Study Of Gukang On The Differentiation Of MSCs In The Different Environment And Effect Of BMP-2 In Osteoblastic Differentiation

Experimental StudyObjective1.In order to study the effects of gukang on the differentiation of mesenchymal stem cells into adipocytes in vitro;2.To investigate osteogenic characteristics of rat bone marrow mesenchymal stem cells(MSCs)co-cultured with osteoblast(OB)indirectlyand effect of bone morphogenetic protein 2(BMP-2)in osteoblastic differentiation of MSCs;3.To observe the influence of pharmacal serum of GuKang and Estradiol-2(E-2) to osteogenic characteristics of rat bone MSCs under co-cultured with OB indirectly,and effect of bone morphogenetic protein 2(BMP-2)in osteoblastic differentiation of MSCs.Methods1.MSCs were isolated from 2 months old rats marrow,then separated by lymphocyte separating medium and proliferated in culture medium,detected in cell purity by flow cytometry analyzing cell phenotypes.The 4th passage MSCs were digested with 0.25%trypsin for 3-5 minutes and made into monoplast suspension.Cells were adjusted into 1×10~5 and then inoculated in 6 holes plates,divided into gukang group and control group.All groups were induced to differentiate into adipocytes in induction medium containing desamethasone,insulin and 1-methyl-3-isobutylxanthine.The induction medium of gukang contained the rabbits pharmacal serum of GuKang,and the control group' s induction medium contained the normal rabbits serum.All groups were observed under microscopic lens.After 4days,7 days and 10days of adipocyte inducing,all groups were stained by Oil Red O;2.MSCs were isolated from normal 2 months old rats marrow,then separated by lymphocyte separating medium and proliferated in culture medium,detected in cell purity by immunohistochemistry method analyzing cell phenotypes. Osteoblast was got from 1 day old rat' s skull by enzyme digestion method and the cells were observed through inverted microscope.ALP staining was made through Gomori' s method and alizarin red staining of mineralized nodus to do the functional indentification.MSCs were adjusted into 1×10~5 and then inoculated in plates,divided into 7 groups:A group,MSCs group;B group, MSCs combine with pharmacal serum of E-2;C group,MSCs combine with pharmacal serum of GuKang;D group,MSCs co-cultured with OB group;E group, co-cultured combine with pharmacal serum of control rabit;F group, co-cultured combine with pharmacal serum of E-2;G group,co-cultured combine with pharmacal serum of GuKang;and control group.The change of morphology and mineralization ability of bone marrow MSCs were observed,and the contents of BMP-2 and bone alkaline phosphatase(BALP)and osteocalein(OC)in supernatant fluid was detected with enzyme-linked immunosorbent assay(ELISA) method.And the correlation between activity of mineralization ability and contents of BMP-2 in supernatant fluid was analyzed.3.All the data was processed and analyzed with SPSS11.5.Results1.The formation of lipid drop lets was in time-dependent manner;2.The formation of lipid drop lets in gukang groups were retarded,compared with control group.The adipocytes diferentiation rate of the control group at 4days,7 days and 10days were24.31±12.53%,39.19±15.64%,50.13±15.13%, and the rate of the gukang group were 11.73±9.09%,20.95±8.05%,30.73±9.52%,as detected by Red Oil O stain.There were significant difference between two groups in different time(P＜0.05).3.the contents of OC in supernatant fluid of A group and C group was no significant difference(P=0.229);A group and B group was significant difference(P＜0.05);D,E groups had very significant difference compared with A group(P＜0.01),and there had no significant difference between D,E groups(P＞0.05);F,G groups had very significant difference compared with B,C,D groups(P＜0.01),and there had no significant difference between F,G groups(P＞0.05).The change of contents of B-ALP in supernatant fluid of all groups liked OC,A group and C group was significant difference(P＜0.05).4.The activity of mineralization ability of A group and B,C group was significant difference(P＜0.05),B,C group also had significant difference(P＜0.05);B,D,E groups had very significant difference compared with A group(P＜0.01),and there had no significant difference between B,D,E groups(P＞0.05);F,G groups had very significant difference compared with B,C,D,E groups(P＜0.01),and there had no significant difference between F,G groups(P＞0.05).The activity of mineralization ability was positively correlated with BMP-2(γP=0.923,P=0.000).Conclusions1.The formation of lipid drop lets was in time-dependent manner;2.The pharmacal serum of GuKang could inhibit differentiation of rat MSCs leading to differentiation into adipocytes,which maybe one of the mechanisms of GuKang in treatment of osteoporosis;3.The co-cultured model partly imitates osteogenic surroundings in vivo.Osteoblastic differentiation/proliferation of MSCs were improved when co-cultured with OB;4.The pharmacal serum of GuKang or E-2 combine with co-cultured increase the ability of osteoblastic differentiation/proliferation of MSCs.5.The activity of mineralization ability was positively correlated with BMP-2,The higher the contents of BMP-2 in supernatant fluid,the more of the activity of mineralization ability were.The Clinical StudyObjectiveTo observe the correlation between lumbar vertebrae(lateral position), menopausal years,serum BMP-2 in the postmenopausal osteoporosis(PMOP).Methods88 PMOP cases were taken part in the study,other 10 healthy young women whose age ranged from 20 to 30 years old were choosen as control group.88 PMOP cases were derided into 6 groups based on each 5 years of menopausal years.Bone mineral density(BMD)of lumbar vertebrae(lateral position)were tested through dual-photon sbsorptiometry(DXA),and the level of BMP-2 in serum were testd by ELISA method.The correlation between lumbar vertebrae(lateral position),menopausal years,serum BMP-2 in the PMOP who were difined as kidney-yang deficiency were observed. ResultsThe level of BMP-2 in serum of PMOP had significant difference compared with healthy control group(P＜0.01),and was negative correlated with menopause years(γP=-0.570,P=0.000).The level of BMP-2 of 6 groups of PMOP had no significant difference(P＞0.05).The level of BMP-2 was negative correlated with menopause years(γP=-0.471,P=0.000).There was positively correlated between BMD and BMP-2(γP=0.638,P=0.000).Conlusions1.BMD decreased as the durations of menopause increased,there was negative correlated between menopause years and BMD.The bone loss rate in lumbar varies among diferent groups of menopause years,and the rate was fastest in 2-10 years postmenopausal.2.the level of BMP-2 in serum of PMOP had significant difference compared with healthy young women,and was negative correlated with menopause years.3.BMD of lumbar vertebrae was positively correlated with BMP-2.the expression of BMP-2 decreased in the postmenopausal women may be one of the mechanisms of PMOP.