怀化医学高等专科学校医学形态学实验中心

目的:探讨抑癌基因p16对肝癌细胞生长的抑制作用及其机制。方法:将p16 cDNA亚克隆至pcDNA3.1真核表达载体上,并经脂质体介导转染至人肝癌细胞株SMMC-7721。用MTT法和Western blot分析转染细胞的生长情况。结果:成功构建重组表达质粒pcDNA3.1-p16,转染pcDNA3.1-p16的SMMC-7721细胞生长速度受到明显抑制;转染后有外源p16蛋白的表达,且伴随Bax上调,Bcl-2和cIAP2的下调。结论:重组pcDNA3.1-p16质粒能在人肝癌细胞SMMC-7721内表达,且能抑制SMMC-7721的生长,其机理与诱导肿瘤细胞凋亡相关。     

抑癌基因p16对人肝癌细胞生长抑制的机制研究

目的：探讨抑癌基因p16对肝癌细胞生长的抑制作用及其机制。方法：将p16cDNA亚克隆至pcDNA3．1真核表达载体上,并经脂质体介导转染至人肝癌细胞株SMMC-7721：用MTT法和Western blot分析转染细胞的生长情况。结果：成功构建重组表达质粒pcDNA3．1-p16,转染pcDNA3．1-p16的SMMC-7721细胞生长速度受到明显抑制;转染后有外源p16蛋白的表达,且伴随Bax上调,Bcl-2和cIAP2的下调。结论：重组pcDNA3．1-p16质粒能在人肝癌细胞SMMC-7721内表达,且能抑制SMMC-7721的生长,其机理与诱导肿瘤细胞凋亡相关。     

pcDNA3.1-Canstatin-3Flag真核表达载体的构建及转染HepG2细胞中的表达

目的:构建pcDNA3.1-Canstatin-3Flag载体并稳定转染肝癌HepG2细胞,检测canstatin在mRNA水平的表达。方法:胎盘中提取总RNA,RT-PCR法获得canstatinDNA,克隆至pcDNA3.1(-)载体中,并测序,重组质粒pcDNA3.1-Canstatin-3Flag转染肝癌HepG2细胞,G418筛选出稳定转染细胞,RT-PCR检测canstatin mRNA表达。结果:1.成功构建出pcDNA3.1-Canstatin-3Flag重组质粒;2.获得稳定转染pcDNA3.1-Canstatin-3Flag的肝癌HepG2细胞;3.发现转染后的肝癌HepG2细胞canstatin在mRNA水平比未转染细胞有明显的增强。结论:获得了稳定转染pcDNA3.1-Canstatin-3Flag的肝癌HepG2细胞,为后期canstatin在肝癌中的研究提供了支持。     

人同源盒基因NKX3.1对前列腺癌细胞的诱导凋亡作用

构建人同源盒基因NKX3.1 cDNA真核表达载体,研究其在前列腺癌细胞PC-3、LNCaP 中的表达及对细胞的促凋亡作用.以人前列腺癌细胞LNCaP细胞中的总RNA为模板,RT-PCR扩增NKX3.1基因全长编码片段,将NKX3.1 cDNA重组到真核表达载体pcDNA3.1（+）中; 将pcDNA3.1-NKX3.1表达载体瞬时转染前列腺癌细胞PC-3和LNCaP 细胞,用RT-PCR和Western印迹检测NKX3.1 cDNA在转录水平和蛋白水平的表达;绘制细胞生长曲线,观察NKX3.1对前列腺癌细胞增殖的抑制作用;用DNA/ladder和流式细胞术检测NKX3.1对前列腺癌细胞凋亡的影响,进一步用RT PCR检测凋亡相关基因caspase3、caspase8、caspase9、Apaf1、survivin和Bcl2表达的变化.人同源盒基因NKX3.1 cDNA真核表达载体pcDNA3.1-NKX3.1经酶切及测序鉴定正确. pcDNA3.1-NKX3.1转染PC-3和LNCaP细胞后,经RT-PCR和Western印迹证明能有效表达NKX3.1.生长曲线显示,前列腺癌细胞转染NKX3.1 cDNA后细胞增殖受到抑制;前列腺癌细胞转染NKX3.1 cDNA 48 h后,DNA电泳呈现具有凋亡特征的DNA ladder;流式细胞术检测出现明显凋亡峰;RT-PCR检测凋亡相关基因.结果显示,caspase3、caspase8、caspase9基因表达明显增加,Bcl2基因表达明显减少.本研究成功构建了真核表达载体pcDNA3.1 NKX3.1, 转染PC3和LNCaP细胞后能有效表达,并对细胞具有诱导凋亡作用     

幽门螺杆菌ureB基因转染胃上皮细胞及其对细胞的作用

研究幽门螺杆菌 (Helicobacterpylori,Hp)ureB基因重组子转染胃上皮细胞后对胃上皮细胞的作用。用PCR方法从Hp标准株NCTC116 37中获取ureB全长基因 ,将其开放读码框架定向克隆入真核表达载体pcDNA3 1;获得的重组子转染SGC 790 1细胞 ,筛选耐潮霉素的细胞克隆 ,用RT PCR方法检测细胞内ureB基因在转录水平的表达 ;分别用荧光染色技术、MTT、流式细胞术检测UreB对细胞表型、增殖、凋亡及细胞周期的影响。UreB阳性表达的细胞 (SureB)胞膜出芽、细胞皱缩 ;用MTT法检测细胞增殖 ,结果表明 ,SureB细胞与SpcDNA3 1细胞比较 (pcDNA3 1转染的细胞 ) ,生长增殖无显著性差异 (P >0 0 5 ) ,流式细胞术检测细胞凋亡结果显示 ,SureB的凋亡率显著高于SpcDNA3 1(P值为 0 0 0 7) ;细胞周期分析显示 ,SureB细胞有S期比率增高、G2 M、G0 G1 期比率下降的趋势。ureB在培养细胞内的表达可促进细胞凋亡     

Expression of Bcl—2 inhibited Fas—mediated apoptosis in human hepatocellular carcinoma BEL—7404 cells

Apoptosis plays an important role in embryonic development,tissue remodeling,immune regulation and tumor regression.Two groups of molecules(Bcl-2 family and “Death factor” family) are involved in regulating apoptosis.In order to know about the effect of Bcl-2 on apoptosis induced by Fas,a typical member of “Death factor” family,the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells,a human hepatocellular carcinoma cell line which expresses endogenous Fas,but not FasL and Bcl-2.The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells.However,the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis.Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis.These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.     

STAT3小干扰RNA的构建及对缺氧复氧人肾小管上皮细胞凋亡的影响

目的:构建信号转导与转录因子3(STAT3)小干扰RNA(siRNA)表达载体,并观察其对缺氧复氧后人肾小管上皮细胞(HKC)凋亡的影响。方法:设计3对人STAT3 siRNA靶序列,用DNA重组技术克隆至质粒pRNAT-U6.1/neo中,构建重组质粒pRNAT-U6.1-STAT3 siRNA,检测并筛选出最佳抑制效率的siRNA质粒载体。重组质粒转染至缺氧复氧后HKC细胞,Western blotting和Real Time-PCR测定STAT3蛋白和mRNA表达量,流式细胞仪测定细胞凋亡,间接荧光法测定Bcl-2和Bax表达的变化。结果:靶向STAT3基因表达的质粒载体构建成功,并筛选出抑制效率最佳的重组质粒。缺氧复氧后HKC细胞STAT3表达、凋亡率和Bax/Bcl-2比值增加;缺氧复氧后HKC细胞转染重组质粒后STAT3表达、凋亡率和Bax/Bcl-2比值明显降低。结论:成功构建并筛选最佳抑制效率的靶向STAT3的重组质粒载体。该载体可有效抑制缺氧复氧后HKC细胞中STAT3信号转导通路的活化,并进一步通过上调Bcl-2、下调Bax蛋白的表达,从而抑制细胞凋亡。     

In vitro chondrogenesis of human bone marrow-derived mesenchymal progenitor cells in monolayer culture: activation by transfection with TGF-beta2

Bone marrow-derived mesenchymal progenitor cells are capable of chondrogenesis, making them a possible source of cells for cartilage tissue engineering. Because of this, we studied the effect of human transforming growth factor beta2 (TGF-beta2) on mesenchymal progenitor cell chondrogenesis in monolayer culture using gene transfection technology. A recombinant pcDNA3.1(+)/TGF-beta2 construct containing a full-length TGF-beta2 from a human placental cDNA library was created through gene cloning and DNA recombination. The construct was then lipofected into mesenchymal progenitor cells isolated from human bone marrow. RT-PCR, Western blotting, and immunohistochemistry analyses were performed to identify the expression of TGF-beta2 and cartilage-associated genes and proteins. The results showed that TGF-beta2 was expressed throughout the culture period. The transfected progenitor cells expressed and produced collagen type II and aggrecan 48 h after transfection, and the expression and synthesis were upregulated after 4 weeks. In contrast, the tested genes and proteins were not detected in non-transfected cells. This shows that transfection of pcDNA3.1(+)/TGF-beta2 into mesenchymal progenitor cells is able to provide transient and persistent expression of cartilage-specific genes and proteins, and suggests that the differentiation of human marrow-derived mesenchymal progenitor cells into chondrocytes in monolayer culture is feasible and may be induced by TGF-beta2.     

pcDNA3.1/myc-His-DJ-1^M26I重组载体构建及其对NIH3T3细胞增殖和凋亡研究

目的构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组表达载体,为研究DJ-1M26I突变与细胞增殖、凋亡的关系及建立转基因动物模型奠定基础。方法采用突变试剂盒将DJ-1蛋白第26位氨基酸进行突变,分别构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组表达载体,并采用脂质体介导的方法分别将其转染入NIH3T3细胞,500μg/mL G418压力筛选稳定克隆,对2种转染细胞在DNA水平、RNA水平和蛋白质水平进行鉴定,采用MTT染色方法和AnnexinV-FITC试剂盒进行转染阳性克隆细胞的细胞活力与细胞凋亡检测。结果 pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组质粒转染NIH3T3细胞经G418筛选后,PCR方法检测分别获得1个和3个阳性细胞克隆,RT-PCR及western blot方法进行DJ-1-His基因表达检测,结果均证明外源插入基因的表达,MTT实验结果初步证明转染DJ-1M26I基因的NIH3T3阳性细胞组细胞增殖速率低于正常NIH3T3细胞组（P〈0.05）,转染DJ-1基因的NIH3T3阳性细胞组细胞增殖速率与正常NIH3T3细胞相比无明显差别;细胞凋亡检测表明转染DJ-1M26I基因的NIH3T3阳性细胞组细胞凋亡率高于正常NIH3T3细胞,转染DJ-1基因的NIH3T3阳性细胞组细胞凋亡率低于正常NIH3T3细胞（P〈0.05）。结论成功构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1 M26I重组表达载体,成功筛选出稳定表达人DJ-1及DJ-1 M26I的NIH3T3细胞。DJ-1 M26I基因突变更易导致NIH3T3细胞的凋亡。     

NALP1 基因与骨肉瘤细胞的相关研究

目的:探讨NALP1基因在骨肉瘤细胞株MG-63、U-2OS中的表达,以及高表达NALP1基因对于骨肉瘤细胞体外凋亡的影响。方法:使用RT-PCR、Western-blot法检测骨肉瘤细胞株MG-63、U-2OS中的mRNA及蛋白表达水平并与人成骨细胞株hFOB1.19比较。将NALP1基因转染质粒PcDNA3.1,将重组质粒转染骨肉瘤细胞,分成高表达基因组、空质粒组及对照组,加入抗肿瘤药物顺铂及甲氨蝶呤促使肿瘤细胞凋亡,使用流式细胞仪测定各组肿瘤细胞凋亡率。结果:通过统计分析,骨肉瘤细胞株MG-63、U-2OS中的mRNA及蛋白表达水平均低于人成骨细胞株hFOB1.19(P<0.05),NALP1高表达组的肿瘤细胞凋亡率明显高于空白质粒组及对照组。结论:上调骨肉瘤细胞株MG-63、U-2OS中的NALP1的表达量可以促进肿瘤细胞凋亡。     

盐酸罗哌卡因对骨肉瘤细胞增殖、侵袭、凋亡的影响及其机制*

目的:探讨盐酸罗哌卡因对骨肉瘤细胞增殖、侵袭、凋亡的影响及分子机制。方法:采用逐步增加药物剂量诱导法建立骨肉瘤多柔比星耐药细胞株(U2OS/DOX),用浓度分别为0、20、50、100 μg/ml的盐酸罗哌卡因处理U2OS/DOX细胞,作为不同浓度盐酸罗哌卡因处理组;将pcDNA3.1、pcDNA3.1-Livin转染至U2OS/DOX细胞中再用浓度为100 μg/ml的盐酸罗哌卡因处理,记为盐酸罗哌卡因100 μg/ml+pcDNA3.1组、盐酸罗哌卡因100 μg/ml+pcDNA3.1-Livin组。MTT检测细胞增殖抑制率及细胞半数抑制浓度(IC₅₀);蛋白质印迹(Western blot)法检测细胞周期蛋白依赖性激酶抑制剂1A(P21)、活化的半胱氨酸天冬氨酸蛋白酶-3(Cleaved Caspase-3)、上皮钙黏蛋白(E-cadherin)、基质金属蛋白酶2(MMP-2)、Livin蛋白表达;克隆形成实验检测细胞克隆形成数;流式细胞术检测细胞凋亡;Transwell检测细胞迁移和侵袭;实时荧光定量PCR(RT-qPCR)检测Livin mRNA表达水平。结果:多柔比星浓度大于1 μg/ml时,骨肉瘤细胞U2OS增殖抑制率显著升高,且具有剂量依赖性(P＜0.05);多柔比星浓度大于10 μg/ml时,骨肉瘤细胞骨肉瘤耐药细胞U2OS/DOX增殖抑制率显著升高,且具有剂量依赖性(P＜0.05)。盐酸罗哌卡因处理的U2OS/DOX细胞中P21、Caspase-3、E-cadherin表达水平显著升高,MMP-2表达水平显著降低,细胞增殖抑制率显著升高,克隆形成数显著降低,细胞凋亡率显著升高,细胞迁移、侵袭数显著降低,Livin表达水平显著降低,且呈浓度依赖性(P＜0.05)。过表达Livin部分逆转了盐酸罗哌卡因对细胞U2OS/DOX增殖、迁移、侵袭的抑制作用及凋亡的促进作用。结论:盐酸罗哌卡因能明显抑制对多柔比星具有耐药性的骨肉瘤细胞的增殖,迁移和侵袭,明显促进骨瘤细胞凋亡,其机制可能与Livin有关。     

pcDNA3.1(+)-CTGF真核表达质粒构建及其在人成骨样细胞SaOS-2细胞中的表达*

目的:构建结缔组织生长因子(CTGF)的pcDNA3.1(+)真核表达质粒(pcDNA3.1(+)-CTGF),并检测其在人成骨样细胞SaOS-2中的表达,为进一步研究CTGF基因在骨发育和骨修复中的机制提供技术支撑。方法:采用PCR方法体外克隆CTGF基因全序列,将其用同源重组技术连接到线性pcDNA3.1(+)载体上,构建pcDNA3.1(+)-CTGF真核表达质粒,并对该质粒进行测序鉴定;鉴定无误后转染至SaOS-2细胞中,观察其48 h的表达情况。结果:基因测序证实pcDNA3.1(+)-CTGF真核表达重组质粒构建成功,与对照组相比,转染SaOS-2细胞48 h后的CTGF表达水平显著上调,达到对照组的4.8×10⁵倍(P＜0.01)。结论:成功构建了pcDNA3.1(+)-CTGF真核表达质粒,并能在人成骨样细胞SaOS-2中稳定表达,为深入研究CTGF基因对骨生成的调控机制奠定了基础。     

人sTNFR1基因的克隆以及在NIH3T3细胞中的稳定表达

以He1a细胞的总RNA为模板,用RT—PCR方法扩增sTNFR1全编码区基因片段,构建含有目的片段的T载体克隆及真核表达载体pcDNA3．1（-）重组质粒亚克隆,将重组质粒和脂质体共同转染NIH3T3细胞系,G418筛选稳定转染细胞株．经核苷酸序列测序和酶切鉴定,成功构建了pcDNA3．1（-）-sTNFR1真核表达质粒,脂质体法建立了高效表达sTNFRI的稳定转染细胞系,并经RT—PCR和Western Blotting鉴定．人sTNFR1基因能在NIH3T3细胞系中稳定表达,为今后的研究打下了基础．     

抑制Beclin1促进H2O2诱导的神经胶质瘤U251细胞凋亡

氧化应激能够引起细胞自噬和凋亡同时发生,但其中细胞自噬的作用仍不十分明确,研究表明Beclin 1作为调节前自噬体形成的关键基因,参与了胶质瘤氧化应激的损伤过程.为了探讨自噬在H2O2引起的神经胶质瘤U251细胞损伤中的作用,本文应用真核细胞转染技术将Psilencer3.1-siRNA-Beclin 1重组质粒转入人...     

Construction of a full-length iASPP expression plasmid pcDNA3.1(+)/iASPP and its biological activity

In order to obtain a full-length expression plasmid for the p53 inhibitor protein, iASPP, fractional amplification was used to clone its full-length coding sequence (CDS) region. The amplified PCR product was then digested and inserted into the pMD19-T simple vector and subcloned into the pCDNA3.1(+) vector. A recombinant eukaryotic expression vector containing the complete CDS region of iASPP was successfully constructed. pcDNA3.1(+)/iASPP was able to express iASPP protein in an in vitro translation system and in cells. Its biological activity was verified using Western blotting, immunoprecipitation and cell apoptosis analysis. This successful preparation of a full-length iASPP expression plasmid lays the foundations for further studies on the function of iASPP.     

Mechanisms of HuR in regulation of epithelial cell apoptosis in rat ulcerative colitis

ObjectiveTo investigate the influence of HuR on the apoptosis rate of epithelial cells in rats with ulcerative colitis (UC) and its mechanism.MethodsUC cell models were established in LPS induced Caco-2 cells. After transfection of si-HuR, pcDNA3.1-HuR, pcDNA3.1-HMGB1, miR-29a-3p mimic or miR-29a-3p inhibitor and their negative controls, apoptosis rate and apoptosis-related proteins (Bcl-2, Bax and cleaved-caspase-3) were tested by flow cytometry, qRT-PCR and Western blot. Actinomycin D treatment was applied to verify the effect of HuR in Caco-2 cells. The binding of HMGB1 to HuR/miR-29a-3p was measured by RIP and dual luciferase reporter gene assays. Experimental UC rat models were established by rectum administration of TNBS/ethanol. The colonic weight/length ratio was calculated at the day 15. The morphology of colon tissues and the apoptosis of tissues were separately detected by H&E staining and TUNEL staining. qRT-PCR and Western blot were conducted to determine the levels of HuR, miR-29a-3p and HMGB1 in colon tissues.ResultsThe apoptosis of LPS-treated Caco-2 cells was inhibited following transfection of si-HuR or miR-29a-3p mimic while facilitated following transfection of pcDNA3.1-HMGB1 or miR-29a-3p inhibitor. RIP and dual luciferase reporter gene assays showed that both HuR and miR-29a-3p can bind HMGB1. Overexpression of HuR in Caco-2 cells results in less HMGB1 that can be bind to miR-29a-3p. The degradation rate of HMGB1 mRNA was increased after transfection of si-HuR in Caco-2 cells. Additionally, miR-29a-3p overexpression can abolish the increases of HMGB1 mRNA induced by HuR, therefore consequently suppress the HMGB1 mRNA that can be bind to HuR. Knockdown of HuR can alleviate TNBS-induced UC in rats and inhibit the apoptosis of colon tissues.ConclusionHuR competitively binds HMGB1 with miR-29a-3p to promote apoptosis of colonic epithelia in rats with UC.     

LMTK2基因沉默抑制人上皮性卵巢癌细胞生长和转移的作用机制

摘要 目的：探讨狐猴酪氨酸激酶2（LMTK2）基因沉默对人上皮性卵巢癌(EOC)细胞生长和转移的抑制作用及其可能的机制。方法：通过RT-qPCR和Western-blot检测了人正常卵巢上皮细胞IOSE80和人上皮性卵巢癌细胞系（SKOV3、ES2、OVCAR-3和HEY）中LMTK2的表达,使用Lipofectamine 3000转染试剂将LMTK2的短发夹RNA（shRNA）、阴性对照shRNA、LMTK2过表达重组pcDNA3.1质粒或阴性对照质粒转染到SKOV3细胞中,并分为LMTK2-shRNA组、NC-shRNA组、LMTK2-pcDNA3.1组或NC-pcDNA3.1组。另外,使用PI3K/Akt抑制剂LY294002处理SKOV3细胞1 h。通过CCK-8法测定细胞增殖,Annexin V-FITC/PI染色法测定细胞凋亡,划痕实验评价细胞迁移,Transwell实验评价细胞侵袭。对BALB/c雌性裸鼠皮下注射转染NC-shRNA或LMTK2-shRNA的SKOV3细胞建立体内移植瘤模型,并记录接种28 d内的肿瘤体积。结果：与人正常卵巢上皮细胞IOSE80相比,卵巢癌细胞系（SKOV3、ES2、OVCAR-3和HEY）中LMTK2的mRNA和蛋白表达水平均显著升高,其中SKOV3的LMTK2 mRNA和蛋白表达水平最高（P<0.05）。与NC-shRNA组相比,LMTK2-shRNA组SKOV3细胞活力、相对迁移面积、侵袭细胞数均显著降低,而细胞凋亡率显著升高（P<0.05）。此外,与NC-shRNA组相比,LMTK2-shRNA组SKOV3细胞中Bax的蛋白表达水平显著升高,而Bcl-2、MMP2、MMP9、p-Akt的蛋白表达水平显著降低（P<0.05）。LY294002处理逆转了上调LMTK2对SKOV3细胞生长和转移的影响（P<0.05）。在接种第21天和28天时,与NC-shRNA组相比,LMTK2-shRNA组裸鼠的肿瘤体积显著降低（P<0.05）。结论：LMTK2基因沉默通过抑制PI3K/Akt信号通路降低了人上皮性卵巢癌细胞的生长和转移能力。