Ⅱ
Ⅱ Ⅱ. MATRIALS AND METHODS
1. Materials
Human bone marrow-derived mesenchymal stem cells (hBM-MSCs; FCB-Pharmicell co.Ltd, Sungnam, korea) were culture-expanded from the bone marrow aspirate from the persons who agree to use his cells for research purpose. We obtained written informed consent from all persons who provide their BM aspirates according to the guideline of institutional Review board(AJIRB-04015) and Korea Food and Drug administration (KFDA-CEPT Approval No.37). And human neuroblastoma cell line(#22266, Korea cell line Bank, Seoul) were used.
For this experiments, High glucose and Low glucose Dulbecco's modified Eagle's medium (DMEM;Gibco-BLR,Grand Island, NY), trypsin-EDTA (Gibco BLR, NY), fetal bovine serum (FBS; Gibco BLR, NY), penicillin/streptomycin(Gibco BLR, NY), Cell culture dish (Corning), and Staurosporine (STS; Sigma, St Louis, MO) were used.
The cell titer 96 [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-ulfophenyl)-2H-tetrazolium, inner salt (MTS), Fluorescein isothiocyanate (FITC)-labeled annexin V (BD Bioscience), Protein assay (Bio-rad), rotease inhibitor cocktail(Sigma), and nitrocellulose membrane(bio-rad) were also used for evaluation procedures,
For Western blot analysis, antibody-Bax (1:1000, Stressgen), antibody -Bcl-2 (1:1000,
- ６ -
Stressgen) were used. HRP-goat anti-mouse IgG(H+L) conjugate (zymed) antibody (1:2000; Amersham) were also used.
2. Isolation of hBM-MSCs and Culture maintenance
We obtained written informed consent from all the persons who agree to use his cells for research purpose. Their BM aspirates were prepared and mononuclear cells were isolated by Ficoll density centrifugation. Mononuclear cells were placed in a 10 cm dishes and were cultivated in low-glucose DMEM containing 10% FBS and 1%
penicillin/streptomycin in a humidified incubator at 37℃ under 5% CO2. Medium containing non-adherent cells were then replaced every 4days of culture. When dish was reached 70-80% confluence, the cells were trypsinized and subcultured. Culture-expanded hBM-MSCs were provided for these trials. Passage 6 hBM-MSCs were used in all experiments [14, 15].
3. Culture of human SH-SY5Y neuroblastoma cell culture and induction of apoptotic cell death with staurosporine treatment
SH-SY5Y cells were cultured in 6-well plate and maintained in high glucose DMEM containing 10% FBS and 1% penicillin/streptomycin in a humidified incubator at 37℃ under 5% CO2 [11]. SH-SY5Ys were plated in a number of 3x104 cells in 24-well plate for these trials. After three days of culture, several different concentration of STS ranging from 0.25 to 2µM were treated for different incubation period of 3, 6, 12 and 24 hours to induce apoptotic cell death. For further trials, SH-SY5Y cells were
- ７ - preconditioned with 0.25 µM STS for 24 hours.
4. Experimental design to effect of hBM-MSCs against apoptosis
4.1 part 1. Co-culture of STS treated SH-SY5Ys with media of hBM-MSCs
For these trials, SH-SY5Ys were plated to density of 3x104 cells. After 3days, SH-SY5Ys were treated with 0.25µM STS for 24hrs to induce apoptotic cell death.
Subsequently, medium of STS-treated SH-SY5Ys was replaced with 1) fresh DMEM containing 10% FBS, 2) 100% of hBM-MSCs media, and 3) media composed of fresh media and hBM-MSCs culture media (1:1 fresh media hBM-MSCs media ratio). These culture was maintained for 5 days and was repeated in triplicate.
4.2 part 2. Co-culture of STS treated SH-SY5Ys with transwell hMSCs inserts After three day culture of SH-SY5Ys in 24-well plate, those were treated with 0.25µM
STS for 24hrs to induce cell death. Cultured hBM-MSCs, and SH-SY5Ys were maintained in each transwell insert for 24 hrs. Each transwell insert with Cultured hBM-MSCs, or SH-SY5Ys, subsequently, was dipped in the basal plate of STS-treated SH-SY5Ys, to make share the same culture environment without direct contacts between hBM-MSCs and STS-treated SH-SY5Ys.
5. Cell viability assay
After addition of culture media of MSCs and trans-well application of hBM-MSCs, the viability of co-cultured SH-SY5Ys was assessed at day 1, 2, 3, and 5 over
- ８ -
time. The viability of SH-SY5Ys was assessed by two different methods including trypan blue dye exclusion method and measurement of metabolic activity of the SY5Y cells using MTS assay. The metabolic activity was measured by cell titer 96[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxylmethoxyphenyl)-2-(4-ulfophenyl)-2H-tetrazolium, innersalt(MTS)]. The harvested cells at the different time points were incubated in serum-free medium containing MTS solution (20 v/v) at 37℃ for 1hrs. Following incubation, MTS were quantified at 490nm by ELISA plate reader (Bio-Tek instruments.
Winooski, VT). The viability of the harvested cells, determined as the ratio between dead (blue) and viable (unstained) cells was also determined by the trypan blue dye exclusion method. Viable and nonviable (blue cells) cells were counted on a hematocytometer.
6. Morphological analysis
To observe morphological changes overtime, cultured SH-SY5Ys at day 1, 2, 3, and 5 were fixed in 4% paraformaldehyde in 0.2M phosphate buffer for 10 min at room temperature. After fixation of cells, they were washed with PBS three times. SH-SY5Ys morphology was visualized by phase contrast microscopic optics (Nicon, Tokyo, Japan) fitted with a model D70s (Nicon, Tokyo, Japan).
7. Flow cytometric measurement of cell death using Annexin-V/PI
Cultured and STS treated SH-SY5Ys with or without hBM-MSCs were harvested by trypsinization and pelleted by centrifugation at 1500rpm for 5min. Cell pellets were
- ９ -
washed once in ice cold PBS, followed by gentle re-suspension in 100µl of annexin-V
binding buffer containing 5µl of FITC-labeled annexin V and 5µl propidium iodide (PI;
100g/ml stock in PBS) solution for 15min. And then add 400µl 1x binding buffer.
Samples were immediately kept on ice and analyzed on a FACScan at 530nm(FL-1) for FITC, and red PI at above 600nm (FL-2). FL-1and FL-2 were collected on log-scale with voltages of 450 and 458, respectively. Signal overlap was compensated for electronically. Data was acquired and analyzed using Winmdi software. Acquisition gates were wet using the forward and side light scatter of the cells and a minimum of 1,000 events were collected for each sample (Martin, Reutelingsperger et al. 1995;
Azuhata, Scott et al. 2006; Zafar, Inayat-Hussain et al. 2006)
8. Caspase -3 activity assay
The caspase-3 activity was measured by caspase-3 activity assay kit(Chemicon) Caspase-3 activity was determined by monitoring proteolysis of corresponding colorimetric substrates. Cells were collected and washed in ice-cold PBS pH 7.0. Cells were subsequently lysed in 1x lysis buffer for 10min in ice and the lysates were clarified by centrifugation at 13000rpm. After centrifugation for 10mins, cytosolic extracts of SH-SY5Ys were transferred to a fresh tube and putted on ice. Then 30µg of the caspase-3-specific colorimetric substrate acetyl-Asp-Glu-Val-Asp-7-p-nitroaniline (Ac-DEVD-pNA) was added in cytosolic extracts. They were incubated for 1hrs at 37℃. The release of DEVD-pNA were quantified at 405nm by ELISA plate reader.(Lopez and Ferrer 2000)
- １０ - 9. Immunobloting
SH-SY5Ys were lysed in phosphate-buffered saline containing 1%, triton X-100, 1%
DOC, 0.1% SDS and 1% protease inhibitor cocktail. The lysed cells were centrifuged at 13000rpm to remove cellular debris. Protein concentration of the extracts was determined by the Bradford assay. Equal amounts of protein were separated by SDS polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and blocked with 5% skim milk in TBST for 2hrs. The membranes were incubated with Bax(Stressgen, 1:1000), Bcl-2(Stressgen, 1:1000) which were diluted with 5% skim milk in TBST at 4℃ with gentle shaking, overnight. After washing, the membrane was incubated with a HRP-goat anti-mouse IgG (H+L) conjugate antibody(zymed) for 1hr.
After several washes, the blots were developed. (Lombet, Zujovic et al. 2001; Azuhata, Scott et al. 2006)
10. Statistical analysis
Statistical analyses were performed using a t-test on SigmaStat software (SigmaStat version 2.03 for Windows). A p value<0.05 was considered statistically significant.
- １１ -