Abercrombie outlet,Matrix metalloproteinase- _

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Matrix metalloproteinase

Key words Intestinal neoplasms; matrix metalloproteinase; tumor necrosis factor receptor
Effects of matrix metalloproteinase9 on the expression of tumor necrosis factor receptor1 in human colon carcinoma cell lines
】【Abstract AIM: To study the effects of matrix metalloproteinase9 (MMP9) on the expression of tumor necrosis factor receptor1 (TNFR1) and migratory and invasive potentials in human colon carcinoma cells. METHODS: Immunofluorescence staining was applied to examine the expression of TNFR1 in SW1116 in human colon carcinoma cell lines. The expression of TNFR1 was assayed by flow cytometry in human colon carcinoma SW1116 cells treated with MMP9 at different concentrations and at different time periods of culture. RESULTS: TNFR1 was expressed in SW1116 cells and MMP9 downregulated the expression of TNFR1 on cell surface in a dose and timedependent manner. CONCLUSION: MMP9 decreases the expression of TNFR1 on the surface of human colon carcinoma cells, which may be a factor associated with invasive and metastasis potentials of colon carcinoma.
【Keywords】 intestinal neoplasms; matrix metalloproteinase; TNFR Abstract Objective: To study the matrix metalloproteinase 9 (MMP9) in colorectal cancer tumor necrosis factor receptor 1 (TNFR1) expression and the promotion of invasion and metastasis of colorectal possible ways. METHODS: The immunofluorescence studies in colorectal cancer cells SW1116 in TNFR1 expression; interfere with MMP9 SW1116 colon cancer cells in culture, flow cytometry using different doses and different role in MMP9 TNFR1 expression time. Results: ① SW1116 cells express TNFR1; ② MMP9 on the cell surface TNFR1 expression SW1116 down effect, but a dose and time dependent. CONCLUSIONS: MMP9 decreased TNFR1 surface expression of colorectal cancer may be related to invasion and metastasis of colorectal cancer closely related.
Key words intestinal neoplasms; matrix metalloproteinase; tumor necrosis factor receptor
0 Introduction
Tumor necrosis factor (tumor necrosis factor, TNF) is a major by the monocyte-macrophage cells to produce the polypeptide cytokines, endotoxin treatment because of its anti-tumor cells has been named as tumor necrosis factor. The biological activity of TNF is present in the cell surface through membrane receptors, namely tumor necrosis factor receptor 1 (tumor necrosis factor receptor 1, TNFR1) and tumor necrosis factor receptor 2 (tumor necrosis factor receptor 2, TNFR2)-mediated . vitro and in vivo experiments have confirmed that tumor necrosis factor receptor (tumor necrosis factor receptor, TNFR)-mediated anti-tumor effect of tumor necrosis factor, in vivo activity of a variety of immune factors (interferon, interleukin, etc.) that is regulated by activation of TNFR in order to achieve remove the primary tumor [1], which induce and stabilize the tumor cell membrane TNFR tumor on the body play a very important role. Other studies also confirmed the existence of colorectal cancer tissue matrix metalloproteinase (matrix metalloproteinase, MMPs) expression or activity of high enhancement. some of the activity of matrix metalloproteinase enzyme may function by promoting the formation of tumor cell membrane TNFR release high levels of soluble tumor necrosis factor receptor [2] (soluble tumor necrosis factor receptor, sTNFR), is to achieve immune escape of tumor cells important ways. In this study, the tumor cell membrane by detecting the expression of TNFR1 and TNFR1 MMP9 expression on tumor cell membrane, and to explore MMP9 TNFR1 to promote colorectal cancer by regulating the transfer of the possible mechanisms.
1 Materials and methods
1.1 Materials
Colorectal cancer cell line (SW1116) kept by the Laboratory of Gastroenterology, Nanfang Hospital; calf serum (Evergreen, Hangzhou Institute of Biological Products); RPMI1640 medium, blue, chain,[link widoczny dla zalogowanych], double anti-, 2.5 g / L trypsin EDTA ( biological agents in Guangzhou Jia Wei Corporation); mouse anti-human TNFR1 monoclonal antibody (Santa Cruz Company); FITC-conjugated goat anti-mouse secondary antibody (Wuhan Boster Company); fluorescein isothiocyanate (FITC) labeled mouse anti- human TNFR1 monoclonal antibody and recombinant human MMP9 was purchased from R & D companies; FITC labeled mouse IgG1 control antibody (Bio Co., Ltd. Beijing Ding States).
1.2 main instrument inverted phase contrast microscopy (Olympas company); flow cytometer (BECTON DICKINSON company).
1.3 Methods
1.3.1 Cell culture of colorectal cancer cell line SW1116 cells adherent growth. With RPMI1640 culture based on 25 cm2 flasks at 37 ℃, saturated humidity, 50 mL / L CO2 incubator, the medium containing 100 mL / L fetal calf serum,[link widoczny dla zalogowanych], penicillin and streptomycin each 100 U / mL. Department of pieces and dumped the culture medium with 2.5 g / L EDTA trypsin digestion and cells. cells were seeded in 25 cm2 flasks, passaged once every 3 d.
1.3.2 Cell climbing films and SW1116 cells using immunofluorescence 2.5 g / L trypsin digestion, containing 100 mL / L fetal calf serum in RPMI1640 cell culture medium will be transferred into a 5 × 104/mL the cell suspension seeded in 24-well plates, each well pre-placement 6 mm × 6 mm with a coverslip until the cells grew well when the termination of culture. 4 g / L paraformaldehyde 30 min after climbing film 0.1 mL / L PBS wash, with 10 mL / L Triton X100 treatment normal goat serum after 15 min at 37 ℃ for 30 min, thrown to the serum. dropwise 1:50 diluted monoclonal mouse anti-human TNFR1 antibody, 4 ℃ overnight, after dropping the wet box 1:100 dilution of FITC-conjugated goat anti-mouse secondary antibody, 37 ℃ incubation 1 h, washed with distilled water, vibration-free glycerol were mounted fluorescent buffer. negative control antibody was replaced with PBS were stained. TNFR1 positive cells under a fluorescence microscope green fluorescence.
1.3.3 experimental human SW1116 colon cancer intervention to 2 × 105/mL seeded in 24-well plates, containing 100 mL / L fetal calf serum RPMI1640 medium at 37 ℃, 50 mL / L CO2 incubator 12 h the cells adsorbed on the culture plate for serum-free medium cultured for 24 h, adding MMP9 intervention, grouped as follows: ① MMP9 0 ng / mL (A group); ② MMP9 154 ng / mL (B group); ③ MMP9 385 ng / mL (C group); ④ MMP9 770 ng / mL (D group); ⑤ MMP9 1155 ng / mL (E group), intervention 3 h; then MMP9 was 770 ng / mL concentrations,[link widoczny dla zalogowanych], respectively, the role of 0,[link widoczny dla zalogowanych],1.5, 3,6 and 9 h.
1.3.4 colorectal cancer by flow cytometry the expression of TNFR1 SW1116 cells by direct immunofluorescence labeling cells into single cell suspension and counting, 20 μL test tube to take a single cell suspension (containing 1 × 105 cells ) and 10 μL FITC labeled mouse anti-human TNFR1 monoclonal antibody, independent of the control tube to join the corresponding control mouse monoclonal antibody at 4 ℃ for a total of 10 μL sterile 45 min, containing 5 g / L BSA in isotonic PBS buffer wash cells two times after the supernatant and add 400 μL 4 g / L paraformaldehyde fixed at 4 ℃ 30 min, ie the machine testing. determination of each sample 10 000 cells, calculating the percentage of cells expressing TNFR1.
Statistical analysis: repeated 3 times independently of each experiment, the application SPSS 10.0 statistical software. Data with x ± s indicated by the single factor analysis of variance and Dunnettt pairwise comparison test, P
2 results
2.1 TNFR1 immunofluorescence staining was stronger in the SW1116 cell fluorescence, no expression of the control group (Figure 1). A: SW1116 cells; B: negative control. Figure 1TNFR1 in SW1116 cells
2.2 with different concentrations of MMP
9 after the role of TNFR1 expression by flow cytometry showed that of the effect of different concentrations of 3 h, and MMP9 0 ng / mL group (expression rate of 90.92%) compare, MMP9 770 ng / mL group (expressed rate of 69.96%), MMP9 1155 ng / mL group (expression rate of 65.06%) TNFR1 expression was significantly decreased (P 0.05); MMP9 154 ng / mL group and MMP9 385 ng / mL group was significantly higher than that of TNFR1 expression levels of MMP9 770 ng / mL and MMP9 1155 ng / mL group (P 0.05); MMP9 770 ng / mL group and MMP9 1155 ng / mL between the expression levels of TNFR1 similar (P> 0.05, Figure 2).
2.2MMP
9 different time after the expression of TNFR1 in the MMP9 770 ng / mL concentration, under different time, with the 0 h group (expression rate of 86.36%) compare, 3 h (expression rate of 67.68%), 6 h (expression rate of 18.08%), 9 h (expression rate of 14.38%) group were significantly lower TNFR1 (P 0.05); 3 h TNFR1 levels were significantly higher than that of group 6 h and 9 h groups (P 0.05, Figure 3). aP <0.05 vs A.
3 Discussion
TNF in vivo on the killing effect of tumor cells, TNF treatment of cancer that people had great expectations. But because it is in the treatment of cancer, but also on the body to produce very serious side effects, thus affecting the It is widely used in clinical. TNF is a biological effect of two different receptors TNFR1 (P55) and TNFR2 (P75) mediated. TNF many of the effects work through the TNFR1, TNFR2 plays a signal transduction is effect. Chen et al [3] reported, TNFR1 oligomerization through its death domain TRADD, FADD molecules, activation MACH / FLICE, caspase cascade effect starting the route of apoptosis. Thus TNFR1 was high in a variety of malignant tumors expression. This study shows that in the colorectal cancer cell surface of the high expression of TNFR1. According to [4] reported that TNFR1 in colorectal cancer was significantly higher than that of normal tissue paraneoplastic, with serosal invasion and lymph node metastasis negative correlation; Japanese scholars in 2003, Dukes C colorectal cancer reported in patients with high expression of TNFR1 disease-specific survival rate was significantly higher than those with low expression, multivariate analysis showed that TNFR1 expression is an independent prognostic predictor of colorectal cancer [ ],[and play an important role in the development process, and through different channels of the biological effects mediated by TNF. have found that blocking TNFR1 can induce cellular apoptosis inhibitory protein (FLIP) in regulation, thereby inhibiting apoptosis; while blocking TNFR2 turn can cause the reduction in FLIP promotes cell apoptosis [7] speculated that TNFR function, one as a carrier to internalize TNF,[link widoczny dla zalogowanych], the second is to activate specific intracellular signal transduction pathway. Some people think that TNF binding to its receptor, the receptor-mediated The proactive role within the TNF into the cell, causing cell lysis and cytotoxicity.

john2072

How to Look for your Perfect Fashion Dress Shoes

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* Style and Fashion
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* Frequency
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