| Objective:To reaseach whether the total flavonoids from arachniodes exilis(TFAE)can induce the osteogenic differentiation of human umbilical cord mesenchymal stem cells(hUCMSCs),and then explore the role of estrogen receptor(ER)signaling pathway in osteogenic differentiation of hUCMSCs.Methods:(1)hUCMSCs were isolated and cultured by tissue explants adherent method,cellular morphologic changes and growth were observed by inverted phase contrast microscope.hUCMSCs at passage 3 in logarithmic growth phase were used to do the experiment.(2)The effect of TFAE on the viability of hUCMSCs: the experiment was diveded into five groups: 0μg/mLTFAEgroup(control group),1μg/mLTFAEgroup,5μg/mLTFAE group,10μg/m LTFAEgroup and 20μg/mLTFAE group,cellular viability was measured by CCK 8 assay.(3)The effect of TFAE on the osteogenic differentiation of hUCMSCs :according to the result of cellular viability measured by CCK 8 assays,the experiment was diveded into four groups:control group(routine medium),0μg/mLTFAEgroup(0μg/mLTFAE of osteogenicinducing medium),1μg/mLTFAEgroup(1μg/mLTFAE of osteogenic inducing medium),5μg/mLTFAE(5μg/mLTFAE of osteogenic inducing medium),1)after 3,7 days of induction,alkaline phosphatase(ALP)activity was tested by AMP method;2)after 14 days of induction,calcium nodule formation was detected by alizarin red staining;3)after 14 days of induction,expression level of osteogenesis-related gene collagen type I alpha1(Col1a1),osteopontin(OPN),Runx2,Osterix(Osx)mRNA was measured by RT-PCR.(4)The role of ER of TFAE on the osteogenic differentiation of hUCMSCs:the experiment was diveded into four groups: control group(0μg/mLTFAE ofosteogenicinducing medium),TFAE group(5μg/mLTFAE of osteogenicinducing medium),TFAE+S group(5μg/mLTFAE of osteogenic inducing medium+S1191),S group(S1191);1)after 3,7 days of induction,ALP activity was tested by AMP method;2)after 14 days of induction,calcium nodule formation was detected by alizarin red staining;3)after14 days of induction,expression level of osteogenesis-related gene Col1a1,OPN,Runx2,Osx mRNA was measured by RT-PCR.Results:(1)hUCMSCs growed when umbilical cord tissue cultured by tissue explants adherent method after 3-5days,fibroblast-like cells overgrowed around the umbilical cord tissue after 14,cells were homogeneous and growed with adherence well cultured for several generations.(2)The results of celluar viability : compared with control group,celluar viability of 1μg/mLTFAE group and 5μg/mLTFAE group was enhanced significantly(P<0.05 or P<0.01),while celluar viability of 10μg/m LTFAE group,20μg/mLTFAE group was weakened significantly(P<0.05 or P<0.01).(3)The results of osteogenic differentiation: 1)the results of ALP was showed that,compared with control group,cellular viability of 0μg/mLTFAE group,1μg/mLTFAE group,5 μg/mL TFAE group was enhanced significantly(P<0.05 or P<0.01),compared with 0μg/mLTFAE group,cellular viability of 1μg/mLTFAE group,5 μg/mL TFAE group was enhanced significantly(P<0.05 or P<0.01);2)the results of alizarin red staining was showed that calcium nodules had formed in different concentration of TFAE groups,but the calcium nodules were not in control group,compated with 0μg/m LTFAE group,there were more calcium nodules in1μg/mLTFAE group,5 μg/mL TFAE group(P < 0.01);3)the results of RT-PCR was showed that,compared with control group,expression level of osteogenesis-related gene Col1a1,OPN,Runx2,Osx m RNA of 0μg/mLTFAE group,1μg/mLTFAE group,5 μg/mL TFAE group was increased significantly(P<0.01),compared with0μg/mLTFAE group,expression level of osteogenesis-related gene Col1a1,OPN,Runx2,OsterixmRNA of 1μg/m LTFAE group,5μg/mLTFAE group was increased significantly(P<0.05 or P<0.01).(4)The results of ER inhibitor on osteogenic differentiation:1)the results of ALP was showed that,compared with control group,cellular viability of TFAE group was enhanced significantl(P<0.01),compared with TFAE group,cellular viability of TFAE+Sgroup was weakened significantly(P<0.05 or P<0.01),no significant different between TFAE+Sgroup and Sgroup;2)the results of alizarin red staining was showed that calcium nodules had formed in each groups,compated with control group,there were more calcium nodules in TFAE group(P < 0.01),compated with TFAE group,there were less calcium nodules in TFAE+Sgroup(P < 0.01),no significant different between TFAE+Sgroup and Sgroup;3)the results of RT-PCR was showed that,compared with control group,expression level of osteogenesis-related gene Col1a1,OPN,Runx2,Osx mRNA of TFAE group was increased significantly(P<0.01),compared with TFAE group,expression level of osteogenesis-related gene Col1a1,OPN,Runx2,OsterixmRNA of TFAE+S group was decreased significantly(P<0.05 or P<0.01),no significant different between TFAE+Sgroup and Sgroup.Conclusion:(1)A certain concentration of TFAE enhanced viability and promoted osteogenic differentiation of hUCMSCs.(2)TFAE promoted osteogenic differentiation of hUCMSCs by ER signaling pathway. |
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