The Study On The Proliferation And Differentiation Of Osteoblast, The Expression Of BMP-2and TRPV5/TRPV6Channel Induced By BUSHENHUOXUEGUCHIFANG

Periodontitis is a common stomatological diseases, characterized byperio-dontal pocket formation, bag wall inflammation, alveolar boneresorption, and loose teeth, which lead to premature teeth loss and systemicdisease. Both prevent alveolar bone resorption and promote alveolar boneregeneration should be consider as the key to cure periodontitis. Ourdepartment made a significant efficacy in treatment of periodontitis by the useof BUSHENHUOXUEGUCHIFANG. Bone Morphogenetic Proteins (BMPs)is the most important inducible factor during the process of bone repair, whichinclude more the20subtypes. BMP2is the most efficient subtype amongothers, which induces osteoblasts express diberse specific genes, such as Ⅰcollagen, alkaline phosphatase(ALP), osteocalcin, produce the correspondingprotein secreted into the extracellular matrix, mineralize the extracellularmatrix, then complete the osteogenesis. By adjust calcium levels in bone cells,Calcium channel TRPV5/TRPV6activate p38MAPK pathway, and therefore,promote osteoblast differentiation process. ALP is an early osteoblastdifferentiation marker enzyme, its activity represent the differentiation activityand differentiation Level of bone cells.Objective: In case of introducing “BUSHENHUOXUEGUCHIFANG”into culture of preosteoblasts from mice calvarium （MC3T3-E1）, by themean of inverted microscope observation on the cellular morphology of thiscell line, also by tests on cell proliferation and ALP level, expression of BMP2and TRPV5/TRPV6channel, and then reveal the osteogenic mechanisminduced by BUSHENHUOXUEGUCHIFANG.Methods:1. Culture MC3T3-E1cell line in vitro. 2. Preparing contained-herb serum from rats by gavage with "BushenHuoxue Gu Chi Fang" of body surface area converted equivalent dose;obtaining blank-contained serum as control group by rate gavage with normalsaline and10%FBS as blank control group.3. Culturing the cell line MC3T3-E1for24hs,48hs and72hs with10%contained-herb serum、blank-contained serum and FBS, respectively, and testthe effect of "BUSHENHUOXUEGUCHIFANG" on this MC3T3-E1byobserving the cell morphology under inverted microscope.4. Culturing MC3T3-E1with10%contained-herb serum blank-containedserum and FBS, respectively, and test the ALP contents in the cell at24hs,48hs and72hs, respectively.5. Culturing MC3T3-E1with5%，10%，15%，20%contained-herb serum,10%blank-contained serum and FBS, respectively, and test the proliferativeactivity in the cell by MTT at12hs,24hs,36hs,48hs,60hs and72hs, respectively.6. Culturing MC3T3-E1with10%contained-herb serum and blank-containedserum,and test the mRNA expression of BMP2,TRPV5/TRPV6in the cell byRT-PCR at24hs,48hs and72hs, respectively.Results:1.Under the inverted microscope,“Bushen Huoxue Gu Chi Fang” showedunobvious effect on the morphology of cell line MC3T3-E1.2.After applying “Bushen Huoxue Gu Chi Fang”, the Contained-herbserum showed no significant effect on cell proliferation of MC3T3-E1（P＞0.05）.3.Comparing to Blank-contained serum and FBS, ALP level increasedsignificantly（P＞0.05） in Contained-herb serum group, and the drug efficacygrew as the time went by, showing–to some extent-time-dependent effect.4.Comparing to Blank-contained serum, mRNA expression of BMP2increased significantly（P<0.05）in Contained-herb serum group, and the drugefficacy grew as the time went by, showing–to some extent-time-dependenteffect.5.The experimental result shows that, the expression of TRPV6increased significantly (P<0.05) as time goes by in herb-contained serum, compare toBlank-contained serum. And the experiment did not detect any TRPV5expressionof MC3T3-E1in neither herb-contained serum, nor Blank-contained.Conclusions:BUSHENHUOXUEGUCHIFANG get no effect on MC3T3-E1morphology.Its effect on MC3T3-E1proliferation is not evident, but as time goes by, ALPlevel and expression of BMP2and TRPV6in MC3T3-E1increase significantly.Its effect on MC3T3-E1proliferation is not evident, so that its mechanism forpromoting osteoblast differentiation can be attribute to the following two points:induce osteoblast differentiation by increase expression of BMP-2, andpromote osteoblast differentiation through calcium channel TRPV6.