Primary Study On The Influence Of Low-dose Radiation And RNAi On The Related Factor Bone Sialoprotein Of Mouse Osteoblast-like MC3T3-E1 Cells

Object: Bone sialoprotein (BSP) is a noncollagenous protein which is present in bone extracellular matrix,rich with phosphorylation and glycosylation major secreted by osteoblast,osteoclast and hypertrophic chondrocytes.As a new osteoblast inducer,bone sialoprotein(BSP) plays a very important role in the osteoblast proliferation and differentiation.This paper aims to study the variation of mouse osteoblast-like MC3T3-E1 cells BSP expression obtained by the application of low-dose radiation and RNA interference,and study the influence on the cell including the related gene expression,even determine the function of BSP gene in the osteoblast proliferation and differentiation.Methods:1. MC3T3-E1 cells were treated with low-dose radiation 0Gy, 0.1Gy, 0.5Gy and 1.0Gy:The absorbance values were detected by MTT after 24 hours,the cell proliferation difference was observed during five days.The cell cycle and apoptosis difference were detected by flow cytometry afere 24 hours.The cell was culcured in the fresh medium after 24 hours for 14 days.And then the expression difference of gene cbfa1,BSP and OCN on 4 day,7 day,10 day and 14 day was detected by RT-PCR.2. Four possible small interfering RNA(RNAi) was designed for BSP gene mRNA,these four small interfering RNA were recombined with the vector pGPU6/ GFP/Neo,and four vectors of RNA interference pGPU6/GFP/Neo-siBSP-1,2,3,4 were built.The plasmids of four vectors were transfected into cell mediated by liprofectamine 2000 in the different concentration. With the highest transfection efficiency and the lowest mortality rate in principle,the system of expression vector plasmid transfected into MC3T3-E1 cells was optimized.3. Four vector plasmids were transfected into MC3T3-E1 cells mediated by liprofectamine2000 according to the selected optimal transfection system.The total RNA was extracted from cells after transfection was finished for 48 hours.The variation of BSP gene in transcription level was detected by RT-PCR.Depend on this,one vector which could inhibit BSP expression distinctly was screened out.The selected vector pGPU6/GFP/Neo-siBSP was transfected into MC3T3-E1 cells. The total RNA was extracted from cells after transfection was finished for 48 hours.The variation of BSP gene in transcription level was detected by RT-PCR.In the same time,the mRNA expression variation of gene cbfa1,BSP and OCN was detected by RT-PCR.Results:1. Compared with the blank control group,0.1Gy and 0.5Gy radiation could not inpact on the proliferation of MC3T3-E1 cells significantly(P>0.05). However,1.0Gy radiation could celebrate the proliferation of MC3T3-E1 cells distinctly(P<0.05). Compared with the blank control group, 1.0Gy could clearly promote cell into the G2/M phase,proliferation celebrated.And cell apoptosis could be inhibited significantly.Compared with the blank control group,the 0.1Gy,0.5Gy and 1.0Gy radiation all could up-regulate the mRNA expression of gene cbfa1,BSP and OCN ,and the radiation dose more,the expression higher.During those,the expression of 1.0Gy radiation group was higher clearly than those of blank control group(P<0.05).2. Succeed in constructing four BSP interference expression vector.Compare the transfection efficiency of these four transfection system, 0.8μg plasmid / 2μl liposome was the final choice with the highest transfection efficiency and the lowest mortality rate in principle.3. Succeed in screening out one siRNA expression vector pGPU6/GFP/ Neo-siBSP-1 that could inhibit BSP gene most efficiently.The result of RT-PCR showed that the mRNA expression of gene cbfa1,BSP and OCN was all down-regulated with the efficient interference vector pGPU6/GFP/Neo-siBSP-1 transfected into cell.Conclusions:1. After MC3T3-E1 cells were treated with 1.0Gy radiation,cell mRNA expression of gene BSP was up-regulated,with cell proliferation promoted, the mRNA expression of gene cbfa1 and OCN up-regulated.All these provide the possibility to study the relation of BSP and other related genes,and the up-regulation of BSP is helpful for the research of bone injure disease. 2. Succeed in constructing four BSP interference expression vector.Succeed in screening out one efficient BSP siRNA vector by use of transient transfection method under the most optimal transfection condition,and then the selected vector could be transfected into the cell,inhibit the expression of the related gene cbfa1 and OCN .All these provide technical support for the research of BSP involved in the cell proliferation and differentiation,and provide the experimental basis and theoretical support for the further research that siRNA blocking BSP expression was applied in the clinical.