Biological Effects Of OA Chondrocytes By Blocking SDF-1/CXCR4 Signaling Pathway With Three Antagonists

Part 1 The Culture and Identification of Human Osteoarthritis Chondrocytes in vitro Objective To set up a good basis for following experiment in vitro by proliferation,amplification and identification of human osteoarthritis chondrocytes.Methods Cartilage tissues in knee from patients diagnosed osteoarthritis (OA) and accepted knee arthroplasty were collected. Then the human chondrocytes were cultured in vitro after combined digestion of trypsin and type ? collagenase. The growth curve of chondrocytes was detected by MTT when P1 OA chondrocytes were cultured on the 1st, 2nd, 4th, 6th, 8th, 10th and 12th day. After P1 chondrocytes were cultured for 2 days, ( i ):Chondrocytes were performed to identify by toluidine blue staining and type ? collagen immunohistochemical staining; (ii) Chondrocytes were stained with Annexin?/PI apoptotic kit and then their apoptosis rates were detected by flow cytometry.Results (1) OA primary chondrocytes had small polygon shape, coated stretch and good refraction. Chondrocytes gathered into germinal center like paving stone. The growth rate of chondrocytes was so slow that they covered all the bottom of the culture bottle until 14-18 days. With the prolongation of culture time, passage cells gradually got big, lenient and extended, and cells displayed a low refraction and cell proliferation rate. (2) MTT assay showed that PI chondrocytes cultured in the 1-2 days were in the period of cell proliferation latency, 4-8 days were in the logarithmic growth phase, and after 10 days were in the plateau. The entire growth curve displayed a shape of "S".(3)Toluidine blue staining and type II collagen immunohistochemical staining showed that the cultured cells were all positive staining, it demonstrated that cultured cells were identified as chondrocytes.(4) The early apoptosis rates of chondrocytes were less than 5% by flow cytometry when P1 cells were cultured for 2 days.Conclusions (1) Human OA chondrocytes grew slowly.(2) P1 chondrocytes had a good activity so that they could be used for related experimental research. (3) The time of 2 days in culture of P1 chondrocytes was an ideal time point with intervention and experimental study.Part 2 The Biological Effects of Three Antagonists by Blocking SDF-1 / CXCR4 Signaling Pathway on OA ChondrocytesObjective To observe the biological effects of three antagonists named TN14003,T140 and AMD3100 by targeted blocking the SDF-1/CXCR4 signaling pathway on OA chondrocytes for finding an effective and safe OA treatment drug.Methods P1 OA chondrocytes were randomly divided into four groups: A, B, C and D. Chondrocytes in group A, B and C were added into 1000nmol / L TN14003, T140 and AMD3100 for 1 hour in advance, respectively, then 100ng/ml SDF-1 was added into every group at the same time. After ten days with drug intervention, the morphological changes of four groups were observed using optical microscope. On the 10th day, immunohistochemical staining of type ? collagen was performed to observe the expression of type ? collagen in OA chondrocytes. At 1, 2,4, 6, 8 and 10 days, chondrocytes were detected the proliferation with MTT. After 2 days, the apoptosis rates of chondrocytes were detected by flow cytometry. At the 2, 4, 6 and 8 days, (i)the levels of matrix metalloproteinases 3, 9, 13 (MMP- 3, MMP-9, MMP-13)were quantified by ELISA;(ii)The relative expression of type ? collagen and aggrecan mRNA in chondrocytes was examined by q-PCR;(iii)The protein level of type ? collagen in chondrocytes was detected by Western blot.Results (1)There were no significant differences and abnormalities in the morphology of OA chondrocytes in four groups under optical microscope.(2) Type ? Collagen immunohistochemical staining showed that the nucleus in the cytoplasm of group A was deepest staining, and the staining in group B, C and D was lighter orderly. (3)MTT assay showed that the cell proliferation trends of four groups were close, and the OD values of the four groups were not significantly different at the same time (P>0.05). (4) After 2 days drug intervention, there were no significant differences in the apoptotic rates between the four groups (P> 0.05). (5) The levels of MMP-3 and MMP-13 in the four groups were significantly increased with the prolongation of culture time after the detecting of ELISA. The production of MMP-3 was increased faster at the front 6 days with drug intervention, however, the production was increased slowly after 6 days. Compared to the production at the same time, the levels of MMP-3 and MMP-13 secreted by group A were least, and the contents of A, B, C and D were increased in each group, the differences were statistically significant (P<0.05). The levels of MMP-9 in four groups were not detected.(6) The expression of type ? collagen (Col ?) and aggrecan (ACAN) mRNA in four groups of chondrocytes decreased gradually with the prolongation of culture time, but the expression level of group A was slower than that of other groups. At the same time point, the expression levels of A, B, C and D were decreased, and the difference was statistically significant (P <0.05). (7) Western blot was used to detect the gray value of type ? collagen in four groups of chondrocytes, and the gray value of group 4 decreased gradually with the prolongation of drug intervention, but the gray value in group A was the slowest. At the same time point, the gray values in group A, B, C and D decreased orderly, and the difference was statistically significant (P <0.05).Conclusions (1)TN 14003, T140 and AMD3100 at 1000nmol/ L had no significant effect on the growth and proliferation of OA chondrocytes.(2)TN 14003, T140 and AMD3100 could reduce the secretion of MMP-3 and MMP-13 in OA chondrocytes,among which TN14003 was the most obvious. (3)TN 14003, T140 and AMD3100 could upregulate the expression of type II collagen and aggrecan mRNA in OA chondrocytes, but TN14003 was the most obvious.(4)TN14003,T140 and AMD3100 could delay the degradation of type II collagen in OA chondrocytes, and TN14003 was the most obvious.Part 3 Molecular Target Docking study between Three Antagonists and CXCR4 by Computer SimulationObjective By computer simulation for target docking technology to further investigate the site and mechanism of CXCR4 and its antagonists(TN14003, T140,AMD3100), thus find the core groups of their interactions and lay the foundation for the probe design and structural optimization for this three small molecular drugs.Methods (1)The crystal structure of CXCR4 was searched in the PDB crystal database and optimumed structure by software of Discovery Studios.(2)The molecular structure of T140, TN14003 and AMD3100 were constructed by ChemDraw15.0 and then loaded the obtained structure into the SYBYL-X 2.0 software to optimize.(3)The combination models between TN4003, T140, AMD3100 and membrane protein receptor CXCR4 were simulated by GOLD5.2.2 docking software, then scored the pharmacophore of TN4003, T140, AMD3100.(4)The binding mode and interaction of molecular docking structure were analysed visually by softwares of Discovery Studio 4.5 visualize and Pymol.Results (1)TN14003 and CXCR4 formed hydrogen bond at the sites of Arg2, Nal3,Cys4, Tyr5, Cit6, Arg11, Citl2.(2)T140 and CXCR4 formed hydrogen bond at the sites of Arg1, Arg2, Nal3, Cys4, Tyr5, Arg6, Tyr10, Arg11, Cit12.(3)The one of the sites of hydrogen bonds between AMD3100 and CXCR4 is four nitrogen heterocyclic ring 4, 8 nitrogen groups in AMD3100, the other one is the fourth nitrogen groups of another four nitrogen heterocyclic ring. (4)The core sites of hydrogen bonding between CXCR4 and TN14003/T140 are Ser178, Glu32, Gln272 and Asp262, Asp187,Asp 193, respectively; The core sites of hydrogen bonding between CXCR4 and AMD3100 are Arg188, Asp 187, Asp97.(5)The numbers of hydrogen bonds formed by TN14003, T140, AMD3100 and CXCR4 are 13, 12,4, respectively; the molecular docking score of TN14003, T140, AMD3100 with CXCR4 are 120.35,110.43,62.69,respectively.Conclusions (1)Computer simulation technology could predict the specific target of the small molecule drug group and its ligand.(2)7,8 Amino acid of TN14003 and T140 were non-core groups, one of the four nitrogen heterocyclic ring 8 nitrogen group in AMD3100 was non-core group too.(3)TN14003, T140, AMD3100 and the receptor molecule CXCR4 binding strength were: TN14003>T140>AMD3100, consecutively.Part 4 Research on Small Molecule Probe Design and Biological Characteristics Based on Computer SimulationSubjective This study sought to design and synthesize photo-affinity labeling probes of three small molecules named TN14003, T140 and AMD3100, and to verify their specific biological targets, and explore their mechanism, then set the early theoretical foundation for structural optimization of the three antagonists for the future.Methods (1)Photo-affinity labeling probes of TN14003, T140 and AMD3100 were designed without changing their pharmacophores, P-benzoyl-L-phenylalanine (Bpa)as the covalent binding group, Polyethylene glycol (PEG) as the connection group and Biotin as reporter group connection were collected to the non-core group of these three small molecule drugs. (2)These three small molecular probes were experimented to define their sites of action by chondrocyte photo cross-linking experiment.(3) The antagonistic efficiency between TN14003, T140, AMD3100 and their corresponding probes was compared by chemotaxis experiment.Results (1)Photo affinity labeling probes of TN14003, T140 and AMD3100 were successfully designed and synthesized.(2)Under the fluorescence microscope,TN14003,T140 and AMD3100 probes showed clear green fluorescence signal on the cell membrane.(3) In the chemotaxis experiment of SDF-1-induced chondrosarcoma cells, there were no significant differences in the number of transmembrane cells between the three small molecule drugs and their corresponding probes (P>0.05).Conclusions (1)The active sites of three small molecules named TN14003, T140,ANMD3100 were cell membrane.(2) There were no significant diferences on antagonistic effect between TN14003, T140, AMD3100 and their corresponding probes.