Objective:To study the effect of seeding cells of tissue-engineered skins,keratinocytes and fibroblasts,on the phenotypes of langerhans cells (LC).Methods:Peripheral blood mononuclear cells were induced by cytokines,granulocyte macrophage-colony stimulating factor (GM-CSF),interleukin 4 (IL-4),transforming growth factor β1 (TGF-β1) to generate LCs,which were subsequently cocultured with allogenic keratinocytes or fibroblasts.Then the phenotypes of langerhans cells was detected by flow cytometer (FCM),and the degree of proliferation of autogeneic lymphocytes was assessed after stimulation with LCs.Results:Cultured LCs had low level expression of HLA-DR,CD80,CD86 and no expression of CD83.Cocultured with allogenic keratinocytes or fibroblasts,phenotypes of LCs did not change significantly.Simultaneously,LCs could not stimulate autogeneic lymphocytes proliferation.Conclusion:These data indicate that LCs cocultured with seeding cells of tissue-engineered skins remain immature state.It is suggested that seeding cells of tissue-engineered skins have low immunogenicity and are unable to induce significant immunological rejection of host after transplantation.

Objective:Marrow mesenchymal stem cells differentiates into neuron-like cells when induced by Gastrodia,which verifies the multipotentiality of the stem cells.Here to detect the immunogenic properties of the neuron-like cells after differentiation.Methods:Human mesenchymal stem cells were isolated from bone marrow by wall sticking method,amplified by in vitro culture,and differentiated into neuron-like cells by oriented induction with Gastrodia.The morphology of cells was observed under light microscopy.Neuron-specific enolase (NSE),nestin and glial fibrillary acidic protein (GFAP) were detected by imnunocytochemistry.The expression of HLA-DR protein after induction was tested by flow cytometry(FCM).The immunogenic properties of the neuron-like cells induced from hMSCs were detected by one-way mixed lymphocyte reaction(MLR).Results:Human mesenchymal stem cells could be separated and amplified in vitro.After being induced by Gastrodia for 3 hours,most of the cells would be differentiated into neuron-like cells,revealing cytodendrite.By immunochemical staining,the cells showed positive of NSE,nestin,and negative of GFAP.HLA-DR was not detectable on differentiated hMSCs by FCM.MLR assays demonstrate that differentiated hMSCs fail to stimulate proliferative response of hPBLs.Conclusion:Human mesenchymal stem cells could be induced to differentiate into neuron-like cells by Gastrodia.MLR assays demonstrate that differentiated hMSCs fail to stimulate proliferative response of hPBLs.They are low immunogenic.

Objective:To study the antitumor effect and mechanism of cocultured CIK cells modified with IL-24 gene and autologous DCs on HL-60 cells in vitro.Methods:DCs and CIK cells were prepared routinely from human peripheral blood mononuclear cells ( PBMC).IL-24 gene was transferred into CIK cells via electroporation.The cells obtained were named CIK-IL24.RT-PCR and ELISA were used to evaluate expression of IL-24 gene in transfected CIK cells.The phenotypic changes of CIK cells were identified by flowcytometry analysis.The concentration of IFN-γ and TNF-α in supernatant of CIK was determined by ELISA.FCM was used to determine the cytotoxicity of cocultured CIK cells modified with IL-24 gene and autologous DCs against HL-60 cells.Results:Eukaryotic expressing plasmid pcDNA3.0-IL24 was transferred into CIK cells successfully via electroporation.The expressing rate of CD3~+、CD3~+CD56~+ cells had no significant change in CIK cells.However,the rate of CD4~+CD25~+ cells was significantly decreased compared with that of the control group.Expression of adhesion molecules CD54,CXCR4 were significantly increased on CD3+CD56+ cells.CIK-IL24 cells produced markedly higher levels of IFN-γ and TNF-α as compared with the CIK cells.By comparison with non-transfected CIK cells co-cultured with DCs,transfected CIK cells co-cultured with DCs had a significantly higher lytic activity against HL-60 cells.Conclusion:IL-24 gene modification can enhance the anti-tumoral immunity of CIK cells,the mechanism of which might be related to the increased secretion of IFN-γ,TNF-α,up-regulation of adhesion molecule expression,and reduction of the rate of CD4~+CD25~+ cells in CIK cells.

Objective:To study the inhibitory effect and anti-cancer mechanisms of adenovirus-mediated ING4 gene on the MG-63 osteosarcoma xenografts in nude mice.Methods:Ad-ING4 was transfected into QBI-293 cells and harvested.15 nude mice of the subcutaneous tumor models were established with MG-63 osteosarcoma cells and were randomly divided into PBS,Ad-GFP and Ad-ING4 groups.Then PBS(100 μl),Ad-GFP(100 μl,10~9pfu/ml) and Ad-ING4 (100 μl,10~9pfu/ml) for each one were given respectively QOD for 5 times,with intratumor injections.Tumor volume changes were monitored;and the 15 mice were sacrificed 2 weeks after treatment,the tumors were removed,weighed and ratios of tumor-suppression were calculated.The morphological changes of apoptotic tumor cells were observed under microscope.Bcl-2,Bax,Caspase-3,VEGF,CD34 expression was tested by immumohistochemistry.Results:High titer(10~9pfu/ml)adenoviral vector of ING4 gene were obtained.In nude mice bearing MG-63 osteosarcoma xenografts,the growth of MG-63 tumors treated by intratumoral injecting of Ad-ING4 was significantly suppressed,compared with PBS group and Ad-GFP group.The ratios of tumor weight-suppression of Ad-ING4 group was 59.3%(P＜0.05).Immumohistochemistry displayed that the expression of Bax,Caspase-3 was up-regulated and the expression of Bcl-2,VEGF,CD34 was down-regulated by Ad-ING4.Conclusion:Ad-ING4 can inhibit the growth of MG-63 osteosarcoma xenografts in nude mice,which may be via activating the apoptosis pathway and inhibiting tumor angiogenesis.

Objective:To investigate the effect of 12(S)-HETE on the p27~(kip1) expression in mesangial cells and glomeruli.Methods:Mesangial cells were exposed to 12(S)-HETE.12(S)-HETE was infused to rats by osmotic mini-pump.Total protein content measurement for cell hypertrophy,RT-PCR for mRNA expression and Western blot for protein expression were performed respectively.Results:12(S)-HETE stimulation induced mesangial cell hypertrophy and p27~(kip1) protein expression,but not p27~(kip1) mRNA expression.Furthermore,p27~(kip1) mRNA and protein expression in the glomeruli were significantly increased by 12(S)-HETE stimulation using osmotic mini-pump.Conclusion:12(S)-HETE plays an important role in the pathogenesis of glomerular cell hypertrophy and senescence through upregulation of p27~(kip1) expression.

Objective: The study was designed to evaluate the changes and significance of circulating CD4~+CD25~+ and CD8~+CD28~- regulatory T cells (Tregs) in patients with multiple myeloma (MM).Methods:CD4~+CD25~+ and CD8~+CD28~-Tregs in peripheral blood of 38 patients with MM and of 20 healthy doners were measured by flow cytometry.Serum albumin and β_2-MG in patients with MM were measured using bromocresol green method,transmission turbidimetry respectively.Results:Compared to those of the controls,the proportions of CD4~+CD25~(+/high),CD4~+CD25~(high) CD127~(low) and CD8~+CD28~-Treg cells in newly diagnosed MM patients were elevated.Furthermore,the proportions of CD4~+CD25~(high) and CD4~+CD25~(high)CD127~(low) Tregs in each clinical stage were elevated when compared to those of the controls.The number of the Tregs were increasing with clinical stages and were significantly higher in stage Ⅲ MM than in stageⅠ MM;In stageⅡand Ⅲ MM,there were also elevated proportions of CD8~+CD28~- Tregs,increasing with clinical stages.However,there were no differences when compared between stage Ⅰ MM and the controls;Both the proportions of CD4~+CD25~(+/high) and CD4~+CD25~(high)CD127~(low) Tregs in active MM were not different from stable MM,although all of them were higher than those of controls.The proportion of CD8~+CD28~- Tregs was higher in active MM than in stable MM and controls,but there were no differences when compared between active and stable MM.The proportions of both CD4~+CD25~(high) Tregs and CD4~+CD25~(high)CD127~(low)Tregs had negative correlation with the levels of serum albumin.Conclusion:MM patients have elevated levels of circulating CD4~+CD25~+ and CD8~+CD28~-Tregs,which may be an important mechanism of MM immune evasion,and may be associated with clinical stages,disease progression and prognosis of MM to some extent.

Objective:To investigate the expression of a new cytokine,thymic stromal lymphopoietin (TSLP) and its receptor TSLPR in the villi of human first trimester gestation.Methods:Villi were collected from women who had undergone an artificial abortion at 7-11 weeks of normal gestation.The trophoblast cells (Tros) were isolated and cultured.The total RNA was extracted using TRIzol reagent,from both villi and the Percoll-gradient-isolated Tros,then the DNA fragments of hTSLP and hTSLPR were amplificated by RT-PCR.Villous tissues were detected for TSLP by immunohistochemistry (IHC) and Western blot.Immunocytochemistry (ICC) was carried out on cultured trophoblast cells for TSLP/TSLPR expression.Levels of TSLP in the supernatants were detected by ELISA.Results:Normal villi and the cultured Tros transcript were found to express TSLP/TSLPR mRNA and secreted TSLP protein.In addition,TSLP receptor was also expressed on trophoblasts.Conclusion:Both TSLP and its receptor are expressed on villi and trophoblast cells,which suggests that TSLP plays an important role in maternal-fetal immuno-tolerance in human early pregnancy.

Objective:To investigate the association between anxiety and the change of humoral immune functions and its correlation with HLA-DQB1 polymorphisms.Methods:Total 31 resident doctors were selected randomly and tested by State-Trait Anxiety Inventory(STAI).IgG,IgA,IgM,complement C3 and complement C4 were detected with BECKMAN array360 system;HLA-DQB1*02、*03、*04、*05 and*06 alleles were individually amplified by polymerase chain reaction(PCR)using exon2 group-specific primers.The correlation between immune function and HLA-DQB1 polgmorphisms were investigated.Results:Statistical analysis showed that there was positive correlation with State Anxiety (Ta) and complement C3,either Trait Anxiety (Tc) and complement C3.There was significant difference between HLA-DQB1*02 positive and negative in Ta (P＜0.05),while no difference in complement C3(P＞0.05).There was significant difference between HLA-DQB1*04 positive and negative in Ta and Tc(P＜0.05),while no difference in complement C3(P＞0.05).Conclusion:Anxiety could change some humoral immune functions and this is related with HLA-DQB1 polymorphism.

Objective:To investigate the relation among platelet activation marker(GPⅡb/Ⅲa,CD62p) and amounts of fibrinogen (FG) and of D-dimer (DD) in elderly patients with chronic cor pulmonale exacerbation.Methods:Subjects were divided into four groups (42 elderly patients with chronic cor pulmonale exacerbation,42 elderly patients with chronic cor pulmonale remission stage,30cases of healthy elderly controls and 30 cases of healthy non-elderly controls).Positive rates of GPⅡb/Ⅲa and CD62p were measured with tricolor flow cytometry.We also determined FG and DD in patients with chronic cor pulmonale and in normal controls.Results:Compared with those of chronic cor pulmonale remission stage group,healthy elderly group and healthy non-elderly group,the levels of GPⅡb/Ⅲa,CD62p,FG and DD increased significantly in elderly patients with chronic cor pulmonale exacerbation (all P<0.001).There was a positive correlation between the amount of GPⅡb/Ⅲa or CD62p and the amount of FG and DD in elderly patients with chronic cor pulmonale exacerbation.Conclusion:There is increased coagulation and platelet activity in elderly patients with chronic cor pulmonale exacerbation,and there is a significant correlation between platelet activity and hypercoagulability.

Objective:To observe the changes and significance of pulmonary vascular endothelial cells (VEC),ICAM-1 and E-selectin in sepsis rats.Methods:60 SD rats were randomly divided into 2 groups:the control group and the sepsis group.The sepsis model was prepared by injection of lipopolysaccharide(4 mg/kg).The expression of ICAM-1 and E-selectin in pulmonary VEC of rats was detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical method.The VEC apoptosis in lung was analyzed with Hoechest-33258 staining.The ultramicrostructure of pulmonary VEC was observed under electron microscope.Results:Compared with the control group,the expression of ICAM-1 was significantly increased in the sepsis group (P<0.01),the expression of ICAM-1 was increased gradually and achieved the peak value at 24 h.The expression of E-selectin was achieved the peak value at 6 h and decreased gradually during 24 h.The apoptosis and necrosis of pulmonary VEC was increased gradually and achieved the peak value at 24 h (P<0.01).Conclusion:The expression of ICAM-1 and E-selectin in sepsis rats is significantly increased,probably leading to both necrosis and apoptosis of pulmonary VEC,resulting in the occurrence of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS).