Research On The Biological Characteristics Of Human Chondrocytes Cultured Influence In Vitro Effect By P-15 Polypeptide

Objective: To investigate the effect of different concentration of P-15 peptide on the influence of the biological characteristics of human chondrocytes, explore the the biological characteristics of human chondrocytes, under the influence of the P-15 polypeptide in vitro.Methods: 1、Chondrocytes; Specimens taken from the Affiliated Hospital of North Sichuan Medical College orthopedics, the total hip arthroplasty of patients. Using the joint digestion method of type II collagenase and trypsin, acting on the femoral head cartilage. Obtaing and culturing the chondrocytes in vitro. Observation of cell morphology in the use of the light microscope, it is about 15 days that the culturing cells passage to the 3rd generation, and these cells for subsequent experiments. 2 、 Preparation containing P-15 peptide nutrient solution; Joing the fetal bovine serum into the DMEM medium(as much as 20% fetal bovine serum), so do the P-15 peptide, preparation to 0mg/L, 100mg/L, 200mg/L, 400mg/L four ladder concentration cell cultures containing P-15 peptide(A culture). Similarly, preparation to 0mg/L, 100mg/L, 200mg/L, 400mg/L four ladder concentration cell cultures containing P-15 peptide, but without the fetal bovine serum(B culture). 3、Use the technology of immunohistology, toluidine blue staining, identification of the chondrocytes. By ELISA technology, to investigate the type I and type II collagen content in the culture supernatant. The cell vitality were determinated by CCK-8 kits, Human Premixed Multi-Analyte be used to quantitative detection the FGF、BMP-2、PDGF-BB、IGFBP-3 cytokines. The results be analyzed by the SPSS statistical software analysis.Results:(1)Cell morphology was observed under a microscope: The cartilage cell jues to digest were round, suspended; adherent cells were polygonal, bigger and elongation. The 1rd to 3rd generation of chondrocytes shape changed little overall, proliferation activity is strong, shorter time be spent with cell passaging.(2)Investigate the type I and type II collagen by ELISA technology: P-15 polypeptide concentration of 100 mg/L,200 mg/L group supernatants type I collagen content of the protein content lower than the control group, the test result has statistically significant difference(P<0.05); the 100 mg/L group supernatants of type II collagen content(13.22±4.37pg/ml) more than other groups, and the difference was statistically significant(P<0.05).(3)CCK-8 analysis: OD value increases with time overall upward trend, the difference was statistically significant, wherein the first 4-6 days with the biggest increase; Measured on the sixth day OD value differences between different concentration group was statistically significant(P<0.05), wherein the P-15 was 100 mg/L when the strongest cell proliferation(1.12±0.03).(4)Quantitative test of 4 cytokines: with the increase of incubation time, FGF、BMP-2、PDGF-BB、IGFBP-3 four cytokines content is higher, P-15 stimulate human cartilage cells secrete FGF、PDGF-BB, of which 100 mg/L effect is best.Conclusion:1、P-15 polypeptide can promote human cartilage cell proliferation in vitro, it is 100 mg/L that the strongest effect on cartilage cell amplification.2、P-15 polypeptide can prolong the time, human cartilage cells to differentiate in vitro culture phenomenon,wherein P-15 to 100 mg/L, the effect is a plus.3、P-15 polypeptides stimulate the human cartilage cells secrete FGF、 PDGF-BB in vitro, it may be one of mechanism of P-15 peptide to cultivate human cartilage cells in vitro.