第二節 研究目的
This study has examined the polymorphism of the gene IL-10, to determine whether it was a marker of susceptibility to SLE by focusing on single
nucleotide polymorphisms (SNPs). We compared allelic and genotypic
frequencies between 119 Chinese patients with SLE and 100 healthy individuals in Taiwan.
第二章 研究方法 第一節 研究材料
Subjects
One hundred nineteen patients with definite SLE according to the 1982 revised American College of Rheumatology criteria for SLE [10],and 100 unrelated, healthy individuals living in central Taiwan who served as control subjects were enrolled in this study. Informed consent was obtained from all patients involved. Clinical and serological data were available on these patients, including malar rash, Photosensitivity, antinuclear antibody (ANA), central nervous involvement, and renal disease (defined as proteinuria>1 g/day). The central nervous system involvement was evaluated by a neurologist.
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第二章 研究方法 第二節 研究設計
Methods
The genomic DNA was prepared from peripheral blood by use of a genomic DNA isolation reagent kit (Genomaker, Taiwan).
Division of IL-10 gene polymorphism
The Polymerase chain reaction (PCR) for IL-10 gene polymorphism was carried out to a total volume of 25 μl, containing genomic DNA (2-6 pmole of each primer); 1X Taq polymerase buffer (1.5 mM MgCl2); and 0.25 units of AmpliTaq DNA polymerase (Perkin Elmer, Foster City, CA, USA). The primer for the IL-10 promoter gene at position -627 was
5’-CCTAGGTCACAGTGACGTGG-3’ and
5’-GGTGAGCACTACCTGACTAGC-3. The polymerase chain reaction(PCR) amplification was performed in a programmable thermal cycler Gene Amp PCR System 2400 (Perkin- Elmer, Foster City, Calif., USA). The cycling conditions for IL-10 polymorphism were set as follows: one cycle at 96°C for 5 min, 35 cycles at 96° C for 30 sec, 35 cycles at 60°C for 30 sec, and 35 cycles at 72°C for 45 sec, and one final cycle of extension at 72°C for 7 min. The PCR product of 412-bp was mixed with two units Rsa I (New England Biolabs, Beverly, USA) and two fragments of 236-bp and 176-bp were present when the product was able to be digested (AA homozygote). The reaction was incubated for 3 hours at 37°C. Then, 10 µl of the product was loaded onto a 3% agarose gel containing
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ethidium bromide for electrophoresis. The polymorphism was divided into digestible (AA homozygote), indigestible (CC homozygote) and A/C heterozygote.
Carriage rate and allelic frequency
The carriage rate of an allele is the number of individuals carrying at least one copy of the allele relative to the total number of individuals. Allelic frequency was expressed as a percentage of the total number of alleles.
第二章 研究方法 第三節 統計方法
Results from the control subjects and the SLE patients were compared using the χ2 test (3 x 2 and 2 x 2 contingency tables) for statistical significance. When the assumption of the chi-squared test was violated and 1 cell had an expected count of <1, or > 20% of the cells had an expected count of <5, Fisher’s exact test was used. The distributions of the IL-10 gene polymorphisms in each group were evaluated. A Pvalue less than 0.05 was considered statistically
significant. The odds ratios (OR) were calculated from allelic frequency with a 95% confidence interval (95% CI) for the polymorphism of the IL-10 gene.
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第三章 研究結果
第一節 描述性統計分析
3-1-1 紅斑性狼瘡患者與健康人兩組基因型資料比較
The frequencies of the genotype in the SLE and control groups are shown in Table 1. It shows that among the 119 SLE patients, 66 patients (55.5%) had IL-10 genotype AA, 48 patients (40.3%) had A/C, and 5 patients (4.2%) had CC.
Among the 100 control volunteers, the IL-10 genotype AA was found in 40 (40.0%), A/C in 45 (45.0%), and CC in 15 (15.0%). There were significant differences in the distribution of the IL-10 gene polymorphism between the healthy control subjects and the SLE patients (p=0.007). In addition, there was a significant difference in the allelic frequency of the IL-10 between the SLE patients and healthy controls (p=0.003), giving an odds ratio of 1.210 for A allele (95% confidence interval 1.063-1.377). There was no significant
association in the carriage rates of the IL-10 (p=0.077), giving an odds ratio of 1.164 for A allele (95% confidence interval 0.981-1.382, respectively).
3-1-2 紅斑性狼瘡患者與健康人臨床症狀及實驗數據差異性
Clinical manifestations and laboratory findings of the SLE patients are shown in Table 2. The associations of IL-10 with particular clinical features of SLE were examined in the 119 Chinese patients. We did not detect any association of IL-10 genotype with the antinuclear antibody (ANA),malar rash,
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photosensitivity, discoid lupus, mucosal ulcer, arthritis, serositis, hematology, immunology, central nervous system involvement and renal disease involvement in the SLE patients (all p＞0.05).
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第三章 研究結果
第二節 推論性統計分析
3-2-1 紅斑性狼瘡影響腎臟病變和IL-10基因型的探討
Relationship between IL-10 genotype and renal involvement in patients with SLE are shown in Table 3 & 4.
3-2-2 狼瘡性腎病變的表現與IL-10基因多型性的關連性
In comparison with different genotype , there are no significant difference (all p＞0.05) in microhematuria, serum Cr and hypertension association, despite patients with CC-genotype had less proteinuria manifestation than AA- &
AC-genotype. In addition, only few patients accepted renal biopsy examination.
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第四章 討論
第一節 結果討論
4-1-1 紅斑性狼瘡的臨床表現受基因所控制
Systemic lupus erythematosus (SLE) is a prototype of autoimmune diseases that affects practically every organ in the body. There are strong epidemiological evidences that genes contribute to the risk of developing many common diseases.
However, the genetic background of patients with SLE is still mostly unknown.
To date, genetic researches of multi-factorial diseases have been studied with difficulty, due to the vast uncertainty surrounding the presence of a polygenic trait. In the present study, we chose the IL-10 gene polymorphism to examine whether IL-10 gene polymorphism was a marker of susceptibility to SLE in Chinese patients in Taiwan.
4-1-2 有關IL-10基因型的差異影響紅斑性狼瘡各系統疾病表現的文獻報 告
IL-10 is a 36 kDa homodimeric cytokine, which is mainly produced by
macrophage, monocytes and lymphocytes. The IL-10 gene maps to the junction of 1q31-q32 [11]. IL-10 production appears to be controlled at the
transcriptional level [12]. The IL-10 5’ flanking region, which controls
transcription, is polymorphic, with 2 microsatellites between -4000 and -1100 and three single base pair substitutions that have been described in the IL-10