ABSTRACT
Objective To investigate the effect of different concentrations of magnesium-calcium alloy extract on the expression of matrix metalloproteinase-9 (MMP9) and tissue inhibitor of metalloproteinase-3 (TIMP3) in human colonic epithelial NCM460 cells.Methods The different concentrations of extracts (the volume fraction was 10％,50％ and 100％ respectively) were made with magnesium-calcium alloy.The 5 × 106 L-1 NCM460 suspension was randomly divided into control group,experimental group 1,experimental group 2 and experimental group 3.The cells in the control group were cultured by 2 000 μL high glucose Dulbecco's modified Eagle's medium (containing 10％ volume fraction of fetal bovine serum).The cells in the experimental group 1,2 and 3 were cultured by 2 000 μL magnesium-calcium alloy extract with volume fraction of 10％,50％ and 100％ respectively.The expressions of MMP9 and TIMP3 mRNA in NCM460 cells was detected by real-time fluorescence quantitative polymerase chain reaction,and the expression of MMP9 and TIMP3 protein in NCM460 cells was detected by Western blot at after one,three and five days of cultivation respectively.Results The expression of MMP9 mRNA and TIMP3 mRNA in NCM460 cells of the experimental group 1,2 and 3 was significantly lower than that in the control group after one day of cultivation (P ＜ 0.05).After three and five days of cultivation,the expression of MMP9 mRNA in NCM460 cells of the experimental group 1 was significantly lower than that in the control group (P ＜ 0.05),but the expression of MMP9 mRNA in the NCM460 cells of the experimental group 2 and 3 was significantly higher than that in the control group and the experimental group 1 (P ＜ 0.05).The expression of MMP9 mRNA in NCM460 cells of the experimental group 3 was significantly higher than that in the experimental group 2 after five days of cultivation (P ＜ 0.05).The expression of MMP9 mRNA in NCM460 cells of the experimental group 1,2 and 3 after three and five days of cultivation was significantly higher than that after one day of cultivation(P ＜ 0.05).There was no significant difference in the expression of MMP9 mRNA in NCM460 cells of the experimental group 1 between three and five days of cultivation (P ＞ 0.05).The expression of MMP9 mRNA in NCM460 cells of the experimental group 2 and 3 after five days of cultivation was significantly higher than that after three days of cultivation(P ＜ 0.05).The expression of TIMP3 mRNA in NCM460 cells of the experimental group 2 and 3 was significantly higher than that in the experimental group 1 after one day of euhivation (P ＜ 0.05).After three days of cultivation,the expression of TIMP3 mRNA in NCM460 cells of the experimental group 1,2 and 3 was significantly lower than that in the control group (P ＜ 0.05);the expression of TIMP3 mRNA in NCM460 cells of the experimental group 2 was significantly lower than that in the experimental group 1 and 3 (P ＜ 0.05).After five days of cultivation,the expression of TIMP3 mRNA in NCM460 cells of the experimental group 1,2 and 3 was significantly higher than that in the control group (P ＜ 0.05).The expression of TIMP3 mRNA in NCM460 cells after three and five days of cultivation was significantly higher than that after one day of cultivation (P ＜ 0.05),and the expression of TIMP3 after five days of cultivation was significantly higher than that after three days of cultivation in the experimental group 1 (P ＜ 0.05).The expression of TIMP3 mRNA in NCM460 cells after three days of cultivation was significantly lower than that after one day of cultivation (P ＜ 0.05),and the expression of TIMP3 after five days of cultivation was significantly higher than that after one and three days of cultivation in the experimental group 2 (P ＜ 0.05).The expression of TIMP3 mRNA in NCM460 cells after five days of cultivation was significantly higher than that after one and three days of cultivation in the experimental group 3 (P ＜ 0.05).After five days of cultivation,there was no significant difference in the expression of MMP9 protein in NCM460 cells between the experimental group 1 and control group (P ＞ 0.05),the expression of MMP9 protein in NCM460 cells of the experimental group 2 and 3 was significantly higher than that in the control group and the experimental group 1 (P ＜ 0.05),but there was no significant difference in the expression of MMP9 protein in NCM460 cells between the experimental group 2 and 3 (P ＞ 0.05).After five days of cultivation,the expression of TIMP3 protein in NCM460 cells of the experimental group 1,2 and 3 was significantly higher than that in the control group (P ＜0.05);but there was no significant difference in the expression of TIMP3 protein in NCM460 cells among the experimental group 1,2and 3 (P ＞ 0.05).Conclusions The high concentration of magnesium-calcium alloy extract has certain influence on the expression of MMP9 and TIMP3 gene in NCM460 cells,which may lead to the early inflammatory reaction,and the mechanism may be related to the calcium ion concentration in the extract.
ABSTRACT
Objective To study the effect of intra-articular injection of dehydroepiandrosterone (DHEA) on the balance of ADAMTS/TIMP-3 system in a rabbit osteoarthritis models.Methods Sixty rabbits were underwent bilateral anterior cruciate ligament transection (ACLT).Rabbits were randomizedn to the following treatment:one knee of each rabbit was treated with 100 μmol/L DHEA dissolved in dimethylsulphoxide (the experimental group) and the other knee was treated under the same schedule using dimethylsulphoxide (the control group) 4 weeks after transection,once a week for eight weeks.Twelve weeks after ACLT,all rabbits were killed after X-ray assessment and the knee joints were evaluated by gross morphology and histology.The concentration of hydroxyproline and glycosaminoglycan in the cartilage were analyzed.The mRNA expression of ADAMTS-4,ADAMTS-5,tissue inhibitor of metalloproteinases-3 (TIMP-3),transforming growth factor (TGF)-β1.Aggrecan and Collagen Ⅱ in the cartilage were analyzed using reverse transcription polymerase chain reaction (RT-PCR).The protein expression of aggrecan ARGxx and Collagen Ⅱ in the cartilage were analyzed by Western blot.Results By Mann-Whitney test,Gross morphologic scores on femoral condyle and tibial plateau in the control group were significantly higher than the experi-mental group (Z=-3.517,P＜0.01 ; Z=-2.518,P＜0.05).By unpaired Student's t test,histological evaluation showed that the grade of cartilage damage in the experimental group [(5.3±1.2) μg/ml] were less severe than that in the control group (10.1 ± 1.3,P＜0.01).The concentration of hydroxyproline [(5.7±0.3,23.6± 1.7) μg/ml] and glycosaminoglycan (30±4) in the experimental group increased significantly when compared with the control group [(4.6±0.5),(18.5±1.4),(24±4) μg/ml,P＜0.01].The mRNA expression of ADAMTS-4 (0.15±0.03)and ADAMTS-5 (0.10±0.04) in the experimental group decreased significantly compared with the control group (0.29±0.08,0.15±0.05; all P＜0.05).The mRNA expression of TIMP-3 (0.85±0.10),TGF-β1(1.2±0.4),Aggrecan (0.87±0.31) and Collagen Ⅱ (2.74±0.59) in the experimental group increased significantly when compared with the control group (0.70±0.13,0.8±0.4,0.49±0.16,2.2±0.5; all P＜0.05).The protein expression of Aggrecan ARGxx (0.53±0.10) in the experimental group decreased significantly when compared with the control group (0.81±0.12,P＜0.01).The protein expression of Collagen Ⅱ (2.3±0.7) in the experimental group increased significantly when compared with the control group (1.7±0.5,P＜0.05).Conclusion DHEA protects against cartilage degradation and inhibits the progression of OA,TGF-β1,Aggrecan and Collagen Ⅱ in cartilage may be the mechanism of the protective effect of DHEA on OA.
ABSTRACT
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 study the expression of matrix metalloproteinase(MMP)-2,9 and tissue inhibitor of metalloproteinase(TIMP)-1,2 protein in human endometrial carcinoma tissue and its relation to the invasion and metastasis of endometrial carcinoma. Methods Immunocytochemistry and zymography techniques were used to measure the MMP-2,MMP-9,TIMP-1,TIMP-2 protein levels and activities in endometrial carcinoma tissue of 37 patients and control group composed of 7 normal postmenstrual endometrial samples. Results The MMP-2,MMP-9,TIMP-1 and TIMP-2 proteins mainly expressed in endometrial carcinoma cells, glandular cells and endothelial cells. The strongly positive expression proportions of MMP-2,9 and TIMP-1 proteins in grade Ⅲ carcinoma cells were respectively 73%, 20% and 67%, which were higher than those in gradeⅡ (13%, 0, 27%) and gradeⅠ (0) ones ( P