山羊骨髓间充质干细胞体外分离培养与鉴定的实验研究
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摘要
背景与目的
骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)来源于中胚层,主要存在于全身的结缔组织与器官间质中,以骨髓中的含量最多。骨髓间充质干细胞具有自我更新的能力与多向分化、增殖的潜能。骨髓间充质干细胞因为具有可于自体获得、易培养、移植后能够长期存活并且不会出现排斥反应,可被外源基因转染并长期表达的优点,近年来成为组织工程、细胞治疗、基因治疗及器官移植的研究热点,在多学科多领域做为一种理想的种子细胞得到了广泛应用。但是骨髓间充质干细胞虽然在体内的来源比较广泛,但是数量极少,并且随着身体机能的下降与个体的衰老,数量会逐渐减少。而组织工程与细胞工程开展的首要条件是种子细胞的获取和培养传代,如何能够在体外获得足够数量和较高纯度的种子细胞极其重要。而且目前尚没有对骨髓间充质干细胞进行直接鉴定的方法。通过本研究拟建立一种体外分离培养、扩增骨髓间充质干细胞的理想方法,并且观察其生长特性,探讨其鉴定方法,为其在组织工程学与细胞工程学等学科的应用奠定基础。材料与方法
选用健康中国青山羊,骨髓穿刺法抽取髂骨骨髓,应用密度梯度离心法从中分离出骨髓间充质干细胞,结合贴壁筛选法进行培养,显微镜下观察细胞形态与生长情况,测定细胞成活率及传代细胞贴壁率,对第2、4、6代细胞采用连续计数法绘制出生长曲线,计算细胞倍增时间,免疫细胞化学染色法鉴定细胞表面抗原标志。结果
1.骨髓间充质干细胞原代细胞培养的形态观察：培养后24h可见部分细胞贴壁,贴壁细胞多呈椭圆形。48 h后可见更多的细胞贴壁,贴壁细胞有伸展变形现象,细胞伸出伪足,形态渐变为梭形、多角形。4～6d后可见数个细胞集落形成,显微镜下见贴壁细胞的细胞膜及细胞核清晰。随培养进行细胞集落进一步增多,细胞生长逐渐融成片状,12～14d时可长满培养瓶底80%。
2.骨髓间充质干细胞传代细胞培养的形态：细胞传代后6～8d即可长满瓶底,细胞融合率可达100%。传代细胞镜下呈均一长梭形,细胞膜及细胞核清晰,细胞密度较高时呈放射状、漩涡状排列。
3.骨髓间充质干细胞的鉴定结果：第3代骨髓间充质干细胞免疫组化染色,细胞表面抗原CD29,CD44呈阳性表达(胞浆呈黄褐色染色),CD34,CD45呈阴性表达。
4.骨髓间充质干细胞的生长曲线与倍增时间：生长曲线呈S型,1-3d为潜伏期,生长速度较为缓慢；2-3d后进入对数增长期,生长速度明显加快；6-7d后进入平台期,生长速度趋于稳定。第2、4、6代细胞平均倍增时间分别为(25.37±1.04)h,(27.45±0.91)h,(29.49±2.02)h,总体平均倍增时间为(27.44±2.18)h。结论
1.本研究采用密度梯度离心法从山羊骨髓中分离骨髓间充质干细胞,结合贴壁培养法进行体外培养扩增,细胞生长状态良好,体外增殖能力较强。
2.通过鉴定细胞表面抗原标志,结合形态学观察,证实研究中所分离培养细胞是骨髓间充质干细胞。
Background and Objective
Bone marrow mesenchymal stem cells (BMSCs) derived from the mesoderm, mainly exists in the body of the interstitial connective tissue and organs, to a maximum content of the bone marrow. Bone marrow mesenchymal stem cells have self-renewal capacity and multi-differentiation and proliferation potential. Bone marrow mesenchymal stem cells in a specific culture conditions can be differentiated into mesoderm origin of bone cells, stromal cells, cartilage cells, tendon cells and fat cells, bone marrow mesenchymal stem cells can differentiate into endoderm source of hepatic oval cells and ectodermal origin of the glial cells, nerve cells. Bone marrow mesenchymal stem cells because of simple isolation and culture, wide variety of sources, no rejection after transplantation and many other advantages. Bone marrow mesenchymal stem cells in recent years become a tissue engineering cell therapy, gene therapy and organ transplant research focus, in the multi-disciplinary multi-domain as an ideal seed cells have been widely applied. However, bone marrow mesenchymal stem cells, although the source of more widely in the body, but the number of very small, and with the decline in physical function and individual aging, the number will be gradually reduced. Tissue engineering and cell engineering to carry out the first condition is the isolation and cultivation of seed cells. There is now did not find bone marrow mesenchymal stem cells in the direct identification method.The experiment ready to find an ideal method to separate in vitro cultured bone marrow mesenchymal stem cells. Observation of bone marrow-derived mesenchymal stem cell growth characteristics and to explore identification methods. We hope that the experiment can to carry out foundation in tissue engineering and cell engineering.
Materials and methods
Cells were isolated from healthy male goat iliaca bone marrow by density gradient centrifugation, and cultured with attachment method in vitro cell culture technique. The characteristics of the proliferating and growing BMSCs were observed with phase contrast microscope. The growth curve was drawn to determine doubling time, determination seeding efficiency and survival rate of cells, immunocytochemical method identified the BMSC activity and phenotype.
Results
1. Primary cell culture morphology:Primary cultured BMSC were oval, spindle-shaped or polygonal, and adhered to plastic surface within 48 hours, cell growth in good condition and 95% cells were alive in passage first to fifth.
2. Passaged cell culture morphology:Passage cells showed spindle-shaped under the microscope, swirling radial arrangement at high cell density, the passage cells rate of cell adhesion within 12h above 90%, cell can covered bottom of tissue culture flask in 6-8 days.
3. Bone marrow mesenchymal stem cells were identified by Immunocytochemistry: Immunocytochemistry showed the third passage of BMSC were positive for CD29 and CD44,while negative for CD34 and CD45.
4. Cell growth curve and doubling time:Cell growth curve growth curve showed S-type, and were in latent phase at 1-3 days, slow cell growth during this period, in logarithmic growth phase 2-3 days later, and growth rate has accelerated noticeably during this period, cells came into platform phase at 6-7 days later. The mean doubling time was (27.44±2.18) hours.
Conclusion
1. BMSC were successfully isolated from goat bone marrow by density gradient centrifugation and adherent culture method, cell growth in good condition.
2. Identification results confirm that the cultured cells is goat bone marrow mesenchymal stem cells(BMSC).
骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)来源于中胚层,主要存在于全身的结缔组织与器官间质中,以骨髓中的含量最多。骨髓间充质干细胞具有自我更新的能力与多向分化、增殖的潜能。骨髓间充质干细胞因为具有可于自体获得、易培养、移植后能够长期存活并且不会出现排斥反应,可被外源基因转染并长期表达的优点,近年来成为组织工程、细胞治疗、基因治疗及器官移植的研究热点,在多学科多领域做为一种理想的种子细胞得到了广泛应用。但是骨髓间充质干细胞虽然在体内的来源比较广泛,但是数量极少,并且随着身体机能的下降与个体的衰老,数量会逐渐减少。而组织工程与细胞工程开展的首要条件是种子细胞的获取和培养传代,如何能够在体外获得足够数量和较高纯度的种子细胞极其重要。而且目前尚没有对骨髓间充质干细胞进行直接鉴定的方法。通过本研究拟建立一种体外分离培养、扩增骨髓间充质干细胞的理想方法,并且观察其生长特性,探讨其鉴定方法,为其在组织工程学与细胞工程学等学科的应用奠定基础。材料与方法
选用健康中国青山羊,骨髓穿刺法抽取髂骨骨髓,应用密度梯度离心法从中分离出骨髓间充质干细胞,结合贴壁筛选法进行培养,显微镜下观察细胞形态与生长情况,测定细胞成活率及传代细胞贴壁率,对第2、4、6代细胞采用连续计数法绘制出生长曲线,计算细胞倍增时间,免疫细胞化学染色法鉴定细胞表面抗原标志。结果
1.骨髓间充质干细胞原代细胞培养的形态观察：培养后24h可见部分细胞贴壁,贴壁细胞多呈椭圆形。48 h后可见更多的细胞贴壁,贴壁细胞有伸展变形现象,细胞伸出伪足,形态渐变为梭形、多角形。4～6d后可见数个细胞集落形成,显微镜下见贴壁细胞的细胞膜及细胞核清晰。随培养进行细胞集落进一步增多,细胞生长逐渐融成片状,12～14d时可长满培养瓶底80%。
2.骨髓间充质干细胞传代细胞培养的形态：细胞传代后6～8d即可长满瓶底,细胞融合率可达100%。传代细胞镜下呈均一长梭形,细胞膜及细胞核清晰,细胞密度较高时呈放射状、漩涡状排列。
3.骨髓间充质干细胞的鉴定结果：第3代骨髓间充质干细胞免疫组化染色,细胞表面抗原CD29,CD44呈阳性表达(胞浆呈黄褐色染色),CD34,CD45呈阴性表达。
4.骨髓间充质干细胞的生长曲线与倍增时间：生长曲线呈S型,1-3d为潜伏期,生长速度较为缓慢；2-3d后进入对数增长期,生长速度明显加快；6-7d后进入平台期,生长速度趋于稳定。第2、4、6代细胞平均倍增时间分别为(25.37±1.04)h,(27.45±0.91)h,(29.49±2.02)h,总体平均倍增时间为(27.44±2.18)h。结论
1.本研究采用密度梯度离心法从山羊骨髓中分离骨髓间充质干细胞,结合贴壁培养法进行体外培养扩增,细胞生长状态良好,体外增殖能力较强。
2.通过鉴定细胞表面抗原标志,结合形态学观察,证实研究中所分离培养细胞是骨髓间充质干细胞。
Background and Objective
Bone marrow mesenchymal stem cells (BMSCs) derived from the mesoderm, mainly exists in the body of the interstitial connective tissue and organs, to a maximum content of the bone marrow. Bone marrow mesenchymal stem cells have self-renewal capacity and multi-differentiation and proliferation potential. Bone marrow mesenchymal stem cells in a specific culture conditions can be differentiated into mesoderm origin of bone cells, stromal cells, cartilage cells, tendon cells and fat cells, bone marrow mesenchymal stem cells can differentiate into endoderm source of hepatic oval cells and ectodermal origin of the glial cells, nerve cells. Bone marrow mesenchymal stem cells because of simple isolation and culture, wide variety of sources, no rejection after transplantation and many other advantages. Bone marrow mesenchymal stem cells in recent years become a tissue engineering cell therapy, gene therapy and organ transplant research focus, in the multi-disciplinary multi-domain as an ideal seed cells have been widely applied. However, bone marrow mesenchymal stem cells, although the source of more widely in the body, but the number of very small, and with the decline in physical function and individual aging, the number will be gradually reduced. Tissue engineering and cell engineering to carry out the first condition is the isolation and cultivation of seed cells. There is now did not find bone marrow mesenchymal stem cells in the direct identification method.The experiment ready to find an ideal method to separate in vitro cultured bone marrow mesenchymal stem cells. Observation of bone marrow-derived mesenchymal stem cell growth characteristics and to explore identification methods. We hope that the experiment can to carry out foundation in tissue engineering and cell engineering.
Materials and methods
Cells were isolated from healthy male goat iliaca bone marrow by density gradient centrifugation, and cultured with attachment method in vitro cell culture technique. The characteristics of the proliferating and growing BMSCs were observed with phase contrast microscope. The growth curve was drawn to determine doubling time, determination seeding efficiency and survival rate of cells, immunocytochemical method identified the BMSC activity and phenotype.
Results
1. Primary cell culture morphology:Primary cultured BMSC were oval, spindle-shaped or polygonal, and adhered to plastic surface within 48 hours, cell growth in good condition and 95% cells were alive in passage first to fifth.
2. Passaged cell culture morphology:Passage cells showed spindle-shaped under the microscope, swirling radial arrangement at high cell density, the passage cells rate of cell adhesion within 12h above 90%, cell can covered bottom of tissue culture flask in 6-8 days.
3. Bone marrow mesenchymal stem cells were identified by Immunocytochemistry: Immunocytochemistry showed the third passage of BMSC were positive for CD29 and CD44,while negative for CD34 and CD45.
4. Cell growth curve and doubling time:Cell growth curve growth curve showed S-type, and were in latent phase at 1-3 days, slow cell growth during this period, in logarithmic growth phase 2-3 days later, and growth rate has accelerated noticeably during this period, cells came into platform phase at 6-7 days later. The mean doubling time was (27.44±2.18) hours.
Conclusion
1. BMSC were successfully isolated from goat bone marrow by density gradient centrifugation and adherent culture method, cell growth in good condition.
2. Identification results confirm that the cultured cells is goat bone marrow mesenchymal stem cells(BMSC).
引文
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[1]Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cell in human umbilical cord blood[J]. Br J Haematol,2000,109(1):235～242
[2]Bedada FB,Gunther S, Kubin T, et al. Differentiation versus plasticity:fixing the fate of undertermined adult stem cells[J]. Cell Cycle,2006;5(3):223-226
[3]Van Damme A, Vanden Driessehe T,Collen D,et al. Bone marrow stromal cells as targets for gene therapy[J].Curr Gene Ther.2002,2(2):195-209
[4]Niemeyer P,Kornacker M, Mehlhorn A, et al. Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenicdifferentiation in vitro [J]. Tissue Eng,2007; 13(1):111～121
[5]李焕萍,刘春蓉,张永亮.骨髓间充质干细胞在医学领域的研究与应用[J].中国组织工程研究与临床康复,2007,11(28),5622～5625
[6]Kadyala S.Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro[J].Cell Transplant,1997,6(2):125-134
[7]Vaquero J, Zurita M, Oya S, et al. Cell therapy using bone marrow stromal cells in chronic paraplegic rats:systemic or local administration [J]. Neurosci Lett,2006; 398(1～2): 129～134
[8]Tropel P, Noel D, Platet N, et al. Isolation and characterization of mesenchymal stem cells from adult mouse bone marrow[J]. Exp Cell Res,2004,295(2):335～340
[9]Reyes M,Lund T,Lenvik T, et al.Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells[J].Blood 2001:98(9):2615-2625
[10]Majumdar MK, Thiede MA,Mosca JD, et al. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells(MSCs) and stromal cells[J]. J Cell Physiol 1998;176(1):57～66
[11]周德存,严中亚.骨髓间充质干细胞分离培养和应用[J].安徽医学,2003,4(4)：78～79
[12]Delorme B, Chateauvieux S, Charbord P. The concept of mesenchymal stem cells [J]. Regen Med,2006,1(4):497～509
[13]Li CD,Zhang WY,Li HL.Mesenchymal stem cells derived from human placenta suppress allogeneic umbilical cord blood lymphocyte proliferation[J].Cell Res,2005,15(7):539～ 547
[14]Baddoo M, Hill K, Wilkinson R, et al. Characterization of mesenchymal stem cells from murine bone marrow by negative selection[J]. J Cell Biochem,2003,89(6):1235～1249
[15]Tondreau T, Lagneaux L, Dejcneffe M, et al. Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection:phenotype, proliferation kinetics and differentiation potential [J]. Cytotherapy,2004,6(4):372～379
[16]Foster LJ, Zeemann PA, Li C.Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation[J].Stem Cells,2005,23(9):1367～1377
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