成年SD大鼠胃窦Cajal间质细胞分离、培养及鉴定
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摘要
目的探讨成年SD大鼠胃窦起搏细胞Cajal间质细胞(interstitial cells of cajal,ICC)的原代分离、培养及鉴定方法,为深入研究其生理功能提供条件。方法 8~9周龄SD成年大鼠,禁食24 h,乙醚吸入麻醉,颈椎脱臼处死。无菌条件下采用精细解剖方法快速取出胃窦平滑肌组织,将其剪成1~2 mm~3小块后,使用Ⅱ型胶原酶消化法于37℃培养箱内消化60 min,离心后过200目筛,接种于DMEM/F12完全培养基(含5 ng/mL干细胞因子)中进行培养。用酪氨酸蛋白激酶受体ckit特异性抗体免疫荧光染色鉴定细胞类型。结果培养72 h后,于倒置显微镜下观察,可见细胞贴壁,呈不规则三角形、星形或梭形,有多个突起;随着培养时间的延长,各突起彼此相互连接形成网络;此原代培养细胞c-kit抗体免疫荧光染色呈阳性。结论成功建立了一套由成年SD大鼠胃窦组织采用Ⅱ型胶原酶消化法原代培养ICC的方法,为ICC的生理功能、病理改变以及胃肠道生理与病理关系的研究奠定了基础。
Objective In order to provide the conditions for further study of its physiological function,the method of isolation,culture and identification of interstitial cells of Cajal(ICC) in the gastric antrum of adult SD rats was investigated.Methods SD rats at 8-9 weeks,were anesthetized with ether and killed by cervical dislocation.The specimens were cut into 1-2 mm~3 small pieces,digested with collagenase type Ⅱ in a 37 ℃ incubator for 60 min,centrifuged and passed through a 200-mesh sieve.The tissues were inoculated in DMEM/F12 complete medium(containing 5 ng/m L stem cell factor).Cell type was identified by immunofluorescence staining using tyrosine protein kinase receptor c-kit specific antibody.Results After culture for 72 h,the cells adhered to the wall under the inverted microscope,which were irregular triangular,star-shaped,or spindle-shaped,with multiple protrusions.With the prolongation of culture period,the protrusions were connected to each other to form a network.The cells were positive for immunofluorescence staining of c-kit antibody.Conclusion This study established a method for primary culture of ICC from type Ⅱ collagenase digestion of gastric antral smooth muscle in adult SD rats.It laid a foundation for the study of the relationship between ICC and gastrointestinal tract in physiological function and pathologic changes.
Objective In order to provide the conditions for further study of its physiological function,the method of isolation,culture and identification of interstitial cells of Cajal(ICC) in the gastric antrum of adult SD rats was investigated.Methods SD rats at 8-9 weeks,were anesthetized with ether and killed by cervical dislocation.The specimens were cut into 1-2 mm~3 small pieces,digested with collagenase type Ⅱ in a 37 ℃ incubator for 60 min,centrifuged and passed through a 200-mesh sieve.The tissues were inoculated in DMEM/F12 complete medium(containing 5 ng/m L stem cell factor).Cell type was identified by immunofluorescence staining using tyrosine protein kinase receptor c-kit specific antibody.Results After culture for 72 h,the cells adhered to the wall under the inverted microscope,which were irregular triangular,star-shaped,or spindle-shaped,with multiple protrusions.With the prolongation of culture period,the protrusions were connected to each other to form a network.The cells were positive for immunofluorescence staining of c-kit antibody.Conclusion This study established a method for primary culture of ICC from type Ⅱ collagenase digestion of gastric antral smooth muscle in adult SD rats.It laid a foundation for the study of the relationship between ICC and gastrointestinal tract in physiological function and pathologic changes.
引文
[1]王曙逢,仇广林,赵志浩,等.大鼠小肠Cajal间质细胞的体外分离、培养及鉴定[J].中国普通外科杂志,2014,23(4):494-498.
[2]刘登群,龙爽,王军平,等.成年C57BL/6小鼠空肠Cajal间质细胞的分离、培养及鉴定.胃肠病学和肝病学杂志[J].2011,20(6):538-541.
[3]柳利明,农晰婷,秦榕,等.体外分离培养小鼠胃Cajal间质细胞的实验方法研究[J].昆明医学院学报,2010,31(3):11-13.
[4]田姣,王宝西,江逊.胃肠道Cajal间质细胞与干细胞因子/ckit信号系统的研究进展[J].临床儿科杂志,2013,31(4):385-388.
[5]Battaglia E,Bassotti G,Bellone G,et al.Loss of interstitial cells of Cajal network in severe idiopathic gastroparesis.World J Gastroenterol[J].2006,12(38):6172-6177.
[6]Negreanu LM,Assor P,Mateescu B,et al.Interstitial cells of Cajal in the gut-a gastroenterologist's point of view.World J Gastroenterol[J]2008,14(41):6285-6288.
[7]Streutker CJ,Huizinga JD,Driman DK,et al.Interstitial cells of Cajal in health and disease.Part I:normal ICC structure and function with associated motility disorders.Histopathology[J].2007,50(2):176-189.
[8]Chen J,Du L,Xiao YT,et al.Disruption of interstitial cells of Cajal networks after massive small bowel resection.World J Gastroenterol[J].2013,19(22):3415-3422.
[9]李泽培,邱野,彭燕.Cajal间质细胞与胃肠动力关系的研究进展[J].胃肠病学和肝病学杂志,2014,23(9):983-986.
[10]王红梅,张立平,陈丽如,等.基于胃食管反流病的胃肠动力与脾虚实质探究[J].中国中西医结合消化杂志,2015,23(3):196-198.
[11]周恒,郭璇,王小娟,等.舒胃汤对功能性消化不良大鼠胃肠动力、血清干细胞因子及Cajal间质细胞修复与再生的影响[J].中华中医药杂志,2015,30(3):863-867.
[12]张旭,徐华.Cajal间质细胞与慢传输型便秘之间关系研究进展[J].临床消化病杂志,2011,23(3):188-191.
[13]祝炼,余保平,吴志轩,等.小鼠空肠Cajal间质细胞的分离、培养和亚甲蓝活染选择性标记[J].世界华人消化杂志,2007,15(32):3439-3442.
[14]刘勇,齐清会.大鼠胃Cajal间质细胞的分离和培养[J].世界华人消化杂志,2005,13(4):73-76.
[15]Shahi PK,Choi S,Jeong YJ,et al.Basal c GMP regulates the resting pacemaker potential frequency of cultured mouse colonic interstitial cells of Cajal[J].Naunyn Schmiedebergs Arch Pharmacol[J].2014,387(7):641-648.
[16]Han S,Kim JS,Jung BK,et al.Effects of ginsenoside on pacemaker potentials of cultured interstitial cells of Cajal clusters from the small intestine of mice[J].Mol Cells,2012,33(3):243-249.
[17]Li X,Xue H,Kang Q,et al.Alterations of the interstitial cells of Cajal and the microstructure of the gastrointestinal tract in KIT distal kinase mutant mice[J].Cell Tissue Res,2014,355(1):49-58.
[18]Zuo DC,Choi S,Shahi PK,et al.Inhibition of pacemaker activity in interstitial cells of Cajal by LPS via NF-κB and MAP kinase[J].World J Gastroenterol,2013,19(8):1210-1218.
[19]Gong YY,Si XM,Lin L,et al.Mechanisms of cholecystokinininduced calcium mobilization in gastric antral interstitial cells of Cajal[J].World J Gastroenterol,2012,18(48):7184-7193.
[2]刘登群,龙爽,王军平,等.成年C57BL/6小鼠空肠Cajal间质细胞的分离、培养及鉴定.胃肠病学和肝病学杂志[J].2011,20(6):538-541.
[3]柳利明,农晰婷,秦榕,等.体外分离培养小鼠胃Cajal间质细胞的实验方法研究[J].昆明医学院学报,2010,31(3):11-13.
[4]田姣,王宝西,江逊.胃肠道Cajal间质细胞与干细胞因子/ckit信号系统的研究进展[J].临床儿科杂志,2013,31(4):385-388.
[5]Battaglia E,Bassotti G,Bellone G,et al.Loss of interstitial cells of Cajal network in severe idiopathic gastroparesis.World J Gastroenterol[J].2006,12(38):6172-6177.
[6]Negreanu LM,Assor P,Mateescu B,et al.Interstitial cells of Cajal in the gut-a gastroenterologist's point of view.World J Gastroenterol[J]2008,14(41):6285-6288.
[7]Streutker CJ,Huizinga JD,Driman DK,et al.Interstitial cells of Cajal in health and disease.Part I:normal ICC structure and function with associated motility disorders.Histopathology[J].2007,50(2):176-189.
[8]Chen J,Du L,Xiao YT,et al.Disruption of interstitial cells of Cajal networks after massive small bowel resection.World J Gastroenterol[J].2013,19(22):3415-3422.
[9]李泽培,邱野,彭燕.Cajal间质细胞与胃肠动力关系的研究进展[J].胃肠病学和肝病学杂志,2014,23(9):983-986.
[10]王红梅,张立平,陈丽如,等.基于胃食管反流病的胃肠动力与脾虚实质探究[J].中国中西医结合消化杂志,2015,23(3):196-198.
[11]周恒,郭璇,王小娟,等.舒胃汤对功能性消化不良大鼠胃肠动力、血清干细胞因子及Cajal间质细胞修复与再生的影响[J].中华中医药杂志,2015,30(3):863-867.
[12]张旭,徐华.Cajal间质细胞与慢传输型便秘之间关系研究进展[J].临床消化病杂志,2011,23(3):188-191.
[13]祝炼,余保平,吴志轩,等.小鼠空肠Cajal间质细胞的分离、培养和亚甲蓝活染选择性标记[J].世界华人消化杂志,2007,15(32):3439-3442.
[14]刘勇,齐清会.大鼠胃Cajal间质细胞的分离和培养[J].世界华人消化杂志,2005,13(4):73-76.
[15]Shahi PK,Choi S,Jeong YJ,et al.Basal c GMP regulates the resting pacemaker potential frequency of cultured mouse colonic interstitial cells of Cajal[J].Naunyn Schmiedebergs Arch Pharmacol[J].2014,387(7):641-648.
[16]Han S,Kim JS,Jung BK,et al.Effects of ginsenoside on pacemaker potentials of cultured interstitial cells of Cajal clusters from the small intestine of mice[J].Mol Cells,2012,33(3):243-249.
[17]Li X,Xue H,Kang Q,et al.Alterations of the interstitial cells of Cajal and the microstructure of the gastrointestinal tract in KIT distal kinase mutant mice[J].Cell Tissue Res,2014,355(1):49-58.
[18]Zuo DC,Choi S,Shahi PK,et al.Inhibition of pacemaker activity in interstitial cells of Cajal by LPS via NF-κB and MAP kinase[J].World J Gastroenterol,2013,19(8):1210-1218.
[19]Gong YY,Si XM,Lin L,et al.Mechanisms of cholecystokinininduced calcium mobilization in gastric antral interstitial cells of Cajal[J].World J Gastroenterol,2012,18(48):7184-7193.