腺病毒介导IL-24基因诱导人脑胶质瘤U87MG细胞的凋亡

目的：研究腺病毒介导IL-24基因表达载体(Ad-IL-24)对人脑胶质瘤U87MG细胞的抑制作用,初步探讨其作用机制。方法：将本科室构建的Ad-IL-24感染U87MG细胞,RT-PCR法检测IL-24基因的表达,MTT法和流式细胞术检测U87MG细胞的生长和凋亡,激光共聚焦显微镜观察U87MG细胞凋亡的形态学变化,RT-PCR法检测Bax、Bcl-2基因mRNA的表达,Western blotting检测caspase-3的活化。结果：Ad-IL-24感染U87MG细胞后,IL-24在U87MG细胞中有明显表达,并抑制U87MG细胞生长、诱导其凋亡、出现典型的凋亡细胞核形态学改变。Ad-IL-24可上调U87MG细胞中Bax基因、下调Bcl-2基因的表达,并诱导caspase-3蛋白的活化。结论：Ad-IL-24可诱导U87MG细胞凋亡,其机制可能与上调Bax基因、下调Bcl-2基因表达,并活化caspase-3有关。     

Leptin促进人脑胶质瘤U87MG细胞的迁移和侵袭

目的:探讨瘦素(leptin)对人脑胶质瘤U87MG细胞迁移及侵袭能力的影响及其机制。方法:Leptin处理U87MG细胞,采用细胞划痕实验检测U87MG细胞的迁移能力,Transwell实验检测U87MG细胞的侵袭能力,RT-PCR及Western blot-ting法检测U87MG细胞中MMP-2及MMP-9 mRNA和蛋白的表达。结果:Leptin明显促进U87MG细胞迁移能力[(152.42±3.29)vs(83.24±2.61)μm,P<0.05]和侵袭能力[(31.78±5.04)vs(17.03±2.41)个细胞,P<0.05],leptin能显著上调U87MG细胞中MMP-2、MMP-9 mRNA[(0.76±0.04)vs(0.35±0.02),(0.84±0.02)vs(0.41±0.06);均P<0.05]及蛋白[(0.79±0.03)vs(0.23±0.01),(0.81±0.05)vs(0.39±0.03);均P<0.05]的表达。MMP抑制剂GM6001(10μmol/ml)可以逆转leptin对U87MG细胞迁移[(82.05±2.98)vs(81.76±3.25)μm,P>0.05]和侵袭能力[(19.23±2.46)vs(18.02±1.98)个细胞,P>0.05]的影响。结论:Leptin可以促进人脑胶质瘤U87MG细胞的侵袭及迁移,其机制可能与上调MMP-2、MMP-9的表达有关。     

积雪草酸通过抑制耐药相关蛋白表达增强U87MG胶质瘤细胞对紫杉醇 的敏感性

目的： 探索积雪草酸（asiatic acid,AA）对紫杉醇（paclitaxel, PTX）耐药性胶质瘤细胞的抑制作用及其可能的作用机 制。 方法： CCK-8实验、实时荧光定量PCR、Western blotting检测AA对成胶质细胞瘤U87MG细胞的增殖、凋亡的影响。浓度递 增法构建PTX耐药性细胞株PR-U87MG,以U87MG细胞为对照,CCK-8实验验证PR-U87MG细胞对PTX的耐药性,实时荧光定 量PCR、Western blotting检测PR-U87MG细胞中MDR1、LRP mRNA及蛋白的表达水平。AA和PTX单独或联合处理PR-U87MG 细胞,CCK-8实验、实时荧光定量PCR、Western blotting检测各组细胞增殖活力及凋亡的变化。 结果： 成功构建PTX耐药性细胞 株PR-U87MG。AA可以剂量依赖方式抑制U87MG细胞和PR-U87MG细胞的增殖活力（P<0.01）,并明显促进其凋亡（P<0.01）。 与AA或PTX单独处理组相比,联合处理组中PARP1的蛋白水平显著减少（P<0.01）,caspase 3的裂解量显著增加（P<0.01）,耐药 相关蛋白P-糖蛋白1 （P-dycoprotein 1, Pgp-1）和LRP表达水平显著减少（P<0.01）。 结论： AA可有效增强U87MG胶质瘤细胞株 对PTX的敏感性,其机制可能与AA抑制具有药物排出功能的耐药蛋白Pgp-1和LRP表达有关。     

溶瘤新城疫病毒抑制IL-6诱导的人胶质母细胞瘤U87MG细胞的迁移和侵袭

目的：探讨溶瘤新城疫病毒（NDV）对IL-6诱导的人胶质母细胞瘤U87MG细胞增殖、迁移和侵袭的作用及其可能的机制。方法：将U87MG细胞分为对照组、IL-6组、NDV组、NDV+IL-6组,其中IL-6组与NDV+IL-6组用75 ng/mL IL-6预处理1 h,其余组用DMEM预处理1 h,后分别用DMEM、75 ng/mL IL-6、1 HU NDV、1 HU NDV+75 ng/mL IL-6处理24 h。MTT法、细胞划痕实验和Transwell侵袭实验分别检测IL-6、NDV对U87MG细胞增殖、迁移和侵袭的影响,WB法检测各组细胞JAK2、p-JAK2、STAT3、p-STAT3和MMP2蛋白的表达水平。结果：与对照组相比,IL-6组细胞迁移率显著升高（P<0.05）,侵袭细胞数目显著增多（P<0.01）;与IL-6组相比,NDV+IL-6组U87MG细胞增殖率显著降低（P<0.05）,细胞迁移率和侵袭细胞数目均显著降低（均P<0.01）。WB实验结果显示,与对照组相比,IL-6组p-STAT3/STAT3比值显著升高（P<0.01）,ND...     

The L84F polymorphic variant of human O6-methylguanine-DNA methyltransferase alters stability in U87MG glioma cells but not temozolomide sensitivity

First-line therapy for patients with glioblastoma multiforme includes treatment with radiation and temozolomide (TMZ), an oral DNA alkylating chemotherapy. Sensitivity of glioma cells to TMZ is dependent on the level of cellular O(6)-methylguanine-DNA methyltransferase (MGMT) repair activity. Several common coding-region polymorphisms in the MGMT gene (L84F and the linked pair I143V/K178R) modify functional characteristics of MGMT and cancer risk. To determine whether these polymorphic changes influence the ability of MGMT to protect glioma cells from TMZ, we stably overexpressed enhanced green fluorescent protein (eGFP)-tagged MGMT constructs in U87MG glioma cells. We confirmed that the wild-type (WT) eGFP-MGMT protein is properly localized within the nucleus and found that L84F, I143V/K178R, and L84F/I143V/K178R eGFP-MGMT variants exhibited nuclear localization patterns indistinguishable from WT. Using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] proliferation and clonogenic survival assays, we confirmed that WT cells expressing eGFP-MGMT are resistant to TMZ treatment compared with control U87MG cells, and that each of the polymorphic eGFP-MGMT variants confers similar resistance to TMZ. However, upon exposure to O(6)-benzylguanine (O(6)-BG), a synthetic MGMT inhibitor, the L84F and L84F/I143V/K178R variants were degraded more rapidly than WT or I143V/K178R in a proteasome-dependent manner. Despite the increased O(6)-BG- stimulated protein turnover caused by the L84F alteration, cells expressing L84F eGFP-MGMT did not exhibit altered sensitivity to the combination of O(6)-BG and TMZ compared with WT cells. In conclusion, we demonstrated that the L84F polymorphic variant has altered protein turnover without modifying sensitivity of U87MG cells to TMZ or combined TMZ and O(6)-BG. These findings may provide a clue to determining the clinical significance of MGMT coding-region polymorphisms.     

神经生长因子（NGF）对H2O2诱导胶质瘤A—172细胞凋亡的抑制效应及其机制

目的：研究神经生长因子（ＮＧＦ）对氧化氢（Ｈ２Ｏ２）诱导神经胶质瘤细胞凋亡的抑制作用及其机制。方法：抑制细胞增殖的检测采用ＭＴＴ法,吖啶橙（ＡＯ）染色荧光显微观察细胞的形态学变化,ＤＮＡ琼脂糖电泳检测ＤＮＡ断裂,二硫硝基苯甲酸（ＤＴＮＢ）法检测细胞内不原型谷胱甘肽（ＧＳＨ）水平的变化。结果：１００～８００μｍｏｌ／Ｌ的Ｈ２Ｏ２明显抑制Ａ－１７２细胞增殖,２００μｍｏｌ／Ｌ Ｈ２Ｏ２作用１２小时后形     

人脑髓鞘碱性蛋白对H2O2诱导人肺癌细胞YTLMC-90凋亡的抑制作用

背景与目的：人脑髓鞘碱性蛋白（myelin basic protein,MBP）广泛分布于神经系统及多种非神经组织之中,而且在肺癌、乳腺癌、神经胶质瘤等多种肿瘤细胞中均检查到MBP的表达。但MBP与癌细胞侵犯神经组织的活性是否相关、与肺癌细胞的生物学行为是否相关尚未见报道。本研究主要探讨MBP对过氧化氢（H2O2）诱导人肺癌细胞YTLMC-90凋亡的影响。方法：实验分为MBP cDNA微基因pSVCEPMBPCAT转染组（试验组）、空质粒pSVCEPCAT转染组和未转染组（对照组）。将含人1型胶原al链（COLⅠA1）基因启动子和增强子元件、并在其3’端接MBP cDNA的微基因,转染YTLMC-90细胞,并驱动MBP异位表达：采用MTT法检测细胞增殖,吖啶橙荧光染色显微镜和电镜观察细胞形态及其超微结构的改变;琼脂糖凝胶电泳检测染色质DNA断裂。结果：200μmol／L H2O2作用24h后、转染组、未转染组和空载体pSVCEPCAT转染组YTLMC-90细胞的生长抑制率分别为36．67％、84．00％和78．67％（P〈0．001）;对照组YTLMC-90细胞普遍可见凋产细胞特有的形态学及生物化学改变,如胞核固缩、染色质断裂,DNA电泳呈梯状条带;而MBP cDNA微基因转染的YTLMC-90细胞未发现上述凋亡特征。结论：MBP促进YTLMC-90细胞增殖和拮抗H2O2诱导的凋亡,明显减少H2O2对YTLMC-90的细胞毒作用。     

H2O2 scavenging inhibits G1/S transition by increasing nuclear levels of p27KIP1

The aim of the present study was to evaluate cell cycle regulation by scavenging H(2)O(2) in tumor cells. A significant arrest in the G1 phase of the cell cycle was demonstrated in CH72-T4 carcinoma cells exposed to catalase, associated with a decrease in cyclin D1 and an increase in the CDK inhibitory protein p27(KIP1). Moreover, we found a differential intracellular distribution of p27(KIP1), which remained in the nucleus after catalase treatment. In vivo experiments showed an increase in nuclear levels of p27(KIP1) associated with the inhibition of tumor growth by H(2)O(2) scavenging, confirming in vitro results. To conclude, H(2)O(2) scavenging may induce cell cycle arrest through the modulation of cyclin D1 and p27(KIP1) levels and nuclear localization of p27(KIP1). To our knowledge, this is the first report that demonstrates that the modulation of ROS alters the intracellular localization of a key regulatory protein of G1/S transition.