A Study Of The Effect Of GYD ON Cartilage Chondrocytes In Vitro

A Study of the Effect of GYD ON Cartilage Chondrocytes in vitro Osteoarthritis(OA) is the prevalent articular disease that is characteristic of joint cartilage degeneration.Lt is known that chondrocytes play a important role in the patbogenesis of osteoarthritis.so it is necessary for preventing chondrocytes from being impaired in reducing artieular cartilage degeneration.The U YAN DING ,GYDan alleviate suffering of OA,promote the restorment of joint function. In order to expound the pharmacologic mechanisms of GYD ,the effect of GYD on articular chondrocyte was exmmed in vitro. Test 1 :The isolation and culture of articular cbondrocytes. Articular chondrocytes were obtained from the cartilage of Japanese rabbit aged 3 months adopting method of sequential digestion by 0.2% collagenase II Primary culture and subculture of chondrocytes were performed respectively The morphologic changes and growth feature were recorded under the inverted microscope each day .Ihstochemical staining was used to determine proteoglycans synthesis of chondrocytes.Result :the time for chondrocyte s to develop a monolayer was about one week in primary culture ,while four to five days in subculture Rounded and epithelioid chondrocytes occupied most of cells .The proteoglycans secreted by chondrocytes were stained to red violet by toluride blue staining .The metacbromasia location was confined to large aggregate of rounded cells and multilayered areas.lt was more prominent in primary cultures,compared with subculture. Test 2: Chondrocyte proliferation and total protein assay. The first subculture chondrocytes were plated in one 96 well plates and one 24-well plates respectively. After 24 hours incubation ,when cell attachment was determined ,cultured plates were diviede into groups for different studys of GYD. Part 1 :chondrocyte proliferation assay. The culture 32 wells in 96 well plate were divided into 8 groups,eacb group occupied 4 wells ,0.2 ml medium was added in per well.GroupA:5%- serum DMIEM medium ;Group B-G:5%serurn DMI~M medium with GYL) (end concentration ranged from 0.625% to 20%);AoGroup:only 5%serum DMEM medium without chondrocytes.After 48 hours incubation,one plate were detected for cell viability and proliferation respectively assessed by MTT colormetric assay .Result demonstrated that GYI) can promote the proliferation of chondrocytes (p<0.O 1). Part 2:chondrocyte total protein assay. 24 well plate were divided into 6 groups ,4 wells per group.After 24 hours incubation,when cell attachment was deterniined,the medium in every well was changed respectively.GroupA:5% DMEM medium;Group B-F: 5% serum DMEM medium with (IYD(5%\l0%\20%)and DANG GUI(5%)\FANG FENG(5%).After 72 hours incubation,ceIl total protein was assessed by Coomassie Brilliant Blue G-250 (CBG)colormetnc assay.Result showed that GYD(5%j0%,20%)can promote the protein synthesis of chondrocytes (p