摘要
目的：研究甲状腺激素释放相关主要水解酶基因在H22肝癌小鼠早期不同证候中的表达特征。方法采用标准化诊法技术筛选出早期邪毒证、气虚证H22肿瘤小鼠，先采用Affeymetrix基因芯片技术，初步获得甲状腺球蛋白（Tg），即甲状腺激素前体蛋白，及其相关的水解酶基因表达特征，并筛选主要差异表达基因；进而重复实验，RT-PCR检测主要差异表达基因的变化，同时ELISA检测血清甲状腺激素T3和T4水平。结果①初次实验芯片结果，Tg及其释放相关重要的水解酶：组织蛋白酶B（Ctsb）、组织蛋白酶D（Ctsd）、组织蛋白酶l（Ctsl）、天冬氨酸肽酶（Napsa）、三肽酶I （Tpp1）在肝癌小鼠早期邪毒证、气虚证呈下调趋势[Tg（邪毒0.77、气虚0.84）、Ctsb（邪毒0.83、气虚0.91）、Ctsd（邪毒0.79、气虚0.95）、Ctsl（邪毒0.95、气虚0.65）、Napsa（邪毒0.78、1.05）、Tpp1（邪毒0.75、0.94）]，以邪毒证下降尤甚。②酶联免疫吸附试验（ELISA）表明，在邪毒证[T3为（1.519±0.162）ng/ml、T4为（2.194±0.305）μg/dl]和气虚证[T4为（4.366±0.727）μg/dl]均出现下调，且邪毒证尤甚（P＜0.01），与两批次Tg转录水平的变化相一致；③对Ctsb等基因的RT-PCR检测结果表明，Tg（邪毒0.22、气虚0.38）、Ctsb（邪毒0.31、气虚0.55）、Ctsd（邪毒0.36、气虚0.78）、Napsa（邪毒0.24、气虚0.59）基本一致，Ctsl（邪毒1.24、气虚2.11）和Tpp1（邪毒2.85、气虚0.85）虽趋势相同，但在气虚证表达量大于同期邪毒证上则始终一致。结论综合不同批次实验结果，H22肝癌小鼠早期甲状腺功能受到抑制，且邪毒证抑制程度更重。
Objective To study the expression features of hydrolase genes related to the secretion of thyroid hormone of H22 hepatoma mice with different symptoms in early stage. Methods Firstly, The quantitative diagnosis and syndrome differentiation methods were used in H22 tumor-bearing mice in early stage, the expression profile of Tg and related hydrolase genes in poisonous pathogenic factors syndrome group （PPFS） and qi-deficiency syndromes （QDS） were got, and the major differential expression were selected. Secondly, the experiment was repeated and ELISA were used to detect T3 and T4 in serum, RT-PCR were applied to detect gene transcription level of genes including Tg, Ctsb, Ctsd, Ctsl, Napsa and Tpp1. Results ① Based on gene chip, the expression of Tg, Ctsb, Ctsd, Ctsl, Napsa and Tpp1were decreased in the first batch of experiment, the exactly ratio was Tg（0.77 in PPFS;0.84 in QDS）, Ctsb（0.83 in PPFS, 0.91 in QDS）, Ctsd（0.79 in PPFS;no notable change in QDS）, Ctsl（no notable change in PPFS; 0.65 in QDS）, Napsa（0.78 in PPFS; no notable change in QDS）, and Tpp1 （0.75 in PPFS; no notable change in QDS）, respectively. ② T3 and T4 downregulated in PPFS （the T3 value was 1.519±0.162ng/ml, T4 value was 2.194±0.305mg/dl） and in QDS （the T4 value is 4.366±0.727μg/dl） in early stage （P〈0.01）, especially in PPFS, which was in accordance with the change of Tg in both batches. ③the same trend happened in the validation of Tg（0.22 in PPFS;0.38 in QDS）, Ctsb（0.31 in PPFS;0.55 in QDS）, Ctsd（0.36 in PPFS;0.78 in QDS） and Napsa（0.24 in PPFS;0.59 in QDS） ,while ctsl（1.24 in PPFS;2.11 in QDS） and Tpp1 （2.85 in PPFS;0.85 in QDS）werethe opposite;even this, the total trend of the expression in QDS was still higher than that in PPFS. Conclusion All the results showed that the thyroid function of H22 hepatoma mice was inhibited in early stage especially in PPFS.
出处
《国际中医中药杂志》
2014年第7期623-627,共5页
International Journal of Traditional Chinese Medicine
基金
国家科技重大专项（项目编号：2009ZX09502-018）
上海市教委预算内项目（项目编号：2010JW06）
关键词
肝癌
证候
小鼠
甲状腺激素
水解酶
基因
Hepatoma
Symptoms and signs
Mice
Thyroid hormone
Hydrolase
Gene
