Vascular endothelial growth inhibitor affects the invasion, apoptosis and vascularisation in breast cancer cell line MDA-MB-231

Background Breast cancer is one of the most common malignant female diseases worldwide.It is a significant threat to every woman＇s health.Vascular endothelial growth inhibitor （VEGI） is known to be abundant in endothelial cells.According to previous literature,overexpression of VEGI has been shown to inhibit tumor neovascularisation and progression in cellular and animal models,but there has been limited research on the significance of VEGI in the breast cancer.Methods In our study,cell lines MDA-MB-231 were first constructed in which VEGI mediated by lentivirus over-expressed.The effects of VEGI over-expression on MDA-MB-231 cells were investigated both in vitro and in vivo.The expression of VEGI in the MDA-MB-231 cells after infection of lentivirus was analyzed using real-time PCR and Western blotting.The effect of the biological characteristics of MDA-MB-231 cells was assessed by growth,invasion,adhesion,and migration assay with subcutaneous tumor-bearing nude mice models.Then the growth curves of the subcutaneous tumors were studied.Expressions of VEGI,CD31 and CD34 in the tumors were analyzed by immunohistochemistry and apoptosis was detected by flow cytometry and immunohistochemistry.Results Infection of MDA-MB-231 cells within the lentivirus resulted in approximately a 1 000-fold increase in the expression of VEGI.As can be seen in the invasion,adhesion and migration assay,the over-expression of VEGI can inhibit the ability of MDA-MB-231 cells during migration,adhesion and invasion.The volume of the subcutaneous tumor in the over-expression group was distinctly and significantly less than that of the control groups.Immunohistochemistry analysis of the tumor biopsies cleady showed the expression of VEGI in the over-expression group increased while CD31 and CD34 decreased significantly.In vitro and in vivo,the early apoptosis rate and the apoptosis index were increased within the VEGI over-expression group as compared with the control group.Conclusions Taken together,recombinant lentivirus that were successfully constructed,demonstrated up-regulated VEGI gene expression in breast cancer cells.Lentivirus-mediated over-expression of VEGI weakened the ability of the breast cancer cell migration,adhesion and invasion.Over-expression of VEGI diminished the tumorigenic capacity of breast cancer cells in vivo.Up-regulation of VEGI gene expression however inhibited breast cancer MDA-MB-231 cell in the early apoptosis.     

Epigenetic regulation of putative tumor suppressor TGFBI in human leukemias

Background Both in vitro and in vivo data have demonstrated the TGFBI gene functions as a putative tumor suppressor and is frequently downregulated in human tumors of different histological types.The hypermethylation of the TGFBI promoter,as one of the main regulatory mechanisms,is associated with TGFBI silencing.In this study,we used a methylation-specific PCR （MSP） method to evaluate the methylation status of the TGFBI promoter in human leukemias.Methods Real-time RT-PCR and methylation-specific PCR approaches were performed to define the TGFBI expression and promoter methylation in human leukemia call lines and clinical samples.Genomic DNA was isolated from peripheral blood mononuclear cells from leukemia patients,bisulfite-converted,and analyzed by the MSP method.Results Hypermethylation of the TGFBI promoter occurred in leukemia cell lines and demethylation treatment reexpressed TGFBI at a substantially increased level in most of leukemia cell lines tested.Furthermore,a much higher level of CpG island methylation and a significantly lower TGFBI expression were also identified in clinical leukemia samples.Conclusion The results suggest an important role of promoter methylation in regulating TGFBI expression in leukemia,which provides a useful diagnostic marker for clinical management of human leukemias.     

基于GS筛选系统的动物细胞高效表达载体的构建及应用

目的：通过插入GS筛选系统和替换启动子改造载体以获得高效表达细胞系。方法将pIRES2-EGFP多克隆位点上的BamHⅠ位点突变,构建一个新的载体pIRES2-EGFP-B,通过AseⅠ和NheⅠ双酶切其上的巨细胞病毒（CMV）启动子,加入谷氨酰胺合成酶（ GS）基因筛选标志,再通过PacⅠ和NheⅠ双酶切,插入人巨细胞病毒（hCMV）启动子,构建载体pHGS1．0。将目的基因分别插入载体pHGS1．0和pIRES2-EGFP中,通过比较重组蛋白TEM8（1-227）-VEGFR1domain2-IgG2（TV-IgG2）表达来分析新构建载体的优越性。结果成功插入GS筛选标志,并加入hCMV启动子,人免疫球蛋白定量试剂盒分析发现,重组蛋白表达量提高了5倍。结论通过GS系统筛选,得到高效表达目的蛋白载体系统,为后续的高效表达细胞系筛选奠定了基础。     

微小RNA-21表达在卵巢癌细胞生长和凋亡过程中的作用

目的;探讨微小RNA-21( miR-21)在卵巢癌细胞生长、凋亡中的作用及调节机制。方法构建表达miR-21的重组干扰载体pSIREN-miR-21质粒,采用酶切鉴定和测序法证实。实验分为3组,即转染组：卵巢癌细胞株OVCAR3细胞转染重组干扰载体pSIREN-miR-21质粒;阴性对照组：OVCAR3细胞转染阴性对照pSIREN-miR-21-Neg质粒;空白对照组：OVCAR3细胞不转染质粒。采用茎环实时荧光定量逆转录(RT)-PCR技术检测3组细胞中miR-21的表达,蛋白印迹法检测3组细胞中程序性细胞死亡因子4( PDCD4)蛋白的表达,四甲基偶氮唑蓝(MTT)比色法及流式细胞仪检测3组细胞的增殖及凋亡情况。结果成功构建了表达miR-21的重组干扰载体pSIREN-niR-21质粒,并经酶切鉴定和测序法证实。转染组、阴性对照组和空白对照组OVCAR3细胞中niR-21的表达水平分别为0.26±0.08、1.26±0.21和1.00,转染组明显低于阴性对照组和空白对照组,差异有统计学意义(P＜0.01)。转染组、阴性对照组和空白对照组OVCAR3细胞中PDCD4蛋白的灰度值分别为1443 ±33、858±19和846±16,转染组明显高于阴性对照组和空白对照组,差异有统计学意义(P＜0.01)。转染组、阴性对照组和空白对照组OVCAP3细胞增殖的A值分别为0.661±0.015、0.848±0.150、0.935±0.133,转染组明显低于阴性对照组和空白对照组,差异有统计学意义(P＜0.01)。转染48 h后,转染组细胞的早期凋亡率明显高于阴性对照组和空白对照组[分别为(25.821±0.763)％、(0.010 ±0.003)％、(0.238±0.023)％;P ＜0.01];转染72 h后,转染组细胞的早期凋亡率和晚期凋亡率均明显高于阴性对照组和空白对照组[早期凋亡率分别为( 30.480±0.821)％、(7.792±0.312)％、(7.033±0.257)％,P＜0.01;晚期凋亡率分别为(3.558±0.211)％、(1.557±0.067)％、(1.049±0.028)％,P＜0.05]。结论miR-21参与调节卵巢癌细胞的生长和凋亡过程,可能通过调控PDCD4蛋白的表达而发挥作用。     

TrkA-siRNA增强人乳腺癌MCF-7细胞对紫杉醇的敏感性

目的 探讨靶向抑制TrkA基因表达后,人乳腺癌细胞MCF-7对化疗药物紫杉醇敏感性的变化.方法 8 μmol/L紫杉醇作用于乳腺癌MCF-7亲本细胞株和TrkA-siRNA转染细胞株24、48小时后,MTT法检测细胞增殖抑制效应,Western blot检测凋亡蛋白caspase-3的活化.倒置显微镜观察细胞株生长的形态学变化.结果 紫杉醇作用24、48小时后,其对TrkA-siRNA细胞株的生长抑制均高于MCF-7亲本细胞株(P＜0.05),且48小时抑制率高于24小时(P＜0.05).Western blot结果显示,caspase-3蛋白在紫杉醇作用24小时后被激活,其在TrkA-siRNA细胞株中的表达高于MCF-7亲本细胞株(P＜0.05).结论 TrkA-siRNA能增加乳腺癌细胞对化疗药物紫杉醇的敏感性.     

Inhibition of PI3K/Akt/mTOR signaling pathway enhances the sensitivity of the SKOV3/DDP ovarian cancer cell line to cisplatin in vitro

The activation of the PI3K/AKT/m TOR pathway plays a key role in ovarian cancer tumorigenesis, progression and chemotherapy resistance. This study aimed to explore the possible mechanism that PI-103, a dual inhibitor of phosphatidylinositide 3-kinase and m TOR, enhances the sensitivity of SKOV3/DDP ovarian cancer cell line to cisplatin chemotherapy. The results showed that PI-103 could significantly increase the sensitivity of SKVO3/DDP cells to cisplatin through inhibiting the activation of PI3K/Akt/m TOR signaling pathway and inducing cell cycle arrest and apoptosis.     

缺失线粒体DNA的人喉癌细胞系的建立

目的为了研究线粒体DNA在人喉癌细胞的增殖、分化及凋亡中的影响及作用,培养线粒体DNA缺失的喉癌细胞系,为构建融合喉癌细胞,研究线粒体DNA突变和线粒体功能改变与喉癌发生之间的关系奠定基础.方法人喉癌细胞系JHUo11加入10%FBS的RPMI1640培养基中,然后在培养基中加入丙酮酸、尿苷、溴化乙锭(EtBr)以去除线粒体DNA,不加溴化乙锭的o11细胞作为对照.经过90天培养,线粒体DNA缺失的喉癌细胞通过健那绿染色后光镜观察和PCR法鉴定.结果 o11细胞经过75ng/μl溴化乙锭持续作用90天后可见细胞肿胀变圆,透光度增加,线粒体DNAPCR扩增未见目的条带,细胞可扩增出定位于mtDNA的500bp片段,健那绿染色对照细胞,可见散在蓝绿色线粒体,而加EB的细胞未见线粒体.结论喉癌细胞o11经过溴化乙锭持续作用90天培养出线粒体DNA缺失的ρ0o11喉癌细胞,这种缺失线粒体DNA的细胞系需补充外源性丙酮酸和尿苷来维持其生存,否则细胞很快死亡.通过观察细胞的增殖和分化发现,ρ0o11细胞生长速度比未经EB处理的对照组慢的多.     

Mechanism underlying drug resistance of HepG2/ADM cells silently reversed by lentivirus-mediated BC047440 gene during chemotherapy

目的 利用siRNA方法沉默HepG2/ADM细胞系中BC047440基因,观察其受抑制后对细胞多药耐药性的影响及其分子机制.方法 建立BC047440蛋白在HepG2/ADM细胞系siRNA干扰模型,采用Western blot检测BC047440蛋白的表达;实验分3组:BC047440-shRNA组、control-shRNA组和HepG2/ADM组.CCK-8法分析阿霉素对细胞生长抑制率的影响,流式细胞仪检测细胞凋亡及细胞周期分布;Western blot检测BC047440沉默后NF-κB蛋白表达的变化;Realtime PCR检测Survivin、CCNL1基因mRNA水平的变化.结果 成功建立BC047440蛋白siRNA干扰模型;NF-κB蛋白表达BC047440-shRNA组约为HepG2/ADM组67.69％,约为control-shRNA组67.39％;细胞毒性实验中24、48 h阿霉素对BC047440-shRNA组细胞毒性作用明显增强;流式细胞仪检测结果显示BC047440-shRNA组细胞凋亡明显增多,细胞周期分布多停留于G1/G0期,S期细胞明显减少.Real-time PCR检测BC047440-shRNA组Survivin基因mRNA表达约为control-shRNA组37％,约为HepG2/ADM组42％;检测BC047440-shRNA组CCNL1基因mRNA表达约为control-shRNA组3.5倍,约为HepG2/ADM组2.4倍.结论 沉默BC047440基因可通过NF-κB信号通路及其下游Survivin、CCNL1信号逆转HepG2/ADM细胞的多药耐药性,提示BC047440基因可能是形成肝癌多药耐药的重要分子之一.     

柿叶黄酮对人肝癌SMMC-7721细胞形态学影响的初步研究

目的 观察柿叶黄酮(persimmon leaf flavonid,PLF)对人肝癌SMMC-7721细胞形态学的影响作用,初步探讨柿叶黄酮对人肝癌细胞的抑制机理.方法 采用浓度0.32mg/ml、0.64mg/ml的柿叶黄酮相同时间处理人肝癌SMMC-7721细胞;采用倒置显微镜观察SMMC-7721细胞的形态学变化及生长情况.结果 倒置显微镜下可见,正常组细胞贴壁生长,形态较规则,生长迅速,胞浆丰富.柿叶黄酮处理组的贴壁细胞的数量减少,体积变小,部分细胞崩解,而悬浮细胞量增多.结论 柿叶黄酮对人肝癌SMMC-7721细胞的形态有一定的诱变作用及一定的抑制增殖作用.     

YKL-40在肿瘤中的研究进展

YKL-40又名人类软骨糖蛋白-39(HCgp-39),一项对新生骨蛋白的研究中发现在人骨肉瘤细胞系MG63的体外培养中发现一种蛋白的大量分泌,因为其一条肽链氨基端的起始3个氨基酸:酪氨酸、赖氨酸和亮氨酸的符号分别为Y、K和L,其相对分子质量约为40 000而被称为YKL-40.1993年,报道公布了人类软骨糖蛋白的完整氨基酸序列及cDNA序列,YKL-40含383个氨基酸,编码的蛋白序列与糖基水解酶相似,基于其氨基酸序列,发现YKL-40属于哺乳动物18糖基水解酶家族的成员.YKL-40结合不同长度的壳多糖的形式类似壳多糖酶18家族,但没有壳质酶活性.