The In Vitro Experiments Of PNS On The Osteoblast

Objective The aim of this study was to study the effect of PNG main components of PNS on the osteoblast in vitro,and to explore the mechanism of the action of the PNSMethods The osteoblastic progenitor cellsMC3T3-E1 cultured in alpha-MEM culture medium containing 10%FBS(Gibco TM Invitrogen),Each culture holewasput in different kinds and different concentrations of PNS for searching the effect of PNS on osteoblasts in vitro,The followings are needed to measure in this experiment: quantitative fluorescence method to detect cell proliferation;detection of cell osteogenic differentiation alkaline phosphatase(ALP)activityby colorimetric;to assay OCN content in the medium through enzyme-linked immunosorbent assay(ELISA);and measurement of cell culture matrix mineralization mineralized nodule area with alizarin red staining;To calibrate the expression level of each gene through real-time fluorescence quantitative RT-PCR detection,and take the expression level of housekeeping beta-actin gene as an internal reference.Results 1,When the concentration of PNS was 50mg/L,it can significantly promote cell proliferation,but the effect of increasing concentration decreased,more than 100 have the function of inhibiting cell proliferation;2,PNS at the concentration of 50mg/L can significantly promote the activity of ALP,with the increase of PNS concentration,the activity of ALP increased,and reached a plateau after the fall,when the concentration of PNS reached 1000mg/L,it inhibited the expression of ALP;3,Early PNS upregulate the expression of OCN was not significantly different,only at concentration of 1000mg/L the expression increased significantly,upregulation of the expression of OCN increased when the concentration of PNS(5-1000mg/L)increased;4,Alizarin red staining indicated that the mineralized nodule area and when PNS at the concentration of 1000mg/L induced largest mineralized nodule area.The blank group referred no mineralization,mineralization area of PNS induced on 28 thday is nearly 3 times as 21thday;5,Limited the concentration range of PNS in 5-1000mg/L,the gene expression Comparison as follow:(1)PNS concentration-effect relationship between the level of ALP gene expression upregulated and PNS concentration showed a bell shaped curve,concentration of 50mg/L reached a peak;(2)The expression of OCN gene increased significantly with the increase of the concentration of PNS;(3)with the induction time increased until the 4th day,the concentration-effectrelationship between the expression of COL1 gene induced by PNS and PNS concentrationshowed the approximate bell shaped curve,peak value was reached in 50mg/L;(4)On the 4th day,PNS concentration was 50mg/L,the expression level of Runx2 was significantly higher than the other group,with inducing time grew,there was no significant difference in the overall expression on the 7th day,however On the 7th day at the concentration of 200mg/L induced the most significant effect.Conclusion Early PNS significantly promoted the proliferation of osteoblasts,and gradually increased with the increase of the concentration of PNS;low concentration PNS can promote osteoblast ALP activity;with the increase of PNS concentration and the prolonged time,the extracellular matrix of mineralized nodule area increased;when PNS concentration in the 5-1000mg/L range,the expression of OCN gene by PNS increased with the increase of PNS concentration,the expression of COL1 gene by PNS was also increased with the induction time,there was no significant difference between the expression of Runx2 gene expression on the 7th day and the 4th day.