1.The role of type 3 muscarinic acetylcholine receptor polypeptide in the pathogenesis of Sj(o)gren's syndrome
Jing HE ; Hui WANG ; Zhanguo LI
Chinese Journal of Rheumatology 2008;12(7):-
Objective To immunize BALB/c mice with type 3 musearinic acetylcholine receptor polypeptide (M3RP) and to evaluate the role of M3RP in SS. Methods Four-week-old BALB/e mice were immunized with M3R polypeptide 213-228 (M3RP) on days 0, 14, 35, 56, and were re-immuniged on days 65, 84, 105, and one mouse was killed every 2 to 3 weeks. The mice of the control group were immunized with submaxillary gland homogenate, GST, and PBS. The animals were analyzed for the presence of anti-SSA,anti-SSB, RF, ANA, anti-a-fodrin and anti-M3RP in sera by immunofluorescenee or ELISA. The cytokines of IFN-γ IL-2 and IL-10 were measured with ELISA. Salivary glands were examined by H&E staining and immunohistochemical analysis. Volume of water drinking by each groups were calculated. Results BALB/e mice immunized with M3RP and submaxillary gland homogenate developed an immune response directed against M3RP, α-fodrin and ANA, but no antibodies against SSA, SSB and RF were found. Furthermore, lym-phocytic infiltrates in the salivary glands of immunized animals were observed 50 days after first immunization of M3RP and submaxillary gland homogenate. The serum IFN-α in mice of M3RP, submaxillary gland ho-mogenate, GST and PBS was (62±6), (89±5), (30±5) and (19±6) pg/ml respectively, and IL-2 was (12.6±1.6), (19.8±0.4), (3.9±0.9), and (4.9±1.1) pg/ml respectively (P<0.05). No difference was found in the level of serum IL-10 among the four group. Expression of α-fodrin was found only in submaxillary gland in M3RP and submaxillary gland homogenate groups of mice, but not in PBS and GST controls when studied by immunohistochemical analysis. Conclusion These results suggest that BALB/c mice immunized with M3RP are reminiscent of human SS, and M3RP as an autoantigen participates the development of SS.
2.The homing of allogenic mesenchymal stem cells transplantation on rats with collagen induced arthritis
Fang LI ; Xiaofeng LI ; Liyun ZHANG ; Yanli YANG ; Hongguang MENG ; Lihui MA ; Ke XU ; Huiying GAO
Chinese Journal of Rheumatology 2011;15(1):12-16,后插1
Objective To observe homing of mesenchymal stem cells (MSCs) in immune organs and inflammatory joints in collagen induced arthritis(CIA) rats. MethodsRats MSCs were isolated and expanded from bone marrow cells by density gradient centrifugation and adhering to the culture cell walls, and the phenotypes were assessed by flow cytometry. MSCs were labeled by PKH-26 and Brdu. Sixty-four rats were randomly divided into normal group and CIA group. Every 8 rats were sacrificed at 3, 11, 30, 42 days after transplantation of MSCs. At the end of the experiment, the specimens of thymus gland, spleen, ankle joints were exposed, fixed, decalcified, wrapped and cut into slices. Confocal laser scanning microscope and immunohistochemical method were used to observe migration and distribution of MSCs in different organs. Independent samples group t test with SPSS 12.0 software package was used for statistical analysis. ResultsIt was found that allogenic MSCs could stay in spleen, thymus gland and joints of CIA rats for a relatively long period (42days). Forty-two days after transplantation of MSCs, the average grey scale values of spleen and thymus gland in CIA group(37.5±8.8, 29.9±5.9 respectively) were significantly higher than the normal group(16.0±2.3,13.2±4.3 respectively), the average grey scale values of ankle joints in CIA group 78±8 was significantly lower than the normal group 93±14(P<0.05). ConclusionIt has been found that MSCs can stay in the injured tissue and organs preferentially.
3.Immunoregulatory effects of mesenchymal stem cells on T-lymphocytes of lupus nephritis
Yingying QIU ; Jing LI ; Jianqiang HE ; Yujun YIN ; Yu TANG ; Haiyan YOU
Chinese Journal of Rheumatology 2009;13(6):-
Objective To investigate the immunoregulatory effects of mesenchymal stem cells (MSCs) on the peripheral T-lymphocytes of lupus nephritis in vitro. Methods MSCs were isolated and expanded from human bone marrow cells. The purity of MSCs was identified by flow cytometry (FCM). The MSCs (4×104, 1×104, 2×103) were added into wells containing peripheral blood lymphocytes (2×105) from lupus nephritis in the presence of phytohemagglutinin [PHA). Samples were divided into the following groups: group A:T-lymphocytes alone; group B: MSCsl with T-lymphocytes(MSCsl:T=1:5); group C: MSCs2 with T-lymphocytes (MSCs2:T=1:20); group D: MSCs3 with T-lymphocytes (MSCs3:T=1:100). The proliferation of T-lymphocytes was assessed by MTT. FCM was used to analyze the apoptosis of T-lymphocytes and surface markers of CD28 and CD152. The gene expression of interferon γ (IFN-γ), interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1) were detected by real-time RT-PCR. Results In vitro, with the presence of MSCs, peripheral blood T-lymphocytes from lupus nephritis were statistically significantly decr-eased in their proliferative activities , apoptosis and CD28 expression in a dose-dependent manner. No inhibitory effects on CD152 expression of T-lymphocytes had been observed . MSCs promoted the gene expression of TGF-β1 and inhibited the gene expression of IL-10, IFN-γ. Conclusion MSCs can inhibit the proliferative activities, apoptosis and CD28 expression of peripheral blood T-lymphocytes of lupus nephritis, increase gene expression of TGF-β1 and lower the gene expression of IL-10, IFN-γ, which may play an important role in it's immunosuppressive effects on lupus nephritis.
4.Expression and significance of stem cell factor in renal tissue of patients with lupus nephritis
Xuemei LIU ; Ruixia MA ; Haiyan ZHOU ; Hui DONG ; Liqiu LIU
Chinese Journal of Rheumatology 2009;13(6):-
Objective To investigate the renal expression of stem cell factor (SCF) in lupus nephritis (LN) and its correlation with disease activity and renal injury parameters. Methods Histochemical stain was used to examine all renal specimens (LN group n=34, chronic glomerulonephritis n=16, control group n=8). Hyhridization in situ and immunohistochemistry were used to detect the expression of SCF and infiltration of mast cells, macrophages , α-SMA (+) cells in renal tissues of the two groups. SPS software was used for tissue of the control group. However, they increased markedly in lupus nephritis and CGN (t=6.03~14.25, P< 0.01). But there was no significant difference between LN and CGN in SCF and mast cells in renal interstitium. Positive correlation was observed among the expression of SCF and α-SMA and the number of mast cells and macrophages (r=0.47~0.84, P<0.01) at their corresponding locations. The expression of SCF and ot-SMA and the number of macruphages were positively correlated with renal pathological active index, chronic index, albuminuria and the injury of renal interstitium (r=0.34~0.93, P<0.05 or 0.01); meanwhile, it was negatively correlated with Ccr(r=-0.39~0.61, P<0.01). There was significant correlation between SCF, macrophages and anti-dsDNA antibody, complement C3 level, SLE disease activity index (SLEDAI). The number of mast cells in renal interstitium was positively correlated with chronic indexes and the injury of renal interstitium (r=-0.86, r=0.93, P<0.01) and negatively correlated with Ccr (r=-0.56, P<0.01), but not correlated with active index and albuminuria (r=0.27, r=0.23, P>0.05). Conclusion The expression of SCF is widespread in kidney, and it is markedly eorrelated with various kinds of inflammatory cells, renal inherent cells, renal function, and urine protein levels. SCF may be an critical participant in the initiation and progression of renal injuries in human lupus nephritis.
5.Hydroxychloroquine treatment for primary Sj(o)gren's syndrome:a prospective,open labeled clinical trial
Qun SHI ; Yan ZHAO ; Ling LI ; Zhaowen WANG ; Yi DONG
Chinese Journal of Rheumatology 2008;12(4):258-260,插2
Objective To evaluate the efficacy and safety,particularly eye safety of hydroxychloro-quine(HCQ)treatment in primary Sj(o)gren's syndrome(pSS)patients.Methods Forty pSS patients were en-rolled and treated with HCQ 400 mg/day for 12 months.This is a prospective open-label study.Clinical mani-festations,clinical efficacy,biochemical and immunoserological parameters as well as ophthalmological exami-nations were investigated every three months to assess the safety and tolerability.Results There were signifi-cant decrease in the erythrocyte sedimentation rate(ESR),immunoglobulin G(IgG),immunoglobulin M (IgM)and rheumatoid factor(RF)level after 6 months treatment with HCQ(P<0.01 or P<0.05).No changewas detected in serum antinuclear antibody(ANA),anti-SSA/SSB antibodies after treated for 12 months.Somepatients had partial improvement in symptoms such as dry mouth,dry eyes and arthralgia.During the treat-ment,no significant effect on serum alanine aminotransferase (ALT),blood urea (BUN),serum creatinine (Cr),whole blood count(WBC)or hemoglobin(Hb)could be discovered.Central semus retinopathv(CSR)was found in one patient after 6 months treatment with HCQ.However,its association with HCQ could not be confirmed since it was not compatible with the usual HCQ retinopathy.Conclusion HCQ can improve svmp-toms of some pSS patients and can significantly decrease ESR,IgG,IgM and RF level.The safety profile of HCQ is generally good.However,ophthalmological examination before and after a 6-month interval may be necessary in long term HCQ treatment.
6.Leflunomide active metabolite inhibites the expression of phorbol-12-myristate-13-acetate-induced CD147,matrix metallo-proteinase-2 and matrix metallo-proteinase-9 on THP-1 cells
Shiyao WU ; Jianlin HUANG ; Baozhao XIE ; Mingxia WANG ; Jieruo GU
Chinese Journal of Rheumatology 2011;15(3):183-187,后插1
Objective To investigate the effects of the leflunomide active metabolite (A771726) on the expression of phorbol-12-myristate-13-acetate (PMA) -induced CD147, matrix metallo-proteinase (MMP)-2 and MMP-9 on THP-1 cells. Methods THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. For all experiments, THP-1 cells were cultured at an initial density of 5×105/ml. Before A771726 treatment, cells were cultured with serum-free RPMI-1640 medium for 12 h, THP-1cells were co-cultured with PMA at three different concentrations of A771726 (5, 15 , 45 μg/ml) for 24 h.The mRNA expression of CD147, MMP-2 and MMP-9 was measured by real-time PCR. CD147 expression on the cells were evaluated by flow cytometric analysis. The activity of MMP-2 and MMP-9 were evaluated by gelatin zymography. Statistical differences among the groups were tested by one-way ANOVA or KruskalWallis test. Results The expression of CD147, MMP-2 and MMP-9 were upgraded by the PMA. The expression of CD147 on THP-1 cells was inhibited significantly by A771726 in a dose-dependent pattern (P<0.01). The mean fluorescence intensity (MFI) of CD147 in positive control group was 109.5±3.8, the MFI in A771726 (5, 15, 45 μg/ml) group were 73.3±2.5, 64.5±2.3, 40.9±2.7, respectively. The expression of MMP-2, MMP-9 mRNA and the activity of MMP-2, MMP-9 in the supernatant was inhibited significantly by A771726 (P<0.01). The expression of CD147 mRNA was not inhibited significantly by A771726 (P>0.05).Conclusion Leflunomide active metabolite (A771726) can inhibit the expression of PMA-induced CD147,MMP-2 and MMP-9 on THP-1 Cells.
7.The effect of umbilical cord mesenchymal stem cells transplantation for the MRL/Ipr mice
Zhifeng GU ; Ouyang JIN ; Ting XU ; Shengnan ZHAO ; Lingyun SUN
Chinese Journal of Rheumatology 2009;13(1):4-7,后插一
Objective To investigate the efficacy of umbilical cord mesenchymal stem cells (UC-MSCs) transplantation in the treatment of the MRL/lpr mice. Methods Twenty four 18-week-old MRL/lpr female mice were divided into 3 groups:group 1 (G1) were transplanted with 1×106 UC- MSCs through caudal vein, group 2 (G2) were transplanted with 1×106 UC- MSCs three times and group 3 (G3) were treated with 0.5 ml normal saline as controls. Enzyme linked immunosorbent assay (ELISA) was used to measure the levels of serum anti-dsDNA antibodies. Twenty-four hours proteinuria and body weight were assessed every two weeks. The histopathology changes of the kidneys and lungs were observed. Results ① At the 25th weeks, the 24 hours proteinuria in group G1 (2.3±1.9) mg and G2 (1.8±1.4) mg was decreased than that in the control group (3.8±2.1) mg (P<0.05), and at the 27th weeks, that of groups G1 (2.5±1.5) mg and G2 (1.9±1.2) mg was also significantly decreased than in the control group (5.4±2.4) mg (P<0.01); ② From the 24th week, the body weight of groups G1 and G2 increased significantly than that of the control group (P< 0.05). At week 29, serum creatinine decreased significantly in both groups G1 (7.2±3.2) μmol/L and G2 (6.2±2.8) μmol/L than in the control group (12.5±2.3 ) μmol/L (P<0.05); ③One week after transplantation, the levels of anti-dsDNA antibodies in group G1 (46±11)×102 U/ml and G2(49×43)×102 U/ml were bothsignificantly decreased than those of the control groups (99±42)×102 U/ml (P<0.05) and the difference between group G2 (36±15)×102 U/ml and the controls (68±32)×102 U/ml was statistically significant; ④The nephron crescent formation in group G1 (0.12±0.07) and G2 (0.08±0.02) was significantly lower that of the control group (0.20±0.06) (P<0.05) and that of group G2 was significantly less that of froup G1 (P<0.05); ⑤ The interstitial pneumonitis was singnificantly milder in group G1 than group G2. Conclusions UC- MSCs is very effective in treating MRL/lpr mice. It is safe and free of rejection reactions.
8.The efficacy of rhTNFR:Fc and its effect on osteopontin in the collagen-induced arthritis mice model
Zhifen LV ; Yi TAO ; Ruilin CHEN ; Wenhui HUANG
Chinese Journal of Rheumatology 2011;15(1):17-21,后插2
Objective To investigate the protection effects of recombinant human tumor necrosis factor-α receptor Ⅱ :IgG Fc fusion protein for injection (rhTNFR:Fc) on rats with collagen-induced arthritis (CIA) and analyze osteopontin (OPN) changes following therapy in order to understand its primary mechanism of action. Methods CIA was induced by bovine Ⅱ collagen (B Ⅱ C) injection. Rats were treated with rhTNFR:Fc from the 13th day after the first injection of B Ⅱ C till the 36th day. The anterior-posterior diameters of ankle joints and weight were measured weekly. The pathological score was evaluated by HE staining and toluidine blue staining. The blood plasma TNF-α and OPN levels were measured by ELISA and the histology expression was evaluated by immuno-histochemistry. Comparisons between groups were performed with one-way ANOVA. Results Quantitative analysis showed pathological score in the model group and treatment group was significantly reduced in joint pathology (8.2±1.0 vs 4.8±1.4, P<0.05). The mean plasma levels of TNF-α and OPN values were (713±146) pg/ml, (4.3±0.6) ng/ml respectively in the model group,but those of the treatment group were (68±20) pg/ml, (4.2±0.6) ng/ml. Serum TNF-α values were significantly different (P<0.05) between the two groups, while no significant difference was found in the value of plasma OPN (P=0.688) between the two groups. rhTNFR:Fc could reduce the cells OPN expression in the interface layer of the synovium and cartilage (P<0.05). Conclusion Pathology scores and ELISA results haveshown that rhTNFR:Fc has good therapeutic efficacy. It can significantly reduce the bone and cartilage damage of CIA mouse model, and can significantly reduce the expression of OPN in the sliding joints, thereby delay disease progression. However, it can not reduce the expression of OPN in the peripheral blood plasma.OPN may be involved in bone destruction and resorption rather than in inflammatory process.
9.Treatment of pentoxifyline on collagen-induced arthritis in rats
Lisha WANG ; Feng HUANG ; Yanyan WANG ; Zheng ZHAO
Chinese Journal of Rheumatology 2008;12(9):622-625,插一
Objective To evaluate the efficacy and safety of pentoxifyline (PTX) on the treatment of collagen-induced arthritis in rats. Methods Wistar rat arthritis model was induced by bovine Ⅱ collagen (BⅡC) . The rats were randomly divided into four groups including normal control group, CIA control group treated with normal saline, indometaein group and PTX group. The body weight, the hind paw volumes, the arthritic index of all rats at different time points were observed and measured. At the end of the experiment, the radiographic changes and the synovial pathology score of rat ankle joints were evaluated to analyze the treatment role of PTX on CIA inflammation. Results The collagen-induced arthritis model was induced successfully at 11~13 days after first immunization with type Ⅱ collagen. After administration, the rat weights of PTX group were higher than that of CIA group (P<0.01) and no significant difference was found between PTX group and normal control group since week three. The degree of swelling of ankle joints in PTX group was decreased since the 17th day. At week five, the volumes of hind paw in PTX group and indometacin group was not different from that in normal control group (P>0.05). Compared with CIA group, the degree of synovial swelling of PTX group and indometacin group was decreased, synovial proliferation and inflammatory cell infiltrations was mild, and vascular hyperplasia and pannus was significantly declined. No cartilage erosion and necrosis was found. The pathological score of PTX group and indometacin group was significantly decreased(P<0.01). The parenchyma of ankle joint was swollen and no osteoporosis and bone erosion was found in PTX group and indometacin group. Conclusion PTX can ameliorate the symptoms and inhibit the swollen of rat arthritis. It is effective and with good safety profile.
10.Chondrogenesis of rabbit mesenchymal stem cells by overexpressing Sox9 gene
Ziquan YANG ; Junren HE ; Gang LI ; Shuhua YANG ; Xiaochun WEI
Chinese Journal of Rheumatology 2008;12(7):-
Objective To observe the effect of Sox9 gene overexpressian on chondrogenesis of esenchymal stem ceils (MSCs) in vitro. Methods Rabbit MSCs were obtained and purified by gradient centrifuge and adhesion culture in vitro. MSCs were transfected by recombinant Sox9 plasmid. RT-PCR and Western blot analysis were used for transfection confirmation. Chondrogenic molecules such as type I1 collagen,aggrecan and Sox9 were detected by immunohistology, RT-PCR and western blot. Cell proliferation was tested by MTT assay. Results MSCs were successfully transfected with Sox9 gene and verified by RT-PCR and Western blot analysis. The overexpression of Sox9 had no effect on the proliferation of MSCs. Transfeeted cells expressed more chondrogenic markers than the control groups. Conclusion Sox9 gene overexpression can enhance chondrogenic differentiation of MSCs.