人脂肪干细胞基本生物学特性、表面抗原及成骨、成脂分化潜能的研究
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摘要
目的研究人脂肪干细胞的基本生物学特性、表面抗原特点及其向成骨细胞和脂肪细胞诱导分化潜能。
方法①取健康人腹部皮下脂肪组织,胶原酶消化法分离出脂肪干细胞,进行体外培养,观察其形态特征。②cck—8比色法检测1、3、6、10代脂肪干细胞活性,并绘制细胞生长曲线,计算群体倍增时间。③流式细胞仪检测第1、3、6代脂肪干细胞的细胞周期及表面抗原,SPSS11.0软件分析数据。④取第3代人脂肪干细胞,分别用成骨和成脂诱导培养液诱导其向成骨细胞和脂肪细胞分化,cck—8比色法检测诱导后的细胞活性,并绘制细胞生长曲线,计算群体倍增时间;RT-PCR检测人脂肪干细胞诱导后成骨细胞特异性骨钙蛋白(osteocalcin,OCN)、骨桥蛋白(osteopontin,OPN)及骨保护素(Osteoprotegerin,OPG)基因的表达和脂肪细胞特异性过氧化物酶体增殖物激活受体γ(PPARγ)基因与CCAAT增强子结合蛋白(C/EBPa)基因的表达,Western-blot进一步检测OPG蛋白的表达;碱性磷酸酶(ALP)染色、冯库萨(Von Kossa)染色、油红O染色定性鉴定其成骨和成脂分化潜能。
结果①形态特征:原代及传代的人脂肪干细胞形态均类似成纤维细胞,生长能力旺盛。②生长曲线及倍增时间:1、3、6、10代人脂肪干细胞的生长曲线大致相同,呈近似“S”形,群体倍增时间约为36h。③细胞周期分析:第1、3、6代人脂肪干细胞中,超过80%的细胞都处于G0/G1期。④表面抗原:第1、3、6代脂肪干细胞表面抗原表达无明显区别,P＞0.05。其中,CD10、CD13、CD55、CD59及CD71表达阳性,CD11b、CD14、CD18、CD34、CD45、CD56及HLA-DR表达阴性。⑤诱导分化:人脂肪干细胞经成骨诱导培养后,细胞增殖速度变慢,群体倍增时间延长至约66h,诱导14d后,RT-PCR检测到OCN、OPN基因的表达,同时,RT-PCR、Western-blot检测到OPG基因与蛋白的表达,ALP染色和Von Kossa染色阳性;人脂肪干细胞经成脂诱导后,细胞增殖速度同样变慢,群体倍增时间延长至约70h,诱导14d后,RT-PCR检测到PPARγ与C/EBPa基因的表达,油红O染色阳性。
结论人腹部皮下脂肪组织可分离培养出脂肪干细胞,大多细胞处于静止期,表面抗原CD10、CD13、CD55、CD59、CD71表达呈阳性,CD11b、CD14、CD18、CD34、CD45、CD56、HLA-DR表达呈阴性,体外培养和传代对其表面抗原的表达无明显影响,能向成骨细胞和脂肪细胞诱导分化,有望成为组织工程理想的种子细胞来源之一。
Objective To investigate the basic biological characteristics of human adipose-derived stromal cells(hADSCs) and their surface protein expression,and to explore their osteogenic and adipogenic potential in vitro.
Methods Human adipose tissue was obtained from the abdominal subcutaneous adipose tissue of health patients under the aseptic condition,then the hADSCs were isolated with typeⅠcollagenase digesting method and adherent method and cultured in DMEM containing 10%FBS.The phase-contrast microscope was used to observe hADSCs every day and the viability of 1st,3rd,6th,and 10th passages was assessed by cck-8.The growth cure and doubling time were drawn.Cell cycle and surface proteins include CD 10, CD11b,CD13,CD14,CD18,CD34,CD45,CD55,CD56,CD59,HLA-DR and CD71 on the 1st,3rd,and 6th passage were analyzed by flow cytometry technique,and the data obtained was statistically analyzed by computer with the software SPSS 11.0 for windows,hADSCs from the 3rd passages were induced into osteogenic and adipogenic lineages by different revulsant for 14 days.The viability of hADSCs after induced was also assessed by cck-8.To confirm osteogenesis,cells were examined by RT-PCR for the expression of several genes,including OCN,OPN and OPG and by von Kossa/alkaline phosphatase staining,and the OPG protein expression was further confirmed by Western blotting.Adipogenic differentiation was identified by RT-PCR for the expression of PPARγ,and C/EBPa genes and by Oil Red O staining of intracellular lipid droplets.
Results①Morphous of primary and passage hADSCs:hADSCs were fibroblast-like and could rapidly expand.②Growth curve and doubling time of hADSCs:The growth curve of 1st,3rd,6th,and 10th passages were similar,and like "s" shape.The doubling time of hADSCs was about 36 hours.③The Cell cycle of hADSCs:The majority of cells under undifferentiated conditions were in G0/G1 phase(80%).④Surface marks of hADSCs:No significant difference among the 1st,3rd,and 6th passages could be demonstrated concerning the expression of these surface marks.Expressed proteins include CD10,CD13,CD55,CD59 and CD71,while CD11,CD14,CD18,CD34,CD45, CD56 or HLA-DR are not expressed by undifferentiated hADSCs,P＞0.05.⑤After osteogenic induction for 14 days,the OCN,OPN and OPG genes were detected by RT-PCR, and the OPG protein expression was verified by Western-blot assay,too.As we know,no literature regarding OPG gene and protein expression has been published.The results for alkaline phosphatase and Von Kossa staining were also positive.In the adipogenic induction,PPARγ,and C/EBPa genes can also be detected by RT-PCR,and the results for Oil red O staining were positive.
Conclusion hADSCs can be isolated from human abdominal subcutaneous adipose tissue and most stayed in G0/G1 phase,hADSCs can express the proteins include CD10, CD13,CD55,CD59 and CD71,and the present method we used to isolate and culture hADSCs has no obviously influence on the expression of the surface proteins.It has the potential to differentiate into osteogenic and adipogenic lineage and may be an ideal seed cell source for tissue engineering.
方法①取健康人腹部皮下脂肪组织,胶原酶消化法分离出脂肪干细胞,进行体外培养,观察其形态特征。②cck—8比色法检测1、3、6、10代脂肪干细胞活性,并绘制细胞生长曲线,计算群体倍增时间。③流式细胞仪检测第1、3、6代脂肪干细胞的细胞周期及表面抗原,SPSS11.0软件分析数据。④取第3代人脂肪干细胞,分别用成骨和成脂诱导培养液诱导其向成骨细胞和脂肪细胞分化,cck—8比色法检测诱导后的细胞活性,并绘制细胞生长曲线,计算群体倍增时间;RT-PCR检测人脂肪干细胞诱导后成骨细胞特异性骨钙蛋白(osteocalcin,OCN)、骨桥蛋白(osteopontin,OPN)及骨保护素(Osteoprotegerin,OPG)基因的表达和脂肪细胞特异性过氧化物酶体增殖物激活受体γ(PPARγ)基因与CCAAT增强子结合蛋白(C/EBPa)基因的表达,Western-blot进一步检测OPG蛋白的表达;碱性磷酸酶(ALP)染色、冯库萨(Von Kossa)染色、油红O染色定性鉴定其成骨和成脂分化潜能。
结果①形态特征:原代及传代的人脂肪干细胞形态均类似成纤维细胞,生长能力旺盛。②生长曲线及倍增时间:1、3、6、10代人脂肪干细胞的生长曲线大致相同,呈近似“S”形,群体倍增时间约为36h。③细胞周期分析:第1、3、6代人脂肪干细胞中,超过80%的细胞都处于G0/G1期。④表面抗原:第1、3、6代脂肪干细胞表面抗原表达无明显区别,P＞0.05。其中,CD10、CD13、CD55、CD59及CD71表达阳性,CD11b、CD14、CD18、CD34、CD45、CD56及HLA-DR表达阴性。⑤诱导分化:人脂肪干细胞经成骨诱导培养后,细胞增殖速度变慢,群体倍增时间延长至约66h,诱导14d后,RT-PCR检测到OCN、OPN基因的表达,同时,RT-PCR、Western-blot检测到OPG基因与蛋白的表达,ALP染色和Von Kossa染色阳性;人脂肪干细胞经成脂诱导后,细胞增殖速度同样变慢,群体倍增时间延长至约70h,诱导14d后,RT-PCR检测到PPARγ与C/EBPa基因的表达,油红O染色阳性。
结论人腹部皮下脂肪组织可分离培养出脂肪干细胞,大多细胞处于静止期,表面抗原CD10、CD13、CD55、CD59、CD71表达呈阳性,CD11b、CD14、CD18、CD34、CD45、CD56、HLA-DR表达呈阴性,体外培养和传代对其表面抗原的表达无明显影响,能向成骨细胞和脂肪细胞诱导分化,有望成为组织工程理想的种子细胞来源之一。
Objective To investigate the basic biological characteristics of human adipose-derived stromal cells(hADSCs) and their surface protein expression,and to explore their osteogenic and adipogenic potential in vitro.
Methods Human adipose tissue was obtained from the abdominal subcutaneous adipose tissue of health patients under the aseptic condition,then the hADSCs were isolated with typeⅠcollagenase digesting method and adherent method and cultured in DMEM containing 10%FBS.The phase-contrast microscope was used to observe hADSCs every day and the viability of 1st,3rd,6th,and 10th passages was assessed by cck-8.The growth cure and doubling time were drawn.Cell cycle and surface proteins include CD 10, CD11b,CD13,CD14,CD18,CD34,CD45,CD55,CD56,CD59,HLA-DR and CD71 on the 1st,3rd,and 6th passage were analyzed by flow cytometry technique,and the data obtained was statistically analyzed by computer with the software SPSS 11.0 for windows,hADSCs from the 3rd passages were induced into osteogenic and adipogenic lineages by different revulsant for 14 days.The viability of hADSCs after induced was also assessed by cck-8.To confirm osteogenesis,cells were examined by RT-PCR for the expression of several genes,including OCN,OPN and OPG and by von Kossa/alkaline phosphatase staining,and the OPG protein expression was further confirmed by Western blotting.Adipogenic differentiation was identified by RT-PCR for the expression of PPARγ,and C/EBPa genes and by Oil Red O staining of intracellular lipid droplets.
Results①Morphous of primary and passage hADSCs:hADSCs were fibroblast-like and could rapidly expand.②Growth curve and doubling time of hADSCs:The growth curve of 1st,3rd,6th,and 10th passages were similar,and like "s" shape.The doubling time of hADSCs was about 36 hours.③The Cell cycle of hADSCs:The majority of cells under undifferentiated conditions were in G0/G1 phase(80%).④Surface marks of hADSCs:No significant difference among the 1st,3rd,and 6th passages could be demonstrated concerning the expression of these surface marks.Expressed proteins include CD10,CD13,CD55,CD59 and CD71,while CD11,CD14,CD18,CD34,CD45, CD56 or HLA-DR are not expressed by undifferentiated hADSCs,P＞0.05.⑤After osteogenic induction for 14 days,the OCN,OPN and OPG genes were detected by RT-PCR, and the OPG protein expression was verified by Western-blot assay,too.As we know,no literature regarding OPG gene and protein expression has been published.The results for alkaline phosphatase and Von Kossa staining were also positive.In the adipogenic induction,PPARγ,and C/EBPa genes can also be detected by RT-PCR,and the results for Oil red O staining were positive.
Conclusion hADSCs can be isolated from human abdominal subcutaneous adipose tissue and most stayed in G0/G1 phase,hADSCs can express the proteins include CD10, CD13,CD55,CD59 and CD71,and the present method we used to isolate and culture hADSCs has no obviously influence on the expression of the surface proteins.It has the potential to differentiate into osteogenic and adipogenic lineage and may be an ideal seed cell source for tissue engineering.
引文
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