新基因AngRem104与Bardet-Biedl综合征2蛋白的相互作用

目的 筛选与新基因AngRem104相互作用的蛋白,并验证AngRem104和Bardet-Biedl综合征2(BBS2)蛋白在哺乳动物细胞中的相互作用,为进一步研究AngRem104的生理功能奠定基础。方法构建AngRem104的酵母真核表达载体AngRem104-pGBKT7／c-myc,用以转化酵母菌AH109,并与转化了成人肾脏cDNA文库的酵母菌Y187接合,筛选与AngRem104相互作用的蛋白。从中挑选Bardet-Biedl综合征2蛋白进行免疫共沉淀,构建AngRem104-pcDNA3．1／V5-His和BBS2-pCMV／c-myc融合表达载体,共转染HEK293T细胞。分别用小鼠抗人c-mvc单克隆抗体和小鼠抗人V5-HRP抗体进行免疫沉淀和免疫印迹,以验证AngRem104和BBS2蛋白问的相互作用。结果以AngRem104为诱饵蛋白在人肾脏cDNA文库中共筛选到包括BBS2在内的7个与之存在相互作用的蛋白。免疫共沉淀结果显示,AngRem104-V5融合蛋白能够在c-myc抗体的免疫沉淀物中检测出来：在V5抗体的免疫沉淀物中也能检测到BBS2-c-myc融合蛋白。结论AngRem104和BBS2蛋白在哺乳动物细胞中存在某种相互作用,为新基因AngRem104功能探讨和Bardet-Biedl综合征发病机制的研究提供了有价值的线索。     

采用基因表达谱芯片技术筛选新基因AngRem104的功能相关基因

目的 采用基因芯片技术来筛选基因AngRem104的功能相关基因，为进一步的基因功能研究提供线索。方法 建立新基因AngRem104的正义和反义表达的系膜细胞模型，应用含有4000个人类基因的cDNA表达谱芯片，对过量表达AngRem104基因和抑制表达AngRem104基因的人肾小球系膜细胞RNA表达进行检测。部分基因的差异表达由RT－PCR方法来验证。结果 筛选出的差异表达基因共96个，其中94个基因上调表达，2个基因下调表达。差异表达的基因涉及到细胞外基质和受体蛋白、细胞信号传导相关基因、DNA结合及转录相关蛋白、免疫相关蛋白、细胞骨架和动力蛋白和细胞合成及代谢相关蛋白等。特别是纤连蛋白（FN）及整合素β1(integrin β1）表达明显上调。RT－PCR的方法也证实了AngRem104与FN表达的相关性。结论 应用作为研究基因功能有效手段的基因表达谱芯片技术来筛选新基因AngRem104的功能相关基因，发现AngRem104与FN的表达有关，为其功能研究提供了重要线索。     

瘢痕疙瘩分泌性卷曲相关蛋白2与骨母细胞特异性因子2的相互作用

目的 验证分泌性卷曲相关蛋白2(SFRP2)与骨母细胞特异性因子2(OSF-2)的相互作用.方法 构建能在哺乳动物细胞中表达带人膜联蛋白(HA)标签的OSF-2融合蛋白重组载体pCMV-HA-OSF-2,经酶切鉴定确认后,与Myc-SFRP2重组真核表达载体pCMV-HA-SFRP2单独或共转染人HK293细胞,利用免疫共沉淀技术及蛋白质印迹法验证OSF-2与SFRP2的相互作用.结果 重组载体经酶切电泳后,显示近800 bp处的条带为酶切后的目的 片段SFRP2编码基因,近2500 bp处的条带为酶切后目的 基因OSF-2.表明pCMV-Myc-SFRP2和pCMV-HA-OSF-2均构建成功.HK293细胞单独转染pCMV-Myc-SFRP2或pCMV-HA-OSF-2后,未检测到HA-OSF-2的表达;当pCMV-Myc-SFRP2和pCMV-HA-OSF-2共转染后,经Myc抗体进行免疫沉淀可见HA-OSF-2表达.结论 HA-OSF-2的重组载体能在哺乳动物细胞中表达,HA-OSF-2与SFRP2间存在相互作用.     

小干扰RNA特异性沉默c-myc基因对人胰腺癌SW1990细胞生物学影响

目的 观察小干扰RNA(siRNA)沉默c-myc基因的表达对人胰腺癌细胞株SW1990细胞生物学影响.方法 用siRNA沉默胰腺癌SW1990细胞中c-myc基因,用实时定量反转录聚合酶链反应(RT-qPCR)及Western blot技术检测c-myc mRNA及蛋白的表达量;噻唑蓝(MTT)法检测siRNA沉默c-myc基因对SW1990细胞增殖的影响;膜联蛋白V/碘化丙锭(Annexin V/PI)双染流式细胞术检测沉默c-myc基因细胞凋亡水平;Transwell细胞迁移实验检测siRNA沉默c-myc基因对SW1990细胞迁移能力的影响.结果 靶向c-myc的特异性siRNA可以高效抑制人胰腺癌SW1990细胞c-myc基因表达,在mRNA水平(0.263±0.048)较转染对照质粒组(0.970±0.012)明显降低,c-myc蛋白质表达量及细胞增值率均较转染对照质粒组明显降低;转染后48 h c-myc siRNA组细胞凋亡率为(19.90±2.09)％,明显高于siRNA阴性对照组(4.93±0.25)％和空白对照组(4.40±0.34)％;Transwell实验结果示细胞穿膜数c-myc siRNA组[(34.3±1.2)个]较siRNA-NC组[(68.3±5.8)个]和空白组[(72.3±1.2)个]均明显降低.结论 c-myc siRNA能够显著抑制c-myc基因在人胰腺癌SW1990细胞中的表达,降低细胞的增殖和迁移能力,促进细胞的凋亡.     

SFRP2与Periostin的相互作用在瘢痕疙瘩形成机制中的研究

目的利用pull-down技术验证凋亡相关蛋白基因SFRP2（Secreted frizzled-related protein 2）和骨母细胞特异性因子-2 Periostin（Osteoblast-specific factor2）间的相互作用。方法构建能在哺乳动物细胞中表达带HA标签的Periostin融合蛋白（HA—Periostin）的重组载体pCMV—HA—Periostin,经酶切鉴定正确后,和表达带Myc标签的融合蛋白（Myc—SFRP2）的重组真核表达载体pCMV—HA—SFRP2,单独或共转染人293细胞,利用pull-down技术验证Periostin与SFRP2间的相互作用。结果成功构建重组载体pCMV—HA-Periostin．与抗Myc单克隆抗体沉淀Myc—SFRP2相互作用蛋白复合物后,可以检测到HA—PeMostin的表达。通过体外蛋白质结合实验证实了Periostin与SFRP2间的相互作用。结论成功构建带HA标签的Periostin融合蛋白（HA-Periostin）的重组载体,利用pull—down技术证实了Periostin与SFRP2间的存在相互作用。     

胰腺癌中bcl-2的表达与细胞凋亡及癌基因c-myc表达的关系

目的：探讨人胰腺癌中bcl-2的变化情况与细胞凋亡及c-myc表达的关系。方法：采用免疫组织化学（SP法）和分子原位杂交法检测bcl-2及c-myc在胰腺癌病人癌组织（50例）和胰腺癌转移灶组织（15例）中的表达。采用TUNEL法检测细胞凋亡。结果：细胞凋亡的变化规律与胰腺癌的发生发展和临床病理特征有着内在联系。在mRNA和蛋白水平上bcl-2基因和c-myc基因分布一致。bcl-2蛋白的表达影响胰腺癌中细胞凋亡,并与c-myc蛋白在胰腺癌和胰腺癌转移灶组中的表达协同关系。结论：bcl-2和c-myc基因在胰腺癌发生和发展过程中发挥着重要作用。     

HBV表面抗原大蛋白与C53蛋白在肝细胞内的相互作用研究

目的 采用酵母双杂交方法筛选出肝细胞内HBV表面抗原大蛋白(LHBs)的结合蛋白,并验证LHBs与C53蛋白在肝细胞内的相互作用.方法 构建pGBKT7-LHBs作为诱饵质粒,从人肝脏cDNA文库中筛选与其相互作用蛋白的编码基因,发现其中包括C53基因.分别构建pCMV-5a-C53和pACT-C53质粒,采用哺乳动物双杂交及免疫共沉淀的实验方法验证肝癌细胞系HepG2内这两种蛋白之间的相互作用.结果 成功从肝脏cDNA文库筛选出C53蛋白;成功构建了pCMV-5a-C53和pACT-C53质粒,并通过哺乳动物双杂交及免疫共沉淀方法明确了LHBs及C53在肝细胞内的相互作用.结论 肝癌细胞系HepG2中LHBs及C53存在明确的相互作用,为进一步研究二者的相互作用及对相关生物学功能的影响奠定了前期基础.     

多价抗人精浆蛋白/抗人CD3双特异性单链抗体的构建及表达

目的　构建多价抗人精浆蛋白 (γ Sm) /抗人CD3 双特异性单链抗体 (scFv)基因 ,并进行真核表达和活性测定。　方法　利用递归PCR法扩增人IgG3上游铰链区与人p5 3四聚功能域融合基因 ,克隆入pUC1 9载体中构建pUC1 9/IgG3/p5 3克隆载体。将抗人CD3 scFv(CD3 scFv)和抗人γ SmscFv(γ SmscFv)依次克隆入pUC1 9/IgG3/p5 3载体中 ,构建多价双特异性γ SmscFv/CD3 scFv[scFv2 (CD3 /γ Sm) ]融合基因。将融合基因克隆入真核表达载体pSecTag2 B中 ,转染HeLa细胞进行表达 ,表达产物纯化后流式细胞仪进行活性测定。　结果　获得了多价scFv2 (CD3 /γ Sm)融合基因 ,基因全长 1 6 38bp ,可编码 5 4 6个氨基酸 ,与已发表的γ SmscFv、CD3 scFv和人p5 3四聚功能域基因cDNA序列一致。表达产物经SDS PAGE和Western印迹实验证实为约 6 70 0 0的特异蛋白条带 ,纯化后经流式细胞仪检测可以特异性地结合PC 3细胞和人外周血单个核细胞 (PBMC) ,亲和力高于双特异性scFv。　结论　获得了可与PBMC和PC 3细胞特异结合的多价scFv2 (CD3 /γ Sm) ,为进一步临床应用提供了实验依据     

血管紧张素Ⅱ上调人肾小球系膜细胞硬化相关基因的克隆

目的：筛选和鉴定培养的人肾小球系膜细胞（MsC)在血管紧张素Ⅱ（Angiotensin Ⅱ）作用下，产生以纤连蛋白（Fibronection,FN)为代表的细胞外基质成分的过程中上调表达的基因，寻找AngⅡ致细胞外基质堆积作用的相关基因，为探讨AngⅡ在肾小球硬化发展过程的分子机制奠定基础。方法：人MsC经AngⅡ（10^-6mol/L)刺激24h后，采用抑制性消减杂交（Suppression subtrative hybridization,SSH)获得AngⅡ相关的差异表达的cDNA，经纯化克隆到pGEM-Teasy Vector并转化大肠杆菌；随机选择120个克隆，经反向Norhern筛选上调表达的基因cDNA片段，并以Northern杂交验证，然后进行120个克隆，经反向Northern筛选上调的基因cDNA片段，并以Northern杂交验证，然后进行DNA序列测定和同源性比较，采用5′和3′－RACE及长距离PCR的方法获得新基因的全长cDNA。结果：120个随机选择的克隆中，反向Northern结果表明有55个克隆表达明显上调，挑选其中20个克隆进行DNA序列测定，结果显示18个为独立的基因片段序列（有两个序列为双拷贝），其中15个为已知基因，包括细胞外基质成分：如血小板反应素I，I型胶原α2，细胞骨架及结合蛋白：如平滑肌肌动蛋白α，钙调蛋白1，γ－胞浆型肌动蛋白等；合成和代谢相关蛋白；如醛缩酶A，延长因子1－γ，arnesyl pyrophosphate synthetase等，蛋白分解相关蛋白：如组织蛋白酶，泛素蛋白连接酶等，3个克隆（克隆104，52，46）与已知序列没有明显同源性，提示为新基因，3个新基因分别命名为AngRem(AngiotensinⅡ related gene in mesangial cells)104,AngRem52,AngRem46,GenBank登录号分别为AF367870，YA040225，AY040224，结论：对已知基因的分析表明，Ang II对细胞外基质具有双重作用。即：既刺激培养的人MsC细胞外基质成分表达上调，也通过刺激PAI－I等分子的表达而制抑细胞外基质成分的降解，此外，我们还发现了目前尚未报告的与AngⅡ对MsC的生物学效应－细胞外基质堆积和增生等细胞相关的基因（包括3个新基因），为揭示AngⅡ介导的MsC在肾小球硬化进展过程的可能分子机制提供了新的线索。     

一种新型的具有免疫调节和介导DNA基因治疗的蛋白质载体的研制

目的:研制一种具有调节宿主免疫系统,同时又能包装核酸进入肿瘤细胞的新型蛋白质载体。方法:利用基因工程技术,将人乙肝病毒核心抗原(HBcAg)的-COOH端和靶向肿瘤的整和素配体RGD肽与谷胱甘肽还原酶(GST)在大肠埃希菌中融合表达,经分子筛和GST亲和纯化后,获得DNA包装蛋白GST-RGD-ΔHBcAg。此包装蛋白用FITC标记后与前列腺癌PC-3细胞孵育,观察进入细胞情况;并用此包装蛋白包装含绿色荧光蛋白GFP基因的DNA载体pEGFP-N1,转染PC-3细胞,观察绿色荧光蛋白表达。此外,用包装蛋白GST-RGD-ΔHBcAg免疫6周龄雌性BALB/c小鼠,检测血清IgG1和IgG2a抗体水平。结果:本研究应用原核表达系统成功地表达并纯化出DNA包装蛋白GST-RGD-ΔHBcAg;荧光显微镜下观察到包装蛋白GST-RGD-ΔHBcAg能进入到PC-3细胞,并能携带绿色荧光蛋白基因进入PC-3细胞并表达;该包装蛋白免疫BALB/c小鼠后,小鼠血清IgG1和IgG2a抗体水平同时升高。结论:本研究研制的DNA包装蛋白GST-RGD-ΔHBcAg可作为基因治疗载体,并能对宿主的免疫系统起调节作用,为肿瘤的免疫——基因治疗载体提供一个新的选择。     

AngRem104, an angiotensin II-induced novel upregulated gene in human mesangial cells,is potentially involved in the regulation of fibronectin expression

Accumulation of extracellular matrix (ECM) in the glomerular mesangium is a common feature of many progressive renal diseases. Angiotensin II (AngII) plays important roles in the proliferation of glomerular mesangial cells (MC) as well as the synthesis of ECM such as fibronectin (FN) and collagens. However, the precise molecular signals responsible for these effects are unknown. To explore possible molecule mechanism of ECM accumulation related to AngII, suppression subtractive hybridization (SSH) was performed to screen and identify upregulated genes induced by AngII in cultured human MC. A novel gene, AngRem104 (GenBank accession number, AF367870), was isolated. The full-length cDNA of AngRem104 is 1690 bp, and it contains a 1041-bp open reading frame (ORF) encoding 347 amino acid residues with a predicted molecular mass of 37.2 kD. AngRem104 widely expressed in human heart, placenta, liver, muscle, kidney, and pancreas. Moreover, AngRem104 was found in human glomeruli and tubule by in situ hybridization. In human MC, the upregulation of AngRem104 induced by AngII was time-dependent, and it was dose-dependently blocked by AngII type 1 receptor antagonist (AT1RA), Losartan. The subcellular localization detected by AngRem104-pEGFP fusion protein revealed that AngRem104 was a nuclear protein. Interestingly, when AngRem104 was overexpressed by transfection of its sense construct, cDNA Microarray showed that two of the ECM-related genes, i.e., human mRNA for FN and integrin-beta-1 (FN receptor), dramatically upregulated their expressions. Furthermore, AngRem104 could regulate the expression of FN induced by AngII, which were detected by RT-PCR and quantitative real-time PCR, when AngRem104 was overexpressed. It is concluded that AngRem104 is a novel human gene potentially involved in the regulation of FN induced by AngII in human MC. These findings may provide new insights into mechanisms of glomerular sclerosis associated with AngII.     

表皮生长因子受体阻断对前列腺癌细胞增殖的影响

目的：探讨阻断表皮生长因子受体（EGFR）对前列腺癌（PCa）细胞增殖的影响。方法：人PCa细胞株DU－145，分别加或不加抗EGFR单抗C225（20nmol/L)阻断EGFR体外细胞培养7d，收集细胞、计数以观察对PCa雄激素非依赖增殖的影响，并采用免疫沉淀和Western blot方法，探讨阻断EGFR后不同时相点磷酸化有丝分裂原激活下蛋白激酶MAPK，及p27^kipl的表达变化。结果：阻断EGFR与对照相比较可使DU－145细胞增殖被抑制达35％，8h后磷酸化MAPK的表达水平开始降低，p27^kip1表达开始升高，至24h最明显。结论：阻断EGFR可抑制PCa细胞增殖，可能的机制是MAPK的活性降低，使p27^kip1的表达升高而细胞周期被阻。     

Characterization of induction of protooncogene c-myc and cellular growth in human vascular smooth muscle cells by insulin and IGF-I

Insulin and insulin-like growth factor I (IGF-I) are structurally related polypeptides that stimulate DNA synthesis and cellular proliferation, probably through a common pathway. Human arterial smooth muscle cells in culture demonstrated the presence of high-affinity receptors for both these hormones. Insulin and IGF-I both exhibited cross-reactivity to each other's receptors but with an affinity that is 100-fold less than for the homologous receptor. To examine more closely the receptor responsible for producing the growth effects, we used the polyclonal antibody against the insulin receptor, B2, and a monoclonal antibody to the IGF-I receptor, alpha IR3. We studied the growth effects of insulin and IGF-I as measured by stimulation of c-myc, DNA synthesis, and cellular proliferation in the presence and absence of these antibodies. F(ab') fragments of the anti-insulin-receptor antibody at a concentration of 10 micrograms/ml were capable of displacing greater than 90% of the bound insulin, thus establishing an effective insulin-receptor blockade. Under such blockade, insulin and IGF-I were both capable of doubling the amount of DNA synthesis and cell number in cultured human arterial smooth muscle cells. However, in the presence of a 1:2500 dilution of the monoclonal antibody alpha IR3, which caused a 90% displacement of IGF-I bound to its receptor, both the insulin and IGF-I effects on stimulating DNA synthesis or cellular proliferation were inhibited by greater than 90%. These findings demonstrate that the IGF-I receptor is the common pathway for the growth effects of both insulin and IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of porcine UL16-binding protein 1 endothelial cell surface expression

Abstract: Background: Natural killer (NK) cells participate in the immune response against solid organ allo‐ and xenografts and are tightly regulated through signals mediated by inhibiting and activating receptors expressed on their cell surface. Human NK cytotoxicity against porcine endothelial cells (pEC) is mediated by the interaction of the activating human NK receptor hNKG2D and its corresponding ligand on pEC, porcine UL‐16 binding protein 1 (pULBP1). The aim of the present study was to characterize the regulation of pULBP1 cell‐surface expression on primary porcine aortic endothelial cells (PAEC). Methods: A monoclonal antibody (mAb; aE5‐63) directed against pULBP1 was generated by immunizing C57BL/6 mice with the pEC line PEDSV.15, and used in cellular ELISA to determine pULBP1 cell surface expression. PAEC were either left untreated or stimulated with human or porcine cytokines [interferon gamma (IFN‐γ), tumor necrosis factor alpha (TNF‐α)], human serum, cultured under hypoxic conditions, or infected with human or porcine cytomegalovirus (CMV). Results: Neither human nor porcine IFN‐γ stimulation changed pULBP1 expression, whereas both human and porcine TNF‐α stimulation as well as human and porcine CMV infection significantly decreased pULBP1 expression on PAEC. Coculture of PAEC with human serum strongly increased pULBP1 expression depending on the binding of human anti‐pig antibodies. Exposure of PAEC to hypoxia only slightly increased pULBP1 expression. Conclusions: In conclusion, (i) the novel anti‐pULBP1 IgM mAb aE5‐63 represents a useful tool to study pULBP1/hNKG2D‐mediated responses in xenotransplantation, and (ii) the expression of pULBP1, a human‐pig cross‐species functional hNKG2D ligand, on the surface of PAEC is modulated by various stimuli associated with transplantation.     

Comparison of antigen expression on normal urothelial cells in tissue section and tissue culture

Antigenic characterization of urothelial cells cultured from normal adult ureter was performed. These cells were cultured using a simplified isolation and culture technique and a commercially available serum-free medium. The cells growing in these cultures had epithelioid morphology and normal quantities of DNA. The antigen expression on these cultured normal urothelial cells was evaluated using a panel of monoclonal antibodies: 5G6.4, AN43, URO-5, anti-keratin and anti-blood group antibodies, and 425 (anti-epidermal growth factor receptor). Lower levels of anti-A and AN43 binding on cultured cells were observed than are seen on urothelial cells in sections of normal ureter, while the binding of anti-blood group H, 5G6.4, and URO-5 was unchanged. Binding of anti-epidermal growth factor receptor antibody 425 was improved if the cells were grown in medium lacking epidermal growth factor. These results confirm the urothelial origin of these cultured urothelial cells but indicate that some antigenic differences between cultured normal urothelial cells and urothelial cells in situ in the normal ureter exist.     

The expression of epidermal growth factor receptor on human bladder cancer: potential use in radioimmunoscintigraphy

Monoclonal antibody 425, which binds to an extracellular domain of the epidermal growth factor receptor, was used to evaluate the expression of this antigen on bladder cancer cells. Epidermal growth factor receptor was found on all bladder cancer cell lines tested. Immunoperoxidase staining of fourteen invasive human bladder cancers with monoclonal antibody 425 demonstrated that ten showed strong staining, one showed weak staining and three were negative. Five noninvasive tumors were similarly examined. Four of these were negative and one showed weak staining. Biodistribution experiments with human bladder tumor xenografts in athymic nude mice using radiolabeled monoclonal antibody 425 and an isotype matched control antibody demonstrated specific tumor localization at five and seven days following antibody injection. Successful imaging of a human bladder tumor xenograft was achieved five days post antibody injection. These data confirm that epidermal growth factor receptor expression correlates with bladder cancer stage and suggests that epidermal growth factor receptor may serve as a target antigen for radioimmunoscintigraphy.     

Preclinical Efficacy and Immunological Safety of FR104, an Antagonist Anti‐CD28 Monovalent Fab′ Antibody

Antagonist anti‐CD28 antibodies prevent T cell costimulation and differentiate from CTLA4Ig since they cannot block CTLA‐4 and PDL‐1 coinhibitory signals. They demonstrated efficacy in suppressing effector T cells while enhancing regulatory T cells function and immune tolerance. However, anti‐CD28 antibodies devoid of immunotoxicity and with a good pharmacokinetic profile have not yet been developed. Here, we describe FR104, a novel humanized pegylated anti‐CD28 Fab′ antibody fragment presenting a long elimination half‐life in monkeys. In vitro, FR104 failed to induce human T cell proliferation and cytokines secretion, even in the presence of anti‐CD3 antibodies or when cross‐linked with secondary antibodies. Furthermore, in humanized NOD/SCID mice adoptively transferred with human PBMC, whereas superagonist and divalent antibodies elicited rapid cytokines secretion and human T cell activation, FR104 did not. These humanized mice developed a florid graft‐versus‐host disease, which was prevented by administration of FR104 in a CTLA4‐dependent manner. Interestingly, administration of high doses of CTLA4‐Ig was ineffective to prevent GVHD, whereas administration of low doses was partially effective. In conclusion, we demonstrated that FR104 is devoid of agonist activity on human T cells and thus compatible with a clinical development that might lead to higher therapeutic indexes, by sparing CTLA‐4, as compared to CD80/CD86 antagonists.