新生大鼠胰腺干细胞的分离和培养及初步鉴定
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摘要
目的探讨分离、培养及初步鉴定新生大鼠胰腺干细胞的方法。方法取30只无特定病原体(SPF)级新生大鼠的胰腺组织,用完整胰腺胶原酶消化法分离胰岛。以低糖改良Eagel培养液为基础培养液,于不同时期分别添加血清、碱性成纤维细胞生长因子、表皮细胞生长因子、胰岛素-转铁蛋白-硒添加剂、白血病抑制因子、神经干细胞原代培养的无血清培养液中的营养因子B27、神经元培养的无血清培养液中的营养因子N2等进行培养,倒置相差显微镜下观察其形态学特征及生长特性。应用免疫细胞化学法对培养不同时期的细胞进行巢蛋白(Nestin)及细胞角质蛋白19(CK-19)检测,并进行初步鉴定。结果 1新生大鼠胰腺内纤维组织少,体积小,完整胰腺胶原酶消化法简化了胰岛分离步骤,减少了污染机会,有利于进一步培养。2培养24 h可见细胞贴壁,但贴壁细胞数量较少,改用无血清培养后,贴壁细胞快速生长,10~15 d形成单层细胞汇集,细胞形态较一致。3培养所得细胞一直较多表达胰腺干细胞的标志物Nestin,随培养时间延长,表达CK-19的细胞数量逐渐增多。结论新生大鼠胰腺消化后,获得的细胞于不同时期表达胰腺干细胞的标志物Nestin、CK-19。
Objective To explore the method of isolation,cultivation and identification of pancreatic stem cells in newborn rats. Methods A total of 30 newborn rats were bought from Experimental Animal Center,Inner Mongolia University. Get the rats 1 h before experiments. The whole pancreas of newborn rats were digested with collagenase,and cultivated with the basic Eagel culture solution modified with low. Serum,b FGF,EGF,ITS,LIF,N2,B27 were added to culture solution during different phases,the digested tissue fragments were cultured. The whole formation process of new pancreatic stem cells was observed under the inverted phase contrast microscope. The expression of Nestin and CK-19 was tested using immunocytochemical method. Results 1Because there was little fibrous tissue in newborn rat,its pancreas and the volume of pancreas were tiny,the opportunity of contamination was greatly reduced,so as to make further experiments.2After 24 hours,some cells adhered to the surface of the culture plates,but the number of them was small,then we removed the liquid,and added medium of free serum,the number of them increased obviously,10-15 days later,a newly formed monolayer of cells could be observed,their shapes were uniform. 3 During the cultivation period,the cells came from the pancreas of newborn rats expressed large amount of Nestin,and as the cultivation time prolonged,the expression of CK-19 increased. Conclusion After digestion of newborn rat's pancreas,the cells express Nestin and CK-19,which are the markers of the expression of pancreatic stem cells,during different periods.
Objective To explore the method of isolation,cultivation and identification of pancreatic stem cells in newborn rats. Methods A total of 30 newborn rats were bought from Experimental Animal Center,Inner Mongolia University. Get the rats 1 h before experiments. The whole pancreas of newborn rats were digested with collagenase,and cultivated with the basic Eagel culture solution modified with low. Serum,b FGF,EGF,ITS,LIF,N2,B27 were added to culture solution during different phases,the digested tissue fragments were cultured. The whole formation process of new pancreatic stem cells was observed under the inverted phase contrast microscope. The expression of Nestin and CK-19 was tested using immunocytochemical method. Results 1Because there was little fibrous tissue in newborn rat,its pancreas and the volume of pancreas were tiny,the opportunity of contamination was greatly reduced,so as to make further experiments.2After 24 hours,some cells adhered to the surface of the culture plates,but the number of them was small,then we removed the liquid,and added medium of free serum,the number of them increased obviously,10-15 days later,a newly formed monolayer of cells could be observed,their shapes were uniform. 3 During the cultivation period,the cells came from the pancreas of newborn rats expressed large amount of Nestin,and as the cultivation time prolonged,the expression of CK-19 increased. Conclusion After digestion of newborn rat's pancreas,the cells express Nestin and CK-19,which are the markers of the expression of pancreatic stem cells,during different periods.
引文
[1]Wang W,Walker JR,Wang X,et al.Identification of small-molecule inducers of pancreatic beta-cell expansion[J].Proc Nati Acad Sci U S A,2009,106(5):1427-1432.
[2]Suen PM,Leung PS.Pancreatic stem cells:a glimmer of hope for diabetes?[J].JOP,2005,6(5):422-424.
[3]Yatoh S,Dodge R,Akashi T,et al.Differentiation of affinity-purified human pancreatic duct cells toβ-cells[J].Diabetes,2007,56(7):1802-1809.
[4]史恺,徐立,邓仪昊.昆明小鼠胰腺导管上皮样细胞的培养方法研究[J].四川解剖学杂志,2006,14(4):72-73.
[5]白日星,田井琦,张海燕.新生鼠胰岛干细胞分离、培养和分化的实验研究[J].中国普通外科杂志,2004,13(10):746-749.
[6]Kajiyama H,Hamazaki TS,Tokuhara M,et al.Pdx1-transfected adipose tissue-derived stem cells differentiate into insulin-producing cells in vivo and reduce hyperglycemia in diabetic mice[J].Int J Dev Biol,2010,54(4):699-705.
[7]Claiborne KC,Stoffers DA.Toward a cell-based cure for diabetes:advances in production and transplant of beta cells[J].Mt Sinai J Med,2008,75(4):362-371.
[8]Gioviale MC,Bellavia M,Damiano G,et al.Beyond islet transplantation in diabetes cell therapy:form embrymic stem cells to transdifferentiation of adult cells[J].Transplant Proc,2013,45(5):2019-2024.
[2]Suen PM,Leung PS.Pancreatic stem cells:a glimmer of hope for diabetes?[J].JOP,2005,6(5):422-424.
[3]Yatoh S,Dodge R,Akashi T,et al.Differentiation of affinity-purified human pancreatic duct cells toβ-cells[J].Diabetes,2007,56(7):1802-1809.
[4]史恺,徐立,邓仪昊.昆明小鼠胰腺导管上皮样细胞的培养方法研究[J].四川解剖学杂志,2006,14(4):72-73.
[5]白日星,田井琦,张海燕.新生鼠胰岛干细胞分离、培养和分化的实验研究[J].中国普通外科杂志,2004,13(10):746-749.
[6]Kajiyama H,Hamazaki TS,Tokuhara M,et al.Pdx1-transfected adipose tissue-derived stem cells differentiate into insulin-producing cells in vivo and reduce hyperglycemia in diabetic mice[J].Int J Dev Biol,2010,54(4):699-705.
[7]Claiborne KC,Stoffers DA.Toward a cell-based cure for diabetes:advances in production and transplant of beta cells[J].Mt Sinai J Med,2008,75(4):362-371.
[8]Gioviale MC,Bellavia M,Damiano G,et al.Beyond islet transplantation in diabetes cell therapy:form embrymic stem cells to transdifferentiation of adult cells[J].Transplant Proc,2013,45(5):2019-2024.