Objective To investigate current situation of otoscope use by practitioners at community health-care service facilities in Hainan and Shenzhen and explore its importance and feasibility of spread of its use in community health-care service facilities in China.Methods Group discussion, telephone personal interview, otoscope market survey and literature research all were used among 112 practitioners at 67 community health-care service facilities in Hainan, 47 practitioners at 23 community health-care service facilities in Shenzhen, Guangdong, and 11 representatives involved in general practice training from 11 districts.Results In Hainan, only one community health-care service facility ( 1.5% ) was equipped with otoscopes and only one practitioner (0.9% ) could use it.In Shenzhen, two community health-care service facilities (8.7%) were equipped with it, but which had never be used.All the practitioners at community health-care service facilities surveyed have not been trained for its use, so most practitioners there did not have basic skills to use otoscope.Conclusions Otoscope is not equipped at most community health-care service facilities in Hainan and Shenzhen, and it is little used there even equipped with it.Otoscopy should be used as an adequate and feasible technique at community health-care service and be spread among general practitioners in China.

Objective To examine the expression of angiopoietin-like protein(ANGPTL)3 in kidneys from children with primary nephrotic syndrome. Methods Immunohistochemistry for ANGPTL3 was performed in kidney biopsies from patients with nephrotic syndrome or hematuria, including MCD (n=31), MN(n=6), FSGS (n=6), TBMN (n=10), IgA nephropathy (IgAN) with mesangial proliferation (n=16). Normal renal tissue of 2 cases with nephrectomy for tumor were used as control. According to the episode, four groups were divided ("12 months"). The expression was quantitatively examined with IMS color image analysis system, using positive index (PI) as sediment degree of ANGPTL3 in glomeruli or tubules. Immunofluorescence for ANGPTL3 co-labeling with WT1 and perlecan was applied to show the distribution of ANGPTL3. Results (1) The PI levels of ANGPTL3 in glomeruli of MCD(7.49?1.96) and MN (6.27?0.98) were significantly higher than those of TBMN (0.02?0.001), FSGS (3.14?0.49) or normal control(0.02?0.001) respectively (all P

Objective To construct a new recombinant immunotoxin expression vector fused with a murine interleukin18(IL18) gene and a truncated pseudomonas exotoxin (PE38) gene, and examine the expression of IL-18-PE38 fusion protein in Escherichia coli (E. coli). Method Murine IL-18 (mIL-18) cDNA was cloned from murine liver tissue through reverse transcription-polymerase chain reaction (RT-PCR). The mIL-18 cDNA was ligased with a PE38 gene carried by PRKL expression vector through T4 DNA ligase and constructed into fusion protein expression plasmid PRKL-IL18-PE38. The recombinant vector was identified by restriction endonucleases digestion, PCR and DNA sequencing. After transformed into E.coli BL21 and induced by IPTG, the expressed product was obtained and the molecular weight and specificity were determined by SDS-PAGE and Western-blotting. Result The new recombinant immunotoxin expression vector was constructed successfully. DNA sequencing revealed that the mIL-18 and PE38 gene were consistent with NCBI Gene Bank. The IL-18-PE38 fusion protein was expressed in E.coli BL21, and Western-blotting analysis indicated that the molecular weight of the expression product is about 56 kDa, and could react with the specific antibody against mIL-18. Conclusion IL-18-PE38 recombinant immunotoxin expression vector will provide the basis for study on the targeted cytotoxic activity to Th1 cells and may have some potential value in the treatment of Th1 cell-mediated autoimmune diseases.

Infectious bursal disease virus (IBDV) VP4 plays an important role in immunosuppression of host. In order to develop monoclonal antibodies (McAbs) against VP4, we vaccinated BALB/c mice with His-VP4, screened and subcloned positive clones. We established 4 hybridoma cell lines that stably secreted McAbs against VP4 and named these cell lines 3B3, 3H11, 4C8 and 4G6, respectively. We tested the dissociation constant (Kd) of these McAbs, and found that their K(d)s were 4.61 x 10(-11), 1.71 x 10(-10), 4.26 x 10(-11), 5.02 x 10(-11), respectively. The isotypes of these McAbs were determined to be IgG1, IgG1, IgG2b and IgG1. These McAbs specifically bound to VP4 in IBDV infected DF-1 cells as demonstrated by Western blotting analysis and fluorescence antibody assay. These McAbs would help to detect IBDV infection and to analyze the biological activities of IBDV VP4.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Blotting, Western ; Cell Line ; Fluorescent Antibody Technique ; Hybridomas ; Infectious bursal disease virus ; Mice ; Mice, Inbred BALB C ; Viral Structural Proteins ; immunology

In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus (A/PR/8/34) and studied their expression in HEK293 cells. HA and HA1 genes were amplified by RT-PCR and cloned into pcDNA3.1(+) to generate pcDNA3.1(+)/HA and pcDNA3.1(+)/HA1, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3.1(+)/HA and pcDNA3.1(+)/HA1 were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3.1(+)/HA or pcDNA3.1(+)/HA1. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HA1, which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine.

Objective To examine the expression of response gene to complement 32 (RGC-32) in renal tissue of children with IgA nephropathy (IgAN), and to explore its significance. Methods The subjects were 45 children diagnosed as IgAN by renal biopsy. The expression of RGC-32, α-smooth muscle actin (α-SMA) and transforming growth factor β1 (TGF-β1) was examined by immunohistochemistry staining. The correlation of RGC-32 expression with α-SMA,TGF-β1, degree of renal pathological lesions and clinical index in IgAN was assessed by Spearman correlation analysis. Results RGC-32 protein located in renal tubular epithelial cells in normal and IgAN renal tissues. The positive expression index of RGC-32 in nomal group, IgAN mild group, moderate group and severe group was (18.29±6.22)%, (23.90±9.65)%, (31.23±9.86)%,and (34.52±10.63)% respectively. With more severity of renal pathological lesions, the expression of RGC-32 in IgAN was enhanced. The RGC-32 expression was positively correlated with the score of glomerulus and renal interstitium in children with IgAN (r=0.385, 0.347, P＜0.05), as well as α-SMA, TGF-β1 (r=0.594, 0.521, P＜0.01), but was not correlated with Scr, urinary NAG/Cr,Alb/Cr, IgG/Cr, and α1-M/Cr (r =0.117, -0.115, -0.138, -0.176, -0.028, all P ＞0.05).Conclusions RGC-32 protein locates in renal tubular epithelial cells in normal and IgAN renal tissues. RGC-32 may participate in the course of renal tubulointerstitial lesions in children with IgAN, especially in the course of epithelial-mesenchymal transition (EMT) induced by TGF-β1.

Objective To observe ultramicro pathologic change of rabbit brain central damaged tissue and peripheral tissue after LOT, to evaluate the changed structure of blood brain barrier (BBB) of peripheral tissue in acute stage. Method Seventy Newzealand rabbits were randomly screened from Zhejiang University Animal Experiment Center. By stereotaxic technique, semiconductor surgica laser fibers were inserted into right frontal lobes and heat treated to randomly build LITT Group A (2 W, 600 s, n = 20) and LITT Group B (15 W, 100 s, n = 20) brain damaged models successfully. Other 15 nomal rabbits were randomly distributed as mannitol perfusion group and fake operation group. The ultramicro structures in central thermodamaged tissue were observed with transmission electro microscope after LITT 3 h,6 h,12 h,24 h. In peripheral tissue, ultramicro morphologic changes of brain vessels and BBB were evaluated. S100B protein in serum and BBB indexe were measured at different stages post LITT. Experimental data were treated as one-factor analysis of variance and q test. Results The brain damage center connected the tip of laser fiber and turn into thermodamage tissue. The main structure changes were cytoclasis, damnification of cell membrum, swelling of cell organelle such as mitochondrion, endoplasmic reticulurn,disappearance of mitochondrion and sparseness of cytoplasm in local tissure. Heat energy conducted to damage peripheral tissue, some cells occured apoptosis in different stage. In acute stage after LITT, contracted capillary vessel, oncreted red cell, swell endothelium cell, broken base membrum, wide around clearance and destroyed aperture structure were identified. The levels of serum S100B and BBB indexe dramatically rised. The opening time of BBB in peripheral tissue was longer than mannital perfusion group. However at 24 h post LITT, they began to recover in Group A. The difference of serum S100B and BBB indexe between Group A and Group B has statistical significance ( P =0.0087). Conclusions With semiconductor laser heat treatment and stereotaxic techniqe, definite cells cytoclasis, cell membrance structures and chondriosome damage could be performed obviously in rabbit brain thermotherapy point. Apoptosis could be found in peripheral tissue, BBB could be opened in an acute stage. The opening time course of BBB was shortened in those LITT cases with small power. It shew us a new method to perform a safe and exact damage zone of brain for functional neurosurgery.

Objective: To investigate the immunological mechanism of influenza A virus for murine S180 ascites sarcoma. Methods: After inoculation with S180 sarcoma cells, mice were i. p. injected with influenza A virus or vehicle 15 days. The average living time and survival rate of the mice were examined. The levels of IL-2, IL-6 and TNF-? were detected. The sarcoma cell's apoptosis was detected by DNA ladder, flow cytometry (FMC) , fluorescent microscope and e-lectron microscope (EM). Results: The average living time and survival rate of the mice injected with Influenza A virus were significantly longer or higher than that of the controls. The levels of IL-2, IL-6 and TNF-? also had the same differences. The apoptosis cells were detected by EM and fluorescent microscope. Sub-diploid peaks were observed by FCM a-nalysis and DNA ladder was seen after electrophoresis in the ascites cells. Conclusion: Our results demonstrated that the feasibility and potential of delivery of influenza A virus as a general means for the treatment of S180 ascites sarcoma.

Aim To construct eukaryotic expressing plasmid of hi FGF2 ( high molecular weight isoform fi-broblast growth factor-2,hi FGF2) gene and to investi-gate its effect on apoptosis after its overexpression in HEK293 cells. Methods The DNA template primer was designed and synthesized. The pDsRed1-N1 plas-mids were digested by the restriction enzymes of Nhel and Hind III. The hi FGF2 was ligated with linearized pDsRed1-N1 by T4 DNA Ligase. The recombinant plasmid was identified by endonuclease digestion and sequenced. The recombinant hi FGF2 plasmid was transient transfected into HEK293 cells by Lipofectami-neTM 2000 Reagent. The transfection efficiency was de-tected by fluorescence inversion microscope. The cell apoptosis was detected by Annexin V-FITC/PI apopto-sis detection kit with flow cytometry analysis. Results The pDsRed1-N1 eukaryotic expression vector was consistent with the design. The recombinant hi FGF2 plasmid was transfected in HEK293 cells. The trans-fection rate was more than 70%. The FITC/PI dyeing rate in hi-FGF2 over-expression HEK297 cells was a-bout ( 29. 12 ± 2. 81 )%. Conclusions pDsRed1-N1 eukaryotic expression vector is successfully constructed and transfected into HEK293 cells. Over-expression of hi FGF2 induces cell apoptosis.

Clinical data of 46 patients with trans-sphincter anal fistula treated by fistulectomy with external anal sphincter bareness in Department of Anorectal Surgery, Jiaxing TCM Hospital from July 2018 to July 2019 were retrospectively analyzed. All operations were performed successfully. There were no significant differences in Wexner incontinence scores (2.00±0.68 vs.1.99±0.70, P<0.05), mean anal resting pressure [(75.60±8.60) vs.(73.60±8.20)mmHg(1 mmHg=0.133 kPa), P<0.05] and maximum systolic pressure [(109.60±7.80) vs.(107.20±8.30)mmHg, P<0.05] before and 6 months after operation. There were 1 case with postoperative incision bleeding and 2 cases with postoperative infection. All patients were followed up for 6 months and there was no recurrence and changes in anal shape during the follow-up. Results indicate that the fistulectomy external anal sphincter bareness is safe, efficient with well preserved sphincter function for patients with trans-sphincter anal fistula.