脂肪干细胞移植对大鼠脑出血模型细胞凋亡的影响
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摘要
干细胞是体内存在的一类具有可以自我更新,同时具有多向分化潜能的特殊细胞。随着近期组织工程和基因治疗技术的研究进展,寻找合适的种子细胞及细胞替代治疗已成为当前生命科学研究的热点,胚胎干细胞与神经干细胞的发现为神经修复开辟了一个有效途径,并且对中枢神经系统退行性疾病治疗采取的细胞替代治疗即细胞移植已取得疗效肯定。但是无论是胚胎干细胞还是神经干细胞都存在着取材来源少,耗材多,生长繁殖慢等缺点以及受法律、道德、伦理的限制,制约它们的发展及应用。
骨髓基质细胞来源于中胚层间充质多能干细胞,具有自我复制及多向分化潜能。骨髓基质细胞移植研究日益深入,已应用于临床治疗心肌梗塞,并且移植到脑内可改善神经退行性疾病的神经功能。然而骨髓基质细胞获取时需通过麻醉后抽取骨髓或取肋骨,给病人带来痛苦,获取数量有限,培养时间长等问题限制其发展。
脂肪组织与骨髓一样来源于中胚层,脂肪间充质干细胞量多且可以多向分化。脂肪组织可以自体抽取,来源丰富,可自体细胞移植,无免疫排斥反应,避免疾病传播,是干细胞生物工程研究的理想来源。
目的:
本实验应用酶消化法,对SD大鼠腹股沟脂肪组织间充质干细胞分离培养,ADSC大鼠脑内出血模型移植后进行神经功能测评,观察ADSC脑内移植后脑内血肿周围神经细胞凋亡的情况,探讨ADSC脑内移植对脑内血肿周围神经细胞凋亡的影响及机制。
方法:
脂肪干细胞培养、增殖、鉴别及诱导
健康成年Sprague-Dawley(S-D)大鼠,过量麻醉处死,无菌条件下取腹股沟脂肪,用PBS反复冲洗,胶原酶消化脂肪组织进行ADSC分离培养。对ADSC在体外进行传代培养,观察其增殖能力。对培养的脂肪干细胞进行成脂诱导。
脂肪干细胞的脑内移植:
在立体定向仪定位下行SD大鼠右侧尾状核脑内血肿模型制作。Ⅱ-Ⅲ代ADSC在立体定向仪定位下行右侧脑室内移植。约10~6个细胞,细胞悬液约10ul。缓慢注入右侧脑室。
脑出血模型ADSC脑内移植后神经功能测评比较
采用longa等的5分评分法观察模型组、对照组、移植组3天、1周、2周、3周、4周神经功能测评。
ADSC脑内移植后Tunel染色:
将ADSC脑内移植3天、1周、2周、3周、4周后按时间点捕杀大鼠,灌注后取脑标本,送病理科制切片,行Tunel染色,观察脑出血灶周围凋亡细胞。
结果:
脂肪组织经胶原酶消化,接种细胞24小时后贴壁生长,原代培养7—10天,即达70—80%融合。2—4天细胞可再次传代。经过频繁换液、传代,细胞得以纯化。加入成脂诱导培养基诱导2—4天后培养液就出现含脂滴的细胞。对照组培养后未见脂肪细胞形成。
大鼠脑出血模型内移植ADSC后神经功能改善明显,对照组模型组无差异,而移植组与模型组,移植组与PBS组间差异有统计学意义。
大鼠脑出血模型脑内移植ADSC组脑出血周围凋亡细胞数小于模型组,从第3天开始随时间增加凋亡数越来越少。
结论:
1从S-D大鼠腹股沟脂肪组织中可分离、培养出ADSCs。ADSCs在10%FBS的低糖DMEM培养基中体外增殖。ADSCs经成脂诱导剂诱导分化为脂肪细胞。
2、ADSC脑内移植能改善大鼠脑出血所致的神经功能缺损。
3、ADSC脑内移植减少脑出血周围细胞凋亡。
4、ADSC脑内移植神经功能可能是由于减少神经细胞凋亡而起到脑保护作用。
Objective
Stem cells are a category of cells in vivo which are capable of self-renewal and possess multi-directional differentiation potency. With the development of the study on tissue engineering and gene therapy, to search suitable seeding cells and cell replacement therapy have come more and more into the focus of life science research. The discovery of embryo stem cells and neural stem cells established a utility path for nerve restore. It's well known that cell replacement therapy (cell transplantation) adopted to central nervous system degenerative diseases is positive. Not only embry stem cells but also neural stem cells are restricted their development and application by their shortcomings.
Bone marrow stromal cells derive from mesenchyme multipotent stem cells which are capable of self-renewal and possess multi -directional differention potency. The research on MSCs celluar transplantation has become profound more and more with each passing day, and has been applied to treat myocardium infarction. However in order to harvest the MSCs, it need draw off bone marrow or recipe ribs after anesthetize. The MSCs development is limited because patients have to suffer pain.
Adipose tissue origins from mesoderm as same as bone marrow. Adipose tissue-dirived stromal cells are rich in adipose tissue and is capable of multi-directional differentiation. The source of adipose tissue is plentiful. ADSCs can autoimmune hepatitis cellular transplantation, having no immuno-rejection reaction and avoiding the propagation disease. Adipose tissue is a ideal resource of stem cells bioengineering research.
In this experimentation, we have disassociated, cultivated, induced and differentiated ADSCs originated from SD rat's inguinal groove. By observing ADSCs bionomics characteristic, and the effect of ADSCs transplantation in cerebral hemorrhage model on neural cells apoptosis.
Methods
The healthy adult Sprague-Dawley rats were sacrificed under overdose anesthesia. The raw adipose tissue in inguinal groove of rat was obtained on the asepsis condition. To isolate stromal cells, samples were washed extensively with equal volumes of phosphate-buffered saline(PBS), and digested at 37°C for 30minutes with 0.075% collagenase. Enzyme activity was neutralized with L-DMEM, containing 10%FBS and centrifuged at 1200 rpm for 10 minutes to obtain a high-density cell pellet. The stromal cell pellet was resuspended in L-DMEN medium containing 10%FBS, and incubated overnight at 37°C /5% CO_2. Medium was replaced first at 24 hours and then every second-third day thereafter.
For adipogenic differentiation, ADSCs were induced by passing cells at a 1:10 dilution in control medium and supplemented 10ng/ml insulin and 10~(-9)M dexamethasone.
The intracerebral hemotoma model of SD rat's right cauate nucleus was maden fixed with stereotaxic apparatus. ADSCs were transplanted into right Ventricle, about 10~6cells/10ul.
Scores of neurological deficits were used to assess neurological function
Tunel stain of brain tissue pathological section:
Sprague-Dawley rats were euthanized at 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks after they were transplanted into ADSC. These animals were subjected to infuse into 4% paraformal dehydeond. The brain specimens were maken into frozen section the terminal deoxynucleotidyl transferrase-mediated duTP nick end labeling(TUNEL) staining was used to observe the apoptotic neurons in the cerebral hemorrhage model.
Result
Adipose tissue was digested by collagenase and subcultured in 50ml culture dish. After 24 hours the seeding cells grew in the culture dish .After 2-4 days it was thus clear that these cells spread out. Most of them were fusiform shape, with ellipse nucleus. The primary cells were growth to at least 80% confluence at 7 days. The passaging cell got purification by frequently replacing medium and passaging. The nucleuses of ADSCs were prunosus and cytoplasms were pink by Giemsa stain.
Under the adipogenic inducation media condition in the media at 2-4 day ADSCs were visualized by the presence of highly refractive intracellular lipid droplets in phase contrast microscopy of staining by oil-Red O.
After transplantation in lateral cerebral ventricle, in ADSC transplantation group neurological functional recovery were improved significantly compared with untreated group and PBS group at the same time point. In the cerebral hemorrhage model after transplantation the numble of neural apoptosis cell obviously was significantly fewer compared with untreated group and control group.
Conclusions
1 .The adipose tissue from S-D rat's inguinal groove can isolate and culture the adipose tissue-devived mesenchymal stem cells. ADSCs can proliferate in vitro in L-DMEM medium containing 10% FBS. ADSCs can be induced into adipose cell in adipogenic inducation media.
2. Neurological functional recovery was improved significantly after ADSC transplantion.
3. The number of neuron apoptosis cells was fewer after ADSCs transplantation.
4. ADSC transplantation in cerebral hemorrhage model may contribute to that it can decrease Tunel positive neurons and protect brain.
骨髓基质细胞来源于中胚层间充质多能干细胞,具有自我复制及多向分化潜能。骨髓基质细胞移植研究日益深入,已应用于临床治疗心肌梗塞,并且移植到脑内可改善神经退行性疾病的神经功能。然而骨髓基质细胞获取时需通过麻醉后抽取骨髓或取肋骨,给病人带来痛苦,获取数量有限,培养时间长等问题限制其发展。
脂肪组织与骨髓一样来源于中胚层,脂肪间充质干细胞量多且可以多向分化。脂肪组织可以自体抽取,来源丰富,可自体细胞移植,无免疫排斥反应,避免疾病传播,是干细胞生物工程研究的理想来源。
目的:
本实验应用酶消化法,对SD大鼠腹股沟脂肪组织间充质干细胞分离培养,ADSC大鼠脑内出血模型移植后进行神经功能测评,观察ADSC脑内移植后脑内血肿周围神经细胞凋亡的情况,探讨ADSC脑内移植对脑内血肿周围神经细胞凋亡的影响及机制。
方法:
脂肪干细胞培养、增殖、鉴别及诱导
健康成年Sprague-Dawley(S-D)大鼠,过量麻醉处死,无菌条件下取腹股沟脂肪,用PBS反复冲洗,胶原酶消化脂肪组织进行ADSC分离培养。对ADSC在体外进行传代培养,观察其增殖能力。对培养的脂肪干细胞进行成脂诱导。
脂肪干细胞的脑内移植:
在立体定向仪定位下行SD大鼠右侧尾状核脑内血肿模型制作。Ⅱ-Ⅲ代ADSC在立体定向仪定位下行右侧脑室内移植。约10~6个细胞,细胞悬液约10ul。缓慢注入右侧脑室。
脑出血模型ADSC脑内移植后神经功能测评比较
采用longa等的5分评分法观察模型组、对照组、移植组3天、1周、2周、3周、4周神经功能测评。
ADSC脑内移植后Tunel染色:
将ADSC脑内移植3天、1周、2周、3周、4周后按时间点捕杀大鼠,灌注后取脑标本,送病理科制切片,行Tunel染色,观察脑出血灶周围凋亡细胞。
结果:
脂肪组织经胶原酶消化,接种细胞24小时后贴壁生长,原代培养7—10天,即达70—80%融合。2—4天细胞可再次传代。经过频繁换液、传代,细胞得以纯化。加入成脂诱导培养基诱导2—4天后培养液就出现含脂滴的细胞。对照组培养后未见脂肪细胞形成。
大鼠脑出血模型内移植ADSC后神经功能改善明显,对照组模型组无差异,而移植组与模型组,移植组与PBS组间差异有统计学意义。
大鼠脑出血模型脑内移植ADSC组脑出血周围凋亡细胞数小于模型组,从第3天开始随时间增加凋亡数越来越少。
结论:
1从S-D大鼠腹股沟脂肪组织中可分离、培养出ADSCs。ADSCs在10%FBS的低糖DMEM培养基中体外增殖。ADSCs经成脂诱导剂诱导分化为脂肪细胞。
2、ADSC脑内移植能改善大鼠脑出血所致的神经功能缺损。
3、ADSC脑内移植减少脑出血周围细胞凋亡。
4、ADSC脑内移植神经功能可能是由于减少神经细胞凋亡而起到脑保护作用。
Objective
Stem cells are a category of cells in vivo which are capable of self-renewal and possess multi-directional differentiation potency. With the development of the study on tissue engineering and gene therapy, to search suitable seeding cells and cell replacement therapy have come more and more into the focus of life science research. The discovery of embryo stem cells and neural stem cells established a utility path for nerve restore. It's well known that cell replacement therapy (cell transplantation) adopted to central nervous system degenerative diseases is positive. Not only embry stem cells but also neural stem cells are restricted their development and application by their shortcomings.
Bone marrow stromal cells derive from mesenchyme multipotent stem cells which are capable of self-renewal and possess multi -directional differention potency. The research on MSCs celluar transplantation has become profound more and more with each passing day, and has been applied to treat myocardium infarction. However in order to harvest the MSCs, it need draw off bone marrow or recipe ribs after anesthetize. The MSCs development is limited because patients have to suffer pain.
Adipose tissue origins from mesoderm as same as bone marrow. Adipose tissue-dirived stromal cells are rich in adipose tissue and is capable of multi-directional differentiation. The source of adipose tissue is plentiful. ADSCs can autoimmune hepatitis cellular transplantation, having no immuno-rejection reaction and avoiding the propagation disease. Adipose tissue is a ideal resource of stem cells bioengineering research.
In this experimentation, we have disassociated, cultivated, induced and differentiated ADSCs originated from SD rat's inguinal groove. By observing ADSCs bionomics characteristic, and the effect of ADSCs transplantation in cerebral hemorrhage model on neural cells apoptosis.
Methods
The healthy adult Sprague-Dawley rats were sacrificed under overdose anesthesia. The raw adipose tissue in inguinal groove of rat was obtained on the asepsis condition. To isolate stromal cells, samples were washed extensively with equal volumes of phosphate-buffered saline(PBS), and digested at 37°C for 30minutes with 0.075% collagenase. Enzyme activity was neutralized with L-DMEM, containing 10%FBS and centrifuged at 1200 rpm for 10 minutes to obtain a high-density cell pellet. The stromal cell pellet was resuspended in L-DMEN medium containing 10%FBS, and incubated overnight at 37°C /5% CO_2. Medium was replaced first at 24 hours and then every second-third day thereafter.
For adipogenic differentiation, ADSCs were induced by passing cells at a 1:10 dilution in control medium and supplemented 10ng/ml insulin and 10~(-9)M dexamethasone.
The intracerebral hemotoma model of SD rat's right cauate nucleus was maden fixed with stereotaxic apparatus. ADSCs were transplanted into right Ventricle, about 10~6cells/10ul.
Scores of neurological deficits were used to assess neurological function
Tunel stain of brain tissue pathological section:
Sprague-Dawley rats were euthanized at 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks after they were transplanted into ADSC. These animals were subjected to infuse into 4% paraformal dehydeond. The brain specimens were maken into frozen section the terminal deoxynucleotidyl transferrase-mediated duTP nick end labeling(TUNEL) staining was used to observe the apoptotic neurons in the cerebral hemorrhage model.
Result
Adipose tissue was digested by collagenase and subcultured in 50ml culture dish. After 24 hours the seeding cells grew in the culture dish .After 2-4 days it was thus clear that these cells spread out. Most of them were fusiform shape, with ellipse nucleus. The primary cells were growth to at least 80% confluence at 7 days. The passaging cell got purification by frequently replacing medium and passaging. The nucleuses of ADSCs were prunosus and cytoplasms were pink by Giemsa stain.
Under the adipogenic inducation media condition in the media at 2-4 day ADSCs were visualized by the presence of highly refractive intracellular lipid droplets in phase contrast microscopy of staining by oil-Red O.
After transplantation in lateral cerebral ventricle, in ADSC transplantation group neurological functional recovery were improved significantly compared with untreated group and PBS group at the same time point. In the cerebral hemorrhage model after transplantation the numble of neural apoptosis cell obviously was significantly fewer compared with untreated group and control group.
Conclusions
1 .The adipose tissue from S-D rat's inguinal groove can isolate and culture the adipose tissue-devived mesenchymal stem cells. ADSCs can proliferate in vitro in L-DMEM medium containing 10% FBS. ADSCs can be induced into adipose cell in adipogenic inducation media.
2. Neurological functional recovery was improved significantly after ADSC transplantion.
3. The number of neuron apoptosis cells was fewer after ADSCs transplantation.
4. ADSC transplantation in cerebral hemorrhage model may contribute to that it can decrease Tunel positive neurons and protect brain.
引文
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[1] Barry FP, Murphy JM. Mesenchymal stem cells: Clinical applications and biological characterization. Int J Biochem Cell Biol 2004; 36:568-584.
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[5] Wagner W, Wein F, Seckinger A et al. Comparative characteristics of mesenchymal stem cells from human one marrow, adipose tissue, and umbilical cord blood. Exp Hematol 2005;33:1402-1416.
[6] Kern S, Eichler H, Stoeve J et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. STEM CELLS 2006;24:1294-1301.
[7] Weisberg SP, McCann D, Desai M et al. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 2003;112:1796-1808.
[8] Xu H, Barnes GT, Yang Q et al. Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest 2003; 112: 1821-1830.
[9] Caspar-Bauguil S, Cousin B, Galinier A et al. Adipose tissues as an ancestral immune organ: Site-specific change in obesity. FEBS Lett2005;579:3487-3492.
[10] Prunet-Marcassus B, Cousin B, Caton D et al. From heterogeneity to plasticity in adipose tissues: Site-specific differences. Exp Cell Res 2006;312:727-736.
[11] Casteilla L, Planat-Benard V, Cousin B et al. Plasticity of adipose tissue: A promising therapeutic avenue in the treatment of cardiovascular and blood diseases. Arch Mal Coeur Vaiss 2005;98:922-926.
[12] Zuk PA, Zhu M, Ashjian P et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002; 13:4279-4295.
[13] Katz AJ, Tholpady A, Tholpady SS et al. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hA DAS) cells. STEM CELLS 2005;23:412-423.
[14] Rodriguez AM, Elabd C, Amri EZ et al. The human adipose tissue is a source of multipotent stem cells. Biochimie 2005;87:125-128.
[15] Oedayrajsingh-Varma M, van Ham S, Kmppenberg M et al. Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure. Cytotherapy 2006;8:166-177.
[16] Izadpanah R, Trygg C, Patel B et al. Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue. J Cell Biochem 2006;99:1285-1297.
[17] Zuk PA, Zhu M, Mizuno H et al. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 2001;7:211-228.
[18] Dicker A, Le Blanc K, Astrom G et al. Functional studies of mesenchymal stem cells derived from adult human adipose tissue. Exp Cell Res 2005;3008:283-290.
[19] Lee RH, Kim B, Choi I et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem 2004; 14:311-324.
[20] Xu Y, Malladi P, Wagner DR et al, Adipose-derived mesenchymal cells as a potential cell source for skeletal regeneration. Curr Opin Mol Ther 2005;7:300-305.
[21] Smith P, Adams WP Jr., Lipschitz AH et al. Autologous human fat grafting: Effect of harvesting and preparation techniques on adipocyte graft survival. Plast Reconstr Surg 2006; 117:1836-1844.
[22] Peptan IA, Hong L, Mao JJ. Comparison of osteogenic potentials of visceral and subcutaneous adipose-derived cells of rabbits. Plast Reconstr Surg 2006;117: 1462-1470.
[23] De Ugrte DA, Morizono K, Elbarbary A et al. Comparison of multilineage cells from human adipose tissue and bone marrow. Cells Tissues Organs 2003; 174:101-109.
[24] Shi YY, Nacamuli RP, Salim A et al. The osteogenic potential of adipose-derived mesenchymal cells is maintained with aging. Plast Reconstr Surg 2005;116:1686-1696.
[25] Pu LL, Cui X, Fink BF et al. The viability of fatty tissues within adipose aspirates after conventional liposuction: A comprehensive study. Ann Plast Surg 2005;54:288-292.
[26] Nakagami H, Morishita R, Maeda K et al. Adipose tissue-derived stromal cells as a novel option for regenerative cell therapy. J Atheroscler Thromb 2006;117:1845-1850.
[27] Lin TM, Tsai JL, Lin SD et al. Accelerated growh and prolonged lifespan of adipose tissue-derived human mesenchymal stem cells in a medium using reduced calcium and antioxidants. Stem Cells Dev 2005;14:92-102.
[28] Jun ES, Lee TH, Cho HH et al. Expression of telomerase extends longevity and enhances differentiation in human adipose tissue-derived stromal cells. Cell Physiol Biochem 2004; 14:261-268.
[29] Wang M, Crisostomo P, Herring C et al. Human progenitor cells from bone marrow or adipose tissue produce VEGF, HGF and IGF-I in response to TNF by a p38 mitogen activated protein kinase dependent mechanism. Am J Physiol Regul Integr Comp Physiol 2006;291:R880-R884.
[30] Kang SK, Lee DH, Bae YC et al. Improvement of neurological deficits by intracerebral transplantation of human adipose tissue-derived stromal cells after cerebral ischemia in rats. Exp Neurol 2003; 183:355-366.