骨髓间充质干细胞体外定向分化为神经样细胞的研究
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摘要
脊髓损伤(spinal cord injury, SCI)后所引起的神经源性膀胱症状严重影响患者的生活质量,但目前尚无有效的治疗手段,并且多数研究集中在尿流动力学及神经功能损伤等方面。随着近年来干细胞研究的突飞猛进,干细胞移植用于治疗血液系统恶性肿瘤、神经系统自身免疫疾病并取得有效成果,甚至已经应用于临床,这使通过干细胞联合组织工程移植增加脊髓神经数量、减少胶质瘢痕和空洞的形成成为可能,采用干细胞接种至组织工程进行移植可望成为一种有效治疗SCI后神经源性膀胱的新方法。然而,在组织工程构建中选择合适的种子细胞成为治疗的重要环节。全能干细胞和多能干细胞具有高度自我更新能力和多向分化的潜能,是组织工程中重要的靶细胞。胚胎干细胞移植存在伦理道德和免疫排斥等问题,神经干细胞、嗅鞘细胞及雪旺细胞的来源均有限,且取材不便,很难应用于临床。BMSCs在体外具有强大的增殖能力,而且可以在不同的理化环境和细胞因子的诱导下向骨、脂肪、腱和肌肉等组织分化,还可以跨胚胎分化为神经组织,展示出良好的研究和应前用景。
实验目的:
通过对SD大鼠BMSCs的体外分离纯化和培养扩增,采用免疫荧光染色法对BMSCs进行形态学观察及表型鉴定,并利用其生物学特性进行Nestin免疫细胞化学分析、诱导分化及分化鉴定,探讨BMSCs定向分化为神经样细胞的潜能,为利用BMSCs治疗SCI所引起的神经源性膀胱提供一种理想的种子细胞和一定的理论依据。
实验方法:
1.采用全骨髓贴壁分离法体外提取、分离及纯化大鼠BMSCs,并进行传代扩增;
2.绘制BMSCs的生长曲线,并进行免疫荧光染色观察其形态结构;
3.流式细胞分析仪检测细胞表面抗原CD29、CD34、CD105的阳性表达率;
4.在成骨细胞诱导剂和神经细胞诱导剂条件下定向诱导BMSCs向成骨细胞和神经样细胞分化,并分别行茜素红、Nestin免疫细胞化学染色鉴定;
5.将诱导形成的神经球样细胞团进一步培养分化,并进行MAP-2、GFAP免疫细胞化学染色鉴定分化形成的神经元、星形胶质细胞结构。
实验结果:
1.全骨髓贴壁分离法获得的BMSCs在接种后24～36小时贴壁,梭形,核质比大,呈漩涡状生长,增殖能力强;
2.细胞生长经过潜伏适应期、对数增长期、平台期阶段;免疫荧光染色后在荧光显微镜下可见全部细胞均呈绿色荧光,细胞为梭形或多边形,细胞核未染荧光,保持了良好的形态;
3.流式细胞分析仪显示P3～P5代细胞表面抗原CD29、CD105表达阳性,而CD34表达阴性;
4.在两种不同诱导剂诱导下BMSCs能分化形成成骨细胞和神经样细胞并表达相应的特异性抗原;
5.进一步培养分化所形成的细胞能表达神经元标志物MAP-2或神经胶质细胞标志物GFAP。
结论:
1.全骨髓贴壁分离法可分离出较纯的BMSCs,经培养扩增后能获取大量的BMSCs,细胞呈贴壁生长,增殖能力强;
2.BMSCs具有多向分化的潜能,在体外可诱导分化为成骨细胞及神经样细胞;
3.分化形成的神经球样细胞能进一步分化为神经元和神经胶质细胞,是干细胞移植治疗的理想种子细胞,具有极大的应用前景。
Symptoms of neurogenic bladder caused by spinal cord injury affect the quality of patient life severely, but there is no effective treatment currently, futhermore, most studies focused on the area of urodynamic and neurological damage. With the rapid development of stem cell research, not only stem cell transplantation has been used to treat the blood system cancer and autoimmune disease of nervous system, but also has achieved effective results, Even has been used in clinical. It is possible that the addition of the number of spinal nerves and the reduction of glial scar transplanted by stem cell together with tissue engineering, which will become the effective method of treatment of neurogenic bladder caused by spinal cord injury. However, suitable choice on seeding cell become important treatment in the construction of tissue engineering. All stem cells and pluripotent stem cells have a high degree of self-renewal ability and potential capability of differentiation, which is an important target cells in tissue engineering. Embryonic stem cells exist ethical issues and immune rejection, source of neural stem cells and schwann cell are limited, and cells derived inconvenience. Therefore , clinical application is difficult. BMSCs have the power ability of proliferation in vitro, moreover, can be differentiated into bone, fat, tendon and muscle in different physical and chemical environment, even can be differentiated into nerve tissue across embryos, which show the favorable prospect of research and application.
Objective
Used the whole bone marrow adherent method, BMSCs of SD rat were isolated, purified and amplified in vitro. To observe the structure of morphology and phenotypic identification, we conduct immunofluorescence dyeing and detection of flow cytometry. Biological characteristics of BMSCs were used to conduct analysis of immunocytochemistry, induction and identification of differentiation. then we explore potential ability of BMSCs which differentiate into neural-like cells. The experimental study provides an ideal seeding cells and a theoretical basis for the treatment of neurogenic bladder caused by spinal cord injury.
Methods
1. BMSCs of SD rat were extracted, isolated, purified and amplified by the whole bone marrow adherent method in vitro;
2. We drew the growth curve of BMSCs, and conduct immunofluorescence dyeing for observation on the structure of BMSCs;
3. The positive expression of cell surface antigens CD29, CD34 and CD105 were detected by flow cytometry;
4. BMSCs were induced to differentiate into osteoblasts and nerve cell under the inducer of osteoblast and nerve cell conditions, moreover,were stained by alizarin red and nestin immunocytochemistry staining respectively;
5. The neurospheres of induced formation were cultured and differentiated furtherly. To identify the formation of neurons and glial cells, BMSCs were stained by MAP-2, GFAP immunocytochemistry staining.
Results
1. BMSCs obtained by the whole bone marrow adherent method attachs the bottle after 24～36 hours, showed the growth of spindle whirlpool and the power ability of proliferation, nuclear-cytoplasmic ratio of BMSCs is large;
2. Cell growth pass the potential adaptation period, the increased logarithmic phase and plateau phase, the whole cells show green fluorescent and good shape, nucleus was not stained fluorescence;
3. Flow cytometry showed the positive expression of the 3～5 passage cell surface antigens CD29 and CD105,while CD34 expression was negative;
4. Under the different inducer conditions, BMSCs can be differentiated into osteoblast and nerve-like cells, moreover, expressed the corresponding antigen;
5. The neurospheres of induced formation furtherly were able to express neuronal markers MAP-2 or the glial marker GFAP.
Conclusion
1. The Whole bone marrow adherent method can separate BMSCs of high purity and obtain a large number of BMSCs adhered to the wall and owned the power ability of proliferation;
2. BMSCs have more potential to differentiate, and can be differentiated into osteoblast and nerve-like cells in vitro;
3. The neurospheres of induced formation can be differentiated into osteoblast and nerve-like cells futherly, BMSCs is an ideal seed cells in the transplantation of stem cell, which possess glorious prospects.
实验目的:
通过对SD大鼠BMSCs的体外分离纯化和培养扩增,采用免疫荧光染色法对BMSCs进行形态学观察及表型鉴定,并利用其生物学特性进行Nestin免疫细胞化学分析、诱导分化及分化鉴定,探讨BMSCs定向分化为神经样细胞的潜能,为利用BMSCs治疗SCI所引起的神经源性膀胱提供一种理想的种子细胞和一定的理论依据。
实验方法:
1.采用全骨髓贴壁分离法体外提取、分离及纯化大鼠BMSCs,并进行传代扩增;
2.绘制BMSCs的生长曲线,并进行免疫荧光染色观察其形态结构;
3.流式细胞分析仪检测细胞表面抗原CD29、CD34、CD105的阳性表达率;
4.在成骨细胞诱导剂和神经细胞诱导剂条件下定向诱导BMSCs向成骨细胞和神经样细胞分化,并分别行茜素红、Nestin免疫细胞化学染色鉴定;
5.将诱导形成的神经球样细胞团进一步培养分化,并进行MAP-2、GFAP免疫细胞化学染色鉴定分化形成的神经元、星形胶质细胞结构。
实验结果:
1.全骨髓贴壁分离法获得的BMSCs在接种后24～36小时贴壁,梭形,核质比大,呈漩涡状生长,增殖能力强;
2.细胞生长经过潜伏适应期、对数增长期、平台期阶段;免疫荧光染色后在荧光显微镜下可见全部细胞均呈绿色荧光,细胞为梭形或多边形,细胞核未染荧光,保持了良好的形态;
3.流式细胞分析仪显示P3～P5代细胞表面抗原CD29、CD105表达阳性,而CD34表达阴性;
4.在两种不同诱导剂诱导下BMSCs能分化形成成骨细胞和神经样细胞并表达相应的特异性抗原;
5.进一步培养分化所形成的细胞能表达神经元标志物MAP-2或神经胶质细胞标志物GFAP。
结论:
1.全骨髓贴壁分离法可分离出较纯的BMSCs,经培养扩增后能获取大量的BMSCs,细胞呈贴壁生长,增殖能力强;
2.BMSCs具有多向分化的潜能,在体外可诱导分化为成骨细胞及神经样细胞;
3.分化形成的神经球样细胞能进一步分化为神经元和神经胶质细胞,是干细胞移植治疗的理想种子细胞,具有极大的应用前景。
Symptoms of neurogenic bladder caused by spinal cord injury affect the quality of patient life severely, but there is no effective treatment currently, futhermore, most studies focused on the area of urodynamic and neurological damage. With the rapid development of stem cell research, not only stem cell transplantation has been used to treat the blood system cancer and autoimmune disease of nervous system, but also has achieved effective results, Even has been used in clinical. It is possible that the addition of the number of spinal nerves and the reduction of glial scar transplanted by stem cell together with tissue engineering, which will become the effective method of treatment of neurogenic bladder caused by spinal cord injury. However, suitable choice on seeding cell become important treatment in the construction of tissue engineering. All stem cells and pluripotent stem cells have a high degree of self-renewal ability and potential capability of differentiation, which is an important target cells in tissue engineering. Embryonic stem cells exist ethical issues and immune rejection, source of neural stem cells and schwann cell are limited, and cells derived inconvenience. Therefore , clinical application is difficult. BMSCs have the power ability of proliferation in vitro, moreover, can be differentiated into bone, fat, tendon and muscle in different physical and chemical environment, even can be differentiated into nerve tissue across embryos, which show the favorable prospect of research and application.
Objective
Used the whole bone marrow adherent method, BMSCs of SD rat were isolated, purified and amplified in vitro. To observe the structure of morphology and phenotypic identification, we conduct immunofluorescence dyeing and detection of flow cytometry. Biological characteristics of BMSCs were used to conduct analysis of immunocytochemistry, induction and identification of differentiation. then we explore potential ability of BMSCs which differentiate into neural-like cells. The experimental study provides an ideal seeding cells and a theoretical basis for the treatment of neurogenic bladder caused by spinal cord injury.
Methods
1. BMSCs of SD rat were extracted, isolated, purified and amplified by the whole bone marrow adherent method in vitro;
2. We drew the growth curve of BMSCs, and conduct immunofluorescence dyeing for observation on the structure of BMSCs;
3. The positive expression of cell surface antigens CD29, CD34 and CD105 were detected by flow cytometry;
4. BMSCs were induced to differentiate into osteoblasts and nerve cell under the inducer of osteoblast and nerve cell conditions, moreover,were stained by alizarin red and nestin immunocytochemistry staining respectively;
5. The neurospheres of induced formation were cultured and differentiated furtherly. To identify the formation of neurons and glial cells, BMSCs were stained by MAP-2, GFAP immunocytochemistry staining.
Results
1. BMSCs obtained by the whole bone marrow adherent method attachs the bottle after 24～36 hours, showed the growth of spindle whirlpool and the power ability of proliferation, nuclear-cytoplasmic ratio of BMSCs is large;
2. Cell growth pass the potential adaptation period, the increased logarithmic phase and plateau phase, the whole cells show green fluorescent and good shape, nucleus was not stained fluorescence;
3. Flow cytometry showed the positive expression of the 3～5 passage cell surface antigens CD29 and CD105,while CD34 expression was negative;
4. Under the different inducer conditions, BMSCs can be differentiated into osteoblast and nerve-like cells, moreover, expressed the corresponding antigen;
5. The neurospheres of induced formation furtherly were able to express neuronal markers MAP-2 or the glial marker GFAP.
Conclusion
1. The Whole bone marrow adherent method can separate BMSCs of high purity and obtain a large number of BMSCs adhered to the wall and owned the power ability of proliferation;
2. BMSCs have more potential to differentiate, and can be differentiated into osteoblast and nerve-like cells in vitro;
3. The neurospheres of induced formation can be differentiated into osteoblast and nerve-like cells futherly, BMSCs is an ideal seed cells in the transplantation of stem cell, which possess glorious prospects.
引文
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5. Nirmalanandhan VS, Dressler MR, Shearn JT, Juncosa-Melvin N, Rao M, Gooch C, Bradica G, Butler DL. Mechanical stimulation of tissue engineered tendon constructs: effect of scaffold materials.J Biomech Eng. 2007 Dec;129(6):919-23.
6. Lemon G, Waters SL, Rose FR, King JR. Mathematical modelling of human mesenchymal stem cell proliferation and differentiation inside artificial porous scaffolds.J Theor Biol. 2007 Dec 7;249(3):543-53.
7. Okumoto K, Saito T , Hattori E , et al. Differentiation of bone marrow cells into cells that express liverspecific genes in vitro : implication of the notch signals in differentiation. Biochem Biophys ResCommun , 2003 , 304 (4) : 691-695.
8. Lee KD , Kuo TK, WhangPeng J , et al. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology , 2004 , 40(6) : 1275-1284.
9. Woodbury D, Schwarz EJ, Prockop DJ, et al. Adultrat and human marrow stromal cells differentiate into neurons[J] . J Neurosci Res ,2000 ,61 :364– 370
10. Eslaminejad MB, Mirzadeh H, Mohamadi Y, Nickmahzar A. Bone differentiation of marrow-derived mesenchymal stem cells using beta-tricalciumphosphate-alginate-gelatin hybrid scaffolds.J Tissue Eng Regen Med. 2007 Nov-Dec; 1(6):417-24.
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16. Del Popolo G, Panariello G, Del Corso F, De Scisciolo G, Lombardi G. Diagnosis and therapy for neurogenic bladder dysfunctions in multiple sclerosis patients. Neurol Sci. 2008 Dec;29 Suppl 4:S352-5.
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25. Doyeob Kim, Gregory R, Dressler. Nephrogenic Factors Promote Differentiation of Mouse Embryonic Stem Cells into Renal E pithelia. JAm Soc Nephrol, 2005, 16: 3527 - 3534.
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1. Guo X, Zheng Q, Kulbatski I, Yuan Q, Yang S, Shao Z, Wang H, Xiao B, Pan Z, Tang S. Bone regeneration with active angiogenesis by basic fibroblast growth factor gene transfected mesenchymal stem cells seeded on porous beta-TCP ceramic scaffolds.Biomed Mater. 2006 Sep;1(3):93-9.
2. Baron F, Gothot A. Mesenchymal stem cells: a new versatile therapeutic option. Rev Med Liege. 2007;62 Spec No:9-14.
3. Meseguer-Olmo L, Bernabeu-Esclapez A, Ros-Martinez E, Sánchez-Salcedo S, Padilla S, Martín AI, Vallet-RegíM, Clavel-Sainz M, Lopez-Prats F, Meseguer-Ortiz CL.In vitro behaviour of adult mesenchymal stem cells seeded on a bioactive glass ceramic.Acta Biomater. 2008 Jul;4(4):1104-13.
4. Tae SK, Lee SH, Park JS, Im GI. Mesenchymal stem cells for tissue engineering and regenerative medicine.Biomed Mater. 2006 Jun;1(2):63-71.
5. Nirmalanandhan VS, Dressler MR, Shearn JT, Juncosa-Melvin N, Rao M, Gooch C, Bradica G, Butler DL. Mechanical stimulation of tissue engineered tendon constructs: effect of scaffold materials.J Biomech Eng. 2007 Dec;129(6):919-23.
6. Lemon G, Waters SL, Rose FR, King JR. Mathematical modelling of human mesenchymal stem cell proliferation and differentiation inside artificial porous scaffolds.J Theor Biol. 2007 Dec 7;249(3):543-53.
7. Okumoto K, Saito T , Hattori E , et al. Differentiation of bone marrow cells into cells that express liverspecific genes in vitro : implication of the notch signals in differentiation. Biochem Biophys ResCommun , 2003 , 304 (4) : 691-695.
8. Lee KD , Kuo TK, WhangPeng J , et al. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology , 2004 , 40(6) : 1275-1284.
9. Woodbury D, Schwarz EJ, Prockop DJ, et al. Adultrat and human marrow stromal cells differentiate into neurons[J] . J Neurosci Res ,2000 ,61 :364– 370
10. Eslaminejad MB, Mirzadeh H, Mohamadi Y, Nickmahzar A. Bone differentiation of marrow-derived mesenchymal stem cells using beta-tricalciumphosphate-alginate-gelatin hybrid scaffolds.J Tissue Eng Regen Med. 2007 Nov-Dec; 1(6):417-24.
11. Gao K, Lu Y, Li S, Cong C, Yuan Y, Zhang J, Cheng J, Li H, Wang L. Isolation, culturing and growth characteristics of mesenchymal stem cells from bone marrow of Rhesus monkey, Macaca mulatta.Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2007 Dec;24(6):1343-7.
12. Sanchez-Ramos J, Song S, Cardozo-Pelaez F. Adult bone marrow stomal cells differentiate into neural cells in vivo.Exp Neurol,2000,164:247.
13. ItoT, SuzukiA,ImaiE,et al.Bone marrow is a reservoir of repopulating mesangial cells during glomerular remodeling. [J]. J Am SocNephrol, 2001, 12(12): 2625- 2635.
14. Rookmaaker MB, Smits AM, Tolboom H,et al. Bone - marrow- derived cells contribute to glomerular endothelial repair in experimental glomerulonephritis. [J]. Am J pathol, 2003, 163(2):553– 562
15. Kwon BK,Fisher CG,Dvorak MF,et al. Strategies to promote Neural repair and regeneration after spinal cord injury. Spine,2005,30(17 Suppl):3-13.
16. Del Popolo G, Panariello G, Del Corso F, De Scisciolo G, Lombardi G. Diagnosis and therapy for neurogenic bladder dysfunctions in multiple sclerosis patients. Neurol Sci. 2008 Dec;29 Suppl 4:S352-5.
17.黎鳌,盛志勇,王正国主编.现代战伤外科学.北京:人民军医出版社,1998:856.
18. Guo JS, Zeng YS, Li HB. Cotransplant of neural stem cells and NT-3 gene modified schwann cells promote the recovery of transected spinal cord injury. Spinal Cord, 2007,45(1): 15-24.
19. Hofstetter CP, Schwarz EJ , Hess D, et al. Marrow stromal cellsfrom guiding strands in the injured spinal cord and promo terecovery. Proc NatlA cad Sci USA , 2002, 99 (4) : 2199-2204.
20. Satake K, Lou J , Lenke L G. Migration of mesenchymal stemcells through cerebro spinal fluid into injured spinal cord tissue. Spine, 2004, 29 (18) : 1971-1979.
21.步星耀,张永福.骨髓间质干细胞移植治疗脊髓损伤.实用诊断与治疗杂志,2004,18 (14) : 259-261.
22. Ren T, Ren J, Jia X. The bone formation in vitro and mandibular defect repair using PLGA porous scaffolds. Biomed Mater Res A 2005,74:562-569.
23. Day RM, Boccaccini AR, Maquel V. In vivo characterisution of a novel bioresorbable poly (lactide-co-glycolide) tubular foam scaffold for tissue engineering applications. Mater Sci Mater Med,2004,15:729-734.
24. Badami AS, Kreke MR, Thompson MS. Effect of fiber diameter on spreading, proliferation,and differentiation of osteoblastic cells on electrospun poly(lactic acid) substrates. Biomaterials,2006,27:596-606.
25. Doyeob Kim, Gregory R, Dressler. Nephrogenic Factors Promote Differentiation of Mouse Embryonic Stem Cells into Renal E pithelia. JAm Soc Nephrol, 2005, 16: 3527 - 3534.