氨基醇类化合物HT24对多房棘球蚴原头节微管蛋白表达水平的影响

Affect of aminoalcohol compound HT24 on the expression of tubulin in Echinococcus multilocularis protoscoleces

目的探索氨基醇类化合物HT24对多房棘球蚴原头节微管蛋白表达水平的影响。方法从感染多房棘球蚴的BALB/c小鼠体内分离原头节,体外培养1 d后分为对照组和低、中、高浓度组,分别加入终浓度0.5%的二甲基亚砜(DMSO)和25、50、100μmol/L的HT24共培养,每组设3个平行孔,每孔含原头节约2000个。HT24作用后第1~3天观察虫体活性及形态改变,第3天透射电镜下观察原头节焰细胞超微结构的变化情况,第3、5、7天实时荧光定量PCR(qPCR)检测原头节中α9-,β2-,β4-和β6-微管蛋白基因的mRNA相对转录水平,蛋白质免疫印迹(Western blotting)检测原头节中β-微管蛋白的相对表达量。组间比较使用双因素方差分析方法。结果氨基醇类化合物HT24作用后第3天,低浓度组多房棘球蚴原头节轮廓模糊,虫体胀大,透明度降低,部分钩突脱落;中浓度组原头节明显胀大,钩突脱落;高浓度组原头节全部死亡,虫体缩小。透射电镜观察结果显示,中、高浓度组焰细胞胞膜破裂,胞质内纤毛断裂,出现大的缺口或断裂口,部分纤毛溢出,游离于细胞外。qPCR结果显示,相较于对照组(100),低、中、高浓度组第7天α9-微管蛋白基因mRNA相对转录水平分别为8.01±1.54、9.84±0.40和7.85±2.60,3组间差异有统计学意义(P<0.01);β2-微管蛋白基因mRNA相对转录水平分别为10.97±0.69、11.83±0.97和6.48±1.52,3组间差异有统计学意义(P<0.01);β4-微管蛋白基因mRNA相对转录水平分别为10.61±1.24、8.31±1.05和7.47±0.96,3组间差异有统计学意义(P<0.01);β6-微管蛋白基因mRNA相对转录水平分别为8.83±0.80、7.89±1.30和5.63±2.25,3组间差异有统计学意义(P<0.01)。Western blotting分析结果显示,HT24作用后第3天低、中、高浓度组β-微管蛋白的相对表达量分别为0.36±0.06、0.23±0.03和0.61±0.06,均低于对照组(0.67±0.05)(P<0.01);第5天分别为1.12±0.03,0.55±0.04,0.30±0.02,均高于对照组(0.26±0.03)(P<0.01);第7天分别为0.73±0.05,0.36±0.04,0.40±0.04,其中中、高浓度组低于对照组(0.73±0.05)(P<0.01)。结论氨基醇类化合物HT24能降低多房棘球蚴原头节微管蛋白基因mRNA和蛋白的表达水平,影响微管蛋白功能,从而抑制多房棘球蚴的生存。

Objective To understand the affect of aminoalcohol compound HT24 on the expression of tubulin in Echinococcus multilocularis protoscoleces.Methods Protoscoleces were isolated from BALB/c mice infected with E.multilocularis metacestode,and cultured in vitro for 1 day.Then they were assigned into groups of low,medium and high concentration of HT24 treatment,which were added with 0.5%DMSO(control group)and 25μmol/L,50μmol/L or 100μmol/L HT24,respectively.Each group was set with 3 replicates,and 2000 protoscoleces in each well.The viability and morphological changes of the protoscoleces were observed in first 3 days after addition of HT24.Samples were collected on the third day and subjected to transmission electron microscopy(TEM)to observe the ultrastructural changes of flame cells.Samples were collected at 3,5,and 7 days for real-time fluorescence quantitative PCR(qPCR)to examine the mRNA expression levels ofα9-,β2-,β4-andβ6-tubulin,and Western blotting to examine the protein levels ofβ-tubulin.Two-way ANOVA was applied for between-group comparisons.Results After 3 days of HT24 treatment,in the low-concentration group,the E.multilocularis protoscoleces had unclear outline,swollen body,decreased transparency,and detachment of some hooks.In the medium-concentration group,the protoscoleces were obviously enlarged and hooks were detached.All protoscoleces in the high-concentration group died and dwindled in size.TEM showed that in the medium-and high-concentration groups,the membranes of flame cells were broken,ciliary in the cytoplasm ruptured with large notches or fracture openings,and some cilia spilled out and were free outside the cells.qPCR showed that compared to the control group,the mRNA expression ofα9-tubulin on day 7 in the low-,medium-and high-concentration groups was 8.01±1.54,9.84±0.40 and 7.85±2.60,respectively,there was significant difference between the 3 groups(P<0.01);the mRNA expression ofβ2-tubulin in the low-,medium-and high-concentration group on day 7 was 10.97±0.69,11.83±0.97 and 6.48±1.52,respectively,there was significant difference between the 3 groups(P<0.01);the mRNA expression ofβ4-tubulin on day 7 in the low-,medium-and high-concentration groups was 10.61±1.24,8.31±1.05 and 7.47±0.96,respectively,there was significant difference between the 3 groups(P<0.01);the mRNA expression ofβ6-tubulin on day 7 was 8.83±0.80,7.89±1.30 and 5.63±2.25 in the low-,medium-and high-concentration groups,respectively,there was significant difference between the 3 groups(P<0.01).Western blotting results showed that on day 3 after treatment,theβ-tubulin protein levels at the low-,medium-and high-concentration groups were 0.36±0.06,0.23±0.03,and 0.61±0.06,respectively,all lower than the control group(0.67±0.05)(P<0.01);on day 5 after treatment,theβ-tubulin protein levels in the low-,medium-and high-concentration groups were 1.12±0.03,0.55±0.04,and 0.30±0.02,respectively,all higher than the control group(0.26±0.03)(P<0.01);on day 7 after treatment,they were 0.73±0.05,0.36±0.04,and 0.40±0.04,respectively,The latter two were lower than control group(0.73±0.05)(P<0.01).Conclusion HT24 can lower the mRNA and protein levels ofβ-tubulin and affect the function of microtubules,thereby suppressing the viability of E.multilocularis metacestodes.