体外共培养方法诱导兔脂肪干细胞向软骨细胞方向分化的实验研究
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摘要
目的
由于软骨的组织学特点及其无神经和血管的营养,在正常的生理环境中,关节软骨缺损的修复是很困难的。应用生物相容性聚合物与细胞复合构建组织功能性软骨以及刺激诱导软骨形成都是修复软骨缺损的方法。在软骨再生中,自体软骨细胞是很有用的,但是其作用却受制于终末分化的软骨细胞增殖能力和纤维性软骨形成等因素。因此,在软骨组织工程中,在干细胞的应用方面,开展了许多研究。近来,脂肪源性干细胞(adipose-derived stem cells, ADSCs)因其具有自我更新能力、长时间的细胞生存能力以及多向分化潜能而成为关节透明软骨修复和再生的种子细胞。另外,ADSCs还具有能够在局麻下从身体不同部位大量获取的优势。本实验研究的目的是通过一种创新的脂肪干细胞、软骨细胞和滑膜细胞共培养方法,诱导兔脂肪干细胞向软骨细胞方向分化,通过各项指标的检测,在实践中检验该共培养方法的可行性,并对实验结果进行评价。
材料和方法
1.取3-4月龄健康新西兰大白兔,3%戊巴比妥耳缘静脉麻醉,无菌取出肾周脂肪组织,I型胶原酶酶解法分离出脂肪源性干细胞(adipose-derived stem cells, ADSCs),进行体外原代培养,传代后差速贴壁培养纯化。流式细胞仪鉴定细胞表面标志CD49d,CD105,CD34,CD106的表达。
2.无菌切取3-4月龄新西兰大白兔膝关节非负重区关节软骨及关节内滑膜,将软骨剪成1—3mm3薄片,先以0.25%胰蛋白酶消化30-35分钟,再以0.1%的Ⅱ型胶原酶消化6-8小时,过滤、离心收集、计数,以2×105个／ml的细胞密度接种于培养瓶内；将滑膜剪碎,4mg／m1Ⅰ型胶原酶消化、过滤、离心收集、计数后,以1×105个／ml的细胞密度接种于细胞培养瓶中。将两种细胞分别体外常规培养与扩增,第三代细胞用于实验。
3.取第三代的ADSCs,根据诱导方式不同分为三组：Ⅰ组空白对照组,不加任何诱导；Ⅱ组软骨细胞诱导组滴加软骨细胞诱导液(组成：TGF-β1 10n g／ml、bFGF-25ng／ml、Vc 50μg/ml、Dexamethasone10-7M／L)；Ⅲ组共培养组取35mm×10mm培养皿培养的第三代脂肪干细胞、软骨细胞和滑膜细胞,将三个皿放入一个200mm×20mm培养皿中,成“品”字形摆放,在大皿中加入培养基(加10%胎牛血清)培养。收集三组中的脂肪干细胞,提取mRNA,反转录酶-聚合酶链锁反应(RT-PCR)产物电泳及荧光定量PCR检测SOX-9、Aggrecan、TypeⅡcollagen的表达。
结果
1.细胞形态学改变倒置相差显微镜观察发现,原代细胞接种后24小时可见少量细胞贴壁,呈短梭形。48小时后多数细胞已贴壁并开始伸展,分裂,出现由单个细胞分裂形成的集落。换液2-3次之后,大多数漂浮细胞被清除。5-7天后,细胞逐渐分裂、增殖,形成多个细胞克隆。传代细胞贴壁快,7-8天形成单层,细胞核大,胞浆内颗粒多,呈平行或漩涡状生长。
2.ADSCs免疫表型流式测定流式细胞学分析显示,CD49d和CD105均呈阳性表达,CD34和CD106均呈阴性表达。
3.RT-PCR电泳及荧光定量PCR检测：在分组培养后的第7天和第14天,三个目的基因中只有SOX-9、Aggrecan表达,第21天,三个目的基因都有表达。共培养组SOX-9和TypeⅡcollagen的表达水平均高于诱导组,且有统计学差异；两组在Aggrecan的表达水平上无统计学差异。
结论
1.ADSCs适合成为软骨组织工程的种子细胞。取材方便,Ⅰ型胶原酶酶解法分离可以获取大量脂肪干细胞,体外培养性能稳定,易于传代扩增,在体外增殖多代之后仍具有完好的细胞特异性标志,并继续保持分化的能力。
2.软骨细胞和滑膜细胞可分泌多种生长因子如：转化生长因子TGF-β、胰岛素样生长因子IGF等。这些因子是公认的间充质干细胞(MSCs)软骨分化诱导因子,这些可溶性因子可能在ADSCs软骨形成过程中发挥了重要作用。
3.本实验所采用的脂肪干细胞、软骨细胞和滑膜细胞共培养方法,可以使软骨细胞和滑膜细胞分泌的多种生长因子对ADSCs进行诱导,使其向软骨细胞方向分化,并具有软骨细胞表型,诱导效果优于外源性转化生长因子TGF-β的诱导。
Objective
The self-healing of articular cartilage defects under normal physiological circumstances is difficult due to the histological characteristics of cartilage and the aneural and avascular tissue. One of the treatments of cartilage defects involves the use of engineered functional cartilage produced using living cells, which requires the use of a biocompatible polymer and stimulation to induce chondrogenesis. Although autologous chondrocytes are useful in cartilage regeneration, their wider application is limited due to the proliferative capacity of terminally differentiated chondrocytes, the formation of fibrous cartilage, and the morbidity of donor sites. Thus, there have been many studies conducted to evaluate the use of adult stem cells in cartilage tissue engineering. Recently, adipose tissue-derived stem cells (ADSCs) have been regarded as good candidates for the repair and regeneration of articular hyaline cartilage due to their self renewal, long term cell viability and multilineage differentiation potential. In addition, ADSCs have advantages such as the ability to be obtained in large quantities from various body parts under local anesthesia with minimal discomfort. The goal of our study is to induce adipose tissue-derived stem cells chondrogenesis by means of an innovative co-culture way, to inspect the feasibilty in practice through detecting some targets,and to evaluate the results.
Materials and Methods
1.Using healthy New Zealand white rabbits approximately 3-4 months of age, adipose tissue was obtained from regions surrounding kidney after anesthesis by 3% pentobarbital and adipose tissue-derived stem cells (ADSCs) could be harvested through type I collagenase digestion.ADSCs were cultured primarily and incubated in vitro and purified by differential adhesion. CD49d, CD105, CD34 and CD106 were identified by flow cytometry analysis.
2.Articular cartilage and intra-articular synovium were obtained from the non-weight regions of healthy New Zealand white rabbits,approximately 3-4 months of age with aseptic technique. The articular cartilage mass was cut into 1-3mm3 thin pieces,digested for 30 to 35 minutes by 0.25% trypsase,and then digested for 6-8 hours by 0.1% typeⅡcollagenase.ADSCs were filtered,centrifuged,collected and counted,then,inoculated into culture bottles by 2×105/ml cell density.Intra-articular synovium was cut into chips, digested by 4mg/ml type I collagenase.Synovialcells were filtered,centrifuged,collected and counted,then,inoculated into culture bottles by 1 X 105ml cell density. Chondrocytes and synovialcells were cultured and amplificated in vitro conventionally and the third generation cells were used in experiment.
3.The third generation ADSCs were classified into three groups according to different inducement methods.The first group was blank control,which without inducement.The second group was induced by chondrocyte induction medium constituted of TGF-β1 lOn g/ml, bFGF-2 5ng/ml,Vc 50μg/ml and Dexamethasone 10-7M/L.The last group was coincubated with the third generation ADSCs, the same generation chondrocytes and synovialcells in 35mm X 10mm culture dishes.Three dishes were put into the same 200mm X 20mm culture dish in the shape of "three mouths stacking".Culture medium(added 10% fetal calf serum)was added into the large dish. ADSCs were collected from the three groups,which mRNA were extracted to detect the expression of SOX-9,Aggrecan and Type II collagen by electrophoresis of the products of reverse transcription-polymerase chain reaction (RT-PCR) detection and real time PCR.
Results
1.Changes of cell morphology The growth of the ADSCs was observed with the phase contrast microscope. During the beginning of 24 hours, sparse primary cells fastened wall, the shape of the primary ADSCs was short spear-like.Most cells had fastened wall and began to extent and split up,morover, clusters which formed of single cell division appeared after 48 hours. After changing medium of 2-3 times, most of the floating cells were cleared.5-7 days later, the clone cluster of the cells showed up along with gradual division and proliferation of cells. The subcultured cells grew faster. After 7-8 days, monolayer cells, with high nuclears and many grains in cytoplasm, were observed in the shape of parallel or swirl.
2. Flow cytometry analysis of ADSCs immune phenotype Flow cytometry analysis showed that the cell surface maker CD49d and CD 105 were positive, but CD 106 and CD34 were negative.
3. RT-PCR and real time PCR detection In three target genes, only SOX-9 and aggrecan expressed in the 7th and 14th day after grouping.But three genes all expressed in the 21st day.The levels of SOX-9 and type II collagen genes expression in co-culture group were higher than that in induction group,which shew statistical difference.While the level of aggrecan gene expression in above two groups had no statistical difference.
Conclusion
1. ADSCs can be easily obtained,generated and amplificated, a mass of which can be harvested through type I collagenase digestion and which characteristics were stable in vitro. After proliferation multi-generation in vitro, ADSCs can have specific cell makers in good condition and continuously keep the abilty of differentiation. Therefore, ADSCs are the suitable seed cells for cartilage tissue engineering.
2. Chondrocytes and synovialcells can secrete multiple growth factors,such as TGF- βand IGF.These factors are accepted as induction factors that can promote MSCs to induce chondrogenesis,which play a important role in the process of ADSCs chondrogenesis possibly.
3.ADSCs can be induced differentiation to chondrocytes with latter phenotypes by multiple growth factors secreted by chondrocyte and synovialcells through the innovative coincubation method in our study,which effect of induction is better than that of exogenous transforming growth factor (TGF-β) induction.
由于软骨的组织学特点及其无神经和血管的营养,在正常的生理环境中,关节软骨缺损的修复是很困难的。应用生物相容性聚合物与细胞复合构建组织功能性软骨以及刺激诱导软骨形成都是修复软骨缺损的方法。在软骨再生中,自体软骨细胞是很有用的,但是其作用却受制于终末分化的软骨细胞增殖能力和纤维性软骨形成等因素。因此,在软骨组织工程中,在干细胞的应用方面,开展了许多研究。近来,脂肪源性干细胞(adipose-derived stem cells, ADSCs)因其具有自我更新能力、长时间的细胞生存能力以及多向分化潜能而成为关节透明软骨修复和再生的种子细胞。另外,ADSCs还具有能够在局麻下从身体不同部位大量获取的优势。本实验研究的目的是通过一种创新的脂肪干细胞、软骨细胞和滑膜细胞共培养方法,诱导兔脂肪干细胞向软骨细胞方向分化,通过各项指标的检测,在实践中检验该共培养方法的可行性,并对实验结果进行评价。
材料和方法
1.取3-4月龄健康新西兰大白兔,3%戊巴比妥耳缘静脉麻醉,无菌取出肾周脂肪组织,I型胶原酶酶解法分离出脂肪源性干细胞(adipose-derived stem cells, ADSCs),进行体外原代培养,传代后差速贴壁培养纯化。流式细胞仪鉴定细胞表面标志CD49d,CD105,CD34,CD106的表达。
2.无菌切取3-4月龄新西兰大白兔膝关节非负重区关节软骨及关节内滑膜,将软骨剪成1—3mm3薄片,先以0.25%胰蛋白酶消化30-35分钟,再以0.1%的Ⅱ型胶原酶消化6-8小时,过滤、离心收集、计数,以2×105个／ml的细胞密度接种于培养瓶内；将滑膜剪碎,4mg／m1Ⅰ型胶原酶消化、过滤、离心收集、计数后,以1×105个／ml的细胞密度接种于细胞培养瓶中。将两种细胞分别体外常规培养与扩增,第三代细胞用于实验。
3.取第三代的ADSCs,根据诱导方式不同分为三组：Ⅰ组空白对照组,不加任何诱导；Ⅱ组软骨细胞诱导组滴加软骨细胞诱导液(组成：TGF-β1 10n g／ml、bFGF-25ng／ml、Vc 50μg/ml、Dexamethasone10-7M／L)；Ⅲ组共培养组取35mm×10mm培养皿培养的第三代脂肪干细胞、软骨细胞和滑膜细胞,将三个皿放入一个200mm×20mm培养皿中,成“品”字形摆放,在大皿中加入培养基(加10%胎牛血清)培养。收集三组中的脂肪干细胞,提取mRNA,反转录酶-聚合酶链锁反应(RT-PCR)产物电泳及荧光定量PCR检测SOX-9、Aggrecan、TypeⅡcollagen的表达。
结果
1.细胞形态学改变倒置相差显微镜观察发现,原代细胞接种后24小时可见少量细胞贴壁,呈短梭形。48小时后多数细胞已贴壁并开始伸展,分裂,出现由单个细胞分裂形成的集落。换液2-3次之后,大多数漂浮细胞被清除。5-7天后,细胞逐渐分裂、增殖,形成多个细胞克隆。传代细胞贴壁快,7-8天形成单层,细胞核大,胞浆内颗粒多,呈平行或漩涡状生长。
2.ADSCs免疫表型流式测定流式细胞学分析显示,CD49d和CD105均呈阳性表达,CD34和CD106均呈阴性表达。
3.RT-PCR电泳及荧光定量PCR检测：在分组培养后的第7天和第14天,三个目的基因中只有SOX-9、Aggrecan表达,第21天,三个目的基因都有表达。共培养组SOX-9和TypeⅡcollagen的表达水平均高于诱导组,且有统计学差异；两组在Aggrecan的表达水平上无统计学差异。
结论
1.ADSCs适合成为软骨组织工程的种子细胞。取材方便,Ⅰ型胶原酶酶解法分离可以获取大量脂肪干细胞,体外培养性能稳定,易于传代扩增,在体外增殖多代之后仍具有完好的细胞特异性标志,并继续保持分化的能力。
2.软骨细胞和滑膜细胞可分泌多种生长因子如：转化生长因子TGF-β、胰岛素样生长因子IGF等。这些因子是公认的间充质干细胞(MSCs)软骨分化诱导因子,这些可溶性因子可能在ADSCs软骨形成过程中发挥了重要作用。
3.本实验所采用的脂肪干细胞、软骨细胞和滑膜细胞共培养方法,可以使软骨细胞和滑膜细胞分泌的多种生长因子对ADSCs进行诱导,使其向软骨细胞方向分化,并具有软骨细胞表型,诱导效果优于外源性转化生长因子TGF-β的诱导。
Objective
The self-healing of articular cartilage defects under normal physiological circumstances is difficult due to the histological characteristics of cartilage and the aneural and avascular tissue. One of the treatments of cartilage defects involves the use of engineered functional cartilage produced using living cells, which requires the use of a biocompatible polymer and stimulation to induce chondrogenesis. Although autologous chondrocytes are useful in cartilage regeneration, their wider application is limited due to the proliferative capacity of terminally differentiated chondrocytes, the formation of fibrous cartilage, and the morbidity of donor sites. Thus, there have been many studies conducted to evaluate the use of adult stem cells in cartilage tissue engineering. Recently, adipose tissue-derived stem cells (ADSCs) have been regarded as good candidates for the repair and regeneration of articular hyaline cartilage due to their self renewal, long term cell viability and multilineage differentiation potential. In addition, ADSCs have advantages such as the ability to be obtained in large quantities from various body parts under local anesthesia with minimal discomfort. The goal of our study is to induce adipose tissue-derived stem cells chondrogenesis by means of an innovative co-culture way, to inspect the feasibilty in practice through detecting some targets,and to evaluate the results.
Materials and Methods
1.Using healthy New Zealand white rabbits approximately 3-4 months of age, adipose tissue was obtained from regions surrounding kidney after anesthesis by 3% pentobarbital and adipose tissue-derived stem cells (ADSCs) could be harvested through type I collagenase digestion.ADSCs were cultured primarily and incubated in vitro and purified by differential adhesion. CD49d, CD105, CD34 and CD106 were identified by flow cytometry analysis.
2.Articular cartilage and intra-articular synovium were obtained from the non-weight regions of healthy New Zealand white rabbits,approximately 3-4 months of age with aseptic technique. The articular cartilage mass was cut into 1-3mm3 thin pieces,digested for 30 to 35 minutes by 0.25% trypsase,and then digested for 6-8 hours by 0.1% typeⅡcollagenase.ADSCs were filtered,centrifuged,collected and counted,then,inoculated into culture bottles by 2×105/ml cell density.Intra-articular synovium was cut into chips, digested by 4mg/ml type I collagenase.Synovialcells were filtered,centrifuged,collected and counted,then,inoculated into culture bottles by 1 X 105ml cell density. Chondrocytes and synovialcells were cultured and amplificated in vitro conventionally and the third generation cells were used in experiment.
3.The third generation ADSCs were classified into three groups according to different inducement methods.The first group was blank control,which without inducement.The second group was induced by chondrocyte induction medium constituted of TGF-β1 lOn g/ml, bFGF-2 5ng/ml,Vc 50μg/ml and Dexamethasone 10-7M/L.The last group was coincubated with the third generation ADSCs, the same generation chondrocytes and synovialcells in 35mm X 10mm culture dishes.Three dishes were put into the same 200mm X 20mm culture dish in the shape of "three mouths stacking".Culture medium(added 10% fetal calf serum)was added into the large dish. ADSCs were collected from the three groups,which mRNA were extracted to detect the expression of SOX-9,Aggrecan and Type II collagen by electrophoresis of the products of reverse transcription-polymerase chain reaction (RT-PCR) detection and real time PCR.
Results
1.Changes of cell morphology The growth of the ADSCs was observed with the phase contrast microscope. During the beginning of 24 hours, sparse primary cells fastened wall, the shape of the primary ADSCs was short spear-like.Most cells had fastened wall and began to extent and split up,morover, clusters which formed of single cell division appeared after 48 hours. After changing medium of 2-3 times, most of the floating cells were cleared.5-7 days later, the clone cluster of the cells showed up along with gradual division and proliferation of cells. The subcultured cells grew faster. After 7-8 days, monolayer cells, with high nuclears and many grains in cytoplasm, were observed in the shape of parallel or swirl.
2. Flow cytometry analysis of ADSCs immune phenotype Flow cytometry analysis showed that the cell surface maker CD49d and CD 105 were positive, but CD 106 and CD34 were negative.
3. RT-PCR and real time PCR detection In three target genes, only SOX-9 and aggrecan expressed in the 7th and 14th day after grouping.But three genes all expressed in the 21st day.The levels of SOX-9 and type II collagen genes expression in co-culture group were higher than that in induction group,which shew statistical difference.While the level of aggrecan gene expression in above two groups had no statistical difference.
Conclusion
1. ADSCs can be easily obtained,generated and amplificated, a mass of which can be harvested through type I collagenase digestion and which characteristics were stable in vitro. After proliferation multi-generation in vitro, ADSCs can have specific cell makers in good condition and continuously keep the abilty of differentiation. Therefore, ADSCs are the suitable seed cells for cartilage tissue engineering.
2. Chondrocytes and synovialcells can secrete multiple growth factors,such as TGF- βand IGF.These factors are accepted as induction factors that can promote MSCs to induce chondrogenesis,which play a important role in the process of ADSCs chondrogenesis possibly.
3.ADSCs can be induced differentiation to chondrocytes with latter phenotypes by multiple growth factors secreted by chondrocyte and synovialcells through the innovative coincubation method in our study,which effect of induction is better than that of exogenous transforming growth factor (TGF-β) induction.
引文
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