骨髓基质细胞体外分离培养和分化为

摘要 目的：观察体外培养大鼠骨髓基质细胞（BMSCs bone marrow stromal cells）的生物学特性并探讨其分化为Schwann细胞表达S100的机制,为组织工程学修复周围神经损伤奠定一定的实验基础。方法：从成年SD大鼠的股骨和胫骨中分离培养BMSCs,倒置显微镜下动态观察细胞的生长状态,采用MTT法检测细胞的活力,FCM检测细胞周期等方法研究BMSCs的生物学特性。应用复合诱导因子（BME, RA ,FSK, bFGF, PDGF, HRG）体外诱导BMSCs向Schwann细胞分化。诱导分化后采用免疫荧光细胞化学染色,流式细胞仪,逆转录PCR方法分别检测检测胶质细胞标记蛋白S100蛋白和mRNA的表达。结果：BMSCs在体外容易扩增,传1－8代以内的细胞增殖能力无明显变化。未经诱导的BMSCs大部分处于G0和G1期。诱导分化后的细胞形态上类似Schwann细胞,并且表达胶质细胞的标记蛋白S100蛋白及其mRNA明显升高。结论：BMSCs在体外可以向类Schwann细胞诱导分化,并且表达胶质细胞的标记蛋白S-100蛋白及其mRNA。     

周围神经细胞悬液对体外培养的骨髓基质干细胞诱导分化的影响

目的应用周围神经细胞悬液、中脑条件培养基与细胞因子联合诱导骨髓基质干细胞(MSCs)转化为多巴胺(DA)能神经元。探索体外诱导MSCs定向分化为DA能神经元的最佳条件。方法取雄性SD大鼠股骨和胫骨骨髓,进行MSCs的体外培养和传代扩增。碱性成纤维细胞生长因子预诱导后,依据处理因素不同分为对照组及实验组(周围神经细胞悬液组、周围神经细胞悬液 中脑条件培养基组)。倒置显微镜下观察细胞形态变化,在诱导第7天进行神经元特异烯醇化酶(NSE),酪氨酸羟化酶(TH)免疫细胞化学检测。计数NSE和TH阳性细胞数,并计算阳性细胞百分比。结果对照组及实验各组7d NSE阳性细胞数分别为2.304±0.767,37.411±2.89.37.836±2.836(细胞数/每视野)。各组以周围神经细胞悬液 中脑条件培养基组NSE阳性细胞率最高,差异有统计学意义(P<0.01)。对照组及实验各组7d TH阳性细胞数分别为0,10.44±0.511,16.671±0.544(细胞数/每视野)。以周围神经细胞悬液 中脑条件培养基组TH阳性细胞数量较多,差异非常显著(P<0.01)。结论周围神经细胞悬液、中脑条件培养基与细胞因子联合可明显促进MSCs向神经元样细胞分化,并促进细胞表达TH。     

Differentiation of mesenchymal stem cells to support peripheral nerve regeneration in a rat model

Mesenchymal stem cells (MSCs) support axon regeneration across artificial nerve bridges but their differentiative capacity and ability to promote nerve regeneration remains unclear. In this study, MSCs isolated from bone marrow of Sprague–Dawley rats were characterized by plastic adherence and pluripotency towards mesodermal lineages. Isolated undifferentiated MSCs (uMSCs) were stimulated towards a Schwann cell (SC) phenotype using specific growth factors, and cell marker analysis was performed to verify SC phenotype in vitro. Differentiation resulted in temporally dependent positive immunocytochemical staining for the SC markers, glial fibrillary acidic protein (GFAP), S100, and nerve growth factor receptor (NGFR), with maximal marker expression achieved after 6 days of treatment with differentiation media. Quantitative analysis demonstrated that ~ 50% of differentiated MSCs (dMSCs) have a SC phenotype. Using an indirect co-culture system, we compared the ability of dorsal root ganglion (DRG) cells to extend neurites in indirect contact with uMSCs and dMSCs as compared to SCs. The mean values of the longest length of the DRG neurites were the same for the dMSCs and SCs and significantly higher than the uMSC and DRG mono-culture systems (p < 0.05). In vivo, compared to an empty conduit, dMSC seeded collagen nerve conduits resulted in a greater number of sciatic motoneurons regenerating axons through the conduit into the distal nerve stump. We conclude that bone marrow-derived MSCs differentiate into a SC-phenotype that expresses SC markers transiently and sufficiently to support limited neurite outgrowth in vitro and axonal regeneration equivalent to that of SCs in vitro and in vivo. The nerve autograft remains the most effective conduit for supporting regeneration across nerve gaps.     

Peripheral glial cell differentiation from neurospheres derived from adipose mesenchymal stem cells

Mesenchymal stem cells derived from bone marrow and adipose tissue are being considered for use in neural repair because they can differentiate after appropriate induction in culture into neurons and glia. The question we asked was if neurospheres could be harvested from adipose-derived stem cells and if they then could differentiate in culture to peripheral glial-like cells. Here, we demonstrate that adipose-derived mesenchymal stem cells can form nestin-positive non-adherent neurosphere cellular aggregates when cultured with basic fibroblast growth factor and epidermal growth factor. Dissociation of these neurospheres and removal of mitogens results in expression of the characteristic Schwann cell markers S100 and p75 nerve growth factor receptor and GFAP. The simultaneous expression of these glia markers are characteristic features of Schwann cells and olfactory ensheathing cells which have unique properties regarding remyelination and enhancement of axonal regeneration. When co-cultured with dorsal root ganglion neurons, the peripheral glial-like cells derived from adipose mesenchymal stem cells aligned with neuritis and stimulated neuritic outgrowth. These results indicate that neurospheres can be generated from adipose-derived mesenchymal stem cells, and upon mitogen withdrawal can differentiate into peripheral glial cells with neurotrophic effects.     

Repair of extended peripheral nerve lesions in rhesus monkeys using acellular allogenic nerve grafts implanted with autologous mesenchymal stem cells

Despite intensive efforts in the field of peripheral nerve injury and regeneration, it remains difficult in humans to achieve full functional recovery following extended peripheral nerve lesions. Optimizing repair of peripheral nerve injuries has been hindered by the lack of viable and reliable biologic or artificial nerve conduits for bridging extended gaps. In this study, we utilized chemically extracted acellular allogenic nerve segments implanted with autologous non-hematopoietic mesenchymal stem cells (MSCs) to repair a 40 mm defect in the rhesus monkey ulnar nerve. We found that severely damaged ulnar nerves were structurally and functionally repaired within 6 months following placement of the MSC seeded allografts in all animals studied (6 of 6, 100%). Furthermore, recovery with the MSC seeded allografts was similar to that observed with Schwann cell seeded allografts and autologous nerve grafts. The findings presented here are the first demonstration of the successful use of autologous MSCs, expanded in culture and implanted in a biological conduit, to repair a peripheral nerve gap in primates. Given the difficulty in isolating and purifying sufficient quantities of Schwann cells for peripheral nerve regeneration, the use of MSCs to seed acellular allogenic nerve grafts may prove to be a novel and promising therapeutic approach for repairing severe peripheral nerve injuries in humans.     

Peripheral nerve regeneration by bone marrow stromal cells

Adult bone marrow contains stem cells that have attracted interest through their possible use for cell therapy in neurological diseases. Bone marrow stromal cells (MSCs) were harvested from donor adult rats, cultured and pre-labeled with bromodeoxyuridine (BrdU) previously to be injected in the distal stump of transected sciatic nerve of the rats. Distal nerve stump of control rats received culture medium solution. MSCs-treated rats exhibit significant improvement on walking track test at days 18 and 33 compared to controls. Dual immunofluorescence labeling shows that BrdU reactive cells survive in the injected area of transected sciatic nerve at least 33 days after implantation, and almost 5% of BrdU cells express Schwann cell-like phenotype (S100 immunoreactivity). Because MSCs injected in a lesioned peripheral nerve can survive, migrate, differentiate in Schwann cells, and promote functional recovery, they may be an important source for cellular therapy in several neurological diseases.     

富血小板血浆诱导骨髓间充质干细胞修复坐骨神经缺损：并与自体神经修复的效果相比较

目的：通过植入经PRP诱导的BMSCs结合化学萃取的去细胞神经修复坐骨神经缺损,观察其对周围神经的修复作用。 方法：32只新西兰大耳白兔,随机分成4组,即单纯的化学萃取的去细胞神经、BMSCs结合化学萃取的去细胞神经、经PRP诱导的BMSCs结合化学萃取的去细胞神和自体神经修复坐骨神经缺损,检测指标包括形态学观察、靶肌肉肌湿重恢复率、运动神经传导速度（MNCV）及轴突直径和髓鞘厚度等。 结果：结果显示,靶肌肉肌湿重恢复率、MNCV、轴突直径和髓鞘厚度和形态学观察在经PRP诱导的BMSCs结合化学萃取的去细胞神经组明显优于单纯的化学萃取的去细胞神经组和BMSCs结合化学萃取的去细胞神经组,而与自体神经修复组结果相似。 结论：经诱导后的BMSCs在体内具有SC的部分功能,可作为组织工程化外周神经的种子细胞,用于周围神经缺损的修复。     

Phenotypic and functional characteristics of mesenchymal stem cells differentiated along a Schwann cell lineage

We have investigated the phenotypic and bioassay characteristics of bone marrow mesenchymal stromal cells (MSCs) differentiated along a Schwann cell lineage using glial growth factor. Expression of the Schwann cell markers S100, P75, and GFAP was determined by immunocytochemical staining and Western blotting. The levels of the stem cell markers Stro-1 and alkaline phosphatase and the neural progenitor marker nestin were also examined throughout the differentiation process. The phenotypic properties of cells differentiated at different passages were also compared. In addition to a phenotypic characterization, the functional ability of differentiated MSCs has been investigated employing a co-culture bioassay with dissociated primary sensory neurons. Following differentiation, MSCs underwent morphological changes similar to those of cultured Schwann cells and stained positively for all three Schwann cell markers. Quantitative Western blot analysis showed that the levels of S100 and P75 protein were significantly elevated upon differentiation. Differentiated MSCs were also found to enhance neurite outgrowth in co-culture with sensory neurons to a level equivalent or superior to that produced by Schwann cells. These findings support the assertion that MSCs can be differentiated into cells that are Schwann cell-like in terms of both phenotype and function.     

Nanoparticles carrying neurotrophin-3-modified Schwann cells promote repair of sciatic nerve defects

Schwann cells and neurotrophin-3 play an important role in neural regeneration,but the secretion of neurotrophin-3 from Schwann cells is limited,and exogenous neurotrophin-3 is inactived easily in vivo.In this study,we have transfected neurotrophin-3 into Schwann cells cultured in vitro using nanoparticle liposomes.Results showed that neurotrophin-3 was successfully transfected into Schwann cells,where it was expressed effectively and steadily.A composite of Schwann cells transfected with neurotrophin-3 and poly(lactic-co-glycolic acid) biodegradable conduits was transplanted into rats to repair 10-mm sciatic nerve defects.Transplantation of the composite scaffold could restore the myoelectricity and wave amplitude of the sciatic nerve by electrophysiological examination,promote nerve axonal and myelin regeneration,and delay apoptosis of spinal motor neurons.Experimental findings indicate that neurotrophin-3 transfected Schwann cells combined with bridge grafting can promote neural regeneration and functional recovery after nerve injury.     

骨髓间充质干细胞神经分化的体内外研究

目的研究骨髓间充质干细胞在体内外向神经细胞分化的潜能。方法(1)分离培养：从水囊引产胎儿的四肢骨中分离培养骨髓基质细胞，再通过贴壁方法将间充质干细胞从骨髓来源的单个核细胞中分离出，利用流式细胞仪进行间充质干细胞免疫表型测定。(2)体外分化观察：将间充质干细胞在二甲基亚砜(DMSO)和丁羟茴醚(BHA)中作用5h后，行神经元特异性烯醇化酶、神经微丝等免疫组化染色。(3)体内分化观察：将用5一溴脱氧尿核苷(BrDU)标记的间充质干细胞通过立体定向的方法移植于顶叶皮质区，然后再行烯醇化酶、神经微丝及胶质纤维酸性蛋白免疫组化染色。结果(1)生物学特性：间充质干细胞贴壁生长，细胞表现为梭形细胞形态，骨髓来源的间充质干细胞CD34、HLA—DR的表达为阴性，而CD44、CD29、CD13的表达为阳性。(2)体外分化：间充质干细胞在体外被DMSO／BHA处理后，胞浆向细胞核方向收缩，形成一个收缩的胞体，外周是细胞膜的突起，呈现出典型的神经元表型，并且免疫组化染色呈现神经元特异性烯醇化酶及神经微丝染色阳性。(3)体内分化：间充质干细胞被立体定向移植后，在移植部位可见BrDU染色阳性的细胞团，同时可见BrDU阳性细胞迁移到周围宿主的皮质中。免疫组化染色显示，在移植物中及邻近宿主皮质中的移植细胞呈神经元标记烯醇化酶染色阳性，星形细胞标记胶质纤维酸性蛋白染色阳性。结论问充质干细胞有能力分化为非间充质细胞，特别是神经元。     

Comparison of different biogenic matrices seeded with cultured Schwann cells for bridging peripheral nerve defects

Tissue-engineering as laboratory based alternative to human autografts and allografts provides "custom made organs" cultured from patient's material. To overcome the limited donor nerve availability different biologic nerve grafts were engineered in a rat sciatic nerve model: cultured isogenic Schwann cells were implanted into acellular autologous matrices: veins, muscles, nerves, and epineurium tubes. Autologous nerve grafts, and the respective biogenic material without Schwann cells served as control. After 6 weeks regeneration was assessed clinically, histologically and morphometrically. The PCR analysis showed that the implanted Schwann cells remain within all the grafts. A good regeneration was noted in the muscle-Schwann cell-group, while regeneration quality in the other groups (with or without Schwann cells) was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. All venous and epineurium grafts had a more disorganized regeneration. Seemingly, the lack of endoneural tube like structures in vein grafts lead to impaired regeneration. And, apparently, the beneficial effects of implanted Schwann cells into a large luminal structure can only be demonstrated to a limited extent if endoneural like structures are lacking. A tube offers less area for Schwann cell adhesion and it is more likely to collapse. This underlines the role of the basal lamina, or at least an inner structure acting as scaffold in axonal regeneration. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biogenic graft with basal lamina tubes as pathway for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

骨髓间充质细胞构建组织工程神经修复坐骨神经缺损

背景：许旺细胞是周围神经组织工程的种子细胞,但体外分离、培养、纯化许旺细胞较困难。脱细胞同种异体神经移植物具有较强的修复外周神经缺损的能力,且可诱导骨髓间充质细胞分化为类许旺细胞,理论上骨髓间充质细胞可替代许旺细胞作为种子细胞应用于周围神经组织工程。 目的：观察骨髓间充质细胞构建组织工程神经修复坐骨神经缺损的效果,评估骨髓间充质细胞作为种子细胞修复周围神经缺损的可行性。 设计、时间及地点：随机对照动物实验,于2008-07/12在大理学院基础医学院实验室完成。 材料：将30只SD大鼠按随机数字表法分为3组,每组10只。骨髓间充质细胞+异体移植组将骨髓间充质细胞复合脱细胞同种异体神经移植物培养的组织工程神经与两断端用10/0 无创线端端吻合;异体移植组将脱细胞同种异体神经移植物桥接;自体移植组将切断的坐骨神经旋转180°端端吻合。 方法：运用骨髓间充质细胞构建的组织工程神经修复大鼠10 mm坐骨神经缺损,移植后12周通过坐骨神经功能指数、腓肠肌湿质量恢复率、S-100免疫组织化学染色、电镜等方法观察移植物修复效果。 主要观察指标：复合物培养时观察细胞形态的变化;移植后观察坐骨神经功能指数及腓肠肌湿质量恢复率;通过甲苯胺蓝染色观察新生髓鞘形成和轴突生长及神经纤维的分布情况,结合透射电镜及S-100蛋白免疫组织化学染色,观察许旺细胞生长和神经纤维再生情况。 结果：坐骨神经功能指数及腓肠肌湿质量恢复率的检测结果显示骨髓间充质细胞+异体移植组优于异体移植组(P < 0.05)。骨髓间充质细胞+异体移植组复合物中S-100的表达明显高于异体移植组,有髓神经纤维数量、有髓纤维直径和髓鞘厚度均大于异体移植组(P < 0.05),修复效果接近自体移植组。 结论：骨髓间充质细胞构建的组织工程神经修复周围神经缺损的效果优于单纯的脱细胞同种异体神经移植物,骨髓间充质细胞作为种子细胞在周围神经组织工程中具有较强的应用价值。     

PC12 neurite regeneration and long-term maintenance in the absence of exogenous nerve growth factor in response to contact with Schwann cells

We have investigated mouse and rat ganglionic Schwann cells as possible sources of neurite outgrowth-promoting factors by co-culturing Schwann cells with nerve growth factor (NGF)-responsive PC12 pheochromocytoma cells primed by pretreatment with NGF. NGF-primed PC12 cells are capable of neurite regeneration when provided with an appropriate neurite promoting factor such as NGF. When primed PC12 cells were co-cultured with Schwann cells in the absence of exogenous NGF, PC12 cells that directly contacted Schwann cells became enlarged and flattened, attaining a neuron-like morphology within one day. When contact with Schwann cells was established, PC12 cells regenerated neurites by the first day of co-culture and these were maintained throughout the experiments (7 weeks). Most PC12 cells cultured in the same collagen-coated dishes with Schwann cells, but not directly in contact with them, failed to regenerate neurites. Instead, they began to proliferate, forming cell clusters. Neurite regeneration by PC12 cells in contact with Schwann cells was not blocked by antibody to NGF. These results demonstrate the presence of a neurite-promoting activity localized to the vicinity of the Schwann cell surface which is capable of eliciting regeneration and long-term maintenance of PC12 neurites in the absence of exogenous NGF. This activity does not appear to be due to NGF.     

Synthetic nerve graft containing collagen and synthetic Schwann cells inproves functional, electrophysiological, and histological parameters of peripheral nerve regeneration

Current methods of peripheral nerve repair are to directly suture cut nerve stumps, or to bridge large gaps with an autograft repair. Autograft-associated problems include donor site morbidity and limited supply. Many of the present limitations of nerve repair might be overcome by expanding the patients own Schwann cells in vitro, then combining the cells with other neuro-tropic and -trophic materials into an Artificial Nerve Graft (ANG) for bridging a nerve gap. In this 4.5 month experiment, a rat peroneal nerve model with a 10 mm gap was used to evaluate the effect of live Schwann cells on peripheral nerve regeneration. Nerve gaps were repaired with cellular ANGs containing live Schwann cell, dead Schwann cell, or mixed fibroblast/Schwann cell populations suspended in a collagen I matrix, and with sutured autografts or ANGs containing just collagen or medium. Regenerated nerves were evaluated by walking track analysis, qualitative and quantitative histology, and electrophysiology. Overall, the autograft was the best repair method, while the ANG containing live Schwann cells was statistically superior to other ANG repair methods. This study demonstrates that an ANG containing cultured syngeneic Schwann cells improves functional, histological, and electrophysiological parameters of peripheral nerve regeneration.     

Insulin and the insulin-like growth factors I and II are mitogenic to cultured rat sciatic nerve segments and stimulate [3H]thymidine incorporation through their respective receptors

The factors that control proliferation of Schwann cells during peripheral nerve regeneration are not yet known. In this study we investigated the effects of insulin, insulin-like growth factor I and II (IGF-I and IGF-II), IGF-I analogues, and factors that interfere with their respective receptors, on [³H]thymidine incorporation into cultured nerve segments from the rat sciatic nerve. Segments cultured in nM (0.1–1.7 nM) concentrations of insulin, truncated IGF-I (tIGF-I), long R³IGF-I, or IGF-II exhibited an increase in [³H]thymidine incorporation compared with control segments. IGF-II was most potent. JB1, an IGF-I antagonist, counteracted the effects of tIGF-I and insulin. The results suggest that non-neuronal cells in the nerve segment, probably Schwann cells, possess distinct receptors for insulin, IGF-I, and IGF-II and that these receptors may be involved in the control of Schwann cell proliferation during peripheral nerve regeneration. © 1996 Wiley-Liss, Inc.     

Myelinated nerve fibre regeneration in diabetic sensory polyneuropathy: correlation with type of diabetes

Observations were made on myelinated fibre regeneration in diabetic sensory polyneuropathy assessed in sural nerve biopsy specimens. These confirmed that regenerative clusters initially develop within abnormally persistent Schwann cell basal laminal tubes. The number of regenerating fibres, identified by light microscopy, was found to decline in proportion to the reduction in total myelinated fibre density. The relative number of regenerating fibres was significantly greater in patients with insulin-dependent as compared with those with non-insulin-dependent diabetes after correction for age. There was a slight negative correlation between the relative proportion of regenerating fibres and age, but this was not statistically significant. The progressive reduction in the number of regenerating fibres with declining total fibre density indicates that axonal regeneration fails with advancing neuropathy. The production of nerve growth factor (NGF) and NGF receptors by denervated Schwann cells is likely to be important for axonal regeneration. To investigate whether the failure of axonal regeneration could be related to a lack of NGF receptor production by Schwann cells, we examined the expression of p75 NGF receptors by Büngner bands immunocytochemically. In comparison with other types of peripheral neuropathy, p75 NGF receptor expression appeared to take place normally. It is concluded that failure of axonal regeneration constitutes an important component in diabetic neuropathy. Its explanation requires further investigation.These results were presented in part at a meeting of the European Association for the Study of Diabetes held in Düsseldorf in September 1994