参环毛蚓肠上皮细胞体外培养及其生物学特性
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摘要
该研究采用混合酶消化法获得参环毛蚓(Pheretima aspergillum)肠上皮细胞(intestine epithelial cell,IEC),并进行体外培养,成功传代培养至第8代,命名为IEC-P.A.。通过光学显微镜和透射电镜技术对IEC-P.A.细胞形态、超微结构进行了鉴定,采用MTT法绘制了细胞生长曲线,应用流式细胞术检测了DNA含量并分析其细胞周期和增殖活性。体外培养的参环毛蚓肠上皮细胞汇合后呈典型的"铺路石样"形态,细胞直径为8±2μm,表面具有大量的微绒毛,胞质内含丰富的线粒体及粗面内质网。细胞周期检测结果显示,G0/G1期细胞约占总细胞数的93%。染色体核型分析结果表明,参环毛蚓肠上皮细胞为二倍体,2n=22,核型为n(♂)=x=6m+3sm+2st。该研究成功获得了离体培养的参环毛蚓肠上皮细胞及其生物学特性的相关资料。
The intestine epithelial cell(IEC), designated IEC-P.A., was digested by complex enzyme from Pheretima aspergillum(E. Perrier), which has been continuously subcultured 8 times in vitro. IEC-P.A. was identified by morphological and ultrastructure observation under inverted microscope and transmission electron microscope. The growth curve of IEC-P.A. was determined by MTT assay. Cell cycle and cell proliferation were observed by flow cytometry. IEC-P.A., cultured in vitro, was about 8±2 μm in diameter, and appeared some distinct typical morphological characteristics of epithelial cells, such as cobblestone-like appearance, numerous microvilli, rough endoplasmic reticulum and mitochondria. Flow cytometric test result showed that more than 93% IEC-P.A. was in G0/G1 phase. Chromosome typing of IEC-P.A. revealed that the diploid chromosome number was 2n=22, and the chromosomal karyotype was n(♂)=x=6m+3sm+2st. IEC-P.A. was successfully cultured in vitro and its basic information on biological characteristics was investigated in this study.
The intestine epithelial cell(IEC), designated IEC-P.A., was digested by complex enzyme from Pheretima aspergillum(E. Perrier), which has been continuously subcultured 8 times in vitro. IEC-P.A. was identified by morphological and ultrastructure observation under inverted microscope and transmission electron microscope. The growth curve of IEC-P.A. was determined by MTT assay. Cell cycle and cell proliferation were observed by flow cytometry. IEC-P.A., cultured in vitro, was about 8±2 μm in diameter, and appeared some distinct typical morphological characteristics of epithelial cells, such as cobblestone-like appearance, numerous microvilli, rough endoplasmic reticulum and mitochondria. Flow cytometric test result showed that more than 93% IEC-P.A. was in G0/G1 phase. Chromosome typing of IEC-P.A. revealed that the diploid chromosome number was 2n=22, and the chromosomal karyotype was n(♂)=x=6m+3sm+2st. IEC-P.A. was successfully cultured in vitro and its basic information on biological characteristics was investigated in this study.
引文
1 彭芙.蚯蚓(地龙)综合利用的现状与展望.中药与临床(Peng Fu.The comprehensive utilization of earthworms.Pharmacy and clinics of Chinese material medica)2012;3(3):56-9.
2 欧阳高亮,肖俐,李祺福,洪水根.分子生物学技术在水产养殖中的应用.海洋科学(Ouyang Gaoliang,Xiao Li,Li Qifu,Hong Shuigen.The application of molecular biological techniques in the aquaculture.Marine sciences)2000;24(3):31-4.
3 洪智敏,贾永杰,瞿明仁,黎观红,刘思国.鸡胚小肠上皮细胞的分离及原代培养研究.江西农业大学学报(Hong Zhimin,Jia Yongjie,Qu Mingren,Li Guanhong,Liu Siguo.Isolation and primary culture of chicken embryo small intestinal epithelial cells.Acta Agriculturae Universitatis Jiangxiensis)2011;33(6):1164-70.
4 Chopra DP,Dombkowski AA,Stemmer PM,Parker GC.Intestinal epithelial cells in vitro.Stem Cells Dev 2010;19(1):131-42.
5 王莉,段相林.大鼠小肠上皮细胞的体外原代培养.军事医学(Wang Li,Duan Xianglin.In vitro primary culture of rat intestinal epithelial cells.Military Medical Sciences)2004;28(1):61-3.
6 匡伟,王海霞,邵桃玉,赵国琦.新生山羊小肠上皮细胞株的建立及鉴定.中国畜牧杂志(Kuang Wei,Wang Haixia,Shao Taoyu,Zhao Guoqi.Establishing and identification of neonate goat intestinal epithelial cells.Chinese Journal of Animal Science)2007;43(19):11-4.
7 刘飞飞,车东升,穆成龙,张海全,王波,孙泽威,等.兔小肠上皮细胞体外分离培养.吉林农业大学学报(Liu Feifei,Che Dongsheng,Mu Chenglong,Zhang Haiquan,Wang Bo,Sun Zewei,et al.Isolation and primary culture of rabbit intestinal epithelial cell in vitro.Journal of Jilin Agricultural University)2011;33(4):429-32.
8 杨文平,高峰,许辉堂,施传信,赵国刚,周光宏.鸡肠上皮细胞体外原代培养研究.江西农业学报(Yang Wenping,Gao Feng,Xu Huitang,Shi Chuanxin,Zhao Guogang,Zhou Guanghong.In vitro primary culture of intestinal epithelial cells of chicken.Acta Agriculturae Jiangxi)2007;19(5):113-5.
9 林小桦,李薇,侯雪芹,李巧容,韦建红,李维.参环毛蚓细胞原代培养灭菌方法的研究.广州中医药大学学报(Lin Xiaohua,Li Wei,Hou Xueqin,Li Qiaorong,Wei Jianhong,Li Wei.Disinfection methods for primary cell culture of Pheretima aspergillum.Journal of Guangzhou University of Traditional Chinese Medicine)2011;28(3):264-7.
10 侯雪芹,林小桦,李薇.参环毛蚓不同部位细胞原代培养及鉴定.辽宁中医药大学学报(Hou Xueqin,Lin Xiaohua,Li Wei.Study on primary culture and identification of different organizations cells from Pheretima aspergillum.Journal of Liaoning University of TCM)2011;13(4):38-40.
11 Gong L,Lin X,Lu R,Yu L,Hou X,Li W.establishment of an in vitro culture system for intestinal epithelial cells from Pheretima aspergillum(E.Perreir).In Vitro Cell Dev Biol Anim 2014;50(1):16-21.
12 Fugère N1,Brousseau P,Krzystyniak K,Coderre D,Fournier M.Heavy metal-specific inhibition of phagocytosis and different in vitro sensitivity of heterogeneous coelomocytes from Lumbricus terrestris(Oligochaeta).Toxicology 1996;109(2-3):157-66.
13 熊良伟,王建国,封琦,黄爱军.中华鳖太湖种群淋巴细胞培养及染色体核型分析.中国农学通报(Xiong Liangwei,Wang Jianguo,Feng Qi,Huang Aijun.The lymphocytic cell culture and chromosome karyotype of Trionyx sinensis in Taihu lake population.Chinese Agricultural Science Bulletin)2013;29(5):68-70.
14 王海霞.山羊小肠上皮细胞系的建立及其对氮素的利用(硕士论文).扬州大学(Wang Haixia.Establishment of goat intestinal epithelial cell line and nitrogen metabolism of IEC.Yangzhou University)2008.
15 Perreault N,Beaulieu JF.Use of the dissociating enzyme thermolysin to generate viable human normal intestinal epithelial cell cultures.Exp Cell Res 1996;224(2):354-64.
16 张道杰,蒋建新,陈永华,熊健琼,段朝霞,朱佩芳.用嗜热菌蛋白酶进行人肠上皮细胞分离培养.第三军医大学学报(Zhang Daojie,Jiang Jianxin,Chen Yonghua,Xiong Jianqiong,Duan Zhaoxia,Zhu Peifang.Isolation and culture of human intestinal epithelial cells using dissociating enzyme thermolysin.Acta Academiae Medicinae Militaris Tertiae)2004;26(11):1016-8.
17 孙秀梅,程帆,刘维,孙涛,薛秀恒,李培英,等.4种小鼠肠上皮细胞分离培养方法的比较.西北农林科技大学学报(自然科学版)(Sun Xiumei,Cheng Fan,Liu Wei,Sun Tao,Xue Xiuheng,Li Peiying,et al.Comparison of four primary culture methods for mouse intestinal epithelial cells.Journal of Northwest A&F University,Natural Science Edition)2013;41(5):25-31.
18 王晓亮,顾宪红,郑冬梅.鸡小肠上皮细胞的分离及体外培养研究进展.中国畜牧兽医(Wang Xiaoliang,Gu Xianhong,Zheng Dongmei.The advance on isolation and primary culture of chicken intestine epithelial cell in vitro.China Animal Husbandry&Veterinary Medicine)2010;37(8):34-8.
19 贾永蕊.流式细胞术在DNA检测中的应用.中国医学装备(Jia Yongrui.Application of flow cytometer in detection of DNA.China Medical Equipment)2010;7(4):4-6.
20 Tan X,Chen G.DNA content and cell cycle phase analysis in uterine sarcomas and its clinical significance.Zhonghua Fu Chan Ke Za Zhi 1999;34(9):551-4.
21 许智芳,张德宁,陈钢,张会.三种蚯蚓染色体组型的初步研究.南京大学学报(自然科学版)(Xu Zhifang,Zhang Dening,Chen Gang,Zhang Hui.On karyotype of the earthworms.Journal of Nanjing University,Natural Sciences)1983;2:308-16.
22 许智芳,Brinkhurst RO,张德宁.两种水栖寡毛类染色体核型的研究.南京大学学报(自然科学版)(Xu Zhifang,Brinkhurst RO,Zhang Dening.The aryotype of Lumbriculus variegates(Muller)and Elseniella tetraedra typica(Savigny)(Lumbriculidae,Lumbricidae,Oligochaeta).Journal of Nanjing University,Natural Sciences)1990;26(4):675-82.
23 Bakhtadze NG,Bakhtadze GI,Sh E.The chromosome numbers of Georgian earthworms(Oligochaeta:Llumbricidae).Comp Cytogenet 2008;2(1):79-83.
24 Kashmenskaka MN,Polyakov AV.Karyotype analysis of five species of earthworms(Oligochaeta:Lumbricidae).Comp Cytogenet 2008;2(2):121-5.
25 Murchie WR.Chromosome numbers of some diplocardian earthworms(Megascolecidae-Oligochaeta).Am Midl Nat 1967;78(2):534-7.
26 Muldal S.The chromosomes of the earthworms I.The evolution of polyploidy.Heredity 1952;6:55-76.
27 Vlasenko RP,Mezhzherin SV,Garbar AV,Kotsuba Y.Polyploid races,genetic structure and morphological features of earthworm Aporrectodea rosea(Savigny,1826)(Oligochaeta:Lumbricidae)in Ukraine.Comp Cytogenet 2011;5(2):91-103.
2 欧阳高亮,肖俐,李祺福,洪水根.分子生物学技术在水产养殖中的应用.海洋科学(Ouyang Gaoliang,Xiao Li,Li Qifu,Hong Shuigen.The application of molecular biological techniques in the aquaculture.Marine sciences)2000;24(3):31-4.
3 洪智敏,贾永杰,瞿明仁,黎观红,刘思国.鸡胚小肠上皮细胞的分离及原代培养研究.江西农业大学学报(Hong Zhimin,Jia Yongjie,Qu Mingren,Li Guanhong,Liu Siguo.Isolation and primary culture of chicken embryo small intestinal epithelial cells.Acta Agriculturae Universitatis Jiangxiensis)2011;33(6):1164-70.
4 Chopra DP,Dombkowski AA,Stemmer PM,Parker GC.Intestinal epithelial cells in vitro.Stem Cells Dev 2010;19(1):131-42.
5 王莉,段相林.大鼠小肠上皮细胞的体外原代培养.军事医学(Wang Li,Duan Xianglin.In vitro primary culture of rat intestinal epithelial cells.Military Medical Sciences)2004;28(1):61-3.
6 匡伟,王海霞,邵桃玉,赵国琦.新生山羊小肠上皮细胞株的建立及鉴定.中国畜牧杂志(Kuang Wei,Wang Haixia,Shao Taoyu,Zhao Guoqi.Establishing and identification of neonate goat intestinal epithelial cells.Chinese Journal of Animal Science)2007;43(19):11-4.
7 刘飞飞,车东升,穆成龙,张海全,王波,孙泽威,等.兔小肠上皮细胞体外分离培养.吉林农业大学学报(Liu Feifei,Che Dongsheng,Mu Chenglong,Zhang Haiquan,Wang Bo,Sun Zewei,et al.Isolation and primary culture of rabbit intestinal epithelial cell in vitro.Journal of Jilin Agricultural University)2011;33(4):429-32.
8 杨文平,高峰,许辉堂,施传信,赵国刚,周光宏.鸡肠上皮细胞体外原代培养研究.江西农业学报(Yang Wenping,Gao Feng,Xu Huitang,Shi Chuanxin,Zhao Guogang,Zhou Guanghong.In vitro primary culture of intestinal epithelial cells of chicken.Acta Agriculturae Jiangxi)2007;19(5):113-5.
9 林小桦,李薇,侯雪芹,李巧容,韦建红,李维.参环毛蚓细胞原代培养灭菌方法的研究.广州中医药大学学报(Lin Xiaohua,Li Wei,Hou Xueqin,Li Qiaorong,Wei Jianhong,Li Wei.Disinfection methods for primary cell culture of Pheretima aspergillum.Journal of Guangzhou University of Traditional Chinese Medicine)2011;28(3):264-7.
10 侯雪芹,林小桦,李薇.参环毛蚓不同部位细胞原代培养及鉴定.辽宁中医药大学学报(Hou Xueqin,Lin Xiaohua,Li Wei.Study on primary culture and identification of different organizations cells from Pheretima aspergillum.Journal of Liaoning University of TCM)2011;13(4):38-40.
11 Gong L,Lin X,Lu R,Yu L,Hou X,Li W.establishment of an in vitro culture system for intestinal epithelial cells from Pheretima aspergillum(E.Perreir).In Vitro Cell Dev Biol Anim 2014;50(1):16-21.
12 Fugère N1,Brousseau P,Krzystyniak K,Coderre D,Fournier M.Heavy metal-specific inhibition of phagocytosis and different in vitro sensitivity of heterogeneous coelomocytes from Lumbricus terrestris(Oligochaeta).Toxicology 1996;109(2-3):157-66.
13 熊良伟,王建国,封琦,黄爱军.中华鳖太湖种群淋巴细胞培养及染色体核型分析.中国农学通报(Xiong Liangwei,Wang Jianguo,Feng Qi,Huang Aijun.The lymphocytic cell culture and chromosome karyotype of Trionyx sinensis in Taihu lake population.Chinese Agricultural Science Bulletin)2013;29(5):68-70.
14 王海霞.山羊小肠上皮细胞系的建立及其对氮素的利用(硕士论文).扬州大学(Wang Haixia.Establishment of goat intestinal epithelial cell line and nitrogen metabolism of IEC.Yangzhou University)2008.
15 Perreault N,Beaulieu JF.Use of the dissociating enzyme thermolysin to generate viable human normal intestinal epithelial cell cultures.Exp Cell Res 1996;224(2):354-64.
16 张道杰,蒋建新,陈永华,熊健琼,段朝霞,朱佩芳.用嗜热菌蛋白酶进行人肠上皮细胞分离培养.第三军医大学学报(Zhang Daojie,Jiang Jianxin,Chen Yonghua,Xiong Jianqiong,Duan Zhaoxia,Zhu Peifang.Isolation and culture of human intestinal epithelial cells using dissociating enzyme thermolysin.Acta Academiae Medicinae Militaris Tertiae)2004;26(11):1016-8.
17 孙秀梅,程帆,刘维,孙涛,薛秀恒,李培英,等.4种小鼠肠上皮细胞分离培养方法的比较.西北农林科技大学学报(自然科学版)(Sun Xiumei,Cheng Fan,Liu Wei,Sun Tao,Xue Xiuheng,Li Peiying,et al.Comparison of four primary culture methods for mouse intestinal epithelial cells.Journal of Northwest A&F University,Natural Science Edition)2013;41(5):25-31.
18 王晓亮,顾宪红,郑冬梅.鸡小肠上皮细胞的分离及体外培养研究进展.中国畜牧兽医(Wang Xiaoliang,Gu Xianhong,Zheng Dongmei.The advance on isolation and primary culture of chicken intestine epithelial cell in vitro.China Animal Husbandry&Veterinary Medicine)2010;37(8):34-8.
19 贾永蕊.流式细胞术在DNA检测中的应用.中国医学装备(Jia Yongrui.Application of flow cytometer in detection of DNA.China Medical Equipment)2010;7(4):4-6.
20 Tan X,Chen G.DNA content and cell cycle phase analysis in uterine sarcomas and its clinical significance.Zhonghua Fu Chan Ke Za Zhi 1999;34(9):551-4.
21 许智芳,张德宁,陈钢,张会.三种蚯蚓染色体组型的初步研究.南京大学学报(自然科学版)(Xu Zhifang,Zhang Dening,Chen Gang,Zhang Hui.On karyotype of the earthworms.Journal of Nanjing University,Natural Sciences)1983;2:308-16.
22 许智芳,Brinkhurst RO,张德宁.两种水栖寡毛类染色体核型的研究.南京大学学报(自然科学版)(Xu Zhifang,Brinkhurst RO,Zhang Dening.The aryotype of Lumbriculus variegates(Muller)and Elseniella tetraedra typica(Savigny)(Lumbriculidae,Lumbricidae,Oligochaeta).Journal of Nanjing University,Natural Sciences)1990;26(4):675-82.
23 Bakhtadze NG,Bakhtadze GI,Sh E.The chromosome numbers of Georgian earthworms(Oligochaeta:Llumbricidae).Comp Cytogenet 2008;2(1):79-83.
24 Kashmenskaka MN,Polyakov AV.Karyotype analysis of five species of earthworms(Oligochaeta:Lumbricidae).Comp Cytogenet 2008;2(2):121-5.
25 Murchie WR.Chromosome numbers of some diplocardian earthworms(Megascolecidae-Oligochaeta).Am Midl Nat 1967;78(2):534-7.
26 Muldal S.The chromosomes of the earthworms I.The evolution of polyploidy.Heredity 1952;6:55-76.
27 Vlasenko RP,Mezhzherin SV,Garbar AV,Kotsuba Y.Polyploid races,genetic structure and morphological features of earthworm Aporrectodea rosea(Savigny,1826)(Oligochaeta:Lumbricidae)in Ukraine.Comp Cytogenet 2011;5(2):91-103.