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Objective:To examine the roles of follicular helper T(Tfh)cells,serum IL-21 and B cells in the pathogenesis of alcohol abuse non-traumatic osteonecrosis of the femoral head ( NONFH ) .Flow cytometry was used to measure the frequencies of peripheral blood inducible Tfh cells and B cells in alcohol abuse NONFH patients and healthy controls.The disease progression and the extent of femoral head collapse,the serum IL-21 were quantified.Methods: A significantly higher percentages of CD19+B cells(t=3.765,P=0.0005),CD86+CD19+B cells(t=5.506,P<0.0001),and CD95+CD19+B cells(t=4.152,P=0.0002) in patients than those in controls was found.The percentages of CD86+CD19+B cells were positively associated with the index of femoral head collapse in alcohol abuse NONFH(P<0.0001,r=0.536).Results: The frequencies of Tfh cells (t=7.611,P<0.0001),and IL-21+Tfh cells (t=5.281,P<0.0001) were higher than those in controls;The frequencies of Tfh cells were positively associated with the percentages of CD19+B cells(P=0.0002,r=0.455),IL-21+Tfh cells were positively associated with the percentages of CD86+CD19+B cells(P=0.0002,r=0.447).Conclusion: Tfh cells and B cells may participate in the pathogenesis of alcohol abuse NONFH,and increased CD86+CD19+B cells may be associated with the extent of femoral head collapse,the interaction of Tfh cells and B cells may have an im-portant role in pathogenesis of alcohol abuse NONFH.
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Objective To investigate the effects of TLR4 on the radiosensitivity of tumor cells.Methods The cell lines of RAW264.7,Lewis,MFC,Hepal-6,Bl6,and NIH3T3 were irradiated with 5 Gy X-rays or sham-irradiated.24 h after irradiation,the expression of TLR4 was detected by flow cytometry.According to the TKR4 level,cells were divided into three groups:without treatment,LPS stimulation and TAK242 block.CCK-8 kit and Annexin-V Apoptosis Kit were used to detect cell proliferation,apoptosis and cell cycle distribution of each group.Results After 24 h of 5 Gy ionizing radiation,TLR4 was significantly increased in Lewis cells (t =-8.68,P ＜0.01) but decreased in MFC cells (t =25.8,P ＜ 0.01) and had no significant changes in Hepal-6,B16 and RAW264.7 cells.In addition,the proliferation vitality (t =57.62,-6.23,P ＜ 0.01) and survival fraction (t =13.37,19.24,P ＜ 0.01) of the Lewis and MFC cells were reduced especially for the TLR4-blocked cells,and the apoptosis rates of both Lewis (t=-167.85,P＜0.01) and MFC cells (t=-26.45,P＜0.01) were elevated.The percentages of G0/G1 phase and S phase Lewis cells were significant increased (t =8.68,14.89,P ＜ 0.01) but its G2/M phase were reduced (t =-37.48,P ＜ 0.01).However,the percentages of G0/G1 phase and S phase MFC cells were obviously reduced (t =20.31,4.48,P ＜ 0.01) and G2/M phase increased (t =-13.06,P ＜ 0.01).For both cell lines of Lewis and MFC,the cycle distribution of TAK242 and LPS groups didn't change significantly.Conclusions High expression TLR4 in the Lewis cells is related to cell proliferation and apoptosis but not cell cycle distribution,and hence TLR4 could influence the radiosensitivity of tumor cells.
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Objective To explore the role of PLC/PIP2 signal pathway in the changes of mouse thymus CD4 + CD25 + NRP1 + Treg and TGF-β1 after different doses of X-ray irradiation Methods 36 ICR mice were randomly divided into 6 groups according to the irradiation doses of 0,0.5,1.0,2.0,4.0 and 6.0 Gy,respectively.Flow cytometry was used to detect the expression of NRP1 + Treg,and ELISA was used to detect the expression of TGF-β1 in mouse thymocytes at 16 h post-irradiation.The EL-4 cells were irradiated by X-rays at the dose of 4.0 Gy after co-cultured with the PMA and TMB-8 for 2 hours.Flow cytometry was used to detect the expression of NRP1 + Treg,and ELISA was used to detect the changes of TGF-β1 at 48 h post-irradiation.Results The NRP1 + Treg appeared a transient decrease at both 0.5 and 1.0 Gy irradiation and reached its valley value at 1.0 Gy (t =6.96,P ＜ 0.01),then showed a dosedependent increase and reached its peak at 6.0 Gy (t =6.70,P ＜ 0.01).The TGF-β1 level decreased after 0.5 Gy X-rays (t =12.53,P ＜0.01),then increased at a dose-dependent manner and reached its peak at 4.0 Gy (t =10.40-19.56,P ＜ 0.01).Compared with the sham-irradiation,NRP1 + Treg was decreased significantly after PMA treatment (t =3.06,P ＜ 0.01),while it was up-regulated significantly after irradiation in the presence of PMA (t =8.27,P ＜ 0.01).TGF-β1 was reduced in the presence of PMA with or without irradiation (t =10.46-39.69,P ＜ 0.01).NRP1 + Treg and TGF-β1 were increased significantly after TMB-8 treatment (t =5.53-44.26,P ＜ 0.01).Conclusions NRP1 + Treg cells and TGF-β1 were up-regulated after a high dose radiation,and the PLC/PIP2 signal pathway may participate in the regulation.
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Objective To study the effects of human insulin-like growth factor 1(hIGF-1) gene transfection on the proliferation of NIH3T3 fibroblasts.Methods The plasmid of pcDNA3.1-hIGF-1 was transfected into NIH3T3 fibroblasts by using Lipofectin method.The positive cell clones were selected with G418 and cultured for 4 weeks.The stable expression of hIGF-1 in the positive cells was determined by in situ hybridization and immunocytochemical analysis.MTT assay and flow cytometer analysis were used to observe the proliferation of NIH3T3 fibroblasts.Results hIGF-1 mRNA and protein expressed in NIH3T3 fibroblasts transfected with pcDNA3.1-hIGF-1 by in situ hybridization and immunocytochemical analysis.MTT assay showed the A value of transfected NIH3T3 fibroblasts rose,compared with untransfected NIH3T3 fibroblasts group,the difference was significant(P
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Objective To study the dose-and time-effect relationship between lipopolysaccharide(LPS) and CD14 expression in the cell line J774A.1 of mouse macrophages. Methods The cell line J774A.1 was stimulated with different doses of LPS,and the CD14 surface-positive cells from the cell line J774A.1 were detected by flow cytometry(FCM)45 min after stained with FITC-CD14 monoclonal antibody. The changes of CD14 surface expression in the cell line J774A.1 at different time(1,2,4,8 and 16 h)after stimulated with LPS were observed.After the best stimulant time was selected,the cell line J774A.1 was treated with different doses(1,10,50,100,500 and 1 000 ?g?L~(-1))of LPS and the changes of CD14 surface expression were observed.(Results In view) of time-effect,compared with control group(the positive rate was 12.50?3.71),LPS significantly enhanced the CD14 expression at 1,2 and 4 h after stimulation(the positive rates were 23.80?5.07,(23.04?)2.88 and 28.22?1.54,respectively) when the dose of LPS was 1 ?g?L~(-1)(P
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Objective To observe the effect of ionizing radiation on the expressions of TLR4 and adaptor protein MyD88 in mouse peritoneal macrophages in the Toll-mediated signal transduction pathways.Methods 160 male Kunming white mice were randomly divided into 16 groups(ten each group):sham-irradiation and five groups after 4,8,16,24 and 48 h X-ray irradiation for time-course experiments;sham-irradiation and nine groups after 0.05,0.075,0.100,0.200,0.500,1.000,2.000,4.000,6.000 Gy irradiation for dose-effect experiments.Flow cytometry was used to detect the expressions of TLR4 and MyD88 after whole body irradiation (WBI) with X-rays.Results Both of TLR4 and Myd88 expressed more than shamirradiation after 0.075 and 2.000 Gy WBI for 4 h,and the expressions of them reached the peak at 16 h or 4 h after WBI(P
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Objective To study the feasibility of constructing tissue engineered cartilage by differentiated rabbit bone marrow mesenchymal stem cells(MSC) cultured in vitro and in vivo. Methods The MSC were isolated from the nucleated cells fraction of autologous bone marrow by density gradient centrifuge, and then induced into chondrogenic differentiation by adding dexamethasone, transforming growth factor-β1(TGF-β1) and ascorbic acid in vitro. After 3 weeks, some cells turned to round shape and secreted metachromatic matrix. The cartilaginoid grafts composed of chondrogenic MSC. Bovine type Ⅰ collagen and human fibrin were cultured within the chondrogenic medium for 2 weeks in vitro or transplanted subcutaneously adjacent to the knee joint for 3 weeks in vivo. Results The most cells in the grafts were degenerated and disappeared after cultured in vitro. But the residual cells were survival and secreted metachromatic staining proteoglycan with toluidine blue, which was characteristic cartilage matrix. The grafts developed into matured cartilage tissue assessed by histological examination after 3 weeks of transplantation in vivo. Conclusion MSC can be used as functional cells to constructing tissue engineered cartilage.
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Objective To observe the effect of repairing the articular cartilage defects with tissue-engineered cartilaginous grafts in rabbits. Methods A total of 60 rabbits were divided into the cartilage graft group, the pure carrier control group and the blank control group. In the cartilage graft group, the bone marrow mesenchymal stem cells (MSCs) of the rabbits were obtained by isolating and culturing the bone marrow aspirates in vitro. The culture system that facilitates the chondrogenous differentiation of MSCs in rabbits was established. The tissue engineering cartilaginous graft was composed of chondrogenetic MSCs, bovine type Ⅰ collagen and human fibrin. Then transplantations of the cartilaginous grafts were performed to repair articular cartilage defects in rabbits. Results Hyaloid cartilage was formed within the defects repaired with the grafts at 12 weeks after transplantation by analyzing the content of type Ⅱ collagen and metachromatism. In the control groups, the fibrous cartilage repair was observed first, then the fibrous tissues and bone repairs were found. Conclusions The cartilaginous graft through tissue engineering is a promising graft for repairing articular cartilage defects.