The Effect Of EGF-R,IGF-1-R On The Fetal-Bone Mesenchymal Stem Cells To Differentiate Into Epithelial Cells In Vitro

Objective:Bone marrow-derived mesenchymal stem cell(BMSCs) have the characteristic of multi-directional differentiation potential.The results of our study had showed that BMSCs could be induced to differentiate into epithelial and express cyokeratin(CK) by epidermal growth factor(EGF) and insulin-like growth factor-1(IGF-1).There was no report about when BMSCs expression EGF-R,IGF-1-R and the relationship between receptor and epithelial cell _differentiation.This experiment try to find the time of EGF-R and IGF-1-R expression in BMSCs,and the changes of expression EGF-R,IGF-1-R and CK in BMSCs induced by EGF or IGF-1.Then,whether BMSCs ccould differentiate into epithelial cells after blocked the receptors.Try to find the differentiated mechanism of BMSCs induced by EGF or IGF-1,and provide a basic theory and approaches for the future research of cell transplantation therapy and tissue engineering.PARTⅠ 1.Obtained bone marrow cell specimens from fetus at aged eleven to twenty-four weeks.BMSCs had been isolated,cultured and amplificated in vitro.Comparing the growth characteristic between the samples obtained from 11～13 week and 20～24week' fetus,the results show that the numers of P4-BMSCs obtained from 11～13 week' fetus was more than that of 20～24week' fetus,and its first subcultured time was earlier,but its aging appeared late.2.The expression of EGF-R and IGF-1-R from P3-BMSCs was detected with immunohistochemistry staining.The results show that EGF-R was first expressed in P3-BMSCs cultured at 4 day to P5 cultured at 3 day;IGF-1-R was first expressed in P5-BMSCs cultured at 2 day to P6 cultured at 2 day3.The expression of EGF-R,IGF-1-R and CK in P3-BMSCs induced by EGF 30ng/mL,IGF-1 50ng/mL,EGF 30ng/mL+IGF-1 50ng/mL was also be detected.The result was that the expression of EGF-Rand IGF-1-R in groups induced with EGFand IGF-1 were earlier 2d～4d than that of control group.The CK positive cells first appeared in P4 cells cultured at 2 day,and slenderly appeared in P4 cells cultured at 4 day in control group.4.P5-BMSCs was cultured with EGF-R Ab 2mg/mL or IGF-1-R Ab 1.5mg/mL for 2 h,and then added EGF 30ng/ml or IGF-1 50ng/ml in medium.Other 4 control groups were set up,added EGF 30ng/ml,IGF-1 50ng/ml,EGF 30ng/ml+IGF-1 50ng/ml,or nothing in medium respectively.We found that the CK positive cells appeared after the EGF-R or IGF-1-R be blockred for 4 days,but the rate of positive cells were lower than that of control groups(P＜0.05).PARTⅡ1.Ten cases of amnion were removed the epithelium with mechanial and chemical method,to prepare amniotic connective tissue which was no epithelial cells on the surface under light microscope.2.The number of 3.0×10~5 DAPI labeled P4-BMSCs was planted on the surface of amnion connective tissue,and added 30ng/mL EGF or/and 50ng/mL IGF-1 in medium,and other groups without growth factor in medium or without amnion connective tissue.After cultured for 24d～28d, the sample of each group were stained with immunofluor escence.Under CLSM,some cells with DAPI labeled nucelus show green fluor of CK,it indicated that the BMSCs had differentiated into epithelial cells.3.After induced for 10 days,the statistical results of the ratio of CK~+ cells in each group revealed that:①The BMSCs induced only by amnion or amniotic leaching liquor show a better ability to differentiate into epithelial cells than that of control groups,P＜0.05;②the induction in group induced by amnion combined with EGF,IGF-1 was powerful than that of only induced by amnion,P＜0.05;③The final concentration of 30ng/mL EGF had a strong induction than that of 50ng/mL IGF-1,P＜0.05.4.The basement membrane between BMSCs and amnion connective tissue and cell junction between cells growth on amnion had not been seen with transmission electron microscope.Conclusion of whole article1.The bone marrow cells isolated from cell numbers of BMSCs isolated from 11～13 week' fetus has a earlier first subcultured time,and a later aging.The numers of P4-BMSCs obtained from 11～13 week' fetus was more than that of 20～24week' fetus.2.The P4- BMSCs begun to express EGF-R,P5-BMSCs begun to express IGF-1-R.It indicated that this time is the appropriate time to added growth factor such as EGF,IGF-1.3.After inducted by EGF and IGF-1,the expression of EGF-R, IGF-1-R and CK in BMSCs was earlier than that of control group.It manifested that EGF and IGF-1 may promote BMSCs to express EGF-R and IGF-1-R,as well as differentiate into epithelial cells.4.After blocked the EGF-R and IGF-1-R of P3-BMSCs,the time and quantity of P3-BMSCs differentiated into the epithelial cell was lower than that of control group.It proved that EGF-R and IGF-1-R might be participated in the regulation of BMSCs to differentiate into epithelial cells.5.BMSCs could grow on the non-epithelial amnion and differentiate into the epithelial cell that express CK induced by amnion or amniotic leaching liquor,exogenous EGF and IGF-1.Among of them,the most effect was amnion,exogenetic EGF united with IGF-1,and the effect of EGF was powerful than that of IGF-1.6.BMSCs cultured on amnion for 28d could grew as stratified epithelium,and expresses CK-the marker of epithelial cells.But the basement membrane between BMSCs and the amnion connective tissue and cell junction between cells had not been seen under electron microscope.It may be that more time and a better cultured medium was required for the formation of basement membrane and cell junction.