慢性肝炎中与代谢型受体相关基因的肝内表达。
Intrahepatic expression of genes related to metabotropic receptors in chronic hepatitis.
机构信息
Department of Gastroenterology, Hepatology and Infectious Diseases, Jagiellonian University Medical College, 31-521 Kraków, Poland.
出版信息
World J Gastroenterol. 2012 Aug 21;18(31):4156-61. doi: 10.3748/wjg.v18.i31.4156.
AIM
To screen for genes related to metabotropic receptors that might be involved in the development of chronic hepatitis.
METHODS
Assessment of 20 genes associated with metabotropic receptors was performed in liver specimens obtained by punch biopsy from 12 patients with autoimmune and chronic hepatitis type B and C. For this purpose, a microarray with low integrity grade and with oligonucleotide DNA probes complementary to target transcripts was used. Evaluation of gene expression was performed in relation to transcript level, correlation between samples and grouping of clinical parameters used in chronic hepatitis assessment. Clinical markers of chronic hepatitis included alanine and aspartate aminotransferase, γ-glutamyltranspeptidase, alkaline phosphatase and cholinesterase activity, levels of iron ions, total cholesterol, triglycerides, albumin, glucose, hemoglobin, platelets, histological analysis of inflammatory and necrotic status, fibrosis according to METAVIR score, steatosis, as well as anthropometric body mass index, waist/hip index, percentage of adipose tissue and liver size in ultrasound examination. Gender, age, concomitant diseases and drugs were also taken into account. Validation of oligonucleotide microarray gene expression results was done with the use of quantitative real-time polymerase chain reaction (qRT-PCR).
RESULTS
The highest (0.002 < P < 0.046) expression among genes encoding main components of metabotropic receptor pathways, such as the α subunit of G-coupled protein, phosphoinositol-dependent protein kinase or arrestin was comparable to that of angiotensinogen synthesized in the liver. Carcinogenesis suppressor genes, such as chemokine ligand 4, transcription factor early growth response protein 1 and lysophosphatidic acid receptor, were characterized by the lowest expression (0.002 < P < 0.046), while the factor potentially triggering hepatic cancer, transcription factor JUN-B, had a 20-fold higher expression. The correlation between expression of genes of protein kinases PDPK1, phosphoinositide 3-kinase and protein kinase A (Spearman's coefficient range: 0.762-0.769) confirmed a functional link between these enzymes. Gender (P = 0.0046) and inflammation severity, measured by alanine aminotransferase activity (P = 0.035), were characterized by diverse metabotropic receptor gene expression patterns. The Pearson's coefficient ranging from -0.35 to 0.99 from the results of qRT-PCR and microarray indicated that qRT-PCR had certain limitations as a validation tool for oligonucleotide microarray studies.
CONCLUSION
A microarray-based analysis of hepatocyte metabotropic G-protein-related gene expression can reveal the molecular basis of chronic hepatitis.
目的
筛选可能参与慢性肝炎发生的代谢型谷氨酸受体相关基因。
方法
采用微阵列技术对 12 例自身免疫性和慢性乙型、丙型肝炎患者肝穿活检组织中 20 个与代谢型谷氨酸受体相关的基因进行评估。微阵列的完整性等级较低，寡核苷酸 DNA 探针与靶转录本互补。评估基因表达与转录水平、样本相关性以及慢性肝炎评估中使用的临床参数分组有关。慢性肝炎的临床标志物包括丙氨酸和天冬氨酸氨基转移酶、γ-谷氨酰转肽酶、碱性磷酸酶和胆碱酯酶活性、铁离子、总胆固醇、甘油三酯、白蛋白、葡萄糖、血红蛋白、血小板水平，根据 METAVIR 评分的炎症和坏死状态、纤维化、脂肪变性以及超声检查的肝大小的组织学分析。还考虑了性别、年龄、合并症和药物。寡核苷酸微阵列基因表达结果的验证采用实时定量聚合酶链反应（qRT-PCR）。
结果
编码代谢型谷氨酸受体途径主要成分的基因（如 G 蛋白偶联α亚基、磷酸肌醇依赖性蛋白激酶或阻遏蛋白）的表达最高（0.002 < P < 0.046），与在肝脏中合成的血管紧张素原相当。趋化因子配体 4、早期生长反应蛋白 1 和溶血磷脂酸受体等抑癌基因的表达最低（0.002 < P < 0.046），而潜在触发肝癌的转录因子 JUN-B 的表达则高出 20 倍。蛋白激酶 PDPK1、磷酸肌醇 3-激酶和蛋白激酶 A 的基因表达（Spearman 系数范围：0.762-0.769）之间的相关性证实了这些酶之间存在功能联系。性别（P = 0.0046）和丙氨酸氨基转移酶活性测量的炎症严重程度（P = 0.035）的不同，表现出不同的代谢型谷氨酸受体基因表达模式。qRT-PCR 和微阵列的 Pearson 系数范围从-0.35 到 0.99，表明 qRT-PCR 作为寡核苷酸微阵列研究的验证工具具有一定的局限性。
结论
基于微阵列的肝细胞代谢型谷氨酸 G 蛋白相关基因表达分析可以揭示慢性肝炎的分子基础。
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