BACKGROUND:There is a linker between Hsp65 and Esat6 coding a segment of water repellent polypeptide.It is very gentle and easy to be folded,which is profitable to translate the keno-folding correctly between the two proteins and to make the space structure of fusional proteins consistent with those two native ones.Then,a correct structure is formed and the immunogenicity of the fusional proteins is improved.OBJECTIVE:This study was to clone the fusional genes Hsp65-Esat6 from mycobacterium tuberculosis(MTB)H37Rv, then to construct into a eukaryotic expressing vector which contains an enhanced green fluorescent protein(EGFP) reporter gene,and finally to identify the expression of Hsp65 protein and Esat6 protein by IHC methods.DESIGN:Single sample experiment.SETTING:Department of Microbiology ＆ Immunology,Medical College of Jinan University.MATERIALS:Plasmid pVAE was donated by Professor Cao from South China University of Technology.MTB H37Rv,E.coil DH5 α,expressing plasmid pEGFP-C1 and Hela cells were donated by Doctor Hu Ping.METHODS:The experiment was conducted at the Department of Microbiology ＆ Immunology and the Medical Experimental Center of Jinan University between September 2005 and June 2006.The whole-genome was extracted from MTB H37Rv by molecular cloning technique,and used it as template to amplify Hsp65 (no terminator) gene by polyrnerase chain reaction (PCR),to recombine it with pEGFP-C1 vector after purification to construct pEGHsp65(no terminator)recombination vector.pVAE vector was used as template to amplify Linker-Esat6 gene(with terminator)by PCR, and then to recombine it with pEGHSP65(no terminator)to construct an eukaryotic expressing vector pEGHsp65-Esat6 with the fusional genes Hsp65-Esat6 inside.Finally,genes Hsp65-Esat was checked by molecule biology methods such as PCR, restriction endonuclease and DNA sequencing.Hela cells were transfected with pEGHsp65-Esat6 and the expression of EGFP and the efficiency of transfection were observed.The expressions of Hsp65 protein and Esat6 protein were detected by IHC methods.MAIN OUTCOME MEASURES:①The size of pEGHsp65-Esat6.②The DNA sequencing result of the pEGFP-C1.③The expression of EGFP in the transfected Hela cells.④The IHC results of Hsp65 protein and Esat6 protein in Hela cells.RESULTS:①The size of pEGFP-C1 was 4.7 kb,that of pEGHsp65 was 6.4 kb,and that of pEGHsp65-Esat6 was 6.7 kb.There were differences between their speeds In agarose electrophoresis.②The results showed that it was the same as reported Hsp65 sequence and Esat6 sequence of MTB H37Rv.③After transfecting pEGHsp65-Esat6 for 24 hours,EGFP was found in 30% of Hela through Laser scanning confocal microscope. But there was no EGFP in non-transfected Hela.④Hsp65 protein and Esat6 protein with biological activities were detected in transfected Hela cells by IHC methods.CONCLUSION: Using Hsp65 and Esat6 as immunogens,we have successfully cloned and constructed a eukaryotic expressing vector which contain fusionaI genes Hsp65-Esat6 of MTB H37Rv and EGFP.

Objective To observe the cardiac function in acute brain injury patients(ABI)and the relationship between ABI and plasma neuropeptideY(NPY),and to inspect the mechanism and find the evidences for preventing cardiac impairment caused by ABI. Methods 89 patients with acute brain injury within 24 hours after the injury were divided into severe group(n =47)and mild group(n = 42)according to Glasgow Coma Scale(GCS),and 35 normal healthy adults were selected as control group.In 24 hours and 72 hours after the brain injury,all patients were examined with echocardiography to observe cardiac structure,Doppler blood flow velocity and cardiac function,and in the same time the plasma NPY were determined by radioimmunoassay.Then the results were compared with controls. Results The parameters of cardiac function such as EF、 SV.AV、CO、CI had statistical change in 24 hours and 72hours after the brain injury between severe ABI group and mild ABI group,and it also had statistical change between severe ABI group and control group(all P <0.05),but no statistical change between mild ABI group and control group(all P ＜0.05).The level of plasma NPY in ABI patients was significantly higher than that before injury,there was statistically different change between severe ABI group and mild ABI group,and it also had statistical change between severe ABI group and control group(all P＜0.05).The parameters of cardiac function was negatively correlated with the rise of plasma NPY by pearson correlation analysis(EF:r =- 0.79,P <0.01; SV:r =- 0.71,P <0.01;AV:r=-0.67,P ＜0.01 ;E/A:r =-0.63,all P ＜0.01)and(CO:r =- 0.32,P ＜0.05;CI:r =-0.35,all P ＜0.05). Conclusion The parameters of cardiac function were significantly decreased in the patients with acute brain injury,and it was closely related with the level of plasma NPY.

Objective Shkbp is also called Shkbp1,can competitively inhibit binding CIN85 and c-Cbl,thereby blocking the epidermal growth factor receptor (EGFR) endocytosis and degradation,to play a role in tumor promotion.This study aims to explore the changes in blood cell classification and T cell subsets in blood,bone marrow,and spleen in Shkbp1-deletion (Shkbp-1-/-) mice.Methods Shkbp-1-/-transgenic mice were identified by PCR genotyping.Blood cell classification was performed using an automatic classification system.Flow cytometry was used to detect the T lymphocyte subsets in the blood,bone marrow,and spleen of Shkbp-1-/-and control mice.Results Routine blood examination showed that neutrophils and eosinophils tended to increase and showing significant differences,and there was no significant difference in lymphocytes.The flow cytometry results showed that there was a decrease of CD4+CD8+ double positive cells and increase of bone marrow CD3+ and CD4+ cells in the control group.However,there was a decreasing trend of CD3+,CD4+,CD8+,and CD4+CD8+ cells in the spleen tissues.Conclusions Shkbp1 is involved in the maturation and differentiation of blood cells,and affects the number of immune cells.This study lays a foundation for the study of how Shkbp1 is involved in the differentiation of blood cells.

Objective PSGL-1 is specifically expressed in leucocytes.The aim of this study was to explore the changes of myeloid-derived suppressor cells (MDSCs) in the spleen and bone marrow in PSGL-1-deficient mice.Methods PSGL-1 -/-mice were used in the experiment.After identification of the offsprings, flow cytometry was used to test the expression of CD11b and Gr-1 in C57 and PSGL-1 -/-mice.Results Compared with the C57 mice, the expression of MDSCs was up-regulated in the PSGL-1-deficient mice ( P <0.001).Conclusion The expression of MDSCs is upregulated in PSGl-1-deficient mice.

Objective:To compare the efficacy of pars plana vitrectomy (PPV) combined with human amniotic membrane (hAM) plugging technique or internal limiting membrane (ILM) flap insertion technique for high myopia macular hole retinal detachment (MHRD).Methods:A non-randomized controlled clinical study was performed.Sixteen eyes of 15 patients with high myopia MHRD treated in the Ninth People's Hospital of Shanghai Jiao Tong University School of Medicine from July 2020 to August 2021 were included.All patients underwent PPV and were divided into hAM plug group (7 eyes of 7 patients) and the ILM insertion group (9 eyes of 8 patients) based on the different plugging materials.The best corrected visual acuity (BCVA) and intraocular pressure were measured before surgery and at 1 week, 1, 3, and 6 months postoperative, respectively.Slit-lamp microscopy combined with lenses, scanning laser ophthalmoscope and optical coherence tomography (OCT) were used to examine the fundus, the macular hole closure and retinal reposition.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine (No.SH9H-2021-T322-2). Written informed consent was obtained from each subject.Results:The retinal reattachment was achieved in 6 eyes in the hAM plug group and all 9 eyes in the ILM insertion group after initial surgery.The macular hole closure was observed in 5 eyes in the hAM plug group and 8 eyes in the ILM insertion group after initial surgery, and there was no statistical difference in the macular hole closure rate between the two groups ( P>0.05). There were significant differences in the overall comparison of BCVA between the two groups over time ( Ftime=4.420, P<0.05). Postoperative BCVA at different time points was better than preoperative BCVA in each group, but the differences were not significant (all at P>0.05). There was no significant difference in the overall comparison of BCVA between the two groups ( Fgroup=0.183, P>0.05). Two eyes in the hAM plug group and 4 eyes in the ILM insertion group developed transient ocular hypertension, which returned to normal after 1 week of treatment. Conclusions:Both PPV combined with hAM plugging technique and ILM insertion technique are safe and effective for the treatment of MHRD in high myopia.The hAM plugging technique can not only achieve anatomical reduction but also functional recovery of the retina even in complicated fundus conditions.