联合培养体系中hUVECs对hBMSCs Runx2，Smad-1基因表达及成骨作用的影响
发布时间：2018-08-19 19:22
【摘要】：[研究背景及目的] 各种类型的颌骨缺损在临床上十分常见,而传统的修复方法如牵引成骨、自体骨移植、异体骨移植等常受到供体数量不足、供体病变、二次损伤、潜在抗原性、感染等因素的制约而亟待改善。以至于颌骨缺损的修复问题一直是临床上难以解决的问题。组织工程学的发展为骨缺损修复带来了希望,其目地是将功能细胞联合培养,与可降解三维生物支架材料构建成为有活性的组织或器官,然后植入体内,替代病损的组织,恢复其形态、结构和功能。这种方法特别适合修复颌骨缺损。但目前组织工程骨构建方法虽然能够成骨,但存在组织工程骨血管化缓慢,新骨生长迟缓生长不稳定等问题。 人骨髓间充质干细胞(bone marrow Mesenchymal stem cells, hBMSCs)且有多向分化的潜能,在适当条件下不仅可以分化为脂肪细胞、骨细胞、软骨细胞、心肌细胞、神经元细胞、成肌细胞、肌腱细胞及星形胶质细胞等。目前关于诱导单纯hBMSCs向成骨细胞分化的研究取得了较大的进展,其中以BMP2、BMP4、 BMP6及BMP7的功能最具代表性,能引起多种细胞增殖、分化、凋亡,参与组织再生和再建(remodeling),在细胞游走和分裂等多种生命活动中发挥调节作用。然而诱导单纯hBMSCs向成骨细胞分化存在成骨周期长、成骨效率低、细胞易老化等缺点。 内皮细胞可以分泌骨形态发生蛋白(Bone morphogenetic protein, BMP),促进成骨分化的同时刺激成骨细胞及其前体细胞分泌血管内皮生长因子(vascular endothelial growth factor, VEGF),而VEGF在血管发生和形成过程中发挥着重要作用,可以促进内皮细胞增殖。然而目前血管内皮细胞对骨髓问充质干细胞成骨分化作用的具体机制还不清楚,尚缺乏从基因水平验证血管内皮细胞对骨髓间充质干细胞成骨分化的作用。Smad蛋白家族是近年新发现的细胞内信号传导蛋白,直接参与TGF-B超家族成员转化生长因子(ransforming growth, TGF-β)之骨形态发生蛋白(bone morphogenetic protein)诱导骨髓问充质干细胞的成骨分化,在目前的生物界中,一共有9个Smad家族成员。其中,Smad1、2、3、5、8为受体调节的Smad(receptor-regulated-Smad、R-Smad), Smad-4为共同的偶配体Smad(common-partner-Smad, Co-Smad-6、7为抑制性Smad(inhibitory Smad, I-Smad)。 BMPR-I使Smad-1、5、8末端丝氨酸残基(SSXSmotif)磷酸化,随后2个或1个R-Smad与1个Smad-4以异源三聚体或异源二聚体R-Smad-Co-Smad的形式进入核内,作用于成骨细胞特异性转录因子Cbfa1、Osx等的基因序列上上调其表达,从而增强成骨细胞ALP和OC的表达。从而促进成骨细胞的分化和成熟。 Runt related gene2(Runx2)转录因子：Runx2,又称Cbfal,或NMP2,或AML3,是Drosophila Runt蛋白的同源蛋白。现已知,Runx2在骨骼的发育过程中参与间充质细胞的凝聚,间充质干细胞向成骨细胞的分化,软骨细胞的肥大化,骨骼的血管侵入等多个步骤,如血管内皮生长因子(vascular endothelia growth factor, VEGF)表达的组织特异性调节需要Runx2的参与,以促进骨骼的血管侵入。成骨细胞和肥大软骨细胞均表达Runx2,成骨细胞的分化和软骨细胞的成熟均需要它的参与,Runx2是调节软骨细胞最终成熟的关键因子。Runx2缺陷的小鼠实验证明,由于软骨成骨和膜内成骨均被排除,其骨骼结构中完全没有骨形成,小鼠在出生后不久死亡。Runx2突变是先天性颅锁骨发育不良致病的遗传学基础。Runx2不发挥直接的成骨基因表达调节作用,它必须与多种蛋白相配合,在不同阶段或细胞内发挥不同作用,具有时空特征。所以,Runx2被称为成骨的中枢调节因子。目前至少有12种Runx2异构体被发现,其中Runx2-Ⅰ作用于早期成骨细胞的分化和膜内骨化过程,Runx2-Ⅱ作用与成骨细胞的成熟和软骨内骨化过程,其他异构体的功能尚不清楚。 为此,本课题着重探讨人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)在联合培养体系中对人骨髓间充质干细胞(human bone marrow Mesenchymal stem cells, hBMSCs)的形态、生长、细胞分化及其Smad-1基因和Runx2基因表达的影响,从基因水平验证血管内皮细胞对骨髓间充质干细胞成骨分化的作用。为脐静脉内皮细胞和骨髓间充质干细胞联合培养作为骨组织工程的种子细胞提供理论基础。 [方法] (1)抽取志愿者骨髓液,使用密度梯度离心法分离骨髓单个核细胞,并借助hBMSCs黏附于塑料瓶底这一特性进行纯化,相差显微镜观察形态变化。将hBMSCs传代扩增培养至第三代,流式细胞仪检测CD34、CD29、CD44表面抗原表达,鉴定hBMSCs; (2)将订购的hUVECs用ECM+10%新生胎牛血清扩增至第三代后与第三代hUVECs按1：1比例建立以DMEM+10%胎牛血清为培养基的hBMSCs和hUVECs联合培养体系。以单独培养的hBMSCs组及hUVECs组作为阴性对照组,分别于第4、6、8、10天相差显微镜观察形态变化,用计数板计数各组hBMSCs数量； (3)分别于第4、6、8、10天每组每个时间点取6孔检测三组培养体系中碱性磷酸酶(Alkaline phosphatase, ALP)及骨钙素(Osteocalin, OC)含量。用SPSS17.0软件对各项检测值进行统计学分析； (4)采用实时荧光定量PCR (real-time-PCR)检测第4、6、8、10天单独培养的hBMSCs组及联合培养组中hBMSCs的Smad-1和基因Runx2表达的情况,每组每个时间点取6孔。用SPSS17.0软件对各项检测值进行统计学分析。 [结果] (1)采用Ficoll液密度梯度离心法分离、提纯hBMSCs可达到较高的纯度。用流式细胞仪对第3代hBMSCs进行细胞表型分析鉴定,CD34低表达,CD29、CD44高表达；符合hBMSCs培养特征。 (2)新生胎牛血清分离培养的人骨髓问充质干细胞成细长梭形,细胞为典型的成纤维细胞样,呈漩涡状贴壁生长,细胞较细小,倒置显微镜下观察,可见接种后24h内少部分单个核细胞贴壁,贴壁细胞成圆形,有小的胞质突起。原代培养4至5天就可见成团生长的细胞；第三代骨髓间充质干细胞形态单一,成梭形,呈旋涡状分布,有明显的极性,没有细胞重叠现象。hBMSCs在4-6天呈对数生长,8-10天后生长进入平台期,数量变化不太明显。HUVECs约2-3天即可见细胞单层生长,形态呈多角形、鹅卵石状镶嵌排列,边界清楚,胞浆丰富,胞核呈圆形或椭圆形,偶见双核,第5d融合成片,第一代至第四代生长速度较快,2-3天即可传代； (3)各组碱性磷酸酶检测量随时间延长先增高后降低,各时间联合培养组ALP较高,8天时联合培养组碱性磷酸酶最高；骨髓间充质干细胞组和脐静脉内皮细胞组ALP基本没有变化;联合培养组和其它各组之间两两比较均有显著统计学意义(P0.05)；各组骨钙素检测量随时间延长先增高后降低；8天时联合培养组骨钙素最高；联合培养组和其它各组之间两两比较均有显著统计学意义(P0.05)； (4)联合培养组Smad-1、Runx2基因检测量随时间延长逐渐增高,与其他组对比各时间联合培养组Smad-1、Runx2基因表达均较高；第8天时联合培养组Smad-1、Runx2基因检测量最高；联合培养组和骨髓间充质干细胞组之间比较有显著统计学意义(P0.05)。 [结论] (1)采用Ficoll液密度梯度离心法分离、提纯的hBMSCs,经流式细胞仪鉴定为较高纯度的hBMSCs细胞； (2)骨髓间充质干细胞与脐静脉内皮细胞联合培养相容性良好,脐静脉内皮细胞对体外联合培养体系中骨髓间充质干细胞具有促进增殖的作用; (3)在体外联合培养体系中,脐静脉内皮细胞能促进骨髓间充质干细胞Smad1,Runx2因子的表达,诱导其向成骨细胞方向分化。
[Abstract]:[background and purpose]
Various types of jaw defects are very common in clinic, but the traditional repair methods such as distraction osteogenesis, autologous bone transplantation, allograft bone transplantation are often constrained by the insufficient number of donors, donor lesions, secondary injury, potential antigenicity, infection and other factors and need to be improved urgently. The development of tissue engineering has brought hope to the repair of bone defect. Its purpose is to construct active tissue or organ by co-culture of functional cells and biodegradable three-dimensional scaffolds, and then implant them into the body to replace the damaged tissue and restore its morphology, structure and function. However, although tissue-engineered bone can form bone, there are some problems such as slow vascularization of tissue-engineered bone and unstable growth of new bone.
Human bone marrow mesenchymal stem cells (hBMSCs) have the potential to differentiate into adipocytes, osteoblasts, chondrocytes, cardiomyocytes, neurons, myoblasts, tendon cells and astrocytes under suitable conditions. Currently, the induction of simple hBMSCs into osteoblasts is discussed. Among them, BMP2, BMP4, BMP6 and BMP7 are the most representative ones, which can induce many kinds of cell proliferation, differentiation, apoptosis, participate in tissue regeneration and remodeling, and play a regulatory role in many kinds of life activities, such as cell migration and division. The bone cycle is long, the osteogenesis efficiency is low, and the cells are easy to be aged.
Endothelial cells can secrete bone morphogenetic protein (BMP), promote osteogenic differentiation and stimulate osteoblasts and their precursors to secrete vascular endothelial growth factor (VEGF), and vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and angiogenesis, and can promote endothelial fineness. However, the specific mechanism of vascular endothelial cells on osteogenic differentiation of bone marrow-derived mesenchymal stem cells is still unclear, and there is no gene level to verify the role of vascular endothelial cells on osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Bone morphogenetic protein (BMP), a member of the transforming growth factor family, induces osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). At present, there are nine Smad family members in the biological world. Among them, Smad1, 2, 3, 5, 8 are receptor-regulated Smad (R-Smad), Smad-4 are common. BMPR-I phosphorylates the Smad-1,5,8-terminal serine residue (SSXSmotif), and then two or one R-Smad and one Smad-4 enter the nucleus in the form of an allotrimer or a dimer, R-Smad-Co-Smad. Factor Cbfa1, Osx and other genes up-regulate their expression, thereby enhancing the expression of ALP and OC in osteoblasts, thereby promoting the differentiation and maturation of osteoblasts.
Runt related gene 2 (Runx2) transcription factor: Runx2, also known as Cbfal, or NMP2, or AML3, is a homologous protein of Drosophila Runt protein. Runx2 is known to participate in the process of bone development in the condensation of mesenchymal cells, mesenchymal stem cell differentiation into osteoblasts, chondrocyte hypertrophy, bone vascular invasion and other steps, such as blood. The tissue-specific regulation of vascular endothelial growth factor (VEGF) expression requires the involvement of Runx2 in promoting vascular invasion of bone. Both osteoblasts and mast chondrocytes express Runx2, which is involved in the differentiation of osteoblasts and the maturation of chondrocytes. Runx2 regulates the final maturation of chondrocytes. Runx2 mutation is the genetic basis for the pathogenesis of congenital skull and clavicle dysplasia. Runx2 does not play a direct role in the regulation of osteogenic gene expression. Runx2 is known as a central regulator of osteogenesis. At least 12 Runx2 isomers have been found, including Runx2-I, which acts on the differentiation and intramembranous ossification of early osteoblasts, Runx2-II, and the maturation and ossification of osteoblasts. The function of other isomers in the process of endochondral ossification is not clear.
In this study, we focused on the effects of human umbilical vein endothelial cells (HUVECs) on the morphology, growth, cell differentiation and the expression of Smad-1 and Runx2 genes of human bone marrow mesenchymal stem cells (hBMSCs) in a co-culture system. To investigate the effect of vascular endothelial cells on osteogenic differentiation of bone marrow mesenchymal stem cells and to provide theoretical basis for the co-culture of umbilical vein endothelial cells and bone marrow mesenchymal stem cells as seed cells of bone tissue engineering.
[method]
(1) Bone marrow mononuclear cells were isolated from the bone marrow of volunteers by density gradient centrifugation and purified by hBMSCs adherence to the bottom of plastic bottles. The morphological changes were observed by phase contrast microscope.
(2) The co-culture system of hBMSCs and hUVECs with DMEM+10% fetal bovine serum as medium was established by amplifying the ordered hUVECs with ECM+10% fetal bovine serum to the third generation and the third generation of hUVECs at a ratio of 1:1. The number of hBMSCs in each group was counted by counting board.
(3) Alkaline phosphatase (ALP) and osteocalcin (OC) were measured at 6 holes at each time point on the 4th, 6th, 8th and 10th day in each group.
(4) Real-time quantitative PCR (real-time-PCR) was used to detect the expression of Smad-1 and Runx2 in hBMSCs cultured on the 4th, 6th, 8th and 10th day respectively and in co-cultured groups. Six holes were taken from each group at each time point.
[results]
(1) High purity of hBMSCs was obtained by density gradient centrifugation with Ficoll solution. The phenotype of the third generation of hBMSCs was analyzed by flow cytometry. The results showed that the expression of CD34 was low, and that of CD29 and CD44 was high.
(2) Human bone marrow mesenchymal stem cells (BMSCs) isolated from fetal bovine serum (FBS) were spindle-shaped, and the cells were typical fibroblast-like cells, which grew in a whirlpool-like manner. The cells were smaller and smaller. Under inverted microscope, a small number of mononuclear cells adhered to the wall within 24 hours after inoculation. The adherent cells were round and had small cytoplasmic processes. The third generation of bone marrow mesenchymal stem cells showed single morphology, spindle-shaped, vortex-like distribution, obvious polarity, no cell overlap. hBMSCs grew logarithmically in 4-6 days, and entered plateau stage after 8-10 days, the number of cells did not change significantly. HUVECs could see cell monolayer growth in 2-3 days, morphology was polygonal. The nuclei were round or oval, occasionally binuclear, and fused into pieces on the 5th day. The growth rate of the first generation to the fourth generation was faster, and it could be subcultured in 2-3 days.
(3) Alkaline phosphatase (ALP) level in the combined culture group was higher than that in the combined culture group. ALP level in the combined culture group was the highest at 8 days. ALP level in the bone marrow mesenchymal stem cell group and umbilical vein endothelial cell group was almost unchanged. There was significant difference between the combined culture group and other groups (P Osteocalcin levels in each group increased first and then decreased with time prolonging, osteocalcin levels in the combined culture group were the highest at 8 days, and there was significant difference between the combined culture group and other groups (P 0.05).
(4) Smad-1, Runx2 gene detection increased gradually with time in the co-culture group, compared with other groups at all times in the co-culture group Smad-1, Runx2 gene expression were higher; on the eighth day in the co-culture group Smad-1, Runx2 gene detection was the highest; there was significant difference between the co-culture group and the bone marrow mesenchymal stem cell group (P 0.05). ).
[Conclusion]
(1) hBMSCs were purified by Ficoll density gradient centrifugation and identified as high purity hBMSCs by flow cytometry.
(2) Bone marrow mesenchymal stem cells and umbilical vein endothelial cells have good compatibility in co-culture. Umbilical vein endothelial cells can promote the proliferation of bone marrow mesenchymal stem cells in vitro co-culture system.
(3) Umbilical vein endothelial cells can promote the expression of Smad1 and Runx2 in bone marrow mesenchymal stem cells and induce them to differentiate into osteoblasts in vitro.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
本文编号：2192592
[Abstract]:[background and purpose]
Various types of jaw defects are very common in clinic, but the traditional repair methods such as distraction osteogenesis, autologous bone transplantation, allograft bone transplantation are often constrained by the insufficient number of donors, donor lesions, secondary injury, potential antigenicity, infection and other factors and need to be improved urgently. The development of tissue engineering has brought hope to the repair of bone defect. Its purpose is to construct active tissue or organ by co-culture of functional cells and biodegradable three-dimensional scaffolds, and then implant them into the body to replace the damaged tissue and restore its morphology, structure and function. However, although tissue-engineered bone can form bone, there are some problems such as slow vascularization of tissue-engineered bone and unstable growth of new bone.
Human bone marrow mesenchymal stem cells (hBMSCs) have the potential to differentiate into adipocytes, osteoblasts, chondrocytes, cardiomyocytes, neurons, myoblasts, tendon cells and astrocytes under suitable conditions. Currently, the induction of simple hBMSCs into osteoblasts is discussed. Among them, BMP2, BMP4, BMP6 and BMP7 are the most representative ones, which can induce many kinds of cell proliferation, differentiation, apoptosis, participate in tissue regeneration and remodeling, and play a regulatory role in many kinds of life activities, such as cell migration and division. The bone cycle is long, the osteogenesis efficiency is low, and the cells are easy to be aged.
Endothelial cells can secrete bone morphogenetic protein (BMP), promote osteogenic differentiation and stimulate osteoblasts and their precursors to secrete vascular endothelial growth factor (VEGF), and vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and angiogenesis, and can promote endothelial fineness. However, the specific mechanism of vascular endothelial cells on osteogenic differentiation of bone marrow-derived mesenchymal stem cells is still unclear, and there is no gene level to verify the role of vascular endothelial cells on osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Bone morphogenetic protein (BMP), a member of the transforming growth factor family, induces osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). At present, there are nine Smad family members in the biological world. Among them, Smad1, 2, 3, 5, 8 are receptor-regulated Smad (R-Smad), Smad-4 are common. BMPR-I phosphorylates the Smad-1,5,8-terminal serine residue (SSXSmotif), and then two or one R-Smad and one Smad-4 enter the nucleus in the form of an allotrimer or a dimer, R-Smad-Co-Smad. Factor Cbfa1, Osx and other genes up-regulate their expression, thereby enhancing the expression of ALP and OC in osteoblasts, thereby promoting the differentiation and maturation of osteoblasts.
Runt related gene 2 (Runx2) transcription factor: Runx2, also known as Cbfal, or NMP2, or AML3, is a homologous protein of Drosophila Runt protein. Runx2 is known to participate in the process of bone development in the condensation of mesenchymal cells, mesenchymal stem cell differentiation into osteoblasts, chondrocyte hypertrophy, bone vascular invasion and other steps, such as blood. The tissue-specific regulation of vascular endothelial growth factor (VEGF) expression requires the involvement of Runx2 in promoting vascular invasion of bone. Both osteoblasts and mast chondrocytes express Runx2, which is involved in the differentiation of osteoblasts and the maturation of chondrocytes. Runx2 regulates the final maturation of chondrocytes. Runx2 mutation is the genetic basis for the pathogenesis of congenital skull and clavicle dysplasia. Runx2 does not play a direct role in the regulation of osteogenic gene expression. Runx2 is known as a central regulator of osteogenesis. At least 12 Runx2 isomers have been found, including Runx2-I, which acts on the differentiation and intramembranous ossification of early osteoblasts, Runx2-II, and the maturation and ossification of osteoblasts. The function of other isomers in the process of endochondral ossification is not clear.
In this study, we focused on the effects of human umbilical vein endothelial cells (HUVECs) on the morphology, growth, cell differentiation and the expression of Smad-1 and Runx2 genes of human bone marrow mesenchymal stem cells (hBMSCs) in a co-culture system. To investigate the effect of vascular endothelial cells on osteogenic differentiation of bone marrow mesenchymal stem cells and to provide theoretical basis for the co-culture of umbilical vein endothelial cells and bone marrow mesenchymal stem cells as seed cells of bone tissue engineering.
[method]
(1) Bone marrow mononuclear cells were isolated from the bone marrow of volunteers by density gradient centrifugation and purified by hBMSCs adherence to the bottom of plastic bottles. The morphological changes were observed by phase contrast microscope.
(2) The co-culture system of hBMSCs and hUVECs with DMEM+10% fetal bovine serum as medium was established by amplifying the ordered hUVECs with ECM+10% fetal bovine serum to the third generation and the third generation of hUVECs at a ratio of 1:1. The number of hBMSCs in each group was counted by counting board.
(3) Alkaline phosphatase (ALP) and osteocalcin (OC) were measured at 6 holes at each time point on the 4th, 6th, 8th and 10th day in each group.
(4) Real-time quantitative PCR (real-time-PCR) was used to detect the expression of Smad-1 and Runx2 in hBMSCs cultured on the 4th, 6th, 8th and 10th day respectively and in co-cultured groups. Six holes were taken from each group at each time point.
[results]
(1) High purity of hBMSCs was obtained by density gradient centrifugation with Ficoll solution. The phenotype of the third generation of hBMSCs was analyzed by flow cytometry. The results showed that the expression of CD34 was low, and that of CD29 and CD44 was high.
(2) Human bone marrow mesenchymal stem cells (BMSCs) isolated from fetal bovine serum (FBS) were spindle-shaped, and the cells were typical fibroblast-like cells, which grew in a whirlpool-like manner. The cells were smaller and smaller. Under inverted microscope, a small number of mononuclear cells adhered to the wall within 24 hours after inoculation. The adherent cells were round and had small cytoplasmic processes. The third generation of bone marrow mesenchymal stem cells showed single morphology, spindle-shaped, vortex-like distribution, obvious polarity, no cell overlap. hBMSCs grew logarithmically in 4-6 days, and entered plateau stage after 8-10 days, the number of cells did not change significantly. HUVECs could see cell monolayer growth in 2-3 days, morphology was polygonal. The nuclei were round or oval, occasionally binuclear, and fused into pieces on the 5th day. The growth rate of the first generation to the fourth generation was faster, and it could be subcultured in 2-3 days.
(3) Alkaline phosphatase (ALP) level in the combined culture group was higher than that in the combined culture group. ALP level in the combined culture group was the highest at 8 days. ALP level in the bone marrow mesenchymal stem cell group and umbilical vein endothelial cell group was almost unchanged. There was significant difference between the combined culture group and other groups (P Osteocalcin levels in each group increased first and then decreased with time prolonging, osteocalcin levels in the combined culture group were the highest at 8 days, and there was significant difference between the combined culture group and other groups (P 0.05).
(4) Smad-1, Runx2 gene detection increased gradually with time in the co-culture group, compared with other groups at all times in the co-culture group Smad-1, Runx2 gene expression were higher; on the eighth day in the co-culture group Smad-1, Runx2 gene detection was the highest; there was significant difference between the co-culture group and the bone marrow mesenchymal stem cell group (P 0.05). ).
[Conclusion]
(1) hBMSCs were purified by Ficoll density gradient centrifugation and identified as high purity hBMSCs by flow cytometry.
(2) Bone marrow mesenchymal stem cells and umbilical vein endothelial cells have good compatibility in co-culture. Umbilical vein endothelial cells can promote the proliferation of bone marrow mesenchymal stem cells in vitro co-culture system.
(3) Umbilical vein endothelial cells can promote the expression of Smad1 and Runx2 in bone marrow mesenchymal stem cells and induce them to differentiate into osteoblasts in vitro.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
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