The Biological Influence Of Lentiviral Medlated GGCX In Transfection Of Osteoarthritis Cartilage Cells Of Rabbit

Objective:To investigate the effect of GGCX mediated by lentivirus on rabbit cartilage cell apotosis in osteoarthritis and the expression of MMp13,.Collagen type ?,Collagen type ?.Methods:1 ? choose twelve Japanese big ear rabbits weight about(2.0 � 0.2)kg to constructing animal model with knee anterior cruciate ligament transection.After 6weeks,kill the rabbits and to isolate and culture articular chondrocytes.After that use Alcian Blue and Type II collagen fiber staining method to indentify the cartilage cells.2?The cartilage cells randomly divided into three groups,the blank group(no treatment),the negative group(empty carrier with transduct by lentivirus without GGCX),and the positive group(transduct by lentivirus with GGCX).Using method of q RT-PCR and Western blot method to detect the expression leveleach of GGCX cartilage cells,MMP13,? type collagen,collagen type ? m RNA and protein in each group.3?Using the Annexin V-FITC & PI method to detect apoptosis of cartilage cells in each group.4 ? Using alizarin red staining dyeing cartilage cells for Histological examinationResults:1?It's flat,polygonal and processes of Rabbit osteoarthritis chondrocytes in the first generation.Oval nuclei located in the middle of the cell body.Alcian Blue staining make the chondrocytes become sky Blue,? type collagen fiber staining make the cartilage cells and extracellular matrix become brown or yellow.2?The results of q RT-PCR showed that the expression of GGCX m RNA levels was significantly increased in the transfection group.the expression of MMP13 m RNA levels was significantly decreased in the transfection group.the expression of collagen type ? m RNA levels was significantly increased in the transfection group.the expression of collagen type X m RNA levels was significantly decreased in the transfection group.the difference was statistically significant(P < 0.05);however,the blank group and the negative control group have no significant difference(P >0.05).The Western blot show that there are a significant increase in the expression of GGCX levels in the transfection group,the expression of MMP13 levels was significantly decreased in the transfection group,the expression of collagen type ?levels was significantly increased in the transfection group,the expression of collagen type X levels was significantly decreased in the transfection group.the difference was statistically significant(P < 0.05);however,the blank group and the negative control group have no significant difference(P > 0.05).3?The result of apoptosis rate in each group by Annexin V-FITC & PI method showed that the apoptosis ratios in the positive group was(17.833�2.612)%,revealing a significantly decrease compared with the blank group with(25.467�2.623)% and the negative group with(26.167�2.637)%,the difference was statistically significant(P < 0.05).The blank group and the negative group have no significant difference(P > 0.05).4?The result of Alizarin red staining showed that the color of transfection group is deeper,more cell numbers and less Calcium nodules,but there are more Calcium nodules in the blank group and the negative group.Conclusions:1?we can use anterior cruciate ligament transaction construct the knee osteoarthritis model,and separate and produce rabbit knee osteoarthritis cartilage cells.2?Lentiviral can successfully medlated GGCX in transfection of osteoarthritis cartilage cells of rabbit.It can significantly increase the expression of GGCX,3?Transducting GGCX by lentivirus can significantly increase the expression of collagen type ?.4?Transducting GGCX by lentivirus can significantly decrease the expression of MMP13 and collagen type X.5?Transducting GGCX by lentivirus can inhibit the apoptosis of cartilage cells.6?Transducting GGCX by lentivirus can inhibit the formation of Calcium nodules..