撤除生长因子对hUCMSCs增殖和细胞外基质表达的影响
|
摘要
目的：
人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUCMSCs)具有多向分化潜能和自我更新能力,其诱导植入有望成为应用广泛的细胞治疗手段。为了快速获得足够数量的hUCMSCs,研究人员常在培养基中添加生长因子以促进细胞生长。但是当hUCMSCs输入人体后,没有添加的生长因子的继续作用,细胞的增殖、迁移及定植能力是否会受到影响呢?细胞外基质与细胞的增殖、迁移及定植能力密切相关,因此本实验研究了细胞外基质的表达情况。
本研究的目的是：1.研究完全撤除表皮生长因子(epidermal growth factor,EGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)、血管内皮细胞生长因子(vascular endothelial cell growth factor, VEGF)对细胞增殖及细胞外基质表达的影响。2.保留半量的EGF、bFGF、VEGF是否可以维持细胞增殖及细胞外基质的表达。
方法：
1. hUCMSCs的分离培养及表型鉴定脐带在采集后6 h内用植块法分离培养,每3-4d换液1次,达到80%融合时以细胞密度5×104个/cm2传代培养,取第5代稳定生长的hUCMSCs用流式细胞仪进行表型鉴定。
2.实验分组取第5代hUCMSCs,细胞生长达70%融合时为细胞换液撤除因子,并将细胞分为三组：对照组,生长因子浓度同前；实验组1,将生长因子浓度突然减半；实验组2,所换培养基不添加任何生长因子,仅含正常浓度的血清。
3.瑞氏-姬姆萨法染色观察细胞形态：将第5代hUCMSCs接种至24孔板,细胞生长达70%融合时按分组为细胞换液,继续培养30小时后行瑞氏-姬姆萨法染色观察细胞形态。
4.MTT法细胞计数：按分组将第5代hUCMSCs种植于96孔板,每孔加入5×103个细胞(200μL),在37℃、5%C02条件下孵育30小时后进行MTT法计数。
5.实时定量PCR (real-time Q-PCR)检测细胞凋亡相关基因及胞外基质基因的表达。
5.1 Trizol法提取总RNA将细胞接种至6孔板,细胞生长达70%融合时为细胞换液,分上述3组,继续培养15小时后吸出培养液,每孔加入Trizol 1 ml,提取总RNA。
5.2紫外分光光度法测所提取总RNA浓度,按说明书进行反转录制备cDNA。
5.3实时定量PCR检测胶原蛋白-Ⅰ(Collagen-Ⅰ, Col-Ⅰ),胶原蛋白-Ⅳ(Collagen-Ⅳ, Col-Ⅳ),纤连蛋白(Fibronectin, FN),层粘连蛋白(Laminin, LN),整合素(Integrin),粘胶蛋白(Tenascin-C),基质金属蛋白酶-2(MatrixMetalloprotease-2, MMP-2),基质金属蛋白酶-7(Matrix Metalloprotease-7,MMP-7),基质金属蛋白酶-9 (Matrix Metalloprotease-9, MMP-9), BCL-2,BAX基因的表达情况。
结果：
1.细胞表形检测结果
所获得的贴壁细胞CD34和CD45阴性表达,CD44、CD105、CD71、CD90阳性表达,符合间充质干细胞的特征。
2. hUCMSCs的形态学观察
2.1对照组：细胞扁平或长梭形,可见突起,胞核较大,扁卵圆形,染色质细小稀疏着色浅,核仁明显,胞质较丰富,呈弱嗜碱性,可见嗜碱性颗粒。
2.2实验组1：细胞扁平或长梭形,部分细胞较对照组变大、变圆,核质比减小。
2.3实验组2：多数细胞较对照组变大、变圆,核质比减小
3.MTT法细胞计数结果
与对照组相比,实验组2细胞数目减少,但不具显著差异(P=0.16)；实验组1细胞数目略有增加,亦不具显著差异(P=0.49)。
4.实时定量PCR结果
4.1凋亡相关基因BCL-2和BAX表达情况撤除生长因子后BCL-2表达上调,BAX表达下调,BCL-2/BAX比率增大,实验组1较实验组2 BCL-2表达上调、BAX表达下调幅度更大,BCL-2/BAX比率更高。
4.2胞外基质基因表达情况与对照组相比,半量撤除因子时,Col-Ⅰ、FN、LN、Integrin和Tenascin-C表达增高,完全撤除时Col-Ⅰ、FN表达增高,LN、Integrin和Tenascin-C表达下调。而Col-Ⅳ、MMP-2、MMP-7和MMP-9半量撤除和完全撤除时均减少,完全撤除时减少更多。
结论：
1.撤除EGF、VEGF、bFGF会降低hUCMSCs的增殖能力,保留半量生长因子可以维持hUCMSCs的增殖能力。
2.撤除因子可导致细胞体积变大,核质比减小。
3.撤除因子后,BCL-2/BAX比率增大,细胞的抗凋亡能力增强,半量撤除时细胞的抗凋亡能力较完全撤除时更强。
4.撤除生长因子会影响细胞外基质的表达,而保留半量生长因子可以改善这种情况。
Objective:
Human umbilical cord mesenchymal stem cells (hUCMSCs) have multi-cell differenttiation potential and high self-renewal capacity. The implantation of hUCMSCs is expected to become a widely used treatment. In order to obtain large quantities of hUCMSCs, researchers often add growth factors in the culture medium. But when hUCMSCs enter the human body, without added growth factors, will their proliferation, migration and colonization ability be affected? As extracellular matrix is closely related to these ability, this study examined the expression of extracellular matrix.
The purpose of this study was to explore the effects of removing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) on the proliferation and extracellular matrix expression of hUCMSCs and to observe the effects of half amount of the growth factors on hUCMSCs. Method:
1. Isolation culture and identification of hUCMSCs Human umbilical cords were processed within 6 hours with explantation technique. The medium was changed every 3-4 days. When the cells reached 80% confluency, they were passaged at a density of 5 X 104/cm2. The phenotype of the 5th generation cells were identified with flow cytometry.
2. Experimental groups When hUCMSCs of the 5th generation reached 70% confluency, the growth factors were removed and the cells were divided into three groups. The group without removing growth factors was named as the control group. The half dose withdrawal group was named as experimental group 1. The growth factors completely removed group(The medium does not contain any added growth factors, with only normal levels of serum) was named as experimental group 2.
3. Observe the cells with Wright-Giemsa staining The 5th generation cells were passaged to 24-well plate. When the cells reached 70% confluency, the medium was changed with different concentration of growth factors and the cells were cultured for 30 hours. The cells were observed under microscope with Wright-Giemsa staining.
4. Detect cell proliferation and growth by MTT assay The 5th generation cells were cultured in 96-well plate at 5×103 cells/well(200μL) with different medium for 30 hours. The cell proliferation was detected by MTT assay.
5. The expression of apoptosis-related and extracellular matrix genes was detected with Real-time quantitative PCR.
5.1 RNA extraction The 5th generation cells were passaged to 6-well plate. When the cells reached 70% confluency, the medium was changed with different concentration of growth factors and the cells were cultured for 15 hours.1 ml trizol was added each well and the total RNA was extracted following the instruction.
5.2 The concentration of the extracted RNA was detected with UV spectrophotometer and the cDNA was prepared by reverse transcription according to the instruction.
5.3 The gene expression of Collagen-Ⅰ(Col-Ⅰ), Collagen-Ⅳ(Col-Ⅳ), Fibronectin (FN), Laminin (LN), Integrin, Tenascin-C, Matrix Metalloproteinase-2 (MMP-2), Matrix Metalloproteinase-7 (MMP-7), Matrix Metalloproteinase-9 (MMP-9), BCL-2 and BAX was detected with Real-time quantitative PCR.
Results:
1. Identification of the phenotype
CD44, CD105, CD71 and CD90 were positive while CD34 and CD45 were negative, which coincided with the characteristics of hUCMSCs.
2. Morphological observation
2.1 In control group the cells were in flat or long spindle shape, showing processes. The nucleus were large, flat oval. The chromatin was thin with light color and the nucleoli were clear. The cytoplasm was abundant and weakly basophilic, containing basic particles.
2.2 In experimental group 1, some cells became larger and rounder than in control group.
2.3 In experimental group 2, the majority of cells became larger and rounder, and the nuclear-cytoplasmic ratio became smaller compared with the cells in experimental group 1.
3. Cell proliferation detected by MTT assay
Compared with the control group, the number of cells in experimental group 2 reduced but not significantly (P=0.16). The number of cells in experimental group 1 slightly rose, still not significantly (P=0.49).
4. Real-time quantitative PCR results
4.1 Expression of apoptosis-related gene BCL-2 and BAX After the removal of growth factors, the expression of BCL-2 increased, the expression of BAX decreased, and the BCL-2/BAX ratio increased. The BCL-2/BAX ratio was larger in experimental group 1 than in experimental group 2.
4.2 Gene expression of extracellular matrix The expression of Col-1, FN, LN, Integrin and Tenascin-C increased in experimental group 1, while in experimental group 2 the expression of LN, Integrin, Tenascin-C decreased. The expression of ColIV, MMP-2, MMP-7, MMP-9 decreased after the removal of growth factors and it decreased much more when the growth factors were removed completely.
Conclusion:
1. Removing the growth factors in the medium inhibits the proliferation of hUCMSCs, and half dose of the growth factors could maintain the proliferation.
2. Removal of EGF, VEGF and bFGF leads to larger cell size and smaller nuclear-cytoplasmic ratio.
3. After the removal of EGF, VEGF and bFGF, hUCMSCs upregulate the expression of Bcl-2 and reduce the expression of Bax to enhance the anti-apoptosis ability. The anti-apoptosis ability is stronger when only half amount of the growth factors are removed than they are removed completely.
4. Removal of growth factors could affect the expression of extracellular matrix gene. Half dose of the growth factors could maintain them.
人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUCMSCs)具有多向分化潜能和自我更新能力,其诱导植入有望成为应用广泛的细胞治疗手段。为了快速获得足够数量的hUCMSCs,研究人员常在培养基中添加生长因子以促进细胞生长。但是当hUCMSCs输入人体后,没有添加的生长因子的继续作用,细胞的增殖、迁移及定植能力是否会受到影响呢?细胞外基质与细胞的增殖、迁移及定植能力密切相关,因此本实验研究了细胞外基质的表达情况。
本研究的目的是：1.研究完全撤除表皮生长因子(epidermal growth factor,EGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)、血管内皮细胞生长因子(vascular endothelial cell growth factor, VEGF)对细胞增殖及细胞外基质表达的影响。2.保留半量的EGF、bFGF、VEGF是否可以维持细胞增殖及细胞外基质的表达。
方法：
1. hUCMSCs的分离培养及表型鉴定脐带在采集后6 h内用植块法分离培养,每3-4d换液1次,达到80%融合时以细胞密度5×104个/cm2传代培养,取第5代稳定生长的hUCMSCs用流式细胞仪进行表型鉴定。
2.实验分组取第5代hUCMSCs,细胞生长达70%融合时为细胞换液撤除因子,并将细胞分为三组：对照组,生长因子浓度同前；实验组1,将生长因子浓度突然减半；实验组2,所换培养基不添加任何生长因子,仅含正常浓度的血清。
3.瑞氏-姬姆萨法染色观察细胞形态：将第5代hUCMSCs接种至24孔板,细胞生长达70%融合时按分组为细胞换液,继续培养30小时后行瑞氏-姬姆萨法染色观察细胞形态。
4.MTT法细胞计数：按分组将第5代hUCMSCs种植于96孔板,每孔加入5×103个细胞(200μL),在37℃、5%C02条件下孵育30小时后进行MTT法计数。
5.实时定量PCR (real-time Q-PCR)检测细胞凋亡相关基因及胞外基质基因的表达。
5.1 Trizol法提取总RNA将细胞接种至6孔板,细胞生长达70%融合时为细胞换液,分上述3组,继续培养15小时后吸出培养液,每孔加入Trizol 1 ml,提取总RNA。
5.2紫外分光光度法测所提取总RNA浓度,按说明书进行反转录制备cDNA。
5.3实时定量PCR检测胶原蛋白-Ⅰ(Collagen-Ⅰ, Col-Ⅰ),胶原蛋白-Ⅳ(Collagen-Ⅳ, Col-Ⅳ),纤连蛋白(Fibronectin, FN),层粘连蛋白(Laminin, LN),整合素(Integrin),粘胶蛋白(Tenascin-C),基质金属蛋白酶-2(MatrixMetalloprotease-2, MMP-2),基质金属蛋白酶-7(Matrix Metalloprotease-7,MMP-7),基质金属蛋白酶-9 (Matrix Metalloprotease-9, MMP-9), BCL-2,BAX基因的表达情况。
结果：
1.细胞表形检测结果
所获得的贴壁细胞CD34和CD45阴性表达,CD44、CD105、CD71、CD90阳性表达,符合间充质干细胞的特征。
2. hUCMSCs的形态学观察
2.1对照组：细胞扁平或长梭形,可见突起,胞核较大,扁卵圆形,染色质细小稀疏着色浅,核仁明显,胞质较丰富,呈弱嗜碱性,可见嗜碱性颗粒。
2.2实验组1：细胞扁平或长梭形,部分细胞较对照组变大、变圆,核质比减小。
2.3实验组2：多数细胞较对照组变大、变圆,核质比减小
3.MTT法细胞计数结果
与对照组相比,实验组2细胞数目减少,但不具显著差异(P=0.16)；实验组1细胞数目略有增加,亦不具显著差异(P=0.49)。
4.实时定量PCR结果
4.1凋亡相关基因BCL-2和BAX表达情况撤除生长因子后BCL-2表达上调,BAX表达下调,BCL-2/BAX比率增大,实验组1较实验组2 BCL-2表达上调、BAX表达下调幅度更大,BCL-2/BAX比率更高。
4.2胞外基质基因表达情况与对照组相比,半量撤除因子时,Col-Ⅰ、FN、LN、Integrin和Tenascin-C表达增高,完全撤除时Col-Ⅰ、FN表达增高,LN、Integrin和Tenascin-C表达下调。而Col-Ⅳ、MMP-2、MMP-7和MMP-9半量撤除和完全撤除时均减少,完全撤除时减少更多。
结论：
1.撤除EGF、VEGF、bFGF会降低hUCMSCs的增殖能力,保留半量生长因子可以维持hUCMSCs的增殖能力。
2.撤除因子可导致细胞体积变大,核质比减小。
3.撤除因子后,BCL-2/BAX比率增大,细胞的抗凋亡能力增强,半量撤除时细胞的抗凋亡能力较完全撤除时更强。
4.撤除生长因子会影响细胞外基质的表达,而保留半量生长因子可以改善这种情况。
Objective:
Human umbilical cord mesenchymal stem cells (hUCMSCs) have multi-cell differenttiation potential and high self-renewal capacity. The implantation of hUCMSCs is expected to become a widely used treatment. In order to obtain large quantities of hUCMSCs, researchers often add growth factors in the culture medium. But when hUCMSCs enter the human body, without added growth factors, will their proliferation, migration and colonization ability be affected? As extracellular matrix is closely related to these ability, this study examined the expression of extracellular matrix.
The purpose of this study was to explore the effects of removing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) on the proliferation and extracellular matrix expression of hUCMSCs and to observe the effects of half amount of the growth factors on hUCMSCs. Method:
1. Isolation culture and identification of hUCMSCs Human umbilical cords were processed within 6 hours with explantation technique. The medium was changed every 3-4 days. When the cells reached 80% confluency, they were passaged at a density of 5 X 104/cm2. The phenotype of the 5th generation cells were identified with flow cytometry.
2. Experimental groups When hUCMSCs of the 5th generation reached 70% confluency, the growth factors were removed and the cells were divided into three groups. The group without removing growth factors was named as the control group. The half dose withdrawal group was named as experimental group 1. The growth factors completely removed group(The medium does not contain any added growth factors, with only normal levels of serum) was named as experimental group 2.
3. Observe the cells with Wright-Giemsa staining The 5th generation cells were passaged to 24-well plate. When the cells reached 70% confluency, the medium was changed with different concentration of growth factors and the cells were cultured for 30 hours. The cells were observed under microscope with Wright-Giemsa staining.
4. Detect cell proliferation and growth by MTT assay The 5th generation cells were cultured in 96-well plate at 5×103 cells/well(200μL) with different medium for 30 hours. The cell proliferation was detected by MTT assay.
5. The expression of apoptosis-related and extracellular matrix genes was detected with Real-time quantitative PCR.
5.1 RNA extraction The 5th generation cells were passaged to 6-well plate. When the cells reached 70% confluency, the medium was changed with different concentration of growth factors and the cells were cultured for 15 hours.1 ml trizol was added each well and the total RNA was extracted following the instruction.
5.2 The concentration of the extracted RNA was detected with UV spectrophotometer and the cDNA was prepared by reverse transcription according to the instruction.
5.3 The gene expression of Collagen-Ⅰ(Col-Ⅰ), Collagen-Ⅳ(Col-Ⅳ), Fibronectin (FN), Laminin (LN), Integrin, Tenascin-C, Matrix Metalloproteinase-2 (MMP-2), Matrix Metalloproteinase-7 (MMP-7), Matrix Metalloproteinase-9 (MMP-9), BCL-2 and BAX was detected with Real-time quantitative PCR.
Results:
1. Identification of the phenotype
CD44, CD105, CD71 and CD90 were positive while CD34 and CD45 were negative, which coincided with the characteristics of hUCMSCs.
2. Morphological observation
2.1 In control group the cells were in flat or long spindle shape, showing processes. The nucleus were large, flat oval. The chromatin was thin with light color and the nucleoli were clear. The cytoplasm was abundant and weakly basophilic, containing basic particles.
2.2 In experimental group 1, some cells became larger and rounder than in control group.
2.3 In experimental group 2, the majority of cells became larger and rounder, and the nuclear-cytoplasmic ratio became smaller compared with the cells in experimental group 1.
3. Cell proliferation detected by MTT assay
Compared with the control group, the number of cells in experimental group 2 reduced but not significantly (P=0.16). The number of cells in experimental group 1 slightly rose, still not significantly (P=0.49).
4. Real-time quantitative PCR results
4.1 Expression of apoptosis-related gene BCL-2 and BAX After the removal of growth factors, the expression of BCL-2 increased, the expression of BAX decreased, and the BCL-2/BAX ratio increased. The BCL-2/BAX ratio was larger in experimental group 1 than in experimental group 2.
4.2 Gene expression of extracellular matrix The expression of Col-1, FN, LN, Integrin and Tenascin-C increased in experimental group 1, while in experimental group 2 the expression of LN, Integrin, Tenascin-C decreased. The expression of ColIV, MMP-2, MMP-7, MMP-9 decreased after the removal of growth factors and it decreased much more when the growth factors were removed completely.
Conclusion:
1. Removing the growth factors in the medium inhibits the proliferation of hUCMSCs, and half dose of the growth factors could maintain the proliferation.
2. Removal of EGF, VEGF and bFGF leads to larger cell size and smaller nuclear-cytoplasmic ratio.
3. After the removal of EGF, VEGF and bFGF, hUCMSCs upregulate the expression of Bcl-2 and reduce the expression of Bax to enhance the anti-apoptosis ability. The anti-apoptosis ability is stronger when only half amount of the growth factors are removed than they are removed completely.
4. Removal of growth factors could affect the expression of extracellular matrix gene. Half dose of the growth factors could maintain them.
引文
1. Baksh D, Song L, Tuan RS. Adult mesenchymal stem cell:characteriza-tion, differentiation, and application in cell and gene therapy [J]. Cell Mol Med,2004,8: 301-16.
2. Pereboeva L, Komarova S, Mikheeva G, et al. Approaches to utilize mesenchymal progenitor cells as cellular vehicles [J]. Stem Cells,2003,21:389-404.
3. Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord [J].Stem Cells,2004,22(7):1330-1337.
4. McElreavey KD, Irvine Al, Ennis KT, et al. Isolation, culture and characterisation of fibroblast-like cells derived from the Wharton's jelly portion of human umbilical cord [J]. Biochem Soc Trans,1991,19 (1):29-33.
5. Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular (HUCPV) cells:a source of mesenchymal progenitors [J]. Stem Cells,2005,23(2): 220-229.
6. Mitchell KE, Weiss ML, Mitchell BM, et al. Matrix cells from Wharton's jelly form neurons and glia [J]. Stem Cells,2003,21 (1):50-60.
7. Ma L, Feng XY, Cui BL, et al. Human umbilical cord Wharton's jelly derived mesenchymal stem cells differentiation into nerve-like cells [J]. ChinMed J (Engl), 2005,118(23):1987-1993.
8. CovasDT, Siufi JL, Silva AR, et al. Isolation and culture of umbilical vein mesenchymal stem cells [J]. Braz J Med Biol Res,2003,36 (9):1179-1183.
9. 宋希拿,何祎,韩俊领.人脐带间充质干细胞的生物学特性及其在血液系统疾病中的应用[J].医学综述,2009,15(2)：166-168.
10. Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular (HUCPV) cells:a source of mesenchymal projenitors [J]. Stem Cells,2005,23(2): 220-229.
11. Tian Y, Deng YB, Huang YJ, et al. Bone marrow-derived mesenchymal stem cells decrease acute graft-versus-host disease after allogeneic hematopoietic stem cells transp lantation [J]. Immunol Invest,2008,37(1):29-42.
12. Ma L, Feng XY, Cui BL, et al. Human umbilical cord Wharton's Jelly derived mesenchymal stem cells differentiation into nerve-like cells [J]. Chin Med J (Engl). 2005,118(23):1987-1993.
13. Kadner A, Zund G,Maurus C, et al. Human umbilical cord cells for cardiovascular tissue engineering:a comparative study [J]. Eur J Cardiothorac Surg,2004,25(4): 635-641.
14. Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular (HUCPV) cells:a source of mesenchymal progenitors [J]. Stem Cells,2005,23 (2): 220-229.
15. Romanov YA, SvintsitskayaVA, SmirnovVN. Searching for alternative sources of postnatal human mesenchymal stem cells:candidate MSC-like cells from umbilical cord [J]. Stem Cells,2003,21 (1):105-110.
16.徐燕,李长虹,孟恒星等.人脐带间充质干细胞分离培养条件的优化及其生物学特性[J].中国组织工程研究与临床康复,2009,13(32)：6289-6294.
17.段积华.细胞凋亡及其与细胞因子和癌基因的研究进展[J].重庆医科大学学报,1999,24(3)：318-322.
18. Han JH, Gileadi C, Rajapaksa R, et al. Modulation of apoptosis in human myeloid leukemic cells by GM-CSF [J]. Experimental Hematology,1995,23:265-272.
19.万晓华.撤除生长因子对脐血细胞凋亡的影响及人参皂甙Rgl、Rbl的干预作用[D].天津：天津医科大学,2003：5-31.
20.高唱,吴伟康,王景.周细胞外基质与神经干细胞的增殖、分化和迁移[J].国外医学脑血管疾病分册,2003,11(2)：124-126.
21.宋丹.表皮生长因子的研究进展[J].解剖科学进展,2002,8(4)：340-342.
22.姜琨,秦樾,童坦君.erbB-2表达抑制与表皮生长因子刺激对信号转导与转录激活分子及细胞周期蛋白D1的影响[J].北京医科大学学报.1998,30(3)：205-2081.
23. Gronthos S, Simmons PJ. The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro [J]. Blood,1995,85(4):929-940.
24. Tamama K, Fan VH, Griffith LG, et al. Epidermal growth factor as a candidate for exvivo expansion of bone marrow-derived mesenchymal stem cells [J]. Stem Cells, 2006,24(3):686-695.
25. Krampera M, Pasini A, Rigo A, et al. HB-EGF/HER-1 signaling in bone marrow mesenchymal stem cells:inducing cell expansion and reversibly preventing multilineage differentiation [J]. Blood,2005,106(1):59-66.
26. Mobasheri A, Csaki C, Clutterbuck AL, et al. Mesenchymal stem cells in connective tissue engineering and regenerative medicine:applications in cartilage repair and osteoarthritis therapy [J]. Histol Histopathol,2009,24(3):347-366.
27. Koga H, Muneta T, Nagase T, et al. Comparison of mesenchymal tissues-derived stem cells for in vivo chondrogenesis:suitable conditions for cell therapy of cartilage defects in rabbit [J]. Cell Tissue Res,2008,333(2):207-215.
28. Ornitz DM, Itoh N. Fibroblast growth factors [J]. Genome Biol,2001,2(3):1-12.
29.张德强,王晓东.人骨髓间充质干细胞生长特性及碱性成纤维细胞生长因子对其增殖的影响[J].中国组织工程研究与临床康复,2007,11(20)：3976-3979.
30.杨雷,李薇,姜震宇等.碱性成纤维细胞生长因子对羊骨髓间充质干细胞体外增殖和分化的影响[J].吉林大学学报(医学版),2007,3(3)：510-512.
31.郑磊,王前,魏宽海等.碱性成纤维细胞生长因子对骨髓基质细胞粘附、增殖和分化的影响[J].第一军医大学学报,2000,20(2)：84-85.
32. Ferrara N, HenzelWL. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells [J]. Biochem Biophys Res Commun,1989,161(2):851-858.
33.肖建武.血管内皮生长因子的基础和临床研究进展[J].中国医疗前沿,2009,4(1)：26-27
34.单伟,秦书俭,曾瑞霞等.血管内皮细胞生长因子对人脐静脉间充质干细胞生长增殖的影响[J].中国组织工程研究与临床康复,2009,13(14)：2709-2711.
35. Kerr J F, Wyllie A H, Currie A R. Apoptosis:a basis biological phenomenon with wide-ranging implications in tissue kinetics [J]. Br J Cancer,1972,26:239-257.
36. Kerr JFR, Winterford CM, Harmon BV. Apoptosis. Its significance in cancer and cancer therapy [J]. Cancer,1994,73:2013-2026.
37.李超,伏圣博,刘华玲.细胞凋亡研究进展[J].世界科技研究与发展,2007,29(3)：45-53.
38. Migheli A, Cavalla P, Marino S, et al. A study of apoptosis in normal and pathologic nervous tissue after insitu end-labeling of DNA strand breaks [J]. Neuropathol Exp Neurol Terminal,1994,53:606-608.
39.梁伟,苟三怀,齐进等.低强度脉冲超声波对体外培养的兔骨髓基质细胞成骨作用的影响[J].第二军医大学学报,2007,28(6)：603-606.
40.李超,伏圣博,刘华玲.细胞凋亡研究进展[J].世界科技研究与发展,2007,29(3)：45-53.
41. Roset R, Ortet L, Gil-Gomez G. Role of Bcl-2 familymembers on apoptosis:what we have learned from knock-out mice [J]. Front Biosci,2007,12 (20):4722-4730.
42.王卫东.Bcl-2/Bax比率与细胞“命运”[J].中国肿瘤生物治疗杂志,2007,14(4)：393-396.
43.王拴柱,马秀梅.细胞外基质与肿瘤转移关系的研究进展[J].内蒙古医学院学报,2009,31(1)：75-77.
44.胡国勇.MMP-7在大肠癌中的研究进展[J].临床内科杂志,2002；4(19)：23-25.
1. Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells inn human umbilicalcord blood [J]. Br J Haematol,2000,109:235-242.
2. De BC, Dell AF, Tylzanowski P, et al. Multipotent mesenchymal stem cells from adult human synovial membrane [J]. Arthritis Rheum,2001,44(8): 1928-1942.
3. PittengerMF,Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells [J]. Science,1999,284:143-147.
4. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cell derived from adultmarrow [J]. Nature,2002,418:41-49.
5. Lee K, Majumdar MK, Buyaner D, et al. Human mesenchymal stem cells maintaintransgene expression during expansion anddifferentiation [J]. Mol Ther,2001,3(6):857-866.
6. 金丹,裴国献,王珂,等.人骨形态发生蛋白-7基因转染对骨髓间充质干细胞增殖及成骨分化性能的影响[J].中国临床康复,2005,9(14)：206-208.
7. Dong Hyum Kim, Keon Hee Yoo, et al. Gene expression profile of cytokine and growth factor during differentiation of bone marrow.derived mesenchymal stem cell [J]. Cytokine,2005,31:119-126.
8. Roufosse CA, Direkze NC, OttoWR, et al. Circulating mesenchymal stem cells [J]. Int J Biochem Cell Biol,2004,36:585-597.
9. Mauro Krampera, Annalisa Pasini, Antonella Rigo, et al. HBEGF/HER-1 signaling in bone marrow mesenchymal stem cells:inducing cell expansion and reversibly preventing multilineage differentiation [J]. Blood,2005,106: 59-66.
10. 赵智刚邹萍.骨髓间充质干细胞的研究进展及其应用前景.临床内科杂志[J].2009,23(9)：588-590.
11. 宋希拿,何祎,韩俊领.人脐带间充质干细胞的生物学特性及其在血液系统疾病中的应用[J].医学综述,2009,15(2)：166-168.
12. Tian Y, Deng YB, Huang YJ, et al. Bone marrow-derived mesenchymal stem cells decrease acute graft-versus-host disease after allogeneic hematopoietic stem cells transp lantation [J]. Immunol Invest,2008,37 (1):29-42.
13. Liu J F,Wang B W,Hung H F,et al. Human mesenchymal stem cells improve myocardial performance in a splenectomized rat model of chronic myocardial infarction[J]. J Formos Med Assoc,2008,107 (2):165-174.
14. St rauer BE,Brehm M,Zeus T,et al. Repair of infarcted myocardium by autologous int racoronary mononuclear bone marrow cell transpla ntation in humans[J]. Circulation,2002,106 (15):1913-1918.
15. Mohyeddin-Bonab M,Mohamad-Hassani MR,Alimoghaddam K,et al Autologous in vit ro expanded mesenchymal stem cell t herapy for human old myocardial infarction[J]. Arch Iran Med,2007,10 (4):467-473.
16. Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous admininstr-ation of bone marrow stromal cells after cerebral ischemia in rats[J] Stroke,2001,32 (4):1005-1011.
17. Li Y, Chen J, Chen XG, et al. Human marrows tromal cell therapy for stroke in rat:neurotrophins and functional recovery[J], Neurology,2002,59:514-523.
18. Li Y, Chen J, Wang L. Treatment of stroke in rat with intracarotid administration ofmarrow stromal cells[J]. Neurology,2001,56:1666-1672.
19. Kurozumi K, Nakamura K, Tamiya T, et al. BDNF gene-modified mesenchymal stem cells promote functional recovery and reduce infarct size in the rat middle cerebralartery occlusion model [J]. Mol Ther,2004,9(2): 189-197. Erratum in:Mol Ther,2004,9 (5):766.
20. Matsumoto R, Omura T, Yoshiyama M, etal. Vascular endothelial growth factor-expressing mesenchymal stem cell transplantation for the treatment of acute myocardial infarction [J]. Arterioscler Thromb Vasc Biol,2005,25 (6):1168-1173.
21. El-Am in SF, A ttaw ia M, L u HH, et al. Integrin exp ression by human o steoblasts cultured on degradable po lymeric materials app licable fo r tissue engineered bone[J].JOrthop Res,2002,20 (1):20-28.
22. 史明霞,房佰俊,廖联明,等.FLK1+间充质干细胞减轻四氯化碳导致的肝纤维化的研究[J].生物工程学报,2005,21(3)：396-401.
2. Pereboeva L, Komarova S, Mikheeva G, et al. Approaches to utilize mesenchymal progenitor cells as cellular vehicles [J]. Stem Cells,2003,21:389-404.
3. Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord [J].Stem Cells,2004,22(7):1330-1337.
4. McElreavey KD, Irvine Al, Ennis KT, et al. Isolation, culture and characterisation of fibroblast-like cells derived from the Wharton's jelly portion of human umbilical cord [J]. Biochem Soc Trans,1991,19 (1):29-33.
5. Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular (HUCPV) cells:a source of mesenchymal progenitors [J]. Stem Cells,2005,23(2): 220-229.
6. Mitchell KE, Weiss ML, Mitchell BM, et al. Matrix cells from Wharton's jelly form neurons and glia [J]. Stem Cells,2003,21 (1):50-60.
7. Ma L, Feng XY, Cui BL, et al. Human umbilical cord Wharton's jelly derived mesenchymal stem cells differentiation into nerve-like cells [J]. ChinMed J (Engl), 2005,118(23):1987-1993.
8. CovasDT, Siufi JL, Silva AR, et al. Isolation and culture of umbilical vein mesenchymal stem cells [J]. Braz J Med Biol Res,2003,36 (9):1179-1183.
9. 宋希拿,何祎,韩俊领.人脐带间充质干细胞的生物学特性及其在血液系统疾病中的应用[J].医学综述,2009,15(2)：166-168.
10. Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular (HUCPV) cells:a source of mesenchymal projenitors [J]. Stem Cells,2005,23(2): 220-229.
11. Tian Y, Deng YB, Huang YJ, et al. Bone marrow-derived mesenchymal stem cells decrease acute graft-versus-host disease after allogeneic hematopoietic stem cells transp lantation [J]. Immunol Invest,2008,37(1):29-42.
12. Ma L, Feng XY, Cui BL, et al. Human umbilical cord Wharton's Jelly derived mesenchymal stem cells differentiation into nerve-like cells [J]. Chin Med J (Engl). 2005,118(23):1987-1993.
13. Kadner A, Zund G,Maurus C, et al. Human umbilical cord cells for cardiovascular tissue engineering:a comparative study [J]. Eur J Cardiothorac Surg,2004,25(4): 635-641.
14. Sarugaser R, Lickorish D, Baksh D, et al. Human umbilical cord perivascular (HUCPV) cells:a source of mesenchymal progenitors [J]. Stem Cells,2005,23 (2): 220-229.
15. Romanov YA, SvintsitskayaVA, SmirnovVN. Searching for alternative sources of postnatal human mesenchymal stem cells:candidate MSC-like cells from umbilical cord [J]. Stem Cells,2003,21 (1):105-110.
16.徐燕,李长虹,孟恒星等.人脐带间充质干细胞分离培养条件的优化及其生物学特性[J].中国组织工程研究与临床康复,2009,13(32)：6289-6294.
17.段积华.细胞凋亡及其与细胞因子和癌基因的研究进展[J].重庆医科大学学报,1999,24(3)：318-322.
18. Han JH, Gileadi C, Rajapaksa R, et al. Modulation of apoptosis in human myeloid leukemic cells by GM-CSF [J]. Experimental Hematology,1995,23:265-272.
19.万晓华.撤除生长因子对脐血细胞凋亡的影响及人参皂甙Rgl、Rbl的干预作用[D].天津：天津医科大学,2003：5-31.
20.高唱,吴伟康,王景.周细胞外基质与神经干细胞的增殖、分化和迁移[J].国外医学脑血管疾病分册,2003,11(2)：124-126.
21.宋丹.表皮生长因子的研究进展[J].解剖科学进展,2002,8(4)：340-342.
22.姜琨,秦樾,童坦君.erbB-2表达抑制与表皮生长因子刺激对信号转导与转录激活分子及细胞周期蛋白D1的影响[J].北京医科大学学报.1998,30(3)：205-2081.
23. Gronthos S, Simmons PJ. The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro [J]. Blood,1995,85(4):929-940.
24. Tamama K, Fan VH, Griffith LG, et al. Epidermal growth factor as a candidate for exvivo expansion of bone marrow-derived mesenchymal stem cells [J]. Stem Cells, 2006,24(3):686-695.
25. Krampera M, Pasini A, Rigo A, et al. HB-EGF/HER-1 signaling in bone marrow mesenchymal stem cells:inducing cell expansion and reversibly preventing multilineage differentiation [J]. Blood,2005,106(1):59-66.
26. Mobasheri A, Csaki C, Clutterbuck AL, et al. Mesenchymal stem cells in connective tissue engineering and regenerative medicine:applications in cartilage repair and osteoarthritis therapy [J]. Histol Histopathol,2009,24(3):347-366.
27. Koga H, Muneta T, Nagase T, et al. Comparison of mesenchymal tissues-derived stem cells for in vivo chondrogenesis:suitable conditions for cell therapy of cartilage defects in rabbit [J]. Cell Tissue Res,2008,333(2):207-215.
28. Ornitz DM, Itoh N. Fibroblast growth factors [J]. Genome Biol,2001,2(3):1-12.
29.张德强,王晓东.人骨髓间充质干细胞生长特性及碱性成纤维细胞生长因子对其增殖的影响[J].中国组织工程研究与临床康复,2007,11(20)：3976-3979.
30.杨雷,李薇,姜震宇等.碱性成纤维细胞生长因子对羊骨髓间充质干细胞体外增殖和分化的影响[J].吉林大学学报(医学版),2007,3(3)：510-512.
31.郑磊,王前,魏宽海等.碱性成纤维细胞生长因子对骨髓基质细胞粘附、增殖和分化的影响[J].第一军医大学学报,2000,20(2)：84-85.
32. Ferrara N, HenzelWL. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells [J]. Biochem Biophys Res Commun,1989,161(2):851-858.
33.肖建武.血管内皮生长因子的基础和临床研究进展[J].中国医疗前沿,2009,4(1)：26-27
34.单伟,秦书俭,曾瑞霞等.血管内皮细胞生长因子对人脐静脉间充质干细胞生长增殖的影响[J].中国组织工程研究与临床康复,2009,13(14)：2709-2711.
35. Kerr J F, Wyllie A H, Currie A R. Apoptosis:a basis biological phenomenon with wide-ranging implications in tissue kinetics [J]. Br J Cancer,1972,26:239-257.
36. Kerr JFR, Winterford CM, Harmon BV. Apoptosis. Its significance in cancer and cancer therapy [J]. Cancer,1994,73:2013-2026.
37.李超,伏圣博,刘华玲.细胞凋亡研究进展[J].世界科技研究与发展,2007,29(3)：45-53.
38. Migheli A, Cavalla P, Marino S, et al. A study of apoptosis in normal and pathologic nervous tissue after insitu end-labeling of DNA strand breaks [J]. Neuropathol Exp Neurol Terminal,1994,53:606-608.
39.梁伟,苟三怀,齐进等.低强度脉冲超声波对体外培养的兔骨髓基质细胞成骨作用的影响[J].第二军医大学学报,2007,28(6)：603-606.
40.李超,伏圣博,刘华玲.细胞凋亡研究进展[J].世界科技研究与发展,2007,29(3)：45-53.
41. Roset R, Ortet L, Gil-Gomez G. Role of Bcl-2 familymembers on apoptosis:what we have learned from knock-out mice [J]. Front Biosci,2007,12 (20):4722-4730.
42.王卫东.Bcl-2/Bax比率与细胞“命运”[J].中国肿瘤生物治疗杂志,2007,14(4)：393-396.
43.王拴柱,马秀梅.细胞外基质与肿瘤转移关系的研究进展[J].内蒙古医学院学报,2009,31(1)：75-77.
44.胡国勇.MMP-7在大肠癌中的研究进展[J].临床内科杂志,2002；4(19)：23-25.
1. Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells inn human umbilicalcord blood [J]. Br J Haematol,2000,109:235-242.
2. De BC, Dell AF, Tylzanowski P, et al. Multipotent mesenchymal stem cells from adult human synovial membrane [J]. Arthritis Rheum,2001,44(8): 1928-1942.
3. PittengerMF,Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells [J]. Science,1999,284:143-147.
4. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cell derived from adultmarrow [J]. Nature,2002,418:41-49.
5. Lee K, Majumdar MK, Buyaner D, et al. Human mesenchymal stem cells maintaintransgene expression during expansion anddifferentiation [J]. Mol Ther,2001,3(6):857-866.
6. 金丹,裴国献,王珂,等.人骨形态发生蛋白-7基因转染对骨髓间充质干细胞增殖及成骨分化性能的影响[J].中国临床康复,2005,9(14)：206-208.
7. Dong Hyum Kim, Keon Hee Yoo, et al. Gene expression profile of cytokine and growth factor during differentiation of bone marrow.derived mesenchymal stem cell [J]. Cytokine,2005,31:119-126.
8. Roufosse CA, Direkze NC, OttoWR, et al. Circulating mesenchymal stem cells [J]. Int J Biochem Cell Biol,2004,36:585-597.
9. Mauro Krampera, Annalisa Pasini, Antonella Rigo, et al. HBEGF/HER-1 signaling in bone marrow mesenchymal stem cells:inducing cell expansion and reversibly preventing multilineage differentiation [J]. Blood,2005,106: 59-66.
10. 赵智刚邹萍.骨髓间充质干细胞的研究进展及其应用前景.临床内科杂志[J].2009,23(9)：588-590.
11. 宋希拿,何祎,韩俊领.人脐带间充质干细胞的生物学特性及其在血液系统疾病中的应用[J].医学综述,2009,15(2)：166-168.
12. Tian Y, Deng YB, Huang YJ, et al. Bone marrow-derived mesenchymal stem cells decrease acute graft-versus-host disease after allogeneic hematopoietic stem cells transp lantation [J]. Immunol Invest,2008,37 (1):29-42.
13. Liu J F,Wang B W,Hung H F,et al. Human mesenchymal stem cells improve myocardial performance in a splenectomized rat model of chronic myocardial infarction[J]. J Formos Med Assoc,2008,107 (2):165-174.
14. St rauer BE,Brehm M,Zeus T,et al. Repair of infarcted myocardium by autologous int racoronary mononuclear bone marrow cell transpla ntation in humans[J]. Circulation,2002,106 (15):1913-1918.
15. Mohyeddin-Bonab M,Mohamad-Hassani MR,Alimoghaddam K,et al Autologous in vit ro expanded mesenchymal stem cell t herapy for human old myocardial infarction[J]. Arch Iran Med,2007,10 (4):467-473.
16. Chen J, Li Y, Wang L, et al. Therapeutic benefit of intravenous admininstr-ation of bone marrow stromal cells after cerebral ischemia in rats[J] Stroke,2001,32 (4):1005-1011.
17. Li Y, Chen J, Chen XG, et al. Human marrows tromal cell therapy for stroke in rat:neurotrophins and functional recovery[J], Neurology,2002,59:514-523.
18. Li Y, Chen J, Wang L. Treatment of stroke in rat with intracarotid administration ofmarrow stromal cells[J]. Neurology,2001,56:1666-1672.
19. Kurozumi K, Nakamura K, Tamiya T, et al. BDNF gene-modified mesenchymal stem cells promote functional recovery and reduce infarct size in the rat middle cerebralartery occlusion model [J]. Mol Ther,2004,9(2): 189-197. Erratum in:Mol Ther,2004,9 (5):766.
20. Matsumoto R, Omura T, Yoshiyama M, etal. Vascular endothelial growth factor-expressing mesenchymal stem cell transplantation for the treatment of acute myocardial infarction [J]. Arterioscler Thromb Vasc Biol,2005,25 (6):1168-1173.
21. El-Am in SF, A ttaw ia M, L u HH, et al. Integrin exp ression by human o steoblasts cultured on degradable po lymeric materials app licable fo r tissue engineered bone[J].JOrthop Res,2002,20 (1):20-28.
22. 史明霞,房佰俊,廖联明,等.FLK1+间充质干细胞减轻四氯化碳导致的肝纤维化的研究[J].生物工程学报,2005,21(3)：396-401.