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OBJECTIVE@#To investigate the expression of citrullinated epitopes in articular cartilage protein and whether its expression is sufficient to induce anti-citrullinated protein antibody (ACPA) response in mice.@*METHODS@#The experimental group was treated with different concentrations of lipopolysaccharide (LPS), heat-inactivated bacteria ( and ) or specific monoclonal antibody against type Ⅱ collagen to induce citrullination of articular cartilage protein, with PBS as the control. Immunohistochemistry with the monoclonal antibody ACC4 (IgG1) that specifically binds to the citrullinated epitope of cartilage protein was performed for detecting the expression of citrullinated protein, with ACC1 (IgG2a) as a positive control antibody and L243 (IgG2a) and Hy2.15 (IgG1) as the negative isotype control. In the in vivo experiment, SD rats were subjected to injection of different doses of LPS in the right knee (with PBS as the controls in the left knee), and 3 days later frozen sections were prepared for immunohistochemical detection of the expression of citrullinated protein. Models of collagen-induced arthritis (CIA) established in different mouse strains were observed for incidence and severity of CIA. Serum samples collected from these models and the sera from rheumatoid arthritis patients were examined for anti-citrullinated protein antibody, and immunohistochemistry was performed to detect the expression of citrullinated protein in the cartilage of the mouse.@*RESULTS@#The citrullinated CII epitope-specific antibody ACC4 did not bind to articular cartilage tissues with different treatments as compared with the positive control antibody ACC1. The ACC4 antibody and the antibodies from patients with rheumatoid arthritis with high titers of anti-citrullinated protein antibody were capable of binding to the synovial tissue around the articular cartilage of the CIA. Luminex analysis showed that the anti-citrullinated protein antibody was lowly expressed in mouse serum, but the anti-type Ⅱ collagen triple helix structure peptide antibody exhibited strong reactivity.@*CONCLUSIONS@#Mild acute inflammatory response is not enough to cause citrullination of articular cartilage protein, and the expression of specific epitope requires a high-intensity inflammatory response. Inflammatory articular cartilage protein can express citrullinated epitopes in type Ⅱ collagen-induced arthritis in mice, but the expression of citrullinated epitopes is not sufficient to induce an immune response to anti-citrullinated antibodies. Stronger stimulation signals are required to induce an immune response for producing anti-citrullinated protein antibodies.
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Animals , Humans , Mice , Rats , Arthritis, Experimental , Autoantibodies , Cartilage, Articular , Citrulline , Inflammation , Rats, Sprague-DawleyABSTRACT
<p><b>OBJECTIVE</b>To observe the impacts of thermosensitive moxibustion (TSM) on the expressions of nitric oxide (NO), typeⅠdisintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), typeⅡcollagen and proteoglycan (PG) in the rabbit models of knee osteoarthritis (KOA) and explore the mechanism of TSM on KOA.</p><p><b>METHODS</b>A total of 42 Japanese long-eared male rabbits were divided into a blank group (6 rabbits), a model group (6 rabbits), a moxibustion group (24 rabbits) and a sham-operation group (6 rabbits) according to the random number table. In the blank group, the rabbits were fed normally. In the model and moxibustion groups, the papain injection was given to establish KOA models. The rabbits in the sham-operation group were treated with the intracavity injection of 0.9% NaCl solution. The rabbits were forced to move for 30 min every day, continuously for 15 days during modeling. At the end of modeling, in the moxibustion group, moxibusiton was applied at "Dubi" (ST 35), once a day, 40 min each time, for 14 days totally. According to the temperature changes during moxibustion, the rabbits were divided into a TSM group and a non-TSM group. 6 rabbits were collected randomly from the two groups. The usual feeding was given in the blank group, the model group and the sham-operation group, without any intervention. The body mass and behavioristics changes were observed in each group. At the end of treatment, the nitrate reduction method was adopted to determine NO expression in the serum. The real-time PCR was adopted to determine the expressions of ADAMTS-4, typeⅡcollagen and PG in the cartilage.</p><p><b>RESULTS</b>① After modeling, compared with the blank group, the body mass was all reduced and the Lequesne MG score was increased in the model group, TSM group, non-TSM group and sham-operation group (<0.05, <0.01). After intervention, compared with the blank group, the body mass was decreased and the Lequesne MG score was increased in the model and sham-operation groups (<0.05, <0.01). Compared with the model group, the body mass was increased and the lequesne MG score was decreased in the TSM, non-TSM, and sham-operation groups (<0.05, <0.01). Compared with the non-TSM group, the body mass in the TSM group was increased remarkably (<0.05), but the difference in Lequesne MG score was not statistically significant (>0.05). ② After intervention, compared with the blank group, the expressions of NO and ADAMTS-4 were all increased and the expressions of typeⅡcollagen and PG were decreased in the model group, TSM group, non-TSM group and sham-operation group (<0.05, <0.01). Compared with the model group, the expressions of NO and ADAMTS-4 were all remarkably lower and the expressions of typeⅡcollagen and PG were increased in the TSM group, non-TSM group and sham-operation group (<0.05, <0.01). Compared with the non-TSM group, the expressions of NO and ADAMTS-4 were all remarkably lower and the expressions of typeⅡcollagen and PG were increased in the TSM group after intervention (all <0.05).</p><p><b>CONCLUSION</b>The thermosensitive moxibustion alleviates the inflammatory reactions and protects the joint cartilage through inhibiting the expressions of NO and ADAMTS-4 to achieve the effects in the treatment of KOA.</p>
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Animals , Male , Rabbits , ADAMTS4 Protein , Metabolism , Cartilage , Metabolism , Collagen Type III , Metabolism , Moxibustion , Nitric Oxide , Blood , Osteoarthritis, Knee , Therapeutics , Proteoglycans , Metabolism , Random AllocationABSTRACT
Objective To investigate changes in the serum expression levels of decorin (DCN),chondroitin sulfate (CS) and C-terminal crosslinking telopeptide of type Ⅱ collagen (CTX-Ⅱ) and analyze the relationship between the expression levels and clinical characteristics,aiming to provide evidence for clinical diagnosis and treatment of arthritis.Methods Ninety subjects were divided into the osteoarthritis (OA),rheumatoid arthritis (RA) and control groups (n=30 for each group).The OA and RA patients were diagnosed in Department of Rheumatism Immunity of Shanxi Dayi Hospital,and the healthy volunteers who were doing physical check-up in the Physical Check-up Center of Shanxi Dayi Hospital were recruited as the control subjects.Visual analogue scale (VAS) was utilized to evaluate the degree of pain.X-ray was performed to assess the severity of joint lesions.Enzyme linked immunosorbent assay (ELISA) was adopted to quantitatively measure the serum expression levels of DCN,CS and CTX-Ⅱ.The correlation among these parameters was statistically analyzed.Measurement data among different groups were statistically compared by one-way analysis of variance (ANOVA)and comparison between two groups was conducted by Tambane's T2.The correlation between two factors was analyzed by using Spearman correlation analysis.Results The difference of the serum expression levels of DCN in the OA,RA and control groups [(3.19±1.38) ng/ml,(1.90±0.62) ng/ml,(1.33 ±0.33) ng/ml] was statistically significant,which of the OA group was higher than the control group (P＜0.01),which of the RA group was higher than control group (P＜0.01),which of the OA group was higher than the RA group (P＜0.001).The difference of the serum expression levels of CS in the OA,RA and control groups [(0.57±0.12) ng/ml,(0.95±0.47) ng/ml,(0.36±0.09) ng/ml] was statistically significant,in which,the OA group was higher than the control group (P＜0.01),RA group was higher than control group (P＜0.01),OA group was lower than RA group (P＜0.01).The serum expression levels of CTX-Ⅱ in the OA and RA groups [(3.08±0.86) ng/ml,(3.03±1.14) ng/ml] were significantly up-regulated compared with those in the control group [(2.17±0.82) ng/ml](P＜0.001,P=0.005).The expression level of CTX-Ⅱ did not significantly different between the OA and RA groups (P=0.996).In the OA and RA groups,the serum levels of DCN,CS,CTX-Ⅱ were po-sitively related to X-ray and VAS,the correlation of DCN was the highest,the correlation was high in both OA (r=0.777,P＜0.01;r=0.622,P＜0.01) and RA (r=0.640,P＜0.01;r=0.493,P=0.006).Conclusion The serum levels of DCN,CS and CTX-Ⅱ in arthritis patients are significantly up-regulated compared with those in healthy controls,which are positively correlated with the degree of pain,and the severity of cartilage and bone defects.DCN is probably valuablein the differential diagnosis of early OA and RA.However,the clinical application remains to be further validated by evidence-based studies.
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Arthritis is a common chronic disease characterized by the destruction of joint cartilage and inflammation in the sur-rounding tissues. Although it is known that the pathogenesis of arthritis is influenced by a series of factors, the underlying mechanisms remain unclarified. Recently, increasing attention has been paid to the increase of matrix metalloproteinases (MMPs) in articular cartilage,resulting in an inevitable degra-dation of cartilage and extracellular matrix (ECM). MMP-13, the major functioning enzyme during arthritis development,plays a vital role in the cartilage destruction, thus contributing to the decomposition of type Ⅱ collagen irreversibly. A variety of cellu-lar cytokines such as IL-1β and TNF-α,and Runx2 are assumed to affect the expression of MMP-13 in chondrocytes. The hypom-ethylation of the promoter region of the MMP-13 may induce its expression, while it can be reduced by inhibiting histone acety-lation. Meanwhile, microRNA can reduce the expression of MMP-13. In conclusion, MMP-13 can be used as an important therapeutic target in arthritis. In this review, we focus on the role of MMP-13 in arthritis and its underlying regulatory mecha-nisms.
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Aim To investigate the effect of TaorenHonghua drug pair on intervertebral disc degeneration (IVDD) in rats.Methods Fifty healthy Wistar rats were randomly divided into control group,model group,sham group,meloxicam group and Taoren-Honghua drug pair group,with 10 rats in each group.We established dynamic and static forces imbalance of cervical disc degeneration model or sham surgery in rats.12 weeks later,rats were intragastrically administered with meloxicam,Taoren-Honghua drug pair or saline for 30 days.C4/5 and C6/7 discs were harvested from rats.ABOG staining was used for observation of intervertebral disc morphology,real time PCR for mRNA expressions of type Ⅱ collagen (Col Ⅱ) and type Ⅹ collagen (Col Ⅹ),and immunohistochemical staining for Col Ⅱ and Col Ⅹ.Results Compared with model group,Col Ⅱ expression increased,while Col X expression decreased in chondrocyte of intervertebral disc in Taoren-Honghua-treated group(P ＜ 0.01).Conclusion Taoren-Honghua drug pair could delay the degeneration of cartilage endplate in rat intervertebral disc.
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Objective To explore the effect of radiofrequency heating on type Ⅱ collagen expression in a rabbit model of osteoarthritis.Methods Knee osteoarthritis was induced in the right hind legs of 54 male rabbits using modified Hulth modeling.The rabbits were randomly divided into a model group which was not given any special treatment,a Lugua polypeptide group and a radiofrequency hyperthermia group.The Lugua polypeptide group was injected with Lugua polypeptide;the radiofrequency hyperthermia group was treated with radiofrequency irradiation.Six,12 and 18 days after the treatment,the morphological condition of the rats' right femoral medial condyle cartilages were evaluated using modified Mankins scoring and the type Ⅱ collagen content of the cartilage was detected using a quantitative PCR technique.Results At the same time points after treatment,the average Mankins scores were decreased in all the 3 groups,with that of the model group was significantly higher than those of both of the other groups,and the radiofrequency hyperthermia group's average score was significantly better than that of the Lugua polypeptide group.The average type Ⅱ collagen content was significantly increased in all the 3 groups to various extent (the radiofrequency hyperthermia group ＞ Lugua polypeptide group ＞ model group).For the radiofrequency hyperthermia group,the average Mankins score decreased significantly and the average type Ⅱ collagen content increased significantly as the treatment continued.Conclusion Radiofrequency hyperthermia is superior to Lugua polypeptide for treating knee osteoarthritis,at least in rabbits.Its therapeutic effectiveness may be related to a significant increase of type Ⅱ collagen in the cartilage.
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Aim To investigate the effect of non-T cell binding peptide(FNS007)on collagen type Ⅱ-induced arthritis(CIA)in mice and the possible mechanisms.Methods The CIA model was induced by intradermal injection of bovine CⅡ+Freunds adjuvant.At the clinical onset of CIA,mice were randomly divided into 6 groups: blank control group(Control),model group,ORENCIA(abatacept)group,FNS007 low dose(1.2 mg·kg-1)group,FNS007 middle dose(2.4 mg·kg-1)group and FNS007 high dose(4.8 mg·kg-1)group.FNS007 was given by intravenous injection on the first day of arthritis and every other day until the study was terminated on d 28 after injection of the drug.The paw thickness and the ankle joint width were measured,and the arthritis scores were recorded.At termination,interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and level of anti-CⅡ antibody in serum were examined by enzyme-linked immunosorbent assay(ELISA).Bone injury was analyzed by X-ray imaging,and HE staining was conducted to observe the histopathologic changes and pathological score of ankle tissues.Results CIA models were successfully induced.Compared with CIA group,FNS007 high dose significantly reduced the paw thickness and the ankle joint left-right diameter,lowered arthritis scores in CIA mice,reduced serum concentrations of IFN-γ,IL-6 and anti-CⅡ antibodies,and lowered the radiographic and histologic scores.Compared with CIA group,FNS007 middle dose group showed marked reduction in the arthritis scores,IL-6 content in serum,and inhibion in the radiographic and histologic scores.The arthritis scores,concentration of IFN-γ,the radiographic and histologic scores were significantly reduced in FNS007 low dose group compared with those in model group.Conclusion FNS007 can effectively inhibit the progression of CIA through inhibiting T-cell activation and reducing inflammatory cytokines,anti-CⅡ antibodies,and histoclasia and bone destruction.
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OBJECTIVE:To investigate the effect of total saponins of Man medicine Thladiantha dubia root(TSTR)on the ex-pressions of CD3+,CD4+and CD8+ in spleen tissue of rats with type Ⅱ collagen-induced arthritis (CIA),and explore its mecha-nism in the treatment of rheumatoid arthritis(RA). METHODS:8 rats were taken as normal control group(NC group),the others 72 rats were injected mixture of bovine type Ⅱ collagen and Freund's complete adjuvant in tail and back to induce CIA model. The 50 modeled rats were randomly divided into model group(MC group),tripterygium polyglycoside(TG)group(12 mg/kg,posi-tive control),TSTR low-dose,medium-dose,high-dose groups (20,40,80 mg/kg),10 in each group. Rats in medicine groups were intragastrically administrated for 35 d,once a day;rats in NC group and MC group were intragastrically administrated equal volume of distilled water. Ankle swelling degree of rats was determined,arthritis indexes were calculated,HE staining was used to observe the lesions in synovial tissue,and immunohistochemistry was used to detect the expressions of CD3+,CD4+,CD8+ in spleen tissue of rats. RESULTS:After 35 d of administration,ankle swelling degree,arthritis indexes,CD4+ expression in spleen tissue,and CD4+/CD8+ ratio in MC group were significantly higher than NC group (P<0.05 or P<0.01),expressions of CD3+, CD8+were significantly lower than NC group(P<0.05);and there was congestion and massive inflammatory cell infiltration in sy-novial tissue. The ankle swelling degree,arthritis indexes,expression of CD4+ in spleen tissue,and CD4+/CD8+ ratio in administra-tion groups were significantly lower than MC group(P<0.05);the expressions of CD3+,CD8+ in spleen tissue in administration groups were significantly higher than MC group (P<0.05);TG group,TSTR medium-dose and high-dose groups showed mild congestion and a small amount of inflammatory cell infiltration in synovial tissue of rats,and TSTR low-dose group showed no ob-vious congestion or inflammatory cell infiltration. CONCLUSIONS:Up-regulating the expressions of CD3+,CD8+ and down-regu-lating the expression of CD4+ may be one of the mechanisms of TSTR in the treatment of RA;and with best efficacy when the TSTR dose of 20 mg/kg.
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<p><b>OBJECTIVE</b>To observe the effects of close-to-bone needling (CBN) on the expressions of type-Ⅱcollagen, pro-collagen type Ⅱ C-terminal propeptide (PⅡCP) and C-telopeptide of type Ⅱ collagen (CTX-Ⅱ) in rabbits with knee osteoarthritis (KOA).</p><p><b>METHODS</b>Among 40 New Zealand rabbits, 10 rabbits were selected into a normal group; the remaining 30 rabbits were made into KOA model, and X-ray was used to evaluate the results of model establishment. After the model was successfully made, the rabbits were randomly divided into a model group, a CBN group and a regular acupuncture group, ten rabbits in each one.Rabbits in the CBN group and the regular acupuncture group were treated at "Neixiyan" (EX-LE 4), "Dubi" (ST 35), "Yinlingquan" (SP 9), "Zusanli" (ST 36) and "Liangqiu" (ST 34). The CBN group applied CBN, and the depth of needling was appropriate with needles reaching bone; the regular acupuncture group applied regular acupuncture. The electroacupuncture(EA) device was used in the two groups, 20 min per treatment, once a day.Five days of treatment were taken as one course, and totally 4 courses were given with an interval of 2 days between courses. The normal group received identical fixation as model group. After treatment, magnetic resonance imaging (MRI) was used to perform imaging observation on knee; transmission electron microscopy (TEM) was used to observe the cell structure of knee joint cartilage;HE staining was used to observe the pathological change of knee; TUNEL was used to observe the apoptotic index; the expressions of type-Ⅱ collagen proteins and mRNA were measured by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR); the serum PⅡCP and CTX-Ⅱ levels were measured using enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>After treatment, compared with the model group, in the CBN group and regular acupuncture group the articular cavity effusion was reduced without the subchondral bone edema; the cell structure of knee joint cartilage was regular with less apoptosis; the expressions of type-Ⅱ collagen proteins and mRNA were significantly increased (all<0.05), the PⅡCP levels were significantly increased (both<0.05), but the CTX-Ⅱ levels were significantly decreased (both<0.05).The differences of the expressions of type-Ⅱ collagen proteins and mRNA, the levels of PⅡCP and CTX-Ⅱ between the CBN group and the regular acupuncture group were significant (all<0.05); the differences between the CBN group and the normal group were non-significant (all>0.05).</p><p><b>CONCLUSIONS</b>CBN can significantly improve the pathological status of cartilage of KOA, reduce apoptosis, and is likely to regulate the expressions of PⅡCP and CTX-Ⅱ to promote the type-Ⅱ collagen, which is superior to regular acupuncture.</p>
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Objective To investigate the effect of treadmill running with different intensities on type Ⅱ collagen (Col2) of knee joint articular cartilages in rats.Methods A total of 48 adult Sprague-Dawley rats were randomly divided into a control (C) group,a low-intensity exercise (L) group,a moderate-intensity exercise (M)group and a high-intensity exercise (H) group,each of 12.Rats in three exercises groups were regularly trained on treadmill at low,moderate,and high intensities respectively.Eight weeks later,all the animals were sacrificed.The right tibial plateau samples were collected to observe collagen fibers under polarizing light microscopy,and the collagen Ⅱ content were examined using immunohistochemistry.The mRNA expression of biglycan (BGN),fibromodulin (FMOD) and Col2 was tested using the quantitative real-time reverse transcription-polymerase chain reaction.Results Compared with group C,collagen fibers in group L and M exhibited almost the same organization,whereas,alteration in organization and shape of collagen fibers was found in group H.Significantly lower content of type Ⅱ collagen was found in group H than that in group C.In comparison with group C,group L had significantly higher gene expression of Col2,whereas group H had significantly higher BGN mRNA expression.Conclusion Low-or moderate-intensity treadmill running appears to have beneficial effect on articular cartilages to maintain its integrity.Highintensity exercises induce lower content and disorder of type Ⅱ collagen in articular cartilages,but the self-healing of cartilage may still exist.
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Objective To observe the effect of aerobic exercise of different intensities on type Ⅱ collagen,glycosaminoglycan (GAG) and chondrocyte apoptosis in rabbits modeling knee osteoarthritis (OA),so as to explore the preventive effect and its possible mechanism.Methods Twenty healthy New Zealand white rabbits were randomly divided into Groups A,B,C and D,each of 5.Group A was allowed free activity in a cage for 9 weeks.Group B was allowed free activity for 4 weeks,then an OA model was established using papain and confirmed via MRI 1 week later,Another 4 weeks of free activity were then allowed.Groups C and group D were given running training for 20 minutes a day at 0.5 km/h,3 times a week,and then 20 minutes a day at 1.5 km/h,5 days a week on a treadmill for 4 weeks.Nine weeks later,all 4 groups of rabbits were killed and the articular cartilage damage of each group was compared using Mankin scoring,and expression of type Ⅱ collagen,GAG content and chondrocyte apoptosis in the cartilage.Results After the intervention,the average Mankin score,expression of type Ⅱ collagen and GAG content of groups B,C and D were significantly lower than those of group A,and all of those values in group B were significantly lower than those of group D.After 9 weeks the chondrocyte apoptosis rate of group A was significantly lower than that of the other groups,and that of groups C and D was significantly lower than that of group B.Conclusion Aerobic exercise may prevent knee articular cartilage degeneration through inhibiting reduction in the amount of type Ⅱ collagen and GAG in the cartilage matrix.It may be related to decreasing the chondrocyte apoptosis.
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BACKGROUND: p38 mitogen-activated protein kinase signal transduction pathway is a member of the mitogen-activated protein kinase family. It plays an important role in the development of osteoarthritis. OBJECTIVE: To review the progress of p38 mitogen-activated protein kinase signal transduction pathway in the pathological process of osteoarthritis. METHODS: An online search of CNKI and PubMed databases was performed for articles using keywords of “p38 mitogen-activated protein kinase signal transduction pathway, osteoarthritis, articular cartilage, chondrocyte” in Chinese and English, respectively. Relevant articles were summarized from three aspects of introduction of p38 signal transduction pathway, the role of p38 mitogen-activated protein kinase signal transduction pathway in osteoarthritis and the inhibitor of p38 in osteoarthritis. A total of 90 articles were included. According to inclusion criteria, a number of 46 articles were retained at last. RESULES AND CONCLUSION: p38 mitogen-activated protein kinase signal transduction pathway has a close relation with chondrocyte hypertrophy and calcification, chondrocyte apoptosis, synthesis of cartilage matrix metal oproteinase, production of proinflammatory cytokines, and exerts a significant effect on the development of osteoarthritis. p38 mitogen-activated protein kinase is involved in the formation and development of osteoarthritis through a variety of complex mechanisms and plays a very important role. Therefore, blocking p38 mitogen-activated protein kinase signaling pathway may be a new target in the treatment of osteoarthritis.
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BACKGROUND:Adipose-derived stem cel s and bone marrow mesenchymal stem cel s are used widely in cartilage tissue engineering, and there are many similarities in biological characteristics between two kinds of cel s. OBJECTIVE:To compare the chondrogenic potential of bone marrow mesenchymal stem cel s and adipose-derived stem cel s in vitro. METHODS:Adipose-derived stem cel s were isolated from the 3-month-old New Zealand white rabbits’ abdomen. Bilateral femurs of rabbits were obtained, and then the bone marrow mesenchymal stem cel s were separated with the adherence screening method. The growth curve of the passage 3 adipose-derived stem cel s and bone marrow mesenchymal stem cel s were drawn, and the doubling time of two kinds of cel s was compared. Then the passage 3 adipose-derived stem cel s and bone marrow mesenchymal stem cel s were treated with chondrogenic induction. After induced for 14 days, the adipose-derived stem cel s and bone marrow mesenchymal stem cel s were treated with toluidine blue staining and type Ⅱ immunohistochemistry staining respectively. RESULTS AND CONCLUSION:Primary bone marrow mesenchymal stem cel s showed aggregative growth, while the primary adipose-derived stem cel s were in single and scattered growth. The proliferation speed of adipose-derived stem cel s was faster than that of bone marrow mesenchymal stem cel s, while the doubling time of adipose-derived stem cel s was shorter than that of the bone marrow mesenchymal stem cel s. After chondrogenic induction for 14 days, both adipose-derived stem cel s and bone marrow mesenchymal stem cel s could express glycosaminoglycans and type Ⅱcol agen, and the expression level of type Ⅱ col agen in bone marrow mesenchymal stem cel s after chondrogenic induction was higher than that in the adipose-derived stem cel s. The in vitro proliferation of adipose-derived stem cel s and bone marrow mesenchymal stem cel s was rapid and stable, but the proliferative ability of adipose-derived stem cel s was faster than that of bone marrow mesenchymal stem cel s. When cultured in single layer, both adipose-derived stem cel s and bone marrow mesenchymal stem cel s could transform into chondrocytes under certain conditions, but bone marrow mesenchymal stem cel s seemed to be more potential than adipose-derived stem cel s.
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Objective To detect type Ⅱ collagen synthesis by cultured human chondrocytes under the influence of different intermittent fluid shearing forces.Methods Second passage human monolayer chondrocytes were divided into low-speed (20 rpm/min), mid-speed (40 rpm/min) and high-speed (60 rpm/min) groups according to the rotation speed of the rocking bed. The expression of type Ⅱ collagen was analyzed using immunohistochemistry and RT-PCRs.Results The absorbance of the mid-speed and high-speed groups was significantly greater than that of the low-speed group and a control group. The average optical density of type Ⅱ collagen by RT-PCR was significantly higher in all three sheared groups than in the control group.Conclusions Intermittent shearing can enhance cellular proliferation and the metabolism of human chondrocytes in vitro and promote the expression of type Ⅱ collagen.
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To investigate the influence of recombinant adenovirus carrying tissue inhibitor of metalloproteinase-3 (RAdTIMP-3) on the main compositions of rabbits intervertebral discs and to assess its potential in treatment for intervertebral disc degeneration.[Method]RadTIMP-3 and empty adenovims vector with Lac-Z gene (Rad66) was propagated in 293 Cells and was purified, identified and tittered. Thirty Japanese white rabbits were randomly divided into 5 groups. And 25 μl of various reagents were injected to the L4、5 and L5、6 intervertebral discs of the rabbits as follows:normal saline in group 1, 1.0×1010 OPU/ml of RAd66 in Group 2, and 1.0×1010 OPU/ml of RAdTIMP-3 in group 3, 4 and 5. The intervertebral discs of each group were collected after 2, 2, 1, 2 and 4 weeks after injection respectively.Then X-gal staining, And Group 1, RT-PCR for TIMP-3 and aggrecan core protein,TUNEL staining, immunohistochemical staining for TIMP-3 and type I! Collagen and Safranin O-Fast green staining was carried out to assess the effects of RadTIMP-3 transfection.[Result](1)concentration of RAdTIMP-3 reached 1.9×1012 OPU/ml after propagation and purification. (2)RT-PCR shows that the expression of TIMP-3 was significantly raised in group 3, 4, 5, as compared with group 1 or 2. And the expression of core protein gene in group 3, 4, 5 increased slightly than in group 1 and 2. (3) TUNEL staining revealed that there was not significant difference between the positive-staining rates of any two of the groups. (4)TIMP-3 staining exhibited an obvious increase of positive-staining rates in group 3, 4 and 5 as compared with groupi or 2. The staining density of Safranin O-Fast Green staining and immunohistochemical staining for type II collagen of group 5 was obviously higher than that of group 1 or 2.[Conclusion]RAdTIMP-3 can express widely and safely in rabbit intervertebral discs, and improve the quantity and quality of matrix. It has the potential to be used in treatment for intervertabral disc degeneration.
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Objective To investigate the expression of type Ⅱ collagen in the articular chondrocyte of osteoarthritis (OA) patients and normal human. Methods The samples of articular cartilage were obtained from the patients undergoing total joint replacement, including 8 primary OA patients, 8 secondary OA patients and 9 normal subjects. Type Ⅱ collagen expression in chondrocyte was detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The expressin of type Ⅱ collagen mRNA in normal OA group was higher than that in primary OA group and secondary OA group with a statistical difference (P=0.014), while there was no statistical difference between primary OA group and secondary OA group(P=0.716). Conclusions The reduction of type Ⅱ collagen expression leads to the change of collagen directly and possibly plays an important role in OA, which is the common pathway of the occurrence of both the primary and secondary OA.
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[Objective]To study the effect of sixpetal clematis root injection(WLX)on histology,collagen phenotype and ultramicrostructurc in cartilage of osteoarthritis animal model.[Method]OA model animals were randomly divided into 3 groups:group S,group D and group K.Animals in group S received WLX and animals in group D received sterilized normal saline intramuscularly,group K was control and received nothing.After the animals were killed,cartilage specimen were obtained and sections were made and stained with HE/SOFG for Mankin score;the expression level of collagen Ⅰ and Ⅱ in matrix was tested by immunohistochemistry;change of ultramicrostructure of cartilage was observed with transmission electron microscope.[Result]Mankin score in group S was better than that in group D and K at each test period;result of IHC shows that positive stain area of collagen Ⅰ was lower in group S than that in group D and K,and positive stain area of collagen Ⅱ was higher than that in group D and K at each test period;cartilage impairment degree in group S was lower than in group D and K,and impairment manifestation in group D and K was similar on the whole.[Conclusion]sixpetal clematis root injection may protect articular cartilage by maintaining and promoting the chondrocyte synthesizing proteoglycan and type Ⅱ collagen.
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Aim To establish method and main evaluation indexes of collagen induced arthritis ( CIA) model in DBA/1 mice. Methods CIA was induced by chicken type Ⅱcollagen ( C Ⅱ) in DBA/1mice. Arthritis was evaluated by arthritis index. X-ray of the paws was taken. Histology pathology of ankles and spleen was observed and scored. Results Immunization d31,the paws of CIA mice appeared red and swelling,the scores of arthritis index increased,the period of swelling peak was from d40 to d60; immunization d35,the weight of CIA mice began to decrease. X-ray of paws showed that the paw joints of CIA mice deformed,and there was osteophyte formation associated with osteolysis. Histo-logical pathology of ankle joints showed that the synovium of CIA mice were hyperplasia,cartilage was destroyed,pannus was formed,and inflammatory cells infiltrated into synovium. Histological pathology of spleen showed more germinal centers and lymphoid follicular hyperplasia were observed,cell density of lymphatic sheath increased,scores of ankle joints and spleen histological pathology in CIA mice were higher than those in normal mice significantly. Conclusions The methods of CIA model induced by chicken C Ⅱ in DBA /1 mouse were reliable and reproducible. CIA incidence was high. Arthritis index,X-ray of paws,ankle joints and spleen histological pathology and so on were the principal indexes of evaluation for CIA model.
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ly) and healthy controls respectively. Anti-CⅡ antibody positive group showed higher prevalence rate of rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody (anti-CCP) (83.3%, 90.5%) than those of anti-CⅡ negative group (43.7%, 52.1%, P
ABSTRACT
Objective: To assess the immunological effects by orally administering chicken type Ⅱ collagen(CCⅡ) on collagen-induced arthritis(CIA)mice. To assess the effect on producing IL-1 of peritoneal macrophage in adjuvant arthritis rats by orally administering CCⅡ. Methods: Arthritis were induced in Kunming mice by immunization with chicken type Ⅱ collagen with Freund's complete adjuvant, followed by an interperitoneal injection of CCⅡ 3 weeks later.Chicken type Ⅱ collagen was orally administered from 5 days prior to the induction of arthritis to 14 days after inducing arthritis model. The animals were examined visually twice weekly for polyarthritic signs of swollen and erythemic limbs. Quantitation of antibody level of CIA mice was measured by ELISA method. Subpopulations of T lymphocytes in mice were evaluated by flow cytometry method. IL-1 assay was evaluated by ELISA method. Results: Joint swelling was significantly reduced at a dose of 5 μg.kg-1 and 50 μg.kg-1 of CCⅡ, but not at 250 μg.kg-1. The level of anti-collagen antibodies was also reduced at a dose of 5 μg.kg-1 and 50μg.kg-1 (OD value from CIA model control 0.242±0.073 to CCⅡ 5 μg.kg-1 0.123±0.029 and CCⅡ 50 μg.kg-1 0.110±0.075 respectively). Subpopulations of T-lymphocytes were changed by orally administering of CCⅡ, and the ratio of L3T4+/Lyt-2+ was lowered (the ratio from 1.71 of CIA model control to 1.21, 1.51 of administered CⅡ 5 μg.kg-1, 50μg.kg-1 respectively.) after administering CCⅡ. IL-1 level can be reduced (the value from adjuvant arthritis model control 62.8±0.9 to 43.4±1.3, 49.7±0 ng.L-1 administered CⅡ 5 μg.kg-1, 50μg.kg-1 respectively). Conclusion: Arthritis sign in CIA animal model can be suppressed by oral CCⅡ. The effects may be involved by influencing the mechanisms both humoral and cellular immunity. The effects occurred at lower doses of CCⅡ. These results demonstrated the biologic relevance of by-stander suppression associated with oral tolerance, and the potential use of this approach to treat human inflammatory joint diseases.