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ObjectiveTo study the inhibitory effect of knocking down miR-30a-5p on the U87 human glioma xenograft growth and its possible mechanism. MethodsNude mice bearing subcutaneous U87 human glioblastoma were established and treated with miR-30a-5p antisense oligonucleotides (AS-miR-30a-5p) subcutaneous injection. Tumor size was measured every other day until the observation period ended. Researchers executed the animals after the treatment, stripped tumor tissues and extracted RNA and protein. Real-time PCR were conducted to detect the expression of miR-30a-5p. The histopathological characteristics and proliferation and apoptosis biological characters (including SEPT7, PCNA, cyclinD1, MMP-2, apoptosis related factor P53, bcl-2 and caspase3)were evaluated by HE and immunohistochemical staining, Western blot analysis respectively, and the cell apoptosis was detected by TUNEL method.ResultsIn AS-miR-30a-5p treated group, the tumor growth was delayed and the final tumor volume was smaller than that in the control and scr-ODN treated group (F=7.167, P<0.05), and the expression of miR-30a-5p was knocked down. The expression of PCNA、cyclinD1 were significantly down-regulated while P53、SEPT7 and caspase3 up-regulated. Apoptotic index was increased significantly. ConclusionAs-miR-30a-5p suppresses the growth of U87 human gliomas xenografts significantly. Malignant phenotype of tumors are reversed to a considerable degree. Therefore, miR-30a-5p can be a candidate for targeted therapy of human glioma. 相似文献
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目的应用RNA干扰(RNA interference,RNAi)技术敲低恶性胶质瘤细胞系U251中AKT1和COX-2表达后,在体外对细胞侵袭转移的抑制作用,初步探讨其可能的作用机制。方法构建重组腺病毒载体rAd5-A-C同时搭载靶向AKT1和COX-2的shRNA干扰序列,将其转染至恶性胶质瘤细胞系U251细胞。Realtime PCR检测RNAi后目的基因mRNA的表达水平,Western Blot检测目的基因及MMP-2、MMP-9、TIMP-2的表达情况。酶联免疫吸附试验检测转染前后分泌到细胞外的MMP-2和MMP-9的浓度变化。划痕实验和Transwell实验评价肿瘤细胞转染前后的细胞侵袭能力的变化。结果rAd5-A-C转染组AKT1和COX-2的mRNA和蛋白表达均明显抑制;MMP-2、MMP-9表达下调,TIMP-2表达则上调。ELISA检测胞外MMP-2、MMP-9浓度明显减低;划痕实验显示细胞转移运动能力明显减弱,Transwell体外侵袭实验结果显示穿过细胞数明显减低(P0.001)。结论重组腺病毒介导的RNAi技术可以序列特异性地抑制U251细胞AKT1和COX-2的表达,在体外对U251细胞侵袭转移产生明显抑制作用,可能成为恶性胶质瘤治疗的新策略。 相似文献
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目的 探究敲低microRNA(miR)-19a和miR-19b表达对人脑胶质瘤细胞系SNB19细胞生物学特征的影响.方法 常规培养SNB19细胞,脂质体介导miR-19a、miR-19b抑制物转染SNB19细胞,同时设未转染组(对照组)、无义序列转染组和miR-19a+miR-19b抑制物转染组.应用RT-PCR检测转染后细胞miR-19a、miR-19b的表达,MTT、流式细胞术分别检测转染后细胞增殖能力和细胞周期的变化,Transwell实验检测转染后细胞的侵袭能力.结果 与对照组和无义序列转染组比较,抑制物转染组细胞miR-19a和miR-19b的表达、增殖和侵袭能力均降低,差异有统计学意义(P<0.05),而且miR-19a+miR-19b抑制物转染组细胞miR-19a和miR-19b的表达、转染后2、3、4、5 d细胞的增殖能力、侵袭能力均低于miR-19a、miR-19b抑制物转染组,差异有统计学意义(P<0.05);抑制物转染组较对照组和无义序列转染组细胞周期延迟,且miR-19a+miR-19b抑制物转染组较miR-19a、miR-19b抑制物单独转染组细胞延迟更为明显.结论 miR-19a和miR-19b可能是癌微RNA,有望作为人脑胶质瘤基因治疗的侯选靶点.Abstract: objective To investigate the effects of knocking down of miR-19a and miR-19b on the biological characteristics of SNB19 glioblastoma cells. Methods Oligonucleotides inhibitor of miR-19a and miR-19b (miR-19a inhibitor or miR-19b inhibitor) mediated by lipofectamine2000 were transfected to SNB19 cells to knock down miR-19a and miR-19b; control group (without transfection),group D (performing transfection with nonsense sequence) and group E (performing transfection with both miR-19a inhibitor and miR-19b inhibitor) were established. Real time PCR was conducted to detect the expressions ofmiR-19a and miR-19b in these groups after the transfection. The cell proliferation rate and cell cycle kinetics were detected by 3-(4, 5-Dime- -thylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively; the cell invasive ability was evaluated by Transwell assay.Results As compared with those in control group and group D, the expressions of miR-19a and miR-19b, proliferation activity and invasive ability of cells in the miR-19a/19b inhibitor transfected cells (group A/B) were significantly reduced (P<0.05). The expressions of miR-19a and miR-19b and the proliferation activity and invasive ability of cells 2, 3, 4 and 5 d after the transfection in group E were significantly reduced as compared with those in group A/B (P<0.05). Delayed cell cycle in group A/B and group E was noted as compared with that in control group and group D; and group E enjoyed more obviously delayed eell cycle than group A/B (P<0.05). Conclusion MiR-19a and miR-19b might be oncomiRs, and may be candidate target miRNAs for gene therapy of glioma. 相似文献
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目的 研究重组腺病毒Ad—Ku70shRNA对C6胶质瘤细胞系Ku70蛋白表达的影响,探讨放射治疗过程中基因增敏的新途径。方法以穿梭质粒为基础,构建重组腺病毒Ad—Ku70shRNA载体,扩增并鉴定其病毒滴度。然后转染至C6鼠脑胶质瘤细胞中,应用WestemBlot及免疫荧光观察其Ku70蛋白的表达情况,研究重组腺病毒Ad—Ku70shRNA对C6细胞Ku70表达的抑制效果。结果成功构建并扩增Ad—Ku70shRNA重组腺病毒,感染C6胶质瘤细胞后Ku70的表达明显降低。结论Ad—Ku70shRNA重组腺病毒载体可以明显降低C6胶质瘤细胞Ku70的表达。该结果为放射治疗基因增敏研究提供了有利的工具。 相似文献
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RNAi下调PIK3 CB表达抑制U251胶质瘤细胞生长的体内外研究 总被引:2,自引:2,他引:0
目的 探讨应用RNAi技术靶向磷酸肌醇酯-3-激酶β催化亚单位(PIK3CB)抑制恶性胶质瘤细胞系U251的PIK3CB表达后在体内外对U251细胞生长抑制作用.方法 将短发夹RNA(shRNA)表达载体psiRNA-PIK3CB进行脂质体介导的U251人脑恶性胶质瘤细胞系表达,检测细胞转染前后的细胞增殖能力和凋亡的变化.应用裸鼠皮下荷瘤模型观察脂质体介导shRNA基因治疗对U251细胞生长抑制作用,对肿瘤组织应用免疫荧光双染色和免疫组化的方法分析结果.结果 靶向PIK3CB的shRNA转染后U251细胞生长受到抑制,细胞周期出现G2/M阻滞,细胞明显凋亡.裸鼠皮下荷瘤模型实验显示psiRNA-PIK3CB显著抑制皮下肿瘤生长(P<0.01).结论 靶向PIK3CB的shRNA基因治疗可以成为胶质瘤治疗的新策略. 相似文献
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反义miR-21抑制异种移植U251人脑胶质瘤生长的体内研究 总被引:2,自引:0,他引:2
目的 探讨敲低miR-21表达抑制裸鼠荷皮下U251人腩胶质瘤生长的疗效和机制. 方法 原位注射miR-21反义寡聚核苷酸(AS.miR-21)治疗裸鼠皮下荷U251人脑胶质瘤.定时测量肿瘤大小评估原位注射AS-miR-21治疗效果:使用Real time PCR和原位杂交方法鉴定治疗后miR-21表达水平下调;采用HE染色和免疫组织化学染色(PCNA、PTEN、MMPs、和Septins)评价治疗后肿瘤牛物学性状改变;TUNEL法检测肿瘤细胞凋亡. 结果 肿瘤生长曲线显示AS-miR-21治疗组肿瘤生长速度及体积小于空白对照与无义序列治疗组(F=52.458,P=0.000);Real-time PCR法:AS-miR-21治疗组miR-21表达下调为对照组的0.018±0.009:原位杂交显示AS-miR-21治疗组miR-21表达水平较对照与无义序列治疗组下调:组织病理学检测表明AS-miR-21治疗后肿瘤恶性度降低;TUNEL法检测可见AS-miR-21治疗组细胞凋亡数显著高于对照与无义序列治疗组(F=141.021,P=0.000).结论 以miR-21作为靶点抑制异种移植U251人脑胶质瘤生长,miR-21可能通过上调抑癌基因PTEN抑制胶质瘤的生长,可以作为人脑胶质瘤基因治疗的侯选靶点. 相似文献
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神经上皮组织起源的脑肿瘤中连接蛋白的表达 总被引:7,自引:0,他引:7
目的 探讨连接蛋白 (Cx) 43、3 2在神经上皮组织起源的脑肿瘤中表达及其与肿瘤的病理类型、恶性程度的关系。方法 利用Cx43、Cx3 2和Ki 67单抗或多抗对 8例正常脑组织、5 0例神经上皮组织起源的脑肿瘤和 4个恶性脑瘤体外细胞系进行免疫组化检测。结果 神经上皮组织起源的脑肿瘤中Cx表达阳性率为 5 4% ,Cx3 2仅在低恶度少枝胶质细胞瘤中表达 ,Cx43表达随WHO分级升高而下降 ;与Ki 67标记指数 (LI)呈负相关。表达缺失多见于高Ki 67LI者。结论 神经上皮组织起源的脑肿瘤中CX 表达可下降或缺失 ,Cx43表达与肿瘤的病理类型及恶性程度密切相关 相似文献
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目的 构建人隔蛋白7(SEPT7,hCDC10/SEPT7)的表达载体并对U251人脑恶性胶质瘤细胞系进行转染且检测其表达.方法 RT-PCR方法扩增SEPT7 cDNA片段,并将扩增的片段插入pCDNA3真核表达载体,构建成含SEPT7cDNA的重组载体pCDNA3/SEPT7,以脂质体介导该质粒转染人脑恶性胶质瘤细胞系U251,应用RT-PCR和免疫荧光染色鉴定转染细胞中hCDC10/SEPT7的表达.用流式细胞术对转染前后的U251细胞进行细胞周期分析.结果 成功构建含SEPT7cDNA的重组载体pCDNA3/SEPT7,并使其稳定转染U251细胞,在转染的细胞中,证实有SEPT7mRNA表达上调.细胞周期检测结果G0/G1期细胞增多,SPF降低.结论 成功构建SEPT7表达载体,并在人脑恶性胶质瘤细胞系获得表达,延迟细胞周期进展. 相似文献
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EGFR反义RNA抑制人脑恶性胶质瘤细胞增殖并诱导凋亡的研究 总被引:4,自引:0,他引:4
目的:研究表皮生长因子受体(EGFR)反义RNA对抑制胶质瘤细胞增殖及诱导凋亡的作用。方法:将互补于EGFR 3′端部分序列的反义cDNA转染胶质瘤细胞,检测其生长率、增殖活性、EGFR mRNA及其蛋白表达和细胞凋亡。结果:胶质瘤细胞转染EGFR反义RNA后生长率及增殖活性下降,EGFRmRNA及蛋白表达减低,出现大量细胞凋亡。结论:EGFR有可能成为胶质瘤基因治疗的候选基因。 相似文献