Objective To observe the expressions of extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase 9 (MMP-9) around the prosthesis, and to study the relationship between the expressions of EMMPRIN and MMP-9 and osteolysis around prosthesis. Methods Interface tissues were obtained at three Delee-Charnley acetabular sections and seven Gruen femur sections from 8 cases (8 hips) undergoing revision after total hip arthroplasty between February 2010 and January 2012, and were divided into osteolysis group and non-osteolysis group based on preoperative X-ray film and intraoperative observation; the tissues from another 8 patients with osteoarthritis undergoing total hip arthroplasty as the control group. The immunohistochemical staining and RT-PCR assays were used to determine the expressions of EMMPRIN and MMP- 9. The correlation between the positive cells and the severity of osteolysis were analyzed and compared. Results Histological examination showed that many macrophages, multinucleated giant cells assembled in the membrane of osteolysis zone, but many fibroblasts and synovial cells in non-osteolysis zones. EMMPRIN and MMP- 9 positive cells and gene expressions were observed in every group. The percentage of positive cells and gene expression of EMMPRIN and MMP-9 in osteolysis group were significantly higher than those in non-osteolysis and control groups (P lt; 0.05), but no significant difference was found between non-osteolysis group and control group (P gt; 0.05). The percentage of positive cells of EMMPRIN in zone III of acetabular was higher than that in zone I and zone II of revision hip (P lt; 0.05), but no significant difference between zone I and zone II (P gt; 0.05). The percentage of positive cells of MMP-9 in zone I and zone III was significantly higher than that in zone II of revision hip (P lt; 0.05), but no significant difference between zone I and zone III (P gt; 0.05). The expression of EMMPRIN from high to low in order was zones 1, 7, 4, 2, 3, 5, and 6 at femur; the values of zones 1, 7, and 4 were significantly higher than those of zones 2, 3, 5, and 6 (P lt; 0.05), but no significant difference among zones 1, 7, and 4, and among zones 2, 3, 5, and 6 (P gt; 0.05). The expression of MMP-9 from high to low in order was zones 1, 7, 4, 2, 3, 6, and 5 at femur; the values of zones 1 and 7 were significantly higher than those of zones 4, 2, 3, 6, and 5 (P lt; 0.05), and the values of zones 4 and 2 were significantly higher than those of zones 3, 6, and 5 (P lt; 0.05), but no significant difference between zone 1 and zone 7, between zone 4 and zone 2, and among zones 3, 5, and 6 (P gt; 0.05). Conclusion The expressions of EMMPRIN and MMP-9 have certain coherence. The over-expressions of EMMPRIN and MMP-9 may be one of the key points of inhibiting bone reconstruction and bone resorption at bone-implant interface under the stimulation of wear debris．
Objective It is reported that transforming growth factor β1 (TGF-β1) has the protective effects on the articular cartilage in osteoarthritis (OA). To investigate the significance of the expressions of matrix metalloproteinase 9 (MMP-9), TGF-β1 mRNA and corresponding proteins in OA. Methods The specimens of articular cartilage and synovium were collected from voluntary donators, including 60 cases of OA (experimental group) and 20 cases of traumatic amputation,cruciate l igament rupture, discoid cartilage injury, and menisci injury (normal control group). The pathological changes were observed by HE staining. MMP-9 and TGF-β1 protein expressions were detected by immunohistochemical technique, and the mRNA expressions of MMP-9 and TGF-β1 were detected through in situ hybridization technique; and their correlation was analysed. Results HE staining showed: shrinkage, necrosis, and irregular arrange of the articular chondrocytes, extracellular matrix fracture, hypertrophy and hyperplasia synovium, infiltration of lymphoid and mononuclear cells and prol iferation of many small blood vessels in the experimental group; regular arrangement of the articular chondrocytes, the homogeneously staining matrix, and synovial tissue without chronic inflammation and significant prol iferation in the normal control group. The mRNA and protein expressions of MMP-9 and TGF-β1 were positive in 2 groups. The positive-stained cells included chondrocytes, synovial l ining cells, and vascular endothel ial cells, fibroblasts, and inflammatory infiltrated cells in subsynovial layer. The expressions of mRNA and corresponding protein of MMP-9 and TGF-β1 in the experimental group were significantly higher than those in the normal control group (P lt; 0.01). There was a positive correlation between MMP-9 mRNA and protein expression (r=0.924, P=0.000), and between TGF-β1 mRNA and protein expression (r=0.941, P=0.000) in the experimental group. There was a negative correlation between the expression of MMP-9 protein and TGF-β1 protein (r= — 0.762, P=0.000), and between the expression of MMP-9 mRNA and TGF-β1 mRNA (r= — 0.681, P=0.000) in the experimental group. Conclusion The higher expression of TGF-β1 can protect articular cartilage by down-regulating the expression of MMP-9 of chondrocytes and synoviocytes in OA, which may delay the biological behavior of OA such as occurrence and progress, etc.
Objective To investigate the effect of doxycycline on the proliferation and vasculogenic mimicry in retinoblastoma (RB) cell line in vitro. Methods RB cell line were tested for their ability to form perfusable tubular networks in 3D culture with doxycycline in the concentrations ranging from 5 to 20 mg/L, and CoCl2 was used as chemical hypoxia-inducing reagent to mimic tumor hypoxic microenvironment. The effect of doxycycline on proliferation were detected by MTT assay in vitro, and the effect on tube formation of RB cells were detected by tube-like structure formation assay and PAS staining. The mRNA levels of MMP2 and MMP9 at different hypoxic culture and different doxycycline concentrations were detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Results The micrograph showed that RB cells linked each other to form cavity and network tructure in 3D culture. the number of tubules in doxycycline group were significantly lower than which in the control group in the concentrations ranging from 5 to 20 mg/L (Plt;0.001).OD of doxycycline group was significantly lower than which in the control group (t=15.320，Plt;0.01) , The proliferation of RB cells had a negative correlation with the concentration of doxycycline (r =-0924, Plt;001). The levels of MMP2 and MMP9 mRNA of RB cells under hypoxia were significantly higher than which in the control group (t=16.469，Plt;0.01). As the concentration of doxycycline increased, the expression of MMP-2 and MMP-9 decreased. The result of double staining also showed that VM, formed by CD34negative and PASpositive tumor cells, existed in 12 simples of retinoblastoma. Conclusion RB cells have the capacity of selfmetamorphosing and vasculorizing in 3D culture. Doxycycline can inhibit their proliferation and vasculogenic mimicry formation in vitro by downregulating the expression of MMP-2 and MMP-9 .
ObjectiveTo explore the value of matrix metalloproteinase 9 (MMP-9) in predicting the occurrence of heterotopic ossification by observing the expression of MMP-9 in heterotopic ossification of the early trauma rat model. MethodsA total of 132 male Sprague Dawley rats, aged 4-5 weeks, weighing (135.0±6.5) g, were randomly divided into experimental group and control group (n=66). In experimental group, the Achilles tendon was cut off and clamped to prepare heterotopic ossification model; in control group, only Achilles tendon was exposed by making a incision. The general condition of the rats was observed after operation; at 2, 3, 4, 5, 6, 7, and 8 days after operation, the Achilles tendon tissue was harvested for gross observation, histological observation, and immunohistochemical staining observation; the serum and Achilles tendon tissue were harvested to detect the expressions of MMP-9 protein and mRNA by ELISA and RT-PCR. The X-ray films at 5 and 10 weeks and histological examination at 10 weeks after operation were used to observe heterotopic ossification. ResultsAll rats survived to the end of the experiment. The Achilles tendon had no significant change in control group at each time point, showing normal tendon structure. In experimental group, the hardness of Achilles tendon tissue gradually increased with the time; there were a large number of irregular connective tissue and cartilage cells; and immunohistochemical staining for MMP-9 was positive results. The MMP-9 protein and mRNA expression levels of experimental group were significantly higher than those of the control group at each time point (P < 0.05). MMP-9 protein and mRNA expression levels of experimental group showed an increasing tendency (P < 0.05). According to the results of X-ray films and histological observation, heterotopic ossification occurred at 10 weeks after operation in experimental group, but no heterotopic ossification was observed in control group. ConclusionIn early heterotopic ossification of rat Achilles tendon, the expression of MMP-9 increases significantly, indicating that it has reference significance in predicting heterotopic ossification.
ObjectiveTo study how CD73 is shed from the retinal pigment epithelium (RPE) surface.MethodsCD73 shedding was induced by treating RPE with lipopolysaccharides (LPS) and TNF-α. After Phospholipase C (PLC) or pan matrix metalloproteinase (MMP) inhibitors were added, surface amount of CD73 was evaluated by flow cytometry (FACS). Then selective inhibitors or their corresponding siRNAs of MMP-2 and MMP-9 were applied to the treatments of RPE; and their effects on induced CD73 shedding were evaluated by FACS. By site directed mutagenesis, mutations were introduced to Lys547-Phe548 coding sites of CD73 cDNA, which was cloned in a pcDNA mammalian expression vector. Both wt-CD73 and mutated-CD73 were over expressed in CD73-/- RPE and their induced shedding was compared.ResultsLPS and TNF-α induced CD73 shedding from RPE was completely blocked by the addition of pan MMP inhibitor but not PLC inhibitor. Selective MPP-9, but not MMP-2, inhibitor or its siRNA blocked CD73 shedding. In CD73-/- RPE induced CD73 shedding was happened to overexpressed wt-CD73 but not Lys547-Phe548 sites mutant CD73.ConclusionMMP-9 is responsible for shedding CD73 from RPE through hydrolyzing its Lys547 -Phe548 sites.
ObjectiveTo preliminarily investigate the mechanism of MMP-9 blocking CD73 detachment from RPE cells surface and preventing and treating experimental autoimmune pigment membranitis (EAU).MethodsRPE cells isolated from wild-type C57BL/6 and CD73 gene knockout (CD73-/-) mice were cultured in vitro, and treated with lipopolysaccharide and TNF-α to induce CD73 detachment from RPE surface. According to whether MMP-9 inhibitor CTK8G1150 was added at the same time (the final concentration was 5.0 mol/L) or not, RPE cells cultured in the two types of mice were respectively set as MMP-9 inhibitor intervention group and non-intervention control group. The cells in each group were treated with the intervention of a solvent, 1 μmol/L adenosine monophosphate (AMP), 1 μmol/L AMP, and 3 μmol/L 5' -α,β-methylene adenosine diphosphate (APCP) (AMP+APCP). The stimulating effect of RPE cells in different groups on CD4+ T cell proliferation was detected by tritiated thymidine incorporation. Adoptive immune induced EAU in wild-type B6 mice and CD73-/- mice, respectively. The receptor mice were randomly divided into the MMP-9 inhibitor intervention group and the non-intervention control group, and CTK8G1150 or the solvent were injected into the subretinal cavity 4, 7 and 10 days after adoptive immunity. CD73 mRNA and protein expression in RPE cells of recipient mice were detected by real-time quantitative PCR (RT-PCR) and Western blot. One-way ANOVA was used to analyze all experimental data.ResultsWhen the stimulation mode was AMP, the proliferation of CD4+ T cells in the C57BL/6 MMP-9 inhibitor intervention group decreased significantly compared with the non-intervention group (F=13.28, P＜0.01). When the stimulation mode was solvent and AMP+APCP, there was no statistically significant difference in the proliferation capacity of CD4+ T cells between the two groups (F=7.78, 6.58; P＞0.05). There was no statistically significant difference in the proliferation capacity of CD4+ T cells between the CD73-/- MMP-9 inhibitor intervention group and the non-intervention group (F=5.24, 6.12, 7.04; P＞0.05). RT-PCR results showed that there was no statistically significant difference in the relative expression of CD73 mRNA in RPE cells between the MMP-9 inhibitor group and the non-intervention control group (F=6.54, P>0.05). Western blot results showed that the expression of CD73 protein in RPE cells in the MMP-9 inhibitor group of B6 receptor mice was significantly increased compared with the control group (F=15.24, P＜0.01).ConclusionMMP-9 inhibitor blocks CD73 detachment from RPE cells surface and has a protective effect on EAU.