| 摘要: |
| 目的 建立位点特异性的实时荧光定量PCR技术检测HLA-I类分子（HLA-A、-B和-C）mRNA表达量，获取浙江汉族人群HLA-I类分子（HLA-A、-B和-C）常见等位基因表达谱。方法 收集浙江省血小板基因数据库已知HLA等位基因型的纯合子样本包括HLA-A纯合子样本88例，HLA-B纯合子样本45例，HLA-C纯合子样本38例，应用实时荧光定量PCR技术分别检测样本的目的基因（HLA-A、-B和-C）和内参基因（β2M）mRNA表达水平，获得不同样本的Ct值，通过内参基因对HLA-A、-B和-C mRNA表达量进行均一化分析，比较不同等位基因型的mRNA表达水平。结果 88例HLA-A纯合子样本包含8种等位基因型，HLA-A mRNA表达量范围为20.26-35.39之间，呈现差异性表达，HLA-A*02:01和HLA-A*02:07两组mRNA表达水平明显高于HLA-A*11:01组（p<0.01）；45例HLA-B纯合子样本包含14种等位基因型，HLA-B mRNA表达量范围为7.7-9.21之间，呈现均衡性表达，HLA-B不同等位基因型 mRNA表达水平无明显差别；38例HLA-C纯合子样本包含9种等位基因型，HLA-C mRNA表达量范围为23.1-46.41之间，呈现差异性表达，HLA-C*03:02组mRNA表达量明显低于HLA-C*01:02组、HLA-C*06:02组和HLA-C*08:01:01G组(p<0.05)。 结论 本研究提供了一种检测HLA-I类分子（HLA-A、-B和-C）mRNA表达量的方法，HLA-A和HLA-C mRNA呈现差异性表达与等位基因型相关，HLA-B mRNA表达呈现均衡性表达与等位基因型无关。 |
| 关键词: 实时荧光定量PCR技术 HLA-I类分子 等位基因 mRNA表达水平 |
| DOI： |
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| 基金项目:国家自然科学基金项目（面上项目，重点项目，重大项目）；浙江省医药卫生科技计划 |
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| Locus-specific real-time fluorescent quantitative PCR was used to detect the mRNA expression of class HLA-I molecules |
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heyanmin, zhufaming
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Blood center of Zhejiang Province
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| Abstract: |
| Objective: A locus-specific real-time fluorescent quantitative PCR technique was established to detect the mRNA expression levels of class HLA-A, -B and -C, and to obtain the expression profiles of common alleles of class HLA-A, -B and -C in Zhejiang Han population. Methods: Homozygous specimen of known alleles were collected from the platelet gene database of Zhejiang Province, including 88 HLA-A homozygous specimens, 45 HLA-B homozygous specimens and 38 HLA-C homozygous specimens. Real-time fluorescent quantitative PCR was used to detect mRNA expression levels of target genes (HLA-A, -B, and -C) and reference genes (β2M) in the specimens, and Ct values of different samples were obtained. The mRNA expression levels of HLA-A, -B, and -C were homogenized by reference genes, and the mRNA expression levels of different alleles were compared. Results: The 88 HLA-A homozygous specimens contained 8 alleles, and the mRNA expression levels of HLA-A ranged from 20.26 to 35.39, showing differences. The mRNA expression levels of HLA-A*02:01 and HLA-A*02:07 groups were significantly higher than those of HLA-A*11:01 group, with statistical significance. The 45 HLA-B homozygous specimens contained 14 alleles, and the HLA-B mRNA expression levels ranged from 7.7 to 9.21, showing a balanced expression, and the mRNA expression levels of different HLA-B alleles showed no significant difference. The 38 HLA-C homozygous specimens contained 9 alleles, and the HLA-C mRNA expression ranged from 23.1 to 46.41, showing differential expression. The mRNA expression level of HLA-C*03:02 group was significantly lower than that of HLA-C*01:02 group, HLA-C*06:02 group and HLA-C*08:01:01G group, which had statistical significance. Conclusion: This study provides a method to detect the mRNA expression levels of class HLA-I molecules (HLA-A, -B and -C). The differential expression of HLA-A and HLA-C mRNA expression is correlated with alleles, while the balanced expression of HLA-B mRNA expression is unrelated to alleles. |
| Key words: Real-time fluorescence quantitative PCR technology HLA-Class I Allele mRNA expression level |