关键词： 金属蛋白酶组织抑制剂-1   转化生长因子-β3   表达载体构建   目的基因   病毒包装   生存素   慢病毒   椎间盘退变  

摘要： 腰痛可引起严重活动受限,其中40％的患者存在椎间盘退变.因此椎间盘退变治疗十分重要.与以减轻症状为目的的传统治疗方法比较,应用基因疗法治疗椎间盘退变最具期待[1].基因治疗对目的基因的选择及有效基因表达载体构建提出更高要求.生存素（Survivin）、转化生长因子-β3（TGF-β3）及金属蛋白酶组织抑制剂-1（TIMP-1）分别可在抑制凋亡、促进细胞外基质合成及抑制其降解三个方面影响椎间盘退变,是椎间盘退变基因治疗的潜在目的基因.本研究旨在构建上述三基因重组表达载体并包装病毒.

关键词： transforming growth factor-beta   melanoma   plasma   prognosis   metastasis  

摘要： Melanoma is an aggressive type of cutaneous malignancy. Transforming growth factor (TGF)-beta has been demonstrated to be an important mediator of tumor progression. However, to the best of our knowledge, the systemic roles of plasma TGF-beta and TGF-beta in situ have not been investigated in Han Chinese melanoma patients. The results of the present study demonstrated that the in situ and plasma levels of TGF-beta 1, TGF-beta 2 and TGF-beta 3 protein and messenger RNA were significantly elevated in tumor tissues compared with those of normal tissues. The survival rates of the patients which were triple-positive (TGF-beta 1(+), TGF-beta 2(+) and TGF-beta(3+)) were found to be markedly decreased compared to those which were single- (TGF-beta 1(+), TGF-(beta 2(+) or TGF-beta 3(+)) or double-positive (TGF-beta 1(+), TGF-beta 2(+);TGF-beta(+), TGF-beta 3(+);or TGF-beta 1(+), TGF-beta 3(+)). These results may therefore contribute to the use of TGF-beta as a prognostic biomarker, and to the development of novel therapies for melanoma treatment.

关键词： 羟基红黄色素A   转化生长因子-β1   成纤维细胞   细胞增殖   细胞迁移  

摘要： 目的:探讨羟基红花黄色素A(HSYA)抑制转化生长因子β1(TGF-β1)诱导NIH/3T3细胞活化的作用。方法:NIH/3T3细胞分8组:正常对照组、HSYA对照组、TGF-β1组、TGF-β1+0.5μmol/L HSYA组、TGF-β1+1μmol/L HSYA组、TGF-β1+2μmol/L HSYA组、TGF-1+2μmol/L SB-431542和TGF-β1+SB-431542+2μmol/L HSYA组。采用四甲基偶氮唑蓝(MTT)法检测细胞的增殖;细胞划痕实验观察细胞的迁移。结果:MTT法观察结果表明:与各时间点正常对照组相比,TGF-β1作用24,48及72小时后的OD 570nm值明显升高(均P<0.01),并且同时高于TGF-β1+HSYA各组(均P<0.05)和TGF-β1+SB-43154两组。在TGF-β1作用0,2,8,12及24小时后,与正常对照组相比,细胞迁移距离明显增加(均P<0.001),并且同时高于TGF-β1+HSYA组(均P<0.001)和SB-431542组。结论:HSYA可抑制TGF-β1诱导NIH/3T3细胞活化作用并且呈现时效关系和量效关系。

关键词： 转化生长因子β3   黑色素   黑色素细胞   羊驼  

摘要： 为了研究转化生长因子β3(Transforming growth factor beta3,TGF-β3)对体外培养的羊驼皮肤黑色素细胞表型的影响。本研究在体外培养的羊驼皮肤黑色素细胞中添加不同浓度TGF-β3(6.25、12.5、25、50ng·mL-1),通过实时监测和检测细胞增殖、毛色相关基因小眼畸形相关转录因子(Microphthalmia—associtated transcription factor,MITF)、酪氨酸酶(Tyrosinase,TYR)和酪氨酸酶相关蛋白2(Tyrosinase related protein 2,TYRP2)表达以及黑色素产量的变化。结果表明:(1)在羊驼皮肤黑色素细胞中添加50ng·mL-1浓度的TGF-β3后,在前30h内对细胞增殖有抑制效果,30h后对细胞增殖有明显的长时程维持细胞数量作用,但对TGF-β3添加的剂量没有依赖性;(2)添加TGF-β3后,黑色素细胞内MITF、TYR和TYRP2的表达量均被下调,而且黑色素细胞产生黑色素的量也被下调,主要以添加50ng·mL-1时下调最为显著。结果揭示,TGF-β3通过对羊驼黑色素细胞内MITF、TYR和TYRP2的表达的影响,并调控黑色素的产生,对黑色素细胞的生物学功能具有重要的影响。

关键词： microRNA-10a   human lung cancer   chemotherapy resistance  

摘要： Lung cancer is one of the primary causes of mortality worldwide and drug resistance is the key contributing factor which results in the failure of lung cancer chemotherapy. Previous studies have shown that microRNA (miR)-10a was involved in the reversal of cisplatin (DDP) resistance in numerous types of tumors;however, the underlying mechanism of action of this remains to be fully elucidated. In the present study, miR-10a silencing in human DDP-resistant lung cancer A549/DDP cells was demonstrated to improve DDP sensitivity, apoptosis, intracellular rhodamine-123 content as well as the expression and activity of caspase-3/8. In addition, miR-10a suppressed the cellular expression of P-glycoprotein, multi-drug resistance protein (MDR) 1, MDR-associated protein 1, RhoE, B cell lymphoma-2 and survivin in A549/DDP cells. Furthermore, miR-10a silencing inhibited the secretion of transforming growth factor (TGF)-beta, phosphorylation of Sma- and Mad-related protein (Smad)2, signal transducer and activator of transcription (STAT)3 and STAT5, the transcriptional activity of hypoxia-inducible factor and eukaryotic translation initiation factor 4E in human lung cancer A549/DDP cell line. These results therefore indicated that miR-10a may be a potential target for improving the effectiveness of lung cancer chemotherapy via regulation of the TGF-beta/Smad2/STAT3/STAT5 pathway.

关键词： 生存素   转化生长因子β3   基质金属蛋白酶抑制剂1   慢病毒属   椎间盘   退变  

摘要： 目的应用Gateway技术构建生存素(Survivin)、人转化生长因子β3(TGFβ3)和基质金属蛋白酶抑制剂1(TIMP1)慢病毒多表达质粒,并检测其表达活性。方法应用Gateway技术,以自剪切多肽2A串联Survivin、TGFβ3、TIMP1的3段外源基因,并克隆到慢病毒多表达质粒上,通过RT-PCR和Western-blot技术分别检测质粒转染293T细胞后目的基因mRNA和蛋白质表达水平。结果 RT-PCR和Western-blot检测结果显示,慢病毒多表达质粒成功表达了3种目的基因,且其mRNA和蛋白质表达水平较空载体对照组、PBS对照组明显升高,差异有显著性(F=17.87~69.11,q=6.94~15.47,P<0.05)。结论成功构建了慢病毒多表达质粒pLenti6.3-Survivin-P2A-TGFβ3-T2A-TIMP1,为下一步病毒的制备以及体内实验提供了基础。

关键词： 结肠癌   Runt相关转录因子3   转化生长因子β1   相关性  

摘要： 目的探讨转化生长因子β1(TGF-β1)与人类Runt相关转录因子3(Runx3)在结肠癌中的表达及其两者表达的相关性。方法收集2011年1月至2013年12月安岳县人民医院收治的结肠癌患者34例、结肠腺瘤性息肉患者34例标本以及正常体检的34例结肠黏膜标本,通过免疫组织化学SP法检测Runx3、TGF-β1的表达,分析Runx3与TGF-β1在结肠癌中的相关性。结果 Runx3在结肠癌组织中表达阳性率最低(17.7%,6/34),与结肠黏膜和结肠息肉组织比较差异均有统计学意义(P<0.01)。TGF-β1在结肠癌组织中的阳性表达率为88.3%(30/34),显著高于结肠息肉和结肠黏膜组织,差异均有统计学意义(P<0.01)。结肠癌中TGF-β1与Runx3表达呈负相关(rs=-0.46,P<0.05)。结论 Runx3及TGF-β1在不同结肠组织中表达差异明显,在结肠癌组织中表达有相关性,检测Runx3和TGF-β1可能为结肠癌早期诊断提供一定依据。

关键词： gangliotetraosylceramide   beta 1,3-galactosyltransferase-4   epithelial-mesenchymal transition   Smads   transcription factor  

摘要： The process of epithelial-mesenchymal transition (EMT) is essential for the proliferation and metastasis of tumor cells. Our previous study demonstrated that the expression of gangliotetraosylceramide (Gg4) and the transcription of UDP-Gal: beta 1,3-galactosyltransferase-4 (beta 3GalT4), a gene which controls the expression of Gg4, are reduced during transforming growth factor (TGF)-beta-induced EMT in normal murine mammary gland (NMuMG) cells. The present study revealed that the transcription level of beta 3GalT4 in patients with breast cancer was decreased compared with healthy control subjects. However, the molecular basis underlying these effects remains to be elucidated. Analysis of the beta 3Ga1T4 promoter sequence revealed a putative Smad-binding element (SBE) of Smad4, which is a transcriptional factor in TGF beta responses and forms a complex with Smad3. To clarify the association between the Smad3/4 complex and the beta 3Ga1T4 gene during EMT in NMuMG cells, an electrophoretic mobility shift assay and a chromatin immunoprecipitation assay were performed. The Smad3/4 complex was found to bind directly to the SBE of the beta 3Ga1T4 promoter. Overexpression of Smad3 and Smad4 through stable transfection had no notable effect on cell phenotype, but did alter the expression of various EMT protein markers. Treatment with TGF beta reduced the expression of Gg4 and the mRNA levels of the beta 3Ga1T4 gene in Smad3- and Smad4-overexpressing cells compared with vector-transfected cells. Expression of the epithelial markers E-cadherin and beta-catenin decreased in parallel with the reduction in Gg4. These findings suggested that the activated Smad3/4 complex downregulated the expression of Gg4 and the)beta 3GalT4 gene through translocation into the nucleus and binding to the [beta GalT4 promoter.

关键词： 1,25羟维生素D3   转化生长因子β1   糖尿病肾病   人第10号染色体缺失的磷酸酶及张力蛋白同源基因  

摘要： 目的 探索活性维生素D3对不同转化生长因子β1（TGF-β1）表达水平的糖尿病肾病大鼠模型的保护作用及可能机制.方法 建立SD大鼠糖尿病肾病模型（n=24）,分别注射3种不同慢病毒,同时给予骨化三醇（0.03μg·kg-1·d-1）干预及花生油对照.37 d后采集标本,以实时荧光定量PCR方法检测大鼠肾脏TGF-β1、人第10号染色体缺失的磷酸酶及张力蛋白同源基因（gene of phosphate and tension homology deleted on chromsome ten,PTEN）、胶原蛋白1（collagen-1,COL-1）和结缔组织生长因子（connective tissue growth factor,CTGF） mRNA含量,通过电镜观察大鼠肾脏超微结构的变化.结果 （1）活性维生素D3干预可使TGF-β1过表达、TGF-β1干扰和空载体组大鼠肾脏TGF-β1、COL-1、CTGF mRNA表达均明显下调（均P＜0.05）,使PTEN mRNA表达上调（P＜0.05）.（2）电镜结果显示,TGF-β1表达水平越高,肾脏病变越严重.维生素D3干预可改善各组大鼠的肾脏病变.结论 活性维生素D3可通过TGF-β1途径保护肾脏.

关键词： butaprost   雌激素   奶牛输卵管上皮细胞   转化生长因子β3   白血病抑制因子  

摘要： 目的：研究旨在揭示雌激素、前列腺素类化合物、前列腺素受体途径对奶牛输卵管上皮细胞分泌转化生长因子β3(TGF-β3)、白血病抑制因子(LIF)有无协同调控作用。为临床上合理用药治疗奶牛输卵管机能紊乱引起的不孕症并提高奶牛繁殖能力提供理论依据。方法：1)利用胰酶消化法及机械法成功培养奶牛输卵管上皮细胞,第四代细胞为实验用细胞。2)采用荧光定量PCR技术检测(雌激素、butaprost、吲哚美辛)作用不同时间(2h、4h、8h、16h、24h.48h)对奶牛输卵管上皮细胞TGF-β3、LIFmRNA表达的影响。3)分别用不同浓度Butaprost (109-10-5mol/L)和雌激素10-10mol/L作用于奶牛输卵管上皮细胞,然后用荧光定量PCR技术检测TGF-β3、LIFmRNA表达的变化。4)采用Western Blot法检测(雌激素、butaprost、吲哚美辛)对奶牛输卵管上皮细胞中TGF-β3合成分泌的影响。5)采用ELISA法测奶牛输卵管上皮细胞中LIF的合成分泌。结果：首先,体外成功培养了奶牛输卵管上皮细胞。其次,雌激素作用16、24和48h时对奶牛输卵管上皮细胞TGF-β3的表达量与空白组相比均极显著(P<0.01)升高,4h的表达量显著性的降低(P<0.05)：且EP：受体激动剂butaprost和雌激素有协同调控TGF-β3的效应。加入吲哚美辛后能有效的抑制内源性前列腺素对TGF-β3表达的作用。雌激素(无吲哚美辛预处理)作用2h对奶牛输卵管上皮细胞LIF的表达量与空白组相比极显著性升高(P<0.01),且2h时LIFmRNA的表达量达到最大值。吲哚美辛预处理后,LIF mRNA的表达量与无吲哚美辛处理组相比均升高。第三,用不同浓度的butaprost刺激奶牛输卵管上皮细胞后,TGF-β3、LIFmRNA表达量且呈现浓度依赖性。第四,Western Blot检测TGF-β3的表达与TGF-β3mRNA检测结果基本一致。第五,ELISA检测奶牛输卵管上皮细胞中LIF的结果与LIFmRNA检测结果相符。结论：1雌激素能够促进奶牛输卵管上皮细胞中细胞因子TGF-β3, LIF mRNA的表达。2雌激素和EP2受体激动剂Butaprost对奶牛输卵管上皮细胞中细胞因子TGF-β 3 mRNA的表达有协同调控作用,对奶牛输卵管上皮细胞中细胞因子LIF mRNA的表达有拮抗作用。3内源性前列腺素对奶牛输卵管上皮细胞细胞因子TGF-β3 mRNA的表达有促进作用,对LIF mRNA的表达有抑制作用。