评价空肠弯曲菌外膜蛋白PEB1介导的重组BCG与上皮细胞的结合能力

目的：评价PEBl介导的屋尘螨抗原（Derp2）重纽BCG疫苗（PEBI-Derp2．rBCG）与人上皮细胞的结合能力。方法：采用体外细胞培养的方法,分别将普通BCG、胞壁型Derp2-rBCG和胞壁型融合蛋白PEBl-Derp2-rBCG与HeLa细胞及人类肠粘膜上皮细胞（HIEC）进行共孵育,利用HE和抗酸染色法对各组细胞与疫苗的黏附结果进行染色,光学显微镜下计数各组的黏附率,并进行比较;对以上各组分别加入PEBl蛋白,进行黏附阻断,观察对结合能力的影响。结果：孵育24小时后,无论HeLa细胞还是H匝CPEBl-Derp2．rBCG组较普通BCG组和Derp2-rBCG组的黏附率明显提高,差异有显著性（P〈0．05）;PEBl蛋白的加入对PEBl．Derp2-rBCG的黏附功能有明显抑制作用（P〈O．05）;但是,Derp2-rBCG组与普通BCG组比较没有明显差异（P〉o．05）,PEBl蛋白的加入对二者的黏附亦无影响（P〉0．05）。结论：PEBl具有介导增强PEBl-Derp2-rBCG与上皮细胞黏附的能力。     

结核分枝杆菌海藻糖磷酸磷酸酶诱导小鼠体液和细胞免疫应答

目的 研究结核分枝杆菌(M．tuberculosis)海藻糖磷酸磷酸酶(TPP)诱导小鼠体液和细胞免疫。方法 差速离心分离结核分枝杆菌H37Rv和卡介苗(BCG)的各细胞组分,通过Western杂交检测抗原TPP在结核分枝杆菌H37Rv和BCG中的亚细胞定位情况。分别用5×10~6CFU的BCG和50μg的TPP蛋白免疫C57BL/6小鼠,检测小鼠血清中抗TPP的IgG1和IgG2a抗体效价。取免疫小鼠的脾细胞,体外抗原刺激,用酶联免疫斑点试验(ELISPOT)检测γ干扰素(IFN-γ)分泌细胞。结果 TPP亚细胞定位于结核分枝杆菌H37Rv和BCG的胞壁和细胞膜组分。TPP蛋白免疫后小鼠产生的TPP特异性IgG1和IgG2a抗体效价明显高于BCG免疫小鼠,并且IgG2a的抗体效价高于IgG1。体外抗原刺激TPP蛋白和BCG免疫小鼠的脾细胞,都能诱导较高的IFN-γ分泌。结论 结核分枝杆菌细胞壁蛋白TPP能诱导小鼠Ⅰ型辅助性T细胞介导的免疫反应,可作为抗结核疫苗的候选抗原。     

Ipr1/PPE68重组BCG诱导BALB/C小鼠免疫应答的探讨

目的构建Ipr1/PPE68重组卡介苗(Recombinant BCG,rBCG),探讨其诱导BALB/C小鼠免疫应答的效果。方法将Ipr1和PPE68基因及结核分枝杆菌(mycobacterium tuberculosis,MTB)复制子OriM分别插入pBudCE4.1的多克隆位点,构建共表达穿梭质粒pBIPO,将其电转入BCG,构建Ipr1/PPE68-rBCG并将rBCG免疫BALB/C小鼠,检测小鼠血清中IgG2a、IL-12、IFN-γ及IL-4的水平、特异性脾淋巴细胞增殖和CD4+和CD8+T细胞数量,同时观察脾、肺荷菌量及脾、肺组织病理学变化。结果酶切测序及菌落PCR鉴定Ipr1和PPE68以及OriM基因序列与理论值相符,Western-blotting结果显示Ipr1和PPE68蛋白成功表达。Ipr1/PPE68-rBCG免疫小鼠后,血清中的IgG2a和IL-12水平及脾淋巴细胞增殖情况明显高于对照组,但与BCG组相比没有显著意义;IFN-γ水平显著低于BCG组,与对照组相比无显著性差异;各组别IL-4的水平差异均不明显。脾、肺荷菌实验未见菌落生长,肺、脾组织未见病理学改变。结论成功构建Ipr1/PPE68-rBCG,该重组BCG能诱导BALB/C小鼠的细胞免疫应答。     

M-CSF上调cyclinD1/D3和CDK2/6的表达促进HeLa细胞增殖

非分泌型巨噬细胞集落刺激因子(M-CSF)的表达在肿瘤的发生发展过程中发挥重要作用,为探讨胞质M-CSF对细胞增殖的影响,采用基因重组技术构建胞内稳定表达M-CSF的HeLa细胞系,以空载体(pCMV/myc/cyto)转染HeLa细胞和未转染HeLa细胞作为对照,MTT法及反义寡核苷酸抑制实验分析M-CSF对细胞增殖的影响,并计算细胞倍增时间,RT-PCR观察胞内M-CSF对G1期细胞周期相关蛋白的影响．结果显示,与对照组比较,转染M-CSF的HeLa细胞倍增时间明显缩短、增殖能力显著增强,M-CSF的特异性反义寡核苷酸能抑制转染M-CSF的HeLa细胞的增殖,且抑制率随着反义寡核苷酸浓度的增高而增强,转染M-CSF 的HeLa细胞的cyclinD1/D3和CDK2/6 mRNA表达显著升高(P < 0.05)．提示：M-CSF可上调cyclinD1/D3和CDK2/6的mRNA表达,促进HeLa细胞的增殖．     

HA纳米载体介导转hGM-CSF基因的HepG2疫苗诱导的 抗瘤效应研究

目的:观察HA纳米颗粒载体介导转染hGM-CSF基因的HepG2细胞疫苗体外抗肿瘤效应,为hGM-CSF基因修饰的HepG2细胞疫苗的临床应用提供依据。方法:HA纳米颗粒载体介导hGM-CSF基因转染HepG2细胞制备转GMCSF基因的HepG2细胞疫苗。密度梯度离心法分离人PBMC,体外诱导人PBMC。WST-1法测定PBMC的增殖活性及对HepG2细胞的杀伤效应,流式细胞术分析CD4+和CD8+的阳性表达率,ELISA法测定INF-γ的分泌。结果:WST-1结果显示,转基因HepG2组疫苗能诱导PBMC增殖,其增殖率优于野生型疫苗(p<0.05);其诱导的PBMC对HepG2的杀伤率高于各野生型疫苗组和各空白对照组(p<0.05)。FCM结果显示,转基因HepG2疫苗组PBMC中CD4+和CD8+阳性表达率均高于各野生型疫苗组和各空白对照组(p<0.05)。ELISA结果显示,转基因组PBMC培养上清中IFN-γ含量为1989.76±254.21pg/ml,高于各野生型疫苗组和各空白对照组(p<0.05)。结论:HA纳米颗粒载体介导转染hGM-CSF基因能增加HepG2细胞疫苗的免疫原性,转hGM-CSF基因HepG2细胞疫苗可有效诱导PBMC增殖、分化,增加INF-γ的分泌,提高其对HepG2细胞的杀伤作用。     

抑制Rent1表达对HeLa细胞黏附能力的影响

Rent1是无义介导的mRNA降解(NMD)通路中的关键因子之一,通过引导含有提前出现的终止密码子(PTC)的mRNA至P-body,从而引发mRNA降解.为了进一步研究Rent1的生理功能,应用RNA干扰(RNAi)技术抑制Rent1的表达.试验发现,抑制Rent1的表达能够增强HeLa细胞对纤维粘连蛋白的黏附能力,另外,抑制Rent1的表达能够增加整合素基因ITGA2、ITGA3、ITGA6、ITGB5的表达.     

检测不同毒力结核分枝杆菌感染对巨噬细胞凋亡的影响及其Caspase-3和Bcl-2的表达

该文为探讨不同毒力的结核分枝杆菌感染对巨噬细胞凋亡的调控作用及其机制。实验用结核分枝杆菌国际标准强毒株H37Rv株和卡介苗BCG分别感染巨噬细胞RAW264.7株,同时设空白对照组,在感染后1,6,12,24 h,用流式细胞技术检测各组巨噬细胞的凋亡率,应用Western blot检测细胞Caspase-3和Bcl-2蛋白表达。结果发现,结核分枝杆菌感染组的凋亡率显著高于对照组,差异具有统计学意义(P<0.05);BCG感染组凋亡率高于H37Rv感染组,在感染后1,12,24 h凋亡率显著升高,差异具有统计学意义(P<0.05)。巨噬细胞感染结核分枝杆菌后其Caspase-3蛋白表达增高,结核分枝杆菌感染组的Caspase-3蛋白表达高于对照组:对照组相似文献   

siRNA沉默SIRT1基因对宫颈癌细胞HeLa增殖和凋亡的影响

化学合成靶向SIRT1基因的小干扰RNA,脂质体法转染人宫颈癌细胞株HeLa,观察小干扰RNA沉默SIRT1基因对HeLa增殖及细胞凋亡的影响。在优化siRNA SIRT1转染条件的基础上,应用RT-PCR和Western blot分别检测各组SIRT1 mRNA、SIRT1蛋白及凋亡相关蛋白的表达;CCK-8法检测细胞增殖抑制率;Hoechst荧光染色法和流式细胞仪检测细胞凋亡。结果表明,siRNA SIRT1转染细胞组SIRT1 mRNA水平和蛋白表达量明显低于对照组;siRNA SIRT1转染组细胞增殖受抑制,细胞凋亡率明显增加;凋亡相关蛋白P53、P21表达上调,Survivin表达下调。上述结果表明:siRNA SIRT1诱导的HeLa细胞凋亡与P53、P21、Survivin通路关系密切,但siRNA SIRT1诱导HeLa细胞凋亡的详尽机制有待进一步研究。     

OCT4 介导卵泡刺激素对永生化人卵巢上皮细胞株增殖、 凋亡和侵袭能力的影响

目的:探讨OCT4基因对卵泡刺激素作用下的永生化人卵巢上皮细胞株(Moody细胞)增殖、凋亡和侵袭能力影响。方法:将不同浓度的FSH(0、25、50、100mIU/ml)作用于Moody细胞48小时,应用Western-blot技术检测OCT4表达情况。采用慢病毒介导将重组质粒OCT4稳定转染至人卵巢上皮细胞株中,应用Western-blot法鉴定OCT4蛋白表达情况。FSH以50 mIU/ml作为工作浓度,实验对象分为4组:①siCon组,转染空载体的阴性对照组;②OCT4组:稳定转染OCT4基因的Moody细胞组;③FSH+siCon组:以FSH处理的siCon组;④FSH+OCT4组:以FSH处理的OCT4组。采用MTT比色法检测各组细胞的增殖情况,流式细胞仪检测各组细胞凋亡情况,Transwell侵袭实验检测各组细胞侵袭能力的变化。结果:(1)随着FSH浓度的增加,Moody细胞中OCT4蛋白表达逐渐增高,在FSH浓度为50 mIU/ml时达最高;(2)OCT4基因成功转染至Moody细胞中,经Western-blot检测该基因在细胞中进行蛋白高表达;(3)FSH+OCT4组细胞增殖活性明显增高,同时凋亡率降低,与另外三组相比差异具有统计学意义(P<0.05);(4)在FSH作用下,转染OCT4后明显增强了细胞的侵袭能力,与另外三组相比差异具有统计学意义(P<0.05)。结论:OCT4介导了FSH对人卵巢上皮细胞增殖、凋亡、侵袭活性的调控。     

The Campylobacter jejuni PEB1a adhesin is an aspartate/glutamate-binding protein of an ABC transporter essential for microaerobic growth on dicarboxylic amino acids

The PEB1a protein of the gastrointestinal pathogen Campylobacter jejuni mediates interactions with epithelial cells and is an important factor in host colonization. Cell fractionation and immunoblotting showed that PEB1a is most abundant in the periplasm of C. jejuni, and is detectable in the culture supernatant but not in the inner or outer membrane. The protein is homologous with periplasmic-binding proteins associated with ABC transporters and we show by fluorescence spectroscopy that purified recombinant PEB1a binds L-aspartate and L-glutamate with sub microM K(d) values. Binding of L-14C-aspartate or L-14C-glutamate was strongly out-competed by excess unlabelled aspartate or glutamate but only poorly by asparagine and glutamine. A mutant in the Cj0921c gene, encoding PEB1a, was completely unable to transport 5 microM L-14C-glutamate and showed a large reduction (approximately 20-fold) in the rate of L-14C-aspartate transport compared with the wild type. Although microaerobic growth of this mutant was little affected in complex media, growth on aspartate or glutamate in defined media was completely prevented, whereas growth with serine was similar to wild type. 1H-NMR analysis of the culture supernatants of the Cj0921c mutant showed some utilization of aspartate but not glutamate, consistent with the transport data. It is concluded that in addition to the established role of PEB1a as an adhesin, the PEB1 transport system plays a key role in the utilization of aspartate and glutamate, which may be important in vivo carbon sources for this pathogen.     

Human monoclonal antibodies derived from pleural effusion lymphocytes of a patient with breast carcinoma react with human breast cancer-associated antigens and mouse mammary tumor virus polypeptides

Four human hybridoma cell lines (PEB1-4) were established from a fusion of pleural effusion lymphocytes isolated from a breast cancer patient with metastatic disease, 6 years postmastectomy. The hybridomas secreted IgG-k (3 micrograms/ml/10(6) cells). These monoclonal antibodies (PEB1-4) reacted to different degrees with mouse mammary tumor virus (MMTV) and T47D particles (HuMTV). Immunological cross-reaction was also detected with antigens isolated from body fluids of breast cancer patients (BF-Ag). The binding capacity of the monoclonal antibodies (MAbs) PEB1-4 to the above-mentioned antigens was measured by RIA. The specificity of these antibodies was further demonstrated by radioimmunoprecipitation of MMTV, T47D (HuMTV) and BF-Ag. The binding of PEB1-4 to surface antigens of intact cells grown in culture was measured by RIA. Some of the MAbs were shown to bind more avidly to breast cancer cells than to nonbreast cancer cells or nonmalignant cells. The PEB1-4 human monoclonal antibodies may be found useful in analyzing the virus-breast cancer relationship.     

The virulence factor PEB4 (Cj0596) and the periplasmic protein Cj1289 are two structurally related SurA-like chaperones in the human pathogen Campylobacter jejuni

The PEB4 protein is an antigenic virulence factor implicated in host cell adhesion, invasion, and colonization in the food-borne pathogen Campylobacter jejuni. peb4 mutants have defects in outer membrane protein assembly and PEB4 is thought to act as a periplasmic chaperone. The crystallographic structure of PEB4 at 2.2-? resolution reveals a dimer with distinct SurA-like chaperone and peptidyl-prolyl cis/trans isomerase (PPIase) domains encasing a large central cavity. Unlike SurA, the chaperone domain is formed by interlocking helices from each monomer, creating a domain-swapped architecture. PEB4 stimulated the rate of proline isomerization limited refolding of denatured RNase T(1) in a juglone-sensitive manner, consistent with parvulin-like PPIase domains. Refolding and aggregation of denatured rhodanese was significantly retarded in the presence of PEB4 or of an engineered variant specifically lacking the PPIase domain, suggesting the chaperone domain possesses a holdase activity. Using bioinformatics approaches, we identified two other SurA-like proteins (Cj1289 and Cj0694) in C. jejuni. The 2.3-? structure of Cj1289 does not have the domain-swapped architecture of PEB4 and thus more resembles SurA. Purified Cj1289 also enhanced RNase T(1) refolding, although poorly compared with PEB4, but did not retard the refolding of denatured rhodanese. Structurally, Cj1289 is the most similar protein to SurA in C. jejuni, whereas PEB4 has most structural similarity to the Par27 protein of Bordetella pertussis. Our analysis predicts that Cj0694 is equivalent to the membrane-anchored chaperone PpiD. These results provide the first structural insights into the periplasmic assembly of outer membrane proteins in C. jejuni.     

Sustained activation of MEK1-ERK1/2 pathway in membrane skeleton occurs dependently on cell adhesion in megakaryocytic differentiation

A human megakaryoblastic cell line, CMK, was treated with 12-o-tetradecanoylphorbol-13-acetate (TPA) for differentiation-induction. We examined TPA-induced activation of the MEK1-ERK1/2 pathway in the 100,000g Triton X-insoluble fraction of CMK cells as the membrane skeleton and researched the relation of the MEK1-ERK1/2 activation with integrin expression. We found that this activation was divided into two phases: the first activation occurred transiently in the membrane skeleton fraction of the suspended cell status and diminished after 1h; and the second sustained activation was maintained by cell adhesion. TPA-treated CMK cells revealed increased expression of integrins alphaIIb and beta3 only when the cell adhesion persisted, regardless of the difference of culture substratum. Sustained activation of the MEK1-ERK1/2 pathway is generated in the membrane skeleton by continuous cell adhesion and seems to be essential to TPA-induced megakaryocytic differentiation of CMK cells.     

Effects of antioxidant enzyme overexpression on the invasive phenotype of hamster cheek pouch carcinoma cells.

To examine the role of reactive oxygen species on the invasive phenotype of cancer cells, we overexpressed manganese- and copper-zinc-containing superoxide dismutases (MnSOD, CuZnSOD) and catalase (Cat) in hamster cheek pouch carcinoma (HCPC-1) cells in vitro using adenoviral vector-mediated gene transfer. Hamster cheek pouch carcinoma cells were transduced with these adenoviral vector constructs alone, or in combination, at concentrations [i.e., multiplicity of infectivity (MOI)] of 100 MOI each. The Escherichia coli beta-galactosidase reporter construct was used as a control virus. Protein expression was examined by Western blot analysis and enzymatic activities were measured using spectrophotometry. To observe the effects of transgene overexpression on in vitro tumor cell invasion, we used the membrane invasion culture system, an accurate and reliable method for examining tumor cell invasion, in vitro. This assay measures the ability of tumor cells to invade a basement membrane matrix consisting of type IV collagen, laminin, and gelatin. MnSOD overexpression resulted in a 50% increase in HCPC-1 cell invasiveness (p < .001); co-overexpression of MnSOD with Cat partially inhibited this effect (p < .05). Moreover, co-overexpression of both SODs resulted in a significant increase in invasiveness compared with the parental HCPC-1 cells (p < .05). These changes could not be correlated with the 72 kDa collagenase IV or stromolysin activities using zymography, or the downregulation of the adhesion molecules E-cadherin or the alpha4 subunit of the alpha4beta1 integrin. These results suggest that hydrogen peroxide may play a role in the process of tumor cell invasion, but that the process does not rely on changes in matrix metalloproteinase activity in the cells, or the expression of cell adhesion molecules.     

Molecular organization and pigment composition of R-phycoerythrin from the red alga Callithamnion rubosum

p3phycoerythrin is the major phycobiliprotein of Rhodophyta and endows these algae with the characteristic color. R-phycoerythrin purified from red alga Calithamnion rubosom is composed of four dissimilar polypeptide subunits, alpha, beta, gamma, and delta. In calibrated SDS gel electrophoresis their molecular weights are 21 000, 21 600, 31 000 and 33 000 daltons, respectively. The stoichiometry of the subunits in the native protein is 9 alpha: 9 beta: 2 gamma: 1 delta. R-phycoerythrin carries two covalently linked apoprotein red tetrapyrrol pigments: phycoerythrobilin (PEB) and phycourobilin (PUB). Chemical and spectroscopic data show that alpha subunit carries solely two PEB chromophores, beta subunit--3 PEB and 1 PUB groups, gamma subunit--3 PEB and 2 PUB groups and delta subunit--1 or 2 PEB and 1 PUB groups. The chromophore and polypeptide structure of R-phycoerythrin is mostly composed of all known phycobiliproteins of red and blue-green algae.     

Role of the F-box protein Skp2 in adhesion-dependent cell cycle progression.

Cell adhesion to the extracellular matrix (ECM) is a requirement for proliferation that is typically lost in malignant cells. In the absence of adhesion, nontransformed cells arrest in G1 with increased levels of the cyclin-dependent kinase inhibitor p27. We have reported previously that the degradation of p27 requires its phosphorylation on Thr-187 and is mediated by Skp2, an F-box protein that associates with Skp1, Cul1, and Roc1/Rbx1 to form the SCF(Skp2) ubiquitin ligase complex. Here, we show that the accumulation of Skp2 protein is dependent on both cell adhesion and growth factors but that the induction of Skp2 mRNA is exclusively dependent on cell adhesion to the ECM. Conversely, the expression of the other three subunits of the SCF(Skp2) complex is independent of cell anchorage. Phosphorylation of p27 on Thr-187 is also not affected significantly by the loss of cell adhesion, demonstrating that increased p27 stability is not dependent on p27 dephosphorylation. Significantly, ectopic expression of Skp2 in nonadherent G1 cells resulted in p27 downregulation, entry into S phase, and cell division. The ability to induce adhesion-independent cell cycle progression was potentiated by coexpressing Skp2 with cyclin D1 but not with cyclin E, indicating that Skp2 and cyclin D1 cooperate to rescue proliferation in suspension cells. Our study shows that Skp2 is a key target of ECM signaling that controls cell proliferation.     

Polyoma virus middle-T-transformed PECAM-1 deficient mouse brain endothelial cells proliferate rapidly in culture and form hemangiomas in mice

Wild-type mouse brain endothelial (bEND) cells transformed with the polyoma virus middle-T proliferate rapidly in culture and form hemangiomas in mice. These cells express high levels of platelet/endothelial cell adhesion molecule-1 (PECAM-1), a molecule shown to be important during hemangioma formation. In this study, we have examined the ability of polyoma virus middle-T-transformed mouse bEND cells prepared from PECAM-1-/- mice to proliferate in culture and form hemangiomas in mice. We show that these cells express a number of endothelial cell markers and share a similar morphology with PECAM-1+/+ bEND cells. PECAM-1-/- bEND cells exhibit a limited ability to form tubes in Matrigel and rapidly form hemangioma when injected into nude mice, very similar to PECAM-1+/+ bEND cells. These cells, however, have increased proliferation, slower migration, altered endothelial cell adhesion molecule expression, and are less adherent when compared to PECAM-1+/+ bEND cells. Therefore, lack of PECAM-1 expression impacts polyoma middle-T-transformed endothelial cell proliferative, adhesive, and migratory properties without impacting their ability to rapidly form hemangiomas in mice or poorly organize to capillary-like structures in Matrigel.     

Phosphatidylinositol 3-kinase is a key regulator of early phase differentiation in keratinocytes

The survival and growth of epithelial cells depend on adhesion to the extracellular matrix. Because epidermal keratinocytes differentiate as they leave the basement membrane, an adhesion signal may regulate the initiation of differentiation. Phosphatidylinositol 3-kinase (PI3K) is a fundamental signaling molecule that regulates the adhesion signal. Transfection of a dominant negative form of PI3K into keratinocytes using an adenovirus vector resulted in significant morphological changes comparable to differentiation and the induction of differentiation markers, keratin (K) 1 and K10. In turn, transfection with the constitutively active form of PI3K almost completely abolished the induction of K1 and K10 by differentiation in suspension cultures using polyhydroxyethylmethacrylate-coated dishes. PI3K activity was lost in suspension culture, except by cells bearing the constitutively active form of PI3K. These data demonstrate that blockade of PI3K results in differentiation and that activation of PI3K prevents differentiation. Furthermore, expression of the dominant negative form of PI3K significantly inhibited keratinocyte adhesion to the extracellular matrix and reduced the surface expression of alpha(6) and beta(1) integrins in suspension culture. Moreover, expression of the active form of PI3K restored the mRNA levels of adhesion molecules that were reduced in suspension culture, including alpha(3), alpha(6), and beta(1) integrins, BP180, and BP230. In conclusion, loss of PI3K activity results in keratinocytes leaving the basement membrane and the initiation of a "default" differentiation mechanism.