Regulation Of BFGF For Collagen Expression Of Human Umbilical Cord Mesenchymal Stem Cell And Its Effect On5-Aza Induced Differentiation To Myoblost Cell

Objective1. To establish a simple,fast,economic,and efficient procurement method frommesenchymal stem cells of Wharton jelly of human umbilical cord.2. To investigate hUCMSCs biological characteristics and phenotypic characteristicsin vitro,and study the differentiation to fat cells and osteoblast in vitro.3. To further discuss hUCMSCs culture conditions, to augment effectively hUCMSCsby bFGF, establish a stable and efficient umbilical cord mesenchymal stem cellculture system,provides quantity guarantee for umbilical cord mesenchymal stemcell research and clinical application,laid the foundation for the related research andapplication.4. To observe the growth pattern and surface markers of human umbilical cordmesenchymal stem cells and explore the influences of basic fibroblast growthfactor (bFGF) on the proliferation of human umbilical cord mesenchymal stemcells (hUCMSCs) and collagen production of hUCMSCs cultured in vitro. toprovide the data for bFGF combined in hUCMSCs PFD treatment.5. Intends to discuss the method that5-Aza induce hUCMSCs to muscle cell,andmuscle differentiation regulation of bFGF.Resulted confirmed that5-Aza caninduce orientational differentiation of hUCMSCs into myoblasts, which providenew sources for myoblast, to arrise a new method for the treatment of diseases ofpelvic floor functional obstacles.Method1. Human umbilical cord mesenchymal stem cells (hUCMSCs) were separated byenzyme digest method. Adherent culture hUCMSCs. Observe cell morphology.2. Flow cytometry instrument analysis its surface markers, the cells were stained withOil red-O and Alizarin red solution to reveal osteogenic and adipogenicdifferentiation for the identification of mesenchymal stem cells.3.Confirming optimal concentration to promote the proliferation is20ng/ml. The control group were cultured in growth medium consisting of Dulbecco’s ModifiedEssential Media/F12(DMEM/F12) and10%volume fraction of fetal bovine serum.The experiment group were cultured in growth medium including20ng/mL bFGFbesides these in control group.2groups were determined hUCMSCs growth curve,MTT method to analyze the effects of basic fibroblast growth factor on proliferationhUCMSCs.4. mRNA expression of collagen I and III were determined by RT-PCR. Proteinexpression of collagen I and III were determined by Western Blot.5.hUCMSCs were cultured in four different systems. Group A:Control group:medium alone;Group B: medium with5-aza;Group C: medium with bFGF;GroupD:medium with5-aza+bFGF. The morphological changes of hUCMSCs wereobserved, the growth curve of each group of hUCMSCs was described.6.5-azacytidine induced culture: spread the2th generation, respectively, with5-Aza5,10,20μmol/L induction medium induced culture. determined by MTT method todetect each group cell proliferation.7. Induced after24-hour and48-hour, mRNA expression of Myf5and myogenin weredetermined by RT-PCR; induced after5-day, protein expression of desmin andmyosin were determined by immunohistochemical staining.Results1. Establish stable hUCMSCs culture system. Recovery of cells can be very goodgrowth, cell morphology, growth characteristics are consistent before freezing.2. Phenotype of hUCMSCs: the hUCMSCs expressed CD29、CD105unanimously,and did not express CD34、CD45and HLA-DR in the presence and absence ofbFGF unanimously.The cells were Alizarin red staining positive and Oil red Ostaining positive.3. Observation of the growth curves indicated that bFGF promoted the proliferation ofhUCMSCs.4. Compared with control group,the expression of typeⅠand type III collagenssignificantly deceased at mRNA and protein levels in experiment group(P＜0.05). 5.Cell morphology shows that bFGF medium (group C),hUCMSCs proliferationsignificantly than the other group,5-Aza+bFGF medium (group D) and pure5-Azamedium (group B) compared to the number of cells:cell cavitation is less,cellnumber is more,cell death is less. Growth curve hint: three days before each groupculture medium of hUCMSCs effect is not obvious, four days later, the proliferationof group C fastest, group B the slowest proliferation, group D faster than group B,the statistics analysis showed that difference was statistically significant (P＜0.05).6. MTT test result display:5μmol/L and10μmol/L of the concentration is theoptimal concentration induced by5-Aza.7. Myoblast differentiation experiments, RT-PCR results showed that the mRNA ofMyf5and myogenin express significantly by the experimental group, however, thecontrol group did not see. Induced by5-Aza, immunohistochemical staining suggestdesmin and myosin of the experimental group are positive expression, the controlgroup express negatively.Conclusion1.Successfully established the method of culture and amplification MSCs isolatedfrom human umbilical cord, the enzyme digestion economy, simple, hUCMSCs havebasic biological characteristics of MSCs and multipotent differentiation potential.2. bFGF can promote significantly the proliferation of hUCMSCs and does notchange the expression of the surface marker.3.bFGF inhibite the expression of typeⅠand Ⅲ type collagens at mRNA proteinlevels.4. hUCMSCs have myoblast differentiation potential, induced by5-Aza, expressed asa regulator of muscle cell differentiation process, hUCMSCs can differentiate intomyoblasts. bFGF have not direct adjustment about5-Aza induced hUCMSCs intomuscle cells differentiation,cell toxicity caused by the latter is reduced.