兔脂肪干细胞BrdU标记情况的初步研究
摘要
目的:研究5-溴脱氧尿嘧啶核苷(BrdU)标记脂肪间充质干细胞(ADSCs)的效果,确定体外标记的最佳浓度和时间,评价BrdU标记的ADSCs的适用性。
方法:取6月龄新西兰白兔颈后皮下脂肪,采用Ⅰ型胶原酶消化方法体外分离培养脂肪干细胞,利用成骨、成脂诱导培养基定向向成脂、成骨细胞诱导并用组织化学方法鉴定其诱导结果,从而鉴定其具有多向分化的干细胞潜能。选取第3代ADSCs,分别采用5、10、15、20μmol/L浓度的BrdU体外标记干细胞24、48、72小时,利用免疫组化实验计算不同浓度和标记时间下脂肪干细胞的标记率,确定BrdU对ADSCs的最佳标记方法。通过台盼蓝染色和细胞倍增时间的测量确定最佳标记方法的安全性。对第3代脂肪干细胞采用最佳标记方法标记后更换为普通培养液继续培养,适时传代,连续检测第5、7、9、11代脂肪干细胞的BrdU标记率,了解该标记方法在脂肪干细胞体外扩增培养过程中的衰减情况。同时将标记后的第3代脂肪干细胞作为实验组,未标记细胞作为对照组,分别种植于乳酸-羟基乙酸共聚物(PLGA)支架材料上,体外培养3天后自体回植于动物皮下,4周后取出支架材料,制作石蜡切片采用免疫组化法鉴定BrdU标记的脂肪干细胞在体内标记的效果。
结果:兔脂肪干细胞体外培养具有成纤维细胞样外形,增埴迅速,连续传代14代细胞形态无明显改变,无自发性向脂肪细胞分化现象。成脂诱导12天后油红O染色胞浆内发现红染的油滴,成骨诱导14天后Von kossa染色阳性,由此证明在体外定向诱导后脂肪干细胞可以向脂肪细胞和成骨细胞分化。BrdU可标记脂肪干细胞的核,采用10μmol/L的BrdU体外标记48小时是适宜的标记方法,该方法对脂肪干细胞的增殖无明显影响。初测标记率达95%,标记率随传代次数而降低,传代8次(约体外培养4周)后标记率仍达41%。同期进行的体内实验证实,BrdU标记后的脂肪干细胞移植入体内4周后免疫组化检测有BrdU阳性细胞存在,从而证明移植的脂肪干细胞可以在体内存活。
结论:兔脂肪干细胞易于体外分离培养,具有较强的自我更新能力及多向分化的潜能.BrdU标记脂肪干细胞的操作简单易行,10μmol/L的BrdU标记脂肪干细胞48h后,体外标记效果满意.标记细胞移植体内1月后BrdU仍显示较好的示踪作用,因此BrdU可以用于脂肪干细胞的标记。
Objective:Investigating the effect of labeling rabbit adipose-derived stem cells (ADSCs) with 5-bromo-2'-deoxyuridine(BrdU),and making sure of the optimal method for BrdU labeling in intro to estimate the applicability of labeling ADSCs with BrdU.
Method:Adipose tissue was dessected from the back of 6-months-old New Zealand rabbit neck,then the stem cells were isolated from the adipose tissue and cultured with collagenase digestion.The passaged cells were exposed to different defined mediums for adipocyte- and osteoblast-differentiation,and the biological characteristics of adipocyte and osteoblast were identified by using histochemistry. The third passage ADSCs were incubated with BrdU at different concentrations(5、10、15、20μmol/L) for different incubating time(24、48、72h).Immunohistochemistry was employed to identify the result of labeling,and the labeling rates of different labeling processes were calculated to make sure of the optimal labeling process.The security of the optimal labeling process was surveyed by trypan blue exclusion and calculating cell doubling time.Then the third passage ADSCs with the optimal labeling process were cultured in minimal essential medium without BrdU.Calculate the labeing rates of 5、7、9、11 passsages ADSCs in vitro to observe the attenuation of BrdU.At the third passage,ADSCs labeled with the optimal labeling process and ADSCs which were not labeled were seeded separately in PLGA scaffolds.We auto-transplanted the seeded scaffolds subcutaneously after grow them for 3 days in vitro.4 weeks after the operation,we observed the result of labeling ADSCs with BrdU in vivo through immunohistochemistry.
Result:The stem cells isolated from the rabbit's adipose tissue demonstrated fibroblastlike morphology and could rapidly proliferate.We did not observe obviously change in cell morphology and spontaneous adipocyte-differentiation in serial passages.The ADSCs grown in adipogenic media demonstrated intracellular lipid vacuoles by Oil red-O staining at 12 days,and grown in osteogenic media demonstrated intense positive staining of calcium nodules with Von kossa at 14 days, so ADSCs had the ability to differentiate into adipocytes and osteoblast when treated with defined mediums.BrdU labeled ADSCs nuclei.Incubateing ADSCs with BrdU at 10μmol/L for 48h is the optimal labeling process.It makes no difference in the activity and proliferation of ADSCs whether they were incubated with the optimal labeling process or not.Labeing rates decreased the passages growing.It was about 95%in the preliminary survey,and 41%after 10 passage(about 4 weeks in vitro). BrdU positive staining stem cells can be detected 4 weeks after transplantation in vivo, which demonstrated transplanted stem cells could survive in vivo.
Conclusion:ADSCs of rabbits,isolated easily in this trial,were characterized by quick self-proliferation and multi-directional differentiation in vitro.BrdU at 10μmol/L for 48h could label ADSCs effectively in intro and the process was easy to do.In addition,ADSCs labeled by BrdU can be traced in vivo one month after transplantation.BrdU could be used to label ADSCs.
方法:取6月龄新西兰白兔颈后皮下脂肪,采用Ⅰ型胶原酶消化方法体外分离培养脂肪干细胞,利用成骨、成脂诱导培养基定向向成脂、成骨细胞诱导并用组织化学方法鉴定其诱导结果,从而鉴定其具有多向分化的干细胞潜能。选取第3代ADSCs,分别采用5、10、15、20μmol/L浓度的BrdU体外标记干细胞24、48、72小时,利用免疫组化实验计算不同浓度和标记时间下脂肪干细胞的标记率,确定BrdU对ADSCs的最佳标记方法。通过台盼蓝染色和细胞倍增时间的测量确定最佳标记方法的安全性。对第3代脂肪干细胞采用最佳标记方法标记后更换为普通培养液继续培养,适时传代,连续检测第5、7、9、11代脂肪干细胞的BrdU标记率,了解该标记方法在脂肪干细胞体外扩增培养过程中的衰减情况。同时将标记后的第3代脂肪干细胞作为实验组,未标记细胞作为对照组,分别种植于乳酸-羟基乙酸共聚物(PLGA)支架材料上,体外培养3天后自体回植于动物皮下,4周后取出支架材料,制作石蜡切片采用免疫组化法鉴定BrdU标记的脂肪干细胞在体内标记的效果。
结果:兔脂肪干细胞体外培养具有成纤维细胞样外形,增埴迅速,连续传代14代细胞形态无明显改变,无自发性向脂肪细胞分化现象。成脂诱导12天后油红O染色胞浆内发现红染的油滴,成骨诱导14天后Von kossa染色阳性,由此证明在体外定向诱导后脂肪干细胞可以向脂肪细胞和成骨细胞分化。BrdU可标记脂肪干细胞的核,采用10μmol/L的BrdU体外标记48小时是适宜的标记方法,该方法对脂肪干细胞的增殖无明显影响。初测标记率达95%,标记率随传代次数而降低,传代8次(约体外培养4周)后标记率仍达41%。同期进行的体内实验证实,BrdU标记后的脂肪干细胞移植入体内4周后免疫组化检测有BrdU阳性细胞存在,从而证明移植的脂肪干细胞可以在体内存活。
结论:兔脂肪干细胞易于体外分离培养,具有较强的自我更新能力及多向分化的潜能.BrdU标记脂肪干细胞的操作简单易行,10μmol/L的BrdU标记脂肪干细胞48h后,体外标记效果满意.标记细胞移植体内1月后BrdU仍显示较好的示踪作用,因此BrdU可以用于脂肪干细胞的标记。
Objective:Investigating the effect of labeling rabbit adipose-derived stem cells (ADSCs) with 5-bromo-2'-deoxyuridine(BrdU),and making sure of the optimal method for BrdU labeling in intro to estimate the applicability of labeling ADSCs with BrdU.
Method:Adipose tissue was dessected from the back of 6-months-old New Zealand rabbit neck,then the stem cells were isolated from the adipose tissue and cultured with collagenase digestion.The passaged cells were exposed to different defined mediums for adipocyte- and osteoblast-differentiation,and the biological characteristics of adipocyte and osteoblast were identified by using histochemistry. The third passage ADSCs were incubated with BrdU at different concentrations(5、10、15、20μmol/L) for different incubating time(24、48、72h).Immunohistochemistry was employed to identify the result of labeling,and the labeling rates of different labeling processes were calculated to make sure of the optimal labeling process.The security of the optimal labeling process was surveyed by trypan blue exclusion and calculating cell doubling time.Then the third passage ADSCs with the optimal labeling process were cultured in minimal essential medium without BrdU.Calculate the labeing rates of 5、7、9、11 passsages ADSCs in vitro to observe the attenuation of BrdU.At the third passage,ADSCs labeled with the optimal labeling process and ADSCs which were not labeled were seeded separately in PLGA scaffolds.We auto-transplanted the seeded scaffolds subcutaneously after grow them for 3 days in vitro.4 weeks after the operation,we observed the result of labeling ADSCs with BrdU in vivo through immunohistochemistry.
Result:The stem cells isolated from the rabbit's adipose tissue demonstrated fibroblastlike morphology and could rapidly proliferate.We did not observe obviously change in cell morphology and spontaneous adipocyte-differentiation in serial passages.The ADSCs grown in adipogenic media demonstrated intracellular lipid vacuoles by Oil red-O staining at 12 days,and grown in osteogenic media demonstrated intense positive staining of calcium nodules with Von kossa at 14 days, so ADSCs had the ability to differentiate into adipocytes and osteoblast when treated with defined mediums.BrdU labeled ADSCs nuclei.Incubateing ADSCs with BrdU at 10μmol/L for 48h is the optimal labeling process.It makes no difference in the activity and proliferation of ADSCs whether they were incubated with the optimal labeling process or not.Labeing rates decreased the passages growing.It was about 95%in the preliminary survey,and 41%after 10 passage(about 4 weeks in vitro). BrdU positive staining stem cells can be detected 4 weeks after transplantation in vivo, which demonstrated transplanted stem cells could survive in vivo.
Conclusion:ADSCs of rabbits,isolated easily in this trial,were characterized by quick self-proliferation and multi-directional differentiation in vitro.BrdU at 10μmol/L for 48h could label ADSCs effectively in intro and the process was easy to do.In addition,ADSCs labeled by BrdU can be traced in vivo one month after transplantation.BrdU could be used to label ADSCs.
引文
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