Effect Of Pilose Antler Polypeptides On Cataplasia And Senescence Of Rat Chondrocyte In Vitro

Object: To study the mechanism of replicative senescence of rat chondrocyte in vitro andthe mechanism of pilose antler polypeptides(PAP) by which to resist senescence, weinvestigated the effect of PAP on the replicative senescence of rat articular chondrocyte invitro with the sensitive molecular and celluar techniques. Methods: 1. The rat articularchondrocytes were isolated with the method of enzyme digestion. The cytoactive weredetected with Typan blue test. 2. The chondrocytes of different generation were observedwith light-microscope and transmission electron microscope for cellular growth andultromicrostructure, with the method of MTT assay for grow curve and proliferation, withalcian blue test for GAG(glycosaminoglycan) of ECM(extracellular matrix), withimmuocytochemistry and RT-PCR (reverse transcript -polymerase chain reaction)for typeⅡcollagen, with flow cytometry for cell life cycle, histochemistry for S-A-β-galand so onby which to identify cataplasia and senescence of chondrocytes cultured in vitro. 3. The 3rdpassage chondrocytes were divided into blank group, different concentration PAP groups,different concentration glucosaminsalfate groups and were sequently passaged to 4thgeneration. The 2nd passage chondrocytes was contrasted as young cells group. Thechondrocytes of different groups were detected with the methods of histochemistry forS-A-β-gal, and with alcian blue test for the content and constructure of GAG of ECM,immuocytochemistry for typeⅡcollagen and PCNA, MTT assay for proliferation, RT-PCRfor typeⅡcollagen and Aggrecan, flow cytometry for cell life cycle and proliferationindex,by which to observe PAP's function regarding to the appearance and functional status inthe process of chondrocyte's cataplasia and senescence. 4. The successive tert-generation (2ndpassage, 3rd passage, 4th passage) chondrocytes and the 4th passage cells intervented by PAPwere studied for senenscence mechanism. In this course, immuocytochemistry was applied for p16, pRb, E2F, CyclinD, CDK4 and TRAP-ELISA was applied for telomerase activation toobserve targets' changing regarding to cataplasia and senescence. The function of PAP wasdetected too. Results: 1. The method of enzyme digestion is practicable for harvestingconsiderable and better activity cells, which were identified that had good phenotype anddifferentiation. 2. From 4th passage, the chondrocytes emerging some cataplasia-senescencechanges such as the expression of S-A-β-gal raising to large extent, cell life cycle beingdetented on G1 phase, dedifferentiation and so on. 3. PAP has better anti-senenscence functionthan GS on several respect such as inhibiting express of S-A-β-gal, promoting chontrocyteproliferating, reducing cell content on G1 phase, promoting cell energy metabolism, makingcell growth active, enhancing chondrocyte differentiating and so on. 4. In the course ofchondrocyte's cataplasia and senescence, factors controlling cell life cycle and cell growthchanges as follow: p16↑—pRb↑—E2F↓—CyclinD↑—CDK4↓—telemorase↓. 5. PAP hasfunction of reversing the tendency of the express of factors controlling cell life cycle and cellgrowth changing. Conclution: 1. The method of enzyme digestion is practicable and can offeroptimal chondrocytes. 2. From 4th passage, the chondrocytes onset senescence. 3. PAP hassignificant anti-senenscence function which is better than GS'. 4. In the corse of chondrocyte'scataplasia and senescence, the factors which controlling cell life cycle and cell growthchanges as follow:↑—pRb↑—E2F↓—CyclinD↑—CDK4↓—telemorase↓. 5. The function ofPAP postponing chondrocyte's senenscence is fairly by means of down-regulating the factor'sexpression of p16, pRb and CyclinD, up-regulating the factor's expression of E2F, CDK andtelemorase.