Immunomodulatory Effect Of Bone Marrow Mesenchymal Stem Cells On T Lymphocytes In Patients With Decompensated Liver Cirrhosis

Part one: Isolation,culture and biological characteristics of bone marrow mesenchymal stem cells from patients with decompensated liver cirrhosisObjective: The purpose of the sudy is to establish the culture system of bone marrow mesenchymal stem cells(BMSCs) from liver cirrhosis patients in vitro,observe the biological characteristics. Methods: BMSCs were isolated and purified from the patients with decompensated cirrhosis by density gradient centrifugation combined with adherent method,and cultured with mesenchymal stem cells(MSCs)medium containing autologous serum. The culture ratio and primary culture time were observed,the growth curve were drawn,then the purity of MSCs was identified by flow cytometry. Results: The culture ratio was 100%(9/9). The primary passage time of MSCs was(13.0±2.3)days, The passage culture time of MSCs was(8.4±1.7)days. All BMSCs appeared similar to fibroblasts, with a characteristic spindle shaped fusiform morphology. As shown by flow cytometry: MSCs at third passage were uniformly positive for CD44(95.01%)and CD29(92.79%),while negative for CD34(1.31%)and CD45(1.08%),The purity of MSCs was 95%,in line with the immunological characteristics of the MSCs. Conclusion: BMSCs derived from patients with cirrhosis have common biological characteristics of MSCs. which cultured in vitro using autologous serum medium have characters of growth stability,proliferative prosperity,continuous passage. BMSCs may be an ideal alternative "seed cells " for orthotopic liver transplantation(OLT).Part two: Immunomodulatory effect of bone marrow mesenchymal stem cells on T lymphocytes in patients with decompensated liver cirrhosisObjective: To explore the immunomodulatury effects of bone marrow mesenchymal stem cells on autologous peripheral blood T lymphocytes in patients with hepatitis B-associated cirrhosis in decompensation stage.Methods: Peripheral blood lymphocytes were isolated from the patients with decompensated cirrhosis for carboxy- fluorescein diacetate succinimidyl ester(CFDA SE) fluorescent staining. Being stimulated by phytohemagglutinin(PHA), autologous peripheral blood lymphocytes were co-cultured with the passage third BMSCs in autologous serum, and they were divided into four groups: ①BMSCs+lymphocyte+PHA contact culture group; ②BMSCs+lymphocyte+PHA non-contact culture group;③ lymphocyte+PHA positive control group; ④ lymphocytes alone negative control group. Lymphocyte proliferation of each group and the frequency of CD4+ CD25+CD127- regulatory T cells(Tregs) and CD4+CD8-IL-17+ T helper 17 cell(Th17) were detected by flow cytometry. The m RNA expression levels of interleukin-10( IL-10)and transforming growth factor-β1(TGF-β1)in BMSCs were detected by using reverse transcription-polymerase chain reaction(RT-PCR).Results:As shown by flow cytometry: 1. Cell proliferation of contact and non-contact culture group significantly declined when compared with that of positive control group(P<0.01), but notably higher than negative control group(P<0.01), and no significant difference was found between groups(P>0.05); 2. Tregs frequencies of contact and non-contact culture group were higher than that of positive and negative control group, but no significant difference was found between groups(P>0.05); 3. Th17 cell frequencies of contact and non-contact culture group were higher than that of negative cintrol group(P<0.01), but lower than positive control group(P<0.01), and no significant difference was found between groups(P>0.05); 4. Intra-group comparison before and after culture: the Treg/Th17 ratio of contact and non-contact culture group raised up remarkably(P<0.01), while that of positive and negative control group did not change significantly(P>0.05). Comparison between groups after culture: the Treg/Th17 ratio of contact and non-contact culture group got higher than that of positive and negative control group( P<0.01), and there was no significant difference between groups(P>0.05). As shown by RT-PCR: Intra-group comparison before and after culture: The expression levels of IL-10 mRNA and TGF-β1mRNA in BMSCs of contact and non-contact culture group raised up significantly(P<0.01),but no significant difference was found between groups after culture(P>0.05). Conclusion: BMSCs from cirrhotic patients can inhibit proliferation of autologous peripheral blood T lymphocytes, up-regulate the expression of Tregs, and improve Treg/Th17 imbalance, the immunomodulatory mechanism may be associated with the secretion of Inhibitory cytokine IL-10 and TGF-β1 in BMSCs.