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Gallic Acid Sulfonamide Derivatives JEZTC Bone Cartilage Degeneration Suppression

Posted on:2015-03-04Degree:MasterType:Thesis
Country:ChinaCandidate:L LiuFull Text:PDF
GTID:2264330431952827Subject:Orthopedic trauma hand surgery
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Background:tissue engineering technology provides a new idea totackle the articular cartilage disease which is always difficult to berepaired.However, so far in order to obtain a sufficient amount of seedcells used in cartilage tissue engineering remains challenge.Objective:To establish the methods for isolation and culture ofrabbit articular cartilage and to observe the biological characteristics ofarticular chondrocyte.Method:To isolate the articular cartilage from white rabbits andculture the cartilage with the method of digestion in vitro. Culturedchondrocytes were identified with HE staining and toluidine blue staining.The cell growth curve was depicted according to MTT assay. The shapesof chondrocyte were observed by inverted microscope and the biologicalcharacteristics were then observed by immunohistochemistry staining andreverse transcript polymerase chain reaction (RT-PCR).Result and Conclusion:With the two-step digestion method, thechondrocytes were successfully isolated. After having been cultured fortwenty-four hours, the internal adhering wall potentially became a shortspindle. After two weeks the cells gathered like “stones on the pavement”.With MTT assay the chondrocyte growth curve was close to an “s” shape.The result of toluidine blue staining was positive with a decreasing trend as the number of generation increased. It was also demonstrated byimmunohistochemistry staining and the reverse transcript polymerasechain reaction (RT-PCR) that the expression of typeⅠcollagen becamestronger gradually with the increase number of generation, while theexpression of typeⅡ collagen became weaker. In this research, a greatquantity of active and pure chondrocytes can be harvested by adoptingthe method of two-step enzymatic digestion. The chondrocytes of the firstthree generations can be grown quite well and their biologicalcharacteristics can be effectively maintained, therefore, they are suitablefor applications in cartilage tissue engineering. After the third generation,chondrocytes could start to dedifferentiate. Objective:To observe the effect of JEZTC on the biologicalcharacteristics of rabbit articular chondrocytes in vitro.Method: Cultured Rabbit articular chondrocytes in vitro weretreated with different concentrations of JEZTC (0,2.344,4.688,9.375ug/ml). The proliferation of rabbit chondrocytes were detectedrespectively at day2,4,6by MTT assay at different concentrations ofJEZTC. The change of characteristics of cartilage cells were observedunder different concentrations with different methods including dimethylylidene Blue (DMMB) detection, safranin O-fast green staining, Hoechst33258staining, calcein/PI staining, eosin (HE) staining, phalloidinstaining and Ⅰ, Ⅱ collagen immunization staining. And furtherchanges in the biological characteristics of the cells was observed byusing the method of inspection glycosaminoglycan, Ⅰ collagen genetype Ⅱ collagen Ⅹ collagen gene expression RT-PCR,, and Sox9gene.Result: Good chondrocyte proliferation was detected by MTT assaywhen the concentration of JEZTC was4.688ug/ml. In terms of thecartilage cell morphology there was no difference between the controlgroup and the JEZTC group treated with HE staining, Safranin O-fastgreen staining and Phalloidin staining. By the method of type Ⅰ, Ⅱcollagen immunohistochemical staining, it was found that cultured chondrocytes at2,4,6days had stronger expression with type II collagenthan that in the control group, while weaker of expression with type Icollagen. The gene expression in both JEZTC group and the control group,was found that SOX9gene, the gene proteoglycan and type Ⅱ collagengenes were significantly up-regulated (p <0.05), but the expression of theJEZTC group Ⅰ collagen gene on average less than in the control group (p<0.05). The expression of SOX9gene, aggrecan gene and type II collagengene was were the strongest when chondrocytes were cultured at aconcentration of4.688ug/ml at day4and day6.Conclusion: JEZTC can not only promote the generation of theextracellular matrix of cartilage cells but also can maintain the biologicalcharacteristics of cartilage cell function, which is expected to becomenew therapeutic drugs to further delay the degenerative osteoarthritis. drugs of intra-articular injection of osteoprotegerin on articular cartilagein a rabbit model of osteuarthritis(OA).Method: Eighteen male New Zealand rabbits were randomlydivided into3groups,6rabbits each group: In JEZTC synthetic drugsgroup,each rabbit underwent anterior cruciate ligament transection(ACLT)in both two knee joints,and then0.5ml JEZTC synthetic drugs wereinjected into the two knee joints after surgery in8weeks, one time eachweek. Arthritis group did not give any treatment after cruciate ligamenttransection. In sham-operated group,the anterior cruciate ligament wasjust exposed without transection,and then the incision was closed. Allrabbits were batchesed to sacrifice12and16weeks after operation, andknee joints were obtained. The Pelletier score and Mankin score wereused to evaluate the result, By PCR detected MMP-1, MMP-3, MMP-13,TIMP-1-related gene expression.Result:The score of femorl condyle cartilage is (2.17±0.41),lowerthan Arthritis group(2.83±0.75).There was not statistical difference inPelletier score between the sham-group and Arthritis group(P=0.101).Mankin score of JEZTC synthetic drugs group(2.50±0.55)was lower than Arthritis group (3.33±0.52),There was statistical difference in Pelletier score between the sham-group and Arthritis group(P=0.046)12weeks after operation..Consistent with the macroscopicdata, the data differences was meaningful in cartilage structure,chondrocyte, Safranin O staining, tidemark integrity in JEZTC syntheticdrugs group and Arthritis group in12weeks (P=0.003;P=0.010;P=0.004;P=0.018). and16weeks after operation, there wasstatistical difference in cartilage structure, chondrocyte, tidemark integrity(P=0.001;P=0.018;P=0.003). PCR Detection found gene expression levelof JEZTC synthetic drugs group in MMP-1, MMP-3, MMP-13lowerthan Arthritis group in12W and16W, Gene expression in MMP-1,MMP-3, MMP-13between JEZTC synthetic drugs group and Arthritisgroup was statistically significant(P=0.000;P=0.000;P=0.003;P=0.045).Conclusion JEZTC synthetic drugs has a protective effect onarticular cartilage and slow the progress of osteoarthritis.
Keywords/Search Tags:chondrocyte, cell culture, tissue engineeringJEZTC, cultured in vitro, chondrocytesJEZTC, osteoarthritis, cartilage, rabbit knee
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