MSCs多向分化潜能及对失神经肌萎缩的作用
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摘要
第一部分MSCs多向分化潜能(成骨、成肌以及神经定向诱导分化)的研究
目的①探讨间充质干细胞(MSCs)体外诱导分化为骨样细胞、肌样细胞以及神经元(NCs)样/星形胶质样细胞的有效方法。②评价体外诱导分化的神经前体样细胞(NPCs)、神经元(NCs)样细胞以及星形胶质样细胞的体外存活和增殖特性,为神经细胞再生寻找良好的种子来源。
方法参考有关文献,采用贴壁筛选法自大鼠骨髓中分离培养间充质干细胞,进行传代纯化,收集得到第5代(P5)间充质干细胞单细胞悬液(1×10~(5~6)/ml)。①成骨诱导分化方案:DMEM/10%FBS培养基+0.1μmol/l地塞米松+50ng/ml维生素C+2.16mg/mlβ-甘油磷酸钠。诱导分化2周以上。行Van Kossa银染色和茜素红染色。②成肌诱导分化方案:DMEM/20%FBS+3μmol/l5-氮胞苷(5-azayttidine)。诱导分化24h,培养2～3周以上。观察形成的肌样细胞及肌管样结构。③神经定向诱导分化方案:采用多因子、分阶段逐步诱导方法定向诱导MSCs分化为神经前体细胞(NPCs),进而分化为NCs样/星形胶质样细胞。即:MSCs以1×10~6/L置入6孔培养板中,加入生长培养基:DMEM/20%FBS培养基,24h后更换为诱导分化培养基:DMEM/F12+2%B27+40μg/L碱性成纤维生长因子(bFGF)+20μg/L表皮生长因子诱导分化培养基(EGF),培养10～20d,定向诱导MSCs分化为NPCs。下一个阶段更换诱导分化培养基为生长培养基:DMEM/5%FBS,分两组进行诱导:一组向神经元样细胞诱导分化:全反式维甲酸(RA)连续加样,首次浓度0.5μmol/L,以后每天半量加样;二组向星形胶质样细胞诱导分化:10μg/L血小板衍生生长因子BB(PDGF-BB)首次加样,以后每天半量加样,均连续诱导10～14d,制作两组细胞爬片均行纤维丝蛋白200(NF200)、胶质纤维酸性阳性蛋白(GFAP)免疫组织化学荧光染色。④神经元样/星形胶质样细胞共同培养:收集诱导分化的神经元样/星形胶质样细胞,各自浓度1×106/L,以1:1混合在DMEM/5%FBS培养基中共同培养。观察两组细胞生长情况。⑤应用活细胞计数试剂盒CCK-8检测单独诱导分化的NCs样/星形胶质样细胞的增殖及存活情况,绘制生长曲线及细胞存活曲线。
结果①经DMEM/10%FBS培养基+0.1μmol/l地塞米松+50ng/ml维生素C+2.16mg/mlβ-甘油磷酸钠诱导培养2周以上,MSCs细胞聚集呈结节状,钙盐沉积,Van Kossa银染色法阳性。茜素红染色阳性。②经3μmol/l 5-氮胞苷诱导处理大鼠MSCs,24小时部分细胞胞体收缩变为瘦长形状, 1~2周后细胞相互形成肌管样结构。2周后可以观察到自发搏动。③bFGF+EGF连续诱导7d后MSCs分化为球团样的神经前体样细胞,巢蛋白(Nestin)表达阳性。RA、PDGF-BB分别连续诱导10d后MSCs逐步分化出神经元样、星形胶质样细胞,分别表达NF200、GFAP蛋白。④神经元样/星形胶质样细胞可以共同培养、生长,呈现各自的细胞形态。⑤活细胞计数试剂盒CCK-8检测显示:神经元样细胞早期生长较缓慢,具有一定的增殖能力,6～7d后达到高峰,此后增殖能力明显下降,细胞存活率明显下降,14～15d之后细胞存活率下降到7.6%以下,难以传代培养。星形胶质样细胞初期生长慢,5d后生长加速,进入对数生长期,7～8d后进入平台期,仍保持较高的细胞存活率。
结论①MSCs是具有自我增殖和多向分化潜能的干细胞。可以诱导分化为骨样细胞、骨骼肌样细胞、神经元样细胞及星形胶质样细胞。②诱导分化的神经元样/星形胶质样细胞可以共同培养、生长。③单独诱导分化的神经元样细胞可继续存活13-15d后发生自然死亡,难以传代,星形胶质样细胞生长较稳定,具有较强的存活和增殖能力,可传代培养至3代以上。
第二部分MSCs归巢性及对失神经肌萎缩的作用
目的检测大鼠间充质干细胞(MSCs)经静脉移植后在周围神经损伤模型内的迁移、分布情况,评价其对轴突、靶器官的保护作用。
方法制作80只大鼠坐骨神经损伤模型,收集P5代MSCs行BrdU(10μmol/L)掺和标记48h。经尾静脉注射BrdU标记的MSCs(2×10~7/ml)入体内。术后8周内评价坐骨神经指数(SFI)。术后神经断端、肌肉组织取材均行BrdU免疫组织化学荧光染色。肌肉组织行HE染色、TUNEL组织化学染色。Image-Pro Plus 5.0专业图像分析软件分析肌纤维横截面积并计算以及实验侧与正常侧肌纤维横截面积之比,分析肌细胞调亡数量。所得数据均用均数±标准差(x——±s)表示,结果用SPSS统计软件行t检验。
结果MSCs体外可以被BrdU掺和标记,阳性标记率为62.5%。术后4、8周神经断端、肌肉组织内检测到BrdU阳性细胞。实验组SFI、肌纤维横截面积明显高于对照组,细胞凋亡数量低于对照组,均有统计学意义﹙4周P<0.05, 8周P<0.01﹚。
结论MSCs静脉移植至体内能归巢到损伤的神经断端及肌肉组织,可以存活至少8周,具有促进轴突再生、延缓肌萎缩的作用。
第三部分骨髓源性神经前体样细胞对失神经肌萎缩的作用
目的探讨大鼠间充质干细胞(MSCs)诱导分化的神经前体样细胞(NPCs)移植在体内存活情况,评价其及对失神经肌萎缩的作用。
方法联合应用EGF、bFGF诱导MSCs分化为NPCs。行Nestin免疫组织化学荧光染色鉴定,并行5-溴脱氧尿苷(BrdU)掺和标记实验和单细胞克隆实验。建立90只大鼠坐骨神经损伤模型。BrdU掺和标记的NPCs以5×106/ml注入神经损伤断端。术后4、8周神经断端、肌肉组织取材,神经断端分别行Nestin、BrdU单标免疫荧光染色检验。肌肉组织行HE染色、TUNEL免疫组织化学染色。Image-Pro Plus 5.0专业图像分析软件分析肌纤维横截面积并计算以及实验侧与正常侧肌纤维横截面积之比。所得数据结果用SPSS统计软件行t检验。
结果MSCs体外诱导分化可获得大量活性的NPCs,Nestin蛋白染色阳性,体外可以被BrdU掺和标记,阳性标记率为87.9%,单细胞克窿率为2.7%。移植术后4、8周实验组神经断端均检测到Nestin、BrdU阳性细胞。实验组肌纤维横截面积明显高于对照组,肌细胞调亡数量明显低于对照组,均有统计学意义﹙4周P<0.05,8周P<0.01﹚。
结论骨髓源性NPCs移植至神经断端可以存活至少8周,具有延缓失神经肌萎缩、抑制失神经肌肉细胞调亡的作用。
第四部分骨髓源性神经样细胞对失神经肌萎缩的作用
目的探讨间充质干细胞(MSCs)诱导分化的神经元(NCs)样、星形胶质样细胞联合移植后在体内存活情况,评价其联合移植对失神经肌萎缩的作用。
方法多因子、分阶段逐步诱导MSCs分化为NCs样、星形胶质样细胞。行NF200、GFAP免疫组织化学荧光染色鉴定。建立60只大鼠失神经骨骼肌模型。行细胞联合移植,以5×10~6/ml肌肉注射入失神经骨骼肌内。术后4、8周肌肉组织取材行HE染色、免疫组织化学染色检测NF200、GFAP阳性细胞。Image-Pro Plus 5.0专业图像分析软件分析肌纤维横截面积并计算以及实验侧与正常侧肌纤维横截面积之比。所得数据结果用SPSS统计软件行t检验。
结果MSCs逐步诱导分化出NCs样、星形胶质样细胞。分别表达NF200、GFAP阳性蛋白。移植术后4、8周实验侧肌肉组织检测到NF200、GFAP阳性细胞。实验侧肌纤维横截面积明显高于对照组,有统计学意义(4周P<0.05,8周P<0.01)。
结论骨髓源性NCs样、星形胶质样细胞可以体外大量获得,联合移植至肌肉内可以存活至少8周,可以增加失神经肌肉面积,有效改善失神经肌萎缩。
PartⅠThe multipotency of mesenchymal stem cells in rats.
Objective To investigate multipotentcy of MSCs differentiation into osteoblasts,skeletal muscle, neuron-like cells and astrocyte-like cells in vitro and evaluate the proliferation and survival capacity of MSCs-differentiated neural progenitor cells, neuron-like cells and astrocyte-like cells in vitro.
Methods Adult rat MSCs were isolated and cultured for 5 passages in vitro. Osteogenic differentiation was induced by plating the cells at 1×10~5cells/ml in DMEM medium containing 10%FBS,supplenmented with 0.1μmol/l dexamethasone,50ng/ml ascorbic acid, and 2.16mg/mlβ–glycerophosphate. Cells were cultured for 14days and the medium was changed twice a week. Skeletal muscle differentiation was induced at 1×10~5cells/ml by DMEM/20%FBS supplenmented with 3μmol/l 5-azayttidine for 24h, then cultured for 2~3weeks.Neural differentiation were selectively and progressively induced into neuron-like cells and astrocyte-like cells.by multi-cytokines, first DMEM/F12/2%B27 with EGF(20μg/L) and bFGF(40μg/L) for 10～20days, then DMEM/5%FBS with retinoic acid (0.5μmol/L) or PDGF-BB(10μg/L) for another 10～20days. The differentiated neuron-like cells and astrocyte-like cells were co-cultured at 1×10~5cells/ml at the ratio of 1:1 in DMEM medium containing 10%FBS. Immunohistochemistry stainings were selectively carried out for CD44,CD45,nestin,NF200 and GFAP. The cell proliferation and survival assays were made using cell counting Kit-8.
Results The MSCs showed a fibroblast-like morphology, and could self-renew and survive at least 15 passages. In the presence of selected cytokines MSCs were effectively differentiated into osteoblasts for 4 weeks. The cells morphology changed after treatment with 5-azacytidine for 1 week. MSCs connected with adjoining cells after one week, formed myotube-like structures, began spontaneously beating after two weeks, and beat synchronously after three weeks. MSCs were differentiated into neurospheres -like cells with EGF and bFGF for 7 days. Then neurospheres-like cells were differentiated into neuron-like cells with retinoic acid and astrocyte-like cells with PDGF-BB for 10～14 days, respectively positive for nestin, NF200 and GFAP immunohistochemistry stainings. The differentiated neuron-like cells and astrocyte-like cells could survive the next co-culture procedure.The CCK-8 assays demonstrated that the differentiated neuron-like cells could proliferate and survive until the next 13～15 days. However the astrocyte-like cells proliferated and survived better and could be cultured for at least 3 passages.
Conclusions MSCs were self-renewing and mutipotent cells which maintain the capacity of differentiation into osteoblasts,skeletal muscle-like cells,neuron-like cells and astrocyte-like cells in vitro by multi-cytokines induction protocols. And the MSCs-derived astrocyte-like cells had a better proliferation and survival capacity than the neuron-like cells.
PartⅡTherapeutic benefits of intravenous administration of mesenchymal stem cells on peripheral nerve regeneration in rats
Objective To investigate the migration and distribution of rat bone mesenchymal stem cells in vivo after peripheral nerve injury and evaluate the beneficial effects on peripheral nerve regeneration and delaying denervation muscle atrophy.
Methods Eighty female Sprague-Dawley rats were randomly divided into 2 groups, with 40 in each. All the sciatic nerves were transected and sutured with 8-0 nylon suture. The transplanted group received an infusion of BrdU-labelled MSCs at 2×10~7cells/ml cells/ml through the tail vein. Every week until 12 weeks after surgery the sciatic function index (SFI) was assessed by walking tract test. Sections were carried out for HE staining, BrdU and TUNEL immunohistochemical staining.
Results BrdU-labeled cells were detected housing the nerve stumps and denervation muscles at 1 week and 8 weeks. MSCs transplantation group had a promotion of sciatic nerve functional recovery from weeks 4 to 12 and the number of denervation muscle apoptotic cells significantly decreased compared with that of control group at 8 weeks(P<0.05, P<0.01).
Conclusions MSCs is capable in vivo of homing to the nerve stumps and denervation muscles after peripheral nerve injury via the blood circulation, and had therapeutic benefits on promoting peripheral nerve regeneration and delaying denervation muscle atrophy.
PartⅢThe effect of neural progenitor cells differentiated from bone marrow mesenchymal stem cells on denervation muscle atrophy
Objective To investigate the effect on delaying denervation muscle atrophy after peripheral nerve injury by transplantion of neural progenitor cells differentiated from bone marrow mesenchymal stem cells.
Methods Isolated and purified MSCs were induced into neural progenitor cells by EGF (20ng/ml) and bFGF (40ng/ml) for 10～20d. Differentiated cells were analyzed by single cell clone technique and immunohistochemistry staining for nestin. Ninety female Sprague-Dawley rats were randomly divided into 2 groups, with 45 in each. All the sciatic nerves were transected and sutured with 8-0 nylon suture. Before transplantation the neural progenitor cells were labeled with BrdU for 48h and then were injected into the sciatic nerve stumps at 5×10~6cells/ml. At weeks 4, 8 the nerve stump frozen sections were carried out for BrdU immunofluorescence staining, and muscle paraffin sections were carried out for HE staining and TUNEL immunohistochemical staining. Muscle cross-sectional areas and apoptotic cells were measured by an image analyzer.
Results MSCs were positive for CD44, however negative for CD45 and nestin. After 10～14d differentiation, neurospheres were observed and mostly of them were positive for nestin. The rate of label of BrdU was 87.9% and the single-clone formed rate was 2.7%.At weeks 4 and 8 after surgery BrdU positive cells were detected at the nerve stumps. Muscle cross-sectional areas of cell transplantation group significantly increased and the number of the denervation muscle apoptotic cells significantly decreased compared with that of control group (P<0.05, P<0.01).
Conclusions MSCs can be efficiently differentiated into neural progenitor cells with EGF and bFGF. Differentiated neural stem cell-like cells can survive the next transplantation into nerve stumps and had beneficial effects on delaying denervation muscle atrophy after injury.
PartⅣThe effect of differentiated neuron-like cells and astrocyte-like cells from bone marrow mesenchymal stem cells on denervation muscle atrophy
Objective To investigate the survive of MSCs-differentiated neuron-like cells and astrocyte-like cells after co-transplantation in vivo and evaluate the beneficial effects on delaying denervation atrophy.
Methods Isolated and purified rMSCs were selectively and progressively induced into neuron-like cells and astrocyte-like cells by multi-cytokine, EGF and bFGF first, followed by retinoic acid or PDGF. Immunohistochemistry staining was carried out for CD44, CD45, nestin, NF200 and GFAP. Sixty female Sprague-Dawley rat sciatic nerve transection models were made and then randomly divided into 2 groups, with 25 in each. In the experimental group, differentiated neuron-like cells and astrocyte-like cells were co-injected into the denervated skeletal muscle at 5×10~6cells/ml. At weeks 4 and 8 after cell transplantation muscle frozen sections were carried out for NF200 and GFAP immunofluorescence staining and muscle paraffin sections were carried out for HE staining Muscle cross-sectional areas were measured by an image analyzer.
Results MSCs were effectively differentiated into neurospheres -like cells , then neuron-like cells and astrocyte-like cells ,respectively positive for nestin , NF200 and GFAP. At 4 ,8 weeks after transplantation NF200 and GFAP -positive cells were detected in denervated skeletal muscle .Muscle cross-sectional areas of cell transplantation group were significantly increased,compared with that of the control group. There was a statistical significance (P < 0.05, P < 0.01). Conclusions Differentiated neuron-like cells and astrocyte-like cells can be acquired in vitro by effective induction protocols. After co-transplantation into the denervated skeletal muscle cells can survive in vivo and have therapeutic benefits on delaying denervation atrophy.
目的①探讨间充质干细胞(MSCs)体外诱导分化为骨样细胞、肌样细胞以及神经元(NCs)样/星形胶质样细胞的有效方法。②评价体外诱导分化的神经前体样细胞(NPCs)、神经元(NCs)样细胞以及星形胶质样细胞的体外存活和增殖特性,为神经细胞再生寻找良好的种子来源。
方法参考有关文献,采用贴壁筛选法自大鼠骨髓中分离培养间充质干细胞,进行传代纯化,收集得到第5代(P5)间充质干细胞单细胞悬液(1×10~(5~6)/ml)。①成骨诱导分化方案:DMEM/10%FBS培养基+0.1μmol/l地塞米松+50ng/ml维生素C+2.16mg/mlβ-甘油磷酸钠。诱导分化2周以上。行Van Kossa银染色和茜素红染色。②成肌诱导分化方案:DMEM/20%FBS+3μmol/l5-氮胞苷(5-azayttidine)。诱导分化24h,培养2～3周以上。观察形成的肌样细胞及肌管样结构。③神经定向诱导分化方案:采用多因子、分阶段逐步诱导方法定向诱导MSCs分化为神经前体细胞(NPCs),进而分化为NCs样/星形胶质样细胞。即:MSCs以1×10~6/L置入6孔培养板中,加入生长培养基:DMEM/20%FBS培养基,24h后更换为诱导分化培养基:DMEM/F12+2%B27+40μg/L碱性成纤维生长因子(bFGF)+20μg/L表皮生长因子诱导分化培养基(EGF),培养10～20d,定向诱导MSCs分化为NPCs。下一个阶段更换诱导分化培养基为生长培养基:DMEM/5%FBS,分两组进行诱导:一组向神经元样细胞诱导分化:全反式维甲酸(RA)连续加样,首次浓度0.5μmol/L,以后每天半量加样;二组向星形胶质样细胞诱导分化:10μg/L血小板衍生生长因子BB(PDGF-BB)首次加样,以后每天半量加样,均连续诱导10～14d,制作两组细胞爬片均行纤维丝蛋白200(NF200)、胶质纤维酸性阳性蛋白(GFAP)免疫组织化学荧光染色。④神经元样/星形胶质样细胞共同培养:收集诱导分化的神经元样/星形胶质样细胞,各自浓度1×106/L,以1:1混合在DMEM/5%FBS培养基中共同培养。观察两组细胞生长情况。⑤应用活细胞计数试剂盒CCK-8检测单独诱导分化的NCs样/星形胶质样细胞的增殖及存活情况,绘制生长曲线及细胞存活曲线。
结果①经DMEM/10%FBS培养基+0.1μmol/l地塞米松+50ng/ml维生素C+2.16mg/mlβ-甘油磷酸钠诱导培养2周以上,MSCs细胞聚集呈结节状,钙盐沉积,Van Kossa银染色法阳性。茜素红染色阳性。②经3μmol/l 5-氮胞苷诱导处理大鼠MSCs,24小时部分细胞胞体收缩变为瘦长形状, 1~2周后细胞相互形成肌管样结构。2周后可以观察到自发搏动。③bFGF+EGF连续诱导7d后MSCs分化为球团样的神经前体样细胞,巢蛋白(Nestin)表达阳性。RA、PDGF-BB分别连续诱导10d后MSCs逐步分化出神经元样、星形胶质样细胞,分别表达NF200、GFAP蛋白。④神经元样/星形胶质样细胞可以共同培养、生长,呈现各自的细胞形态。⑤活细胞计数试剂盒CCK-8检测显示:神经元样细胞早期生长较缓慢,具有一定的增殖能力,6～7d后达到高峰,此后增殖能力明显下降,细胞存活率明显下降,14～15d之后细胞存活率下降到7.6%以下,难以传代培养。星形胶质样细胞初期生长慢,5d后生长加速,进入对数生长期,7～8d后进入平台期,仍保持较高的细胞存活率。
结论①MSCs是具有自我增殖和多向分化潜能的干细胞。可以诱导分化为骨样细胞、骨骼肌样细胞、神经元样细胞及星形胶质样细胞。②诱导分化的神经元样/星形胶质样细胞可以共同培养、生长。③单独诱导分化的神经元样细胞可继续存活13-15d后发生自然死亡,难以传代,星形胶质样细胞生长较稳定,具有较强的存活和增殖能力,可传代培养至3代以上。
第二部分MSCs归巢性及对失神经肌萎缩的作用
目的检测大鼠间充质干细胞(MSCs)经静脉移植后在周围神经损伤模型内的迁移、分布情况,评价其对轴突、靶器官的保护作用。
方法制作80只大鼠坐骨神经损伤模型,收集P5代MSCs行BrdU(10μmol/L)掺和标记48h。经尾静脉注射BrdU标记的MSCs(2×10~7/ml)入体内。术后8周内评价坐骨神经指数(SFI)。术后神经断端、肌肉组织取材均行BrdU免疫组织化学荧光染色。肌肉组织行HE染色、TUNEL组织化学染色。Image-Pro Plus 5.0专业图像分析软件分析肌纤维横截面积并计算以及实验侧与正常侧肌纤维横截面积之比,分析肌细胞调亡数量。所得数据均用均数±标准差(x——±s)表示,结果用SPSS统计软件行t检验。
结果MSCs体外可以被BrdU掺和标记,阳性标记率为62.5%。术后4、8周神经断端、肌肉组织内检测到BrdU阳性细胞。实验组SFI、肌纤维横截面积明显高于对照组,细胞凋亡数量低于对照组,均有统计学意义﹙4周P<0.05, 8周P<0.01﹚。
结论MSCs静脉移植至体内能归巢到损伤的神经断端及肌肉组织,可以存活至少8周,具有促进轴突再生、延缓肌萎缩的作用。
第三部分骨髓源性神经前体样细胞对失神经肌萎缩的作用
目的探讨大鼠间充质干细胞(MSCs)诱导分化的神经前体样细胞(NPCs)移植在体内存活情况,评价其及对失神经肌萎缩的作用。
方法联合应用EGF、bFGF诱导MSCs分化为NPCs。行Nestin免疫组织化学荧光染色鉴定,并行5-溴脱氧尿苷(BrdU)掺和标记实验和单细胞克隆实验。建立90只大鼠坐骨神经损伤模型。BrdU掺和标记的NPCs以5×106/ml注入神经损伤断端。术后4、8周神经断端、肌肉组织取材,神经断端分别行Nestin、BrdU单标免疫荧光染色检验。肌肉组织行HE染色、TUNEL免疫组织化学染色。Image-Pro Plus 5.0专业图像分析软件分析肌纤维横截面积并计算以及实验侧与正常侧肌纤维横截面积之比。所得数据结果用SPSS统计软件行t检验。
结果MSCs体外诱导分化可获得大量活性的NPCs,Nestin蛋白染色阳性,体外可以被BrdU掺和标记,阳性标记率为87.9%,单细胞克窿率为2.7%。移植术后4、8周实验组神经断端均检测到Nestin、BrdU阳性细胞。实验组肌纤维横截面积明显高于对照组,肌细胞调亡数量明显低于对照组,均有统计学意义﹙4周P<0.05,8周P<0.01﹚。
结论骨髓源性NPCs移植至神经断端可以存活至少8周,具有延缓失神经肌萎缩、抑制失神经肌肉细胞调亡的作用。
第四部分骨髓源性神经样细胞对失神经肌萎缩的作用
目的探讨间充质干细胞(MSCs)诱导分化的神经元(NCs)样、星形胶质样细胞联合移植后在体内存活情况,评价其联合移植对失神经肌萎缩的作用。
方法多因子、分阶段逐步诱导MSCs分化为NCs样、星形胶质样细胞。行NF200、GFAP免疫组织化学荧光染色鉴定。建立60只大鼠失神经骨骼肌模型。行细胞联合移植,以5×10~6/ml肌肉注射入失神经骨骼肌内。术后4、8周肌肉组织取材行HE染色、免疫组织化学染色检测NF200、GFAP阳性细胞。Image-Pro Plus 5.0专业图像分析软件分析肌纤维横截面积并计算以及实验侧与正常侧肌纤维横截面积之比。所得数据结果用SPSS统计软件行t检验。
结果MSCs逐步诱导分化出NCs样、星形胶质样细胞。分别表达NF200、GFAP阳性蛋白。移植术后4、8周实验侧肌肉组织检测到NF200、GFAP阳性细胞。实验侧肌纤维横截面积明显高于对照组,有统计学意义(4周P<0.05,8周P<0.01)。
结论骨髓源性NCs样、星形胶质样细胞可以体外大量获得,联合移植至肌肉内可以存活至少8周,可以增加失神经肌肉面积,有效改善失神经肌萎缩。
PartⅠThe multipotency of mesenchymal stem cells in rats.
Objective To investigate multipotentcy of MSCs differentiation into osteoblasts,skeletal muscle, neuron-like cells and astrocyte-like cells in vitro and evaluate the proliferation and survival capacity of MSCs-differentiated neural progenitor cells, neuron-like cells and astrocyte-like cells in vitro.
Methods Adult rat MSCs were isolated and cultured for 5 passages in vitro. Osteogenic differentiation was induced by plating the cells at 1×10~5cells/ml in DMEM medium containing 10%FBS,supplenmented with 0.1μmol/l dexamethasone,50ng/ml ascorbic acid, and 2.16mg/mlβ–glycerophosphate. Cells were cultured for 14days and the medium was changed twice a week. Skeletal muscle differentiation was induced at 1×10~5cells/ml by DMEM/20%FBS supplenmented with 3μmol/l 5-azayttidine for 24h, then cultured for 2~3weeks.Neural differentiation were selectively and progressively induced into neuron-like cells and astrocyte-like cells.by multi-cytokines, first DMEM/F12/2%B27 with EGF(20μg/L) and bFGF(40μg/L) for 10～20days, then DMEM/5%FBS with retinoic acid (0.5μmol/L) or PDGF-BB(10μg/L) for another 10～20days. The differentiated neuron-like cells and astrocyte-like cells were co-cultured at 1×10~5cells/ml at the ratio of 1:1 in DMEM medium containing 10%FBS. Immunohistochemistry stainings were selectively carried out for CD44,CD45,nestin,NF200 and GFAP. The cell proliferation and survival assays were made using cell counting Kit-8.
Results The MSCs showed a fibroblast-like morphology, and could self-renew and survive at least 15 passages. In the presence of selected cytokines MSCs were effectively differentiated into osteoblasts for 4 weeks. The cells morphology changed after treatment with 5-azacytidine for 1 week. MSCs connected with adjoining cells after one week, formed myotube-like structures, began spontaneously beating after two weeks, and beat synchronously after three weeks. MSCs were differentiated into neurospheres -like cells with EGF and bFGF for 7 days. Then neurospheres-like cells were differentiated into neuron-like cells with retinoic acid and astrocyte-like cells with PDGF-BB for 10～14 days, respectively positive for nestin, NF200 and GFAP immunohistochemistry stainings. The differentiated neuron-like cells and astrocyte-like cells could survive the next co-culture procedure.The CCK-8 assays demonstrated that the differentiated neuron-like cells could proliferate and survive until the next 13～15 days. However the astrocyte-like cells proliferated and survived better and could be cultured for at least 3 passages.
Conclusions MSCs were self-renewing and mutipotent cells which maintain the capacity of differentiation into osteoblasts,skeletal muscle-like cells,neuron-like cells and astrocyte-like cells in vitro by multi-cytokines induction protocols. And the MSCs-derived astrocyte-like cells had a better proliferation and survival capacity than the neuron-like cells.
PartⅡTherapeutic benefits of intravenous administration of mesenchymal stem cells on peripheral nerve regeneration in rats
Objective To investigate the migration and distribution of rat bone mesenchymal stem cells in vivo after peripheral nerve injury and evaluate the beneficial effects on peripheral nerve regeneration and delaying denervation muscle atrophy.
Methods Eighty female Sprague-Dawley rats were randomly divided into 2 groups, with 40 in each. All the sciatic nerves were transected and sutured with 8-0 nylon suture. The transplanted group received an infusion of BrdU-labelled MSCs at 2×10~7cells/ml cells/ml through the tail vein. Every week until 12 weeks after surgery the sciatic function index (SFI) was assessed by walking tract test. Sections were carried out for HE staining, BrdU and TUNEL immunohistochemical staining.
Results BrdU-labeled cells were detected housing the nerve stumps and denervation muscles at 1 week and 8 weeks. MSCs transplantation group had a promotion of sciatic nerve functional recovery from weeks 4 to 12 and the number of denervation muscle apoptotic cells significantly decreased compared with that of control group at 8 weeks(P<0.05, P<0.01).
Conclusions MSCs is capable in vivo of homing to the nerve stumps and denervation muscles after peripheral nerve injury via the blood circulation, and had therapeutic benefits on promoting peripheral nerve regeneration and delaying denervation muscle atrophy.
PartⅢThe effect of neural progenitor cells differentiated from bone marrow mesenchymal stem cells on denervation muscle atrophy
Objective To investigate the effect on delaying denervation muscle atrophy after peripheral nerve injury by transplantion of neural progenitor cells differentiated from bone marrow mesenchymal stem cells.
Methods Isolated and purified MSCs were induced into neural progenitor cells by EGF (20ng/ml) and bFGF (40ng/ml) for 10～20d. Differentiated cells were analyzed by single cell clone technique and immunohistochemistry staining for nestin. Ninety female Sprague-Dawley rats were randomly divided into 2 groups, with 45 in each. All the sciatic nerves were transected and sutured with 8-0 nylon suture. Before transplantation the neural progenitor cells were labeled with BrdU for 48h and then were injected into the sciatic nerve stumps at 5×10~6cells/ml. At weeks 4, 8 the nerve stump frozen sections were carried out for BrdU immunofluorescence staining, and muscle paraffin sections were carried out for HE staining and TUNEL immunohistochemical staining. Muscle cross-sectional areas and apoptotic cells were measured by an image analyzer.
Results MSCs were positive for CD44, however negative for CD45 and nestin. After 10～14d differentiation, neurospheres were observed and mostly of them were positive for nestin. The rate of label of BrdU was 87.9% and the single-clone formed rate was 2.7%.At weeks 4 and 8 after surgery BrdU positive cells were detected at the nerve stumps. Muscle cross-sectional areas of cell transplantation group significantly increased and the number of the denervation muscle apoptotic cells significantly decreased compared with that of control group (P<0.05, P<0.01).
Conclusions MSCs can be efficiently differentiated into neural progenitor cells with EGF and bFGF. Differentiated neural stem cell-like cells can survive the next transplantation into nerve stumps and had beneficial effects on delaying denervation muscle atrophy after injury.
PartⅣThe effect of differentiated neuron-like cells and astrocyte-like cells from bone marrow mesenchymal stem cells on denervation muscle atrophy
Objective To investigate the survive of MSCs-differentiated neuron-like cells and astrocyte-like cells after co-transplantation in vivo and evaluate the beneficial effects on delaying denervation atrophy.
Methods Isolated and purified rMSCs were selectively and progressively induced into neuron-like cells and astrocyte-like cells by multi-cytokine, EGF and bFGF first, followed by retinoic acid or PDGF. Immunohistochemistry staining was carried out for CD44, CD45, nestin, NF200 and GFAP. Sixty female Sprague-Dawley rat sciatic nerve transection models were made and then randomly divided into 2 groups, with 25 in each. In the experimental group, differentiated neuron-like cells and astrocyte-like cells were co-injected into the denervated skeletal muscle at 5×10~6cells/ml. At weeks 4 and 8 after cell transplantation muscle frozen sections were carried out for NF200 and GFAP immunofluorescence staining and muscle paraffin sections were carried out for HE staining Muscle cross-sectional areas were measured by an image analyzer.
Results MSCs were effectively differentiated into neurospheres -like cells , then neuron-like cells and astrocyte-like cells ,respectively positive for nestin , NF200 and GFAP. At 4 ,8 weeks after transplantation NF200 and GFAP -positive cells were detected in denervated skeletal muscle .Muscle cross-sectional areas of cell transplantation group were significantly increased,compared with that of the control group. There was a statistical significance (P < 0.05, P < 0.01). Conclusions Differentiated neuron-like cells and astrocyte-like cells can be acquired in vitro by effective induction protocols. After co-transplantation into the denervated skeletal muscle cells can survive in vivo and have therapeutic benefits on delaying denervation atrophy.
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