CAJ | 학술논문

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。
목적：연구갑상선격소석방상관주요수해매기인재H22간암소서조기불동증후중적표체특정。방법채용표준화진법기술사선출조기사독증、기허증H22종류소서，선채용Affeymetrix기인심편기술，초보획득갑상선구단백(Tg)，즉갑상선격소전체단백，급기상관적수해매기인표체특정，병사선주요차이표체기인；진이중복실험，RT-PCR검측주요차이표체기인적변화，동시ELISA검측혈청갑상선격소T3화T4수평。결과①초차실험심편결과，Tg급기석방상관중요적수해매：조직단백매B(Ctsb)、조직단백매D(Ctsd)、조직단백매l(Ctsl)、천동안산태매(Napsa)、삼태매I (Tpp1)재간암소서조기사독증、기허증정하조추세[Tg(사독0.77、기허0.84)、Ctsb(사독0.83、기허0.91)、Ctsd(사독0.79、기허0.95)、Ctsl(사독0.95、기허0.65)、Napsa(사독0.78、1.05)、Tpp1(사독0.75、0.94)]，이사독증하강우심。②매련면역흡부시험(ELISA)표명，재사독증[T3위(1.519±0.162)ng/ml、T4위(2.194±0.305)μg/dl]화기허증[T4위(4.366±0.727)μg/dl]균출현하조，차사독증우심(P＜0.01)，여량비차Tg전록수평적변화상일치；③대Ctsb등기인적RT-PCR검측결과표명，Tg(사독0.22、기허0.38)、Ctsb(사독0.31、기허0.55)、Ctsd(사독0.36、기허0.78)、Napsa(사독0.24、기허0.59)기본일치，Ctsl(사독1.24、기허2.11)화Tpp1(사독2.85、기허0.85)수추세상동，단재기허증표체량대우동기사독증상칙시종일치。결론종합불동비차실험결과，H22간암소서조기갑상선공능수도억제，차사독증억제정도경중。
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.