20
III. Materials and Methods
3-1. Patients and sample collection
A total of 95 children (age range: 4.23 ± 2.51 years old) with perimembranous VSD were recruited from the outpatient base of the Taipei and Hsinchu Mackay Memorial Hospitals.
There were 120 healthy individuals (age range: 5.53 ± 2.87 years old) were enrolled for gene polymorphism analysis. Age and gender distribution had no statistical difference between patient and control group. The study was performed with the approval of the Institutional Review Board of Mackay Memorial Hospital. Informed consent was obtained from the parents of all patients. The defects of all patients enrolled in this study were documented by 2-D echocardiography using a Phillips HP 4500 echocardiography machine with 5-8-MHz probe. Each patient received twice echocardiographys by two pediatric cardiologists for preventing interobserver variability and mean values of VSD sizes and aortic root diameters of patients were extracted. All the patients with defects enrolled in this study were
documented by 2-D echocardiography. The plasma of 12 healthy children donated blood samples as a control group in the MMPs activity assays.
Blood samples were collected by needle aspiration from the antecubital vein. Plasma was isolated by centrifuge 3,000 × g for 10 min, and stored at −80°C for further zymography assay.
3-2. Patient grouping by VSD diameter/diameter of aortic root (Ao) ratio
Clinically, there is a wide range of parameters of a child’s heart that depend on the
patient’s body size and age. Therefore, the VSD size can be normalized by the aortic root (Ao) diameter and the VSD/Ao ratio usually correlated with patients’ clinical severities better than
21
VSD sizes did (Eroglu et al., 2003; Hornberger et al., 1989). Usually, VSD can be classified into three defect sizes: large, median and small sizes. The large defect is defined when the size is larger than two-thirds of the Ao; the median size is between two-thirds and one-third of the Ao; and small if the size is less than one-third of the Ao.
In our experimental design, we followed up the VSD patients regularly but very large VSD patients with poor medical control mandatorily undergo surgery. Therefore, patients in our study were exclusively those without extremely large defects. According to the definition of VSD/ Ao ratio, the patients were classified into three groups: VSD/Ao ratio ≦ 0.2 as Trivial group; 0.2 < VSD/Ao ≦ 0.3 as Small group; and 0.3 < VSD/Ao was considered as Median group.
3-3. Genomic DNA extraction
Genomic DNA for PCR-RFLP genotyping was isolated from the blood of 95 children by using the genomic DNA Mini kit. Up to 200 μL of whole blood was added with 30 μL of proteinase K (10 mg/mL) and mix briefly. After that, the mixture was incubated at 60°C for 15 min. Following then, 200 μL GB buffer was added and mixed by shaking vigorously, and incubated the mixture in a 60°C water bath for 15 min. A volume of 200 μL absolute ethanol was added to the sample lysate and immediately mixed by shaking vigorously. And then, the entire mixture (including any precipitate) was transferred to the GD Column and followed with centrifugation (at 14,000 × g for 5 min). The collections were added with 400 μL of W1 Buffer to the GD Column and centrifuged at 14,000 × g for 1 min. Then added 600 μL of Wash Buffer (ethanol added) to the GD Column and centrifuged at 14,000 × g for 1 min. The supernatant was displaced into new collection tube and centrifuged again at 14,000 × g for 3 min to dry the column matrix. Finally, 30 μL of pre-heated elution buffer (10 mM Tris-HCl,
22
pH 8.5) was added to the matrix and last for 5 min; after that, centrifuged at 14,000 × g for 1 min to elute the purified DNA. The maximum yield was up to 50 μg. Purified genomic DNA was stored in the −80°C.
3-4. Genotyping
3-4-1. MMP-2 -735C>T polymorphism
To analyze the -735C>T polymorphism, we amplified a region of the MMP-2 gene with the primers pair were shown in Table 3-1. The target sequence was amplified in a 50 µL reaction volume containing 1 μL of genomic DNA, 1 µL each primer (10 µM), 5 µL of 10X PCR buffer, 4 µL of 2.5 mM dNTPs, 0.5 µL of Taq Polymerase (Promega, Madison, WI, USA), and 37.5 µL distilled water. The PCR started with an initial activation step of 94°C for 2 min followed by 40 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec, and a final step at 72°C for 7 min. The 297-bp fragment was purified with a purification kit (Invitrogen, Grand Island, NY, USA). Purified PCR products were then digested with Sau 96 I (New England BioLabs, Ipswich, MA, USA) at 37°C overnight and separated on a 2% agarose gel. Sau96 I does not digest the T allele but generates 202-bp and 95-bp fragments for the C allele.
3-4-2. MMP-9 -1562C>T polymorphism
The primers used to detect this polymorphism were shown in Table 3-1, which generated a 435-bp fragment containing the -1562C>T site in a 50 µL reaction volume. The annealing temperature was also shown in this table. The PCR product was then digested with Sph I (New England BioLabs) at 37°C overnight and separated on a 2% agarose gel. Sph I does not digest the C allele but generates 247-bp and 188-bp fragments for the T allele.
23
3-4-3. MMP-9 R279Q polymorphism
The primers used to detect this polymorphism were sense 5’-GGT GGG TGC TTC CTT TAA CA-3’ and antisense 5’ -GCG TTA GAG ACG TTG GAA CC-3’ (Ikeda et al., 2008), which generated a 467-bp fragment containing the R279Q site in a 50 µL reaction volume, and the annealing temperature was shown in Table 3-1. The PCR product was then digested with Sma I (New England BioLabs) at 25°C overnight and separated on a 2% agarose gel.
Sma I does not digest the A allele but generates 296-bp and 171-bp fragments for the G allele.
3-4-4. MMP-9 R574P polymorphism
The primers used to detect this polymorphism were shown in Table 3-1, which
generated a 246-bp fragment containing the R574P site in a 50 µL reaction volume, and the annealing temperature was also shown in this table. The PCR product was then digested with Nla IV (New England BioLabs) at 25°C overnight and separated on a 2% agarose gel. Three
genotypes could be distinguished: GG (246-bp), GC (246, 182 and 65-bp), and CC (182 and 65-bp).
3-5. Gelatin zymography assay
The activities of MMP-2 and MMP-9 in the plasma were measured by gelatin gel zymography as previously had described (Chen et al., 2008). Plasma was mixed with 2 X zymography sample buffer [0.125 M Tris-HCl, pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS, 0.005% bromophenol blue] incubated for 10 min at room temperature, and then loaded into SDS-PAGE which was performed in 7% acrylamide gels containing 0.1% (w/v) gelatin (Sigma-Aldrich, St. Louis, MO, USA). After electrophoresis under power supply of 100 V,
24
the gel was washed twice for 30 min in zymography renaturing buffer (2.5% Triton X-100) with gentle shake at room temperature to remove SDS, then incubated 18 h at 37°C in reaction buffer (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, and 5 mM CaCl2). The gels were then stained with coomassie blue for 30 min prior to destain with destain buffer (50%
methanol, 10% acetic acid, and 40% ddH2O). The presence of enzyme activity was evident by clear or unstained zones, indicating the action of the enzyme on the gelatin substrate
(Stawowy et al., 2004). Gelatinase activity in the gel slab was quantified by Scion Image software (NIH, Bethesda, MD, USA), which quantifies the area of bands hydrolyzed by gelatinase. A MMP-2 or MMP-9 positive control (Chemicon, Temecula, CA, USA) was contained in each gel as a standard intensity value to normalize intensity of samples.
3-6. Determination of the TIMP-3 and BNP concentrations by enzyme-linked immunosorbent assay (ELISA)
TIMP-3 concentrations were measured by using an ELISA kit (cat# MTM100 R&D Systems, Minneapolis, MN, USA). According to the manufacturer’s instructions, 100 μg of protein per sample was used. The plasma BNP concentration was determined by a
commercially available immunoassay kit (Shionoria BNP assay kit; Shionogi Ltd, Osaka, Japan). Results obtained via spectrophotometry were compared against serial dilutions of known concentrations of the respective standards.
3-7. Statistical analysis
The levels of various markers in the VSD samples are expressed as the mean ± standard deviation (SD) or shown as the median (interquartile range; IQR). Data were analyzed by using SPSS for Windows (SPSS statistics package, Chicago, IL). All polymorphisms were
25
tested separately for confirmation with Hardy-Weinberg expectations in both groups.
Differences between VSD patients and healthy control children were examined with the χ2 test for categorical variables. To compare the quantitative MMP activities between two groups, Student’s t-test was used for presumably normally distributed variables. One-way analysis of variance (ANOVA) method was used to test the differences in the MMPs enzyme activities among groups when the group numbers were more than two. A p value less than 0.05 was considered significant.
26
Table 3-1. Sequences of the primers used for MMP-2/-9 genotyping by PCR-RFLP
SNPs Primers sequence (5→3)
PCR
27