小鼠DLL1真核表达载体的构建及其在肿瘤细胞中的表达

目的：构建带绿色荧光蛋白的小鼠DLL1全长基因真核表达载体,并在肿瘤细胞中表达。方法：利用PCR特异性引物扩增出DLL1基因全长,将克隆的基因片段插入带绿色荧光蛋白的真核表达载体pIRES2-EGFP质粒中。然后利用脂质体将重组质粒pIRES2-EGFP-DLL1转染进小鼠B16黑色素瘤细胞中,并通过G418筛选后选取生长良好、荧光强度高的三株单克隆进行mRNA水平DLL1表达的鉴定。结果：成功扩增小鼠DLL1的全长基因。克隆入质粒载体后,通过DNA序列测定证实其序列正确。将构建的pIRES2-EGFP-DLL1质粒转染小鼠B16黑色素瘤细胞,经过G418筛选和荧光显微镜观察后,挑选得到GFP阳性率90%以上的稳定转染细胞株。RT-PCR检测稳定转染细胞的mDLL1的表达显著增加,进一步证实了pIRES2-EGFP-DLL1的表达效能。结论：成功构建了小鼠DLL1基因的真核表达质粒,证实其在真核细胞B16中可以表达。     

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.     

垂体瘤转化基因1(PTTG1)的表达变化对人前列腺癌细胞LNCaP生长增殖的影响

垂体肿瘤转化基因1(PTTG1)具有促进肿瘤生长和转移的作用.通过上调或下调基因表达的策略,观察PTTG1基因对人前列腺癌细胞株LNCaP细胞生长增殖的影响.利用PCR技术分离出PTTG1全长cDNA,分别正向和反向插入真核表达载体pIRES2-EGFP,重组载体分别命名为正义PTTG1-S/pIRES2-EGFP(即pI-P-S)和反义PTTG1-AS/pIRES2-EGFP(即pI-P-AS),将这两种重组载体稳定转染LNCaP细胞,通过流式细胞仪和MTT法分别检测了细胞周期和细胞增殖的情况.转染正义PTTG1后处于S期和G2期的细胞明显增加,细胞生长增殖能力增强;相反,转染反义PTTG1后处于S期和G2期细胞明显减少,细胞生长增殖能力减弱(P<0.05).结果表明,PTTG1能明显改变人前列腺癌细胞株LNCaP的细胞周期和细胞生长增殖能力,它的异常表达可能参与前列腺癌细胞生长增殖过程.     

牛c-myc基因的克隆及其在皮肤成纤维细胞中的表达

为了构建包含牛c-myc基因编码序列的重组载体,以胎牛原始生殖嵴为材料,用RT-PCR方法克隆出牛c-myc 基因的编码序列,将其亚克隆至pMD19-T载体,再从酶切鉴定和测序正确的质粒上切下目的片段,定向克隆到pIRES2-AcGFP1-Nuc表达载体上,挑选序列正确的重组真核表达质粒转染牛皮肤成纤维细胞,用RT-PCR和Western blotting分别检测c-myc mRNA和蛋白的表达。结果表明,从胎牛原始生殖嵴中正确克隆了c-myc基因的全长编码序列,所构建的重组质粒能够在皮肤成纤维细胞中有效     

RNA干扰PLCε诱导人膀胱癌BIU-87细胞凋亡的实验研究

目的探讨以RNA干扰沉默人源磷脂酶Ce(phospholipase Cepsilon,PLCε)基因表达后诱导人膀胱癌细胞株BIU-87细胞凋亡的作用和机制。方法脂质体介导重组阳性质粒pGenesil-PLCε(以下简称P)和阴性质粒pGenesil-NP(以下简称NP)转染BIU-87细胞48h后,用RT-PCR和Western blot检测转染前后PLCεmRNA和蛋白表达,流式细胞术检测细胞周期和细胞凋亡率,电镜观察细胞形态改变。结果转染P质粒后可明显抑制PLCεmRNA和蛋白表达水平,抑制率分别为78.7%和76.6%;流式细胞术显示P质粒转染组细胞周期发生改变呈明显G0/G1期阻滞,并出现亚二倍体凋亡峰,细胞凋亡率增加(P0.05);电镜观察P质粒转染组细胞可见凋亡小体。结论:RNA干扰沉默PLCε基因表达可诱导膀胱癌BIU-87细胞凋亡,其作用机制与细胞周期分布的改变有关。     

小鼠DLL1真核表达载体的构建及其在肿瘤细胞中的表达

目的:构建带绿色荧光蛋白的小鼠DLL1全长基因真核表达载体,并在肿瘤细胞中表达。方法:利用PCR特异性引物扩增出DLL1基因全长,将克隆的基因片段插入带绿色荧光蛋白的真核表达载体pIRES2-EGFP质粒中。然后利用脂质体将重组质粒pIRES2-EGFP-DLL1转染进小鼠B16黑色素瘤细胞中,并通过G418筛选后选取生长良好、荧光强度高的三株单克隆进行mRNA水平DLL1表达的鉴定。结果:成功扩增小鼠DLL1的全长基因。克隆入质粒载体后,通过DNA序列测定证实其序列正确。将构建的pIRES2-EGFP-DLL1质粒转染小鼠B16黑色素瘤细胞,经过G418筛选和荧光显微镜观察后,挑选得到GFP阳性率90%以上的稳定转染细胞株。RT-PCR检测稳定转染细胞的mDLL1的表达显著增加,进一步证实了pIRES2-EGFP-DLL1的表达效能。结论:成功构建了小鼠DLL1基因的真核表达质粒,证实其在真核细胞B16中可以表达。     

双基因真核表达载体pIRES-BMP2-TGFβ3 的构建与鉴定

目的:构建与鉴定骨形态发生蛋白BMP2和转化生长因子TGFβ3双基因真核表达载体pIRES-BMP2-TGFβ3。方法:首先,用PCR方法从质粒pGEMT/BMP2中扩增出BMP2基因全长,并将其连入双基因真核表达载体pIRES,得到质粒pIRES-BMP2,其次,从人胚胎组织提取总RNA,反转录成cDNA,以反转录的cDNA为模板,PCR扩增出TGFβ3基因全长,将TGFβ3基因连入质粒pIRES-BMP2;用酶切的方法筛选出阳性重组质粒,并进行测序鉴定。结果:酶切鉴定证明已将BMP2和TGFβ3两个基因连入载体中,测序结果完全正确。结论:成功构建PIRES-BMP2/TGFβ3双基因真核表达载体。     

双基因真核表达载体pIRES-BMP2-TGFβ3的构建与鉴定

目的：构建与鉴定骨形态发生蛋白BMP2和转化生长因子TGFβ3双基因真核表达载体pIRES-BMP2-TGFβ3。方法：首先,用PCR方法从质粒pGEMT/BMP2中扩增出BMP2基因全长,并将其连入双基因真核表达载体pIRES,得到质粒pIRES-BMP2,其次,从人胚胎组织提取总RNA,反转录成cDNA,以反转录的cDNA为模板,PCR扩增出TGFβ3基因全长,将TGFβ3基因连入质粒pIRES-BMP2;用酶切的方法筛选出阳性重组质粒,并进行测序鉴定。结果：酶切鉴定证明已将BMP2和TGFβ3两个基因连入载体中,测序结果完全正确。结论：成功构建PIRES-BMP2/TGFβ3双基因真核表达载体。     

Small interfering RNA targeting integrin-linked kinase inhibited the growth and induced apoptosis in human bladder cancer cells

Integrin-linked kinase (ILK), an intracellular serine/threonine kinase, is implicated in cell growth and survival, cell-cycle progression, tumor angiogenesis, and cell apoptosis. Recent studies showed that the expression and activity of ILK increased significantly in many types of solid tumors. However, the exact molecular mechanism of ILK underlie tumor has not been fully ascertained. The purpose of our study was to determine whether knockdown of ILK would inhibit cell growth and induce apoptosis in bladder cancer cells using a plasmid vector based small interfering RNA (siRNA). The experiments showed that knockdown of ILK could remarkably inhibit cell proliferation and growth, regulate cell cycle and induce apoptosis of bladder cancer BIU-87 and EJ cells. We demonstrated that knockdown of ILK inhibited phosphorylation of downstream signaling targets protein kinase B/Akt, glycogen synthase kinase 3-beta (GSK-3β), and reduced expression of β-catenin in BIU-87 as well as EJ cells by Western blot and Immunofluorescence analysis. In addition, down-regulation of ILK also could increase expression of Ribonuclease inhibitor (RI), an important acidic cytoplasmic protein with many functions. BALB/C nude mice injected with the BIU-87 cells transfected ILK siRNA showed a significant inhibition of the tumor growth with lighter tumor weight, lower microvessels density and higher apoptosis rate than those in the other two control groups. In conclusion, these results suggest that ILK might be involved in the development of bladder cancer, and could be served as a novel potential therapy target for human bladder cancer. Our study may be of biological and clinical importance.     

乳腺癌细胞株MCF-7-PC-1-46的建立

以人前列腺癌C4-2细胞基因组DNA为模板,扩增出PC-1基因N端编码46个氨基酸残基及其上游非编码区共599bp的DNA序列,将其正向克隆到真核表达载体pIRES2中,并在脂质体介导下,转染人乳腺癌细胞MCF-7,经G418筛选获得阳性单克隆,细胞扩大培养后,进行PCR和RT-PCR分析,检测外源PC-1基因在靶细胞中的整合与转录,PCR和RT-PCR结果表明,稳定转梁细胞株MCF-7-PC-1-46具有外源目的基因的整合和相应mRNA的高表达,说明成功建立了稳定表达外源PC-1基因N端46个氨基酸的人乳腺癌细胞株,为进一步研究PC-1基因的生物学功能提供了实验材料。     

骨形成蛋白9对人膀胱癌BIU-87细胞增殖和迁移的影响

目的:人骨形成蛋白9(bone morphogenetic protein 9,BMP9)对人膀胱癌BIU-87细胞增殖和迁移的影响。方法:使用过表达BMP9基因的腺病毒(AdBMP9)感染BIU-87细胞,采用定量PCR检测BMP9 mRNA的表达,Western blot检测BMP9蛋白及BMP9下游相关信号通路蛋白的表达;MTT及集落形成实验检测BIU-87细胞增殖能力;划痕愈合实验及Transwell ^TM小室迁移实验检测BIU-87细胞迁移能力。结果:感染AdBMP9后,BIU-87细胞中BMP9的mRNA水平和蛋白质水平均显著增加;过表达BMP9后,BIU-87细胞的体外增殖和迁移能力明显增加;Western blot结果显示BMP9可明显激活AKT信号通路。结论:高表达BMP9可能通过激活AKT信号通路促进人膀胱癌BIU-87细胞的增殖和迁移。     

Knockdown of ribonuclease inhibitor expression with siRNA in non-invasive bladder cancer cell line BIU-87 promotes growth and metastasis potentials

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein. RI is constructed almost entirely of leucine-rich repeats, which might be involved in some unknown biological functions like other structurally similar proteins besides inhibiting RNase A and angiogenin activities. Our previous experiments demonstrated that up-regulating RI might effectively inhibit some tumor growth and metastasis. However, the down-regulating RI influence on the tumor does not have any report until now, the mechanisms underlying antitumor of RI have not been fully understood. In this study, the efficient RNA interferences of RI were constructed using a plasmid vector and identified with RT-PCR, Western blot and Immunocytochemistry, then were transfected into non-invasive bladder cancer BIU-87 cells. We demonstrated that knockdown RI expression in BIU-87 cells could obviously change the cell morphology, rearrange the microfilaments and extend the lamellipodia, as well as enhance proliferation, increase migration, invasion and matrix metalloprotease level, and also reduce adhesion in vitro. BALB/C nude mice that were injected with the BIU-87 cells transfected RI siRNA showed a significant facilitation of the tumor with heavier tumor weight, higher density of microvessels, lower nm23-H1 and E-Cadherin expressions than those in the control group. Taken together, these experiments suggest that knockdown of RI could promote growth and metastasis potentials of BIU-87 cells. Our present findings reveal the novel mechanism that anti-tumor effect of RI is also involved in suppressing growth and metastasis, besides antiangiogenesis. The results show that RI may be a therapeutic target protein for bladder cancer and may be of biological importance.     

膀胱癌中内皮素作用的研究

应用Ｎｏｒｔｈｅｒｎｂｌｏｔ杂交技术及放射免疫方法对人膀胱移行细胞癌组织中内皮素－１ｍＲＮＡ及内皮素活性分子的表达水平进行检测,研究了佛波酯对人膀胱移行细胞癌ＢＩＵ－８７细胞中内皮素活性分子表达水平的调节作用,并探讨了内皮素抗血清及内皮素Ａ型受体竞争性拮抗剂ＢＱ_（１２３）对ＢＩＵ－８７细胞ＤＮＡ合成的影响。结果表明,人膀胱移行细胞癌组织中内皮素－ｌｍＲＮＡ及内皮素活性分子的表达水平明显高于相应的癌旁组织,佛波酯可促进ＢＩＵ－８７细胞合成和分泌内皮素,内皮素抗血清及ＢＱ_（１２３）可抑制ＢＩＵ－８７细胞合成ＤＮＡ。提示内皮素在膀胱癌发展及ＢＩＵ－８７细胞恶性增殖中可能起重要作用。     

大鼠μ型阿片受体的pIRES2-EGFP质粒构建及其在HEK293细胞中的表达

提取大鼠脑组织总RNA,通过逆转录巢式PCR,扩增μ型阿片受体全长cDNA,克隆至pMD20-T载体中,测序鉴定,纠正点突变后,经酶切连接克隆入pIRES2-EGFP中,测序及酶切结果表明μ基因正确,μ-pIRES2-EGFP质粒构建成功.用脂质体法将μ-pIRES2-EGFP转染入HEK293细胞中,在荧光显微镜下,转染细胞可以观察到绿色荧光,应用免疫组化荧光可以观察到μ基因的高强度表达.     

miR-92在膀胱癌患者中的表达与膀胱癌细胞侵袭和耐药性的关系

为了考察miR-92在膀胱癌患者中的表达及与膀胱癌细胞侵袭和耐药性的关系,本研究通过RT-PCR检测了膀胱癌患者癌组织和BIU-87细胞中的miR-92表达,通过对BIU-87细胞转染miR-92抑制剂来敲低miR-92的表达。使用10μg/mL的顺铂处理BIU-87细胞24 h、48 h和72 h,Cell Counting Kit-8试剂盒(CCK-8)检测细胞活力。基质胶侵袭实验检测侵袭能力,Annexin V/PI流式细胞仪检测细胞凋亡。RT-PCR和Western blotting检测GSK3β、细胞核β-catenin、Cyclin D1、c-myc和MMP7的表达。研究显示,膀胱癌组织和细胞中miR-92的表达上调且与TNM分期和淋巴结转移相关。敲低miR-92抑制膀胱癌细胞增殖、侵袭和上皮-间质转化,并降低膀胱癌细胞的顺铂耐药性。敲低miR-92导致Cyclin D1、c-myc、MMP7和细胞核β-catenin的表达水平显著降低,而GSK3β的表达水平显著升高。本研究表明,miR-92在膀胱癌患者中明显上调,敲低miR-92可抑制膀胱癌细胞的增殖、转移和上皮-间质转化,并提高化疗药物敏感性。miR-92对膀胱癌细胞生物学行为的调控作用部分由Wnt信号通路相关分子(如GSK3β等)介导。     

外源高表达PC-1蛋白N端43个氨基酸可加速人前列腺癌细胞系C4-2的生长

研究PC-1蛋白N端43个氨基酸表达对人前列腺癌细胞C4—2生长的影响。用DNA重组技术将含PC—1蛋白N端43个氨基酸的DNA序列正向克隆到真核表达载体pIRES2-EGFP中,采用脂质体法将重组质粒稳定转染进C4—2细胞中,RT—PCR分析外源序列的转录情况,固相ELISA法测定PC—1蛋白N端43个氨基酸的表达,MTT实验分析细胞的生长速度。结果获得了稳定转染PC—J基因N端43个氨基酸的前列腺癌细胞株,在该细胞株中外源PC—1蛋白N端43个氨基酸得到高表达,细胞生长速度较对照细胞加快了38％。结果表明外源PC—I基因N端43个氨基酸高表达可提高人前列腺癌细胞C4—2的生长速度,推论PC—J基因高表达可能在人前列腺癌的发展中起一定的促进作用。     

pIRES2-GDNF-NT-3真核表达载体的构建与鉴定(英文)

目的:采用一种简便和高效的方法构建双基因共表达载体pIRES2-GDNF-NT-3。方法:人胶质细胞源性神经营养因子和神经营养素3是采用PCR的方法从人外周血单个核细胞的基因组DNA中获取,将人胶质细胞源性神经营养因子的cDNA片段插入到pIRES2-EGFP多克隆位点构建成为pIRES2-GDNF-EGFP.神经营养素3 cDNA片段通过替换EGFP的方式插入到pIRES2-GDNF-EGFP中构建成为pIRES2-GDNF-NT-3双基因共表达载体。结果:人胶质细胞源性神经营养因子和神经营养素3被克隆,通过测序和酶切鉴定的得知与基因库报道序列一致。结论:人神经生长因子和神经营养素3双基因真核表达载体成功构建,它提供了一个新的表达系统,为进一步研究双基因的功能奠定了基础。     

Construction, characterization and expression of full length cDNA clone of sheep YAP1 gene

RT-PCR, 5′RACE, 3′RACE were used to clone sheep full length cDNA sequence of YAP1 (Yes-associated protein 1), eukaryotic expression plasmid and a mutant that cannot be phosphorylated at Ser42 was successfully constructed. The amino acid sequence analysis revealed that sheep YAP1 gene encoded water-soluble protein and its relative molecular weight and isoelectric point was 44,079.0 Da and 4.91, respectively. Sub-cellular localization of YAP1 was in the nucleus, it is hydrophilic non-transmembrane and non-secreted protein. YAP1 protein contained 33 phosphorylation sites, seven glycosylation sites and two WW domains. The secondary structure of YAP1 was mainly composed of random coil, while the tertiary structure of domain area showed a forniciform helix structure. YAP1 gene was expressed in different tissues, the highest expression was in kidney and the lowest was in hypothalamus. The CDS of sheep YAP1was amplified by RT-PCR from healthy sheep longissimus dorsi muscle, cloned into pMD19-T simple vector by T/A ligation. YAP1 coding region was further sub-cloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant. PCR, restriction enzyme and sequencing were used to confirm the recombinant plasmid. The sheep full-length YAP1 cDNA sequence is 1712 in length encoding 403 amino acids. It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine by PCR, restriction digestion and sequencing. The result showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly, this will help for further studies on the YAP1 protein expression and its biological activities.     

人细胞周期蛋白G2基因真核表达载体构建及其功能研究

构建人cyclin G2基因真核表达载体,进一步研究cyclin G2对体外培养细胞增殖的调节作用及可能的调节机制。以人口腔癌前上皮细胞系POE4总RNA的反转录产物为模板,应用RT-PCR方法克隆cyclin G2基因cDNA,成功构建真核表达载体pIRES -G2;应用脂质体介导的基因转染技术,以体外培养的肿瘤细胞系HeLa细胞和正常细胞系CV-1细胞作为受体细胞,进行转基因表达研究,发现cyclin G2高表达对体外培养细胞的增殖起明显抑制作用;应用p16INK4a、p21WAF1、p27KIP1三种周期蛋白依赖性激酶抑制因子的单克隆抗体对转基因的HeLa细胞进行免疫细胞化学研究,发现转染pIRES-G2的实验组细胞中,p21 WAF1蛋白染色阳性细胞数明显多于转染空载体的对照组,平均光密度值高于对照组,两组间均有显著性差异（p<0.01）,提示cyclin G2抑制细胞增殖作用可能是通过诱导p21WAF1的表达而实现。     

多靶点复合抗原抗肿瘤基因疫苗的构建及真核表达

目的：构建含有人存活蛋白（survivin）-2B主要T细胞表位区域、人和猴绒毛膜促性腺激素β链的核心片段CTP37区域融合基因的真核表达质粒,并在人胚肾293T细胞中进行表达。方法：通过基因合成和搭桥PCR技术构建含有Survivin2B主要T细胞表位区域、人和猴CTP37区域基因的融合基因2PAG,将其插入含有人IgK链前导信号肽（sig）、人IgG-Fc和糖基磷脂酰肌醇（GPI）锚定信号肽融合基因序列的细胞膜锚定修饰真核表达载体pCI—Fc—GPI中,继而又将酶切后的sig2PAG-FC-GPI融合基因导入含有细小病毒内部核糖体结合位点（IRES）基因且可以共表达人GM-CSF和B7．1融合基因的真核表达载体pVAX1-IRES-GM／B7中;将构建的重组质粒pVAX1-sig-2PAG-FC-GPI-GM／B7（简称pVAX1-2PFcGB）转染293T细胞,利用流式细胞仪和免疫荧光检测其表达情况。结果：2PAG融合基因经测序正确,PCR和酶切鉴定证明已成功连入真核表达载体pVAX-IRES-GM／B7中;流式细胞仪和免疫荧光的检测结果显示,重组质粒pVAX1-2PFcGB在293T细胞中得到很好的表达。结论：成功构建了重组质粒pVAX1-2PFcGB,且在293T细胞中可以有效表达,为对该基因疫苗的后续功能研究奠定了基础。