Construction of Expressing Plasmids of Recombinant FN Polypeptides with Bifunctional－domain and the Characterization of the P

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Effect of fragment Asp1961—Glu1978 in fibronectin on the expression of triple-domain polypeptide inE. coli

Summary Two plasmids were constructed and used to express two triple-domain recombinant polypeptide of human fibronectin (FN). The cDNAs in plasmids code for two polypeptides, CH62 (Pro1239-Ser1515 of FN linked with Ala1690-Val2049 through Met) and CH63 (CH62 without Ile1850-Glu1978). The expression level of CH62 inE. coli was very low, but that of CH63 was very high. The results suggests that Asp1961 -Glu1978 in FN is a key sequence influencing the expression of triple-domain polypeptide inE. Coli. After being dissolved and renatured, CH63 can be purified by heparin-agarose affinity chromatography. Both of the cell-binding domains in the recombinant polypeptide were functional. The production of CH63 provides a fundamental basis for further study of recombinant products with better anti-metastasis function. This project was supported by grants from National Natural Science Foundation of China (39370783) and Science Foundation of Ministry of Public Health (No. 94-1-141)     

Comparative Study on the Inhibitory Effect of RecombinantFN Polypeptide CH50 and CH56 on the Metastasis ofMelanoma Cells

Intactmoleculesoffibronectinhavenoinhibitoryeffectonthemetastasisoftumorcells.RecombinantCellIorHepI(singledomain)polypeptidesoffibronectin,expressedinE.colt,arenotabletoinhibitthemetastasesoftumornomattertheywereusedaloneorasamixture.ButtheinhibitoryeffectcanbeachievedwhenCellIandHepIdomainsoffibronectinwerepreparedasarecombinantbifunctional--domainpolypeptideexpressedinE.coliLlj.ItisnotclearuntilnowthatrecombinantCellI-Hep1polypeptideinhibitsthemetastasisoftumorcellsbecauseotherdomains…     

Effect of Fragment Asp1961-Glu1978 in Fibronectin on the Expression of Triple-domain Polypeptide in E. coli

Thebifunctional-domainrecombinantpolypeptldeoffibronectin(FN)ispotentiallyusefulintumortherapy.Thispolypeptideyieldedtheinhibltoryeffectontumormetastasisatseveralstepsoftumorcellsinvadingothertissuesll].ThestrongerfunctionwasobtainedifCell--ndomainwasconnectedtotheC--terminalofCellI--HepIpolypeptide['].Buttheexpressinglevelofthetriple--domainpolypeptideinE.coltwasverylow[ZJ.Wereportedthepreparationofbifunctional--domainrecombinantpolypeptideofFN,whichisProl239Ser1515ofFNlinkedwithAla16…     

Construction of eukaryotic expressing vector pCH503 of CH50 and its chemotaxis and anti-tumor function by expressionin vivo in mice

Summary An eukaryotic expressing vector that expresses CH50, a recombinant polypeptide of human fibronectin, in mice was constructed, and its chemotactic and anti-tumor function byin vivo gene transfection was investigated. The plasmid was constructed by recombination techniques. The cDNA fragment coding CH50 polypeptide from a prokaryotic expressing vector of CH50 was ligated with 5′-terminal noncoding region and coding region of signal peptide of mouse IFN-7 cDNA at 5′ side and 3′-terminal noncoden region of human FN cDNA at 3′ side. The recombinant cDNA was inserted into plasmid pREP8. The resulted expressing plasmid was designated as pCH503. The macrophages transfected with pCH503in vivo and culturedin vitro could produce CH50. The expressed product was identified by heparin-affinity chromatography and SDS-PAGE. By counting and Giemsa-staining of coeliac cells and histotomy and staining of muscle tissue, the chemotaxis on immune cells was observed after transfection of pCH503 either in peritoneal cavity or in muscle. The inhibition of gene transfection of pCH503 on melanoma was observed in mice. The number of melanoma nodes in mice was reduced by 50%–60% after coeliac transfection with pCH503. The pCH503, an eukaryotic expressing vector of CH50, can expressin vivo in mice. The transfection of pCH503in vivo has the chemotaxis on immune cells and can inhibit the formation of tumor nodes, suggesting that plasmid pCH503 is potentially useful in combined treatment of tumor. This project was supported by a grant from the National Natural Science Foundation of China (No. 39870763) and Trans-Century Training Program Foundation for Talents under the Supervision of Ministry of Education of China.     

Construction and expression of eukaryotic expressing vector pCH510 of polypeptide CH50 and its chemotaxis and antitumor function byin vivo transfection

Summary  To construct an eukaryotic expressing vector that expresses CH50, a recombinant Cell I-Hep I bifunctional-domain polypeptide of human fibronectin, and to investigate the chemotaxis to immune cells and the inhibitory effect on the growth of tumor by the expression of the plasmidin vivo, the plasmid was constructed by DNA recombination. Gene transfection was performedin vitro andin vivo. The expressed product was identified by Western blot. The chemotaxis after gene transfectionin vivo was observed by histotomy and staining of muscle tissues. The inhibition of gene transfection on solid tumor was observed in mice. The results showed that plasmid pCH510 was constructed by the recombination of the 5′-terminal noncoding region and signal peptide coding region of human fibronectin cDNA and cDNA fragment coding CH50 polypeptide with a 3′-terminal noncoding region of human FN cDNA, and the insertion of the recombinated fragment into plasmid pcDNA3. 1. After transfection with plasmid pCH510, NIH3T3 cells could produce CH50 polypeptide. The transfection of plasmid pCH510 by the injection in muscle of mouse could produce the effects of chemotaxis on immune cells and the inhibition on the growth of solid tumor. It is concluded that plasmid pCH510 can express in cells andin vivo in mouse. The expression of the plasmidin vivo has a chemotactic effect on immune cells and can inhibit the growth of solid tumor. This project was supported by a grant from the National Natural Science Foundation of China (No. 39870763) and a Funding Program for New-Century Talent of the Ministry of Education of China.     

Effect of polypeptide CH50 on macrophage activationin vivo and antitumor function

Surgery,chemotherapy and radiotherapy aremajor methods used in tumor therapy.After the re-moval of large tumor tissue by surgery,the mainproblem to be faced with is the inhibition and elimi-nation of the residual tumor cells in vivo. Ifmacrophages can be effectively activated,the residualtumor cells could be inhibited and eliminated as earlyas possible,the conditions favorable to the induce-mentof specificimmune response againsttumorcouldalso be created. Recombinant polypeptide CH50 wasprepar…     

Expression of Serum and Glucocorticoid-inducible Kinase1 in Diabetic Rats and Its Modulation by Fluvastatin

The expression of serum and glucocorticoid-induced protein kinase in the renal cortex of diabetic rats was examined, and the function of signal transduction mediated by SGK1 in diabetic nephropathy and its modulation by fluvastatin were also investigated. 24 male Wistar rats were randomly divided into normal control group （n = 8）, diabetic nephropathy group （n = 8） and fluvastatin-treated diabetic nephropathy group （15 mg/kg/d, n=8）. The metabolic parameters were measured at the 8th week. The expression of transforming growth factor β1 （TGF-β1） and fibronectin （FN） was immunohistochemically examined. The expression of SGK1 was detected by RT-PCR and Western blot, and CTGF mRNA was assessed by RT-PCR. As compared to DN, blood glucose, 24-h urinary protein, Cer and kidney weight index were all decreased and the weight was increased obviously in group F. At the same time, mesangial cells and extracellular matrix proliferation were relieved significantly. The levels of cortex SGK1 mRNA and protein were up-regulated, and both TGF-β1 and FN were down-regulated by fluvastatin. The mRNA of SGK1 was positively correlated with the CTGF, TGF-β1 and FN. SGK1 expression is markedly up-regulated in the renal cortex of DN group and plays an important role in the development and progress of diabetic nephropathy by means of signal transduction. Fluvastatin suppressed the increased SGKlmRNA expression in renal cortex and postponed the development of diabetic nephropathy.     

Optimal Expression Condition of Recombinant RAP

In order to construct the expression recombinant of human receptor associated protein (RAP), optimize its expression condition and obtain the recombinant protein after expression with high efficiency, two prokaryotic expression vectors-pT7-PL and pET-28a( ) were used to construct the expression recombinant containing RAP cDNA, and the expression efficiency of two kinds of ex-pression E. coli of BL21 strains was compared. The effect of different induction conditions on the expression of recombinant RAP was observed. After recombinant protein was purified with Ni -nitrilotriacetic acid (Ni -NTA) affinity chromatogram, its binding ability with microphage was observed. The results showed that two recombinant plasmids both obtained high expression of RAP. The expression levels of RAP in plasmid pT7-PL-RAP in BL21 (DE3, plysS) strain were signifi-cantly higher than in BL21 (DE3) strain. The expression of pT7-PL-RAP in the presence of chloramphenicol was higher than in the absence of chloramphenicol, and most of the inducible ex-pressed RAP was soluble. The RAP which was purified by Ni -NTA resin could strongly bind with the RAW264.7 cells rich in low density lipoprotein receptor (LDLR) family receptors. It was con-cluded that the expression condition of recombinant RAP was optimized and functional RAP was ob-tained, which offered a good foundation for the further production of RAP as research tool.     

Augmentation of Recombinant Fibronectin Polypeptide CH50 on the Antitumor Function of Macrophages

Fibronectin(FN)isamulti-domainglycoproteinwithcomplicatedregulatoryfunctionsllj.TheapplicationofFNtothetherapyofdiseaseshasbeentriedsince1983,buthampered'becauseofthecomplexityofitsfunctionslll.Inrecentyears,theresearchonFNwasconcentratedonthefunctionsofitsdomainfragments.IthasbeenprovedthattheFNfragmentcontainingCellIdomaincouldenhancethefunctionofmonocyte--macrophage['--'J,suggestingthatdomainfragmentofFNcouldbeveryusefulinthetherapyofdiseases.WehaverecombinedcDNAfragmentsofCellIdoma…     

Construction and Expression of Eukaryotic Expressing Vector pCH510 of Polypeptide CH50 and Its Chemotaxis and Antitumor Function by in vivo Transfection

Fibronectin ( FN) is a high- molecular glycopro-tein with multiple- domain.FN fragments containingCell domain are chemotactic to macrophages[1] .CH5 0 polypeptide,a recombinant polypeptide weproduced by the recombination of Cell and Hep domains of human fibronectin and expression in E.coli,can activate macrophages and inhibitthe metas-tasis of tumors[2 -4 ] .To investigate the function ofCH5 0 expressed in vivo by gene transfection,explorethe feasibility of applying the polypeptide to gene…     

Inhibitory Effect of Recombinant Fibronectin Polypeptide CH50 on Invasion and Metastasis of Melanoma B16 Cells

In order to investigate the inhibitory effect and mechanism of recombinant polypeptide CH50 on invasion and metastasis of melanoma B16 cells, the recombinant polypeptide CH50 was separated and purified by ion exchange chromatographic technique. The melanoma B16 cells treated with purified CH50 were cultured in vitro, the number was counted at 4, 24, 48 and 72 h and their morphological changes were observed in order to detect their adhesion and spreading abilities. In in vivo study, the melanoma B16 cells were labeled with CFSE and treated with CH50 and then they were injected into mice via mouse-tail veins. After 5 h, the lung tissues were fixed by frozen section. Accumulation and invasion abilities of B16 cells on lung tissues were observed under the fluorescent microscopy. The results showed that the morphological character of B16 cells treated with CH50 changed greatly and the number of B16 cells treated with CH50 decreased significantly (P<0.05). The adhesion and spreading abilities of B16 cells treated with CH50 were weakened obviously and the metastasis foci on lung tissues reduced. It was concluded that the recombinant polypeptide CH50 inhibited invasion and metastasis of melanoma B16 cells on tissues and could be a prospective bio-product in tumor general therapy.     

Augmentation of recombinant CH50 polypeptide on the function of macrophages of mice during chemotherapy

CH50 is a Cell I- Hep Ⅱ bifunctional-domain recombinant polypeptide of human fibronectin expressed in E. colilll. This polypeptide can inhibit theinvasion and metastasis of tumor cells 1'n the[2] andactivate the anti--tumor activity of macrophages[']. Ithas been reported that this polypeptide andchemotherapeutic agent have a synergistic inhibitoryeffect on the metastasis of tumors[4J. In this study,we further investigated the effect of CH50 on thefunction of macrophages of mice during chem…     

基于国家自然科学基金资助的中医院校科研竞争力分析

目的：分析全国24所中医院校的科研竞争力及其变化趋势,为中医药科研创新和发展提供参考。方法：利用2010-2017年国家自然科学基金中医药面上项目数据分析其在地区及院校层面资助的分布,通过综合项目数和金额构建的竞争力指数分析中医院校科研竞争力的变化趋势。结果：2010-2017年,中医院校整体国家自然科学基金面上项目数量年均增幅远高于国家自然科学基金委员会医学科学部和中医药领域,2017年占中医药领域面上项目资助项目数的57.26%。竞争力指数显示,中医院校的科研竞争力可分为5个层次。结论：中医药领域的国家自然科学基金资助整体增长乏力,而中医院校的国家自然科学基金资助则整体增长迅速。地区和中医院校间的科研竞争力虽差异显著,但呈缩小趋势。     

大型综合医院国家自然科学基金管理政策的改革与实践

大型综合医院是国内进行医学科研的重要机构,国家自然科学基金是现阶段倍受重视的项目申报途径,大型综合医院中国家自然科学基金管理政策的改革与实施日趋重要。通过回顾性分析某综合医院2005—2011年国家自然科学基金资助项目数、批准经费额度忮不予资助情况,探讨国家自然科学基金申报管理工作存在的问题及其原因,针对性地提出基金管理政策改革的措施,对初步取得效果进行说明,为大型综合医院中该基金的科研管理提供借鉴。     

猪血浆FN的分离及其性质鉴定

本文用明胶和肝素亲和层析柱等方法,从猪血浆中分离得到大量(0.5mg/ml)的纤维粘连蛋白(FN)。此FN在琼脂糖电泳和聚丙烯酰胺凝胶电泳中呈单一条带,分子量约450000;巯基乙醇还原后呈现分子量约230000的两条带;可以与抗人和抗牛血浆FN抗体发生免疫沉淀反应;并具有强烈促CHO细胞贴壁活性。提示猪血浆是一种分离提取FN的良好资源。     

内源性大麻素及其受体在神经病理痛治疗中的研究进展

在神经病理痛的发生、发展和维持中,内源性大麻素系统在神经系统从定位到表达水平都发生着动态变化。这些变化存在有利的一面,也存在不利的一面。外源性给予内源性大麻素在不同的疼痛模型中都产生了镇痛作用,且通过阻断大麻素受体发现内源性大麻素镇痛作用的发挥主要由大麻素CB1及CB2受体介导。抑制内源性大麻素降解酶在不同的疼痛模型中也表现出了镇痛作用,这种作用主要是因为增加了局部内源性大麻素含量水平。     

Platelet fibrinogen in the patients with idiopathic bleeding platelet disorder

Obscure bleeding platelet disorder with undefined reasons was a new kind of thrombocytopathyadvanced on the basis of Shen Di's disease, whichwas characterized by hemorrhage of skin and mucosa, but blood platelet counts, morphology and plasma coagulation profiles were all normal. The bleedingtendency caused by other reasons had been excepted.The patients usually had one or two defects ofplatelet aggregation response to adenosine diphosphate (ADP), platelet activated factor (PAF), collagen an…     

玉肾汤对IgA肾病大鼠的实验研究

目的:研究玉肾汤治疗IgA肾病的作用机理。方法:56只大鼠随机分为对照组、模型组、保肾康组、玉肾汤组,保肾康组予保肾康药片,玉肾汤组以中药玉肾汤水煎剂,其余两组予等量生理盐水灌服。观察各组尿蛋白定量、尿红细胞计数、血清肌酐、血尿素氮的检测结果;用免疫荧光法检测IgA免疫复合物及免疫组化法检测纤维连接蛋白(FN)在肾组织中的沉积及病理组织学改变。结果:玉肾汤组尿蛋白定量显著下降,与模型组比较有显著差异(P<0.05)。各用药组对尿红细胞数影响无显著性差异(P>0.05),玉肾汤组能降低IgA肾病大鼠血肌酐、血尿素氮水平,与模型组比较,有显著性差异(P<0.05)。对照组无IgA沉积,FN无异常变化,两治疗组IgA与纤维连接蛋白的沉积较模型组明显降低(P<0.01)。两治疗组间比较无显著性差异。结论:玉肾汤能够减轻免疫复合物及纤维连结蛋白在肾组织中的沉积,从而阻止肾组织硬化。     

细胞外基质分子影响人颊癌细胞侵袭力的体外实验研究

为探讨人颊癌细胞侵袭性生物学行为的有关分子机制。采用体外人羊膜侵袭模型,以建株人颊癌细胞系ＢｃａＣＤ８８５ 为研究对象,观察了细胞外基质（ｅｘｔｒａｃｅｌｌｕｌａｒ ｍａｔｒｉｘ ,ＥＣＭ）活性分子层粘连蛋白（Ｌａｍｉｎｉｎ,ＬＮ） 、纤粘连蛋白（ｆｉｂｒｏｎｅｃｔｉｎ,ＦＮ）及其结合位点序列肽ＲＧＤ（Ａｒｇ Ｇｌｙ Ａｓｐ） 对人颊癌细胞侵袭力的影响。结果表明,不同浓度的ＬＮ、ＦＮ（１０～５０μｇ／ｍｌ）均明显地增强了人颊癌细胞的侵袭力,并呈现出一定的剂量效应关系;ＲＧＤ不仅能降低颊癌细胞的侵袭力,而且能阻抑ＦＮ 的促颊癌细胞侵袭力。由此提示,ＬＮ、ＦＮ 可能是人颊癌细胞侵袭过程中的重要调节分子,利用ＥＣＭ分子结合位点序列肽有望成为一种新的抗癌侵袭转移治疗方法。