Expression of PSMA 7 and its effect on proliferation, invasion, migration and tumorigenesis of gastric cancer / 南方医科大学学报

OBJECTIVE@#To study the expression of PSMA7 and its effect on proliferation, invasion and migration of gastric cancer and subcutaneous tumorigenesis in nude mice. >and subcutaneous tumorigenesis in nude mice.@*METHODS@#Specimens of tumor tissues and paired adjacent tissues were collected from 60 patients with gastric cancer for detecting the expression levels of PSMA7 using immunohistochemical method. Gastric cancer cell line SGC7901 was transfected with a lentiviral vector to inhibit PSMA7 expression, and the changes in cell proliferation and invasion were observed using cell counting kit-8 (CCK-8), clone formation assay and Transwell assay. A BALB/c mouse model bearing subcutaneous gastric cancer xenograft was established using SGC7901 cells with stable PSMA7 knockdown to assess the effect of low expression of PSMA7 on xenograft growth.@*RESULTS@#Gastric cancer tissues expressed significantly higher levels of PSMA7 than the paired adjacent tissues ( < 0.05). In SGC7901 cells, interference of PSMA7 expression significantly inhibited the cell proliferation and invasion ( < 0.05). In the tumor-bearing BALB/c mice, the xenografts derived from SGC7901 cells with PSMA7 expression interference showed significant growth suppression as compared with the control xenografts ( < 0.05).@*CONCLUSIONS@#PPSMA 7 is overexpressed in gastric cancer tissues, and PSMA7 knockdown inhibits the proliferation, invasion, migration and subcutaneous tumorigenesis of gastric cancer cells in nude mice.

Construction of latent membrane protein 2A chimeric antigen receptor-T cells and their lethal effects on nasopharyngeal carcinoma cells / 中华耳鼻咽喉头颈外科杂志

Objective@#To produce latent membrane protein 2A (LMP2A) chimeric antigen receptor (CAR)-T cells and detect the lethal effect of LMP2A CAR-T cells on nasopharyngeal carcinoma (NPC) cells.@*Methods@#The study was conducted from September 2016 to December 2017.Genetic engineering technology was used to construct anti-LMP2A CAR lentiviral expression vector and sequencing was identified. The expression of anti-LMP2A CAR in the 293T cells was confirmed by western blot. CCK8 assay was used to evaluate the cytotoxicity of LMP2A CAR-T cells to NPC cells. ELISA assay was performed to test IL-2 and IFN-γ releasing of activated LMP2A CAR-T cells. The inhibition effect of LMP2A CAR-T cells on NPC xenograft tumor was observed in vivo. Statistical analysis was performed by statistical software SPSS 21.0.@*Results@#The results of PCR and sequencing showed that anti-LMP2A CAR lentiviral expression vector was constructed successfully. The result of western blot indicated the expression of anti-LMP2A CAR in the 293T cells effectively. The results of CCK-8 assay showed that the killing activities of LMP2A CAR-T cells to LV-LMP2A-CNE1 cells were (72.11±9.75)%, (54.65 ±5.42)% and (36.68±3.80)% at 20∶1, 10∶1 and 5∶1 ratio of effective cells to target cells, and had a statistical difference compared to CD19 CAR-T cells and T cells (P<0.05). There was no significant difference in the killing activities of LMP2A CAR-T cells to CNE1 cells compared with CD19 CAR-T cells and T cells. The results of ELISA showed that the content of IL-2 and IFN-γ in the co-culture supernatant of LMP2A CAR-T cells and LV-LMP2A-CNE1 cells was significantly higher than that of LMP2A CAR-T cells and CNE1 cells which had statistical difference (P<0.05); In vivo experiment, the volume of LMP2A CAR-T cell group was (80.3±10.0) mm3 which was significantly lower than that of the control groups, and the difference was statistically significant (P<0.05).@*Conclusion@#LMP2A CAR-T cells are successfully prepared and have an obvious targeting cytotoxicity on LMP2A-positive NPC cells.

The progress of the novel human infected H7N9 avain influenza viruses / 医学研究生学报

Chinese Center for Disease Control and Prevention published three confirmed novel H 7N9 avain influenza virus in-fection cases on March 31 ,2013 .The disease then spread across China and caused extremely high morbility and mortality , which trig-gered extensive attention .These cases were caused by a novel H 7N9 avian influenza virus which was highly mutable .Once the virus a-dapted to human respiratory epithelium receptors , a pandemic outbreak would occur immediately .This article summerizes the research progress on the H7N9 avain influenza, including epidemiologic characteristics , etiology features, clinical manifestation, detection tech-niques as well as theraputic and prevention strategy .

Construction, expression and identification of a recombinant immunotoxin of scFv against Schistosoma japonicum / 中国血吸虫病防治杂志

Objective To construct a recombinant immunotoxin expression vector composed of a single-chain Fv fragment of Sehistosorna japomicum and PE38KDEL gene,and identify the binding activity of the purified product with SEA antigen.Methods The V_H and V_L genes were amplified by PCR from the parent monoclonal antibody NP11-4.Then the amplified scFv and PE38KDEL genes were inserted into the expression vector pBAD/gIII A.The fusion protein expressed in E.coli Top10F' induced by L-arabinose.After purification,the activity of the immunotoxin was evaluated by Westem-blot and ELISA.Results The new recombinant immunotoxin expression vector pBAD/gIII A-scfv-PE38KDEL was constructed successfully.The main product was in inclusion bodies.ELISA assay showed that the refolding recombinant immunotoxin remained binding activity with SEA antigen.Conclusion A new recombinant expression plasmid pBAD/gIII A-scfv-PE38KDEL has been constructed and expressed successfully,which is useful in further study of the treatment of schistosomiasis japonica.

Monoclonal Antibody S2C4 Neutralizes The Toxicity of Shiga Toxin 2 and Its Variants / 生物化学与生物物理进展

Shiga toxin 2 (Stx2) toxoid produced by formaldehyde treatment of purified toxin was used to immunize BALB/c mice for monoclonal antibody (MAb) production.The neutralizing activities of positive clones against Stx2 were screened by in vitro cytotoxicity assay.The isotype and specificity of resultant clone was determined,and its efficacy to neutralize the activity of purified Stx2 was evaluated by in vitro and in vivo toxicity model.Lastly,its spectrum of activity against Stx2 variants was also accessed by mouse toxicity model.It was demonstrated that one of the 12 positive MAb clones against Stx2,designating $2C4 had neutralizing activity.S2C4 belongs to the immunoglobulin G1 subclass and has a K light chain,and it reacts with the A subunit of Stx2 and does not bind to Stx2 B subunit or to Stx1.S2CA could efficiently neutralize the cytotoxicity of Stx2 to Veto cells and mice.It also protected mice against lethal doses of Stx2 variants challenge including Stx2c and Stx2vha.S2C4 is a promising candidate molecule in preventing the progression of hemolytic-uremic syndrome (HUS) mediated mainly by Stx2 in Stx-producing Escherichia coli(STEC)infection.

Purification and functional characterization of enterohemorrhagic Escherichia coli O157: H7 Shiga toxinⅡ / 中华传染病杂志

Objective To purify Shiga toxin Ⅱ (STX Ⅱ) of enterohaemorrhagic Escherichia coli (EHEC) O157: H7 by affinity chromatography, and characterize its biological function. Methods The immno-affinity chromatography column was prepared by STX Ⅱ A subunit-specific antibody S1D8 coupling to Sepharose 4B matrix. The purity and specificity of STX Ⅱ molecule secreted by EHEC O157:H7 were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, respectively. The purified toxin was serially diluted and the toxic activities to Vero cell line and mice were observed. The 50% cytotoxic dose (CD50) for Vero cell line and 100% lethal dose (LD100) for mice were calculated. The protection effect of anti-STX Ⅱ polysera to the mice against the purified toxin challenge was also observed. Results STX Ⅱ was successfully purified from culture supernatant of EHEC O157:H7 using affinity chromatography scheme. The relative molecular weights of STX Ⅱ A and B subunits were 32 000 and 7 500 confirmed by SDS-PAGE, respectively. The purified toxin could react with monoclonal antibodies against STX Ⅱ A and B subunits, respectively.The toxin was cytotoxic to Vero cell with CD50 of 20 ng/L and lethal to mice with LD100 of 5 ng.The toxin could be neutralized by anti-STX Ⅱ polysera in vivo. Conclusion STX Ⅱ is successfully purified and its toxic effects are confirmed in both cell line and mouse model.

Antiapoptolic Mechamsms of Cell Transplantation in Cerebral Isehemia / 国际脑血管病杂志

The antiapoptotic mechanisms of cell transplantation have been paid wide attention in the process of the treatment of cerebral ischemic injtry.The transplanted cells play their roles of mtiapoptotic effect through releasing growth factors and nutritional factors,direct bonding,promoting angiogenesis,and anti-inflammation.

Effect of methalcobalamin on down regulation of insulin-like growth factor-1 gene expression and prevention of diabetic peripheral neuropathy / 中国组织工程研究

BACKGROUND: Diabetes causes abnormal insulin like growth factor-1 (IGF-1) gene expression, which contributes to initiation and development of peripheral neuropathy.OBJECTIVE: To investigate the efficacy of a single dose of methylcabalamin on prevention of experimental diabetic neuropathy and the possible molecular mechanism of its involvement in IGF-1 gene expression.DESIGN: Completely randomized and controlled experiment.SETTING: Endocrinology Department of the First Affiliated Hospital of Nanjing Medical University.MATERIALS: The study was carried out in an Animal Center of Nanjing Medical University. Totally 80 male Sprague Dawley rats (sanitary degree)were randomly selected.METHODS: ① Totally 64 rats were chosen to be induced diabetic. They were injected intravenously with alloxan dissolved in saline solutions, at the dose of 240 mg/kg. ② Of 16 rats were chosen as normal control group who were injected intravenously with equivalent volume of saline solution. ③ Of 64 established diabetic rats were treated with daily subcutaneous injection of pork regular insulin in combination of protamine zinc insulin (2:1) then further divided into 2 groups as insulin-treatment diabetic control groups based on different blood glucose levels: group 1 with relatively better control of diabetes, group 2 with relatively worse control of diabetes, with 32 rats in each group. Totally 16 rats of each group were treated with methylcobalamin injection intramuscularly with 500 μg/kg body weight, thus correspondingly divided into insulin +methylcobalamin group 1 and insulin+methylcobalamin group 2. The remaining 16 rats of each group as respective insulin-treatment diabetic control groups were treated with equivalent volume of saline. ④ Initiate weight and end weight were measured at beginning of the experiment and after diabetic model was established. Glucose oxidase was used to detect glucose level. 1-deoxy-1-malin was used to detect fructose level. ⑤ Parameters were measured as follows: Sensory/motor nerve conduction velocity (SNCV, MNCV) and evoked potential amplitude (EPA) of sciatic nerves detected by evoked electromyogram; IGF-I mRNA by reverse-transcriptase polymerase chain reaction (RT-PCR); IGF-1 peptide by enzyme-linked immunosorbent assay (ELISA). ⑥ One-way analysis of variance was used to analyze the Significance of differences among groups.MAIN OUTCOME MEASURES: ① Tissue IGF-1 mRNA/ IGF-1 peptide, electrophysiological data of individual groups at different points of the experiment. ② Comparison between individual groups in glucose metabolic parameters and body weights at different points of the experiment.RESULTS: Three rats died for diabetic infection or other acute complications and only 77 rats were included in the final statistical analysis.① Body weight and glucose metabolic parameter changes: After diabetic model, glucose, fructose level and body weight change between methylcobalamin+insulin treated groups and insulin treated groups were not significant. ② IGF-1 mRNA/peptide changes: Tissue IGF-1 mRNA increased significantly in methylcobalamin + insulin treated groups than that in insulin treated groups, respectively (P ＜ 0.05-0.01). Two weeks after diabetic model was established, the sciatic tissue IGF-1 mRNA contents were obviously higher in methylcobalamin + insulin treated group 1 than that in insulin treated group 1 (P ＜ 0.05), but not significantly different from that in NC group; Similarly, tissue IGF-1 mRNA contents were obviously higher in methylcobalamin + insulin treated group 2 than that in insulin treated group 2 (P ＜ 0.05), but lower than that in NC group (P ＜ 0.01); Month 2, tissue IGF-1 contents in methylcobalamin+ insulin treated groups were lower signiiicantly than NC groups, but higher than insulin treated groups (P ＜ 0.05-0.01). By month 3, IGF-1 mRNA level in methylcobalamin+ insulin treated group 2 was not significantly different from that in insulin treated group 2. The IGF-1 peptide levels in nerve tissue changed approximately parallel to IGF-1 mRNA level over time course. ③ Nerve electrophysiological data changes: Month 2 and 3, SNCV, MNCV and EPA were significantly higher in methylcobal-amin+ insulin treated group 1 than in insulin treated group 1 (P ＜ 0.05);Month 2, SNCV and EPA were higher in methylcobalamin+ insulin treated group 2 than in insulin treated group 2 (P ＜ 0.05); Month 3, SNCV, MNCV and EPA were significantly lower in methylcobalamin + insulin treated group 2 than in control group (P ＜ 0.05-0.01), whereas no difference was observed between methylcobalamin + insulin treated group 2 and insulin treated group 2.CONCLUSION: ① Methylcobal has not effect on blood glucose. ②Methylcobal could prevent occurrence of experimental neuropathy through its effect on nerve IGF-1 gene expression of diabetic rats. ③ A better efficacy could be achieved by Methylcobal with a good control of blood glucose level in prevention of diabetic peripheral neuropathy.

The relationship between KAI1 gene and neoplastic invasion, metastasis, prognosis / 医学研究生学报

KAI1 gene is a metastasis suppressor gene discovered in 1995, the product of which is a member of the transmembrane-4 superfamily. The expression of KAI1 gene correlate with metastasis of almost all cancer. But some results of some study are different. This article reviewed the study of the genomic structure of KAI1 gene and the relationship between KAI1 gene and neoplastic invasion, metastasis, prognosis.

Protective immunity induced by the anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the protective immunity induced by the anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum in mice.</p><p><b>METHODS</b>An orthogonal table L(16) (4 x 2(12)) was selected as the experimental design. Eight-week-old Kunming outbred mice (male and female) were randomly divided into 16 experimental groups and 2 control groups. Control groups were injected with SP2/0 ascites intraperitoneally. Mice from each group were infected with 100 +/- 2 cercariae of Schistosoma japonicum in the abdominal skin and were sacrificed on the thirtieth day postchallenge. Adult worms were recovered and counted by perfusion of the left ventricle-portal vein. The SP2/0 ascites injected mice were used as controls and the percentage of protection was calculated.</p><p><b>RESULTS</b>Active immunization of mice with NP30 could produce protection levels ranging from 22.36% to 50.46% depending on the different immunity protocols. The best immunization protocol was established from the results.</p><p><b>CONCLUSIONS</b>Active immunization with NP30 can induce a degree of protection to infection with Schistosoma japonicum cercariae and NP30 is a potential vaccine candidate against Schistosoma japonicum.</p>

CLONING AND SEQUENCE ANALYSIS OF THE LIGHT CHAIN VARIABLE REGION GENE OF MONOCLONAL ANTI-IDIOTYPIC ANTIBODY NP30 OF SCHISTOSOMA JAPONICUM / 中国寄生虫学与寄生虫病杂志

Objective] To amplify and sequence the light chain of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum. [Methods] By comparing the conserved regions at each end of the nucleotide sequences of murine germ line genes enco ding FR1 and FR4 regions of immunoglobulin light chain variable regions, we designed a set of primers for amplification of V L gene. The hybridoma cells secreting anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum were cultured and their genome DNAs were extracted and used as templates for PCR. The PCR product was then cloned into pUC19 vector. The recombinants were sequenced by Sanger′s method. The V L gene was compared with GenBank and published mouse V L genes. [Results] The full length of V L gene was 318 bp. The V L gene was a member of mouse Ig ? light chain subgroup IV and generated from rearrangement of germ line V and J? 4 genes. The V L gene sequence has been registered by GenBank(accession No. AF206720). [Conclusion] The obtained V L gene was a potentially functional gene of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum .

PROTECTIVE EFFECT OF ASTRAGALUS MEMBRANCE ON MICE OF ACUTE TOXOPLASMOSIS / 中国血吸虫病防治杂志

Objective To study the protective effect of Astragalus membrance on mice infected with tachyzoites of Toxoplasma gondii. Methods ICR mice were infected intraperitoneally with 10 5, 10 3, 10 2 tachyzoites of virulent RH strain of Toxoplasma gondii, and the mice were orally treated with Astragalus membrance 0 075 g/d per mouse or Azithromycin [150 mg/(d?kg)] each day starting from day 1 post-infection for 10 days. The survival rate and period were investigated. The parasite loads of livers and lungs of the mice infected with 10 2 tachyzoites were determined by fluorescence PCR methods at 4 day-post-infection (dpi) and 8 dpi. Results When infected with 10 5, 10 3 tachyzoites, treated with Astragalus membrance, the average survived days of the mice were 5 57 days and 6 23 days, and treated with azithromycin were 6 96 days and 8 12 days respectively. The azithromycin group but not the astragli group survived significantly longer than the control(P

Construction, expression and characterization of a mono-specific bivalent diabody derived from an anti-anti-idiotypic monoclonal antibody NP48 of Schistosoma japonicum / 中国血吸虫病防治杂志

Objective To construct a mono-specific bivalent diabody (scFv dimer) gene derived from an anti-anti-idiotypic monoclonal antibody NP48 of Schistosoma japonicum and to express and characterize the protein.MethodsThe mono-specific diabody gene (D) was constructed by SOE (splicing by overlap extension) and using Gly_4Ser as a linker to join the C-terminus of the V_H to the N-terminus of the V_L.D was linked with prokaryotic expression vector pBAD/g. The target protein expression in E.coli TOP10 was induced by arabinose. Then a purification procedure for the target protein was carried out. The antigen binding activity of expressed product was detected with Dot-ELISA. ResultsThe V_H-G_4S-V_L (D) gene was confirmed by sequencing. The pBAD/g-D recombinant were determined by digesting with endonucleases and expected bands were identified. There were less soluble target proteins in the supernantes and higher target proteins in the pellets as inclusion body when separating the D expression proteins. And the insoluble fraction was recovered as a soluble, correctly processed protein by solubilising with 8 mol/L Urea. The molecular weight of the target protein was about 27 kD. The binding activity of the target protein to anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum was verified by Dot-ELISA. ConclusionThe purified protein from the constructed recombinant pBAD/g-D could interact specifically with antigen NP30. So the constructed mono-specific diabody has the part characteristics of anti-anti-idiotypic monoclonal antibody NP48 of Schistosoma japonicum.

MOLECULAR MECHANISM OF APOPTOSIS OF CELLS IN EGG GRANULOMA INDUCED BY ANTI-IDIOTYPIC ANTIBODY NP30 / 中国血吸虫病防治杂志

Objective To study the molecular mechanism of apoptosis of cells in egg granuloma induced by anti-idiotypic antibody NP30 of Schistosoma japonicum.Methods BALB/c mice were randomly divided into two groups. The mice of the experimental group were immunized by injecting NP30 intraperitoneally for three times, while the mice of control group were injected normal saline intraperitoneally. The mice were sacrificed respectively on the 39th, 49th, 64th, 108th, 112nd day after challenge with schistosome cercariae. The expressions of apoptosis-related gene Bax, Bcl-2, death receptor Fas, FasL (Fas ligand) and c-Fos were examined by the S-P method of immunohistochemistry,and Bax, mRNA and Fas mRNA investigated by the in-situ hybridization. Results The expressions of Bax, Fas, FasL and c-Fos were positive in granuloma cells of both groups. The expressions of Bax and FasL in experimental group were higher than those in control group (P

Effects of Anti Idiotypic Antibody NP30 on Modulation of Egg Granuloma Formation and Hepatic Fibrosis of Schistosomiasis * / 中国寄生虫学与寄生虫病杂志

Objective To study the effects of the monoclonal anti idiotypic antibody NP30 active immunization on egg granuloma formation and hepatic fibrosis in Schistosoma japonicum infection. Methods ICR mice were actively immunized with NP30 100 ?g ?3 ip. every 10 days while the mice in control group were injected with SP2/0 ascites ip. simultaneously. After cercariae challenging,the mice were killed at the 4th, 8th,12th, 16th, 20th and 24th week, respectively.Mouse livers were removed and stained histochemically with VG and subjected to immunohistochemical assay of collagen type Ⅰ,Ⅲ and fibronectin(FN).The volume of egg granulomas and the content of collagen type Ⅰ,Ⅲ and FN were determined quantitatively by NYD 1000 Image Analysis System. Results The volume of egg granulomas in NP30 immunized group was much smaller than that of control group from the 12th week after cercariae challenge. The cellular components of egg granulomas in NP30 immunized group were significantly different from those of the control group,exhibiting two types of atypical egg granulomas were found.VG stain revealed that the average optical density of collagen in hepatic granulomas of experimental group was lower than that of control group.Immunohistochemical assay revealed that the contents of collagen type Ⅰ,Ⅲ and fibronectin in egg granulomas of experimental group were lower than those of control group. Conclusion NP30 vaccination may induce both cellular and humoral protective immunity to modulate egg granulomas and suppress liver fibrosis of schistosomiasis japonica.