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Interstitial cells and dendritic cells in bone marrow of immune dysfunction
         
The first part of the thalidomide correct ITP patients mesenchymal cells induce regulatory dendritic cell function Study Background: immune thrombocytopenia (Immune thrombocytopenia, ITP) is the most common form of clinical autoimmune bleeding diseases (peripheral blood platelet count <100 × 109 / L). ITP clinical symptoms mainly for skin and mucous membrane bleeding, internal bleeding and fatal intracranial hemorrhage. ITP's current research focus is mainly divided into the pathogenesis of increased platelet destruction and inadequate platelet production in two parts. Classic humoral immune cellular mechanisms proposed, ITP patients with platelet glycoprotein (Glycoprotein, GP) specific autoantibodies not only accelerated platelet destruction while also reducing platelet formation. Immunological aspects of cell found, ITP patients T helper cells (T helper lymphocyte, Th) in Thl / Th2 cell subsets imbalance, regulatory T cells (Regulatory T cells, Treg) dysfunction, abnormal number of cytotoxic T cells ( Cytotoxic T lymphocyte, CTL) directly mediated platelet destruction and activation of Th17 cell proliferation were directly involved in the pathophysiology of ITP patients. Recent studies have found that ITP pathogenesis and treatment provides a new way of thinking among patients with ITP mesenchymal cell proliferation and immune function abnormalities. Bone marrow stromal cells (Mesenchymal stem cells, MSC) was found 40 years ago, it is a non-hematopoietic multipotent differentiation and function of stem cells. MSC in vitro expression of a number of major non-specific surface markers such as: CD105, CD73, CD166, CD44, CD90, CD271 and CD29, etc., and differentiate into fat cells, adult cells, the ability of bone cartilage cells. MSC innate immunity and adaptive immunity in concern. MSCs can direct interventions complement innate immunity, Toll-like receptors (Toll-like receptor). Macrophage (Macrophage, MO), dendritic cells (Dendritic cell, DC), mast cells and natural killer cell function. In adaptive immunity, MSC can inhibit the function of T cells, regulate the balance of Th cells induced Treg, and can regulate the function of B cells and antigen-presenting cells (Antigen presenting cell, APC) of. MSC has a significant role, it may block the APC and the T cell antigen presenting between the APC and can induce differentiation into regulatory DC direction, thereby inhibiting the activation of proliferation of immune effector cells in maintaining immune tolerance. MSC whether ITP patients still have the ability to induce differentiation into regulatory APC direction is unclear. ITP is currently the first-line therapy in patients mainly corticosteroids, intravenous immunoglobulin-based (Intravenous immune globulin G, IVIg), but nearly a third of patients with recurrent or invalid. There are second-line therapy MabThera (Rituximab), thrombopoietin (Thrombopoietin, TPO), Romi Secretary Pavilion (Romiplostim) and eltrombopag (Eltrombopag) and other nascent drug composition for the treatment of glucocorticoid drugs, immunoglobulin therapy patients, but the treatment is expensive. Thalidomide (Thalidomide, THD) was used as a non-barbiturate sedative hypnotics drugs as an immunosuppressant, currently thalidomide widely used: rheumatoid arthritis, systemic sclerosis, dried syndrome, graft versus host disease (GvHD), Crohn's disease and other immune-related sexually transmitted diseases. 2004 Falco such as the use of thalidomide successfully cured a concomitant ITP patients with multiple myeloma. So far, no ITP associated with MSC induce regulatory DC research reports and MSC THD impact on immune function in patients with ITP reported. Objective: To detect the proliferation of normal controls and patients with ITP MSC's ability to inhibit lymphocyte activation and induction of regulatory differences in the ability of DC. THD measured before and after the intervention, ITP patients MSC changes and molecular mechanisms of proliferation and changes in the ability of lymphocyte activation and induction of regulatory DC power. TIEG detect gene induce regulatory role in DC in MSC. Discussion THD correct dysfunction in patients with ITP MSC possible mechanisms. Methods: a cell proliferation assay (CCK8):. After the healthy controls and patients with ITP MSC plates with different kinds of concentrated THDC (0,0.05,0.1,0.5,1,5,10,50mg / mL) for 3 days, Application of Cell Counting Kit-8 (CCK8) kit to detect and compare the changes in ITP patients and healthy controls MSC proliferation conditions. 2. Experimental apoptosis (Annexin V / 7AAD): healthy controls and patients with ITP MSC kind THD culture plate was added after 3 days, the application Annexin V fluorescein isothiocyanate / 7-amino-actinomycin D (AnnexinV / 7-AAD) kit detected and compared changes in ITP patients and healthy controls apoptosis of MSC. 3. Cell Cycle Experiment (PI): After the healthy controls and patients with ITP MSC kinds of board added THD cultured for 3 days, propidium iodide (propidium iodide, PI) test kit and compare healthy subjects and patients with ITP MSC cell cycle situation changes. The expression chips: ITP patients after MSC species THD culture plate was added 12 hours, apply Human Genome Array (CapitalBio Corp) ITP patients detected and compared changes in gene expression profile of MSC before and after the intervention THD, looking for differences in gene THD before and after intervention. After adding healthy controls and patients with ITP MSC seed plate THD cultured for 12 hours, and real-time quantitative methods (real-time reverse-tranion polymerase chain reaction, real-time RT-PCR) forensic expression chips: 5 differentially expressed genes verification experiment Screening of differentially expressed genes. 6.MSC inhibition test: healthy controls and patients with ITP THD before and after the intervention through MSC, respectively, with allogeneic phorbol ester (phorbolmyristate acetate, PMA) activated succinimide ester (Carboxyfluorescein diacetate succinimidyl ester, CFSE) marker The CD4 + T cells, the same kind of syngeneic DC co-culture. The proliferation of T cells by flow cytometry to detect the healthy controls and patients with ITP MSC suppression functional differences and THD suppression Functions of the MSC. 7. Regulatory DC suppression function tests: mDC and healthy controls as well as MSC and MSC THD intervention of ITP patients after co-culture, through magnetic activated cell sorting, and then with PMA allogeneic activation of CFSE-labeled CD4 + T cells, the same kind of syngeneic DC co-culture. To detect differences in healthy controls and patients with ITP DC MSC induce regulatory function proliferation of T cells by flow cytometry and THD of MSC induce regulatory function of DC. 8. The culture supernatant cytokines: healthy controls and patients with ITP THD before and after the intervention, after MSC and CD4 + T cells of allogeneic, syngeneic isotype DC co-culture, cell culture supernatant of IL-4 , IFN-r, IL-12, IDO, TGF-β, IL-10 protein enzyme-linked immunosorbent assay test (enzyme-linked immunoabsorbent assay, ELISA) assay. 9.DC maturity test: after the mDC and healthy controls as well as MSC and MSC THD ITP patients after the intervention of co-culture, marked CD80, CD86 antibody flow, the machine detected before and after the intervention THD MSC co-culture system on DC maturation the degree of influence. 10. lentivirus interference test: the healthy control MSC co-cultured with DC TIEG1 virus interferes with the front and rear, respectively. Then after MACS sorted nDC, MSC-DC, MSC-DC, (TIEGl shRNA), MSC-DC (control shRNA) and allogeneic CD4 + T-cells were co-cultured. The proliferation of T cells by flow cytometry to verify TIEG1 inducible gene regulatory role of DC in the MSC. Results: 1.THD can significantly promote the proliferation of MSC ITP patients, and 0.1μg / mL to 10μg / mL concentration range in a concentration-dependent increase. But having inhibitory effect on MSC when THD concentrations greater than 10μg / mL. ITP patients MSC percentage of cells in S + G2 phase was significantly lower than normal, after a significant increase in its intervention THD ratio. MSC apoptosis rate of normal with no significant difference in ITP patients, after THD interventions can significantly reduce the rate of apoptosis both. Results are expressed before and after the intervention 2.THD chip display P27, P16, caspase-8, caspase-10, klf4, c-myc, oct3 / 4 and TGF-β is closely related to 'THD intervention. THD MSC ITP patients after the intervention of caspase-8, caspase-10 gene was significantly reduced, while normal no significant change. Normal and c-myc gene ITP patients were significantly reduced after the intervention THD. After THD intervention, ITP patients oct3 / 4, TGF-β gene significantly increased, while the normal no significant change. Upregulated genes in normal human klf4, p27 genes were down but there was no significant difference in ITP patients. THD before and after the intervention of the p16 gene in normal and ITP patients had no significant change. 3.MSC inhibition test results showed that: ITP patients with normal MSC PMA alone were not inhibit activation of CD4 + T cell proliferation. MSC can inhibit normal mDC to activate CD4 + T-cell proliferation but inhibited lost MSC ITP patients CD4 + T cell proliferation mDC activation, MSC THD recovered after intervention suppression. THD normal subjects and in patients with ITP after intervention MSC can inhibit the activation by PMA DC portion of CD4 + T cells. 4. Regulatory DC suppression test showed: normal MSC-induced regulatory intervention THD DC and ITP patients after induction of regulatory DC MSC are losing the ability to activate CD4 + T, but ITP patients MSC cells induced by DC exception. Although all conditions induced regulatory DC were unable to separate the significantly inhibit the proliferation of CD4 + T PMA activation. But normal MSC-induced regulatory intervention THD DC and ITP patients after induction of regulatory DC MSC can significantly inhibit the proliferation of allogeneic mDC activation of CD4 + T cells, however, the MSC-induced ITP patients lose DC this functionality. 5. MSC and THD normal after the intervention of the MSC and mDC ITP patients after co-culture can be reduced CD80, CD86 expression, whereas no significant change in the DC ITP patients with a total culture. 6. The cell culture supernatant, after the intervention group THD supernatant IL-4, before IDO expression was significantly increased compared with intervention; IL-12, IFN-γ expression was significantly less. In addition to stand-alone DC culture supernatant and THD before intervention MSC / DC co-culture supernatants compared can significantly improve the supernatant IL-10, TGF-β expression THD after the intervention of the MSC and DC co-culture. 7. lentivirus interference experiments showed, MSC and DC interference TIEG1 gene after co-culture, can not be induced to differentiate into the direction of tolerance. MSC induce regulatory DC is TIEG1 dependent. Conclusion: MSC proliferation 1.ITP patients was significantly lower than normal. THD can be significant recovery after the intervention MSC proliferation ITP patients, prompting it to S + G2 convert and reduce their rate of apoptosis. MSC's 2.ITP immune suppression in patients with dysfunction after the intervention THD ITP patients recover part of the MSC direct immune suppression, and the ability to correct ITP patients of MSC induce regulatory DC. 3.MSC induce regulatory DC is TIEG1 dependent. The second part CD205 expression and large doses of dendritic cells in ITP patients in dexamethasone research background and its regulatory role: the dendritic cells (Dendritic cell, DC) in 1973 Steinman was first discovered and reported. As antigen presenting cells, DC widely distributed in body tissues and organs, swallow handle exceptions within the capture antigen and bind to major histocompatibility complex class of molecules and II molecules presented to CD4 + T cells and CD8 + T respectively cells in maintaining the body's own immune balance and prevent pathological autoimmune disease plays a crucial role. DC expressed itself through a different receptor to distinguish between autologous and allogeneic antigen antigen, which can select by C-type receptor (C-type lectin receptors, CLRs) recognize autoantigens, such as: sugar, protein or through Toll-like receptors (Toll -like receptors, TLRs) recognize alien antigens, such as: microbial antigens. Under immune homeostasis, antigen-specific binding CLRs after presenting via DC, usually induce specific immune tolerance. CD205 is expressed mainly in the DC cells, is one of CLRs family members are currently poorly understood, CD205 in maintaining the body's immune tolerance to self-antigens play an important role. Under steady state immunity, tolerance to self-antigens induce specific T cells in CD205 mediated. And CD205 + DC cells can induce Foxp3-CD4 + T to differentiate into Foxp3 + regulatory T cells (regulatory T cell, Treg) direction. Related studies show that patients with immune thrombocytopenia (Immune thrombocytopenia, ITP) of DC activation capacity allogeneic T cells was significantly higher than normal. But the case of CD205 expression in patients with ITP DC has not been reported. ITP is a common autoimmune disease, one of its main features is an anti-platelet glycoprotein antigen itself (platelet glycoproteins, GPs) antibody-mediated platelet destruction increased. CLRs as a member of the family, CD205 may be involved in identifying and capturing their own DC GPs process. At present, glucocorticoids as first-line treatment of ITP, the effect of high-dose dexamethasone (highdose dexamethasone, HD-dexa) has been widely recognized for shock therapy. HD-dexa can reduce monocyte phagocytosis by regulating and adjusting the patient's balance FcγR Th1 / Th2 cell balance to correct abnormal immune status of patients with ITP. But the application of HD-dexa regimen for patients with ITP DC antigen presenting cell function, the impact on the state of DC maturation as well as the role of DC CD205 expression remains unclear. The spleen is an important human immune organs, immune response and immune tolerance are inseparable function of the spleen. In ITP patients, the spleen is one of the main organs of platelet destruction in ITP patients. Recent studies have found that the spleen is the contact activation of lymphoid nodules at autoantigen and T cell ITP patients. ITP patients with splenic DC cells in the case of DC distribution and CD205 expression is not yet clear. Objective: To detect and compare the expression of CD205 in ITP patients and normal cells mature and immature DC difference. Detected and compared HD.dexa ITP patients and healthy impact on the state of DC maturation and CD205 expression. Detect the expression of CD205 relationship between glucocorticoids. ITP patients detected and compared with normal splenic CD205 + DC cells in tissue distribution. Methods: 1.DC cell culture: After extracting normal peripheral blood of patients with ITP, Ficoll method sorting peripheral blood mononuclear cells (Peripheral blood mononuclear cells, PBMC) with MACS CD14 + CD14 + cells were screened, with 10 % FBS (fetal calf serum, FCS) cells were resuspended in RPMI1640 medium and planted boards. Added to each well to a final concentration of 1000u / mL of interleukin-4 (interleukin-4, IL.4) and 1000u / mL granulocyte growth factor (granulocyte.macrophage Colony stimulating factor, GM-CSF). Incubated for 5 days added 1μg / mL lipopolysaccharide (Lipopolysaccharides, LPS) to induce DC maturation. Reserve with cultured seven days. 2. Flow cytometry: ITP patients and healthy controls were used FITC- DC or PE- bound CD80, CD86, and CD205 antibody labeling, while marking isotype control. Applications FACScalibur flow cytometer (BD Biosciences) machine testing and application Cell Quest Pro software (BDBiosciences) analyze data. 3. Real-time quantitative polymerase chain reaction: patients and healthy controls were collected induced DC ITP, total RNA was extracted using the reverse transcription kit TAKARA synthesized cDNA: by TOYOBO kit, Roche 480PCR instruments, quantitative amplification of the target gene. 4. dexamethasone test: inducing normal maturation of immature DC and DC, adding different concentrations of dexamethasone (0nm0l / L, 10nmol / L, 25nmol / L, 50nmol / L, 100nmol / L) and cultured for 3 days. Real-time quantitative polymerase chain reaction to detect the relative expression of CD205.CD80.CD83.CD86 analyzed CD205.CD80.CD83.CD86 glucocorticoid therapy correlation. 5. immunofluorescence experiment: Take ITP patients and normal spleen tissue, embedding, frozen, sliced ​​and stored at -20 ℃. Of tissue sections fixed in acetone and air-dried, with 1% fetal bovine protein (bovine serum albumin, BSA) in phosphate buffer (hosphate-buffered saline, PBS) hydration, antibody stained overnight, PBS buffer, washed 3 times After 1 hour the fluorescent secondary antibody staining. With Molecular Devices Olympus AX70 microscope observations and obtain tissue images using image analysis software METAMORPH Meta. Results: 1 case of normal ITP patients with dendritic cells CD205, CD80, CD83, CD86 expression: 1.1. In normal subjects, as mature dendritic cells, CD205, CD80, CD83, CD86 The expression was significantly increased. 1.2ITP immature dendritic cells in patients with CD205 expression was significantly reduced compared with normal subjects and with the mature DC, the expression of CD205 was not significantly increased. ITP patients with immature dendritic cells CD80, CD83, CD86 was significantly higher than normal, and accompanied by mature dendritic cells CD80, expression of CD83, CD86 was significantly higher than normal. DC ITP patients CD205 2. HD-dexa cells before and after treatment, the situation is CD80, CD83, CD86 expression changes: CD205 expression of mature dendritic cells in patients with HD-dexa significantly increased after treatment, immature dendritic cells expressing CD205 volume did not change significantly. Similarly, mature and immature dendritic cells CD80, CD86 expression was significantly reduced. Although the expression of CD83 immature dendritic cells was significantly reduced in the HD-dexa treatment, but the expression of mature dendritic cells CD83 No significant change. 3. HD-dexa effect on the expression of CD205 DC: CD205 expression of mDC With increasing concentrations of dexamethasone, CD205 expression imDC no significant changes. Expression and concentration dexamethasone CD80, CD83, CD86 was no significant correlation. 4. spleen immunohistofluorescence chemical results: 4.1HE staining showed that: ITP patients with splenic lymphoid nodules than normal number of significantly increased lymphatic nodules significantly increased B cell and T cell zone area. 4.2 normal splenic DC cell phenotype showing CD205 + CD80-CD86-HLA-DR + state, located in the red pulp and white pulp around the junction of the lymphoid nodules with. ITP patients with splenic DC cell phenotype showing CD205low / -CD80 + CD86 + HLA-DR + state, gathered in the T cell zone and B cell zones. Conclusion: DC CD205 expression cells 1.ITP patients was significantly lower than normal. ITP patients and DC cells mature state. CD205 expression 2. HD-dexa patients after treatment significantly increased and maturity decreased DC 3. dexamethasone and mDC cells CD205 is expressed as a concentration-dependent increase. 4.ITP splenic lymphoid nodules in patients with higher than normal number of significantly increased lymphatic nodules increased significantly. DC cell aggregation in T cell zone and B cell area was CD205low / -CD80 + CD86 + phenotype.

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