Immunoregulation Efficacy Of Qa-2of Umbilical Cord Mesenchymal Stem Cells On Transplantation For Treatment Of Mouse Experimental Autoimmune Hepatitis

Part Ⅰ:Expansion, purification and identification of mice umbilicalcord mesenchymal stem cellsObjective: To estanblish the culture system of isolating, identifying and culturingmouse mesenchymal stem cells from umbilical cord Wharton's jelly. And the inhibitoryeffect on T lymphocytes of umbilical cord mesenchymal stem cells (UC-MSCs) in vitro.Methods: UC-MSCs were isolated from C57BL/6pregnant mice umbilical cord bytrypsin-collagenase digestion. The cells were adherent to culture flask and subculture. Thecell growth curve was plotted. After3passages, UC-MSCs were observed under invertedphase contrast microscope, the flowcytometer assay was used to detect the surface markerson UC-MSCs. UC-MSCs were cultured under different induced condition to show theirmultiple differentiation potential respectively. Immunocytochemistry method was used toidentify the adipogenic, ostegenic and chondrogenic induction. Immunomagnetic beadswere used to separate T lymphocytes of mouse.3H-TDR insert assay was used to detect theinhibitory effect of UC-MSCs on proliferation of PHA-stimulated T cells. The expressionlevels of cell cytokine in medium supernatant were tested by ELISA.Results:(1) UC-MSCs showed plastic adherence and typical fibroblastic morphologyand radial or spiral-shaped growth by light microscopy and were gradually purified in theculture flask. The surface makers were expressed positive as CD73, CD90, CD105, whileCD34, CD45, HLA-DR, CD11b and CD19were negative. The differentiation potential ofUC-MSCs could be induced into adipocytes, chondrocytes and osteocyte in vitro.(2)The Tlymphocytes purity was90%separated by immunomagnetic beads from mouse spleen. (3)UC-MSCs could inhibit proliferation of T cell activated by PHA and showed dependenton the number of UC-MSCs in vitro, and regulated the secretion of IL-2, IL-4, IL-10,IFN-γ.Conclusions: UC-MSCs could be successfully isolated and cultured bytrypsin-collagenase digestion. UC-MSCs could inhibit proliferation of T cell activated byPHA, and regulate the secretion of cytokine IFN-γ, IL-2, IL-4, IL-10. Part Ⅱ: Immunomodulation of Qa-2secreted by umbilical cordmesenchymal stem cells inhibited proliferation of activated TlymphocytesObjective: To explore the immunosuppression of UC-MSCs on activated Tlymphocytes, and the possible mechanism of TC-PTP and Lck signaling molecules withinT lymphocytes interferenced by Qa-2secreted by UC-MSCs.Methods: CFSE and DAPI dye stained the UC-MSCs and T lymphocytes in vitro.The expression of H2-Q7gene and Qa-2protein in UC-MSCs was analyzed by ELISA,Flow cytometry, RT-PCR and Western-blot, as well as the change of H2-Q7and Qa-2inMLR system at different time points was analyzed. The blocking effect of Qa-2monoclonal antibody on inhibition of the activation of T lymphocytes by UC-MSCs wasdetected. Meanwhile the levels of cytokines IL-10, IFN-γ in the MLR were tested. TheH2-Q7gene and Qa-2protein in different passages were analyzed by RT-PCR andWestern-blot. The level of Qa-2protein secreted by UC-MSCs simutilated with rmIFN-γwas measured by ELISA.Results:(1) The cytoplasm of UC-MSCs stained by CFSE and DAPI dye was greenand nuclei in blue.(2) UC-MSCs could inhibit the proliferation of T lymphocytessignificantly in the MLR and the inhibitory effect was partially reversed after the Qa-2monoclonal antibody was added into the MLR. Transwell test showed there was inhibitoryeffect even though without contact between them, suggesting there was soluble molecules involved the inhibition process.(3)The level of IFN-γ increased slightly (P=0.0015), whileIL-10decreased slightly (P=0.0034) after Qa-2mAb was added to the MLR.(4) The levelsof H2-Q7and Qa-2decreased gradually with the passages increased of UC-MSCs.(5)Flow cytometry showed Qa-2protein expressed both in cytoplasm and surface ofUC-MSCs, the positive rates were46.21%and58.75%, respectively.(6) The expression ofLck mRNA decreased firstly and then increased gradually to normal level, this change wastransient at1h,12h,24h,36h in the MLR. The p56Lck protein had a similar change at24h,36h,48h,60h. The expression of TC-PTP mRNA increased firstly and then decreasedgradually to normal level, this change was transient at1h,12h,24h,36h in the MLR. TheTC-PTP protein had a similar change at24h,36h,48h,60h.(7) The Qa-2protein secretedby UC-MSCs stimulated by IFN-γ was increased, and showed dependent on the number ofUC-MSCs.Conclusions: UC-MSCs expressed Qa-2protein and showed dependent on thenumber of rmIFN-γ. The levels of Qa-2secreted by UC-MSCs were different and Qa-2was decreased while the passages were increased. Qa-2protein was involved the inhibitoryeffect of UC-MSCs on the proliferation T lymphocytes, and may produceimmunomodulatory effect on TC-PTP and p56Lck signaling molecules in T lymphocytes. Part Ⅲ: Immunomodulatory treatment of umbilical cord mesenchymalstem cells to mouse experimental autoimmune hepatitisObjective: To establish a murine experimental autoimmune hepatitis model and toobserve the treatment effectiveness infused UC-MSCs via tail vein.Methods: The liver of six-to-eight male mice of C57BL/6strain was obtained. Theportal vein was cannulated and the liver flushed with cold phosphate buffer saline (PBS).All further steps were done at4℃. The liver was cut into small pieces, homogenized in aPotter Homogenizer and centrifuged at150g for10min to remove the nuclear fraction.The supernatant was subsequently centrifuged for1hr at100,000g. The supernatant was called S-100. We separated S-100using a90cm CL-6B sephrose column, then the proteinpeaks could be collected from the column (called peaks1,2and3). We collected the1stand3rd peak, and that was LSP.48mice were randomly divided into2groups. Animalswere immunized with protein concentrations1.25g/L LSP0.5ml and emulsified with0.5mlcomplete Freund's adjuvant. Immunizations were done twice at weekly intervals.6micewere sacrificed at7,14,21,28days, respectively, and peripheral serum was collected for thepurpose of detecting ALT, TBIL, ALB. The liver of mice was obtained in order to stainwith HE. After the models were identified successfully, the models were reproduced. Atotal72EAH mice were treated with UC-MSCs after the2ndintraperitoneal injection.These mice were divided into3groups, that is, group A (experimental group): infusion ofUC-MSCs(stimulated by rmIFN-γ)1×106plus PBS0.5ml via tail vein (CFSElabeled,n=6); group B(transplant control group): infusion of UC-MSCs1×106plus PBS0.5ml via tail vein(CFSE labeled,n=6); group C (black control group): infusion of saline0.5ml (n=6). Liver function and liver pathology of mice and the levels of IFN-γ, IL-4,IL-10were detected at1,2,3,4weeks after treatment.Results: The EAH was characterized with decreaed activity, downcast, low responseto stimulus and urine yellow discoloration, weight gained slowly. Liver function wasseverely damaged, and the histological examination shows hepatocyte necrosis along withthe inflammatory cell infiltrate. The histological examination found hepatocyte lesions inthe central vein, sinusoidal and portal aera along with the inflammatory cell infiltrate,mainly lymphocytes. Hepatic lobules were destructed partially, and some areas appearedspotty necrosis and slice necrosis, even ballooning at1,2,3,4weeks. Liver function wasmoderately damaged, the levels of TBIL, ALT were increased gradually, and the levels ofALB was decreased, and all these index were statistically significant before and aftermodeling (P <0.05). The ALT, TBIL were decreased from2weeks after transplantationin group A and B, while was still increased in group C. Compared with group C, thedifference was statistically significant(P <0.05). The levels of ALB were increased ingroup A and B to some degrees from3rd weeks, conversely, ALB was reduced gradually in group C. Comparison to group C, the difference was statistically significant (P <0.01). Thelevels of TBIL, ALT in group A and B were decreased remarkably, but group A decreasedquicker than group B, there was a statistical significance(P <0.05). There were still somehepatic necrosis, degeneration, inflammatory cells infiltration after transplantation2weeks,while these index was improved compared with group C. At4weeks, the liver wasrepaired remarkably in group A and B, the inflammatory cell infiltration was reducedsignificantly, and hepatic lobule structure was completed. There was no significantdifference between those two groups, but the lesions in group C had no improvement, therewas still the hepatic lobule structure disorder, as well as inflammatory cells infiltration.UC-MSCs could be observered in the liver tissue sections by fluorescence microscope at1,2,3and4weeks after treatment. One week after treatment, the levels of IFN-γ in group Aand B were decreased dramatically, and IL-4, IL-10levels increased slightly, group A andB compared with group C, the difference was statistically significant (P <0.01).Conclusions: Transplantation of UC-MSCs through the tail vein which can hasten therecovery of liver function and relieve the pathological changes of hepatic histology had agood therapeutic effect on EAH. The UC-MSCs may play a therapeutic role by autocrinesecretion of Qa-2after they reached the local lesion of liver. UC-MSCs transplantation canadjust anti-inflammatory and pro-inflammatory cytokines to reach a new equilibrium. Itmight be one of the mechanisms of UC-MSCs transplantation on the treatment of EAH. Part Ⅳ Efficacy of umbilical cord-derived mesenchymal stem cells intreating the patients with autoimmune liver diseaseObjective: To explore the therapeutic effect and safety of umbilical cord-derivedmesenchymal stem cells (UC-MSCs) on the patients with autoimmune1iver diseases(AILD).Methods: There were7patients with AILD including2males and5females received UC-MSCs transplantation therapy in the1stAffiliated Hospital of Soochow Universityfrom November2009to October2011. The median age was57years old (range from53to68). There were2AIH,1PBC,3PSC and1OLS among them.7AILD patients receivedtwo intravenous infusions of UC-MSCs with a1month interval between injections. Theaverage number of UC-MSCs was8×107, and the drip was100d/min. The follow-upperiod was24months after UC-MSCs administration to observe the therapeutic effects andsafety, including with or without chills, nausea, vomiting, fever, rashes and other allergicsymptoms in the course of treatment and after treatment. The patients' symptoms includingloss of appetite, fatigue, itchy skin, liver discomfort, abdominal distension, abdominal painwere observed, too. Patients were assessed at every three months interval including liverfunction, kidney function, blood cell analysis, abdominal ultrasound, AFP, IL-4, IFN-γ.The differences of the clinical symptoms, the index of curative effect and safety before andafter treatment were compared.Results:(1) The course of treatment was safe. No patient had embolic symptomscaused by UC-MSCs. All operations were successful and adverse reactions did not appearincluding chills, fever, nausea, vomiting, rash and other allergic symptoms.(2)Noabnormal signs of renal function and blood cell analysis were observed in the follow-up.Serious complications did not occer relating to UC-MSCs transplantation. There were noevidence indicating AFP was unusual increased and liver tumor was found by colorDoppler. There were no patients with hepatic encephalopathy and other symptoms afterUC-MSCs therapy.(2)1case was lost at10month in the follow-up, and the other patientshad reached remission criteria after24months. Compared with before, clinical symptomsincluding appetite, fatigue, jaundice, abdominal distension, itchy skin relief wereimproved. The levels of ALB were33.51±5.32g/L,38.85±6.98g/L, ALT were69.95±47.96U/L,39.65±18.87U/L, AST were65.71±43.64U/L,35.52±17.24U/L beforeand after treatment, respectively. Compared with pre-treatment, the differences werestatistically significant (P<0.05). The levels of IFN-γ of patients were decreased(P=0.0368) and the levels of IL-4were increased to some extent (P=0.0418). Conclusions: UC-MSCs transplantation by peripheral intravenous infusion mayeffectively improve the clinical symptoms and promote the liver function turn for the betterin AILD patients. No severe adverse effects appeared, the immune disorders in patientswere partially corrected. In addion, the operation was simple, safe and feasible.