大鼠骨髓基质干细胞的分离培养及分化潜能鉴定的实验研究
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摘要
目的
骨髓中存在一定数量的骨髓基质干细胞(BMSCs，bone marrow stromal stem cells)，这些细胞具有自我复制能力，在特定的诱导条件下能够分化为中胚层起源的组织，如骨、软骨和脂肪等。将骨髓基质干细胞诱导分化为成骨细胞、软骨细胞、脂肪细胞等，为修复重建由于创伤、感染、肿瘤等各种原因形成的组织缺损，提供了理想的治疗手段，是一项非常具有应用前景的技术。
本实验旨在通过对大鼠骨髓基质干细胞进行体外分离、培养，动态观察BMSCs的生长特点、检测细胞的表面分子抗原；观察在诱导条件下，骨髓基质干细胞分化为成骨细胞和脂肪细胞的能力；并通过观察骨髓基质干细胞对荧光色素Hoechst 33342的排出能力，评价BMSCs所处的不同分化状态，以期进一步了解骨髓基质干细胞的生物学特性，为其应用于临床提供可靠的理论依据。
实验方法
1．全骨髓贴壁培养法体外分离、培养大鼠骨髓基质干细胞，倒置相差显微镜下逐日观察细胞形态及生长状况。
2．免疫细胞化学技术测定细胞表面抗原CD34、CD44、CD54、CD106的表达情况，判定所培养的细胞中有无存在造血细胞和内皮细胞的污染。
3．向生长状态良好的三代细胞中加入成骨细胞诱导液(DMEM培养液，15％胎牛血清，10mmol／L β-甘油磷酸钠，10~(-8)M地塞米松，50μg／ml抗坏血酸)培养两周。两周后进行碱性磷酸酶染色和Ⅰ型胶原免疫细胞化学染色。
4．当第3代骨髓基质干细胞达到完全融合时，加入脂肪细胞诱导液(1μg／ml胰岛素、1μmol／L地塞米松、0.5mmol／L的IBMX及10％FBS的DMEM)，逐日观察细胞变化。两周后以油红O染色鉴定诱导后的骨髓基
质干细胞。
5.分别取原代及第5代骨髓基质干细胞，将配制好的H加兄h吐33342工
作液，加于铺满细胞的培养皿上，避光孵育30分钟，除去染液，PBS冲洗3
次，OlymPusBX51荧光相差显微镜观察细胞对荧光色素H吮cllst 33342的着
色强弱。
实验结果
1.大鼠骨髓基质干细胞的形态学观察:骨髓基质干细胞接种后24h开
始贴壁，随着时间的延长贴壁细胞数量明显增加，呈现梭形或多角形形态，
经换液逐步将培养瓶中不贴壁的杂细胞清除;接种后4一sd可见较明显的
集落形成，集落的形态、大小、密度各不相同;约6一sd，贴壁细胞逐渐融合
成单层，细胞多呈梭形，细胞排列呈现明显的辐射状、游涡状。
原代培养4一sd时，倒置相差显微镜下可见到聚集成堆，大个、圆形，内
含折光性强的脂滴的脂肪样细胞，油红0检侧为阳性。
2.骨髓基质干细胞的表型特征:免疫组织化学技术检侧BMSCs的4种
表面分子表达的结果显示:BMScs的CD料(纤连蛋白和透明脂酸盐的受
体)、CD54(基质受体)、CD106(细胞间钻附分子)表达阳性;而CD:抖(造血
细胞系标记物)表达阴性。
3.定向诱导为成骨细胞:在诱导3一4d后，骨髓基质干细胞逐渐变得
扁平，由细长梭形转变为三角形或多角形;8d时，培养细胞大部分变为多角
形，细胞外基质分泌增多;第14d时，培养细胞之间出现黑色结节样结构。
用钙钻法测定细胞内碱性磷酸酶活性，呈阳性反应;免疫细胞化学显示l型
胶原表达阳性。
4.定向诱导成脂肪细胞:在成脂肪诱导5d，细胞原来的多角形突触逐
渐回缩，但仍呈多角形;7一d时出现明显脂滴，脂滴多位于细胞边缘。诱导
14d后，脂肪细胞增加，细胞内脂滴密布，多数细胞形态变圆，多散在分布。
油红O染色为阳性。
5.Hoechst 33342染色结果:原代细胞进行H~h欢33342染色，紫外光
激发后，可见绝大多数细胞核不着色。而第5代细胞染色后，明视场可见大
部分细胞的细胞核周围都有亮晕，暗视野下，即紫外光激发后，可见细胞核
大部分呈现弱着色，着色深浅不一。
结论
1.大鼠骨髓中可以从分离出骨髓基质干细胞，经体外培养可以大量扩
增。
2.大鼠骨髓基质干细胞在Dex、p一甘油磷酸钠和vitc作用下，可以向成
骨细胞分化。
3.大鼠骨髓基质干细胞在胰岛素、Dex和IBMX诱导下，可以定向分化
为脂肪细胞。
4.H慨bst 33342荧光染色技术在多潜能干细胞向单能干细胞转化过
程中，能够准确客观的评价细胞所处的分化状态，可以作为干细胞筛选和检
测干细胞功能状态的新方法。
Bone marrow stromal stem cells ( BMSCs) are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues , including bone, cartilage, and fat et al. Under controlled in vitro conditions , these cells could be induced to differentiate exclusively into the adipocyt-ic, chondrocytic, or osteocytic lineages. Using BMSCs to fabricate engineered tissue is a foreground technology, which offer an ideal therapy for reconstruction of imperfect tissue caused by various reasons.
The purpose of this study were to observe the growth characteris of cultured murine marrow stromal stem cells (BMSCs ) ;to investigate the differentiate potential of MSCs under the induced condition; to evaluate the differentiated state of BMSCs on the basis of the Hoechst 33342 stained degree of cells. Based the experiment, we expect to understand the biological characters of BMSCs further more, and offer reliable bases before they are applied to clinic.
Methods
1. A population of homogeneous murine marrow stem cells were isolated and cultured from bone marrow by their abilities to proliferate in culture with an attached well-spread morphology. And the shapes and structures of the cells were observed by phase contrast microscope.
2. Immunohistochemistry was used to examine the expression of the suface markers: CD34 CD 44 CD54 and CD106.
3. The osteogenic differentiation were induced under the influence of 10-8M dexamethasone, 10 mmol/L p-glycerol phosphate, and 50ug/ml ascorbate and in the presence of 15% v/v FBS. Two weeks later, examine the expression of alkaline phosphatase and type I collagen.
4. Adipogenic differentiation were induced in the expanded marrow stem cell cultures by treatment with 0. 5 mmol/L l-methyl-3-isobutylxanthine, 1 umol/L dexamethasone, and 1 ug/ml insulin. Observe the changes of induced cells. Two weeks later, Oil red-0 stain show adipogenic differentiation.
5. Drop the liquid including Hoechst 33342 into foster which creeped original and passed BMSCs. Incubate 30 min avoided the light. Remove the dye-stuff. Rinse 3 times with PBS. Observe the stained degree of cells by Olympus-BX51 fluorescence phase contrast microscope.
Results
1. Mor phologic observation of BMSCs: 24h after the initial seeding, BMSCs attached the surface of foster. Purification of BMSCs was achieved by removal of the nonadherent cells during subsequent changes of medium. There were distinct formation of clonies 4 ~5 days later, and the shape, the size and the density of the clonies were different; and BMSCs reached confluence 6 ~ 8 days later.
At the fourth and fifth day of initial culture, we observed some lipocytes by phase contrast microscope. Oil red-0 stain show positive.
2. The result of the surface markers expression of BMSCs : the cells were negative for reactivity to antigen CD34 which is common on cells of the hemato-poietic lineages; and positive forCD44,CD54 and CD106.
3. Osteogenic Differentiation of BMSCs : after 3-4 days , the shape of the cells change from slightly shuttle into triangle and polygon; at the eighth day, the isolated BMSCs fonned aggregates or nodules. The expression of alkaline phosphatase and type I collegon were positive.
4. Adipogenic Differentiation of BMSCs: after 5 days, the shape of cells change from polygon to ellipse ; at 7 ~9 days, there were lipid vacuoles within cells. After 14 days, there were apparent and accumulation of lipid-rich vacuoles within cells with an eccentric deviation of the nucleus. The Oil red-0 stained result of the induced cells was positive.
5. The result of Hoechst 33342 staining: most nucleolus of original cells were not stained. But the nucleolus of the fifth generation were stained generally , and the stained degree of cells are different.
Conclusion
1. Bone marrow marrow stromal stem cells( BMSCs) could be isolated from bone marrow, and proliferated in vitro.
2. Bone marrow marrow stromal stem cell
骨髓中存在一定数量的骨髓基质干细胞(BMSCs，bone marrow stromal stem cells)，这些细胞具有自我复制能力，在特定的诱导条件下能够分化为中胚层起源的组织，如骨、软骨和脂肪等。将骨髓基质干细胞诱导分化为成骨细胞、软骨细胞、脂肪细胞等，为修复重建由于创伤、感染、肿瘤等各种原因形成的组织缺损，提供了理想的治疗手段，是一项非常具有应用前景的技术。
本实验旨在通过对大鼠骨髓基质干细胞进行体外分离、培养，动态观察BMSCs的生长特点、检测细胞的表面分子抗原；观察在诱导条件下，骨髓基质干细胞分化为成骨细胞和脂肪细胞的能力；并通过观察骨髓基质干细胞对荧光色素Hoechst 33342的排出能力，评价BMSCs所处的不同分化状态，以期进一步了解骨髓基质干细胞的生物学特性，为其应用于临床提供可靠的理论依据。
实验方法
1．全骨髓贴壁培养法体外分离、培养大鼠骨髓基质干细胞，倒置相差显微镜下逐日观察细胞形态及生长状况。
2．免疫细胞化学技术测定细胞表面抗原CD34、CD44、CD54、CD106的表达情况，判定所培养的细胞中有无存在造血细胞和内皮细胞的污染。
3．向生长状态良好的三代细胞中加入成骨细胞诱导液(DMEM培养液，15％胎牛血清，10mmol／L β-甘油磷酸钠，10~(-8)M地塞米松，50μg／ml抗坏血酸)培养两周。两周后进行碱性磷酸酶染色和Ⅰ型胶原免疫细胞化学染色。
4．当第3代骨髓基质干细胞达到完全融合时，加入脂肪细胞诱导液(1μg／ml胰岛素、1μmol／L地塞米松、0.5mmol／L的IBMX及10％FBS的DMEM)，逐日观察细胞变化。两周后以油红O染色鉴定诱导后的骨髓基
质干细胞。
5.分别取原代及第5代骨髓基质干细胞，将配制好的H加兄h吐33342工
作液，加于铺满细胞的培养皿上，避光孵育30分钟，除去染液，PBS冲洗3
次，OlymPusBX51荧光相差显微镜观察细胞对荧光色素H吮cllst 33342的着
色强弱。
实验结果
1.大鼠骨髓基质干细胞的形态学观察:骨髓基质干细胞接种后24h开
始贴壁，随着时间的延长贴壁细胞数量明显增加，呈现梭形或多角形形态，
经换液逐步将培养瓶中不贴壁的杂细胞清除;接种后4一sd可见较明显的
集落形成，集落的形态、大小、密度各不相同;约6一sd，贴壁细胞逐渐融合
成单层，细胞多呈梭形，细胞排列呈现明显的辐射状、游涡状。
原代培养4一sd时，倒置相差显微镜下可见到聚集成堆，大个、圆形，内
含折光性强的脂滴的脂肪样细胞，油红0检侧为阳性。
2.骨髓基质干细胞的表型特征:免疫组织化学技术检侧BMSCs的4种
表面分子表达的结果显示:BMScs的CD料(纤连蛋白和透明脂酸盐的受
体)、CD54(基质受体)、CD106(细胞间钻附分子)表达阳性;而CD:抖(造血
细胞系标记物)表达阴性。
3.定向诱导为成骨细胞:在诱导3一4d后，骨髓基质干细胞逐渐变得
扁平，由细长梭形转变为三角形或多角形;8d时，培养细胞大部分变为多角
形，细胞外基质分泌增多;第14d时，培养细胞之间出现黑色结节样结构。
用钙钻法测定细胞内碱性磷酸酶活性，呈阳性反应;免疫细胞化学显示l型
胶原表达阳性。
4.定向诱导成脂肪细胞:在成脂肪诱导5d，细胞原来的多角形突触逐
渐回缩，但仍呈多角形;7一d时出现明显脂滴，脂滴多位于细胞边缘。诱导
14d后，脂肪细胞增加，细胞内脂滴密布，多数细胞形态变圆，多散在分布。
油红O染色为阳性。
5.Hoechst 33342染色结果:原代细胞进行H~h欢33342染色，紫外光
激发后，可见绝大多数细胞核不着色。而第5代细胞染色后，明视场可见大
部分细胞的细胞核周围都有亮晕，暗视野下，即紫外光激发后，可见细胞核
大部分呈现弱着色，着色深浅不一。
结论
1.大鼠骨髓中可以从分离出骨髓基质干细胞，经体外培养可以大量扩
增。
2.大鼠骨髓基质干细胞在Dex、p一甘油磷酸钠和vitc作用下，可以向成
骨细胞分化。
3.大鼠骨髓基质干细胞在胰岛素、Dex和IBMX诱导下，可以定向分化
为脂肪细胞。
4.H慨bst 33342荧光染色技术在多潜能干细胞向单能干细胞转化过
程中，能够准确客观的评价细胞所处的分化状态，可以作为干细胞筛选和检
测干细胞功能状态的新方法。
Bone marrow stromal stem cells ( BMSCs) are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues , including bone, cartilage, and fat et al. Under controlled in vitro conditions , these cells could be induced to differentiate exclusively into the adipocyt-ic, chondrocytic, or osteocytic lineages. Using BMSCs to fabricate engineered tissue is a foreground technology, which offer an ideal therapy for reconstruction of imperfect tissue caused by various reasons.
The purpose of this study were to observe the growth characteris of cultured murine marrow stromal stem cells (BMSCs ) ;to investigate the differentiate potential of MSCs under the induced condition; to evaluate the differentiated state of BMSCs on the basis of the Hoechst 33342 stained degree of cells. Based the experiment, we expect to understand the biological characters of BMSCs further more, and offer reliable bases before they are applied to clinic.
Methods
1. A population of homogeneous murine marrow stem cells were isolated and cultured from bone marrow by their abilities to proliferate in culture with an attached well-spread morphology. And the shapes and structures of the cells were observed by phase contrast microscope.
2. Immunohistochemistry was used to examine the expression of the suface markers: CD34 CD 44 CD54 and CD106.
3. The osteogenic differentiation were induced under the influence of 10-8M dexamethasone, 10 mmol/L p-glycerol phosphate, and 50ug/ml ascorbate and in the presence of 15% v/v FBS. Two weeks later, examine the expression of alkaline phosphatase and type I collagen.
4. Adipogenic differentiation were induced in the expanded marrow stem cell cultures by treatment with 0. 5 mmol/L l-methyl-3-isobutylxanthine, 1 umol/L dexamethasone, and 1 ug/ml insulin. Observe the changes of induced cells. Two weeks later, Oil red-0 stain show adipogenic differentiation.
5. Drop the liquid including Hoechst 33342 into foster which creeped original and passed BMSCs. Incubate 30 min avoided the light. Remove the dye-stuff. Rinse 3 times with PBS. Observe the stained degree of cells by Olympus-BX51 fluorescence phase contrast microscope.
Results
1. Mor phologic observation of BMSCs: 24h after the initial seeding, BMSCs attached the surface of foster. Purification of BMSCs was achieved by removal of the nonadherent cells during subsequent changes of medium. There were distinct formation of clonies 4 ~5 days later, and the shape, the size and the density of the clonies were different; and BMSCs reached confluence 6 ~ 8 days later.
At the fourth and fifth day of initial culture, we observed some lipocytes by phase contrast microscope. Oil red-0 stain show positive.
2. The result of the surface markers expression of BMSCs : the cells were negative for reactivity to antigen CD34 which is common on cells of the hemato-poietic lineages; and positive forCD44,CD54 and CD106.
3. Osteogenic Differentiation of BMSCs : after 3-4 days , the shape of the cells change from slightly shuttle into triangle and polygon; at the eighth day, the isolated BMSCs fonned aggregates or nodules. The expression of alkaline phosphatase and type I collegon were positive.
4. Adipogenic Differentiation of BMSCs: after 5 days, the shape of cells change from polygon to ellipse ; at 7 ~9 days, there were lipid vacuoles within cells. After 14 days, there were apparent and accumulation of lipid-rich vacuoles within cells with an eccentric deviation of the nucleus. The Oil red-0 stained result of the induced cells was positive.
5. The result of Hoechst 33342 staining: most nucleolus of original cells were not stained. But the nucleolus of the fifth generation were stained generally , and the stained degree of cells are different.
Conclusion
1. Bone marrow marrow stromal stem cells( BMSCs) could be isolated from bone marrow, and proliferated in vitro.
2. Bone marrow marrow stromal stem cell
引文
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23．卢慧玲，王宏伟，林汉华．调节脂肪细胞分化的分子机制．医学综述．2003，9(5)：257-258．
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29．李海，周虚，高志花，等．用台盼蓝和荧光染色法评价腔前卵泡的活力．中国兽医学报．2003，23(5)：503-504．