The Research Of DHT’s Function On Osteogenesis Of Ankylosing Spondylitis’s Fibroblasts

PartⅠ Primary cultured fibroblasts and cell growth curve assayOBJECTIVE: To master the method of more simplecultured fibroblasts， observe the morphology of primary cell.Understandfibroblast growth trend and compare the experimental group and normalcontrast of difference between fiber cell growth trend.METHODS: Cultivate fibroblasts tissue from Ankylosing Spondylitis(Ankylosing Spondylitis (AS) patients which take from the spine injurypatients that take from the spine ligaments. Retrieve tissue after repeatedwashing, centrifugation, and cut it into pieces to1mm3, the tissue wastiled in culture bottle and add1.5mL H-DMEM (15%FBS)，upright theflasks4h and continue to cultivate it4d then replace the cell culture media.When the cells climb out, spread4passages and freeze some cells, put theremaining cells into96-well cell culture plate as2,500/well.Start timingafter inoculate2h,then add90μL MTT+10μL H-DMEM into the plates atthe same time in the next week.Then continue to cultive4h,add110μLFormazan liquid. Measuring absorbance A value by microplate reader at last.RESULT：Primary cultured fibroblast cells’s successful rate over90%.the cells which can be observed in7d,spindle-shaped, flat,2~3feet.Two cell growth curves are substantially S-shaped and cells’ slowperiod range1~2d,logarithmic phase is about3d, stable time range4~6d,the cells’ decline phase after7d.Results there was no significantdifference(P>0.05).CONCLUSION: To lay the foundation for the next step of theexperiment of fiber cells.The growth curve showed no differences infibroblast AS experimental group and normal control. Part Ⅱ Test osteogenic genic expression in the target cells and thedifference in two groups under DHT interveneOBJECTIVE: Use PCR (RT-PCR) method to test the raw expressionof osteogenic gene such as osteocalcin (OC)，alkaline phosphatase (ALP)，Ⅰ collagenase (COL1A1) and the expression under DHT intervene,thencompare the differences between the two groups.METHODS: The exponential phase of growth of fibroblasts digestioninoculated into24well culture plates, divided into AS group and normalcontrol group, each group according to different culture medium added into2treatment groups, were added to normal medium and containing10-8mmoL/L dihydrotestosterone (DHT) medium. Change the mediumevery48h. Extract RNA of cells by TRIZOL method after cultivated the cells in3,9,12、15、18、21d，and subject to reverse transcription useTAKARA kit. PCR reaction test for CDNA template, roche MIX manual for20uL system (10uL2xsybr Green Master MIX，0.6uL upstream anddownstream primers，2uL CDNA，6.8uL Rnase free ddH2O)，PCR testreaction conditions:95℃for10min，94℃for5s，60℃for1min，40amplification cycle.RESULT：COL1A1gene expression in the original amount of the twogroups showed no difference between cells (P>0.05),Two groups of cells’amplification of ALP, OC gene have no seen. Osteogenic genes in theexpression of two fibroblast were gradually increased with incubation time,reach the maximum in cultured21d. There is no difference of osteogenicgenes’ expression after DHT intervene,the difference was no statisticalsignificance (P>0.05).Three kinds of expression of the gene, ASexperimental group was significantly higher than the normal control groupin the strongest21d, the difference was statistically significant P <0.05CONCLUSION: The osteogenic AS experimental group comparedwith the normal control group, this characteristic may result in AS inpatients with pathological bone formation reasons, DHT does not have thefunction of promote fibroblast osteogenic, remains to be further to study themechanism.