| Objective:Blocking the signaling pathway of SDF-1/CXCR4 with T140 and AMD3100 in vitro and vivo. To compare the effects of T140 and AMD3100 on degenerative joint disease in different envirments and provide the clinical evidence for prevent and cure osteoarthritis earlier.Methods:Part 1:12 clinical patients who has been accepted knee replacement surgery were selected randomly as the experimental group in vitro, called OA group.Then 12 patients amputated because of trauma were randomly selected as control group. There was an experiment on each cartilage tissue in the two groups.The cartilage tissue was cut into 3×3×lmm3 (Mankin score:0 or 1), and then stimulated the joint degenerate by SDF-1. Next T140 and AMD3100 were put into two groups and provided a proper nutrition environment for cartilage tissue with the CO2 content for 5% and the temperature for 37 ℃;the treatment of nutrition liquid contained SDF-1 100ng/ml, the other experiment reagent like T140 1000 nm, AMD3100 1000 nm, MAB310 1000 nm, MAB002 1000 nm. According to the difference of nutrition liquid elements in each group,it will be divided into five groups:(1) T140 group:T140+SDF-1; (2) AMD3100 group:AMD3100+SDF-1; (3) MAB310 group:MAB310+SDF-1. (4) MAB002 group:MAB002+SDF-1. (5) Blank control group:nothing+SDF-1. The cartilage and nutrient solution were cultured in 2 days and 4 days respectively and do some relevant tests:(1) Histological examination. HE staining and sarranine-O staining were used to test the tissue degeneration, then Mankin scoring method to evaluated the changes of cartilage morphology. (2) To text the composition of MMP-3,-9,-13 in nutrient solution were tested with Elisa, the expression of mRNA of MMP-3,9,13 in cartilage tissue were tested with RT-PCR.Part 2:48 male Hartley cavys with the age of 6 months were divided into four groups: group A, group B, group C, and group D. In group A each cavy was injected with micro pumps through subcutaneous injections,the liquid in the pump was T140 which diluted by PBS solution, the speed of pump injection was 180ug/ml.d.In group B the injection was same to group A, and pump into the same amount of AMD3100 with the speed of 180ug/ml. The method of Group C is same to group A, injected into PBS solution with the speed of 180ug/ml. Group D is the blank control group.After 12 weeks, Hartley cavys were killed and the cartilage tissue were got from the tibial plateau for histologic examination, which including HE staining and safranin-O staining, and then parallel IL-1,TNF-a was tested with immunohistochemical. Next, use the fluorescent protein quantitative method to determinate the expression of MMP-3,MMP-9,MMP-13. Finally, summary and analyze the experiment data in the statistics perspective.Part 3:Based on the first two steps, the best blocking reagent of the SDF-1/CXCR4 signal passway was chosen.To test the type Ⅱ collagen expression of articular cartilage in vivo and vitro.Local degeneration changes was observed in the microstructure, the experiment data was calculated with SPSS 17.0, statistically significant was p<0.05.The results:Part 1:(1) The results of histological examination:1) The same group of the cartilage structure at the same time:group A compare with group B, group C, group D and blank control group.The tissue structure was regular and the articulage cells on the surface arranged orderlly, enough cell number closely packed, cutting dyeing were even without necrotic cells. Mankin scores were closely to normal situations,the data had statistically significance.(P<0.05);2)The comparison of same group in different time:there was no changes in group A, The experimental data had no statistical significance.(P>0.05),but in the blank contrl group, the changes was obvious by the time progresses,the cartilage surface began to loose and the surface became uneven with concave and convex, the number of cells was began to decrease, the cell became aging and necrosis. The Mankin score increased, means the tissue structure changed obviously, the research data had statistically significance.(P<0.05); 3)At the same culture time, compare OA cartilage with normal cartilage group:the cartilage tissue cell in OA group arranged more closely with dead and aging cells.Mankin score was increasing and the data analysis had a statistically significance (P< 0.05).(2) ELISA determine the MMP protease content nutrient solution and PCR technique was used to detect the content of mRNA expression in the cartilage:1) At the same time in the same cartilage group:The MMP content is lower in group A than other group, and the lower mRNA expression of the data had a statistically significance (P<0.05);2) the same group and different culture time comparison:MMP content and mRNA expression level in group A closed to the same level, there was no obvious changes, the data had no statistical significance.(P>0.05), but the blank control group with MMPs and the mRNA expression level raised obviously, the data had a statistica significance (P<0.05);3).OA group compared with the normal cartilage group at the same culture time, the MMP-3,9,13 and the mRNA expression in the normal group were lower than the OA group, the data had statistical significance (P<0.05).Part 2:The histology and morghology showed:Mankin scores in group A was lower than group C group D and blank control group, the data had a statistically significance (P<0.05), the degree of cartilage degenerative diseases was lower. Group A showed weak positive expression of mRNA. But the other four groups showed positive expression Real-time fluorescent quantitative PCR detection:group A had a lower expression level of MMPs than other groups of mRNA in the cartilage, the data had a statistically significance (P<0.05).Part 3:1) Compared the histologic microscopic changes by the mRNA expression of type Ⅱ collagen and aggrecan to analyze the cartilage in the same environment:group A was more active than other group, the data had a statistically significance (P<0.05).2) At different culture time and the same cartilage group:group A had no obvious difference, the data had no statistical significance (P>0.05), but there was enough mRNA expression in the blank control group, the data had a statistically significance (P<0.05); 3) Through the comparison between normal cartilage groups with arthritic cartilage group,the results showed that in the same environment, the mRNA expression levels of specific proteins in normal cartilage were lower than OA cartilage group.The data had a statistically significance (P<0.05);2. Western Blotting:type Ⅱ collagen was tested in vivo and vitro:1) In the same culture time of the same cartilage group:The grey value in group A was significantly higher than other groups, it was also higher than blank control group, the data analysis had statistically significance (P< 0.05).2. At different culture time in the same cartilage group:1) the grey value had no statistically significance (P> 0.05), and with the extension time, the grey value in the blank control group was reduced, the data had statistically significance (P< 0.05).3. the comparison of different cartilage group at the same culture time:grey value of OA cartilage group was lower than normal cartilage group, the data had a statistically significance (P< 0.05).Conclusion:1.T 140 could delay the articular cartilage degeneration in vivo and vitro better; 2.T140 could not recovery degenerated articular cartilage; 3. T140 had played a key role in the process of the development of osteoarthritis by blocking SDF-1/CXCR4 signaling pathways; 4. T140 can be used for further study of targeted drugs in the OA treatment. |
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