广灵驴成纤维细胞系的建立及其相关生物学特性研究
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摘要
试验旨在建立广灵驴成纤维细胞库,以期在细胞水平上对广灵驴进行保护。本研究以1月龄广灵驴耳缘皮肤组织为试验材料,采用组织块贴壁法进行成纤维细胞培养,建立了广灵驴耳缘皮肤组织成纤维细胞系,并对其相关生物学特性进行了鉴定。结果发现,试验所建立的广灵驴成纤维细胞系大多数细胞呈长梭形,部分细胞呈三角状或星型。在培养过程中,广灵驴原代成纤维细胞在贴壁4d时开始有细胞从组织边缘游离出来,贴壁14d后,细胞汇合率达到80%,可以进行第一次传代培养;细胞生长态势良好,生长曲线呈典型的S型曲线;细胞冻存复苏后活率有所下降,但生长状态良好。细胞分裂中期染色体数2n=62,说明成功建立了广灵驴成纤维细胞系。通过本方法建立的广灵驴成纤维细胞系为后续广灵驴的相关研究奠定了基础。
This study was aimed to establish the fibroblast cell line of Guangling donkey,and protect Guangling donkey at the cell level.In the experiment,the fibroblast cell culture was performed on auricular skin of Guangling donkey using the method of tissue-sticking.The fibroblast cell line was established,and its biological characteristics were studied.The results showed that the shape of most fibroblasts in Guangling donkey were spindle,however,some of them were triangle or star.During the process of culturing,the primary fibroblasts began to dissociate from the edge of the tissue at 4 days of adherence.After 14 days of adherence,the cell confluence rate reached about 80%,and the cells could be subcultured for the first time.The measured cell growth curve which was the typical S-shaped curve showed that the cell growth was in good con-dition.After cryopreservation,the survival rate decreased.The quantity of chromosomes 2 n=62 indicated that the fibroblast cell line of Guangling donkey was successfully established.The fibroblast cell line established by this kind of method provided the foundation for the related research of Guangling donkey.
This study was aimed to establish the fibroblast cell line of Guangling donkey,and protect Guangling donkey at the cell level.In the experiment,the fibroblast cell culture was performed on auricular skin of Guangling donkey using the method of tissue-sticking.The fibroblast cell line was established,and its biological characteristics were studied.The results showed that the shape of most fibroblasts in Guangling donkey were spindle,however,some of them were triangle or star.During the process of culturing,the primary fibroblasts began to dissociate from the edge of the tissue at 4 days of adherence.After 14 days of adherence,the cell confluence rate reached about 80%,and the cells could be subcultured for the first time.The measured cell growth curve which was the typical S-shaped curve showed that the cell growth was in good con-dition.After cryopreservation,the survival rate decreased.The quantity of chromosomes 2 n=62 indicated that the fibroblast cell line of Guangling donkey was successfully established.The fibroblast cell line established by this kind of method provided the foundation for the related research of Guangling donkey.
引文
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[16]向杨,邢健生,白志明,等.近交系五指山小型猪骨髓间充质干细胞分离培养及生物特性[J].中南大学学报(医学版),2015,40(3):261-268.XIANG Y,XING J S,BAI Z M,et al.Separation culture and biological characteristics analysis for bone marrow-derived mesenchymal stem cells from inbreed line miniature pig of Wuzhishan[J].Journal of Central South University(Medical Science),2015,40(3):261-268.(in Chinese)
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[21]张明.大熊猫皮肤成纤维细胞系选育的初步研究[D].雅安:四川农业大学,2003.ZHANG M.Primary studies on selective culturing and establishment of giant panda’s cutaneous fibroblast line[D].Ya’an:Sichuan Agricultural University,2003.(in Chinese)
[22]张静南.马、驴和骡成纤维细胞培养、核型及其G带分析研究[D].呼和浩特:内蒙古大学,2011.ZHANG J N.Fibroblast cell culture,karyotyping and G-banding of the chromosomes of horse,donkey and mule[D].Hohhot:Inner Mongolia University,2011.(in Chinese)
[23]梁仁敏,张伟永,杨子红,等.小鼠中期染色体制备方法探讨[J].生物技术通讯,2010,21(5):718-720.LIANG R M,ZHANG W Y,YANG Z H,et al.Discussion of the methods for obtaining the mouse metaphase chromosomes[J].Letters in Biotechnology,2010,21(5):718-720.(in Chinese)
[24]LI G C,LIU Y F,LIANG J.Isolation and protective effect in UW solution of human hepatocytes during cold storage[J].International Congress Series,2003,1255:217-218.(in Chinese)
[2]马省强.家畜遗传资源多样性保护[J].西北民族学院学报(自然科学版),2002,23(4):24-26.MA S Q.Protection of genetic resources diversities in livestocks[J].Journal of Northwest Minorities University(Natural Science),2002,23(4):24-26.(in Chinese)
[3]张桂贤,郭传甲,李桢,等.广灵驴及其开发利用价值[J].当代畜牧,2002,20(9):26-27.ZHANG G X,GUO C J,LI Z,et al.Guangling donkey and its development and utilization[J].Contemporary Animal Husbandry,2002,20(9):26-27.(in Chinese)
[4]梁全.广灵驴品种的保护与开发[J].中国畜牧业,2015,11(8):53-54.LIANG Q.Protection and development of Guangling donkey[J].China Animal Industry,2015,11(8):53-54.(in Chinese)
[5]张攀,马杰,韩冰毅,等.我国养驴业现状及趋势分析[J].中国畜牧兽医文摘,2013,29(1):36-38.ZHANG P,MA J,HAN B Y,et al.Analysis on the current situation and tendency of donkey raising in China[J].Zhongguo Xumu Shouyi Wenzhai,2013,29(1):36-38.(in Chinese)
[6]李艳红.我国北方五个驴品种染色体特征的研究[D].太谷:山西农业大学,2005.LI Y H.Studies on chromosomal characteristics in five donkey breeds of North China[D].Taigu:Shanxi Agricultural University,2005.(in Chinese)
[7]朱文进,张美俊,葛慕湘,等.中国8个地方驴种遗传多样性和系统发生关系的微卫星分析[J].中国农业科学,2006,39(2):398-405.ZHU W J,ZHANG M J,GE M X,et al.Microsatellite analysis of genetic diversity and phylogenetic relationship of eight donkey breeds in China[J].Scientia Agricultura Sinica,2006,39(2):398-405.(in Chinese)
[8]李建国.四个驴品种微卫星位点的遗传分析[D].太谷:山西农业大学,2004.LI J G.Genetic analysis on microsatellite loci in four donkey breeds[D].Taigu:Shanxi Agricultural University,2004.(in Chinese).
[9]赵朝霞,刘文忠,朱文进,等.六个家驴品种mtDNA D-Loop部分序列的遗传多样性分析[J].中国草食动物,2008,28(4):3-5.ZHAO C X,LIU W Z,ZHU W J,et al.Analysis on the genetic diversity ofpartial sequences in mitochondriai DNA D-loop of six donkey breeds[J].China Herbivores,2008,28(4):3-5.(in Chinese)
[10]李红梅.六个驴品种血清蛋白多态性的研究[D].太谷:山西农业大学,2004.LI H M.A study on serum protein polymorphism of six type ass breeds(lines)[D].Taigu:Shanxi Agricultural University,2004.(in Chinese)
[11]SCOTT G M,RATNAMOHAN V M,RAWLINSON W D.Improving permissive infection of human cytomegalovirus in cell culture[J].Archives of Virology,2000,145(11):2431-2438.(in Chinese)
[12]ZIMMERMANN T,KUNISCH E,PFEIFFER R,et al.Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary culture-primary culture cells markedly differ from fourth-passage cells[J].Arthritis Research,2001,3(1):72-76.
[13]罗军.水牛胎儿成纤维细胞系的建立及其生物学特性的研究[D].南宁:广西大学,2012.LUO J.Establishment and biological characterization of fibroblast cell lines derived from buffalo(Bubalus bubalis)fetus[D].Nanning:Guangxi University,2012.(in Chinese)
[14]GUASTALI M D,MONTEIRO B A,MAZIERO R R,et al.Influence of culture medium and age of bovine blastocysts in established colonies of embryonic stem cells[J].Journal of Stem Cells,2014,9(4):225-234.
[15]郝柱,王颖,彭静,等.金华猪胎儿成纤维细胞系的建立与生物学特性分析[J].农业生物技术学报,2012,20(5):536-542.HAO Z,WANG Y,PENG J,et al.Establishment and biological characteristic analysis of fibroblast cell line for Jinhua pig[J].Journal of Agricultural Biotechnology,2012,20(5):536-542.(in Chinese)
[16]向杨,邢健生,白志明,等.近交系五指山小型猪骨髓间充质干细胞分离培养及生物特性[J].中南大学学报(医学版),2015,40(3):261-268.XIANG Y,XING J S,BAI Z M,et al.Separation culture and biological characteristics analysis for bone marrow-derived mesenchymal stem cells from inbreed line miniature pig of Wuzhishan[J].Journal of Central South University(Medical Science),2015,40(3):261-268.(in Chinese)
[17]吕春荣,王思宇,赵其毅,等.龙陵黄山羊成纤维细胞系的建立及其生物学特性分析[J].中国草食动物科学,2016,36(6):11-13.LV C R,WANG S Y,ZHAO Q Y,et al.Establishment and the biological characteristics of fibroblast cell line in Yunnan Longling Yellow goat[J].China Herbivores,2016,36(6):11-13.(in Chinese)
[18]LASSITER K,DRIDI S,PIEKARSKI A,et al.Bioenergetics in chicken embryo fibroblast cells:Evidence of lower proton leak in spontaneously immortalized chicken embryo fibroblasts compared to young and senescent primary chicken embryo fibroblast cells[J].Comparative Biochemistry and Physiology,Part A,2014,175:115-123.
[19]冯若飞,马忠仁,关伟军,等.天祝白牦牛肾组织成纤维细胞系的建立与生物学特性研究[J].畜牧兽医学报,2008,39(6):726-732.FENG R F,MA Z R,GUAN W J,et al.Establishment and characteristics of Tianzhu White yaks kidney explant fibroblast[J].Acta Veterinaria et Zootechnica Sinica,2008,39(6):726-732.(in Chinese)
[20]李天达,刘丑生,王志刚,等.马头山羊成纤维细胞系的建立与生物学特性分析[J].生物工程学报,2008,24(12):2056-2060.LI T D,LIU C S,WANG Z G,et al.Establishment of fibroblast cell line and its biological characteristics in Matou goat[J].Chinese Journal of Biotechnology,2008,24(12):2056-2060.(in Chinese)
[21]张明.大熊猫皮肤成纤维细胞系选育的初步研究[D].雅安:四川农业大学,2003.ZHANG M.Primary studies on selective culturing and establishment of giant panda’s cutaneous fibroblast line[D].Ya’an:Sichuan Agricultural University,2003.(in Chinese)
[22]张静南.马、驴和骡成纤维细胞培养、核型及其G带分析研究[D].呼和浩特:内蒙古大学,2011.ZHANG J N.Fibroblast cell culture,karyotyping and G-banding of the chromosomes of horse,donkey and mule[D].Hohhot:Inner Mongolia University,2011.(in Chinese)
[23]梁仁敏,张伟永,杨子红,等.小鼠中期染色体制备方法探讨[J].生物技术通讯,2010,21(5):718-720.LIANG R M,ZHANG W Y,YANG Z H,et al.Discussion of the methods for obtaining the mouse metaphase chromosomes[J].Letters in Biotechnology,2010,21(5):718-720.(in Chinese)
[24]LI G C,LIU Y F,LIANG J.Isolation and protective effect in UW solution of human hepatocytes during cold storage[J].International Congress Series,2003,1255:217-218.(in Chinese)