Synthesis of multiple copies of neurotrophic peptide and their high level expressions in E.coli

AIM: To construct and express containing multiple tandem copies of a peptide (neurotrophic peptide,NP),which was designed according to the NP sequence of prosaposin.METHODS: DNA sequence of peptide NP was synthesized by the preferred codons of E.coli.Two copies of NP fragments were produced by PCR and inserted into pUC18 vector.The fragments from pUC18-2NP by EcoT 14I were ligated into tandem multi-NP fragments through self-ligation,and subcloned into pET-EcoT vector,which has a unique EcoT14I cloning site allowing unidirectional insertion of a desired sequence.Multi-NP clones were screened by PCR-array.RESULTS: The constructs with different repeats of NP were obtained and expressed as fused-proteins at high level in E.coli BL21 (DE3).In order to get monomer peptide,each copy of peptide was interspersed by a unique site where the fused-protein could be cleaved by cyanogens bromide.CONCLUSION: Peptide NP could be highly expressed in E.coli.This work builds a solid foundation for further study on its bioactivity.     

Effects of lipopolysaccharide binding protein on activation of p38 signaling pathway induced by LPS in macrophages

AIM:To investigate the regulatory effects of lipopolysaccharide binding protein(LBP)on activation of p38 signaling pathway induced by lipopolysaccharide(LPS)in alveolar macrophages.METHODS:The LBP from actue phase rat serum was purified by ammonium sulphate precipitation, Bio-Rex70 resin and the MonoQ column. Rat alveolar macrophages were exposed to LPS (0.01 mg/L or 1 mg/L) the various concentrations of LBP(0 mg/L, 0.01 mg/L, 0.1 mg/L, 1 mg/L and 10 mg/L).Western blotting were used to detect phospho-p38 in alveolar macrophages. RESULTS:SDS-PAGE analysis indicated that the purified preparation of rat LBP showed homogeneity and the molecu-lar weight was 60 kD.The binding of lipopolysaccharide to mononuclear cells were enhanced by purified rat LBP.Stimu-lation of rat alveolar macrophages with LPS at concentration of 0.01 mg/L was LBP dependent.LBP at concentrations up to 1 mg/L was able to increase the activation of p38.However, when LBP concentrations were further increased to 10mg/L, the phosphorylation levers of p38 were lower as compared with that in the presence of 1 mg/L.Stimulation of ratalveolar macrophages with LPS at concentrations of 1 mg/L was LBP-independent.CONCLUSION:The activation of p38 induced by LPS at lower concentration(0.01 mg/L) was LBP-dependent, meanwhile, LPS at higher concentration(1 mg/L) was LBP-independent.     

黄瓜脂氧合酶基因CsLOX2 的原核表达载体 的构建及表达分析

以先前克隆到的黄瓜脂氧合酶基因CsLOX2 全长的cDNA 为模板,用含有特异性酶切位点
的Yh1、Yh2 为引物,通过PCR 方法将黄瓜脂氧合酶基因CsLOX2 的ORF 区构建到大肠杆菌表达载体
pGEX-4T-1 上获得原核表达载体p4t-LOX2,将该表达载体p4t-LOX2 转化到大肠杆菌菌株BL21（DE3）
中,获得相应的重组工程菌。在IPTG 诱导下,通过SDS-PAGE 电泳,得到一条约115 kD 的融合蛋白条带,
除去pGEX-4T-1 自身诱导的约26 kD 大小的GST 标签蛋白后,CsLOX2 编码一个约89 kD 的蛋白。 经过
融合蛋白表达体系的优化分析,表明该融合蛋白在 37℃,0.80 mmol·L^-1 IPTG 诱导表达 10.5 h,可获得
融合蛋白的最大表达量。     

Effect of p38 protein kinase on the activation of rat alveolar macrophages by lipopolysaccharide

AIM:To investigate the role of p38 protein kinase in the activation of rat alveolar macrophages(AMs) induced by lipopolysaccharide(LPS).METHODS:Nuclear protein was extracted, p38 protein kinase in nuclear extracts was analyzed by Western blot. The concentrations of TNF-α and IL-8 in supernatant were measured by radioimmunoassay.RESULTS:The concentrations of TNF-α, IL-8 in supernatant and p38 protein kinase in nuclear extracts were increased significantly induced by LPS and blocked by SB203580, a selective inhibitor of p38 protein kinase.CONCLUSION:The inductoin of TNF-α and IL-8 in alveolar macrophages by LPS may be mediated through the activation of p38 protein kinase.     

The activation effect of maize pollen polysaccharides on human thoracic cavity macrophages

AIM: To study the activation effects of maize pollen polysaccharides(PPM) on human thoracic cavity macrophage (hTMΦ). METHODS: Activities of lactate dehydrogenase(LDH) and acid phosphatase (ACP) in the hTMΦ were detected by automatic biochemical analyzer, and the level of tumor necrosis factor alpha(TNF-α) and interleukin-6(IL-6) in the hTMΦ was analyzed by radioimmunoassay, after hTMΦ were cultured with 0.312, 0.625, 1.250, 2.500, 5.000 mg/mL PPM-RPMI 1640 for 24 and 48 hours in vitro. RESULTS: The activities of LDH and ACP increased in the hTMΦ induced by PPM, and the levels of TNF-α and IL-6 in the hTMΦ induced by PPM increased markedly too. And the induced expression effect of TNF-α and IL-6 is associated with the concentration of PPM, and time for PPM inducing. CONCLUSION: PPM can induce cytokines secretion in hTMΦ, and activate hTMΦ in vitro.     

Effects of paecilomyces cicadidae total polysaccharides on macrophages of old rats

AIM: To study the activation and the immune regulation of paecilomyces cicadidae total polysaccharides on macrophages in old rats. METHODS: The ability of devouring neutral red and stimulating index in the alveolar macrophages (AM) and peritoneal macrophages (PM) were observed. The activities of lactate dehydrogenase (LDH), acid phosphatase (ACP) and arginase (Arg) were detected by automatic biochemistry analyzer. The shape and structure of spleen macrophages were observed by electron microscope. RESULTS: Compared with control group, the activity of the LDH/ACP/Arg in the AM or PM and in the culture fluid of the AM or PM were significantly increased (P<0.05 or P<0.01), and the ability devouring neutral red and stimulating index in the PM were strengthened (P<0.01), the ergastoplasm was hyperplasia and the amount of lysosome was manifold in the spleen macrophages of old rats induced by paecilomyces cicadidae total polysaccharides. CONCLUSION: Paecilomyces cicadidae total polysaccharides promote immune function in old rats.     

CCK-8 upregulates B7.1 and B7.2 expressions and enhances the costimulatory activity of murine peritoneal macrophages

AIM: To investigate in vitro effects of cholecystokinin octapeptide(CCK-8) on the expressions of B7.1 and B7.2 and the costimulatory activity of T lymphocytes in unstimulated macrophages.METHODS: Mouse peritoneal macrophages were isolated and incubated with CCK-8（10-12-10-6 mol/L） for indicated time. The B7.1 and B7.2 expressions of murine peritoneal macrophages were analyzed by flow cytometry. CD4+T cells were isolated from mouse spleen using immunomagnetic beads, and cultured with 1/4 numbers of macrophages which were pretreated with CCK-8 and/or anti-B7.1 antibody, anti-B7.2 antibody, CCK1R antagonist CR1409, CCK2R antagonist CR2945 for 24 h. ConA was added into the culture medium to stimulate CD4+T cell proliferation. The proliferation was determined by measuring ［3H］-TdR incorporation in a β-scintillation counter. RESULTS: B7.1 and B7.2 expressions and costimulatory activity of peritoneal macrophages were enhanced by CCK-8 in a dose-dependent manner, and the maximal effects occurred at the concentrations of 10-9 mol/L to 10-7 mol/L. Anti-B7.2 antibody, but not anti-B7.1 antibody, reduced the modulatory role of CCK-8 on costimulatory activity. Both CR1409 and CR2945 reversed the effect of CCK-8 on costimulation, and the role of CR1409 was more significant. CONCLUSION: CCK-8 enhances macrophage costimulatory activity by upregulating B7.2 expression, which is mediated by CCK1R and CCK2R. CCK1R might be the major receptor responsible for the modulation of CCK-8 on costimulation.     

Effect of homocysteine on expression of interleukin-8 mRNA and protein in THP-1 macrophages

AIM: To investigate the effect of homocysteine (Hcy) on expression of interleukin-8 (IL-8) mRNA and protein in THP-1-derived macrophages (THP-1 macrophages). METHODS: Cultured THP-1 monocytes were induced to macrophages by 0.1 μmol/L PMA treatment for 72 hours, then the differentiated THP-1 macrophages were incubated with homocysteine (0.01 mmol/L-0.20 mmol/L) for 24 hours, or with 0.10 mmol/L Hcy for various time up to 48 hours. IL-8 protein in THP-1 supernatants was measured by ELISA, and IL-8 mRNA expression was detected by semiquantitive RT-PCR. RESULTS: Compared with control, Hcy significantly increased the expression of IL-8 protein in a concentration-dependent manner. 0.05 mmol/L, 0.10 mmol/L and 0.20 mmol/L Hcy increased IL-8 production by 1.28 fold, 1.32 fold and 1.55 fold, respectively (P＜0.01). IL-8 production were elevated significantly 3 h after treatment with 0.10 mmol/L Hcy. In addition, Hcy also increased IL-8 mRNA expression in a concentration-and time-dependent manner. CONCLUSION: Hcy may contribute to atherogenesis by inducing IL-8 expression and secretion in THP-1 macrophages.     

山东寿光地区番茄黄化曲叶病毒外壳蛋白基因克隆及其在大肠杆菌中的表达

利用双生病毒简并引物PA/PB,对山东寿光地区保护地疑似感染番茄黄化曲叶病毒（TYLCV）的番茄植株进行PCR检测,结果证明番茄病叶由TYLCV侵染所致。以提取的病叶总DNA为模板,扩增得到长约770 bp的TYLCV-CP基因片段,克隆至pEASY-T1 Simple载体,然后用Xho I和EcoR I将其切下并插入到pET-32a表达载体中,构建了寿光地区TYLCV分离物的外壳蛋白基因原核表达载体,转入大肠杆菌BL21（DE3）,经IPTG（异丙基-β-D-硫代吡喃半乳糖苷）诱导获得了50 kD重组蛋白,Ni2+-NTA亲和层析纯化和Western印迹分析证明目的蛋白为TYLCV外壳蛋白,且具有良好的抗原活性。     

Effects of intracellular expression of Mycobacterium tuberculosis CFP10-ESAT6 fusion protein on proliferation and apoptosis of mouse macrophages

AIM：To establish Mycobacterium tuberculosis culture filtrate protein 10 (CFP10)-early secretory antigenic target 6 (ESAT6) eukaryotic expression vector and investigate the effect of intracellular expression of CFP10-ESAT6 fusion protein on the proliferation and apoptosis of mouse macrophages (RAW264.7 cells). METHODS：The recombinant plasmid pEGFP-N1/CFP10-ESAT6 was constructed by inserting CFP10-ESAT6 fusion gene into eukaryotic expression vector pEGFP-N1, and then transfected into RAW264.7 cells to express CFP10-ESAT6 fusion protein. The viability of RAW264.7 cells was measured by MTT assay. Flow cytometry was applied to measure the apoptotic rate and Toll-like receptor 2 (TLR2) expression in RAW264.7 cells treated with Mycobacterium tuberculosis 19 kD lipoprotein or staurosporine. RESULTS：The recombinant plasmid pEGFP-N1/CFP10-ESAT6 was successfully constructed and transfected into RAW264.7 cells. Compared with the control cells, intracellular expression of CFP10-ESAT6 fusion protein did not affect the viability of RAW264.7 cells, but could inhibit the apoptosis of RAW264.7 cells treated with Mycobacterium tuberculosis 19 kD lipoprotein. Moreover, CFP10-ESAT6-expressing macrophages had markedly lower expression of TLR2 on the surface. CONCLUSION：Intracellular expression of CFP10-ESAT6 fusion protein has no cytotoxicity on mouse macrophages, but can inhibit the apoptosis of the macrophages treated with Mycobacterium tuberculosis 19 kD lipoprotein through down-regulating the expression of TLR2.     

马铃薯卷叶病毒CP基因水溶性原核表达的研究

采用两种策略增强了马铃薯卷叶病毒(PLRV)CP基因的水溶性原核表达。首先,用5种可表达出分子伴侣的质粒分别转化重组菌TOP10(p BAD-LRCP),获得了既含p BAD-LRCP又含分子伴侣质粒的转化子;诱导转化子中PLRVCP基因与分子伴侣基因同时表达,SDS-PAGE结果表明,从转化子提取的水溶性蛋白中有明显的PLRV重组CP条带,即分子伴侣增进了重组CP的水溶性表达。其次,将PLRV-CP基因定向插入p ColdⅠ中构建了该基因的冷激诱导原核表达载体p Cold-LRCP,15℃、IPTG诱导24h,SDS-PAGE分析表明,从重组菌BL21(p Cold-LRCP)提取的上清蛋白中有明显的PLRV重组CP条带。试验结果表明,分子伴侣与冷激蛋白基因csp A的启动子均可提高PLRV-CP基因的水溶性表达,且后者的表达水平高于前者。     

Effects of fibrous mineral dusts on pulmonary alveolar macrophages in vitro

AIM: To assess the role of surface free radicals and electromotive voltage of fibrous mineral dusts in rabbit pulmonary alveolar macrophage injuries induced by fibrous mineral dusts. METHODS: Changes in cell death ratio, malandialdehyde (MDA) and cellur electrophoresis ratio, lactate dehydrogenate (LDH)and superoxide dismitase(SOD) activities were determined, the technique of cell culture and Scanning electron Microscopy were used to examine the change of membranous permeability, charge and cellular shape. RESULTS: Fibrous wollastonite and tabulate clinoptilolite, which had no OH^-, had no cytotoxicity, while fibrous sepiolite, fibrous palygorskite, fibrous brucite and chrysolite asbestos damaged pulmonary alveolar macrophages in various degrees because of the different OH^- levels. All the six fibrous mineral dusts changed the cellular electrophoresis ratio. CONCLUSION: The surface electromotive voltage of fibrous mineral dusts is not an important factor, and the cytotoxicity of them may be related to OH^- levels on the mineral dust surface.     

Effect of quercetin on ox-LDL-induced lipid accumulation and peroxidation in mouse macrophages

AIM：To investigate the effect of quercetin (QUE) preconditioning on oxidized low-density lipoprotein (ox-LDL)-induced lipid accumulation and peroxidation in mouse RAW264.7 macrophages and the underlying molecular mechanisms. METHODS：RAW264.7 cells were pretreated with different concentrations (20, 40 and 80 μmol/L) of QUE for 30 min and then treated with ox-LDL (100 mg/L) for 24 h. Intracellular lipid droplets were assayed by oil red O staining. Extracellular lactate dehydrogenase (LDH) and malondialdehyde (MDA) and intracellular reactive oxygen species (ROS) were determined to characterize the membrane integrity and the lipid peroxidation, respectively. The mRNA and protein levels of CD36, an important scavenger receptor which mediates ox-LDL uptake, were examined by real-time PCR and Western blotting, respectively. RESULTS：Pretreatment with QUE (20, 40 and 80 mmol/L) significantly attenuated ox-LDL-induced lipid accumulation in RAW264.7 cells and foam cell formation in a dose-dependent manner. Ox-LDL induced LDH release in RAW264.7 cells. This cytotoxic effect was significantly inhibited by QUE pretreatment. Compared with ox-LDL group, the intracellular ROS content and MDA level in culture medium decreased markedly in QUE group. In addition, pretreatment with QUE attenuated ox-LDL-induced up-regulation of CD36 at mRNA and protein levels. CONCLUSION: QUE inhibits ox-LDL-induced lipid accumulation and peroxidation in mouse macrophages and the mechanism may partially involve its ability to down-regulate CD36 expression.     

Regulation of ATP-binding acssette transporter A1 on apolipoprotein E secretion from macrophages

AIM: To investigate the regulatory effect of the adenosine triphosphate binding cassette transporter A1 (ABCA1) on apolipoprotein E secretion from human THP1 macrophages.METHODS: Differentiation of THP1 macrophages from monocytes was stimulated with phorbol 12-myristate 13-acetate. The macrophages then were incubated with factors which regulate ABCA1 expression. After periods of incubation, apo E secreted in the medium and synthesized in the cell was determined with ELISA, and apo E mRNA espression was detected with Northern blot.RESULTS: An increase in apo E secretion from THP1 macrophages was observed by 8 h of incubation with 8-Br-cAMP, an activator of ABCA1 expression (P＜0.05). Glyburide, a putative ABCA1 inhibitor, and antisense oligonucleotides specifically against ABCA1 mRNA remarkably decreased apo E secretion from both THP1 macrophages and macrophage foam cells (P＜0.01,respectively). Neither apo E mRNA expression nor intracellular apo E level in THP1 macrophages was changed by inhibition of ABCA1.CONCLUSION: ABCA1 may promote the secretion of apo E from macrophages and macrophage foam cells and the effect may occur at the level of post-translation. The present results reveal a new aspect underlying antiatherogenic properties of ABCA1.     

Polarized distribution of M2 macrophages in marginal region around lung adenocarcinoma and its effect on prognosis

AIM: To explore the polarized distribution of M2 macrophages in the marginal region around lung adenocarcinoma, the marginal/central ratio and their effect on the prognosis. METHODS: Double immunohistochemistry staining was used to determine the distribution and the difference of CD163⁺/CD68⁺ (M2) macrophages in the marginal and central regions in 49 cases of lung adenocarcinoma in situ (AIS), 11 cases of minimally invasive adenocarcinoma (MIA) and 57 cases of invasive adenocarcinoma (IA) in order to explore the effect and mechanism of the polarized distribution and the marginal/central ratio on the progression of lung adenocarcinoma. Single-factor Kaplan-Meier survival curve analysis and multivariate Cox survival analysis were employed to explore the relationship between the polarized distribution of M2 macrophages and the prognosis. RESULTS: Polarized aggregation of M2 macrophages was observed in the marginal region of lung adenocarcinoma compared with that in the central region, and the difference was significant (P<0.01). Based on the median level, they were divided into high polarized group and low polarized group. In low polarized group, M2 macrophage count in AIS was not significantly different from that in MIA or IA. However, in high polarized group, M2 macrophage count in AIS was lower than that in MIA and IA in turn and there were statistically significant differences (P<0.01). Single-factor Kaplan-Meier survival curve analysis and log-rank test result showed that the number of M2 macrophages in the marginal region and marginal/central ratio were negatively correlated to the survival time (χ²=44.71, P<0.01; χ²=21.75, P<0.01). Multivariate Cox survival analysis showed that the high polarized distribution of M₂ macrophages in the marginal region and the marginal/central ratio were independent risk factors for the prognosis (P<0.01). CONCLUSION: There is a polarization effect of M2 macrophages on the marginal region of lung adenocarcinoma. The marginal polarization and the marginal/central ratio are independent risk factors of the prognosis. Therefore, it may be an effective method for the evaluation of the prognosis to judge the marginal polarization by preoperative puncture and to determine the marginal/central ratio of M2 macrophages by postoperative biopsy.     

Study on scavenger receptor A from mouse peritoneal macrophages

AIM: To study the role of scavenger receptor A(SR A) in the uptake of oxidized low density lipoprotein(OxLDL) in mouse peritoneal macrophages(MPM). METHODS: Comparing the difference of the uptake of OxLDL in SR A-deficient and wild-type MPM. RESULTS: The results showed that the binding of OxLDL wasn't apparently reduced in SR A-deficient MPM. The association of OxLDL was reduced by 35.8% and degradation of OxLDL was reduced by 42% in SR A-deficient MPM compared with those in wild-type MPM. CONCLUSION: Studies showed that SR A didn't play an important role in the uptake of OxLDL in MPM. Approximately 70% of the uptake of OxLDL in macrophages was attributable to non-SR A receptor.