ABSTRACT
<p><b>OBJECTIVE</b>To observe the dynamic changes of immune responses of splenocytes in mice immunized with recombinant vaccine Bifidobacterium bifidum (pGEX-Sj32) of Schistosoma japonicum and investigate the immunological mechanism of the vaccine.</p><p><b>METHODS</b>Eighty-eight BALB/c mice were randomized for immunization with 10⁶ CFU recombinant vaccine orally or with 10⁵ CFU recombinant vaccine intranasally. Four mice were selected from each group every two weeks to test the responses of the splenocytes to stimulations with SjAWA or ConA. MTT assay and flow cytometry were used to assess splenocyte proliferation and the distribution of CD4⁺ and CD8⁺ T cells, respectively; the levels of interleukin-10 (IL-10), IL-12 and tumor necrosis factor-α (TNF-α) in the cell culture supernatant were detected by ELISA.</p><p><b>RESULTS</b>Regardless of the stimulations, the splencytes showed significantly enhanced proliferation in weeks 2-16 in oral administration group and in weeks 2-18 in intranasal group (P<0.01). CD4⁺ subsets in both two groups increased obviously in weeks 2-12 (P<0.01) but CD8⁺ subsets remained stable. In oral administration group, the levels of TNF-α, IL-10 and IL-12 increased in weeks 2-14, 2-18 and 2-14, and peaked at week 8, 10 and 6, respectively; in intranasal group, the cytokines increased in weeks 2-14, 2-18 and 2-18, and peaked at week 8, 10 and 8, respectively.</p><p><b>CONCLUSION</b>The recombinant vaccine rBb (pGEX-Sj32) can induce effective immune responses in mice.</p>
Subject(s)
Animals , Mice , Antigens, Helminth , Allergy and Immunology , Bifidobacterium , CD4-Positive T-Lymphocytes , Allergy and Immunology , CD8-Positive T-Lymphocytes , Allergy and Immunology , Interleukin-10 , Allergy and Immunology , Interleukin-12 , Allergy and Immunology , Mice, Inbred BALB C , Schistosoma japonicum , Schistosomiasis japonica , Spleen , Cell Biology , Allergy and Immunology , Tumor Necrosis Factor-alpha , Allergy and Immunology , Vaccination , Vaccines, Synthetic , Allergy and ImmunologyABSTRACT
Objective To construct and express a recombinant plasmid pGEX-Sj 14-3-3-Sj32 of Schistosoma japonicum in Escherichia coli (E.Coli) BL21 (DE3).Methods Sj14-3-3 and Sj32 antigen genes were amplified by PCR from template of plasmids pGEX-Sj14-3-3 and pET28α-Sj32 which were extracted from recombinant bacteria BL21 (pET28α-Sj32) and BL21 (pGEX-Sj14-3-3) stored in Institute of Infectious and Parasitic Disease of the First Affiliated Hospital of Chongqing Medical University.Sj14-3-3-Sj32 fusion gene obtained with gene SOEing was cloned into the vector pGEX-1λT to construct pGEX-Sj14-3-3-Sj32 which was identified by double digestion.The recombinant plasmid pGEX-Sj 14-3-3-Sj32 was transformed into E.Coli BL21 (DE3).The recombinant strains were induced by isopropyl-β-d-thiogalactoside (IPTG),and the expressed products were analyzed and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.Results The 1 750 bp Sj14-3-3-Sj32 fusion gene was successfully amplified by gene SOEing and cloned into the vector pGEX-1 λT verified by restriction analysis,the recombinant plasmid pGEX-Sj 14-3-3-Sj32 was successfully constructed.The molecular mass of the expressed recombinant protein was proximately 73 × 103 as detected by SDS-PAGE.Western blotting confirmed that the expressed protein could be recognized by the immune sera from rabbit infected with Schistosomajaponicum.Conclusion The recombinant plasmid pGEX-Sj14-3-3-Sj32 is successfully constructed and could be highly expressed in E.coli and the expressed recombinant protein has specific antigenicity.
ABSTRACT
Objective To construct and identify recombinant vaccine Bifidobacterium bifidum (pGEX-Sj32) of Schistosomajaponicum (Sj).Methods The Sj32 gene amplified by PCR from template of plasmid pET28α-Sj32 that extracted from recombinant Escherichia coli(E.coli) BL21 (pET28α-Sj32) stored in our laboratory was cloned into E.coli-Bifidobacterium bifidum shuttle expression vector pGEX-1λT to construct recombinant plasmid pGEX-Sj32.The recombinant plasmid was transformed into E.coli BL21 (DE3).The plasmid was extracted and identified by restriction enzyme digestion.The recombinant Bifibacterium bifidum (pGEX-Sj32) vaccine was constructed by electroporating pGEX-Sj32 into Bb.The extracted plasmid was amplified and identified by PCR.Results The gene Sj32 of 1 270 bp in length was amplified by PCR.The restriction endonuclease digestion showed that the length of plasmid vector was 4 947 bp,and Sj32 gene was 1 270 bp.The Sj32 gene amplified by PCR from the template of pGEX-Sj32 extracted from recombinant Bb (pGEX-Sj32) vaccine was 1 270 bp in length which consistent with expected result.Conclusion The recombinant Bb (pGEX-Sj32) vaccine of Sj is successfully constructed.
ABSTRACT
Objective To construct the recombinant plasmid pGEX-Sj32 of Schistosoma japonicum(Sj )and to research its ex-pression in Escherichia coli (E.coli)BL21.Methods Sj32 gene was amplified by PCR from template of plasmid pET28α-Sj32 ex-tracted from recombinant bacterium BL21 (pET28α-Sj32 )stored by our laboratory,and then cloned into the vector pGEX-1λT to construct pGEX-Sj32.The recombinant plasmid pGEX-Sj32 was transformed into E.coli BL21(DE3).The recombinant strains were induced by isopropyl-β-d-thiogalactoside(IPTG),and the expressed products were identified by SDS-PAGE and Western blot.Re-sults Sj32 coding gene was successfully amplified by PCR and cloned into the vector pGEX-1λT,and the recombinant plasmid pGEX-Sj32 was constructed successfully.The molecular mass of the expressed recombinant protein was proximately 58 000 as de-tected by SDS-PAGE.The amount of the expressed protein was about 21% of the total bacterial protein.Western blot confirmed that the expressed protein could be recognized by the immune sera from rabbit infected with Schistosoma japonicum.Conclusion The recombinant plasmid pGEX-Sj32 is successfully constructed.The Sj32 protein was highly expressed in E.coli and the expressed recombinant protein possesses the specific antigenicity.
ABSTRACT
To observe the dynamic changes of splenocyte proliferation ,subsets and apoptosis in mice immunized with re-combinantBifidobacteriumbifidum(pGEX-Sj26GST)ofSchistosomajaponicum,themiceweresubcutaneously(SCgroup) and intranasally (IN group) immunized ,respectively .Four mice from each group were sacrificed in every 2 wk during 0-20 wk after immunization .Splenocyte proliferation was investigated by MTT colorimetric assay ,subsets of CD+4 and CD+8 T cells and apoptosis of splenocytes by FACsort flow cytometry .In SC group ,unstimulated and stimulated with S jAWA ,the level of splenocyte proliferation significantly increased at 4-20 wk after vaccination and increased markedly at 4-18 wk stimulated with ConA ,both of which peaked at 8 wk;in IN group ,the proliferation level of splenocyte cultured with SjAWA and ConA signif-icantly increased during the 4-18 wk ,2-10 wk and 14-18 wk ,2-8 wk and 12-18 wk ,respectively ,and all reached the maximum at the 4 wk after immunization (P＜0 .01 or P＜0 .05) .CD+4 subsets increased obviously during 2-14 wk ,2 wk and 6-16 wk re-spectively ,and reached the peak at 8 wk (P＜0 .01 or P＜0 .05) in both group ,while CD8+ subsets rose lightly during 2-20 wk in both group ,and reached the maximum at 8 wk (SC group) and 6 wk respectively (P>0 .05) .Whether unstimulated or stimulated with ConA ,the level of splenocyte apoptosis of which remarkably increased at 2-4 wk and 2-6 wk separately in SC group ,and both peaked at 2 wk (P＜0 .01 or P＜0 .05 );in IN group ,the level of splenocyte apoptosis all increased at 4 wk and reached the maximum at the same time .In summary ,by inducing the proliferation of splenocytes ,increasing CD4 + T cells and decreasing splenocyte apoptosis ,the rBb (pGEX-Sj26GST ) vaccine plays a critical role in the protective immune response .
ABSTRACT
Objective To observe the clinical effect of umbilical cord blood stem cell transplantation in the treatment of decompensated hepatic cirrhosis .Methods The umbilical cord blood of healthy puerperal women was collected,and stem cells were isolated and injected into liver through hepatic artery to treat 20 patients withdecompensated cirrhosis.Blood,liver function,coagulation changes ,indicators of clinical symptoms and adverse events were observed after transplantation 1 week,2 weeks,4 weeks and 8 weeks.Results 1 week after transplantation ,the fatigue,loss of appetite situation were improved in more than 80% patients,but the changes were not significant ( t=0.88,0.80, 0.20,0.23,0.56,all P>0.05).2 weeks after transplantation,the bloating of 60% patients was improved,ALB in-creased significantly(t=2.03,P<0.05).After 4 weeks,the bloating of 70%patients relieved,50%of patients with ascites subsided.ALB,ALT,PT were significantly improved (t=3.84,P <0.01;t =1.75,3.02,all P<0.05);8 weeks later,75%of patients with ascites subsided .Except for AST was not obvious ,other indicators were improved significantly(t=2.20,2.22,5.93,all P<0.05).Except for one case had low fever ,there were no other adverse reac-tions.Conclusion Cord blood stem cell transplantation in the treatment of decompensated cirrhosis can achieve a certain short-term treatment effects , especially in increasing the level of ALB and reducing PT , could resume liver damage and has relatively high security at the same time .
ABSTRACT
ObjectiveTo establish rabbit knee joint cartilage injury models to evaluate effects of the type Ⅱ collagen sponge in repair of the articular cartilage.MethodsThe type Ⅱ collagen sponge was prepared according to previous method and the pore size of the sponges was measured based on the collagen autofluorescence characteristics.The type Ⅱ collagen sponge was transplanted into the injury lesions of the animal model for experimental study.The regeneration of the cartilage defects was observed by using MRI, histologic HE staining, Safranin O, sirius red polarized light staining, areas determination of the newly grown cartilage and immunohistochemistry of type Ⅱ collagen.ResultsAutofluorescent images of confocal microscope layer scanning showed that the pore size was (93.26 + 38.40) μm in diameter, suitable for chondrocyte growth.Comparison between MRI and H&E staining results showed quicker effusion absorption in the treatment groups than that in the control group, while the level of inflammatory response in the treatment group was lower than that in the control group.The sporadic cartilage signals first appeared at the 6th week.The newly formed cartilage with the expression of glycosaminoglycan and type Ⅱ collagen matrix was confirmed by Safranin O staining and immunohistochemical analysis.The sirius red polarized light staining showed that areas of the newly formed cartilage were significantly larger in the treatment group than that in the control group (P ＜ 0.01).Conclusion The type Ⅱ collagen sponge developed from purification can effectively repair the damaged cartilage tissues of the rabbit knee joints, as has been verified either by MRI or histology.
ABSTRACT
Objective To investigate the diagnosis rate of early gastric calleer(EGC)with mucosa biopsy after compound staining under gastroseopy.Methods Two hundreds and six pafients who suspected tO be EGC by gastroscopy were randomly divided into two groups,the mucosa in control group(n=103)was biopsy directly under gastrescopy,and the experimental group(n=103)was biopsy after compound staining.Compared the diagnosis rate of ECG between these two groups.Results Twelve cases were confirmed to be EGC by surgery plus pathology among all the patients.Two cases were from control group,the other ten cases were from experimental group.Type Ⅱ c+Ⅲ accounted for 58.3%of twelve EGG.Eleven cases(91.7%)were positive with helicobacter pylori(Hp).Conclusion Compound staining may improve diagnosis rate of ECG.There is significant deference between directed and mueosa biopsy after compound staining under gastrescopy(P=0.033).TypeⅡc+Ⅲwere main type in endoscope of EGC.Hp infection is closely related to early gastric cancer.
ABSTRACT
BACKGROUND: Simple polyvinyl alcohol (PVA) has limited ability to cell adhesion. There are not generally accepted studies on improved effects of collagen protein modified polyvinyl alcohol on cell adhesion and proliferation.OBJECTIVE: To investigate the feasibility of PVA/type Ⅰ college (COL-Ⅰ) as anterior cruciate ligament (ACL) scaffolds in tissue engineering.DESIGN, TIME AND SETTING: The controlled observation experiment was performed at the Fourth Affiliated Hospital, Medical College. Ji'nan University, Guangzhou Red Cross Hospital, Guangzhou Institute of Trauma Surgery from August 2006 to October 2007.MATERIALS: COL-Ⅰ gel was produced by Guangzhou Institute of Trauma Surgery.METHODS: PVA filature was used to weave fascicular scaffolds. NIH-3T3 cell line and human ACL cells were in vitro incubated, amplified, and then implanted on the PVA/COL scaffolds.MAIN OUTCOME MEASURES: The growth of NIH-3T3 cell line and human ACL cells on the PVA/COL scaffolds and the secretion of extracellular matrix were observed using scanning electron microscope. Cell compatibility of PVA/COL scaffolds was assessed. Mechanics characteristic of PVA/COL scaffolds was measured by using the electric. tensile force apparatus. Mechanical property of PVA/COL scaffolds was analyzed using the SPSS 11.5 software package.RESULTS: NIH-3T3 cell line and human ACL cells on the PVA/COL scaffolds adhered, proliferated, and secreted extracellular matrix. NIH-3T3 cell line highly grew compared with human ACL cells on the PVA/COL scaffolds. The adhered number of NIH-3T3 cell line and human ACL cells was significantly increased on the PVA/COL scaffolds. NIH-3T3 cell line and human ACL cells presented well morphology on the PVA/COL scaffolds. COL-Ⅰ could promote the secretion of extracellular matrix from NIH-3T3 cells, but its effects on human ACL cells were not significant. Tensile force test showed that load-extension curve of the materials was identical to ACL of human and rabbits, and the scaffolds possessed strong flexibility. The maximal load, ultimate stress and elastic modulus were respectively 52.61 N, 14.96 MPa and 202.08 MPa.CONCLUSION: COL-Ⅰ accelerates the adhesion and proliferation of NIH-3T3 cell line and human ACL cells on the surface and in the pore of the PVA/COL scaffolds, promotes the secretion of extracellular matrix from NIH-3T3, and PVA filature material has mechanical property and good cell compatibility.
ABSTRACT
BACKGROUND: Fibronectin serves not only as the supporter for cells,but also as an important intracellular linkage, possessing opsonic-like functions. It can promote the phagocytic function of mononucleophages and the repair in inflammation and trauma.OBJECTIVE: Type O plasma was freshly obtained from healthy males in search of the optimal preparation method for lower sampling, convenient and higher-yielding of fibronectin.DESIGN: Open experiment.SETTING: Institute of Traumatic Surgery, Fourth Affiliated Hospital of Guangzhou Red Cross Hospital, Jinan University.MATERIALS: This experiment was carried out in the Institute of Traumatic Surgery of Guangzhou Red Cross Hospital between July 2000 and July 2002. The major materials consisted of type O fresh plasma from healthy males, gelatin, sepharose 4B activated with cyanogen bromide,sephadex G-25, urea, and trishydroxymethylaminomethane.METHODS: Affinity column constituted by gelatin coupling with sepharose 4B activated with cyanogen bromide was used to purify fibronectin. ① Human plasma was added: 150 mL type O plasma was freshly obtained from healthy males and added into the column once by 25 mLat an interval of 20 minutes, the speed of flow was 3.5 mL/min. ② Removing mixed protein: the column was rinsed by Tris-sodium citrate equilibrium liquid (pH 7.5) until the absorbency of flow-out fluid decreased to ＜ 0.02 at 280 nm. Again lmol/L NaCl containing Tris-sodium citrate was used to wash off other mixed proteins. ③ Collecting fibronectin: the column was rinsed by urea-Trispurge Fluid (3 mol/L) to collect fibronectin.④ Removing urea from fibronectin collection: fibronectin collection liquid was filtrated by Sephade G-25 to remove urea. ⑤Disinfection: 0.22 μmgermtight filter was used for disinfection.MAIN OUTCOME MEASURES: ①Results of sodium dodecyl benzene sulfonate-polyacrylamide gel electrophoresis. ② The influence of retention time on the yield of purified fibronectin. ③ The influence of plasma quantity on the yield of purified fibronectin.RESULTS: ① Results of sodium dodecyl benzene sulfonate-polyacrylamide gel electrophoresis: The density of separation gel and condensed gel was 7% and 3%, respectively; fibronectin was electrophoresized into single protein lane. ② The influence of retention time on the yield of purified fibronectin: The yield of fibronectin was (65.24±3.45) %, (74.77±4.05) %,(86.99±4.10) %, and (80.47±3.75) %, respectively, when the loading amount of fibronectin was 150 mL with non-retention time and retention time of 10, 20 and 30 minutes. ③ The influence of plasma quantity on the yield of purified fibronectin: The yield of fibronectin was (72.56±3.63) %,(77.61±3.14) %, (86.99±4.12) %, and (74.67±3.05) % when the column retention time was controlled at minute 20 with the loading amount of 100,130, 150 and 180 mL, respectively.CONCLUSION: In a given column volume of gelatin, the quantity of purified fibronectin was closely related with the plasma retention time in column and the total loading amount of plasma. As a result, the optimal loading amount of plasma was 150 mL, and the retention time was 20 minutes.The preparation method, herein, has been proved to require small amounts of plasma and yield large amounts of fibronectin.