Effect Of Recombinant Human Erythropoietin On Differentiation Of Myocardial Processor Cells From Amniotic Fluid Stem Cells And Study The Effect Of Wnt Single In This Process

Objective: To Study wether different concentration of rhEPO canrespectively induce the differentiation of myocardial processor cells fromthe amniotic fluid stem cells.And to study the effect the effect of Wntsingle in this differentiation process.Methods:⑴According to the characteristics of amniotic fluid stem cells,AFSCwere isolated from amniotic fluid of pregnancy women and then havebeen purified.⑵The morphology and expansion of the amniotic fluid stem cell wasobserved by phase microscope.The expression of membrane antigensCD+29and CD+34were detected by flow cytometry (FCM).⑶There were three groups： control group,rhEPOgroup andrhEPO+LiCl group. ⑷The medium for control group is DMEM/LG containing15%characterized FBC. And this medium was named full mediun.DifferentrhEPO groups needed to be intervened with induceced medium containingrhEPO at indicated concentration (1.0、5.0、10.0、20.0U／m1) for24h.Different rhEPO~+LiCl groups needed to be intervened with inducecedmedium together with LiCl(5mmol/L)for24h too.After24h，the mediumof all intervened group was the same as control group.⑸All groups were intervened as aboved mentioned. Two days later, theβ-catenin and p-GSK-3β(Ser~9) were measured with immunohistochemistrystaining.The expression of EPOR was detected byimmunofluorescence(FITC),⑹The morphology and differentiation rate of myocardial processorcells was evaluated by inverted phase contrast microscopy as well as theexpression of cardiac-restricted transcription factors Nkx-2.5and GATA-4atmRNA levels was measured with reverse transcription-polymerase chainreaction (RT-PCR) at day14of differentiation. Also Cardiac specificproteins(β-MHC、cTnT) were measured by westerning blot at day28ofdifferentiation.Result:⑴after being cultured for7days，Amniotic fluid stem cells grow toCloning. These clonings do not grow to the same size.There are someapoptotic cells at the centre of the cloning. The morphology of Amniotic fluid stem cell is just like MSC.It grows fast.after being expansed，Amniotic fluid stem cells seem like Fibroblast-like cells and arranged in aswirling⑵The expression of CD~+29was positive and the CD~+34was negative.⑶EPOR was found expressing before or after being inducted⑷After being inducted14days，Amniotic fluid stem cell graduatlybecomes shorter and wider.Cells mutual fused to form myotube-likestructure.⑸The differentiation rate of myocardial processor cells was higher inrhEPO group than that in control group. The most obvious difference was inrhEPO group at the concentration0f5.0U/ml(0.920±0.003vs0.151±0.002，p<0.05).Compared to control group,rhEP0significantly up-regulated themRNA levels of Nkx2.5and GATA-4at early stage ofdifferentiation(p<0.05)．rhEPO~+LiCl group compared with rhEPO group,the mRNA levels of Nkx2．5and GATA-4is higher. The most obviousdifference was found at the concentration0f5.0U/ml rhEPO. Compared tothe control group,the Production of Cardiac specific proteins β-MHC andcTnT of the Intervention group was significantly up-regulated(p<0.05),Thevariation tendence of protein is the same as the mRNA levels of Nkx2.5andGATA-4.compared to control group,the β-catenin and p-GSK-3β wereincreased(p<0.05) in rhEPO group and rhEPO~+LiCl group,and rhEPO~+LiClgroup is the most. Conclusion:⑴There are amniotic fluid stem cells in the amniotic fluid.The natureof them is similar to the nature of MSC.⑵The exogenous recombinat human erythropoietin can be appliedin stem cell Research field as a kind of Chemical Inducing agent⑶It is demonstrated that exogenous rhEP0can induce amniotic fluidstem cells from pregnancy women to differentiate into myocardial processorcells.⑷The Wnt Signal pathways may play in important in this inductionporcesss.