旋毛虫肌幼虫细胞传代培养及超微结构观察

消化、分离观察旋毛虫(Trichinella spiralis)肌幼虫,获得肌幼虫细胞,用含10%胎牛血清的RPMI-1640培养液培养原代细胞,胰酶(含0.02?TA)消化法进行传代,透射电镜观察培养细胞超微结构,用多重PCR鉴定培养细胞。结果表明,在培养24～72h原代细胞开始贴壁,7～8d形成单层细胞,细胞间融合现象不明显,10～12d传一代。透射电镜显示旋毛虫细胞核为椭圆形,核膜、核仁清晰,核内染色质较丰富,胞浆含丰富的线粒体。细胞主要有两种类型:椭圆形和多角形,以椭圆形为主。多重PCR扩增培养细胞DNA,可见1条与旋毛虫肌幼虫DNA扩增产物相同的条带(173bp)。结果表明,旋毛虫肌幼虫细胞可在含10%胎牛血清的RPMI-1640培养液中传代培养。

Passage Cultivation and Ultrastructure of Trichinella spiralis Muscle Larval Cells Cultured in Vitro

Muscle larvae of Trichinella spiralis were isolated by digestion method,the cells were separated through homogenization,the primary cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum,and passage cultivation was carried out after trypsin(containing 0.02% EDTA) digestion.The cellular ultrastructure was observed by using transmission electron microscope,and the cultured cells were identified by multiplex PCR.The results showed that the primary cells began to adhere to the bottom of culture flask 24-72 hours after inoculation.The cell monolayer formed 7-8 days after culture,and distinct fusion among cells was not observed.Each passage could be completed within 10-12 d.The results of transmission electron microscopy showed that the cell nucleus of T.spiralis muscle larvae was elliptic.There were clear karyotheca,nucleolus and abundant chromatin in the nucleus,and plentiful mitochondria in the cytoplasm.There were mainly two types of cells:elliptic and polygonal cells,and most of them were elliptic.The DNA extracted from the cultured cells was amplified by multiplex PCR and the band(173 bp) was the same as the DNA from T.spiralis muscle larvae.The results show that the passage cultivation of T.spiralis muscle larval cells could be maintained in RPMI-1640 medium containing 10% fetal bovine serum.