The Study Of RhoC/ROCK2 Regulates Chondrocyte Migration Through FAK/PI3K/AKT And Its Influence On Tissue Engineering Cartilage Formation In Vitro

BackgroundAuricle defect caused by trauma,heredity or disease factors is a common disease in plastic surgery.Because of the complex structure of cartilage and the characteristics that chondrocytes are difficult to regenerate,the reconstruction and treatment of auricle defect is very difficult.Although in recent years,many studies have been devoted to the application of tissue-engineered cartilage to reconstruct ear cartilage defects,the difficulty of chondrocyte material infiltration,uneven distribution of cartilage tissue and easy collapse have become the main problems restricting its application.In order to solve this problem,we try to find the molecules that affect the ability of cartilage migration in cells and apply them to obtain better tissue-engineered cartilage.Cell migration is precisely regulated by a variety of signals,among which Rho-associated protein kinase(ROCK)plays an important role,especially Rho-associated protein kinase 2(ROCK2).Emphasizes that ROCK2 affects cell proliferation,migration and apoptosis through regulation of the actin cytoskeleton.In contrast,ROCK2 is directly regulated by Ras Homolog Family Member C(RhoC).In addition,integrin family cell adhesion receptors,as the main receptors between cells and extracellular matrix(ECM),are essential for every step of cell migration.Focal adhesion kinase(FAK)is one of the most significant signaling molecules.ROCK2 has been reported to regulate cell motility through integrin-activated FAK signaling.Studies have also shown that ROCK2 can regulate phosphatidylinositide 3-kinase(PI3K)/protein kinase B(AKT)signaling to regulate cell proliferation and migration of endothelial cells,mouse hepatoma cells,etc.FAK/PI3K/AKT is a very classic migration-related pathway,which has been widely studied in a variety of tumors and normal cells.However,the regulation of RhoC on chondrocyte proliferation and migration has not been reported.Based on these studies,we hypothesized that RhoC/ROCK2 regulates tissue engineered chondrocyte migration through the FAK/PI3K/AKT pathway.Therefore,this experiment is devoted to studying the role of RhoC/ROCK2 in the migration of chondrocytes through the FAK/PI3K/AKT pathway,and attempts to improve the proliferation and migration ability of chondrocytes by interfering with the activity of RhoC or ROCK2,and promote the construction of tissue engineered cartilage.Objective1.To explore the effects of RhoC and ROCK2 on the proliferation and migration of chondrocytes2.To investigate whether RhoC and ROCK2 affect proliferation and migration through FAK/PI3K/AKT3.To clarify the effects of RhoC and ROCK2 on chondrogenic capacity of chondrocytes by constructing tissue engineered cartilage MethodsPart 11.Transfect RhoC overexpression plasmid to construct ROCK2 overexpression chondrocyte model,PMA construct ROCK2 long-term overexpression chondrocyte model,and Y27632 construct ROCK2 inhibition chondrocyte model.2.Western-Blot and Real-time PCR techniques were used to detect the expression changes of cartilage-related proteins and genes.3.Scratch test and Transwell test to detect the changes of chondrocyte migration ability.4.Ki67,Ed U-488 and CCK8 techniques were used to detect the changes of chondrocyte proliferation ability.Part 2.1.To construct RhoC/ROCK2 overexpression chondrocyte model and ROCK2 inhibition chondrocyte model,and Western-Blot to detect RhoC/ROCK2 overexpression and ROCK2 inhibition of FAK/PI3K/AKT pathway activation in chondrocytes.2.Western-Blot and proliferation and migration experiments were used to verify the effect of ROCK2 inhibitor Y27632 on chondrocytes and FAK/PI3K/AKT signaling pathway.3.Add FAK inhibitor PF573228 to detect the effect of FAK inhibition on chondrocyte proliferation and migration.4.Add PI3 K inhibitor LY294002 to detect the effect of PI3 K inhibition on chondrocyte proliferation and migration.5.Add AKT inhibitor MK2206 to detect the effect of AKT inhibition on chondrocyte proliferation and migration.Part 3.1.Tissue engineered cartilage was constructed in vitro,and the cartilage-forming effect under different treatment conditions was analyzed by detecting the contents of DNA,GAG and total collagen in each group.2.Western-Blot and Real-time PCR were used to detect the expression levels of cartilage-related proteins and genes in chondrocytes under different treatment conditions.3.HE staining,Safranin O staining,Toluidine blue staining and Col-II immunohistochemical staining were used to detect the difference of chondrogenic ability of chondrocytes under different treatment conditions.ResultsPart 11.RhoC overexpression plasmid and PMA activator were used to successfully construct ROCK2 overexpressed chondrocytes,and Y27632 was used to construct ROCK2 inhibited chondrocytes.2.The proliferation and migration of chondrocytes were enhanced under RhoC/ROCK2 inhibition.3.The inhibition of RhoC/ROCK2 maintained the normal homeostasis of chondrocytes and promoted the expression of cartilage-related genes and proteins.Part 2.1.Overexpression of RhoC/ROCK2 inhibits the activation of the FAK/PI3K/AKT pathway,and inhibition of RhoC/ROCK2 can promote the activation of the downstream FAK/PI3K/AKT pathway.2.The inhibition of RhoC/ROCK2 promoted the activation of FAK/PI3K/AKT pathway,and finally enhanced the proliferation ability of chondrocytes.3.The inhibition of RhoC/ROCK2 promoted the activation of FAK/PI3K/AKT pathway,which further promoted the migration of chondrocytes.Part 31.The inhibition of RhoC/ROCK2 is beneficial to the secretion of GAG and collagen in tissue engineered cartilage.2.The inhibition of RhoC/ROCK2 promotes the expression of cartilage characteristic genes and proteins,and enhances the chondrogenic ability of chondrocytes.3.The chondrocytes inhibited by RhoC/ROCK2 were more evenly distributed inside the scaffold,with a larger infiltration depth and higher cell density.4.When RhoC/ROCK2 was inhibited,the scaffold cartilage tissue secreted more collagen type II,which was beneficial to the formation of cartilage tissue.Conclusions1.The inhibition of RhoC/ROCK2 can promote the proliferation and migration of chondrocytes,increase the expression of cartilage-related genes and proteins,and is conducive to the maintenance of cartilage homeostasis.2.The inhibition of RhoC/ROCK2 can activate the FAK/PI3K/AKT pathway,resulting in a significant improvement in chondrocyte proliferation and migration.3.The chondrocytes inhibited by RhoC/ROCK2 have better chondrogenic ability,which is beneficial to the distribution of chondrocytes in the scaffold,and can promote the secretion of cartilage-related proteins.