反义miR-30a-5p抑制人脑胶质瘤细胞U87生长的体外实验研究

目的 前期发现miR-30a-5p在胶质瘤中表达明显上调,本实验探究敲低miR-30a-5p对人脑胶质瘤细胞U87细胞生物学特征的影响.方法 real-time PCR检测转染miR-30a-5p I(miR-30a-5p抑制物)后U87细胞的miR-30a-5p表达水平,流式细胞术、MTT、Transwell、Annexin V法检测细胞周期、生长、侵袭及凋亡的改变;Western blot检测相关蛋白.结果 real-time PCR结果显示miR-30a-5p I可有效降低U87细胞中miR-30a-5p表达,抑制细胞增殖活性,使细胞周期阻滞在G0/G1期,侵袭能力明显受抑,凋亡增加,促增殖蛋白PCNA、促细胞周期进展蛋白Cyclin D1及促侵袭蛋白MMP-9的表达明显下调,而抑制侵袭蛋白TIMP-1及凋亡相关蛋白p53表达明显上调.结论 敲低U87细胞的miR-30a-5p表达可抑制胶质瘤的增殖及侵袭,诱导凋亡;miR-30a-5p可成为人脑胶质瘤基因治疗的潜在候选靶点.

Abstract：

Objective Our previous study had shown that there was overexpression of miR- 30a- 5p in malignant glioma cell lines.In the present study, we aim to investigate the effect of knocking -down miR -30a -5p on the biological characteristics of U87 glioblastoma cells.Method The U87 cells were divided into three groups:control cells,cells transfected with scramble oligonucleotides and cells transfected with miR -30a -5p inhibitors(miR-30a-5p I).Oligonucleotides mediated by lipofectamine were transfected to U87 cells.Real -time PCR was conducted to detect the expression of miR- 30a -5p in transfected cells.The cell proliferation was determined by 3- (4,5- Dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide(MTT) assay and flow cytometry.The cell invasion was evaluated by Transwell assay and cell apoptosis was detected with Annexin V staining Moreover, the relevant molecules regulating proliferation, invasion, cell cycle progression and apoptosis were examined by Western blot analysis.Results The expression of miR - 30a - 5p in the cells transfected with miR - 30a - 5p I was significantly reduced.The cell proliferation activity of U87 cell was inhibited.The cell cycle was arrested in G0/G1 phase, cell invasive ability was attenuated and apoptotic cells were increased in cells transfected with miR -30a -5pI as compared to those of the cells transfected with scrambled siRNA and control cells.The expression of proliferating cell nuclear antigen (PCNA), matrix metallopeptidase 9 ( MMP - 9) and Cyclin D1 were downregulated while the tissue inhibitor of metalloproteinase1 (TIMP - 1 ) and p53 were upregulated.Conclusions Transfection of miR - 30a-5p I into glioma cells could inhibit the proliferation activity and invasive ability of U87 cell and induce cell apoptosis, miR -30a -5p is a potential target of gene therapy for glioma.     

RNA干扰敲低Dicer对胶质瘤细胞恶性表型的影响

目的 观察应用RNA干扰(RNAi)技术敲低Dicer酶后,在体外对U251人胶质瘤细胞生物学特征的影响.方法 构建针对Dicer基因的小分子干扰RNA重组腺病毒表达载体(rAd-Dicer)转染至U251细胞.应用RT-PCR检测Dicer mRNA水平的变化,应用免疫荧光和Western blot方法检测Dicer基因在蛋白水平的变化,并对肿瘤细胞中相关功能蛋白的表达进行分析比较.应用MTT法、流式细胞术和Transwell方法评价肿瘤细胞转染前后生物学行为的变化.结果 rAd-Dicer可显著抑制U251细胞Dicer基因的表达;与正常对照组(control)和阴性对照组(rAd-HK)比较,Western blot分析结果显示p-AKT,MMP2/9,PCNA,Cyclin a,VEGF,CD34功能蛋白在rAd-Dicer转染组细胞中表达明显上调;细胞周期结果表明rAd-Dicer转染组进入S期的细胞数增加了 14.1%～15.3%,MTT和Transwell实验结果显示rAd-Dicer转染组细胞增殖速率和体外侵袭能力显著增强.结论 敲低Dicer酶表达后,U251细胞表型具有更为恶性转化的倾向,由此初步推测全面抑制细胞microRNAs表达有可能促进肿瘤生成.

Abstract：

Objective To investigate the effect of knocking down Dicer by RNAi on the biological characteristics of human glioma cells U251 in vitro.Methods The recombinant adenovirus expression vector which contained short hairpin RNA targeting Dicer (rAd-Dicer) was transfected into U251 cells.The silencing effect of RNAi on Dicer expression was identified by RT- PCR,immumofluorecence staining and Western blot.Western blot was also undertaken to analyze the expression of some functional proteins.The biological behavior changes of U251 cells trasfected by rAd- Dicer were evaluated by MTT,FCM and transwell assay.Methods rAd-Dicer dramatically down- regulated the expression of Dier in U251 cells.As compared with parental and rAd- HK transfected cells,the protein expression of p- AKT,MMP2/9,PCNA,Cyclin a,VEGF,CD34 were up- regulated in rAd-Dicer treated cells.Decreased dicer expression in rAd-Dicer transfected cells was accompanied by 14.1% to 15.3% increase in U251 cells in S phase.Meanwhile,the proliferation and invasive activity were significantly enhanced in rAd-Dicer transfected cells by the MTT and transwell assay.Conclusion Knockdown of dicer renders the U251 cells more malignant transformation.This preliminary finding suggests that global lowering expression of microRNAs may have an oncogenic role.     

敲低Yes相关蛋白表达对人脑胶质瘤细胞SNB19生长的抑制作用

目的：探究敲低Yes相关蛋白（YAP）对人脑胶质瘤细胞SNB19细胞生物学特征的影响。方法采用YAP干扰RNA（YAPsiRNA）敲低SNB19细胞中YAP的表达,Western blot法鉴定肿瘤细胞中YAP是否已被敲低;噻唑蓝（MTT）比色法测定YAP敲低后胶质瘤细胞的增殖能力;Transwell实验检测胶质瘤细胞侵袭能力的变化;流式细胞术和AnnexinⅤ标记法分别检测胶质瘤细胞周期及凋亡的变化。应用统计软件SPSS18．0进行统计学处理。结果 Western blot证实转染 YAPsiRNA 可有效敲低SNB19细胞中Yes相关蛋白的表达;敲低Yes相关蛋白的表达可以抑制胶质瘤细胞增殖活性,细胞存活率由（100．00±0．00）％下降为（52．32±3．10）％（F＝33．00,P＜0．01）,细胞周期阻滞于G0-G1期（F＝8．76,P＜0．01）,细胞侵袭能力明显受抑,穿膜细胞数由（163．20±10．10）个下降至（37．71±2．52）个（F＝282．05,P＜0．01）,凋亡增加,凋亡率由（3．56±0．35）％增加至（18．99±0．66）％（F＝931．99,P＜0．01）。结论敲低胶质瘤细胞SNB19中YAP表达可抑制肿瘤细胞增殖和侵袭,诱导细胞凋亡;YAP可成为人脑胶质瘤基因治疗的潜在靶点。     

miRNA海绵在胃癌细胞EMT过程中调控作用的体外研究

  目的   探讨“miRNA海绵”在胃癌细胞系SGC7901上皮-间质转化（EMT）过程中的作用及机制。   方法   将人工合成的ZEB2 3'UTR质粒及siZEB2采用脂质体Lipofectamine 2000转染SGC7901细胞。qRT-PCR检测转染后miR-200a/b/c的表达;Transwell侵袭实验、划痕实验和克隆形成实验检测细胞侵袭迁移增殖能力;Western Blot检测目的蛋白的表达。   结果   与对照组相比,ZEB2 3'UTR转染组miR-200a/b/c的表达均下调,迁移、侵袭、增殖活性均增强;而转染siZEB2后miR-200a/b/c表达均升高,迁移、侵袭、增殖能力则下降。Western Blot显示ZEB2 3'UTR转染导致ZEB2表达升高,E-cadherin表达下降,而Vimentin表达上调;而转染siZEB2后,各项指标则表现出相反的表达趋势。   结论   ZEB2 3'UTR可以通过调控miR-200a/b/c的表达调控胃癌细胞EMT过程,调节肿瘤的侵袭与转移。      

肝开窍于目刍议

一、肝开窍于目之生理目之能视,籍肝阴肝血之营养。如:《素问五脏生成篇》日:“肝受血而能视”。张介宾则云:“肝得血则神聚于目,故能视”。是目赖阴血之养,方能视万物。肝知阴血虚少,则目视障碍。须之阴血之能上承于目,必以肝之气阳为动力,肝脉为运行道路。目之主宰,在肝阳肝气。人身脏腑皆寓阴阳气血,肝亦同例。肝舍一阳生化之气,主升发,达于目,使目睛转动灵活;运阴血,目得所养,视物精明。陈达夫教授在《中医眼科六经法要》曰:“肝气上升,则目中即有主宰……”。在分析“能远视而怯近视,或能近视而怯远视”之病机时,陈达夫教授指出:二者病变都以悬     

浅谈中风病的合理调护

<正> 患中风病之后,若能予以合理调护,不但能补助正气,而且又可药以祛邪,因而是扶正护虚的重要一环。1.调理情志,舒畅气机:中风发病后,在药物治疗的同时,调理情志与本病预后密切相关。调情志稳定患者情绪,可促使气机疏达,血脉流畅,以利于疾病向愈,且可预防中风之复发。     

代谢综合征痰证研究进展

将代谢综合征(MS)和中医理论有机结合已成为关注的焦点,“痰”与MS的发生发展尤显密切相关,并且从中医痰证论治MS有其优势所在。主要通过对MS病因病机研究、MS各组成部分与痰的关系,以及MS从痰论治的近代研究,论述了治痰在MS防治中的重要地位。参考文献22篇。     

从《内经》心身理论谈疼痛的发生机制

疼痛是一种不愉快的感觉和情绪上的感受，是临床最常见的自觉症状之一。基于《内经》对疼痛的论述，后世医家以“不通则痛”与“不荣则痛”分别概括疼痛病机的虚实两端。但临床上常见到一些疼痛患者，疼痛部位不定，临床检查并无实质性损害，频繁出入医院而不见缓解，     

教育部重点学科——中医基础理论学科

山东中医药大学中医基础理论学科，始建于1958年，1978年获硕士学位授予权，1986年获博士学位授予权，为全国较早的中医基础理论博士点。1991年被列为山东省“八五”省级重点学科建设点，1996年由山东省教委组织专家验收，成绩优秀，确定为“山东省重点学科”；2001年被列为国家中医药管理局中医药重点学科建设单位；2001年入选山东省“十五”强化建设A级学科；2002年批准为教育部重点学科。2006年中华中医药学会中医基础理论分会挂靠本学科。本学科奠基人为全国著名中医基础理论专家、博士研究生导师张珍玉教授。学科学术带头人为迟华基教授，学科带头人为乔明琦教授。     

试析肝藏对血液循行和代谢的影响

试析肝藏对血液循行和代谢的影响山东中医药大学（２５００１４）张安玲日照市卫生局（２７６８００）赵为爱关键词肝藏，藏血，生血，泄浊中医学认为，肝对血液的影响主要是贮藏和调节血量及主疏泄推动血行，然细析之，发现肝对血液的循行和代谢均有重要作用，试分析如下...