目的 鉴定血根碱致钉螺肝脏差异表达的基因。方法 浓度梯度法测定血根碱浸杀钉螺的半数致死浓度（LC₅₀）；以80% LC₅₀血根碱浸泡钉螺，分离血根碱组和清水组活钉螺肝脏；提取mRNA，逆转录为cDNA，进行抑制消减杂交；巢式PCR扩增差异cDNA，克隆至pMD-18T载体，PCR和测序鉴定，NCBI中blastx比对分析表达序列标签（expressed sequence tag，EST）。结果 血根碱浸杀钉螺LC₅₀为7.5 mg/L。获得的EST序列大小在150～450 bp，经blastx比对，表达上调的基因有α-4（VI）胶原链、α-5（VI）胶原链、40 S核糖体蛋白S19、假定蛋白（WP_009787 197.1）、kelch样蛋白、颗粒体样蛋白；表达下调的基因有β-微管蛋白、α-淀粉酶1、大亚基核糖体蛋白L23e、几丁质酶-1、捷蛋白-3、假定蛋白（EKC34 262.1）、线粒体乙醛脱氢酶、肽聚糖识别蛋白、真核转录延长因子1A、铁蛋白、去整合素金属蛋白酶、溶菌酶1、1, 3 (4)-β-葡聚糖酶1、血蓝蛋白α-4D亚基，这些基因编码蛋白的功能涉及物质转运、蛋白合成、增殖诱导、营养、抗氧化、抗炎、呼吸、信号转导等。结论 血根碱处理导致钉螺肝脏基因表达发生改变。

Objective To identify the differentially expressed genes in Oncomelania hupensis liver induced by sanguinarine. Methods The median lethal concentration (LC₅₀) of sanguinarine for O. hupensis was determined by concentration gradient method. Livers were isolated from live O. hupensis after being treated by 80% LC₅₀ of sanguinarine or clean water. Then the mRNA of livers was purified and used for RT-PCR. The differentially expressed genes were acquired by suppression subtractive hybridization (SSH) and amplified by nested PCR before cloned into pMD-18T vecter. Positive clones were identified using PCR and DNA sequencing analyses. Then the further study was accomplished by bioinformatic analysis with blastx in NCBI. Results The LC₅₀ of sanguinarine for O. hupensis was 7.5 mg/L. The differentially expressed sequences were between 150 and 450 bp. After blastx homological analysis, the up-regulated genes were obtained as collagen α-4 (VI) chain, collagen α-5 (VI) chain, 40S ribosomal protein S19, hypothetical protein, kelch-like protein, and granulin-like protein. The down-regulated genes were obtained as β-tubulin, α-amylase 1, large subunit ribosomal protein L23e, chitotriosidase-1, Chit3 protein, hypothetical protein, mitochondrial aldehyde dehydrogenase, peptidoglycan recognition protein, eukaryotic translation elongation factor 1A, ferritin, disintegrin, and metalloproteinase with thrombospondin motifs 3, lysozyme 1, endo-1, 2 (4)-β-glucanase 1, and hemocyanin α-4D-subunit. The functions of these genes encoding proteins were related to material transportation, protein synthesis, proliferation induction, nutrition, anti-oxidation, anti-inflamation, respiration, signal transduction, and so on. Conclusion Sanguinarine could cause the significant changes of gene expression in O. hupensis liver.

湖南省自然科学基金资助项目（11JJ3121）