A Preliminary Study Of The Influence Of Sedlin Protein On Human Chondrocyte Proliferation

PART ONE TARGETING ENHANCE OR INTERTERE SEDLGENE ON HUMAN CHONDROCYTES, DETECTING THEEXPRESSION OF BONE DEVELEPMENT RELATED GENESWITHIN THE OSTEOGENESIS PCR ARRAYObjective: Targeting enhance or interfere SEDL gene of humanchondrocytes, detecting the expression of bone develepment related geneswithin the Osteogenesis PCR Array, providing high throughput informationfor studying SEDT pathogenesis.Method: Using the directional cloning of homologous recombinationto construct the adenovirus vector pAd5-SEDL and lentiviral vectorLV-siSEDL, packed into293T cells,and obtain recombinant adenoviruspAd5-SEDL and LV-siSEDL, identify it at last.(Our previous completed).293cell expansion adenovirus, the virus was collected to detect adenovirustiter. Detecting the Mutiplicity of Infection (MOI) of adenovirus andlentivirus for HC-a. The experiment were divided into four groups:overexpression group (HC-a/pAd5-SEDL),RNAi group (HC-a/LV-siSEDL),control group of overexpression (HC-a/pAd5blank carrier), control group of RNAi (HC-a/LV blank carrier), four groups of cells were sended to detectusing Osteogenesis PCR Array.Result:1. Harvesting high titer recombinant adenovirus pAd5-SEDL,the virus titer was2.6×1011pfu/ml.2. Adenovirus and lentivirus infected HC-a successfully, the MOI ofadenovirus for HC-a is50, the MOI of lentiviral for HC-a is20.3. Microarray results: the expression changes of SEDL gene wouldlead to changes in the upstream and downstream gene, wherein the bonedevelopment related genes: COL2A, CD36, FLT1, GDF10, IGF1et al.Conclusion: SEDL gene associated with the development of bone,changes of SEDL gene in chondrocyte will vary the expression of bonerelated gene, eg: COL2A et al. Those provide clue for further study of theSEDT pathogenesis. PART TWO THE INFLUENCE OF SEDLIN ONCHONDROCYTES PROLIFERATION AND PRELIMINARYMECHANISM STUDYINGObjective: Combined with microarray screening, exploring the effectand mechanism of Sedlin protein on the proliferation of human chondrocytes,to further explore the pathogenesis of SEDT.Method: The experiment was divided into three groups:overexpression group (chondrocytes were infected with adenoviruspAd5-SEDL), RNAi group (chondrocytes were infected with lentivirusLV-siSEDL),control group (chondrocytes). Cells were collected on time.Using MTT assay to measure the proliferation of different groups; QPCR todetect mRNA expression of SEDL gene and COL2A gene; Western blot totest the expression of sedlin protein; Cell immunohistochemistry to detectthe expression of collagen type Ⅱ.Result:1. Compared with control group, the proliferation rate ofoverexpression group is higher, and the RNAi group was significantlyinhibited (P <0.05).2. QPCR results showed that the expression of SEDL gene among thegroups, overexpression group is the highest, followed by the control group,the expression of RNAi group was significantly lower (P <0.001). Threegroups expression of COL2A mRNA, overexpression group wassignificantly higher than the control group, and the RNAi group, the expression level is the lowerst(P <0.001).3. The expression of sedlin protein among Three groups,overexpression group is the highest, followed by the control group, theexpression level of the RNAi group is the lowerst (P <0.001).4. immunohistochemistry showed that among three groups of humanchondrocytes, for expression of collagen type Ⅱ, the overexpression groupis highest, control group Secondly, there is no expression of collagen typeⅡon the RNAi group chondrocytes almostly (P <0.001).Conclusion: The proliferation of human chondrocyte is affected bysedlin through regulating the expression of collagen type Ⅱ.