血管内皮细胞生长抑制因子的研究进展

血管内皮细胞生长抑制因子(vascularendothelialgrowthinhibitor,VEGI)是一种新型的血管内皮细胞生长抑制因子,属于TNF超家族,是Ⅱ型跨膜蛋白。重组VEGI不仅可以抑制内皮细胞增殖和诱导血管内皮细胞凋亡,而且可阻止新生血管生成,从而产生抗肿瘤生长的作用。VEGI作为一个内皮细胞产生的血管生成负调控因子可激活JNK、P38MAPA及胱冬肽,也可激活NF-κB,从而诱导内皮细胞的凋亡。VEGI的N段部分缺失可影响其生物活性,具有重要的病理生理意义,在肿瘤生物治疗方面有很大的应用前景

肾脏-胰岛联合移植研究进展

糖尿病肾病是糖尿病最严重的并发症,可通过间质及肾小管纤维化等机制导致肾脏功能衰竭,最终进展为终末期肾脏病（end stage renal disease,ESRD）[1-2]。既往认为,糖尿病肾病及其他慢性肾病患者更容易死于心血管疾病而不是ESRD,但是据一份2型糖尿病（type 2 diabetes mellitus,T2DM）随访统计,ESRD导致的死亡比心血管疾病更为

The preparation and tumor cell apoptosis-inducing activity assay of anti-human DR5 single-chain antibody

作者简介: 张佳锴,男,硕士研究生,主要从事肿瘤免疫学研究, Tel: 0592-2180587，E-mail: baroce@ 163.com; 庄国洪，通信作者，E-mail: [email protected]。[中文摘要]目的构建、表达、纯化抗人死亡受体5(DR5)单链抗体,并检测其诱导肿瘤细胞凋亡的活性。方法采用RT-PCR获取鼠源性抗人DR5单克隆抗体重链和轻链可变区基因序列,以一柔性连接肽连接二者,转入表达载体,以大肠杆菌表达融合蛋白,亲和色谱纯化后用MTT实验和凋亡检测试剂盒检测其凋亡诱导活性。结果获得的序列经比对为抗体重链和轻链可变区基因,表达纯化后的重组蛋白具有接近完整抗体的肿瘤细胞凋亡诱导活性。结论抗人DR5 scFv可作为诱导肿瘤细胞凋亡的候选药物,为肿瘤免疫学研究提供材料。[英文摘要]Purpose To construct，express，purify anti-human DR5 scFv，and to assay its activity of apoptosis induction for tumor cell lines． Methods Variable region sequences of heavy chain and light chain to murine anti-human DR5 monoclonal antibody were acquired by RT-PCR， then they were linked through a flexible linker peptide and was cloned into the expression vector． After being expressed by E．coli and purified using affinity chromatography， the recombinant protein was employed to MTT assay as well as apoptosis assay kit in order to detect its apoptosis-inducing activity． Results The sequences we've got were established as the variable region genes of antibody's heavy and light chain，while the recombinant proteins expressed and purified possessing the apoptosis-inducing activity come near to complete antibody．Conclusion Anti-human DR5 scFv can supply material for tumor immunology research as drug candidate inducing tumor cell apoptosis．福建省自然科学基金资助项目(C0710046

Airborne fine particle decreases the cell viability and induces inflammation in human bronchial epithelial cells

目的:研究大气细颗粒物(fine particulate; matter,PM_(2.5))对人支气管上皮细胞活性的影响及其炎性作用。方法:用PM_(2.5)采样器采集上海地区大气PM_(2.5)样本,扫; 描电镜观察PM_(2.5)形态特征。将人支气管上皮细胞BEAS-2B暴露于不同浓度(0,50,100,200,400,800; mug/mL)的PM_(2.5) 12,24,48 h,细胞活力检测试剂盒(cell counting kit-8,; CCK-8)法检测PM_(2.5)暴露对细胞活性的影响。实时定量PCR(quantitative real-time; PCR,qRT-PCR)检测细胞粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating; factor,GM-CSF)和TNF-alpha mRNA的表达,Western印迹检测; GM-CSF和TNF-alpha蛋白的表达。结果:扫描电镜观察发现,PM_(2.5)形态多样,大小不一,直径大多等于或小于2.5; mum。与同时间点未暴露组比较,各暴露组(50~800; mug/mL)细胞活性呈不同程度的下降,差异具有统计学意义(P<0.05)。与未暴露组比较,暴露于100,400或800 mug/mL; PM_(2.5) 24 h后,GM-CSF和TNF-alpha; mRNA和蛋白表达水平明显升高(P<0.05),且PM_(2.5)暴露浓度越高,GM-CSF和TNF-alpha的mRNA和蛋白升高水平越显著。; 结论:大气PM_(2.5)可引起人支气管上皮细胞的炎症反应,降低细胞活性,这可能与PM_(2.5)促发和加重支气管肺部炎性疾病有关。Objective: To investigate the effects of airborne fine particle on cell; viability and inflammation in human bronchial epithelial cells. Methods:; Atmospheric PM_(2.5) samples were collected by PM_(2.5) sampler.; PM_(2.5) morphology was observed by scanning electron microscope (SEM).; Human bronchial epithelial cells (BEAS- 2B) were treated with PM_(2.5); at different concentrations (0, 50, 100, 200, 400, 800 mug/mL) for 12,; 24 or 48 hours, and the cell activity were evaluated by cell counting; kit-8 (CCK-8). The mRNA expression levels of (granulocyte-macrophage; colony stimulating factor,GM-CSF) and TNF-alpha were detected by; quantitative real-time PCR (qRT-PCR). Western blot was used to detect; the protein expressions of GM-CSF and TNF-alpha. Results: According to; SEM, the shape of PM_(2.5) varied, and the diameter was different and; mostly equal to or less than 2.5 mum. CCK-8 assay showed that different; concentrations of PM_(2.5) exposure for 12 hours, 24 hours and 48 hours; resulted in loss of cell viability of BEAS-2B cells (P<0.05). Different; concentrations of PM_(2.5) increased the mRNA and protein expression of; GM-CSF and TNF-alpha, and the higher concentration of PM_(2.5) induced; higher expression, which have statistical significant difference between; the groups (P<0.05). Conclusion: Atmospheric PM_(2.5) can cause; inflammatory response in human bronchial epithelial cells. They can; reduce cell viability, which may be related to the PM_(2.5) trigger and; aggravation of bronchopulmonary inflammatory diseases

Effect and mechanism of TIPE3 interference plasmid on SW480 colorectal cancer growth

目的:通过TIPE3干扰质粒转染SW480结肠癌细胞,验证干扰TIPE3表达对SW480结肠癌细胞生长的影响并探讨相关机制。方法:构建TIPE3; -shRNA-pSIREN-RetroQ干扰质粒,通过脂质体转染法成功将干扰质粒导入SW480细胞,通过RT-PCR、Western; blot检测重组质粒的干扰效率。应用CCK-8方法检测SW480细胞生存率。AnnexinV-FITC/PI流式细胞法检测细胞凋亡。使用West; ern; blot检测细胞增殖、凋亡相关分子的表达情况。结果:成功设计、构建和筛选具有生物活性且干扰效率最佳的TIPE3-shRNA-pSIREN-Ret; roQ干扰质粒。CCK-8检测证实干扰SW480结肠癌细胞TIPE3表达可以抑制细胞生长。流式结果显示,TIPE3-shRNA3干扰组的凋亡率为; (27. 991. 087) %,显著高于正常细胞组(12. 102. 213) %及转染空载质粒组(11. 440. 277 0); %。证实了降低TIPE3表达可以增加SW480对aDR5ScFv所诱导的细胞凋亡敏感性。Western; blot结果显示干扰TIPE3表达可以活化caspase3蛋白,降低p-AKT、p-PDK1、PCNA等分子的表达。结论:干扰TIPE3的表达对; SW480结肠癌细胞具有促进凋亡,抑制增殖的作用。Objective: To study the effect of interference TIPE3 on the colon cancer; cell growth by transfecting SW480 colon cancer cells with the TIPE3; interference plasmid were detected. Methods: Transfecting the; constructed TIPE3-shRNA-pSIREN-RetroQ plasmid to SW480 cells. To; determine the highest interference efficiency plasmid, the mRNA and; protein levels of recombined plasmid were detected by RT-PCR and Western; blot separately and tested the cell proliferation with CCK8. Meanwhile,; apoptosis rate of SW480 cells was determined by flow cytometry assay; with AnnexinV-FITC/PI. To further determined the effects of recombined; plasmid on cell development, the level of protein involved in; proliferation and apoptosis were detected by Western blot. Results: The; most effecient interference plasmids were successfully constructed. We; found that the cell survival rate decreased when interference TIPE3 gene; expressing in colorectal cancer cells. Flow cytometry indicated that; interefering the expression of TIPE3 would increase the sensitivity of; SW480 cell to apoptosis induced by aDR5ScFv. The results of Western blot; showed that low expression of TIPE3 would activate caspase3 and; downregulate the expression of p-AKT,p-PDK1 and PCNA. Conclusion:; Interference TIPE3 could promote apoptosis and inhibit proliferation in; SW480 colon cancer cells.国家自然科学基金; 福建省属公益类科研院所基本科研专项项

TIPE2在T细胞介导的心脏移植急性排斥反应中的潜在作用

目的探讨作为免疫负调控因子的肿瘤诱导坏死因子-α-诱导蛋白8样因子-2(TIPE2)在T细胞介导的小鼠心脏移植急性排斥反应中的潜在作用。方法通过苏木素-伊红(HE)染色和流式细胞术确定心脏移植物中的主要免疫排斥细胞,免疫荧光染色确定TIPE2与脾细胞间的关系,实时定量荧光PCR(Real-time PCR)检测心脏移植物TIPE2及细胞因子表达量。结果小鼠心脏移植7天后心脏组织出现明显免疫细胞浸润及心肌组织的破坏,流式细胞术检测心脏移植物中CD4~+和CD8~+T细胞增高,免疫荧光显示移植小鼠脾细胞中的CD4~+和CD8~+T细胞表达TIPE2,而心脏移植物在第7天时TIPE2和细胞因子表达量均明显增高。结论 TIPE2与在T细胞介导的小鼠心脏移植急性排斥反应中可能发挥着潜在作用。国家重点科学研究计划(2012CBA01303);;国家自然科学基金(31271038

Enhancement of glioma cell apoptosis induced by anti-human DR5/DR4 monoclonal antibodies by sub-toxic dose of doxorubicin in human

目的探讨亚毒性剂量的阿霉素影响抗DR4、DR5单克隆抗体(mAb)FMU1.4和FMU1.5诱导3株神经胶质瘤细胞株U343(TRAIL敏感株)、U138(TRAIL部分敏感株)及U373(TRAIL耐受株)凋亡的作用及可能的机制。方法采用流式细胞术检测DR4、DR5的表达及神经胶质瘤细胞中DNA倍增。用MTT比色法检测mAbFMU1.4和FMU1.5对3株神经胶质瘤细胞增殖的抑制作用。用共聚焦显微镜观察3株细胞中Ca2+的浓度。以Westernblot检测细胞内色素C、FLIP[FLICE-inhibitoryprotein,为一组含有死亡效应结构域(DED)的胞浆蛋白]的表达。结果亚毒性剂量的阿霉素作用后,DR5、DR4在3株细胞中的表达提高;而mAbFMU1.4、FMU1.5诱导U138和U373细胞凋亡的作用增强,细胞内细胞色素C的表达提高,FLIP的表达降低,Ca2+浓度增加。结论亚毒性剂量的阿霉素与抗DR4、DR5mAb联合应用后,可提高mAb诱导靶细胞凋亡的效应,其作用机制与DR4、DR5、细胞色素C、FLIP的表达及Ca2+的含量有关。 【英文摘要】 AIM: To study the cytotoxic effects of doxorubicin on apoptosis in glioma cell lines U343, U138, U373 induced by anti-human DR4/DR5 monoclonal antibodies (FMU1.4/FMU1.5) and the underlying mechanism. METHODS: Expression of DR4/DR5 was quantitated by flow cytometry. Cytotoxicity exerted by FMU1.4/FMU1.5 on three cell lines was measured by MTT colorimetry and the induced apoptosis was determined by agarose gel electrophoresis. The expression of cytochrome C, FLIP and Ca 2+ concentration were also measured. RE...教育部跨世纪优秀人才培养计划资助项目(2000年

Fas ligand and Anti-human DR5 monoclonal antibody induce tumor cell lines apoptosis

目的探讨FasL、Anti-DR5mAb对肿瘤细胞Hela、BGC823、MCF-7、L342、H9101、D6的杀伤作用及机制。方法采用RT-PCR、MTT比色法、电泳、DNA倍体分析、Westernblot等方法。结果H9101、Hela细胞株DR5mRNA水平有表达,D6细胞无表达;H9101、L342细胞株FasmRNA水平有表达,D6细胞无表达。H9101、L342细胞株对FasL、Anti-DR5mAb敏感并呈剂量依赖性;MCF-7、BGC823细胞株对FasL敏感,对Anti-DR5mAb相对敏感。Hela对FasL相对敏感,对Anti-DR5mAb敏感;D6对两种凋亡诱导剂耐受。结论FasL、Anti-DR5mAb能不同程度地诱导肿瘤细胞凋亡,其机制与Fas、DR5、Caspase-8、Bcl-2的表达有关。 【英文摘要】 Objective:To study the cytotoxic effects on tumor cell lines Hela,BGC823,MCF-7,L342,H9101,D6 induced by Fas ligand and anti-human DR5 monoclonal antibodies(Anti-DR5 mAb) and the underlying mechanism.Methods:Fas/DR5 mRNA were detected by RT-PCR. Cytotoxicity exerted by FasL/Anti-DR5 mAb on tumor cell lines was measured by MTT colorimetry and the induced apoptosis was determined by agarose gel electrophoresis.Results:The expression of DR5 on BGC823 and Hela cells were higher and DR5 didn’t express in D6. The ...厦门大学科研启动基金(No.Z03103)资

重组人RGD-FasL对胶质瘤细胞U138/U343/U373的体外抗肿瘤的活性分析

目的研究重组人RGD-FasL对3株胶质瘤细胞U138/U343/U373的杀伤作用及机制。方法采用RT-PCR方法、MTT比色法、DNA倍体分析、流式细胞术检测融合蛋白对3株胶质瘤细胞的杀伤作用;利用Western blot法探讨其作用机制。结果FasmRNA在U138和U343细胞中有表达,在U373细胞中未有可见表达;DcR3mRNA在3株细胞中均有表达,在U373细胞中强表达。采用流式细胞术检测肿瘤细胞表面两种受体的表达情况,结果与RT-PCR相符。U138细胞株对RGD-FasL敏感并呈剂量依赖性;U343细胞株对RGD-FasL相对敏感;U373细胞对RGD-FasL不敏感。用PI检测细胞周期与凋亡表明RGD-FasL能使U138/U343细胞停留在G1期,推迟进入S期,抑制细胞生长并诱导细胞发生凋亡。Western blot实验表明RGD-FasL作用细胞后caspase-8/3/9的表达升高,Bcl-2的表达降低。结论重组人RGD-FasL可以不同程度的诱导胶质瘤细胞凋亡,其机制与其受体的表达和caspase-8/3/9、Bcl-2的表达有关

Correlation between expression of DcR3 on tumor cells and sensitivity to FasL

To investigate the correlation between sensitivity to Fas ligand (FasL) and expression level of decoy receptor 3 (DcR3) on tumor cell surface, Fas/DcR3 mRNA expression was detected by RT-PCR. Anti-DcR3 mAb was used to detect expression level of DcR3 on surface of tumor cells by flow cytometry. Caspase-8, caspase-9, caspase-3, Bcl-2 expressions were analyzed by Western blot, respectively. Sensitivity to apoptosis induced by FasL was determined by Annexin V apoptosis kit. The expressions of DcR3 on the surface of tumor cells from high to low were approximately 35.3% in BGC823 cells, and 21.6% in MCF-7 cells, respectively. The apoptotic rates induced by FasL from low to high were 15.6% in BGC823 cells, and 58.2% in MCF-7 cells, respectively. There was a significant correlation between the expression levels of DcR3 with FasL-inducing apoptosis