Discussing The Influnce Of Tortoise Shell Gule And Antler Gule On Chondrocytes Proliferation Cultured In Vitro Based On Raf-MEK-ERK Signal Pathway

Objective:By observing the effects of tortoise shell glue and antler glue on cultured OA chondrocytes in vitro,comparative analysis of the differences of Raf-1,MEK,ERK mRNA expression and Raf-1,MEK,p-MEK,ERK,p-ERK protein expression in the Raf/MEK/ERK pathway related genes,It was proved that tortoise shell glue and antler glue which is monarch drug in a prescription of Guiluerxianjiao enhance the expression level of MEK/ERK phosphorylation in this pathway,thereby promoting the proliferation of OA chondrocytes,and further analyzing the mechanism of Guiluerxianjiao in preventing and treating OA cartilage degeneration.Method:1.The OA chondrocytes in the knee joint of 12 week old guinea pigs was cultured in vitro,identified by toluidine blue staining method and immunohistochemical method of type II collagen,then to get guinea pig's F2 generation OA chondrocytes by cell passage.2.Screening the best intervention concentration and the best intervention time of medicated culture medium of tortoise shell glue and antler glue by MTT colorimetric method.3.The experiments were divided into 6 groups:tortoise shell gule group(shorthand tortoise group),antler gule group(shorthand antler group),blank group(shorthand empty group),tortoise shell + PD98059 group(shorthand tortoise suppression group),antler gule +PD98059 group(shorthand antler suppression group),blank control + PD98059 group(shorthand empty suppression group),tortoise shell glue and antler glue are both the optimal concentration for intervention,MEK inhibitor PD98059(shorthand suppression)intervention was 10uM.4.The difference of mRNA content of Raf-1,MEK and ERK in the cells of the experimental groups by qPCR method.5.Determination of the expression of Raf-1,MEK,p-MEK,ERK and p-ERK in the cells of the experimental groups by Western Blot method.Result:1.The OA chondrocytes cultured in vitro in the knee joint of guinea pigs were positive by toluidine blue staining and collagen ? immunohistochemical staining.2.MTT OD results show that:the tortoise shell glue proliferation significantly in the 50ug/ml-400ug/ml concentration range,the best proliferation concentration is 100ug/ml,antler glue proliferation significantly in the 100ug/ml-800ug/ml concentration range,the optimal concentration of 200ug/ml promoting proliferation.And the concentration of both more than 1.6mg/ml can inhibit the trend on the proliferation of chondrocytes,the best intervention time was 72h,There was a significant difference between the control group and the blank control group(P<0.05).3.QPCR results showed that:Tortoise shell glue and antler glue could promote the Raf-1,MEK and Erk mRNA expression in cartilage cells of guinea pigs,the effect of proliferation was tortoise shell glue group>antler glue group>blank control group>tortoise shell +PD98059 group>antler gule + PD98059 group>blank control + PD98059 group(P<0.05).4.Western Blot results showed that:Tortoise shell glue,antler glue can promote the expression of cartilage cells of guinea pigs in Raf-1,MEK,p-MEK,ERK,p-ERK protein,the effect of proliferation was tortoise shell glue group>antler glue group>blank control group>tortoise shell + PD98059 group>antler gule + PD98059 group>blank control + PD98059 group(P<0.05).ConclusionThe Raf/MEK/ERK signaling pathway and its phosphorylation participates in the proliferation of OA cartilage cells of guinea pig,tortoise shell glue and antler glue which is monarch drug in a prescription of Guiluerxianjiao can increase the activity of this pathway and promote the proliferation of chondrocytes in guinea pigs,which in turn slows cartilage degeneration in OA.