Objective:To construct the expression vectors of VH and VL from an anti -CD71 monoclonal antibody (line7579) , and express an anti-CD71 mouse/human chimeric antibody (D2C) in vitro.Methods:The variable regions of heavy and light chains from an anti -CD71 monoclonal antibody (line7579) cloned in the pGEM-T vectors were constructed respectively into the expression vectors,p?-Expr(including human IgG1 constant region) and p?-Expr(including human Ig? constant region).The expression plasmid vectors (Chi7579-p?-Expr and Chi7579- p?-Expr) ,confirmed by a clone-PCR and a further sequence analysis, were co-transfected by electroporation into the non-Ig-producing murine hybridomas SP2/0.Results:Screening for transfectomas secreting chimeric antibody was done by cross sandwich ELISA, which identified the transfectomas to secrete a chimeric antibody (D2C) with the human heavy constant region(C?) and light constant region(C?). FCM analysis of culture supernatants of the transfectomas demonstrated that the binding capacity of the chimeric Ab (D2C) to the antigen (CD71) was retained.Conclusion:The construction of the V H and V L expression vectors and the expression of an anti-CD71 mouse/human chimeric antibody (D2C) were accomplished successfully.

OBJECTIVETo evaluate the short-term and long-term effects on treatment of neck pain caused by cervical spondylosis with the combination of acupuncture and moxibustion with seed-size moxa cone.

METHODSOne hundred and forty-five patients of neck pain were randomly divided into an acupuncture-moxibustion group (49 cases), an acupuncture group (48 cases) and a moxibustion group (48 cases). Acupoints of Bailao (Extra), Dazhui (GV 14), Jianzhongshu (SI15) and Zhongzhu (TE 3) were adopted for all the 3 groups. Acupuncture was applied at all the acupoints with 20 min needling retention for the acupuncture group. Moxibustion with seed-size moxa cone was used with 5 cones on each point for the moxibustion group. And both acupuncture and moxibustion with seed-size moxa cone were adopted for the acupuncture-moxibustion group. The treatment was applied once every 3 days, and 10 treatments should be finished within 4 weeks. Follow-up should be carried out for 3 months. The short-term and long-term effects were evaluated with the scores of Northwick Park Pain Questionnaire (NPQ) and McGill Pain Questionnaire (MPQ) as the indices of therapeutic effect.

RESULTSThe NPQ score and MPQ score of all the 3 groups after the treating course and the 3-month follow-up were both decreased when compared with those before the treatment (all P<0. 05). The scores of NPQ and MPQ the acupuncture-moxibustion group were lower than that of the other two groups. And the difference had obvious significance (P<0. 05). High efficiency of pain relieving for cervical spondylosis could be found in all the 3 groups, which showed that short-term and long-term effects were good for all the 3 groups. And the highest curative effect could be found in acupuncture-moxibustion group.

CONCLUSIONCombination of acupuncture and moxibustion with seed-size moxa cone has reached a superior effect in short-term and long-term for neck pain caused by cervical spondylosis.

Acupuncture Points ; Acupuncture Therapy ; Adult ; Aged ; Combined Modality Therapy ; Female ; Humans ; Male ; Middle Aged ; Moxibustion ; instrumentation ; Neck Pain ; etiology ; therapy ; Spondylosis ; complications ; Treatment Outcome ; Young Adult

Aim To observe the protective effects of breviscapine against lung fibrosis and investigate its possible mechanism.Methods Effects of breviscapine on cell proliferation,activation and extracellular matrix secretion were examined in mouse embryonic lung fibroblast L929 cells in vitro.The mouse model of bleomycin-induced lung fibrosis was used to assess the protective effect of breviscapine against lung fibrosis.Results In vitro,breviscapine had no cytotoxicity directly on L929 cells,however,it could suppress cell proliferation,activation and secretion of laminin(LN) and collagen Ⅰ(ColⅠ) induced by transforming growth factor beta1(TGF-?1) in L929 cells.In vivo,breviscapine could prevent increase in serum TGF-?1 and decrease in superoxide dismutase(SOD),peroxidase(POD) and catalase(CAT) in mice with lung fibrosis caused by bleomycin.In addition,breviscapine was able to reduce pulmonary hydroxyproline,collagen,malondialdehyde(MDA)and TGF-?1 in lung fibrosis mice.Conclusion Breviscapine has protective effect against lung fibrosis and the possible mechanism is to enhance antioxidative defense activities and prevent TGF-? signal.

Objective To investigate the role of glargine in glucose metabolism improvement and antiinflammation of skeletal muscle in Caveolin-1 silenced type 2 diabetic mice.Methods Multiple low doses of streptozotocin (STZ) intraperitoneal injection and high-fat high-glucose (HFHG) were used to induce type 2 diabetic mice model.The mice were divided into normal control group (NC group) and type 2 diabetic model group (T group).Then according to virus injection and glargine treatment,T group were further divided into type 2 diabetes group (T2DM group),type 2 diabetes with insulin treatment group (insulin group),Caveolin-1 silenced with insulin treatment group (LV-CAV1 group),and scramble virus with insulin treatment group (LV-GFP group).Glucose metabolism was accessed by the fluctuation of blood glucose.TNF-α protein expression in skeletal muscle was detected by Western blot.Results The glycemic control of LV-CAV1 group needed more dosages of glargine (P ＜ 0.05).The expression of TNFαin skeletal muscle was elevated in LV-CAV 1 group than that in LV-GFP and insulin group (P ＜ 0.05).Conclusion The anti-inflammation function and glycemic metabolism improvement of glargine may be associated with the expression of Caveoin-1 in skeletal muscle.

Objective Glucose metabolism trend was dynamicly mornitored following liver transplantation, and its affecting factors were assessed. Methods The glucose metabolism status were assessed at four time points respectively after liver transplants, then they were divided into two groups:normal glucose metabolism (NGM) and abnormal glucose metabolism (AGM). The clinical data were univariate analyzed and multivariate analyzed to screen the risk factors. Results At 1 month, 3 months, 1 year and 3 years post-transplantation, the incidence of AGM were 74.0%, 43.9%, 29.4%, 24.1% respectively Between these two groups, age > 45 y had a significant difference at 1 month, 3 months, 1 year and 3years post-transplantation; the use of tacrolimus had a significant difference at 3 months, 1 year and 3years post-transplantation, but the dose of tacrolimus or tacrolimus blood concentration showed no significant difference; high dose of glucocorticoid had significant difference at 1 month , 3 months post-transplantation; high BMI and acute rejection had significant difference at 1 month post-transplantation. Conclusions There is a high incidence of abnormal glucose metabolism (AGM) in the early stage post-transplantation, and a considerable number of patients' glucose metabolism improved in the later period. Age>45 y and tacrolimus affect glucose metabolism for a longer period post-transplants. High BMI and acute rejection have an impact on glucose metabolism only in the early stage post-transplantation. Large dose of glucocorticoid affect glucose metabolism for at least 3 months post-transplantation , and there is no significant difference after 1 year.

Objective To develop a dental crown remover with the strength provided constantly and adjusted freely.Methods The remover was composed of a shell, a crown hook, a conduction mechanism, a strength generating mechanism and a percussion mechanism. The spring had the explosive force formed in the crown head to destruct the cohesion of the cement.Results The remover could remove the crown, with one hand freed for the protection of abutment and mucous membrane.Conclusion The remover has simple structure and easy operation, and is worth popularizing clinically.

This study was aimed to establish a method for sensorial color digitalization of Chinese herbal medicines (CHMs) with the application of spectrocolorimeter. The discussion was focused on difficulties of distinguishing surface and section color of CHMs. Based on uniform color space system of CIE1976L*a*b*, two methods for determination of section and surface color were constructed with two different kinds of spectrocolorimeters taking Glycyrrhizae Radix et Rhizoma as the experimental objective. In this paper, different kinds of sample preparation methods were used. Based on results, the method of scraping and grinding was proposed to prepare samples for section color determination. The method of wet pressing and peeling was proposed to prepare samples for surface color determination. Besides, RSD and dE*ab were served as evaluation indexes. This paper provided a simple, rapid and reliable analysis method for the color determination of CHMs. It also gave insight to future research on digitalization and modernization of CHMs' organoleptic characteristics based on traditional macroscopic identification.

Objective To construct the pshuttle-Egr-1-hSmac plasmid and transfect human breast cancer MDA-MB-435 cells,and to observe its radiotherapy enhancing effect on tumor cells.Methods The empty vector pshuttle and pshuttle-Egr-1-hSmac plasmid were transfected into MDA-MB-435 cells by liposomal.At different time(4,8,12,24 and 48 h)after irradiation with 2.0 Gy X-ray and 24 h after irradiation with 0.5 -5.0 Gy,the total RNA and protein were collected and extracted from these cells to analyze the Smac mRNA and protein expression levels with RT-PCR and Western blotting methods. The cells were divided into control, pshuttle, pshuttle-Egr-1-hSmac,2.0 Gy irradiation group, pshuttle + 2.0 Gy irradiation and pshuttle-Egr-1-hSmac+2.0 Gy irradiation groups.MTT method was used to evaluate cell proliferation,and the cell survival ability was measured with clone formation assay;Annexin Ⅴ/PI double staining and PI single staining were used to examine the apoptosis and cell cycle of MDA-MB-435 cells. Results There was no Smac mRNA expression in MDA-MB-435 cells in control and pshuttle groups,but the Smac mRNA expression levels in MDA-MB-435 cells in pshuttle-Egr-1-hSmac plasmid group were gradually increased with the time prolongation, and reached the maximum at 24 and 48 h;the Smac mRNA expression levels in MDA-MB-435 cells were increased gradually 24 h after irradiation of 0.5 - 5.0 Gy X-ray with the increasing of irradiation doses, and reached the maximum after 2.0 and 5.0 Gy irradiation. The Smac protein expression levels in pshuttle-Egr-1-hSmac plasmid group were increased gradually with the time prolongation,and reached the maximum at 24 h.The Smac protein expression lervels were increased 24 h afer irradiation of 0,0.5,1.0,2.0 and 5.0 Gy X-ray,especially in 5.0 Gy group. The MTT results showed that the A490 values in 2.0 Gy,pshuttle+2.0 Gy and pshuttle-Egr-1-hSmac groups 24, 48,and 72 h after irradiation were lower than those in control group(P<0.01);the A490 values of MDA-MB-435 cells in pshuttle-Egr-1-hSmac group after 1.0-5.0 Gy X-ray irradiation were lower than those in 0 Gy group (P<0.05 or P<0.01);the survival fraction(SF)in pshuttle-Egr-1-hSmac group was lower than those in control group (P<0.01).The percentages of the cells at G0/G1 and S phase in pshuttle-Egr-1-hSmac group were lower than those in 2.0 Gy group(P<0.01),the percentage of the cells at G2/M phase was higher than that in 2.0 Gy group (P<0.01);the apoptotic rate of the cells in pshuttle-Egr-1-hSmac group was higher than that in 2.0 Gy group (P<0.01).Conclusion X-ray irradiation can significantly increase the Smac mRNA and protein expression levels in MDA-MB-435 cells transfected with pshuttle-Egr-1-hSmac plasmid,inhibit the cell survival rate,and induce G2/M arrest and apoptotic increasing;Smac gene combined with radiotherapy could significantly increase the radiosensitivity of breast cancer cells.

To understand the immunogenicity of purified inactivated severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), concentration by ultrafiltration as well as molecular-sieve chromatography (MSC) were used for purification of inactivated SFTSVs. Inactivated viruses in purified samples were analyzed and identified by western blotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the glycoprotein (GP) and nucleoprotein (NP) antigen titers of which were detected using a double-sandwich enzyme-linked immunosorbent assay (ELISA). Purified inactivated SFTSVs were enriched and observed by electron microscopy, and the total protein concentration detected using the bicinchoninic acid assay. Purified inactivated SFTSVs were applied to New Zealand rabbits via two immunization programs to evaluate immunogenicity and to compare the immune effect. After SFTSVs were inactivated and concentrated by ultrafiltration, MSC revealed two typical elution peaks. The sample of one peak was identified as inactivated virions, in which GP and NP were detected by SDS-PAGE, western blotting and ELISA. Main corponent of the other peak was NP. After concentration by ultrafiltration, purified inactivated SFTSVs with purity >90% and total protein concentration of 1. 1 mg/mL were obtained, and the typical electron microscopy of bunyavirus was observed. In the sera of animals immunized with purified inactivated SFTSVs, SFTSV-specific IgG antibody and neutralizing antibody were detected at high titers. However, antibody titers were affected by the immunization program. Effect of immunization on days 0, 14 and 28 was significantly better than that on days 0, 7 and 28. Our work revealed that cultivation of SFTSVs contained intact virus particles and large amounts of free NP. Using MSC, purified inactivated SFTSVs of high purity could be obtained. Purified inactivated SFTSVs induced high titers of neutralizing antibody and virus-specific IgG antibody showing satisfactory immunogenicity, which provides important clues for further study on a vaccine for the inactivated virus.
Animals ; Antibodies, Neutralizing ; immunology ; Antibodies, Viral ; immunology ; Bunyaviridae Infections ; immunology ; virology ; Humans ; Neutralization Tests ; Phlebovirus ; classification ; genetics ; immunology ; isolation & purification ; Rabbits

To prepare monoclonal antibodies (mAbs) against structural proteins of severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), BALB/c mice were immunized using purified inactivated SFTSV virions as the antigens. Subsequently, hybridoma cell lines that secreted monoclonal antibodies against nucleoprotein (NP) and glycoproteins (GP) were obtained using a hybridoma technique. The antigen specificities of prepared mAbs were examined by indirect immunofluorescence and immunoprecipitation assays. Functional analyses were then performed,including the detection of IFA antibody titers,the levels of neutralizing activity and antibody affinities. After cell fusion and cloning,13 hybridoma cell lines secreted mAbs specifically against SFTSV-GP and 7 hybridoma cell lines secreted mAbs specifically against SFTSV-NP. Immunofluorescence and immunoprecipitation assays showed that the mAbs had high levels of antigen specificity. Among the 13 anti-SFTSV-GP mAbs,6 recognized Gn,whereas the others reacted with Gc. IFA titers of most anti-SFTSV-GP mAbs were between 1,280 and 20,480, and four anti-SFTSV-Gn mAbs showed neutralizing activity. Seven of the obtained anti-SFTSV-NP mAbs reacted specifically with NP,of which the IFA titers ranged from 5,120 to 20,480 with no observed neutralizing activity. Furthermore, two anti-SFTSV-GP mAbs, 1C8 and 1G8, showed high levels of affinity via a non-competitive ELISA. Our study lays the foundation for the development of further diagnostic assays and basic research into SFTSV.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antibody Specificity ; Bunyaviridae Infections ; immunology ; virology ; Female ; Humans ; Hybridomas ; immunology ; Mice ; Mice, Inbred BALB C ; Phlebovirus ; immunology ; Viral Structural Proteins ; immunology