With the deepening of the national medical and health system reform,it is particularly important to establish the regional medical cooperative system with the large hospital as the core.The Affiliated Hospital of Xuzhou Medical University establishes interactive coordination relation with county-level hospitals through township hospitals in the cooperation model for collectivize operation,trusteeship and technical support,and it has accumulated some practical experience.The paper investigates and contrasts the core hospital and the member hospital development of the before and after the cooperative relationship,and sums up 6 key measures that the core hospital promoted the regional medical cooperative system construction,that is,improving the member hospital grade,strengthening talent development,support technology,specialty developments,hardware support and public project cooperation in order to provide references to promote the development of the regional medical cooperative system.

As a transmembrane protein, CD47 is widely distributed in a variety of cells. It can bind to signal regulatory protein alpha (SIRPα) on macrophages and release inhibitory signals, thus avoiding phagocytosis of macrophages. In lymphoma cells, the expression of up-regulation of CD47 expression in lymphoma cells is one of the important mechanisms for inducing immune escape, and it is also a potential therapeutic target. This article reviews the research progress of CD47-induced immune escape, monoclonal antibodies targeting CD47 and cellular immunotherapy in the treatment of lymphoma.

Objective:To investigate the expression of CD28 costimulatory molecule on human lymphocytes inhibited by siRNA.Methods:Three different siRNA (siRNA 1,siRNA 2,siRNA 3) were designed and synthysized and transfected into freshly isolated human lymphocytes with cationic liposome.At 24,48 and 72 h post transfection,the changes of CD28 expression were detected by flow cytometry,and the changes of CD28 mRNA levels were determined by semi quantitative RT PCR.Results:Different siRNA showed different reduction in CD28 expression.At 48 h post transfection,the degrees of reduction with siRNA 1,siRNA 2 and siRNA 3 were 22 10%?1 63%,73 50%?1 02% and 42 90%?0 89% respectively compared with the control ( P

Objective To explore the transfection efficiency of primary lymphocytes from human peripheral blood by different methods to acquire the method with higher transfection efficiency.Methods Mononuclear cells from human peripheral blood were isolated using Ficoll-Hypaque.Cell viability was detected by Trypan blue staining.Suspending lymphocytes were sucked out and were incubated in 24-well plate after cultured in 6-well plate for 2 h.Activated lymphocytes were transfected by electroporation with plasmid(PEGFP-N1).Resting or activated lymphocytes were transfected by lentivirus vector(LVGFP) single infection or repeated infection,respectively.Green fluorescence protein (GFP) was detected under the fluorescence microscopy and percentage of positive cells was checked by flow cytometry at different time points after infection.At the same time,the effectiveness of lentivirus infection was compared under different conditions.Results Purity of mononuclear cells isolated by Ficoll-Hypaque was 95 ％ and its viability was over 95 ％.The percentage of lymphocytes obtained with a uniform shape was 90 ％-95 ％.Scattered fluorescence was observed by electroporation under the conditions of voltage 2 100 V,pulse width 10 ms,pulse number 1 for lymphocyte,while fluorescent became weaker over time and no green fluorescent was observed after transfection for 72 h.After resting lymphocytes were infected once for 48 h by lentivirus vector,green fluorescent was not found and positive cells were less than 1％.1％-5 ％ of activated lymphocytes could express GFP after single lentivirus infection and the expression levels were enhanced with concentration increasing,while 5 ％-10 ％ of activated lymphocytes showed strong green fluorescent by repeated lentivirus infection.In contrast with electroporation,the fluorescent with lentivirus infection was stronger over time.Conclusion Repeated lentivirus infection could efficiently transfect exogenous genes into activated lymphocytes for stable expression.

Objective To detect and determine the expression and significance of MHC class Ⅰ chain-related gene A/B (MICA/B) and membrane MIC molecules (mMIC) on the bone marrow mononuclear cells (MNC) of patients with acute leukemia (AL). Methods Expression of MICA/B gene was detected by semi-quantitative reverse transcriptaso polymerase chain reaction (RT-PCR) in MIC-pesitive K562 cell line, bone marrow MNC from 10 healthy people and 69 cases of acute leukemia (AL). Expression of mMIC was detected by Western blotting. The differences of the expression of MIC gene and mMIC between AML and ALL were compared. The prognosis was determined by chromosome type between patients with mMIC+ and mMIC-. Results The expression of MIC gene and mMIC could not be detected in healthy people. The expression rate of MICA gene was 49.28% and the MICB gene was 42.03% and the mMIC was 34.78% in patients with AL. In AML group, the expression rate of MICA gene was 60.00%, and the expression rate of MICB gene was 53.33%, and the expression rate of mMIC was 44.44%. But in ALL group, the expression rate of MICA gene was 29.17%, of MICB gene 20.83%, and of mMIC 16.67%. The expression of MICA/B gene and mMIC in AML group were higher than that in ALL group (P<0.05). The prognosis of patients with mMIC+ is better than the ones with mMIC-. Conclusion The up-regnlation of MIC gene and mMIC in bone marrow MNC from patients of AL may have some relationship with the occurrence of AL The expression of MIC gene and mMIC is high in AML and low or devoid in ALL, which would be an possible mechanism that ALL cells were easy to escape killing from NK and CTL cells. Determined by chromosome type, the prognosis of AL with mMIC positive was better than the ones with mMIC negative. MIC might be one of the factors to determine the prognosis of AL.

Objective To observe the effect of bortezomib on acute graft-versus-host disease (aGVHD) in an aGVHD model of mice and investigate the related mechanism. Methods Male C57BL/6( H-2Kb)mice were used as donors and female Balb/c (H-2Kd) mice used as recipients. Balb/c mice received total body irradiation (TBI) by 7.0 Gy X-radiation, and randomly divided into five groups. normal (group A), TBI (group B), TBI + bortezomib (group C), TBI + bone marrow cells (BMC) + spleen cells (SC) (group D) and TBI + bortezomib + BMC + SC (group E). The physical signs and the pathological damage of aGVHD, mean survival time, and chimerism were observed in recipients. The NF-κB p65 levels in nuclei of the liver and small intestine tissues of groups A,B and C were analyzed by Western blot. Results ( 1 ) The clinical aGVHD score in group D was (7.37±0. 32), significantly higher than in group E (5.85 ± 0.40) (P＜0. 05). Histopathology of the gut, liver and skin illuminated that the Ⅲ-Ⅳ degree GVHD occurred in group D. The occurrence of aGVHD in group E was later than in group D. The symptoms and the pathological damage of aGVHD in group E were milder than in group D. The average survival time in group E was significantly longer than that in group D (P＜0.05). The percentage of donor-derived cells in recipient mice was above 90% at day 12 after transplantation; (2) NF-κB p65 levels in nuclei of the liver and small intestine tissues in group B was significantly higher than in group C on the day 1,3 and 5 (P＜0. 05). Conclusion Bortezomib can inhibit the activation and expression of NF-κB,which may be the underlying mechanism for it to relieve aGVHD.

Objective To investigate the relation and difference of expression phase between classⅡtransactivator (CⅡTA) and HLA-DR antigens after IFN-? incubation, so as to investigate the potential effect of the class Ⅱ trasactivator (CⅡTA) in graft-versus-host disease(GVHD). Methods T lymphocyte from peripheral blood of health was incubated with IFN-? for 1 000 U/ml. RT-PCR was used to detect CⅡTA mRNA and Western blot was used to explore HLA-DR antigen in various time periods. Then Stat1? antisense oligonucleotides (AS) were given to inhibit the expression of CⅡTA, CⅡTA mRNA and HLA-DR antigen were tested again at peak point. Results CⅡTA mRNA was detectable 5 h after IFN-? treatment and peaks at 14 h; HLA-DR protein was detectable 28 h after IFN-? treatment and peaks at 52 h. The expression of CⅡTA mRNA and HLA-DR protein was lower in AS groups than that in control groups. Conclusion CⅡTA expression was positively correlated to HLA-DR expression, and earlier than the HLA-DR expression after IFN-? incubation. CⅡTA might be used as an early predicting marker of GVHD.

Objective To observe the preventive effects of Tripterygium hypoglaucum(Level)Hutch(THH)extract on T lymphocyte subpopulations in mice with graft-versus-host disease(GVHD)and to explore its protective mechanism.Methods 2?107 bone marrow cells mixed with 2?107 spleen cells from C57BL/6 mouse were transplanted into the myeloablative irradiated inbred BALB/c mouse.The experiment groups were designed as follows:model control group,cyclosporine A(CsA,10 mg?kg-1?d-1)group,THH(400 mg?kg-1?d-1)group,combination group(CsA 5 mg?kg-1?d-1 + THH 100 mg?kg-1?d-1).The ocurrence of GVHD was assessed by signs of weight loss,diarrhea,ruffled fur,hunched posture and histologic changes of skin,liver and small intestines.Chimerism was detected by monitoring the H-2b molecule in bone marrow cells of recipient mice with flow cytometer.The percentages of CD+3CD+4 and CD+3CD+8 T cells in peripheral blood were detected by flow cytometer.Results The survival time of CsA group,THH group and combination group were prolonged as compared with that of the model control group(P

The microenvironment of lymphoma is an important factor affecting the development of lymphoma, which is involved in regulating the recognition and immune response of lymphoma cells by the immune system. In the era of immunotherapy of lymphoma, the state of microenvironment also affects the effect of monoclonal antibodies, small molecular compounds and other immune targeting drugs on lymphoma cells. Among them, microenvironment-related immune escape is one of the key factors leading to the failure of lymphoma treatment. This article reviews some microenvironment factors such as stromal immune cell subsets, vascular proliferation, hypoxia, immune checkpoint and the recent research progress of immune escape.

Objective:To construct GST-tagged human NRP-1 fusion protein expression vector and induce its expression in Escherichia coli ( E.coli) ,then carry on inclusion body refolding and purification so as to obtain GST-NRP-1 fusion protein.Methods:NRP-1 gene was amplified by RT-PCR and inserted into pCR-blunt vector.Then the reconstructed plasmid was inserted into prokaryotic expression vector pGEX-4T-1.The constructed pGEX-4T-1-NRP-1 expression vector was transformed into BL21 cells and induced by i-sopropyl-β-D-thiogalactoside ( IPTG).Bacterial bodies were disrupted by sonication.Then the soluble fraction of fusion proteins were verified by Western blot and purified by Glutathione Sepharose 4B after inclusion body refolding.Results: The NRP-1 gene fragment was amplified by RT-PCR and inserted into pCR-blunt vector.Fusion protein expression vector pGEX-4T-1-NRP-1 was constructed suc-cessfully.After transformation, GST-NRP-1 expression vector was detected in BL21 cells and obtained purifying protein after refolding.Conclusion:The plasmid GST-NRP-1 was constructed successfully and laid basis for subsequent studies.