大鼠骨髓间充质干细胞向软骨细胞体外诱导分化过程中microRNA 130a作用的研究
摘要
研究背景和目的：
骨关节炎是最常见的关节炎,随着世界人口的老龄化,骨关节炎患者在逐年增加。骨关节炎是关节的主要致残的原因,大多数药物只是控制疼痛,而不能逆转关节损害。外科手术可以改善关节功能,如关节镜、截骨、关节置换等,但外科手术一般效果不满意。因此,需要更有效的改善功能的治疗方法。
近年来,通过天然或生物工程方法修复受损组织的再生医学成为热点,这些方法包括骨软骨移植、自体软骨细胞移植等。自体软骨细胞移植已经开展十余年,临床中也采用一些改良的自体软骨细胞移植术。但是仍有一些缺陷,如缺少细胞来源、损害正常组织、不能形成原有软骨结构、与周围组织不完全结合等。
近年来间充质干细胞的研究为软骨移植提供了新的方法。在胚胎发展过程中,软骨是有间充质干细胞聚集后分化而成的。目前,间充质干细胞已从骨髓、骨膜、脂肪、滑膜、肌肉等多个组织中分离获得,并证实具有向不同结缔组织分化的能力,如骨、软骨、脂肪、椎间盘、韧带和肌肉。目前,新的研究方向主要是加强和延长体外软骨细胞的分化潜能。
MicroRNAs是19-23个核苷酸的单链RNA,存在于广泛的生物体中。在研究干细胞功能的分子Loquacious和DGCR8时,研究者发现microRNA在干细胞功能和分化过程中起重要作用。产生microRNA的关键酶Dicer敲除后,间充质干细胞分化为软骨的功能受损。以上均证明microRNA是干细胞分化机制中的重要分子。
我们的课题基于文献的复习和前期的骨髓间充质干细胞培养的实验基础,研究microRNA在骨髓间充质干细胞诱导分化为软骨细胞的过程中的调节作用,探索诱导分化的机制,优化骨髓间充质干细胞向软骨细胞分化的条件,诱导出软骨细胞,为软骨移植提供细胞来源,最终解决骨关节炎的治疗问题。
实验方法：
1、提取大鼠骨髓间充质干细胞,体外经TGF-β1诱导其向软骨细胞分化。通过相差显微镜形态学观察,免疫荧光法、免疫组化法检测软骨细胞特异性标志Ⅱ型胶原,阿辛兰染色检测氨基葡聚糖的方法证实大鼠骨髓间充质干细胞经过体外诱导分化培养后是否具有成熟软骨细胞的标志。
2、从大鼠骨髓中分离间充质干细胞,体外经TGF-β1诱导分化为软骨细胞。分别于TGF-β1诱导前(d0)、诱导培养7天(d7-induced)和无TGF-β1诱导培养7天(d7-non-induced)三个时间点,应用实时定量逆转录—聚合酶链反应(real time RT-PCR)方法检测三个时间点细胞的microRNA130a的表达水平。
3、Hulth法建立兔关节炎模型。剪断兔膝关节前后交叉韧带及内侧半月板以改变膝关节的生物力学状态。手术后12周进行Maknin评分。
研究结果：
1、大鼠骨髓间充质干细胞经TGF-β1诱导分化培养后细胞具有成熟软骨细胞的形态。应用Ⅱ型胶原特异性抗体进行的免疫荧光染色显示骨髓基质干细胞在分化第14天、21天,细胞质中充满了大量绿色特异性染色,而在未诱导培养的细胞中,未发现特异性荧光染色。应用Ⅱ型胶原特异性抗体进行的组织化学染色显示骨髓基质干细胞在分化2天、7天、14天、21天后,细胞质中充满了大量棕色特异性染色,而在无诱导培养的细胞中,未发现特异性染色。经诱导培养的细胞于第2、7、14、21天在细胞内均可呈现阿辛兰染色阳性物质,含有软骨细胞分泌的特异的蛋白聚糖。而未诱导培养组细胞无阳性染色物质。
2、软骨细胞分化过程中均表达microRNA130a。骨髓间充质干细胞培养7天后,诱导分化组和未诱导分化组细胞都有不同程度的表达水平下调。经7天培养,TGF-β1诱导分化组细胞表达microRNA130a水平显著低于诱导培养前(P＜0.05)。经7天培养后,诱导分化组细胞表达microRNA130a水平显著低于未诱导分化组(P＜0.1)。
3、Hulth法可建立兔关节炎的模型。12周Maknin评分9-10分。
研究结论：
1、大鼠骨髓间充质干细胞在生长因子TGF-β的诱导培养下可以分化为具有成熟软骨细胞形态和特异标志的软骨细胞样细胞。
2、骨髓间充质干细胞在向软骨细胞分化的早期,从第1天到第7天,microRNA130a表达水平显著下调,microRNA130a参与了软骨形成的过程。推测microRNA130a可能通过Runx3调控软骨细胞分化。
3、Hulth法可建立骨关节炎模型。
Background and Objective
Osteoarthritis is the most common type of arthritis. The number of OA patients steadily rises as the elderly population grows in the world. OA is an important cause of disability. The majority of the drugs for the treatment of OA have been inadequate as they only treat the symptoms of pain and inflammation. The drugs have not the capacity to reverse the molecular changes that occur in OA. A number of surgical methods and procedures have been implemented to restore synovial joint function. These range from minimally invasive procedures such as arthroscopic abrasion and shaving of small cartilage defects, to more extended surgical procedures such as microfracture of the subchondoral bone and mosaicplasty. Surgery for OA, which may involve joint replacement,is generally unsatisfactory. Consequently more effective function-modifying therapeutic strategies will need to be introduced for the clinical treatment of OA.
In recent years, regenerative medicine is an emerging field that seeks to repair or replace injured tissues through natural or bioengineered means. A range of methods have been developed including osteochondral transplantation, microfracture and autologous chondrocyte transplantation(ACT), with or without the assistance of scaffold matrix to deliver the cells. ACT has been in clinical for a decade, and many modifications of the technique are also used in the clinic, but these have several major drawbacks. Challenges in treating cartilage defects with ACT are including paucity of the cell source; damage caused to native tissues by cell harvest; inability to restore the original cartilage structure; lack of adhesion between new repair cartilage and the original tissue.
Recent research on mesenchymal stem cells has provided a new and exciting opportunity for bone and cartilage tissue engineering. During embryogenesis, cartilage is formed from the condensation of MSCs. Thus far, MSCs have been isolated from bone marrow, periosteum, trabecular bone, adipose tissue, synovium, skeletal muscle and deciduous teeth. MSCs possess the capacity to differentiate into cells of connective tissue lineages, including bone, fat, cartilage, intervertebral disc, ligament and muscle. New strategies have to center around enhancing and prolonging the chondrogenic potential of the chondrocytes during their in vitro expansion phase.
MicroRNAs are single-stranded RNAs of 19-23 nucleotides and are found in a wide variety of organisms. Evidence for the requirement of the processing of microRNA in stem cell function and differentiation comes from studies of the Drosha complex partners Loquacious (homolog of human TAR (HIV-1) RNA binding protein 2), which is required for germ-line stem cell maintenance, and DGCR8, which is required for embryonic stem cell selfrenewal. Similarly, Dicer knockouts exhibit defects in stem cell differentiation. Clearly, microRNAs underlie key differentiation mechanisms.
Based on our prophase works, we will study on how microRNA works in the differentiation from MSCs to chondrocytes, imploring the mechanism, optimizing the culture conditions, inducing the chondrocytes and denoting a optimized therapy for OA.
Methods
1. Mesenchymal stem cells were isolated from rat bone marrow and induced into mature chondrocyte in the presence of transforming growth factor-β1 (TGF-β1). Chondrogenesis was assessed by immunohistochemistry and immunofluorescence for typeⅡcollagen, and by alcian blue staining for proteoglycan.
2. BMSCs were induced to differentiate into chondrocytes by TGF-β1 in vitro, immunofluorescence and immunohistochemistry were performed to evaluate MSCs differentiation. Real-time reverse transcription polymerase chain reaction was performed to analyze microRNA 130a expression at different time points (before induced culture,7 days later in induced culture and 7 days later in non-induced culture).
3. We have developed a model of arthritis in rabbits and assess the joint using Maknin score in 12 weeks after surgery.
Results
1. We found clear positive staining of collagenⅡin the cytoplasm in the induced medium during differentian by using immunofluorescence and immunohistochemical performance. And we also found positive staining cells in the induced medium by Alcian blue staing.
2. We found microRNA130a was expressing during the differentiation. MicroRNA130a was down-modulated significantly during chondrogenesis after BMSCs had cultured in the present of TGF-β1 for 7 days (P＜0.05)
3. The joint was assessed 9-10 scores by Maknin method after 12 weeks in operation.
Conclusion
1. BMSCs were induced to differentiate into chondrocytes by TGF-β1 in vitro.
2. These findings show that, during the early stage of BMSC chondrogenic differentiation, mciroRNA130a expression was specifically repressed, suggesting a role in differentiation of rat bone mesenchymal stromal cells.
3. Hulth method can establishes a model of osteoarthritis.
骨关节炎是最常见的关节炎,随着世界人口的老龄化,骨关节炎患者在逐年增加。骨关节炎是关节的主要致残的原因,大多数药物只是控制疼痛,而不能逆转关节损害。外科手术可以改善关节功能,如关节镜、截骨、关节置换等,但外科手术一般效果不满意。因此,需要更有效的改善功能的治疗方法。
近年来,通过天然或生物工程方法修复受损组织的再生医学成为热点,这些方法包括骨软骨移植、自体软骨细胞移植等。自体软骨细胞移植已经开展十余年,临床中也采用一些改良的自体软骨细胞移植术。但是仍有一些缺陷,如缺少细胞来源、损害正常组织、不能形成原有软骨结构、与周围组织不完全结合等。
近年来间充质干细胞的研究为软骨移植提供了新的方法。在胚胎发展过程中,软骨是有间充质干细胞聚集后分化而成的。目前,间充质干细胞已从骨髓、骨膜、脂肪、滑膜、肌肉等多个组织中分离获得,并证实具有向不同结缔组织分化的能力,如骨、软骨、脂肪、椎间盘、韧带和肌肉。目前,新的研究方向主要是加强和延长体外软骨细胞的分化潜能。
MicroRNAs是19-23个核苷酸的单链RNA,存在于广泛的生物体中。在研究干细胞功能的分子Loquacious和DGCR8时,研究者发现microRNA在干细胞功能和分化过程中起重要作用。产生microRNA的关键酶Dicer敲除后,间充质干细胞分化为软骨的功能受损。以上均证明microRNA是干细胞分化机制中的重要分子。
我们的课题基于文献的复习和前期的骨髓间充质干细胞培养的实验基础,研究microRNA在骨髓间充质干细胞诱导分化为软骨细胞的过程中的调节作用,探索诱导分化的机制,优化骨髓间充质干细胞向软骨细胞分化的条件,诱导出软骨细胞,为软骨移植提供细胞来源,最终解决骨关节炎的治疗问题。
实验方法：
1、提取大鼠骨髓间充质干细胞,体外经TGF-β1诱导其向软骨细胞分化。通过相差显微镜形态学观察,免疫荧光法、免疫组化法检测软骨细胞特异性标志Ⅱ型胶原,阿辛兰染色检测氨基葡聚糖的方法证实大鼠骨髓间充质干细胞经过体外诱导分化培养后是否具有成熟软骨细胞的标志。
2、从大鼠骨髓中分离间充质干细胞,体外经TGF-β1诱导分化为软骨细胞。分别于TGF-β1诱导前(d0)、诱导培养7天(d7-induced)和无TGF-β1诱导培养7天(d7-non-induced)三个时间点,应用实时定量逆转录—聚合酶链反应(real time RT-PCR)方法检测三个时间点细胞的microRNA130a的表达水平。
3、Hulth法建立兔关节炎模型。剪断兔膝关节前后交叉韧带及内侧半月板以改变膝关节的生物力学状态。手术后12周进行Maknin评分。
研究结果：
1、大鼠骨髓间充质干细胞经TGF-β1诱导分化培养后细胞具有成熟软骨细胞的形态。应用Ⅱ型胶原特异性抗体进行的免疫荧光染色显示骨髓基质干细胞在分化第14天、21天,细胞质中充满了大量绿色特异性染色,而在未诱导培养的细胞中,未发现特异性荧光染色。应用Ⅱ型胶原特异性抗体进行的组织化学染色显示骨髓基质干细胞在分化2天、7天、14天、21天后,细胞质中充满了大量棕色特异性染色,而在无诱导培养的细胞中,未发现特异性染色。经诱导培养的细胞于第2、7、14、21天在细胞内均可呈现阿辛兰染色阳性物质,含有软骨细胞分泌的特异的蛋白聚糖。而未诱导培养组细胞无阳性染色物质。
2、软骨细胞分化过程中均表达microRNA130a。骨髓间充质干细胞培养7天后,诱导分化组和未诱导分化组细胞都有不同程度的表达水平下调。经7天培养,TGF-β1诱导分化组细胞表达microRNA130a水平显著低于诱导培养前(P＜0.05)。经7天培养后,诱导分化组细胞表达microRNA130a水平显著低于未诱导分化组(P＜0.1)。
3、Hulth法可建立兔关节炎的模型。12周Maknin评分9-10分。
研究结论：
1、大鼠骨髓间充质干细胞在生长因子TGF-β的诱导培养下可以分化为具有成熟软骨细胞形态和特异标志的软骨细胞样细胞。
2、骨髓间充质干细胞在向软骨细胞分化的早期,从第1天到第7天,microRNA130a表达水平显著下调,microRNA130a参与了软骨形成的过程。推测microRNA130a可能通过Runx3调控软骨细胞分化。
3、Hulth法可建立骨关节炎模型。
Background and Objective
Osteoarthritis is the most common type of arthritis. The number of OA patients steadily rises as the elderly population grows in the world. OA is an important cause of disability. The majority of the drugs for the treatment of OA have been inadequate as they only treat the symptoms of pain and inflammation. The drugs have not the capacity to reverse the molecular changes that occur in OA. A number of surgical methods and procedures have been implemented to restore synovial joint function. These range from minimally invasive procedures such as arthroscopic abrasion and shaving of small cartilage defects, to more extended surgical procedures such as microfracture of the subchondoral bone and mosaicplasty. Surgery for OA, which may involve joint replacement,is generally unsatisfactory. Consequently more effective function-modifying therapeutic strategies will need to be introduced for the clinical treatment of OA.
In recent years, regenerative medicine is an emerging field that seeks to repair or replace injured tissues through natural or bioengineered means. A range of methods have been developed including osteochondral transplantation, microfracture and autologous chondrocyte transplantation(ACT), with or without the assistance of scaffold matrix to deliver the cells. ACT has been in clinical for a decade, and many modifications of the technique are also used in the clinic, but these have several major drawbacks. Challenges in treating cartilage defects with ACT are including paucity of the cell source; damage caused to native tissues by cell harvest; inability to restore the original cartilage structure; lack of adhesion between new repair cartilage and the original tissue.
Recent research on mesenchymal stem cells has provided a new and exciting opportunity for bone and cartilage tissue engineering. During embryogenesis, cartilage is formed from the condensation of MSCs. Thus far, MSCs have been isolated from bone marrow, periosteum, trabecular bone, adipose tissue, synovium, skeletal muscle and deciduous teeth. MSCs possess the capacity to differentiate into cells of connective tissue lineages, including bone, fat, cartilage, intervertebral disc, ligament and muscle. New strategies have to center around enhancing and prolonging the chondrogenic potential of the chondrocytes during their in vitro expansion phase.
MicroRNAs are single-stranded RNAs of 19-23 nucleotides and are found in a wide variety of organisms. Evidence for the requirement of the processing of microRNA in stem cell function and differentiation comes from studies of the Drosha complex partners Loquacious (homolog of human TAR (HIV-1) RNA binding protein 2), which is required for germ-line stem cell maintenance, and DGCR8, which is required for embryonic stem cell selfrenewal. Similarly, Dicer knockouts exhibit defects in stem cell differentiation. Clearly, microRNAs underlie key differentiation mechanisms.
Based on our prophase works, we will study on how microRNA works in the differentiation from MSCs to chondrocytes, imploring the mechanism, optimizing the culture conditions, inducing the chondrocytes and denoting a optimized therapy for OA.
Methods
1. Mesenchymal stem cells were isolated from rat bone marrow and induced into mature chondrocyte in the presence of transforming growth factor-β1 (TGF-β1). Chondrogenesis was assessed by immunohistochemistry and immunofluorescence for typeⅡcollagen, and by alcian blue staining for proteoglycan.
2. BMSCs were induced to differentiate into chondrocytes by TGF-β1 in vitro, immunofluorescence and immunohistochemistry were performed to evaluate MSCs differentiation. Real-time reverse transcription polymerase chain reaction was performed to analyze microRNA 130a expression at different time points (before induced culture,7 days later in induced culture and 7 days later in non-induced culture).
3. We have developed a model of arthritis in rabbits and assess the joint using Maknin score in 12 weeks after surgery.
Results
1. We found clear positive staining of collagenⅡin the cytoplasm in the induced medium during differentian by using immunofluorescence and immunohistochemical performance. And we also found positive staining cells in the induced medium by Alcian blue staing.
2. We found microRNA130a was expressing during the differentiation. MicroRNA130a was down-modulated significantly during chondrogenesis after BMSCs had cultured in the present of TGF-β1 for 7 days (P＜0.05)
3. The joint was assessed 9-10 scores by Maknin method after 12 weeks in operation.
Conclusion
1. BMSCs were induced to differentiate into chondrocytes by TGF-β1 in vitro.
2. These findings show that, during the early stage of BMSC chondrogenic differentiation, mciroRNA130a expression was specifically repressed, suggesting a role in differentiation of rat bone mesenchymal stromal cells.
3. Hulth method can establishes a model of osteoarthritis.
引文
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