| Objective:To investigate the effects of PAP on synthetic metabolic functionof normal rabbit articular chondrocytes in vitro; To investigatethe effects of PAP on normal rabbit articular chondrocytesapoptosis; To investigate the effects of PAP on rabbit articularchondrocytes apoptosis induced by NO.Methods:1 The system of rabbit articular chondrocytes in vitro wasestablished. Rabbit articular chondrocytes were obtained fromcartilage chips which were scraped from the knees and hips of 6-month-old rabbits and were washed with PBS. The cells werereleased by sequential digestion at 37oC with 0.25% trypsinase and0.3% collagenase Ⅱ. Parimary culture was plated in DMEM culturemedium with 10%FBS and placed in incubator with 5%CO2 at 37oC. Thecells in vitro were judged to be chondrocytes by toluidine bluestaining. When cells of any group reached near confluence, thechondrocytes were subcultured.2 The model of chondrocytes apoptosis induced by NO wasestablished when rabbit articular chondrocytes were incubated withSNP which generated NO. The chondrocytes apoptosis rate wasanalyzed by Annexin V/PI with flow cytometry and the alterationsof morphology were detected by acridine orange(AO) fluorescencestaining. According to Wang Chuanjia chondrocytes apoptosis rateup to peak at 20h. The chondrocytes were randomly divided intofour groups, they were normal control group, normal group plus PAP,apoptosis group and apoptosis group plus PAP. For any group, thechondrocytes apoptosis was observed by using flow cytometry and AOfluorescence staining at 20h.3 The proliferation function of normal chondrocytes was observedby MTT method after treated with PAP. The secondary chondrocytesof rabbits were incubated with PAP, and MTT method was used toobserve the effects of PAP on the proliferation function ofchondrocytes at 7 days.4 In order to observe the synthetic metabolic change of rabbitsarticular chondrocytes, the DNA content, collage and proteoglycansof chondrocytes were detected by H-TdR incorporation, H-Pro 3 3incorporation and toluidine blue staining respectively. Thecontent of DNA and the synthesis of collage of chondrocytes weredetected by H-TdR incorporation and H-Pro incorporation after 3 3chondrocytes were cultured with H-TdR and H-Pro for 24 hours. The 3 3proteoglycans of chondrocytes were observed by toluidine bluestaining.Result:1 The effects of PAP on normal rabbit articular chondrocytesapoptosis. The apoptosis of normal chondrocytes treated with PAPwas observed by flow cytometry. After chondrocytes were treatedwith PAP, chondrocytes apoptosis rate was 0.158±0.092%, which waslower than those in normal control group(3.660±0.505%), and thedifference was significant (P<0.01). Therefore, PAP could inhibitnormal rabbit articular chondrocytes apoptosis.2 The effects of PAP on rabbit articular chondrocytes apoptosisinduced by NO. The chondrocytes apoptosis induced by NO wasobserved by flow cytometry, and its alterations of morphology weredetected by acridine orange(AO) fluorescence staining. Afterchondrocytes apoptosis induced by NO was treated with PAP, itsapoptosis rate was 9.573±2.047%, which was lower than those inapoptosis control group(24.828 ±8.649%), and the difference wassignificant (P<0.05). PAP could inhibit chondrocytes apoptosisinduced by NO.3 The effects of PAP on synthetic metabolic function of normalrabbit articular chondrocytes in vitro. The proliferation functionof normal chondrocytes was observed by MTT method. MTT analysisresults show that the proliferation function of chondrocytestreated with PAP was obviously higher than that of normal controlgroup. The H-TdR and H-Pro incorporation results show that the 3 3contents of DNA and collage of chondrocytes treated with PAP weremuch more than those of normal control group. These results showPAP could improve the proliferation function and syntheticmetabolic function of the normal chondrocytes.Conclusions:The results of this study suggests that PAP has effects onstimulating cell proliferation function for normal chondrocytes invitro, and could improves synthetic metabolic function of DNA,collagen and proteoglycans of articular chondrocytes. PAP couldinhibit normal chondrocytes apoptosis and chondrocytes apoptosisinduced by NO.Key words : Pilose Antler Peptide; Articular; Chondrocyte;Synthetic metabolism; Apoptosis; Cell culture...
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