温度敏感型干细胞移植联合亚低温治疗脑创伤的实验研究
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摘要
目的：建立一种温度敏感型干细胞系,研究其在亚低温(33℃)和正常体温(37℃)两种环境下的增殖与分化特点,同时利用BDNF的营养和诱导作用增加移植干细胞向神经元方向的分化比例,为脑外伤患者接受亚低温治疗期间实施干细胞原位移植打下基础。
方法：采用原代细胞贴壁培养法培养人源性UCMSCs,流式细胞术进行表面标记蛋白鉴定,体外诱导其向神经细胞分化。用含温度敏感性猿猴病毒40大T抗原(tsSV40LT)的表达质粒转染UCMSCs, PCR检测外源基因的整合,绘制其在33℃和37℃培养条件下的细胞生长曲线；流式细胞术检测细胞周期,PCR-ELISA端粒酶检测法检测端粒酶活性,Western Blot法检测细胞周期调控蛋白---cyclin D1、CDK2和P16蛋白的表达；血清依赖实验及软琼脂克隆形成实验检测该细胞系在两种温度下的致瘤潜能；利用腺病毒载体转染BDNF进入UCMSCs, ELISA方法测定BDNF分泌时相,免疫荧光方法检测转染与非转染细胞表达NSE和GFAP的比例；建立去胸腺小鼠颅脑液压打击模型进行干细胞原位移植,脑组织标本行增殖细胞核抗原(PCNA)免疫组化及原位细胞凋亡检测,实验动物予以神经功能缺陷评分。
结果：利用原代贴壁细胞培养法成功获得UCMSCs,流式细胞结果符合UCMSCs特征,经体外条件诱导,UCMSCs可表达神经元和神经胶质细胞标记蛋白；PCR结果显示tsSV40LT抗原的基因片段被成功整合入干细胞,端粒酶活性高,cyclin D1和CDK2高表达,P16低表达,并高表达nestin,不表达NSE和GFAP,同时该细胞系在33℃时增殖旺盛,可耐受极低的营养条件,并可在软琼脂中形成小的细胞克隆；在37℃条件下细胞增殖缓慢甚至停滞,端粒酶活性低,cyclin D1和CDK2的表达强度明显低于33℃条件,P16则相反,与此同时nestin表达强度明显下降,NSE和GFAP表达水平上升,低营养耐受力下降,无细胞克隆形成；BDNF的表达水平于病毒转染后第72h达到高峰,此期干细胞NSE阳性细胞比例明显增多(P<0.05),相反GFAP阳性细胞比例下降；动物体内结果显示,亚低温治疗期间的脑组织高表达PCNA,凋亡细胞少见；常温组脑组织则低表达PCNA,创伤灶周边见较多凋亡细胞存在。
结论：
(1)借助tsSV40LT抗原建立的温度敏感型UCMSCs细胞系,在细胞增殖和1分化方面具有显著的温度可调控性特征,即在其适宜温度下(33℃)增殖能力旺盛,并维持在前体细胞水平,极少产生细胞分化；当其处于37℃环境中时则停止增长,转而分化为神经细胞；
(2)温度敏感型UCMSCs细胞系在其适宜温度下存在一定的致瘤性(对低营养环境的高耐受力和半固体介质中的强克隆形成能力),但当其处于非适宜温度(37℃)下则几乎完全失去了增殖能力,也不再具有致瘤性。其潜在致瘤性可以通过改变温度得到有效控制；
(3)常温条件下在脑创伤及其近缘部位移植的干细胞,绝大部分因为不耐受创伤灶半损伤带的低营养、高毒素环境而死亡；但在亚低温环境中,在创伤灶半损伤带移植温度敏感型的UCMSCs,可见存活的细胞显著增多,同时凋亡减少,动物神经功能得到有效恢复；
(4)通过对干细胞进行BDNF基因转染可增加干细胞向神经元而非神经胶质细胞的分化比例,结合其自身的神经营养作用,进一步提高干细胞恢复创伤后神经运动功能的潜能。
Objective:To study the proliferation and differentiation character of a temperature-sensitive stem cell line cultured at33℃and37℃respectively for establishing a basement on stem cells transplantation into injured brain area during hypothermia treatment. The brain derived neurotrophic factor was transferred into UCMSCs for promoting the stem cells differentiation into neuron and enhance neuromotor function after head injury.
Methods:Human umbilical cord mesenchymal stem cells were isolated and expanded in culture, and certificated by flow cytometry analysis. After transfecting plasmid containing temperature-sensitive simian virus40large T-antigen (tsSV40LT) into umbilical cord mesenchymal stem cells (UCMSCs), PCR method was used to detect gene integration. Then we cultured these UCMSCs modified by tsSV40LT in33℃and37℃incubators respectively. Cell growth curves were draw, cell cycle was detected by flow cytometry, then telomerase activation analysis by PCR-ELISA telomerase detect kit, and cell cycle regulating protein, including cyclin Dl, cyclin dependent kinase2(CDK2) and P16, detected by western blot. Serum-dependent experimental and soft agar colony assay were used to examine the tumorigenesis of the stem cells. Recombinant adenovirus-mediated brain derived neurotrophic factor gene transferred into human-derived umbilical cord mesenchymal stem cells (UCMSCs). Enzyme linked immunosorbent assay (ELISA) was used to test the expression level of BDNF, and immunofluorescence assay to detect the NSE and GFAP expression, which were characteristic for neuron and glia respectively. After TBI models in thymic mice were established, the stem cells were transplated into injured area. Proliferating cell nuclear antigen expression and in situ apoptosis were detected by immunofluorescence method.
Results:The tsSV40LT gene was integrated into UCMSCs successfully. When cultured at33℃incubator, the new cell line displayed high proliferation activity, strong tolerance to low nutrient conditions, and strong cell clone ability, as well as its telomerase activation, highly expressed cyclin D1and CDK2, and lowly expressed P16, meanwhile, highly nestin, lowly NSE and GFAP. But when cultured at37℃ incubator, the cell line showed a completely converse profile. The BDNF expression achieved its peak at72h after gene transfer, and on the meanwhile, the proportion of the NSE-positive cells was increased significantly, but GFAP-positive cells decreased. In vivo, the results showed that the brain tissue during hypothermia treatment more highly expressed PCNA, and existed lower cell apoptosis, than normothermia group. The mouse obtained a better score of nerve function. In the site of BDNF high expression, the apoptosis cell number was much minor.
Conclusion:
1) The stem cell line modified by tsSV40LT is highly temperature-sensitive, and its proliferation activity can be regulated effectively, and that supports the clinical application of stem cells transplantation into injured brain area during hypothermia treatment.
2) When cultured at33℃incubator, the new cell line displayed strong tolerance to low nutrient conditions, and strong cell clone ability. But when cultured at37℃incubator, the cell line showed a converse profile, and that supports that the temperature may control cell proliferation of the temperature-sensitive stem cell line.
3) When transplanted into injured brain area, a vast majority of stem cells were dead because of the low-nutrition, high-toxic environment. But at a lower temperature environment (33℃), the quantity of survival cells was significantly increased, cell apoptosis was lower, and the scores of mouse nerve function was better.
4) The stem cells modified by BDNF gene may promote the differentiation into neuron, not glia, and enhance neuromotor function after head injury.
方法：采用原代细胞贴壁培养法培养人源性UCMSCs,流式细胞术进行表面标记蛋白鉴定,体外诱导其向神经细胞分化。用含温度敏感性猿猴病毒40大T抗原(tsSV40LT)的表达质粒转染UCMSCs, PCR检测外源基因的整合,绘制其在33℃和37℃培养条件下的细胞生长曲线；流式细胞术检测细胞周期,PCR-ELISA端粒酶检测法检测端粒酶活性,Western Blot法检测细胞周期调控蛋白---cyclin D1、CDK2和P16蛋白的表达；血清依赖实验及软琼脂克隆形成实验检测该细胞系在两种温度下的致瘤潜能；利用腺病毒载体转染BDNF进入UCMSCs, ELISA方法测定BDNF分泌时相,免疫荧光方法检测转染与非转染细胞表达NSE和GFAP的比例；建立去胸腺小鼠颅脑液压打击模型进行干细胞原位移植,脑组织标本行增殖细胞核抗原(PCNA)免疫组化及原位细胞凋亡检测,实验动物予以神经功能缺陷评分。
结果：利用原代贴壁细胞培养法成功获得UCMSCs,流式细胞结果符合UCMSCs特征,经体外条件诱导,UCMSCs可表达神经元和神经胶质细胞标记蛋白；PCR结果显示tsSV40LT抗原的基因片段被成功整合入干细胞,端粒酶活性高,cyclin D1和CDK2高表达,P16低表达,并高表达nestin,不表达NSE和GFAP,同时该细胞系在33℃时增殖旺盛,可耐受极低的营养条件,并可在软琼脂中形成小的细胞克隆；在37℃条件下细胞增殖缓慢甚至停滞,端粒酶活性低,cyclin D1和CDK2的表达强度明显低于33℃条件,P16则相反,与此同时nestin表达强度明显下降,NSE和GFAP表达水平上升,低营养耐受力下降,无细胞克隆形成；BDNF的表达水平于病毒转染后第72h达到高峰,此期干细胞NSE阳性细胞比例明显增多(P<0.05),相反GFAP阳性细胞比例下降；动物体内结果显示,亚低温治疗期间的脑组织高表达PCNA,凋亡细胞少见；常温组脑组织则低表达PCNA,创伤灶周边见较多凋亡细胞存在。
结论：
(1)借助tsSV40LT抗原建立的温度敏感型UCMSCs细胞系,在细胞增殖和1分化方面具有显著的温度可调控性特征,即在其适宜温度下(33℃)增殖能力旺盛,并维持在前体细胞水平,极少产生细胞分化；当其处于37℃环境中时则停止增长,转而分化为神经细胞；
(2)温度敏感型UCMSCs细胞系在其适宜温度下存在一定的致瘤性(对低营养环境的高耐受力和半固体介质中的强克隆形成能力),但当其处于非适宜温度(37℃)下则几乎完全失去了增殖能力,也不再具有致瘤性。其潜在致瘤性可以通过改变温度得到有效控制；
(3)常温条件下在脑创伤及其近缘部位移植的干细胞,绝大部分因为不耐受创伤灶半损伤带的低营养、高毒素环境而死亡；但在亚低温环境中,在创伤灶半损伤带移植温度敏感型的UCMSCs,可见存活的细胞显著增多,同时凋亡减少,动物神经功能得到有效恢复；
(4)通过对干细胞进行BDNF基因转染可增加干细胞向神经元而非神经胶质细胞的分化比例,结合其自身的神经营养作用,进一步提高干细胞恢复创伤后神经运动功能的潜能。
Objective:To study the proliferation and differentiation character of a temperature-sensitive stem cell line cultured at33℃and37℃respectively for establishing a basement on stem cells transplantation into injured brain area during hypothermia treatment. The brain derived neurotrophic factor was transferred into UCMSCs for promoting the stem cells differentiation into neuron and enhance neuromotor function after head injury.
Methods:Human umbilical cord mesenchymal stem cells were isolated and expanded in culture, and certificated by flow cytometry analysis. After transfecting plasmid containing temperature-sensitive simian virus40large T-antigen (tsSV40LT) into umbilical cord mesenchymal stem cells (UCMSCs), PCR method was used to detect gene integration. Then we cultured these UCMSCs modified by tsSV40LT in33℃and37℃incubators respectively. Cell growth curves were draw, cell cycle was detected by flow cytometry, then telomerase activation analysis by PCR-ELISA telomerase detect kit, and cell cycle regulating protein, including cyclin Dl, cyclin dependent kinase2(CDK2) and P16, detected by western blot. Serum-dependent experimental and soft agar colony assay were used to examine the tumorigenesis of the stem cells. Recombinant adenovirus-mediated brain derived neurotrophic factor gene transferred into human-derived umbilical cord mesenchymal stem cells (UCMSCs). Enzyme linked immunosorbent assay (ELISA) was used to test the expression level of BDNF, and immunofluorescence assay to detect the NSE and GFAP expression, which were characteristic for neuron and glia respectively. After TBI models in thymic mice were established, the stem cells were transplated into injured area. Proliferating cell nuclear antigen expression and in situ apoptosis were detected by immunofluorescence method.
Results:The tsSV40LT gene was integrated into UCMSCs successfully. When cultured at33℃incubator, the new cell line displayed high proliferation activity, strong tolerance to low nutrient conditions, and strong cell clone ability, as well as its telomerase activation, highly expressed cyclin D1and CDK2, and lowly expressed P16, meanwhile, highly nestin, lowly NSE and GFAP. But when cultured at37℃ incubator, the cell line showed a completely converse profile. The BDNF expression achieved its peak at72h after gene transfer, and on the meanwhile, the proportion of the NSE-positive cells was increased significantly, but GFAP-positive cells decreased. In vivo, the results showed that the brain tissue during hypothermia treatment more highly expressed PCNA, and existed lower cell apoptosis, than normothermia group. The mouse obtained a better score of nerve function. In the site of BDNF high expression, the apoptosis cell number was much minor.
Conclusion:
1) The stem cell line modified by tsSV40LT is highly temperature-sensitive, and its proliferation activity can be regulated effectively, and that supports the clinical application of stem cells transplantation into injured brain area during hypothermia treatment.
2) When cultured at33℃incubator, the new cell line displayed strong tolerance to low nutrient conditions, and strong cell clone ability. But when cultured at37℃incubator, the cell line showed a converse profile, and that supports that the temperature may control cell proliferation of the temperature-sensitive stem cell line.
3) When transplanted into injured brain area, a vast majority of stem cells were dead because of the low-nutrition, high-toxic environment. But at a lower temperature environment (33℃), the quantity of survival cells was significantly increased, cell apoptosis was lower, and the scores of mouse nerve function was better.
4) The stem cells modified by BDNF gene may promote the differentiation into neuron, not glia, and enhance neuromotor function after head injury.
引文
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58 Ni HT, Hu S, Sheng WS, et al. High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells. Developmental Brain Research 2004; 152(2):159-169.
59 Son BR, Marquez-Curtis LA, Kucia M,et al. Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by stromal-derived factor-1-CXCR4 and hepatocyte growth factor-c-met axes and involves matrix metalloproteinases. Stem Cells 2006; 24(5):1254-1264.
60 Chen ZY, Bath K, McEwen B, Hempstead B, Lee F. Impact of genetic variant BDNF (Val66Met) on brain structure and function. Novartis Found Symp 2008; 28(9):180-188.
61 Spedding M, Gressens P. Neurotrophins and cytokines in neuronal plasticity. Novartis Found Symp 2008;28(9):222-233.
62 Weber JT. Experimental models of repetitive brain injuries. Prog Brain Res. 2007; 161:253-261.
1 Walker PA, Shah SK, Harting MT,et al. Progenitor cell therapies for traumatic brain injury:barriers and opportunities in translation[J]. Dis Model Mech, 2009,2(l):23-38.
2 Yoshinaga T, Hashimoto E, Ukai W, et al. Neural stem cell transplantation in a model of fetal alcohol effects[J]. J Neural Transm Suppl,2007,37(72):331-337.
3 Burns JS, Abdallah BM, Guldberg P, et al. Tumorigenic heterogeneity in cancer stem cells evolved from long-term cultures of telomerase-immortalized human mesenchymal stem cells[J]. Cancer Res,2005,65(8):3126-3135.
4孙洪涛,刘晓智,张赛.脐带间充质干细胞分离、鉴定与神经分化[J].中华神经外科疾病研究杂志,2008,7(2)：132-135.
5 Schrepfer S, Deuse T, Reichenspurner H, et al. Stem cell transplantation:the lung barrier[J]. Transplant Proc,2007,39(2):573-576.
6 Walczak P, Zhang J, Gilad AA, et al. Dual-modality monitoring of targeted intraarterial delivery of mesenchymal stem cells after transient ischemia[J]. Stroke,2008,39(5):1569-1574.
7 Fricker SP. A novel CXCR4 antagonist for hematopoietic stem cell mobilization[J]. Expert Opin Investig Drugs,2008,17(11):1749-1760.
8 Ni HT, Hu S, Sheng WS, et al. High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells[J]. Developmental Brain Research,2004,152(2):159-169.
9 Son BR, Marquez-Curtis LA, Kucia M, et al. Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by stromal-derived factor-1-CXCR4 and hepatocyte growth factor-c-met axes and involves matrix metalloproteinases[J]. Stem Cells,2006,24(5):1254-1264.
10 Spees JL, Olson SD, Ylostalo J, et al. Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma[J]. Proc Natl Acad Sci U S A,2003,100(5):2397-2402.
11 Goncalves MA, Swildens J, Holkers M, et al. Genetic complementation of human muscle cells via directed stem cell fusion[J]. Mol Ther,2008,16(4): 741-748.
12 Qu R, Li Y, Gao Q, et al. Neutrophic and growth factor gene expression profiling of mouse bone marrow stromal cells induced by ischemic brain extracts[J]. Neuropathology,2007,27(4):355-363.
13 Chen X, Katakowski M, Li Y, et al. Human bone marrow stromal cell cultures conditioned by traumatic brain injury extracts:growth factor productionfJ]. J Neurosci Res,2002,69(5):687-691.
14 A Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogenic immune cell responses[J]. Blood,2005,105(4):1815-1822.
15 Chen J, Li Y, Zhang R, et al. Combination therapy of stroke in rats with a nitric oxide donor and human bone marrow stromal cells enhances angiogenesis and neurogenesis[J]. Brain Res,2004,1005(1-2):21-28.
16 Rubio D, Garcia-Castro J, Martin MC, et al. Spontaneous human adult stem cell transformation[J]. Cancer Res,2005,65(8):3035-3039.
17 Shi M, Li J, Liao L, et al. Regulation of CXCR4 expression in human mesenchymal stem cells by cytokine treatment:role in homing efficiency in NOD/SCID mice[J]. Haematologica,2007,92(7):897-904.
18 Li H, Fan X, Kovi RC, et al. Spontaneous expression of embryonic factors and p53 point mutation in aged mesenchymal stem cells:a model of age related tumorigenesis in mice[J]. Cancer Res,2007,67(22):10889-10898.
19 Peters A, Manivel JC, Dolan M, et al. Pulmonary cytolytic thrombi after allogenic hematopoietic cell transplantation:a further histologic description[J]. Biol Blood Marrow Transplant,2005,11(6):484-485.
20邓志锋,汪泱,邓丽影,等.自体骨髓间充质干细胞移植治疗中枢神经系统损伤性疾病[J].实用临床医学,2007,8(6)：62-70.
21 Willerth SM, Arendas KJ, Gottlieb DI, et al. Optimization of fibrin scaffolds for differentiation of murine embryonic stem cells into neural lineage cells[J]. Biomaterials,2006,27(36):5990-6003.
22 Silva GA, Czeisler C, Niece KL, et al. Selective differentiation of neural progenitor cells by highepitope density nanofibers[J]. Science,2004,303(5662): 1352-1355.
23 Thonhoff JR, Lou DI, Jordan PM, et al. Compatibility of human fetal neural stem cells with hydrogel biomaterials in vitro[J]. Brain Res,2008,1187(2):42-51.
24 Park KI, Teng YD, Snyder EY.The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue[J]. Nat Biotechnol,2002,20(11):1111-1117.
25 Mahoney MJ, Saltzman WM.Transplantation of brain cells assembled around a programmable synthetic microenvironment[J]. Nat Biotechnol,2001,19(10): 934-939.
2 Yoshinaga T, Hashimoto E, Ukai W, et al. Neural stem cell transplantation in a model of fetal alcohol effects[J]. J Neural Transm Suppl,2007,37(72):331-337.
3 Burns JS, Abdallah BM, Guldberg P, et al. Tumorigenic heterogeneity in cancer stem cells evolved from long-term cultures of telomerase-immortalized human mesenchymal stem cells[J]. Cancer Res,2005,65(8):3126-3135.
4 Schrepfer S, Deuse T, Reichenspurner H, et al. Stem cell transplantation:the lung barrier[J]. Transplant Proc,2007,39(2):573-576.
5 Walczak P, Zhang J, Gilad AA, et al. Dual-modality monitoring of targeted intraarterial delivery of mesenchymal stem cells after transient ischemia[J]. Stroke,2008,39(5):1569-1574.
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51梁鹏,梁桃,刘恩重等.人胚神经干细胞条件化培养基促进脊髓损伤大鼠皮质脊髓束再生.中华神经外科杂志,2006,22(10)：591-594.
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53邓志锋,汪泱,邓丽影,等.自体骨髓间充质干细胞移植治疗中枢神经系统损伤性疾病[J].实用临床医学,2007,8(6)：62-70.
54 Willerth SM, Arendas KJ, Gottlieb DI, et al. Optimization of fibrin scaffolds for differentiation of murine embryonic stem cells into neural lineage cells[J]. Biomaterials,2006,27(36):5990-6003.
55 Silva GA, Czeisler C, Niece KL, et al. Selective differentiation of neural progenitor cells by highepitope density nanofibers[J]. Science,2004,303(5662): 1352-1355.
56 Park KI, Teng YD, Snyder EY.The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue[J]. Nat Biotechnol,2002,20(11):1111-1117.
57 Shi M, Li J, Liao L, et al. Regulation of CXCR4 expression in human mesenchymal stem cells by cytokine treatment:role in homing efficiency in NOD/SCID mice[J]. Haematologica,2007,92(7):897-904.
58 Ni HT, Hu S, Sheng WS, et al. High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells. Developmental Brain Research 2004; 152(2):159-169.
59 Son BR, Marquez-Curtis LA, Kucia M,et al. Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by stromal-derived factor-1-CXCR4 and hepatocyte growth factor-c-met axes and involves matrix metalloproteinases. Stem Cells 2006; 24(5):1254-1264.
60 Chen ZY, Bath K, McEwen B, Hempstead B, Lee F. Impact of genetic variant BDNF (Val66Met) on brain structure and function. Novartis Found Symp 2008; 28(9):180-188.
61 Spedding M, Gressens P. Neurotrophins and cytokines in neuronal plasticity. Novartis Found Symp 2008;28(9):222-233.
62 Weber JT. Experimental models of repetitive brain injuries. Prog Brain Res. 2007; 161:253-261.
1 Walker PA, Shah SK, Harting MT,et al. Progenitor cell therapies for traumatic brain injury:barriers and opportunities in translation[J]. Dis Model Mech, 2009,2(l):23-38.
2 Yoshinaga T, Hashimoto E, Ukai W, et al. Neural stem cell transplantation in a model of fetal alcohol effects[J]. J Neural Transm Suppl,2007,37(72):331-337.
3 Burns JS, Abdallah BM, Guldberg P, et al. Tumorigenic heterogeneity in cancer stem cells evolved from long-term cultures of telomerase-immortalized human mesenchymal stem cells[J]. Cancer Res,2005,65(8):3126-3135.
4孙洪涛,刘晓智,张赛.脐带间充质干细胞分离、鉴定与神经分化[J].中华神经外科疾病研究杂志,2008,7(2)：132-135.
5 Schrepfer S, Deuse T, Reichenspurner H, et al. Stem cell transplantation:the lung barrier[J]. Transplant Proc,2007,39(2):573-576.
6 Walczak P, Zhang J, Gilad AA, et al. Dual-modality monitoring of targeted intraarterial delivery of mesenchymal stem cells after transient ischemia[J]. Stroke,2008,39(5):1569-1574.
7 Fricker SP. A novel CXCR4 antagonist for hematopoietic stem cell mobilization[J]. Expert Opin Investig Drugs,2008,17(11):1749-1760.
8 Ni HT, Hu S, Sheng WS, et al. High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells[J]. Developmental Brain Research,2004,152(2):159-169.
9 Son BR, Marquez-Curtis LA, Kucia M, et al. Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by stromal-derived factor-1-CXCR4 and hepatocyte growth factor-c-met axes and involves matrix metalloproteinases[J]. Stem Cells,2006,24(5):1254-1264.
10 Spees JL, Olson SD, Ylostalo J, et al. Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma[J]. Proc Natl Acad Sci U S A,2003,100(5):2397-2402.
11 Goncalves MA, Swildens J, Holkers M, et al. Genetic complementation of human muscle cells via directed stem cell fusion[J]. Mol Ther,2008,16(4): 741-748.
12 Qu R, Li Y, Gao Q, et al. Neutrophic and growth factor gene expression profiling of mouse bone marrow stromal cells induced by ischemic brain extracts[J]. Neuropathology,2007,27(4):355-363.
13 Chen X, Katakowski M, Li Y, et al. Human bone marrow stromal cell cultures conditioned by traumatic brain injury extracts:growth factor productionfJ]. J Neurosci Res,2002,69(5):687-691.
14 A Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogenic immune cell responses[J]. Blood,2005,105(4):1815-1822.
15 Chen J, Li Y, Zhang R, et al. Combination therapy of stroke in rats with a nitric oxide donor and human bone marrow stromal cells enhances angiogenesis and neurogenesis[J]. Brain Res,2004,1005(1-2):21-28.
16 Rubio D, Garcia-Castro J, Martin MC, et al. Spontaneous human adult stem cell transformation[J]. Cancer Res,2005,65(8):3035-3039.
17 Shi M, Li J, Liao L, et al. Regulation of CXCR4 expression in human mesenchymal stem cells by cytokine treatment:role in homing efficiency in NOD/SCID mice[J]. Haematologica,2007,92(7):897-904.
18 Li H, Fan X, Kovi RC, et al. Spontaneous expression of embryonic factors and p53 point mutation in aged mesenchymal stem cells:a model of age related tumorigenesis in mice[J]. Cancer Res,2007,67(22):10889-10898.
19 Peters A, Manivel JC, Dolan M, et al. Pulmonary cytolytic thrombi after allogenic hematopoietic cell transplantation:a further histologic description[J]. Biol Blood Marrow Transplant,2005,11(6):484-485.
20邓志锋,汪泱,邓丽影,等.自体骨髓间充质干细胞移植治疗中枢神经系统损伤性疾病[J].实用临床医学,2007,8(6)：62-70.
21 Willerth SM, Arendas KJ, Gottlieb DI, et al. Optimization of fibrin scaffolds for differentiation of murine embryonic stem cells into neural lineage cells[J]. Biomaterials,2006,27(36):5990-6003.
22 Silva GA, Czeisler C, Niece KL, et al. Selective differentiation of neural progenitor cells by highepitope density nanofibers[J]. Science,2004,303(5662): 1352-1355.
23 Thonhoff JR, Lou DI, Jordan PM, et al. Compatibility of human fetal neural stem cells with hydrogel biomaterials in vitro[J]. Brain Res,2008,1187(2):42-51.
24 Park KI, Teng YD, Snyder EY.The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue[J]. Nat Biotechnol,2002,20(11):1111-1117.
25 Mahoney MJ, Saltzman WM.Transplantation of brain cells assembled around a programmable synthetic microenvironment[J]. Nat Biotechnol,2001,19(10): 934-939.