7，8-二羟基香豆素促进大鼠脂肪来源间充质干细胞增殖及成软骨分化的研究
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摘要
在整形美容外科中，经常遇到因软骨移植、外伤所致的软骨损伤和缺损。而软骨内往往因无血管、淋巴管和神经组织，导致软骨细胞的增殖能力相对有限，并且无迁移能力。在临床中遇到软骨缺损，常常采用软组织筋膜代替解决，但并不能真正恢复软骨的组织及生物力学结构。人们一直在探索寻找能够恢复受损软骨的方法。随着组织工程学发展，促进了科学的研究和开发生物替代品的脚步，为修复和替代受损的软骨组织形态，以恢复受损组织的功能打下基础。组织工程学的发展为组织和器官缺损和功能障碍的治疗提供新的治疗方案。
获得组织工程学软骨的先决条件是如何选择和获得种子细胞，这对如何构建组织的结构起到决定性作用。目前，应用于临床的组织工程学软骨的种子细胞只有自体的软骨细胞。但存在很多缺陷，供区有限，分离培育困难，在体外培养时，增殖能力有限等等。所以其应用前景不大。同时由于多潜能干细胞的诱导以及胚胎干细胞的应用在安全性，伦理学等方面与临床的应用还有一定的差距。而自体干细胞尤其是脂肪来源的间充质干细胞在体内的分布广泛，其来源较多，获取方法相对简单，是作为组织工程学种子细胞的理想选择。
但是，如何使干细胞在体外快速并大量的增殖呢？同时，如何使其更有效的向软骨细胞诱导分化呢？
现在实验室常规使用的促进干细胞分化增值的诱导剂是各种细胞因子，而这些细胞因子的提取及分离使其造价比较昂贵，而且人体的内环境是很难靠细胞因子来模拟的。随着对天然药物的深入研究，其促成骨的作用也备受关注。
有研究表明7,8-二羟基香豆素，一种天然药物，具有促进骨形成、增加骨密度的作用。Tang等2008年报道香豆素及其衍生物能够激活大鼠成骨细胞p38-和ERK-依赖型通路，上调BMP-2表达、促进成骨。Tang等2010年报道香豆素类化合物蛇床子素能够激活小鼠成骨细胞Wnt/beta-catenin-BMP2信号通路促进成骨，并且能够大大促进小鼠头骨的骨修复和骨密度。
因此，本研究采用7,8-二羟基香豆素（以下称香豆素，Coumarin）培养大鼠脂肪间质干细胞（ADMSCs），以研究香豆素在促进脂肪干细胞成软骨分化中的作用。
本实验采用了I型胶原酶消化、贴壁培养法对大鼠皮下脂肪组织中分离培养ADMSCs并对其进行纯化。实验证明此法可以在大鼠皮下脂肪组织来源的样本中分离并诱导培养出长梭形并贴壁生长的脂肪间充质干细胞。培养出的细胞密集生长，呈鱼群样，增殖速度较快，倍增时间为46.7h。经过流式细胞技术检测，结果显示培养出的细胞可以表达CD73、CD29、CD90和CD105抗原，而CD14、CD34、CD45、CD79α和HLA-DR未见表达。在鉴定间充质干细胞的功能方面，多向分化潜能是重要指标之一，本研究应用不同种类的诱导剂将间充质干细胞向三种细胞诱导分化，分别为：成骨细胞、软骨细胞和脂肪细胞，用以证实其多向分化的潜能，结果显示符合间充质干细胞的鉴定标准。
完成以上鉴定后，我们采用细胞微团培养的方法，观察香豆素对ADMSCs向软骨分化的促进作用。研究结果显示：只应用香豆素时，诱导成软骨分化作用较微，而香豆素与TGF-β1具有协同效应，对细胞内SOX9、PGC-1α、Col2a1三种基因的表达具有较明显的促进作用，可以促进细胞外基质二型胶原和糖胺聚糖的聚集。对脂肪间充质干细胞向软骨细胞分化具有促进作用。并且本研究还发现：即随着浓度的改变，其成软骨分化的作用能力也有所变化，经过试验证实最为适宜的浓度为50ug/ml-100ug/ml。推测其可能的作用机制为：香豆素内富含的天然细胞因子及寡肽组分能够促进细胞间或细胞与细胞外基质间相互作用来促进ADMSCs向软骨细胞分化。
In the plastic surgery, often meet for cartilage transplantation, trauma ofcartilage damage and defect. The articular cartilage has no nourishment fromblood vessels and surrounding tissues, in addition to the limited proliferationability of chondrocytes not to be transplanted, so it is hard to repair the damageof the articular cartilage. Currently, the clinical treatments of cartilage damageinclude arthroscopic lavage debridement, subchondral bone drilling,microfractures technology, periosteum or perichondrium transplantation, andautologous or allogeneic bone and cartilage transplantation. However, thesemethods have the poor prognosis, not restore the articular cartilage into thenormal tissue and biomechanical characteristics in the true sense. The tissueengineering has applied the biological substitutes to repair the damaged tissues.The autologous chondrocyte is the only clinical cartilage seed cell applied in thetissue engineering. However, the donor source is limited, the cells’ isolation andculture are difficult, the cells are of poor proliferation capacity, and thus theapplication is limited. As to the embryonic stem cells and the inducedpluripotent stem cells, because of the ethical issues and safety issues, there isstill a long-range from the clinical application. Adult stem cells are widelypresent in the body, especially adipose-derived mesenchymal stem cells(ADMSCs), with a wide distribution to obtain easily, are ideal for the use as thetissue engineered seed cells.
For tissue engineering cartilage prerequisite is how to choose and obtainseed cells, this to how to construct the structure of organizations play a decisive role. At present, clinical applications of tissue engineering cartilage seed cellonly autologous cartilage cells. But there are many defects, for area is limited,separation cultivate difficulties, the in vitro culture, proliferation ability islimited, etc. At the same time because pluripotent stem cells and embryonicstem cells induced in the application of safety, ethics, etc and clinical applicationhad a certain gap. And autologous stem cell especially fat source ofmesenchymal stem cells in the body are widely distributed, its source is more,acquisition methods relative simple, as tissue engineering seed cells of the idealchoice.
However, how to make the stem cells in vitro quickly and a large numberof breeder? At the same time, how to make it more effective to the cartilage celldifferentiation?
Now routine use of laboratory to promote stem cells value-added revulsantis all sorts of cell factor, and the cell factor extraction and separation make itscost is expensive, and human internal environment is difficult to rely on cellfactor to simulation. With the deep research of natural drugs, which facilitatedthe role of bone also concern?
Previous studies have shown that7,8-dihydroxy coumarin, a natural drugcan promote bone formation and increase bone mineral density. Tang et al.(2008) reported that coumarin and its derivatives can activate the p38-andERK-dependent pathways in rat osteoblasts, raise the BMP-2expression andpromote bone formation. Tang et al (2010) reported that coumarin compoundsosthole is able to activate the Wnt/beta-catenin-BMP2signaling pathways inmouse osteoblasts, promote osteoblast and contribute substantially to the mouse skull bone repair and bone mineral density
Therefore, this study used7,8-dihydroxy Coumarin (hereinafter referredto as Coumarin, Coumarin) training rats adipose mesenchymal stem cells(ADMSCs), so as to research in Coumarin in promoting fat stem cells into therole of cartilage differentiation.
This experiment adopted type I collagen enzyme digestion, adherent culturemethod rats to fat tissue separation culture ADMSCs and carry on thepurification. Experiments show this method can in rats subcutaneous adiposetissue sources of sample separation and induced cultivate long spindle andattached growth adipose mesenchymal stem cells. Cultivate cells
Intensive growth, a fish sample, multiplication rate faster, doubling time is46.7h. After flow cell technical detection and the results showed that thecultivated cells can express CD73, CD29, CD90and CD105antigen, and CD14,CD34, CD45, CD79alpha and HLA-DR did not see expression. In the appraisalof mesenchymal stem cells function, the multiplex differentiation potential.thisis one of the important indexes, this study applies different kinds of revulsivewill mesenchymal stem cells to three kinds of cell differentiation, respectively:osteoblast, cartilage cells and fat cells to prove its changeover differentiationpotential, the results showed that conform to the mesenchymal stem cellsappraisal standard.
After complete the above appraisal, we use the cell micelle training method,observation coumarin to ADMSCs to cartilage differentiation role. The resultsof the study show that used only coumarin, induced into cartilage differentiationis micro, and coumarin and TGF-β1has synergistic effect, the intracellular SOX9、PGC-1α、Col2a1three kinds of gene expression has more obvious role,can promote the extracellular matrix type ii collagen and glycosaminoglycangathering. Mesenchymal stem cells to fat to the cartilage cell differentiation hasrole in promoting. And this study also found that an interesting phenomenon thatalong with the concentration change, has the function of cartilage differentiationability to change, through test confirmed the most appropriate concentration is50ug/ml-100ug/ml. Speculation its possible mechanism for: coumarin in richnatural cell factor and oligopeptides component can promote cell or cells andextracellular matrix interaction to promote ADMSCs to cartilage celldifferentiation.
获得组织工程学软骨的先决条件是如何选择和获得种子细胞，这对如何构建组织的结构起到决定性作用。目前，应用于临床的组织工程学软骨的种子细胞只有自体的软骨细胞。但存在很多缺陷，供区有限，分离培育困难，在体外培养时，增殖能力有限等等。所以其应用前景不大。同时由于多潜能干细胞的诱导以及胚胎干细胞的应用在安全性，伦理学等方面与临床的应用还有一定的差距。而自体干细胞尤其是脂肪来源的间充质干细胞在体内的分布广泛，其来源较多，获取方法相对简单，是作为组织工程学种子细胞的理想选择。
但是，如何使干细胞在体外快速并大量的增殖呢？同时，如何使其更有效的向软骨细胞诱导分化呢？
现在实验室常规使用的促进干细胞分化增值的诱导剂是各种细胞因子，而这些细胞因子的提取及分离使其造价比较昂贵，而且人体的内环境是很难靠细胞因子来模拟的。随着对天然药物的深入研究，其促成骨的作用也备受关注。
有研究表明7,8-二羟基香豆素，一种天然药物，具有促进骨形成、增加骨密度的作用。Tang等2008年报道香豆素及其衍生物能够激活大鼠成骨细胞p38-和ERK-依赖型通路，上调BMP-2表达、促进成骨。Tang等2010年报道香豆素类化合物蛇床子素能够激活小鼠成骨细胞Wnt/beta-catenin-BMP2信号通路促进成骨，并且能够大大促进小鼠头骨的骨修复和骨密度。
因此，本研究采用7,8-二羟基香豆素（以下称香豆素，Coumarin）培养大鼠脂肪间质干细胞（ADMSCs），以研究香豆素在促进脂肪干细胞成软骨分化中的作用。
本实验采用了I型胶原酶消化、贴壁培养法对大鼠皮下脂肪组织中分离培养ADMSCs并对其进行纯化。实验证明此法可以在大鼠皮下脂肪组织来源的样本中分离并诱导培养出长梭形并贴壁生长的脂肪间充质干细胞。培养出的细胞密集生长，呈鱼群样，增殖速度较快，倍增时间为46.7h。经过流式细胞技术检测，结果显示培养出的细胞可以表达CD73、CD29、CD90和CD105抗原，而CD14、CD34、CD45、CD79α和HLA-DR未见表达。在鉴定间充质干细胞的功能方面，多向分化潜能是重要指标之一，本研究应用不同种类的诱导剂将间充质干细胞向三种细胞诱导分化，分别为：成骨细胞、软骨细胞和脂肪细胞，用以证实其多向分化的潜能，结果显示符合间充质干细胞的鉴定标准。
完成以上鉴定后，我们采用细胞微团培养的方法，观察香豆素对ADMSCs向软骨分化的促进作用。研究结果显示：只应用香豆素时，诱导成软骨分化作用较微，而香豆素与TGF-β1具有协同效应，对细胞内SOX9、PGC-1α、Col2a1三种基因的表达具有较明显的促进作用，可以促进细胞外基质二型胶原和糖胺聚糖的聚集。对脂肪间充质干细胞向软骨细胞分化具有促进作用。并且本研究还发现：即随着浓度的改变，其成软骨分化的作用能力也有所变化，经过试验证实最为适宜的浓度为50ug/ml-100ug/ml。推测其可能的作用机制为：香豆素内富含的天然细胞因子及寡肽组分能够促进细胞间或细胞与细胞外基质间相互作用来促进ADMSCs向软骨细胞分化。
In the plastic surgery, often meet for cartilage transplantation, trauma ofcartilage damage and defect. The articular cartilage has no nourishment fromblood vessels and surrounding tissues, in addition to the limited proliferationability of chondrocytes not to be transplanted, so it is hard to repair the damageof the articular cartilage. Currently, the clinical treatments of cartilage damageinclude arthroscopic lavage debridement, subchondral bone drilling,microfractures technology, periosteum or perichondrium transplantation, andautologous or allogeneic bone and cartilage transplantation. However, thesemethods have the poor prognosis, not restore the articular cartilage into thenormal tissue and biomechanical characteristics in the true sense. The tissueengineering has applied the biological substitutes to repair the damaged tissues.The autologous chondrocyte is the only clinical cartilage seed cell applied in thetissue engineering. However, the donor source is limited, the cells’ isolation andculture are difficult, the cells are of poor proliferation capacity, and thus theapplication is limited. As to the embryonic stem cells and the inducedpluripotent stem cells, because of the ethical issues and safety issues, there isstill a long-range from the clinical application. Adult stem cells are widelypresent in the body, especially adipose-derived mesenchymal stem cells(ADMSCs), with a wide distribution to obtain easily, are ideal for the use as thetissue engineered seed cells.
For tissue engineering cartilage prerequisite is how to choose and obtainseed cells, this to how to construct the structure of organizations play a decisive role. At present, clinical applications of tissue engineering cartilage seed cellonly autologous cartilage cells. But there are many defects, for area is limited,separation cultivate difficulties, the in vitro culture, proliferation ability islimited, etc. At the same time because pluripotent stem cells and embryonicstem cells induced in the application of safety, ethics, etc and clinical applicationhad a certain gap. And autologous stem cell especially fat source ofmesenchymal stem cells in the body are widely distributed, its source is more,acquisition methods relative simple, as tissue engineering seed cells of the idealchoice.
However, how to make the stem cells in vitro quickly and a large numberof breeder? At the same time, how to make it more effective to the cartilage celldifferentiation?
Now routine use of laboratory to promote stem cells value-added revulsantis all sorts of cell factor, and the cell factor extraction and separation make itscost is expensive, and human internal environment is difficult to rely on cellfactor to simulation. With the deep research of natural drugs, which facilitatedthe role of bone also concern?
Previous studies have shown that7,8-dihydroxy coumarin, a natural drugcan promote bone formation and increase bone mineral density. Tang et al.(2008) reported that coumarin and its derivatives can activate the p38-andERK-dependent pathways in rat osteoblasts, raise the BMP-2expression andpromote bone formation. Tang et al (2010) reported that coumarin compoundsosthole is able to activate the Wnt/beta-catenin-BMP2signaling pathways inmouse osteoblasts, promote osteoblast and contribute substantially to the mouse skull bone repair and bone mineral density
Therefore, this study used7,8-dihydroxy Coumarin (hereinafter referredto as Coumarin, Coumarin) training rats adipose mesenchymal stem cells(ADMSCs), so as to research in Coumarin in promoting fat stem cells into therole of cartilage differentiation.
This experiment adopted type I collagen enzyme digestion, adherent culturemethod rats to fat tissue separation culture ADMSCs and carry on thepurification. Experiments show this method can in rats subcutaneous adiposetissue sources of sample separation and induced cultivate long spindle andattached growth adipose mesenchymal stem cells. Cultivate cells
Intensive growth, a fish sample, multiplication rate faster, doubling time is46.7h. After flow cell technical detection and the results showed that thecultivated cells can express CD73, CD29, CD90and CD105antigen, and CD14,CD34, CD45, CD79alpha and HLA-DR did not see expression. In the appraisalof mesenchymal stem cells function, the multiplex differentiation potential.thisis one of the important indexes, this study applies different kinds of revulsivewill mesenchymal stem cells to three kinds of cell differentiation, respectively:osteoblast, cartilage cells and fat cells to prove its changeover differentiationpotential, the results showed that conform to the mesenchymal stem cellsappraisal standard.
After complete the above appraisal, we use the cell micelle training method,observation coumarin to ADMSCs to cartilage differentiation role. The resultsof the study show that used only coumarin, induced into cartilage differentiationis micro, and coumarin and TGF-β1has synergistic effect, the intracellular SOX9、PGC-1α、Col2a1three kinds of gene expression has more obvious role,can promote the extracellular matrix type ii collagen and glycosaminoglycangathering. Mesenchymal stem cells to fat to the cartilage cell differentiation hasrole in promoting. And this study also found that an interesting phenomenon thatalong with the concentration change, has the function of cartilage differentiationability to change, through test confirmed the most appropriate concentration is50ug/ml-100ug/ml. Speculation its possible mechanism for: coumarin in richnatural cell factor and oligopeptides component can promote cell or cells andextracellular matrix interaction to promote ADMSCs to cartilage celldifferentiation.
引文
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