猪带绦虫不同发育阶段14-3-3基因的差异表达研究

Differential expression of 14-3-3 gene in different stages of Taenia solium

目的研究14-3-3基因在猪带绦虫不同发育阶段的表达差异情况,为探讨猪带绦虫14-3-3(Ts14-3-3)基因的生长发育调控机制奠定基础。方法自四川省甘孜藏族自治州雅江县绦虫病流行区的猪带绦虫病患者体内采集成虫虫体,经镜下形态学观察和物种内转录间隔1区(ITS1)鉴定法确定为猪带绦虫后,感染仔猪获得猪囊尾蚴(幼虫);采用反转录PCR法检测猪带绦虫成虫和幼虫阶段Ts14-3-3基因家族成员(Ts14-3-3.1、Ts14-3-3.2、Ts14-3-3.3、Ts14-3-3.4、Ts14-3-3.5、Ts14-3-3.6)的表达情况,实时荧光定量PCR法检测其各自的相对表达量。结果光镜下,可见采集的成虫虫体头节由4个吸盘、顶突和小钩组成,符合猪带绦虫头节典型特征;且虫体样本可经反转录PCR扩增出与ITS1基因大小一致的目的片段,确定其为猪带绦虫。Ts14-3-3基因家族成员在猪带绦虫成虫和幼虫阶段均有表达;Ts14-3-3.1(0.47±0.09比1.01±0.23)、Ts14-3-3.2(0.31±0.09比1.05±0.14)、Ts14-3-3.3(0.64±0.23比1.26±0.23)和Ts14-3-3.4(0.30±0.09比0.79±0.23)在成虫阶段表达水平与幼虫阶段比较显著降低(t=-3.816、-7.093、-3.377、-3.481,P<0.05或<0.01),Ts14-3-3.5(3.59±0.09比0.99±0.12)和Ts14-3-3.6(5.74±2.76比1.03±0.60)的表达水平显著增高(t=30.714、9.718,P均<0.01)。结论Ts14-3-3基因的表达具有阶段特异性,提示Ts14-3-3基因在猪带绦虫的不同发育阶段可能发挥特定的调控作用。

Objective To investigate the expression difference of 14-3-3 gene in different stages of Taenia solium (Ts), and to provide basic information for exploring the regulation mechanism of Ts14-3-3 gene in growth and development of Ts. Methods Adult worms were collected from patients with taeniasis solium in the taeniasis epidemic area of Yajiang County, Ganzi Tibetan Autonomous Prefecture, Sichuan Province. After determining the species through morphological observation under the light microscope and transcribed spacer 1 (ITS1) method, it was identified as Ts, then piglets were infected to obtain the Cysticercus cellulosae of Ts (larvae). Reverse transcription PCR was used to detect the expression of Ts14-3-3 gene in the adult stage and larval stage of Ts, and the relative expression levels of each were detected by real-time fluorescent quantitative PCR. Results Under light microscope, it could be seen that the collected adult worm scolex consisted of 4 suckers, rostellum and hooks, which was consistent with the typical characteristics of the Ts scolex;the worm sample could amplify the target fragment which was consistent with the length of the ITS1 gene by reverse transcription PCR, and was determined to be Ts. The Ts14-3-3 gene family members Ts14-3-3.1, Ts14-3-3.2, Ts14-3-3.3, Ts14-3-3.4, Ts14-3-3.5 and Ts14-3-3.6 were expressed in the Ts adult stage and larval stage. Compared with the larval stage, the expression levels of Ts14-3-3.1 (0.47 ± 0.09 vs 1.01 ± 0.23), Ts14-3-3.2 (0.31 ± 0.09 vs 1.05 ± 0.14), Ts14-3-3.3 (0.64 ± 0.23 vs 1.26 ± 0.23) and Ts14-3-3.4 (0.30 ± 0.09 vs 0.79 ± 0.23) were significantly decreased in the adult stage (t=-3.816,-7.093,-3.377,-3.481, P < 0.01 or < 0.05);the expression levels of Ts14-3-3.5 (3.59 ± 0.09 vs 0.99 ± 0.12) and Ts14-3-3.6 (5.74 ± 2.76 vs 1.03 ± 0.60) were significantly increased (t = 30.714, 9.718, P < 0.01). Conclusion The expression of Ts14-3-3 gene has stage specificity and the Ts14-3-3 gene may play a special regulatory role in different stages of Ts.