| With the rapid development of transportation and sports and the acceleration of aging progress in human society,the incidence of joint cartilage trauma,strain and degenerative diseases increased every year.Because of the lack of blood vessels,nerves and lymph,capability of cartilage to repair itself is extremely limited,and once the cartilage is damaged,it is almost impossible to heal.Relevant studies have showed that brucine can promote the proliferation of chondrocytes.However,the molecular mechanism of brucine regulating chondrocyte proliferation is still not revealed.Therefore,this study aims to explore the effect of brucine sulfate(BS)on the proliferation activity of chondrocytes and its molecular mechanism.The research content mainly includes the following two parts:In the first part,the extraction,isolation and in vitro culture system of rabbit knee chondrocytes were established,and the effect of BS on the proliferation activity of chondrocytes was detected.Firstly,the rabbit knee chondrocytes were isolated and extracted by step-by-step enzyme digestion method,and then the cells were cultured routinely and the morphology of the extracted cells was observed.Next,the cells was identified through toluidine blue staining,immunocytochemistry(ICC)and immunofluorescence(IF).Secondly,the IC50 value and the proliferation concentration range of BS on the chondrocytes were determined by thiazolyl blue(MTT)staining.Subsequently,the effects of BS on the m RNA and protein expressions of chondrocytes were detected by Western blot(WB)and reverse transcription-polymerase chain reaction(RT-PCR).The following conclusions were drawn:(1)The extracted primary cells were paving stone-like after being overgrown,and the cell proliferation activity increased after digestion and passage.However,after passage to the F5 generation,the cells showed dedifferentiation and the proliferation activity decreased.(2)The results of biological identification showed that the extracted cells were consistent with the characteristics of chondrocytes.(3)The IC50 value of BS on rabbit articular chondrocytes for 72 h was 0.086 mg/m L.The proliferation of chondrocytes could be promoted by BS in the concentration range of 0.02 μg/m L to 0.8 μg/m L in concentration and time dependent manner,and the maximum proliferation rate was about 14%.(4)Within a range of concentrations,BS promoted the m RNA and protein expression of Colagen II and aggrecan in chondrocytes.The second part,the mechanism of chondrocyte proliferation induced by BS was explored.Firstly,the effect of BS on the m RNA and protein expression of molecules that related to Wnt/β-catenin signaling were detected through RT-PCR and WB method.Then,the expression of β-catenin in the nucleus was detected by IF and WB methods.Secondly,after treating the chondrocytes with the Wnt/β-catenin signal inhibitor XAV-939,the expression changes of related molecules at the gene and protein level were detected by RT-PCR and WB methods.The following conclusions are obtained:(1)The m RNA and protein expression of Wnt-4,Frizzled-2 β-catenin and Sox9 were promoted by BS,and the m RNA and protein expression of Runx2 was down-regulated.(2)The expression of β-catenin in the nucleus was effectively increased by BS.(3)The expression levels of Wnt-4,Frizzled-2,β-catenin and Sox9 were significantly decreased by XAV-939,while BS can resist this inhibition.These results revealed that the mechanism by which BS promotes the proliferation of chondrocytes may be achieved by activating the Wnt/β-catenin signaling pathway. |
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