差异有显著性。活血复方组Fas/GAPDH、FasL/GAPDH的积分光密度值分别为 0 99± 0 36、0 94± 0 33,与老年对照组比较 ,差异无显著性 (P >0 0 5) ;两个补肾组Fas/GAPDH为 0 78± 0 11和 0 78± 0 2 0 ,与老年对照组比较差异无显著性 (P >0 0 5) ;而两个补肾组FasL/GAPDH的积分光密度值为 0 71± 0 2 1和 0 68± 0 15,低于老年对照组 (P <0 0 1)。结论 :补肾复方对T细胞FasL基因的转录具有一定的负调控作用 。

脑缺血再灌流后Fas及FasL在脑内表达的变化和氟桂嗪的抑制作用

目的 观察全脑缺血再灌流后Fas和FasL基因在脑内表达的变化及氟桂嗪的影响。方法 用大鼠 4血管结扎全脑缺血 30min再灌流模型和免疫组织化学染色法。结果 脑缺血再灌流 6h在皮质及海马区即出现Fas表达阳性细胞 ,2 4～ 48h时达高峰 ;FasL则在 12h后出现 ,48～ 72h时达到高峰。ip氟桂嗪 10mg·kg-1和 2 0mg·kg-1则Fas和FasL的表达明显低于不给药的缺血再灌流对照组 ,并呈剂量依赖性。结论 脑缺血再灌流可诱导促凋亡基因Fas和FasL在缺血易感部位表达 。

Fas/FasL基因多态性与乳腺癌发生的相关性

目的：探讨Fas/FasL基因多态性与汉族人群乳腺癌发病风险的关联性。 方法：提取250例乳腺癌患者和250例体检健康者外周血基因组DNA，用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）分析技术对Fas基因1377G〉A,670A〉G和FasL基因844T〉C位点多态性进行基因分型，用非条件Logistic回归方法计算风险比（OR）及95%可信区间（95%CI）。 结果：与Fas 1377多态性位点GG基因型相比，GA、AA基因型显著降低乳腺癌的发病风险［校正OR（95%CI）分别为0.44（0.29~0.68），0.29（0.17~0.48），P均〈0.01］。与Fas 670多态性位点AA基因型相比，AG、GG基因型显著降低乳腺癌的发病风险［校正OR（95%CI）分别为0.63（0.42~0.94）和0.36（0.21~0.60），P均〈0.01］。与FasL 844多态性位点CC基因型相比，TC基因型显著降低乳腺癌的发病风险［校正OR（95%CI）为0.58（0.40~0.84），P=0.012）。 结论：Fas-FasL基因多态性与乳腺癌发病风险相关。

腺病毒介导的FasL基因转移及对胃癌细胞生长和凋亡的影响

目的:探讨FasL基因对人胃癌的作用以及FasL基因治疗胃癌的可能性。方法:通过腺病毒载体介导FasL基因转移到低水平表达FasL的人胃癌细胞系SGC-7901,对FasL基因的表达,细胞生长的抑制与机制和对肿瘤生长模型的治疗效果进行分析。结果:外源性FasL基因在靶细胞高水平的表达,显著抑制了SGC-7901的生长和集落形成,流式细胞仪检测显示细胞G1期阻滞并发生了凋亡。瘤内注射Ad-FasL对裸鼠体内移植瘤有一定的抑瘤作用。结论:FasL基因参与了肿瘤细胞凋亡的诱导,能抑制胃癌细胞的生长,因此该基因在肿瘤的基因治疗上有着广泛的应用前景。

非小细胞肺癌Fas/FasL基因蛋白的表达及其临床意义

目的 :研究Fas/FasL基因蛋白在非小细胞肺癌 (NSCLC)的表达及其临床意义。 方法 :应用链霉菌抗生物素蛋白 过氧化物酶 (SP)法染色技术测定 6 5例NSCLC组织标本Fas/FasL基因蛋白的表达水平 ,并分析其与病理类型、区域淋巴结转移、病理分级及TNM分期之间的关系。 结果 :NSCLC组织中Fas基因蛋白表达率显著低于正常肺组织 (46 .15 %vs 80 .0 0 % ,P <0 .0 5 ) ,而FasL基因蛋白表达率显著高于正常肺组织 (49.2 3%vs13.33% ,P <0 .0 5 ) ;FasL的表达与肺癌区域淋巴结转移有关 ,但Fas/FasL与肿瘤病理类型、病理分级及TNM分期无关。 结论 :Fas/FasL基因蛋白可能参与了非小细胞肺癌细胞凋亡的调节 ;检测FasL基因蛋白的表达有可能为临床判断NSCLC区域淋巴结转移。

FasL基因关节腔内转染对佐剂性关节炎大鼠滑膜细胞凋亡的影响

目的观察FasL基因关节腔内转染对佐剂性关节炎大鼠滑膜细胞的凋亡诱导作用。方法 SD大鼠分为正常转染对照组、正常转染组、模型转染对照组、模型转染组。向模型转染对照组、模型转染组动物右后足趾皮内注射0.1 mL弗氏完全佐剂致炎,诱导佐剂性关节炎。观察大鼠关节炎指数,大鼠滑膜细胞增生、炎性细胞浸润情况,检测滑膜细胞凋亡水平。结果模型转染组与模型转染对照组比较大鼠关节炎指数(AI)显著降低,差异有统计学意义(P<0.05);模型转染对照组、正常转染组与正常转染对照组比较,大鼠滑膜细胞凋亡指数差异无统计学意义(P>0.05),模型转染组与正常转染对照组、模型转染对照组比较,大鼠滑膜细膜凋亡指数间差异有统计学意义(P<0.01)。结论 FasL基因关节腔内转染可诱导佐剂性关节炎大鼠滑膜细胞凋亡,降低佐剂性关节炎大鼠的关节炎指数。

腺病毒介导的FasL基因诱导肺癌细胞凋亡

目的 研究外源性FasL基因对人肺癌细胞凋亡的影响以及 FasL基因治疗肺癌的可能性。方法 构建腺病毒载体介导FasL基因转移到低水平表达 FasL的人肺腺癌细胞系 A549,逆转录聚合酶链反应(RT- PCR)、流式细胞术(FCM)检测 FasL基因的表达,并观察 A549 肺癌细胞凋亡和生长,对肿瘤生长模型的治疗效果进行分析。结果 外源性FasL基因在A549肺癌细胞获得高水平的表达,FasL能显著抑制A549的生长和集落形成,流式细胞仪检测显示细胞G1 期阻滞并发生了凋亡。瘤内注射Ad- FasL对裸鼠体内移植瘤有较强的抑瘤作用。结论 FasL基因参与了肺癌细胞凋亡的诱导,能抑制肺腺癌细胞的生长,因此该基因在肿瘤的基因治疗上有广泛的应用前景。

几种ACEI对实验性糖尿病大鼠心肌细胞凋亡和Caspase-3及Fas、FasL基因表达影响的类效应

目的:探讨几种血管紧张素转换酶抑制剂(ACEI)卡托普利、培哚普利、咪达普利、贝那普利、福辛普利对糖尿病大鼠左室心功能及心肌细胞凋亡和凋亡相关蛋白影响的类效应。方法:将48只SD大鼠腹腔注射链脲佐菌素(STZ)诱发糖尿病模型,随机分为5个给药组和1个糖尿病组(每组8只),另选8只做正常对照组。给药组每日分别灌胃给予卡托普利(50 mg/kg),培哚普利(雅施达4 mg/kg),咪达普利(达爽10 mg/kg),贝那普利(洛汀新10 mg/kg),福辛普利(蒙诺10 mg/kg),糖尿病组给予生理盐水灌胃,共8周。对照观察心肌细胞凋亡、心肌组织Fas、FasL蛋白、Caspase-3及其Fas mRNA表达的变化。结果:与正常对照组相比,糖尿病给药组及未给药组的心肌细胞凋亡指数明显增高(均为P<0.05);Caspase-3阳性蛋白表达率明显增高(均为P<0.05);Fas蛋白阳性染色指数增高(均为P<0.05);Fas的mRNA表达明显增高(均为P<0.05)。但糖尿病给药组的较未给药组低(均为P<0.05)。所有各组的FasL蛋白阳性染色指数差异不明显(均为P>0.05)。各种ACEI治疗组之间差异无显著性(均为P>0.05)。结论:糖尿病心肌病的发生、发展过程中有心肌细胞凋亡的变化和Caspase家族及Fas、FasL系统的参与;糖尿病大鼠心肌细胞凋亡指数、心肌组织Fas蛋白以及Fas的mRNA表达水平、Caspase-3蛋白表达水平增高。ACEI干预糖尿病心肌病,能够降低细胞凋亡及Fas基因的表达及Caspase-3蛋白表达水平,且几种ACEI具有类效应。

牦牛Fas和FasL基因克隆及其在主要组织器官中的表达

为了进一步理解Fas/FasL系统,本研究拟克隆牦牛Fas和FasL基因,并对2个基因在各组织器官中的表达进行检测。采用RT-PCR方法克隆牦牛Fas和FasL基因,并利用相关软件进行生物信息学分析,结合RTPCR方法及免疫组织化学方法对2个基因在牦牛主要组织器官中的表达进行检测。克隆得到了2 605bp的牦牛Fas基因cDNA和1 572bp的FasL基因cDNA序列,与黄牛的相应序列有很高的同源性(分别为99.1%和99.4%)。除了在心中只检测到Fas mRNA外,Fas和FasL mRNA共同表达于所有检测的组织中,且2个基因均在肝、脾、肾、淋巴结、胸腺和睾丸中强表达。免疫组化检测发现FasL蛋白存在于多种体细胞的胞浆中。结果表明,除了在淋巴器官和免疫赦免位点外,Fas和FasL还强表达于其他器官如肾和肝,表明Fas/FasL系统比以前所了解的要复杂。

Fas/FasL与自身免疫性疾病

细胞凋亡是一种在形态学特征和生物化学特征与坏死截然不同的自主性坏死过程,在生物体发育、防御及维持免疫耐受中有重要作用。Fas/FasL系统介导的细胞凋亡在淋巴细胞的发育、维持内环境稳定方面起重要作用。目前研究发现,此途径介导的细胞凋亡参与类风湿关节炎、系统性红斑狼疮、自身免疫性甲状腺炎、支气管哮喘等疾病的发病过程。

Fas,FasL和Bcl-2在胃癌发生发展中的表达意义

目的 :探讨Fas,FasL和Bcl 2在胃癌发生发展中的表达及临床病理意义 .方法 :收集 93例不同胃粘膜标本 ,应用免疫组化技术检测不同病变阶段胃粘膜的Fas ,FasL和Bcl 2表达 .结果 :在正常粘膜 (n =2 0 )、癌前病变 (n =2 3)、高 中分化胃癌 (n =2 5 )、低分化胃癌 (n =2 5 )组织中Fas阳性表达率分别为 95 .0 % ,91.3% ,6 0 .0 %和 4 0 .0 % .FasL阳性表达率分别为 2 5 .0 % ,5 6 .5 % ,88.2 %和 84 .0 % .Bcl 2的阳性表达率分别 2 0 .0 % ,78.3% ,80 .0 %和 6 4 .0 % .结论 :Fas,FasL ,Bcl

Fas-670 G>A和Fasl-844 T>C基因多态性与肺癌易感性关系研究

背景与目的：肺癌是全球肿瘤相关性死亡的主要病因之一。该研究旨在探索Fas-670 G〉A和Fasl-844 T〉C基因多态性与肺癌易感性关系。方法：2010—2015年,共收集400例具有明确病理诊断的肺癌病例,同时按照同性别,选取年龄±5岁进行匹配健康对照,采用Taq Man荧光探针法进行基因分型检测。对实验结果,采用SPSS 16.0软件进行统计分析。结果：共386例肺癌患者和394例健康对照者分型成功,与Fas基因的AA基因型携带者相比,GA和GG基因型携带者肺癌发病风险无显著性增加,OR值分别为1.05（0.77~1.44）和1.27（0.81~1.99）,与Fasl基因的TT基因型携带者相比,CT和CC基因型携带者患肺癌的风险显著升高,调整年龄、性别和吸烟状态后,OR值分别为1.37（1.01~1.86）和1.74（1.09~2.77）。结论：Fasl-844 T〉C多态性可能与肺癌易感性有关,Fas-670 G〉A多态性可能与肺癌易感性无关。

大鼠FasL基因重组慢病毒载体三质粒包装细胞系统的建立

目的建立真核细胞表达的大鼠FasL基因重组慢病毒载体三质粒包装细胞系统,为进一步研究转FasL基因在同种异体器官移植中诱导免疫耐受、保护移植物的作用奠定基础。方法制备完整的重组慢病毒载体三质粒系统:转移质粒(pLO134-FasL),包装质粒(ΔNRF)及包膜蛋白质粒(VSV-G)。脂质体法将三质粒共转染包装细胞293T,72 h后收集病毒上清,Western-blot法检测293T细胞的FasL蛋白表达。结果转染后的293T细胞表达FasL蛋白。结论成功建立重组慢病毒载体的三质粒包装细胞系统。

人FasL全长cDNA的克隆及其真核表达载体的构建

目的:克隆人FasL全长cDNA并构建其真核表达载体。方法:采用RT鄄PCR法从健康人外周血单核细胞中扩增包含全部阅读框架的FasL全长cDNA,将之与pGEM鄄TEasy质粒连接、测序。构建pcDNA3.1鄄FasL真核表达载体,用脂质体介导方法转染人直肠癌8348细胞,检测FasL表达情况。结果:RT鄄PCR扩增的FasL产物经限制性内切酶酶切后生成的片段符合预期大小。克隆测序证实所得的FasLcDNA序列与GeneBank序列完全一致。pcDNA3.1鄄FasL重组质粒经EcoRI+XhoI双酶切后,电泳显示898bp目的片段和pcDNA3.1载体片段,证明重组质粒正确。转染直肠癌8348细胞后,用RT鄄PCR方法检测FasL表达阳性。结论:采用RT鄄PCR方法成功克隆了人FasL全长cDNA并构建了其真核表达载体,为进一步研究FasL功能奠定了基础。

转染的FasL基因诱导GRC-1细胞凋亡的形态学观察

目的 构建含 Fas L 基因的腺病毒载体 ,转染肾癌GRC- 1细胞株 ,观察 Fas L基因对 GRC- 1细胞株的影响 .方法 以腺病毒作载体 ,把 Fas L基因克隆入腺病毒载体 ,构建成含 Fas L 基因的转基因载体并酶切鉴定 ,用脂质体包裹法把含 Fas L 基因的腺病毒载体转染 GRC- 1细胞株 ,用 RT-PCR方法证实转染后有无 Fas L基因表达 ,采用电镜观察GRC- 1细胞形态学变化 .结果 酶切鉴定证实含 Fas L基因的转基因载体构建成功 ;RT- PCR显示转染后 GRC- 1细胞株Fas L 基因表达水平显著高于未转染细胞株 (P<0 .0 1) ;肾癌细胞株 GRC- 1转染 Fas L基因 72 h后 ,在光镜下可见细胞体积缩小 ,生长不良 ,染色质浓缩 ,密度增高 ,胞核中出现颗粒样物质 ,电镜下可见凋亡小体 .结论 成功构建了 Fas L 基因的转基因载体 ;转染 Fas L 基因可诱导肾癌 GRC-