High Expression of Insulin-like Growth Factor H (IGF-Ⅱ) Using Bac-to-Bac Expression System

Objective In order to obtain mature insulin-like growth factor- Ⅱ ( IGF- Ⅱ ), we used Bac-to-Bac baculovirus expression system. Methods Firstly the IGF- Ⅱ cDNA was cloned into a donor plasmid pFastBac1 and the recombinant pFastBac1 was then introduced into competent cells DH 10Bac. Recombinant bacmids were constructed by transposing a mini-Tn7 element from a donor plasmid pFastBac1 to the mini-attTn7 attachment site on the bacmid where the Tn7 transposition functions were provided in trans by a helper plasmid, and then used to transfect Sf9 insect cells to get recombinant baculovirus. The recombinant baculovirus was used to infect insect cells. Results Agarose gel analysis showed that recombinant donor plasmid pFastBac1 was constructed successfully; Agarose gel analysis of PCR products confirmed recombinant bacmid ; SDS-PAGE and Western Blotting showed that a 7KD protein band appeared. Conclusion The mature IGF- Ⅱ with immunogenecity has been expressed and produced by using Bac-to-Bac expression system.     

High Expression of Insulin-like Growth Factor Ⅱ (IGF-Ⅱ) Using Bac-to-Bac Expression System

Ｈｕｍａｎｉｎｓｕｌｉｎ ｌｉｋｅｇｒｏｗｔｈｆａｃｔｏｒ Ⅱ (ＩＧＦ Ⅱ )ｉｓａ 6 7ａｍｉｎｏａｃｉｄｓｉｎｇｌｅ ｃｈａｉｎｐｅｐｔｉｄｅ ,ｗｈｉｃｈｉｓｉｎｖｏｌｖｅｄｉｎｆｅｔａｌｇｒｏｗｔｈａｎｄｄｅｖｅｌｏｐ ｍｅｎｔ[1] .Ｔｈｅｇｒｏｗｔｈ ｐｒｏｍｏｔｉｎｇｐｒｏｐｅｒｔｉｅｓｏｆＩＧＦ Ⅱ ,ａｓｗｅｌｌａｓｔｈｅｉｒｓｔｒｕｃｔｕｒａｌｈｏｍｏｌｏｇｙｔｏｐｒｏｉｎｓｕｌｉｎａｎｄＩＧＦ Ⅰａｒｅｒｅｆｌｅｃｔｅｄｉｎｔｈｅｎａｍｅｏｆｔｈｅｐｅｐｔｉｄｅｓ .Ｉｔｉｓａｌｓｏｈｅｌｐｆ…     

The immunocytochemical localization of insulin-like growth factor Ⅱ in human cirrhosis

Sections of 30 cases of human cirrhosis were stained with rabbit anti-insulin-likegrowth factor Ⅱ(IGF Ⅱ)by double PAP method.By the serological examination 15 patientsshowed HBV infection and sections of 14 eases were HBsAg postively with a total rate of 67％(20 cases).The IGF Ⅱ was positive in the cytoplasm of all the liver and ductular cells.Binucle-ated,polypoid liver cells and the peripheral cells of the lobules or nodules were distinctly posi-tive,The liver cells which were strongly positive were a kind of thin polygonal cells with asmall oval or a round deeply stained nucleus in each.They might exist sporadically in the lob-ules or in the marginal portion of a nodule.These liver cells are quite different from the so-called oval cells which are derived from the proliferating ductules and are generally believed tobe responsible for the pathogensis of hepatoma.     

Dynamic Expression of Tumor Necrosis Factor-α and Vascular Endothelial Growth Factor in Rat Model of Pulmonary Emphysema Induced by Smoke Exposure

In order to explore the roles of tumor necrosis factor-α(TNF-α) and vascular endothelial growth factor(VEGF) in the pathogenesis of pulmonary emphysema,male Wistar rats were randomized into group A1,group A2.5 and group A4,each with smoke exposure for 1 month,2.5 months or 4 months,respectively.Group B1,group B2.5 and group B4 were used as non smoking controls at corresponding time points.TNF-α in bronchoalveolar lavage fluid(BALF) and expression of VEGF in lung tissue was determined by ELISA or by SABC immunohistochemistry assay either.Lung slices were stained with hematoxylin and eosin(HE).Results showed that in animal with smoke exposure the mean linear interceptor(Lm),an index of pulmonary emphysema and the content of TNF-α in BALF increased gradually,on contrary,the expression of VEGF in lung tissue decreased(P<0.05).This phenomenon was not obvious in animals without smoke exposure.Lm was negatively correlated to the VEGF expression(γ=-0.81,P<0.01) and positively correlated to TNF-α concentration(γ = 0.52,P<0.004),which implies that smoke exposure decreased the expression of VEGF and increased the expression of TNF-α.It is plausible to speculate that the imbalance of TNF-α and VEGF may play an important role in the pathogenesis of smoke-induced pulmonary emphysema.     

Effects of Mycophenolic Acid on High Glucose-induced Expression of TGF-β and CTGF in Mesangial Cells

Mycophenolicacid(MPA),theactivemetab oliteofmycophenolatemofetil(MMF),isapo tent,non competitive,andreversibleinhibitorofinosine5'monophosphatedehydrogenase(IMP DH),therate limitingenzymefordenovopurinesynthesis,andthusinterfereswithcellularbiosyn thesisofguanosineanddeoxyguanosinenucleo tides.BesidesstronglyinhibitingtheproliferationofTlymphocyteandBlymphocyte,MPAhasbeenidentifiedtohavesomeotherbiologicalfunctionsinrecentyears,includinginterferingglycosylationofadhesionmolecule,relievin…     

Cloning and Expression of Rat Transforming Growth Factor β1 cDNA in Osteoblasts

In order to study transforming growth factor81 (TGF51) in molecular level and investigate thefeasibility of TGF51 gene therapy for bone defects,an eukaryotic expression vector containing ratTGF51 cDNA was constructed. The expression ofTGF51 in the transfected osteoblasts was observed.1 MATERIALS AND METHODS1. 1 Animals and Cell CultureSprague--Dawley rats, I adult and 5 newborn,were purchased from Animal Center of Tong nMedical University. Lymphocytes, isolated fromperipheral…     

The expression of the products of IGF-Ⅱ,IGF-Ⅱ receptors and CSF-Ⅰ receptors in human primary hepatocellular carcinoma and juxtacancerous liver tissue

The expression of the products of IGF-Ⅱ,IGF-Ⅱ receptors(IGF-Ⅱ-R)and CSF-Ⅰ re-ceptors(CSF-Ⅰ-R)was observed in 17 cases of human primary hepatocellular carcinoma(PHC)and the juxtacancerous liver tissue with immunohistochemistry(ABC),Western blot and North-ern blot technique,It was found that the expression of IGF-Ⅱ,IGF-Ⅱ-R and CSF-Ⅰ-R was signif-icantly higher in PHC than in normal liver tissue and the expression of IGF-Ⅱ and IGF-Ⅱ-R wasremarkably higher in the juxtacancerous liver tissue from PHC patients than in PHC proper.Itwas noteworthy that the expression of IGF-Ⅱ in both the cancer proper and the juxtacancerousliver tissue was characterized by its fetal type.Besides,the expression of CSF-Ⅰ-R was signifi-cantly higher in PHC than in the juxtacancerous liver tissue.It is believed that the abnormal ex-pression of IGF-Ⅱ,IGF-Ⅱ-R and CSF-I-R in PHC and the juxtacaneerous liver tissue might berelated to the autocrine mechanism of human PHC.     

Effect of Colquhoumia Root on the Expression of Transforming Growth Factor-β in Mesangial Proliferation Glomerulonephritis Model

ThechronicglomerulonephritisremainthemostcommondiseasetocausechronicrenalfailureinChina.Themesangialproliferationglomerulo nephritis(MsPGN)isthemostcommonpathologi caltypeoftheprimaryglomerulonephritis,ac countingforabout50%ofpatientsreceivingrenalpuncture[1].Reducingproteinuriaandmaintainingthekidneyfunctionisveryimportantforthetreat mentofthecondition.InthisstudyMsPGNmodelwasestablishedbyinjectionofanti thymocyteserum(ATS)inSDratsandproteinuria,pathologicalchangesofkid neyandTGFβ1exp…     

Construction of Antisense Transforming Growth Factorβ_1 Gene and Its Effect on the Proliferation by Expression in Osteosarcoma Cells

Summary:To construct the antisense transforming growth factorβ1(TGFβ1)gene and investigatethe effect of TGFβ1 autocrine loop blockage on the proliferation of osteosarcoma cells.TGFβ1 cDNAwas cloned by RT-PCR from human osteosarcoma cells(MG-63)and inserted into pcDNA_3 to con-struct an antisense expression vector,which was dubbed pcDNA_3-TGFβ1(-).MTT was used to de-tect the proliferation of osteosarcoma cells transfected by antisense TGFβ1 gene.Our results showedthat the proliferation of the transfected osteosarcoma cells was suppressed markedly.It is concludedthat TGFβ1 autocrine loop blockage in osteosarcoma cells could inhibit cell proliferation,which mightbe helpful for gene therapy of osteosarcoma.     

Expression of Transforming Growth Factor β1 in Mesenchymal Stem Cells: Potential Utility in Molecular Tissue Engineering for Osteochondral Repair

Summary: The feasibility of using gene therapy to treat full-thickness articular cartilage defects was investigated with respect to the transfection and expression of exogenous transforming growth factor (TGF)-β1 genes in bone marrow-derived mesenchymal stem cells (MSCs) in vitro. The full-length rat TGF-β1 cDNA was transfected to MSCs mediated by lipofectamine and then selected with G418,a synthetic neomycin analog. The transient and stable expression of TGF-β1 by MSCs was detected by using immunohistochemical staining. The lipofectamine-mediated gene therapy efficiently trans fected MSCs in vitro with the TGF-β1 gene causing a marked up-regulation in TGF-β1 expression as compared with the vector-transfected control groups, and the increased expression persisted for at least 4 weeks after selected with G418. It was suggested that bone marrow-derived MSCs were sus ceptible to in vitro lipofectamine mediated TGF-β1 gene transfer and that transgene expression persist-ed for at least 4 weeks. Having successfully combined the existing techniques of tissue engineering with the novel possibilities offered by modern gene transfer technology, an innovative concept, I.e.molecular tissue engineering, are put forward for the first time. As a new branch of tissue engineer-ing, it represents both a new area and an important trend in research. Using this technique, we have a new powerful tool with which: (1) to modify the functional biology of articular tissue repair along defined pathways of growth and differentiation and (2) to affect a better repair of full-thickness artic ular cartilage defects that occur as a result of injury and osteoarthritis.     

Effects of Shenggu Injection (生骨注射液) on mRNA Expression of Vascular Endothelia Growth Factor in Rat Osteoblasts in vitro

Vascular endothelia growth factor(VEGF),one of the most effective growth factor of angio-genesis,is known to have a significant action inpromoting the development of new blood vessels.A great number of research work showed thatVEGF plays an i mportant role in fracture heal-ing(1).In previous studies,much has been repor-ted about ectogenic VEGF in promoting fracturehealing.Yet experi ment in promoting endogeneticVEGF has been seldom reported.Following thedevelopment of molecular biology,…     

Effects on the Tumor Specific Growth Factor and Tumor Necrosis Factor α in Rats' Precancerous Lesion of Primary Hepatocellular Carcinoma by Direct Moxibustion at Ganshu(BL 18) Acupoint

Objective: To investigate the effect of direct moxibustion at Ganshu(BL18) on the serum concentrations of tumor specific growth factor(TSGF) and tumor necrosis factor α(TNF-α) in a rat model with precancerous lesion of primary hepatocellular carcinoma(HCC), so as to explore the mechanism of moxibustion underlying improvement of HCC. Methods: Sixty male Wistar rats were randomly divided into control group(n=10), model group(n=20), prevention group 1(n=15) and prevention group 2(n=15). The normal rats were injected with physiological saline as blank control. At the same time, the rats of other three groups were injected with diethylnitrosamine to establish the HCC model. Direct moxibustion with grain-sized moxa was applied to bilateral Ganshu acupoint of the rats in the prevention group 1(1 treatment course, 20 days) and prevention group 2(2 treatment courses, 40 days), 5 doses for each acupoint, 0.5 mg/dose, once every other day. At each time point(before model establishment, the end of 1st course prevention, the end of 2nd course prevention and the end of model establishment), serum levels of TSGF and TNF-α were detected using enzyme-linked immunosorbent assay. Results: Compared with the control group, there was a remarkably increase of serum TSGF and TNF-α contents in the model group at the end of the experiment(P0.05). At the end of the 1st course of direct moxibustion, the contents of serum TSGF and TNF-α of rats in the prevention group 1 were significantly increased compared with that of the model group(P0.05). At the end of the 2nd course of direct moxibustion, serum TSGF and TNF-α levels of rats in the model group were higher than the normal group with significantly difference(P0.05), and the levels of TSGF and TNF-α in the prevention group 2 were significantly reduced in comparison with the model group(P0.05). Conclusion: It was possible that direct moxibustion could inhibit precancerous lesion and postpone hepatocarcinogenesis, and the therapeutic effect of two courses were better than one course.     

Effect of Kang''''ai Injection(康艾注射液) on Serum Level of Soluble Interleukin-2 Receptor and Vascular Endothelial Growth Factor in Patients with Esophageal Carcinoma during Radiotherapy

Radiotherapy(RT)is an important ap-proach for treatment of esophageal carcinoma(EC),especially for patients who have missedthe chance of surgical section.The efficacy ofRTis not satisfactory for its1-year,3-year and5-year survival rates being merely50%,20%,and10%respectively(1).Researches in recent years showed thatbetter efficacy can be obtained by combiningRTwith Chinese herbal medicines for EC thatincapable to be resected.Kang ai Injection(康艾注射液,KAI)is a Chinese herbal prep-arati…     

Effect of Reduqing (热毒清) on Endotoxin-Induced Production of Tissue Factor and Cytokines in Human Whole Blood

lipopolysaccharide,monocytes,mononuclearcellsReduqinginjection(RDQ,originallynamedAnti--inflammatoryAgentNo.6)isamodernChineseherbalpreparationmodifiedfromtherepresentativedecoctionWuweiXiaoduYin(Eapi6$ts).ItconsistsofextractedmaterialsfromFlosLonicerae,Foliumlsatidis,HerbaTaraxaci,andHerbaHouttuyniae,andbelongstothe"heat--clearinganddetoxifying"categoryofChinesemedicine.Inthepasttwodecades,RDQInfectionhadbeenusedinanumberofChinesehospitalsforthetreatmentofacuteabdomenandotheracuteinf…     

Effect of Huan-shuai Recipe (缓衰方) on Expression of Extracellular Matrix in 5/6 Nephrectomized Rats

Ｈｕａｎ ｓｈｕａｉＲｅｃｉｐｅ（ＨＳＲ，缓衰方），ａＣｈｉｎｅｓｅｈｅｒｂａｌｍｅｄｉｃｉｎａｌｐｒｅｐａｒａｔｉｏｎｅｓｔａｂｌｉｓｈｅｄｂｙｔｈｅａｕ ｔｈｏｒｓ，ｐｏｓｓｅｓｓｅｓｔｈｅｍａｉｎｆｕｎｃｔｉｏｎｏｆｂｅｎｅｆｉｔｉｎｇｖｉｔａｌｅｎｅｒｇｙ，ｎｏｕｒｉｓｈｉｎｇｂｌｏｏｄ，ｐｒｏｍｏｔｉｎｇｂｌｏｏｄｃｉｒｃｕｌａ ｔｉｏｎ，ｐｒｏｍｏｔｉｎｇｄｉｕｒｅｓｉｓｔｏｅｌｉｍｉｎａｔｅｅｖｉｌＷｅｔｎｅｓｓａｎｄｐｕｒｇｉｎｇｔｈｅＴｕｒｂｉｄｎｅｓｓ，ｈａｓｂｅｅｎｕｓｅｄｔ…     

原发性肝癌射波刀(Cyberknife)治疗前后IGF-Ⅱ、CA199、AFP的变化

目的:探讨原发性肝癌患者射波刀(Cyberknife)治疗前后血清胰岛素样生长因子-II(IGF-Ⅱ)、CA199、AFP水平变化及临床意义。方法:对125例原发性肝癌患者Cyberknife治疗前后检测血清中IGF-Ⅱ、CA199、AFP的水平。结果:原发性肝癌患者Cyberknife治疗前血清中IGF-Ⅱ、CA199、AFP水平均显著高于对照组(P<0.01),Cyberknife治疗后患者血清IGF-Ⅱ、CA199、AFP的水平显著低于Cyberknife治疗前的水平,具有统计学意义(P<0.05)。125例肝癌患者CR23例,PR82例,NC14例,PD6例,近期有效率(CR+PR)为84.0%。结论:血清IGF-Ⅱ、CA199、AFP的水平的监测可以作为原发性肝癌的辅助诊断及放射治疗疗效的监测;Cyberknife是治疗不能手术的中晚期肝癌的有效手段,不良反应较轻,值得推广应用。     

Effect of Qi-tonifying and Stasis-eliminating Therapy(益气活血法)on Expression of Vascular Endothelial Growth Factor and Its Receptors FIt-l,FIk-1 in the Brain of Intracerebral Hemorrhagic Rats

Objective:To investigate the effects and mechanism of qi-tonifying and stasis- eliminating(QTSE)therapy on the expression of vascular endothelial growth factor(VEGF)and its receptors Fit-1 and FIk-1 in the brains of intracerebral hemorrhagic(model)rats.Methods:One hundred and eighty Sprague-Dawley rats were randomly divided into six groups:the normal group (n=5),the sham-operative(SO)group(n=35),the model group(n=35),the QTSE group(n=35),the QT group(n=35)and the SE group(n=35).All the rats except those in the normal group and SO group were established into an intracerebral hemorrhage(ICH)model by intracerebral injection of collagenase typeⅦand the latter three were orally administered with Buyang Huanwu Decoction (补阳还五汤,a classical recipe for QTSE)or with some of its components for qi-tonification and for stasis-elimination,respectively.To the other three groups,normal saline solutions were given instead.Behavioral tests were carried out in the animals randomly chosen from each group on days 1, 2,4,7,14,21 and 28 after modeling.The expressions of VEGF,FIk-1 and Fit-1 were determined by immunohistochemistry and the number of vascular segments with positive expression in the injured brain area of the rats was calculated.Results:From day 7 onwards,the asymmetric forelimb use rate in the QTSE group recovered more significantly than that in the other model groups.In the model group,the expressions of VEGF,FIk-1 and Fit-1 appeared on day 1 and reached a peak on day 21, then weakened gradually.In the QTSE group,as compared with the other model groups,a higher level of VEGF expression was shown from day 7(P<0.01)and a higher level of Fit-1 expression was shown from the 7th day to the 21st day(P<0.01).Conclusion:QTSE therapy can up-regulate the expressions of VEGF and its receptors(FIk-1 and Fit-1)and improve the recovery of kinetic function in the ICH rats,which may be correlated with its action in modulating vascular regeneration to promote the reconstruction of microvascular networks in the damaged areas.     

Clinical Biochemical Observation on the Treatment of Postmenopausal Osteoporosis with Gusong (骨松)Ⅱ

Osteoporosisisacommonbonemetabolicandgeriatricdiseasewhichjeopardizesthehealthoftheaged.Since1992,about100casesofosteoporosishavebeentreatedwithGusong(#ac)11,amongwhom35postmenopausalosteoporosiscaseswerethenfollowed-upwithsatisfactoryresults,whichwasreportedasfollows.METHODSOrit6riaforD18gnosisOsteoporosisdiagnosticcriteriareferredtoOrimo'sc.it.ria(1)recommendedbytheCommitteeofFirstNationalConferenceonMetabolicBoneDisease,DoneDensitometryanditsClinicalApplication.Thedifferentialdiagn…