中国寄生虫学与寄生虫病杂志 ›› 2012, Vol. 30 ›› Issue (6): 11-464-467.

• 实验研究 • 上一篇    下一篇

东方田鼠肺成纤维细胞的分离培养及其对日本血吸虫的杀伤作用

成钢*,王京仁,王文彬,李淑红,曾文虎   

  1. 湖南文理学院湖南省高校动物学重点实验室,环洞庭湖生物资源保育与利用研究中心,常德415000
  • 出版日期:2012-12-31 发布日期:2013-02-05

Isolation and Culture of Lung Fibroblasts from Microtus fortis and its Killing Effect on Schistosomula in Vitro

CHENG Gang*,WANG Jing-Hen,WANG Wen-Bin,LI Shu-Hong,ZENG Wen-hu   

  1. Zoology Key Laboratory of Hunan Higher Education; Research Center on Conservation and Utilization of Biological Resources in Dongting Lake Area, Hunan University of Arts and Science, Changde 415000, China
  • Online:2012-12-31 Published:2013-02-05

摘要: 目的  探索和建立东方田鼠肺成纤维细胞体外分离、培养的技术方法,并观察其对日本血吸虫的杀伤作用。  方法  运用含15%小牛血清的Dulbecco改良Eagle培养液(DMEM)和RPMI 1640 两种培养体系,采用组织块贴壁法和胰酶消化法,分别对出生后1、3和5 d的东方田鼠乳鼠肺成纤维细胞(Microtus fortis lung fibroblasts,MfLF)进行原代分离、培养,倒置显微镜下观察MfLF形态和生长特性. 第2代和第3代MfLF培养上清与日本血吸虫童虫共培养96 h,观察其对童虫的杀伤效果。  结果  实验发现,采用胰酶消化法培养,只有少量MfLF细胞缓慢贴壁生长,而组织块贴壁法MfLF贴壁速度较快。不同日龄幼鼠的肺成纤维细胞采用组织块贴壁法培养,细胞活率和细胞纯度以1~3日龄乳鼠为最佳。在DMEM和RPMI 1640 两种培养液中MfLF均可传代培养,两者无明显差异。MfLF体外只可传代4~5次。日本血吸虫童虫经第2代和第3代MfLF培养上清处理后,死亡率分别为9.5%和10.5%,与阴性对照(8.8%)比较差异无统计学意义(P<0.05)。  结论  提示组织块贴壁法适用于MfLF的体外培养,但MfLF培养上清对日本血吸虫童虫无明显杀伤效果。

关键词: 东方田鼠, 肺成纤维细胞, 日本血吸虫

Abstract: Objective  To establish the technique of isolation and culture of lung fibroblasts from Microtus fortis in vitro, and observe the killing effect of lung fibroblasts on schistosomula.  Method  The shape and growth characteristics of lung fibroblasts, isolated from new-born Microtus fortis at the ages of 1, 3 and 5 days and cultured in DMEM and RPMI 1640 culture media respectively, were observed by inverted microscope after tissue adhering and trypsin digesting. The mortality rate of schistosomula was counted after co-culturing with the supernatant of 2nd and 3rd generation of Microtus fortis lung fibroblasts(MfLF)for 96 h respectively, and the schistosomulum-killing effect was observed by inverted microscope.  Results  The fibroblasts grew well by using tissue adherence method. MfLF cultured in both DMEM and RPMI 1640 media showed no difference. One to 3-day-old rats revealed better cell viability and purity. HE staining showed that the MfLF had an oval-shape nucleus and was passaged 4-5 times only in vitro. The culture supernatant of the second and third generations MfLF caused 9.5% and 10.5% death rate of schistosomula in 96 h respectively, with no significant difference to the negative control(8.8%).  Conclusion  The tissue adherence method developed is suitable for culturing the lung fibroblasts of M. fortis but the culture supernatant shows no effect in killing schistosomula.

Key words: Microtus fortis, Lung fibroblasts, Schistosoma japonicum