小鼠巨噬细胞内表达结核分枝杆菌CFP10-ESAT6融合蛋白对细胞增殖和凋亡的影响

 目的：建立培养滤液蛋白10-早期分泌性抗原靶6（CFP10-ESAT6）真核表达质粒并转染RAW2647巨噬细胞,观察胞内表达CFP10-ESAT6对细胞活性及凋亡的影响,并初步探讨其机制。方法：将CFP10-ESAT6融合基因插入真核表达质粒pEGFP-N1,构建重组质粒,转染RAW264.7巨噬细胞。采用MTT的方法测定CFP10-ESAT6融合蛋白对RAW264.7巨噬细胞活性的影响,采用结核分枝杆菌19 kD脂蛋白和十字孢碱（staurosporine）处理RAW264.7巨噬细胞,并以流式细胞术检测细胞凋亡率以及Toll样受体2（TLR2）的表达。结果：成功构建了重组质粒pEGFP-N1/CFP10-ESAT6并转染至RAW264.7巨噬细胞;与对照组相比,胞内表达CFP10-ESAT6不能影响巨噬细胞的活性,但可以明显抑制结核分枝杆菌19 kD脂蛋白所造成的凋亡(P＜0.05),并显著抑制了TLR2的表达(P＜0.05)。结论：巨噬细胞内表达的CFP10-ESAT6融合蛋白不具有细胞毒性作用,但可以通过下调TLR2的表达来抑制结核分枝杆菌19 kD脂蛋白所造成的凋亡。     

干扰P2X_7R基因对RAW264.7细胞增殖和吞噬的影响

目的:应用RNA干扰技术抑制小鼠巨噬细胞RAW264.7细胞P2X7受体(P2X7R)基因的表达,建立稳定干扰细胞株,并观察其对细胞增殖和凋亡的影响。方法:用脂质体法将P2X7R shRNA重组质粒转染至RAW264.7细胞,经G418筛选后获得稳定干扰细胞株。细胞分为野生型(WT)组、阴性对照(NC)组和干扰(sh P2X7R)组。Real-time PCR法检测细胞中P2X7R mRNA的表达,Western blot检测细胞中P2X7R蛋白的表达;CCK-8方法检测细胞生长活性,5-乙炔基-2’-脱氧尿苷(5-ethynyl-2’-deoxyuridine,Ed U)掺入实验检测细胞增殖活性;流式细胞术分析细胞周期的分布和吞噬情况。结果:P2X7R shRNA能明显抑制RAW264.7细胞的P2X7R mRNA和蛋白的表达,抑制率在80%以上。48 h后,sh P2X7R组细胞的生长速度明显高于NC组和WT组(P0.05),增殖期细胞比例明显升高(P0.05),说明下调P2X7R基因能明显促进细胞增殖。sh P2X7R组的细胞周期出现改变,S期和G2/M期的比例明显上升,增殖指数增高(P0.05)。sh P2X7R组的细胞吞噬活性明显高于NC组(P0.05)。结论:本研究成功构建了稳定干扰P2X7R基因表达的小鼠巨噬细胞株RAW264.7,sh P2X7R能够明显促进RAW264.7细胞的增殖,改变了细胞的吞噬活性。     

香菇茯苓银耳复合多糖对小鼠巨噬细胞功能的作用研究

目的：研究香菇茯苓银耳复合多糖对不同状态下巨噬细胞功能的调节作用。方法：将巨噬细胞株RAW264.7细胞分别与复合多糖（CP）、复合多糖联合LPS（CP+LPS）、复合多糖预孵育后联合LPS（Pre-CP+LPS）共孵育,采用荧光显微镜和流式细胞仪检测RAW264.7细胞吞噬荧光微球情况;采用Griess试剂检测RAW264.7细胞分泌NO水平;采用流式细胞术检测巨噬细胞表面活化标志物CD40、CD80和CD86的表达水平。结果：RAW264.7细胞与复合多糖共孵育条件下,复合多糖可以增强RAW264.7细胞对荧光微球的吞噬作用,且随着时间的延长愈加明显;复合多糖低浓度对RAW264.7细胞分泌NO无影响,当浓度达1 000 μg/ml时才能促进其显著分泌NO;复合多糖可以提高RAW264.7细胞CD40、CD80和CD86分子表达水平。在LPS刺激条件下,复合多糖联合LPS与复合多糖预孵育后联合LPS两种处理都可以减轻LPS导致的CD40、CD80和CD86活化标志过表达现象。结论：复合多糖对不同状态下巨噬细胞功能具有调节作用,即可以促进静息状态下巨噬细胞活化和吞噬作用,而对活化状态的巨噬细胞功能具有抑制作用。     

干扰盐皮质激素受体基因对RAW 264.7细胞增殖和凋亡的影响

 目的：应用RNA干扰技术沉默小鼠RAW 264.7巨噬细胞盐皮质激素受体(MR)基因,建立稳定干扰细胞株,并观察其对细胞增殖和凋亡的影响。方法：针对MR基因设计合成重组MR shRNA质粒,脂质体法转染质粒至RAW 264.7细胞,经G418筛选后获得稳定表达细胞株。细胞分为3组：野生型(WT)组、阴性对照（NC）组和干扰（shMR）组。荧光显微镜下观察确定细胞的转染效率;实时定量PCR法检测细胞中MR mRNA的表达;CCK-8方法检测细胞增殖活性;流式细胞术分析细胞周期分布和凋亡情况。结果：(1) MR shRNA能明显抑制RAW 264.7细胞的MR基因表达,抑制率70%以上。(2) 从第3 d开始,shMR组细胞的生长速度明显低于NC和WT组（P<0.05）,说明MR shRNA能明显抑制细胞增殖。(3) WT、NC和shMR组的增殖指数分别为(37.2±0.5)%、(37.5±1.6)%和(31.0±1.3)%,shMR组的细胞周期出现改变,S期和G₂/M期比例明显下降,增殖指数下降（P<0.05）。(4) WT、NC和shMR组的细胞凋亡率分别为(2.18±0.36)%、(6.65±0.81)%和(7.70±1.34)%,shMR组略高于NC组,但二者的差异无统计学意义（P>0.05）。结论：本研究成功构建了稳定干扰MR基因表达的RAW 264.7细胞株,MR shRNA能够明显抑制RAW 264.7细胞增殖,但对其凋亡无明显影响。     

Axl and MerTK receptor tyrosine kinases maintain human macrophage efferocytic capacity in the presence of viral triggers

The requirement to remove apoptotic cells is equally important in homeostasis and inflammatory disease. In particular, during viral infections large quantities of infected cells undergo apoptosis and need to be efficiently cleared by phagocytes to prevent secondary necrosis. Although specific roles of several apoptotic cell sensors, such as the TAM (Tyro3, Axl, MerTK) receptor family, have been characterized in mouse models, little is known about their regulation and involvement in apoptotic cell uptake (efferocytosis) by human macrophages under inflammatory conditions. We show that whereas pro‐inflammatory stimuli consistently downregulated MerTK expression in human monocyte‐derived macrophages (MDMs), stimuli indicative of a viral infection, interferon‐α (IFN‐α) and the TLR3 ligand poly(I:C), specifically induced Axl expression and promoted binding of the bridging molecule Gas6. Axl induction by IFN‐α and poly(I:C) was associated with higher MDM efferocytic capacity compared to cells treated with other pro‐inflammatory stimuli, such as LPS and IFN‐γ. While MerTK blocking antibody uniformly suppressed apoptotic cell uptake by MDMs, Axl blocking antibody significantly reduced efferocytosis by poly(I:C)‐stimulated MDMs, but not by resting MDMs. Our observations demonstrate that Axl induction during viral infections contributes to maintaining macrophage capacity to engulf apoptotic cells, which may have important consequences for resolution of anti‐viral immune responses.     

Functional expression of Nramp1 in vitro in the murine macrophage line RAW264.7

Mutations at the Nramp1 locus in vivo cause susceptibility to infection by unrelated intracellular microbes. Nramp1 encodes an integral membrane protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagosome to control microbial replication is unknown. To devise an in vitro assay for Nramp1 function, we introduced a wild-type Nramp1(G169) cDNA into RAW 264.7 macrophages (which bear a homozygous mutant Nramp1(D169) allele and thus are permissive to replication of specific intracellular parasites). Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane of Salmonella typhimurium and Yersinia enterocolitica containing phagosomes. Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication of S. typhimurium. Studies with a replication-defective S. typhimurium mutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells, Nramp1, and Nramp2 transfectants could all efficiently kill a temperature-sensitive, replication-defective mutant of S. typhimurium. Finally, increased antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele. Together, these results indicate that transfection of Nramp1 cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.     

Aire 对巨噬细胞极化影响的研究

目的:研究自身免疫调节因子(Aire)对巨噬细胞极化的影响。方法:分别用LPS、IL-4 以及LPS 联合免疫复合物刺激小鼠单核巨噬细胞系RAW264郾7 细胞、稳定表达GFP-Aire 的RAW264.7 细胞(A33-3) 细胞和稳定表达GFP 的RAW264.7 细胞(C1-6),使其向M1(LPS)、M2a(IL-4)和M2b(LPS 联合免疫复合物)型巨噬细胞极化。通过Real-time PCR 检测各组细胞中M1 型巨噬细胞特征分子IL-1、iNOS 和IL-6,M2a 型特征分子Arg-1 和M2b 型特征分子IL-10 的表达水平,研究Aire 对各种类型巨噬细胞极化的影响。结果:LPS 在0.5 g/ ml 浓度时,RAW264.7 细胞中M1 型巨噬细胞产物IL-1 、iNOS和IL-6 基因表达量最高;而IL-4 以及LPS 联合免疫复合物的刺激作用有显著的剂量依赖性,都在浓度最高时RAW264.7 细胞中Arg1(M2a)和IL-10(M2b)基因表达量最高。LPS 刺激后,A33-3 细胞中IL-1 和iNOS 表达水平明显高于C1-6 细胞,IL-6 则相反;IL-4 及LPS 联合免疫复合物刺激后,A33-3 细胞中Arg1 和IL-10 的表达水平明显低于C1-6 细胞。结论:Aire 可能促进巨噬细胞向M1 极化,同时抑制其向M2a 和M2b 极化。     

The pH 6 antigen is an antiphagocytic factor produced by Yersinia pestis independent of Yersinia outer proteins and capsule antigen

The pH 6 antigen (pH 6 Ag; PsaA) of Yersinia pestis has been shown to be a virulence factor. In this study, we set out to investigate the possible function of Y. pestis PsaA in a host cell line, RAW264.7 mouse macrophages, in order to better understand the role it might play in virulence. Y. pestis KIM5 derivatives with and without the pCD1 plasmid and their psaA isogenic counterparts and Escherichia coli HB101 and DEta5alpha carrying a psaA clone or a vector control were used for macrophage infections. Macrophage-related bacteria and gentamicin-resistant intracellular bacteria generated from plate counting and direct microscopic examinations were used to evaluate these RAW264.7 macrophage infections. Y. pestis psaA isogenic strains did not show any significant difference in their abilities to associate with or bind to mouse macrophage cells. However, expression of psaA appeared to significantly reduce phagocytosis of both Y. pestis and E. coli by mouse macrophages (P < 0.05). Furthermore, we found that complementation of psaA mutant Y. pestis strains could completely restore the ability of the bacteria to resist phagocytosis. Fluorescence microscopy following differential labeling of intracellular and extracellular Y. pestis revealed that significantly lower numbers of psaA-expressing bacteria were located inside the macrophages. Enhanced phagocytosis resistance was specific for bacteria expressing psaA and did not influence the ability of the macrophages to engulf other bacteria. Our data demonstrate that Y. pestis pH 6 Ag does not enhance adhesion to mouse macrophages but rather promotes resistance to phagocytosis.     

GFP／HBVX融合蛋白重组载体的构建及稳定表达细胞系的建立

目的构建绿色荧光蛋白（GFP）与人乙型肝炎病毒（HBV）X基因的重组表达载体，建立稳定表达HBVX蛋白（HBx）与GFP融合蛋白的HepG2细胞系，以进一步研究HBx的生物学功能及其在肝癌发生中的作用。方法应用PCR法从adr亚型HBV质粒pHBVDNA中扩增HBVX基因片段，PCR产物经HindⅢ和KpnⅠ双酶切后定向插入绿色荧光蛋白真核表达载体pEGFP-C1的相应酶切位点，转化宿主菌DH5α，采用上述双酶切及DNA测序鉴定重组质粒pGFP-HBx；采用脂质体转染法将pEGFP-C1质粒、pGFP-HBx重组质粒DNA转染人肝母细胞瘤细胞系HepG2细胞，G418选择抗性细胞克隆，荧光显微镜下观察GFP表达，挑取表达GFP的抗性克隆扩大培养、传代。采用RT-PCR检测转染细胞HBVX基因的表达。结果经酶切及测序鉴定成功构建了GFP-HBVX重组表达载体pGFP-HBx；将pEGFP-C1、pGFP-HBx重组质粒转染HepG2．，经G418筛选15d获得抗性细胞克隆。将带绿色荧光的抗性克隆细胞扩大培养并经传代70次，细胞仍表达强的荧光蛋白。RT-PCR检测表明转染pGFP-HBx重组体的HepG2／GFP-HBx细胞有HBVX转录、表达。结论成功构建了GFP．HBVX真核重组表达载体pGrP-HBx；获得了稳定表达GFP-HBx融合蛋白的HepG2细胞系，这为进一步研究FIBx的生物学功能以及HBx在肝癌发生中的作用与机制打下了基础。     

丹酚酸B在巨噬细胞胆固醇外排中的作用

目的考察丹酚酸B（SAB）对巨噬细胞胆固醇外排的影响。方法分别采用巨噬细胞RAW264．7和PMA诱导的THP．1细胞,利用流式细胞仪检测丹酚酸B对已摄取DiI—acLDL的细胞外排脂质作用的影响,对RAW264．7细胞表面ABCA1蛋白表达水平的影响。采用ABCAl的抑制剂DIDS及小分子干扰RNA的方法检测ABCA1在丹酚酸B诱导的促进脂质外排过程中的作用。进而利用靶向CD36的小分子干扰RNA,通过抑制CD36的表达,检测CD36在丹酚酸B促进载脂巨噬细胞脂质外排过程中的作用。结果采用DiI荧光标记的脂质检测巨噬细胞对脂质的外排作用,结果显示SAB显著增加了荷脂细胞对DiI－acLDL的外排作用。对脂代谢相关转运蛋白表达的检测结果显示,在荷脂的RAW264．7细胞中,SAB能够以一定剂量依赖的方式增加细胞表面ABCA1蛋白水平的表达。进一步研究显示,上述SAB促进外排的作用可被ABCAl的抑制剂或者siRNA的作用消除,确证了ABCA1在SAB介导的脂质外排过程中的重要作用。同样利用CD36小分子干扰RNA将CD36表达抑制后,SAB促进外排的作用消失,说明SAB介导的细胞脂质的外排作用同样依赖于CD36。结论丹酚酸B通过上调ABCA1的表达,促进巨噬细胞胆固醇或脂质的外排,且该作用依赖于ABCA1及CD36,为SAB抗动脉粥样硬化作用新机制的研究提供了重要依据。     

The Axl receptor tyrosine kinase is a discriminator of macrophage function in the inflamed lung

Much of the biology surrounding macrophage functional specificity has arisen through examining inflammation-induced polarizing signals, but this also occurs in homeostasis, requiring tissue-specific environmental triggers that influence macrophage phenotype and function. The TAM receptor family of receptor tyrosine kinases (Tyro3, Axl and MerTK) mediates the non-inflammatory removal of apoptotic cells by phagocytes through the bridging phosphatidylserine-binding molecules growth arrest-specific 6 (Gas6) or Protein S. We show that one such TAM receptor (Axl) is exclusively expressed on mouse airway macrophages, but not interstitial macrophages and other lung leukocytes, under homeostatic conditions and is constitutively ligated to Gas6. Axl expression is potently induced by granulocyte-macrophage colony-stimulating factor expressed in the healthy and inflamed airway, and by type I interferon or Toll-like receptor-3 stimulation on human and mouse macrophages, indicating potential involvement of Axl in apoptotic cell removal under inflammatory conditions. Indeed, an absence of Axl does not cause sterile inflammation in health, but leads to exaggerated lung inflammatory disease upon influenza infection. These data imply that Axl allows specific identification of airway macrophages, and that its expression is critical for macrophage functional compartmentalization in the airspaces or lung interstitium. We propose that this may be a critical feature to prevent excessive inflammation because of secondary necrosis of apoptotic cells that have not been cleared by efferocytosis.     

趋化因子受体7基因绿色荧光慢病毒载体构建及在未成熟树突状细胞中的表达**

背景：趋化因子受体7(chemokine receptor-7, CCR7)是树突状细胞从外周迁移至淋巴系统发挥作用的最重要的启动和调节者,但未成熟树突状细胞表面不表达CCR7,因此利用携带CCR7基因的未成熟树突状细胞可以更好地诱导免疫耐受。 目的：构建携带小鼠CCR7基因的绿色荧光蛋白重组慢病毒载体,观察其在未成熟树突状细胞中的表达。 方法：采用RT-PCR扩增小鼠CCR7基因并克隆至pCR-Blunt载体。将CCR7 DNA片段及IRES-GFP连入慢病毒转移质粒LV-Lac,生成重组慢病毒质粒LV-CCR7。采用脂质体转染法将慢病毒系统3质粒(重组慢病毒质粒LV-CCR7、包装质粒     ΔNRF及包膜质粒pVSVG)共转染包装慢病毒,重组慢病毒感染未成熟树突状细胞,光学显微镜观察细胞状态,流式细胞术鉴定CCR7蛋白的表达。 结果与结论：实验成功扩增出小鼠CCR7 DNA片段并克隆至pCR-Blunt载体,亚克隆构建慢病毒表达载体LV-CCR7,经3质粒包装系统感染293 FT细胞后,24 h于荧光显微镜下均观察到绿色荧光蛋白阳性表达,病毒滴度为108 U/L以上,获得携带CCR7基因的重组慢病毒。慢病毒颗粒可有效感染未成熟树突状细胞,荧光显微镜可见大量GFP蛋白表达,阳性细胞达50%,流式细胞术检测到CCR7蛋白表达,LV-CCR7基因修饰的未成熟树突状细胞仍保持在未成熟状态。结果证实,实验成功构建携带小鼠CCR7基因绿色荧光慢病毒载体LV-CCR7,并可在未成熟树突状细胞细胞中表达。 关键词：趋化因子受体7;未成熟树突状细胞;慢病毒载体;真核表达;组织工程 doi:10.3969/j.issn.1673-8225.2012.01.029     

Angiopoietin-Like Protein 7 Promotes an Inflammatory Phenotype in RAW264.7 Macrophages Through the P38 MAPK Signaling Pathway

Angiopoietin-like protein 7 (Angptl7) has been extensively studied for decades, but its potential immune functions have not been characterized. Hence, we investigated the relationship between Angptl7 and inflammation by using RAW264.7 monocyte/macrophage cells. The expression of genes encoding inflammation-associated factors cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, IL-10, and transforming growth factor beta 1 (TGF-β1)) decreased after RAW264.7 cells were treated with anti-Angptl7 polyclonal antibody but increased after the cells were transfected with an Angptl7-expressing plasmid. Angptl7 overexpression enhanced phagocytosis and inhibited the proliferation of RAW264.7 cells. In addition, Angptl7 antagonized the anti-inflammatory effects of TGF-β1 and dexamethasone. Pathway analysis showed that Angptl7 promoted the phosphorylation of both p65 and p38, but only the P38 mitogen-activated protein kinase (MAPK) signaling pathway mediated Angptl7-associated inflammatory functions. Additionally, after 1 week of daily intraperitoneal injections of recombinant TNF-α in a mouse model of peripheral inflammation, Angptl7 expression increased in the mouse eyes. Thus, Angptl7 is a factor that promotes pro-inflammatory responses in macrophages through the P38 MAPK signaling pathway and represents a potential therapeutic target for treatment of inflammatory diseases.     

重组大鼠ΔNΔC/VEGF-C/Cys152Ser逆转录病毒载体的构建及表达

目的:构建重组大鼠ΔNΔC/VEGF-C/Cys152Ser(ddVEGF-C)逆转录病毒载体,建立稳定表达ddVEGF-C的PT67包装细胞株,并验证其表达。方法:利用PCR从pSecTag-ddVEGF-C中获取大鼠ddVEGF-C基因,克隆到pLPCX逆转录病毒载体,经PCR、双酶切和测序验证,转染PT67包装细胞,嘌呤霉素筛选出稳定表达目的基因的包装细胞株,用反转录PCR(RT-PCR)和Western blot方法验证其在RAW 264.7细胞中的表达。结果:构建的pLPCX-ddVEGF-C逆转录病毒载体经双酶切和PCR鉴定正确,测序结果与ddVEGF-C序列一致,病毒滴度为2×107CFU/mL,RT-PCR和Western blot结果提示重组病毒感染RAW264.7细胞能正确表达ddVEGF-C。结论:成功构建了pLPCX-ddVEGF-C逆转录病毒载体,并建立了稳定表达ddVEGF-C的PT67包装细胞株,为以VEGF-C基础的实验研究提供了新工具。     

Effects of Lycium barbarum polysaccharides with different molecular weights on function of RAW264.7 macrophages

The present study was aimed at investigating the effects of four LBP fractions with different molecular weights (MWs), designated LBP2, LBP3, LBP4 and LBP5, on RAW264.7 macrophages function. Results showed that LBP fractions could significantly enhance the expression of CD86 and MHC-II molecules on RAW264.7 macrophages. LBP3, LBP4 and LBP5 could enhance the production of ROS, NO, TNF-α and IL-6, and the phagocytosis of RAW264.7 macrophages. LBP2 with an MW of larger than 350?kDa could only enhance the secretion of TNF-α. LBP3 enhanced the RAW264.7 macrophages function in a dose-dependent manner and also enhanced the iNOS mRNA expression in the cells. These results demonstrated that the immunomodulatory activity of LBP on RAW264.7 macrophages was closely related to its MW. It indicated that fractions with an MW smaller than 350?kDa were the main active fractions of LBP in enhancing macrophages function.     

Rab7负向调控巨噬细胞中CpG诱导的细胞因子的表达

目的:研究Rab7过表达及失活突变(Rab7T22N)对CpG刺激的RAW264.7巨噬细胞中分泌细胞因子的影响。方法:采用RT-PCR和Real-time PCR检测RAW264.7细胞中Rab7在CpG刺激下表达模式。将Rab7真核表达质粒及失活突变质粒Rab7T22N通过脂质体法转染RAW264.7细胞,G418稳定筛选,Western法鉴定表达效果。用CpG刺激稳定表达Rab7的RAW264.7细胞系,RT-PCR和Real-time PCR检测细胞因子IL-6、IL-1β、IFN-β的表达量变化。结果:CpG刺激RAW264.7后,Rab7 mRNA表达水平逐渐增高,在8小时达到高峰,表达增高近4倍。Rab7过表达后,CpG刺激后产生的IL-6、IL-1β、IFN-β显著降低。巨噬细胞中Rab7失活突变后,在CpG刺激后IL-6、IL-1β、IFN-β的表达又显著增加。结论:CpG促进RAW264.7巨噬细胞中Rab7 mRNA的表达,Rab7抑制了CpG刺激的巨噬细胞中IL-6、IL-1β、IFN-β的表达,该抑制作用的发挥与其酶活性的GTP结合有关。该研究为进一步阐明Rab7在CpG/TLR9信号通路中的作用奠定了基础。     

同源无启动子DNA片段抑制人胰腺癌细胞株整合基因的表达

目的观察仅转染无启动子绿色荧光蛋白（GFP）的cDNA全长片段能否使整合有GFP基因的胰腺癌细胞内GFP表达降低。方法用pEGFP—C1质粒转染人胰腺癌细胞株PANC-1,G418筛选及流式细胞术分选建立GFP蛋白高表达的稳定细胞株。用PCR方法从pEGFP—C1扩增出GFP基因全长cDNA并将其克隆至无启动子的复制型质粒PUC19,得到重组质粒即PUC—GFP。实验分为4组：空白对照组、空质粒组（PUC组）、GFP重组质粒干扰组（PUC—GFP组）、GFP小RNA干扰组（siGFP组）。分别用稳定表达GFP和未经处理PANC-1细胞进行实验。用Western印迹、流式细胞术与倒置荧光显微镜检测重组质粒对细胞内稳定表达的GFP的影响及对GFP阴性细胞pEGFPC1质粒瞬时转染后绿色荧光表达强度的影响。结果PUC—GFP可使稳定细胞株内的GFP表达下降,并且呈剂量依赖性。PUC—GFP组绿色荧光强度减弱程度和空白对照及空质粒组差异有统计学意义（P〈0．05）和siGFP组差异无统计学意义（P〉0．05）。转染后第4天GFP表达降低并可持续至第6天,第4天后PUC—GFP组和siGFP组对GFP的抑制差异无统计学意义（P〉0．05）。PUC—GFP与pEGFP—C1共转染GFP阴性PANC-1细胞株可使pEGPC1的瞬时转染效率降低。这两种情况下,荧光降低程度均和转染PUC—GFP的量呈正相关。结论仅转染无启动子的某个特定基因的全长cDNA能使哺乳细胞内相应同源性基因的表达降低。DNA干扰可用于哺乳动物细胞。     

Viili多糖对巨噬细胞RAW264.7激活及分泌细胞因子的影响

探讨viili胞外多糖(Viili exopolysaccharides,VEPS)对小鼠巨噬细胞RAW264.7激活和增殖的影响。噻唑蓝(MTT)比色法检测细胞的生长与增殖;中性红吞噬实验检测吞噬活性;Griess试剂盒检测培养上清液中NO分泌量,ELISA法检测VEPS不同浓度及不同作用时间培养上清中IL-6,IL-1β含量;扫描电子显微镜观察VEPS对细胞形态的影响;碘化丙啶(PI)染色检测VEPS对细胞周期的影响。结果显示,VEPS对RAW264.7细胞的增殖、吞噬能力、分泌NO、IL-6、IL-1β等都有显著的促进作用,VEPS为100μg/ml时促进作用最明显,呈剂量相关,作用72h时细胞因子分泌量达到最大,72h后下降。VEPS激活巨噬细胞并使其变得扁平伸展且形成伪足。VEPS促进G1期细胞增多,提高细胞的增殖能力。VEPS免疫调节作用与其激活RAW264.7细胞,促进NO、IL-6、IL-1β等分泌有关,且VEPS与LPS对RAW264.7细胞有相似的作用规律。以上结果证明VEPS能激活巨噬细胞,也可能最终激活淋巴细胞,达到增强非特异性和特异性免疫的作用。