不同培养基与饲养细胞组合对多房棘球绦虫体外培养模型的影响
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摘要
背景:多房棘球绦虫原头节的体外成囊培养是提取与研究其生发层干细胞的实验基础。D-MEM与RPMI-1640是最适宜用于泡球蚴体外培养的商业培养基,HeLa细胞与大鼠肝癌细胞(ATCC No. CRL-1600)也是常用饲养细胞。目的:构建多房棘球绦虫体外培养模型,观察泡球蚴在不同饲养细胞[HeLa细胞与大鼠肝癌细胞(ATCC No.CRL-1600)]及含有体积分数10%胎牛血清的不同培养基[D-MEM(高/低糖型)与RPMI-1640]下生长情况。方法:预先培养HeLa细胞及大鼠肝癌细胞(ATCC No.CRL-1600),从新疆石河子大学第一附属院实验动物中心提供的感染多房棘球绦虫6个月以上的种鼠体内提取原头节后分别与不同饲养细胞及培养基体外共同培养。实验经石河子大学医学院第一附属医院医学伦理委员会批准,批准号:2015-018-01,批准时间:2017-04-07。实验根据饲养细胞与培养基组合分为6组,每组加入1×10~6饲养细胞与50mL含体积分数10%胎牛血清的培养基及5000个原头节,Ⅰ组:HeLa细胞与1640培养基培养;Ⅱ组:HeLa细胞与D-MEM(高糖型)培养基培养;Ⅲ组:大鼠肝癌细胞与1640培养基培养;Ⅳ组:大鼠肝癌细胞与D-MEM(高糖型)培养基培养;Ⅴ组:HeLa细胞与D-MEM(低糖型)培养基培养;Ⅵ组:大鼠肝癌细胞与D-MEM(低糖型)培养基培养,观察泡球蚴原头节的生存、生长和发育,每隔7d计算原头蚴成囊率,使用目镜测微尺测量囊泡平均直径大小。结果与结论:①原头蚴在6种组合中均可长期存活,大部分都形成囊泡,囊泡直径分布在1-6 mm;②培养第56天原头蚴成囊率和囊泡平均直径均为大鼠肝癌细胞与D-MEM(高糖型)培养基组效果最佳(P <0.05),成囊率(72.08±1.79)%,囊泡平均直径(3.379±0.199) mm;③囊泡囊液蛋白定量检测结果显示6组囊液蛋白含量均低于体内囊泡,Ⅳ组蛋白含量高于其他5组(P <0.05);④结果证实:同条件下D-MEM(高糖型)培养基囊泡大小与数量优于其他2种培养基。大鼠肝癌细胞(ATCC No.CRL-1600)作为饲养细胞囊泡大小与数量优于HeLa细胞。D-MEM(高糖型)与大鼠肝癌细胞(ATCC No.CRL-1600)共同作用的环境下原头蚴生长发育的成囊率及囊泡大小最佳,可作为体外培养泡球蚴原头蚴获得囊泡的最优选择。
BACKGROUND: The cystic culture of Echinococcus multilocularis protoscolex in vitro is the experimental basis for extracting and studying its original cells. D-MEM and RPMI-1640 are the most suitable commercial media for in vitro culture of Echinococcus multilocularis, and HeLa cells and rat hepatoma cells(ATCC No. CRL-1600) are also commonly used feeder cells. OBJECTIVE: A culture model of Echinococcus multilocularis in vitro was established to observe the growth of Echinococcus multilocularis in different feeder cells(HeLa cells and rat hepatoma cells(ATCC No.CRL-1600) and different media(D-MEM(High and Low glucose type) and RPMI-1640) containing 10% fetal bovine serum. METHODS: HeL a cells and rat hepatoma cells(ATCC No. CRL-1600) were pre-cultured. The Echinococcus multilocularis metacestode was extracted from mice infected with Echinococcus multilocularis for more than 6 months(provided by the Laboratory Animal Center of the First Affiliated Hospital of Shihezi University, Xinjiang Uygur Autonomous Region) and co-cultured with different feeder cells and culture media. The experiment has been approved by the First Affiliated Hospital of School of Medicine, Shihezi University, approval number: 2015-018-01 on April 7, 2017. The experiment was divided into six groups according to the combination of feeder cells and medium. Each group was added 1 x10~6 feeder cells, 50 mL medium containing 10% fetal bovine serum and 5 000 protoscolexes. Group Ⅰ: HeLa cells and 1640 medium; group Ⅱ: HeLa cells and D-MEM(high glucose type) medium; group Ⅲ: hepatocellular carcinoma cells and 1640 medium; group Ⅳ: hepatocellular carcinoma cells and D-MEM(high glucose type) medium; group Ⅴ: HeLa cells and D-MEM(low glucose type) medium; group Ⅵ: hepatocellular carcinoma cells and D-MEM(low glucose type) medium. The survival, growth and development of the protoscolex were observed. The rate of protoscolex vesicle formation was calculated every 7 days, and the average diameter of vesicle was measured using microscope micrometer. RESULTS AND CONCLUSION:(1) Protoscolex could survive for a long time in all four combinations. The vesicle diameter ranged from 1 to 6 mm.(2) On day 56, the cyst formation rate and average vesicle diameter of protocephalus were the best in rat hepatocellular carcinoma cells and D-MEM(high sugar type) culture medium group(P < 0.05), the cyst formation rate was(72.08±1.79)% and the vesicle diameter was(3.379±0.199) mm.(3) Quantitative analysis of vesicle fluid protein showed that the contents of vesicle fluid protein in six groups were lower than those of vesicles in vivo, and the group Ⅳ was higher than those in the other five groups(P < 0.05).(4) These results indicate that under the same conditions, the vesicle size and quantity of D-MEM medium are better than those of the other two media. Rat hepatocellular carcinoma cells(ATCC No. CRL-1600) are better than HeLa cells in size and number of vesicles as feeder cells. The cyst formation rate and vesicle size of D-MEM and rat hepatocellular carcinoma cells(ATCC No. CRL-1600) are the best, which can be used as the optimal choice for obtaining Echinococcus multilocularis vesicles in vitro.
BACKGROUND: The cystic culture of Echinococcus multilocularis protoscolex in vitro is the experimental basis for extracting and studying its original cells. D-MEM and RPMI-1640 are the most suitable commercial media for in vitro culture of Echinococcus multilocularis, and HeLa cells and rat hepatoma cells(ATCC No. CRL-1600) are also commonly used feeder cells. OBJECTIVE: A culture model of Echinococcus multilocularis in vitro was established to observe the growth of Echinococcus multilocularis in different feeder cells(HeLa cells and rat hepatoma cells(ATCC No.CRL-1600) and different media(D-MEM(High and Low glucose type) and RPMI-1640) containing 10% fetal bovine serum. METHODS: HeL a cells and rat hepatoma cells(ATCC No. CRL-1600) were pre-cultured. The Echinococcus multilocularis metacestode was extracted from mice infected with Echinococcus multilocularis for more than 6 months(provided by the Laboratory Animal Center of the First Affiliated Hospital of Shihezi University, Xinjiang Uygur Autonomous Region) and co-cultured with different feeder cells and culture media. The experiment has been approved by the First Affiliated Hospital of School of Medicine, Shihezi University, approval number: 2015-018-01 on April 7, 2017. The experiment was divided into six groups according to the combination of feeder cells and medium. Each group was added 1 x10~6 feeder cells, 50 mL medium containing 10% fetal bovine serum and 5 000 protoscolexes. Group Ⅰ: HeLa cells and 1640 medium; group Ⅱ: HeLa cells and D-MEM(high glucose type) medium; group Ⅲ: hepatocellular carcinoma cells and 1640 medium; group Ⅳ: hepatocellular carcinoma cells and D-MEM(high glucose type) medium; group Ⅴ: HeLa cells and D-MEM(low glucose type) medium; group Ⅵ: hepatocellular carcinoma cells and D-MEM(low glucose type) medium. The survival, growth and development of the protoscolex were observed. The rate of protoscolex vesicle formation was calculated every 7 days, and the average diameter of vesicle was measured using microscope micrometer. RESULTS AND CONCLUSION:(1) Protoscolex could survive for a long time in all four combinations. The vesicle diameter ranged from 1 to 6 mm.(2) On day 56, the cyst formation rate and average vesicle diameter of protocephalus were the best in rat hepatocellular carcinoma cells and D-MEM(high sugar type) culture medium group(P < 0.05), the cyst formation rate was(72.08±1.79)% and the vesicle diameter was(3.379±0.199) mm.(3) Quantitative analysis of vesicle fluid protein showed that the contents of vesicle fluid protein in six groups were lower than those of vesicles in vivo, and the group Ⅳ was higher than those in the other five groups(P < 0.05).(4) These results indicate that under the same conditions, the vesicle size and quantity of D-MEM medium are better than those of the other two media. Rat hepatocellular carcinoma cells(ATCC No. CRL-1600) are better than HeLa cells in size and number of vesicles as feeder cells. The cyst formation rate and vesicle size of D-MEM and rat hepatocellular carcinoma cells(ATCC No. CRL-1600) are the best, which can be used as the optimal choice for obtaining Echinococcus multilocularis vesicles in vitro.
引文
[1]Brunetti E, Kern P, Vuitton DA. Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans. Acta Trop.2010;114(1):1-16.
[2]刘平,李金花,李印,等.包虫病病原在我国的流行现状及成因分析[J].中国动物检疫,2016,33(1):48-51.
[3]Dieckmannschuppert A, Ruppel A, Burger R, et al. Echinococcus multilocularis:in vitro secretion of antigen by hybridomas from metacestode germinal cells and murine tumor cells. Exp Parasitol.1989;68(2):186-191.
[4]Toledo A, Cruz C, Fragoso G, et al. In vitro culture of taenia crassiceps larval cells and cyst regeneration after injection into mice. J Parasitol.1997;83(2):189-193.
[5]Galindo M, Paredes R, Marchant C, et al. Regionalization of DNA and protein synthesis in developing stages of the parasitic platyhelminth Echinococcus granulosus. J Cell Biochem. 2010;90(2):294-303.
[6]Koziol U, Rauschendorfer T, Rodríguez LZ, et al. The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis. Evodevo. 2014;5(1):10.
[7]Spiliotis M, Lechner S, Tappe D, et al. Transient transfection of Echinococcus multilocularis primary cells and complete in vitro regeneration of metacestode vesicles. Int J Parasitol. 2008;38(8-9):1025-1039.
[8]Hemphill A, Gottstein B. Immunology and morphology studies on the proliferation of in vitro cultivated echinococcus multilocularis metacestodes. Parasitol Res. 1995;81(7):605-614.
[9]赵阶峰,夏海洋,郁晓峰,等.不同培养基及不同温度对体外培养泡球蚴原头节的影响[J].中国人兽共患病学报,2015,31(3):244-246.
[10]米晓云,古努尔·吐尔逊,张壮志,等.多房棘球绦虫-泡球蚴的体外培养研究[J].中国动物传染病学报,2011,19(4):67-70.
[11]Brehm K, Koziol U. Echinococcus-host interactions at cellular and molecular levels. Adv Parasitol. 2017;95:147-212.
[12]王季武.泡球蚴体外厌氧培养模型建立及影响体外培养泡球蚴生长发育因素研究[D].厦门:厦门大学,2011.
[13]张传山,李朝旺,范金亮,等.多房棘球绦虫原头蚴对体外培养宿主肝细胞MAPK信号通路影响的初步研究[J].中国病原生物学杂志,2011,6(8):574-577.
[14]Brehm K, Wolf M, Beland H, et al. Analysis of differential gene expression in Echinococcus multilocularis larval stages by means of spliced leader differential display. Int J Parasitol. 2003;33(11):1145-1159.
[15]张怀,张示杰,陈骞,等.缺氧对肝泡状棘球蚴原头节血管内皮生长因子和CD34表达的影响研究[J].中国全科医学,2017,20(14):1702-1706.
[16]曾红春,王俊华,刘文亚,等.早期大鼠肝泡状棘球蚴病模型的超声表现与病理对照[J].中国介入影像与治疗学,2015,12(8):508-511.
[17]李玉鹏,吐尔洪江·吐逊,沙地克·阿帕尔,等.多房棘球蚴感染BALB/c小鼠转录因子T-bet、GATA-3 mRNA表达的实验研究[J].中国病原生物学杂志,2016,11(6):530-534.
[18]李红军,张艳琼.肿瘤细胞培养中的问题及采取措施[J].教育教学论坛,2013,(13):131-132.
[19]唐岩,唐军民.肿瘤细胞培养、冻存及复苏过程中的工作体会[J].解剖学杂志,2009,32(4):561-562.
[20]郝广萍.人肝癌细胞HepG2培养方法探究与分析[J].华北理工大学学报(医学版),2018,20(1):7-10.
[21]周启锋,任利,张灵强,等.肝泡状棘球蚴病动物模型的研究进展[J].临床肝胆病杂志,2017,33(11):208-211.
[22]武坚坚.多房棘球绦虫Sox2基因的克隆与功能鉴定[D].厦门:厦门大学,2014.
[23]商桂华.多房棘球绦虫OCT4基因的克隆及功能鉴定[D].厦门:厦门大学,2014.
[24]袁丽英,张壮志,石保新,等.细粒棘球蚴-原头蚴体外培养成囊模型的建立[J].畜牧与兽医,2009,41(7):29-31.
[25]马海龙,李兆勇,孙世安,等.肝癌细胞及其上清液体外培养多房棘球蚴的实验研究[J].武警后勤学院学报(医学版),2012,21(11):864-867.
[26]马海龙.泡球蚴体外培养模型的建立及细胞亲和性观察[D].乌鲁木齐:新疆医科大学,2008.
[27]Spiliotis M, Tappe D, Sesterhenn L, et al. Long-term in vitro cultivation of Echinococcus multilocularis metacestodes under axenic conditions.Parasitol Res. 2004;92(5):430-432.
[28]Spiliotis M, Brehm K. Axenic in vitro cultivation of Echinococcus multilocularis metacestode vesicles and the generation of primary cell cultures. Methods Mol Biol. 2009;470(1):245-262.
[29]赵翔,张沙,肖军军,等.RPMI-1640和D-MEM培养基中肝癌细胞系BEL-7402与HepG-2的生长状态比较[J].中国组织工程研究, 2011,15(11):2002-2005.
[30]张迪,夏薇,芦瑶.RPMI-1640培养基和DMEM培养基中HepG-2细胞生长状况[J].现代养生,2017,12(24):35-36.
[2]刘平,李金花,李印,等.包虫病病原在我国的流行现状及成因分析[J].中国动物检疫,2016,33(1):48-51.
[3]Dieckmannschuppert A, Ruppel A, Burger R, et al. Echinococcus multilocularis:in vitro secretion of antigen by hybridomas from metacestode germinal cells and murine tumor cells. Exp Parasitol.1989;68(2):186-191.
[4]Toledo A, Cruz C, Fragoso G, et al. In vitro culture of taenia crassiceps larval cells and cyst regeneration after injection into mice. J Parasitol.1997;83(2):189-193.
[5]Galindo M, Paredes R, Marchant C, et al. Regionalization of DNA and protein synthesis in developing stages of the parasitic platyhelminth Echinococcus granulosus. J Cell Biochem. 2010;90(2):294-303.
[6]Koziol U, Rauschendorfer T, Rodríguez LZ, et al. The unique stem cell system of the immortal larva of the human parasite Echinococcus multilocularis. Evodevo. 2014;5(1):10.
[7]Spiliotis M, Lechner S, Tappe D, et al. Transient transfection of Echinococcus multilocularis primary cells and complete in vitro regeneration of metacestode vesicles. Int J Parasitol. 2008;38(8-9):1025-1039.
[8]Hemphill A, Gottstein B. Immunology and morphology studies on the proliferation of in vitro cultivated echinococcus multilocularis metacestodes. Parasitol Res. 1995;81(7):605-614.
[9]赵阶峰,夏海洋,郁晓峰,等.不同培养基及不同温度对体外培养泡球蚴原头节的影响[J].中国人兽共患病学报,2015,31(3):244-246.
[10]米晓云,古努尔·吐尔逊,张壮志,等.多房棘球绦虫-泡球蚴的体外培养研究[J].中国动物传染病学报,2011,19(4):67-70.
[11]Brehm K, Koziol U. Echinococcus-host interactions at cellular and molecular levels. Adv Parasitol. 2017;95:147-212.
[12]王季武.泡球蚴体外厌氧培养模型建立及影响体外培养泡球蚴生长发育因素研究[D].厦门:厦门大学,2011.
[13]张传山,李朝旺,范金亮,等.多房棘球绦虫原头蚴对体外培养宿主肝细胞MAPK信号通路影响的初步研究[J].中国病原生物学杂志,2011,6(8):574-577.
[14]Brehm K, Wolf M, Beland H, et al. Analysis of differential gene expression in Echinococcus multilocularis larval stages by means of spliced leader differential display. Int J Parasitol. 2003;33(11):1145-1159.
[15]张怀,张示杰,陈骞,等.缺氧对肝泡状棘球蚴原头节血管内皮生长因子和CD34表达的影响研究[J].中国全科医学,2017,20(14):1702-1706.
[16]曾红春,王俊华,刘文亚,等.早期大鼠肝泡状棘球蚴病模型的超声表现与病理对照[J].中国介入影像与治疗学,2015,12(8):508-511.
[17]李玉鹏,吐尔洪江·吐逊,沙地克·阿帕尔,等.多房棘球蚴感染BALB/c小鼠转录因子T-bet、GATA-3 mRNA表达的实验研究[J].中国病原生物学杂志,2016,11(6):530-534.
[18]李红军,张艳琼.肿瘤细胞培养中的问题及采取措施[J].教育教学论坛,2013,(13):131-132.
[19]唐岩,唐军民.肿瘤细胞培养、冻存及复苏过程中的工作体会[J].解剖学杂志,2009,32(4):561-562.
[20]郝广萍.人肝癌细胞HepG2培养方法探究与分析[J].华北理工大学学报(医学版),2018,20(1):7-10.
[21]周启锋,任利,张灵强,等.肝泡状棘球蚴病动物模型的研究进展[J].临床肝胆病杂志,2017,33(11):208-211.
[22]武坚坚.多房棘球绦虫Sox2基因的克隆与功能鉴定[D].厦门:厦门大学,2014.
[23]商桂华.多房棘球绦虫OCT4基因的克隆及功能鉴定[D].厦门:厦门大学,2014.
[24]袁丽英,张壮志,石保新,等.细粒棘球蚴-原头蚴体外培养成囊模型的建立[J].畜牧与兽医,2009,41(7):29-31.
[25]马海龙,李兆勇,孙世安,等.肝癌细胞及其上清液体外培养多房棘球蚴的实验研究[J].武警后勤学院学报(医学版),2012,21(11):864-867.
[26]马海龙.泡球蚴体外培养模型的建立及细胞亲和性观察[D].乌鲁木齐:新疆医科大学,2008.
[27]Spiliotis M, Tappe D, Sesterhenn L, et al. Long-term in vitro cultivation of Echinococcus multilocularis metacestodes under axenic conditions.Parasitol Res. 2004;92(5):430-432.
[28]Spiliotis M, Brehm K. Axenic in vitro cultivation of Echinococcus multilocularis metacestode vesicles and the generation of primary cell cultures. Methods Mol Biol. 2009;470(1):245-262.
[29]赵翔,张沙,肖军军,等.RPMI-1640和D-MEM培养基中肝癌细胞系BEL-7402与HepG-2的生长状态比较[J].中国组织工程研究, 2011,15(11):2002-2005.
[30]张迪,夏薇,芦瑶.RPMI-1640培养基和DMEM培养基中HepG-2细胞生长状况[J].现代养生,2017,12(24):35-36.
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