收稿日期: 2021-11-08
修回日期: 2022-01-25
网络出版日期: 2022-07-06
基金资助
国家自然科学基金(8176120052)
Effect of osteopontin expression level on the growth and development of Echinococcus multilocularis protoscoleces
Received date: 2021-11-08
Revised date: 2022-01-25
Online published: 2022-07-06
Supported by
National Natural Science Foundation of China(8176120052)
目的 探讨骨桥蛋白（OPN）表达水平对多房棘球蚴原头节生长发育的影响。 方法 从感染多房棘球蚴的长爪沙鼠体内分离原头节,体外培养3 d后,分为沉默EmOPN（LV-EmOPN-734）组、沉默对照（LV3-NC）组、过表达EmOPN（LV-EmOPN-0423）组、过表达对照（LV5-NC）组等4组,每组设置3个平行孔,每孔含原头节约5 000个。LV-EmOPN-734组中加入LV-EmOPN-734慢病毒稀释液（终浓度为1.4 × 107 TU/ml）、LV-EmOPN-0423组中加入LV-EmOPN-0423慢病毒稀释液（终浓度为4.83 × 108拷贝/ml）,相应对照组加入等量的慢病毒稀释液（空质粒）。感染后72 h,荧光显微镜下观察各组原头节的荧光强度,蛋白质免疫印迹（Western blotting）检测原头节中OPN的相对表达量,半胱天冬氨酸蛋白酶-3（Caspase-3）检测试剂盒检测原头节Caspase-3活性,EdU细胞成像试剂盒检测原头节增殖能力。感染后72 h,将原头节与大鼠肝癌细胞共培养8~12周,扫描电镜下观察多房棘球蚴囊泡生发层的显微结构。采用t检验对组间差异进行统计学分析。 结果 慢病毒感染原头节后72 h,荧光显微镜下可见慢病毒侵入原头节内,呈环状或点状分布。Western blotting结果显示,LV-EmOPN-734组EmOPN相对表达量为0.43 ± 0.04,低于LV3-NC组的0.80 ± 0.07（t = 8.623, P < 0.01）;LV-EmOPN-0423组EmOPN相对表达量为1.18 ± 0.21,高于LV5-NC组的0.73 ± 0.06（t = 3.333, P < 0.05）。Caspase-3活性检测结果显示,LV-EmOPN-734组Caspase-3活性为（61.55 ± 1.64）μmol/L,高于LV3-NC组的（28.20 ± 2.16）μmol/L（t = 24.57, P < 0.01）;LV-EmOPN-0423组Caspase-3活性为（50.11 ± 6.45）μmol/L,低于LV5-NC组的（78.22 ± 16.43）μmol/L（t = 3.185,P < 0.05）。EdU细胞成像试剂盒检测结果显示,LV-EmOPN-734组EdU+原头节数/总原头节数为0.47 ± 0.06,低于LV3-NC组的0.72 ± 0.10（t = 3.663, P < 0.05）;LV-EmOPN-0423组EdU+原头节数/总原头节数为0.81 ± 0.09,高于LV5-NC组的0.54 ± 0.06（t = 4.309, P < 0.05)。扫描电镜观察结果显示,LV-EmOPN-734组囊泡生发层细胞出现塌陷、皱缩、脱落,失去正常结构;LV-EmOPN-0423组囊泡生发层细胞膜完整,形态饱满,形成生发小囊并长出小蒂与生发层相接。 结论 OPN表达水平的升高/降低可促进/抑制多房棘球蚴原头节的生长发育。
卓怡呈 , 杨海成 , 刘程豪 , 张宝财 , 多小勇 , 张示杰 . 骨桥蛋白表达水平对多房棘球蚴原头节生长发育的影响[J]. 中国寄生虫学与寄生虫病杂志, 2022 , 40(3) : 299 -304 . DOI: 10.12140/j.issn.1000-7423.2022.03.004
Objective To investigate the effect of osteopontin (OPN) expression level on the growth and development of Echinococcus multilocularis protoscoleces. Methods Protoscoleces were isolated from E. multilocularis-infected Meriones unguiculatus and cultured in vitro for 3 days. The E. multilocularis protoscoleces were assigned into four groups: LV-EmOPN-734 (knockdown EmOPN) group, LV3-NC (knockdown control) group, LV-EmOPN-0423 (overexpression EmOPN) group, and LV5-NC (overexpression control) group. Each group was tested in parallel triplicate wells, with 5 000 protoscoleces in each well. LV-EmOPN-734 lentivirus diluent (final concentration 1.4 × 107 TU/ml) was added to the silenced EmOPN group, LV-EmOPN-0423 lentivirus diluent (final concentration 4.83 × 108 copies/ml) was added to the EmOPN overexpression group, and the corresponding control group was added with the same amount of lentivirus diluent (blank plasmid). After 72 hours of lentivirus infection, the fluorescence intensity of each group of protoscoleces was observed under fluorescence microscope, the relative expression of OPN in protoscoleces was detected by Western blotting, Caspase-3 activity was detected by applying cysteine aspartate-3 (Caspase-3) assay kit, and the proliferation ability of protoscoleces was detected by applying EdU Imaging kit (Cy3). 72 hours after infection, the protosegment was co-cultured with rat hepatoma cells for 8-12 weeks, and the morphology of the germinal layer of E. multilocularis vesicles was observed by scanning electron microscopy. Statistical analysis of differences between groups using t-test. Results After 72 hours of lentivirus infection, it can be seen that the E. multilocularis protoscoleces were infected by lentivirus successfully, which is distributed in a circle or dots. Western blotting results showed that the relative expression of EmOPN in the LV-EmOPN-734 group was (0.43 ± 0.04), which was lower than that in LV3-NC group (0.80 ± 0.07) (t = 8.623, P < 0.01); the relative expression of EmOPN in LV-EmOPN-0423 group was (1.18 ± 0.21), which was higher than that in the LV5-NC group (0.73 ± 0.06) (t = 3.333, P < 0.05). Caspase-3 activity assay showed that the Caspase-3 activity was (61.55 ± 1.64) μmol/L in the LV-EmOPN-734 group, higher than (28.20 ± 2.16) μmol/L in the LV3-NC group (t = 24.57, P < 0.01); Caspase-3 activity in the LV-EmOPN-0423 group was (50.11 ± 6.45) μmol/L, which was lower than (78.22 ± 16.43) μmol/L in the LV5-NC group (t = 3.185, P < 0.01). The results of EdU Imaging Kits (Cy3) assay showed that the protoscoleces of EdU+ in the LV-EmOPN-734 group were(0.47 ± 0.06), which was lower than that of the LV3-NC group (0.72 ± 0.10) (t = 3.663, P < 0.05); the protoscoleces of EdU+ in the LV-EmOPN-0423 group had of EdU+ protoscolecesof (0.81 ± 0.09), which was higher than that of the LV5-NC group (0.54 ± 0.06) (t = 4.309, P < 0.05). Scanning electron microscopy observations showed that most of the germinal layer cells of the LV-EmOPN-734 group were collapsed, shrank, shed to a large extent, and lost their normal structure; while the germinal layer cells of the LV-EmOPN-0423 group had intact cell membranes and full morphology, forming germinal layer vesicles and growing small tips to connect with the germinal layer. Conclusion The OPN expression level upon rising-up or lowering-down may promote or inhibit the growth and development of the E. multilocularis protoscoleces.
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