骨髓间充质干细胞与施万细胞联合移植对大鼠周围神经 损伤端侧吻合的修复效果研究

目的:探究骨髓间充质干细胞(MSCs)与施万细胞(SCs)联合移植对大鼠周围神经损伤端侧吻合的修复效果。方法:选取SD雌性大鼠60只均制作成坐骨神经损伤端侧吻合模型,并将其随机分为联合移植组、MSCs组和SCs组,分别对吻合端进行骨髓间充质干细胞与SCs联合移植、MSCs移植、SCs移植。观察分析三组大鼠的神经电生理学指标和腓神经功能指数(PFI)和神经传导速度(NCV)。结果:三组大鼠的PFI和NCV均有所改善,且联合移植组的PFI和NCV均优于其他两组,并随着时间推移损伤坐骨神经功能恢复越来越好。结论:MSCs与SCs均具有促进大鼠周围神经身上修复的功能,且两种细胞联合移植效果更加明显。     

担载神经生长因子(NGF)的乳液法涂层纤维体外缓释研究

目的:目前周围神经修复中,神经导管是研究热点,本文研究乳液法涂层纤维制备的神经导管在神经修复中应用的可能性。方法:本文采用乳液法制备担载NGF的丝素-聚乳酸(PLLA)涂层电纺纤维,观察纤维的形态,测定NGF的体外释放动力学参数,并考察纤维释放液对于PC12细胞增殖的影响。结果:担载NGF的涂层纤维具备类似于细胞外基质(ECM)的三维结构和多孔形态;涂层纤维中NGF体外有效缓释10天;细胞实验中,在含有释放液的培养基中生长的PC12细胞,与空白对照组相比,荧光强度平均多了2000-4000个荧光强度,所以释放液可以更好地促进PC12细胞的增殖。结论:担载NGF的乳液法涂层纺丝纤维具备促进缺损周围神经修复的条件,可以进一步研究在动物体内修复缺损周围神经中的效果,为以后的临床应用打下基础。     

成体干细胞向类许旺细胞诱导分化研究进展

周围神经损伤修复的黄金标准是自体神经移植,但由于遗留供区部位感觉功能障碍及可供移植神经数量有限,致使该项技术在临床上的应用受到限制,组织工程学的发展为此提供了一种新的解决途径.许旺细胞是周围神经组织工程重要的种子细胞,在神经再生过程中发挥重要作用,移植许旺细胞修复周围神经损伤有广阔的应用前景,但异体移植常面临免疫排斥反应,这些均导致了许旺细胞作为周围神经组织工程的种子细胞在临床应用中受到限制.近年来,随着对具有自我更新和多向分化潜能的间充质干细胞的深入研究,为组织工程化人工神经种子细胞的发展提出新的思路,本文将各种成体干细胞作为许旺细胞的替代细胞的研究进展作一综述.     

超选择性股动脉内骨髓单个核细胞移植治疗股骨头坏死187例

目的 研究自体骨髓单个核细胞移植对股骨头坏死患者缺血状态的改善程度和治疗效果.方法 选取2004 年7 月至2010 年11 月期间187 例252 髋股骨头坏死患者,应用自体骨髓单个核细胞移植治疗,分别采集 187 例患者骨髓200 -360 ml,采用 Ficoll 密度梯度离心法分离单个核细胞,单个核细胞总数为（2.4 ~ 7.8）× 108 个,流式细胞仪检测 CD34＋ 细胞和 CD133＋ 细胞在所分离出的干细胞悬液中的含量分别为2.47﹪± 0.58﹪和1.29﹪± 0.35﹪,然后将单个核细胞用生理盐水制备成细胞悬液 20 - 30 ml,使用数字减影血管造影技术（DSA）行超选择性股骨头供血动脉内干细胞移植术.按世界骨循环研究学会（ARCO）对骨坏死分期,设自身前后对照观察疗效.移植术后第 3、6、12、24、36 和48 个月,根据髋关节 Harris 评分评价疗效,移植术后6 个月通过复查患者股骨头供血动脉 DSA,观察其新生血管形成情况,以后每隔 6 个月采用影像学方法观察股骨头形态学变化.结果 （1）临床疗效：对接受自体骨髓单个核细胞移植治疗的187 例患者随访 3 ~ 48（24.2 ± 4.5）个月,其中髋关节疼痛缓解者 158 例（占患者总数的 84.5﹪）,髋关节功能改善者 146 例（占患者总数的 78.1﹪）,行走间距延长者 149 例（占患者总数的 79.7﹪）;（2）影像学检查：干细胞移植术后 6 个月187 例患者中54 例行股骨头供血动脉 DSA 检查,48 例显示供血动脉较移植术前明显增多、增粗,血流速度增快,12 ~ 24个月后 72 例患者股骨头区骨质病变获得改善.结论 超选择性动脉内骨髓单个核细胞移植方法简便、安全有效,对因缺血导致坏死的股骨头无再次损伤,能够有效治疗缺血性股骨头坏死.     

鞘内药物注射神经修复方法临床应用五例

中枢神经损伤或病变后的可修复性是神经修复学的核心概念,是近年来基础和临床研究的重要成果。它不但为中枢神经损伤和变性疾病的自身恢复提出合理解释,更为鼓励医生采取各种积极干预策略,以增加神经功能恢复的速度和程度,减轻残障,提供理论基础和探索方向。细胞移植发挥作用的机制之一,是分泌多种神经修复营养因子,而临床使用的神经修复营养药物,如神经生长因子、神经节苷脂等,对于体外神经细胞和周围神经的修复具有营养支持作用。     

干细胞治疗周围神经损伤研究进展

周围神经损伤是临床常见的疾病。损伤后神经的修复和再生是复杂又漫长的过程。严重的神经损伤其预后效果并不令人满意,相应支配区域的功能难以恢复,这给患者及家人带来了极大的痛苦。因此如何更好的对周围神经损伤进行治疗一直是医学界的难题。在神经修复机制的研究中,科学家发现施万细胞对周围神经的修复和再生起到了非常重要的作用,但获取和扩增的困难限制了其临床的应用。随着生物医学的发展,人们把目光投向了干细胞,经实验发现干细胞不仅具有旺盛的增殖能力,而且可以分化为神经系细胞,还能分泌相关的神经营养因子促进神经的修复和再生,这为周围神经损伤后的治疗带来了新的希望。本文就近些年来应用于修复周围神经的干细胞及促进修复机制的研究做以综述。     

胶原蛋白-壳聚糖支架的构建及其与骨髓基质干细胞复合的实验研究

目的:构建一种组织工程神经支架,并观察体外培养的骨髓基质干细胞在其内部的生长情况,为后续种子细胞的移植提供阶段性实验数据.方法:以Ⅰ型胶原蛋白和壳聚糖为原料通过冷冻干燥技术制备神经支架,扫描电镜观察其内部结构,测量其孔径大小、孔隙率等指标.将体外培养的骨髓基质干细胞与Ⅰ型胶原蛋白-壳聚糖神经支架复合,共培养2天;扫描电镜观察细胞在支架内部的生长情况.结果:构建的神经支架均为圆柱状,内部为纵向平行排列的孔径均匀的微管样结构,细胞紧密贴附在支架微孔内壁上,细胞生长状况良好.结论:Ⅰ型胶原蛋白-壳聚糖支架具有良好的内部三维结构和生物相容性,可与细胞复合后用于修复周围神经缺损.     

RunX3对小鼠骨髓造血干细胞功能的影响

目的研究RunX3基因对造血干细胞自我更新和分化能力的影响。方法流式细胞术测定小鼠骨髓干细胞和外周血单个核细胞的比例;通过竞争性骨髓移植实验检测RunX3转基因小鼠骨髓干细胞的功能。结果移植后来源于RunX3-/-小鼠骨髓干细胞供体的外周血细胞占总外周血细胞的比例与野生对照鼠相比无明显差异,移植后来源于RunX3-/-小鼠骨髓干细胞供体的外周血中髓系细胞占总外周血髓系细胞的比例较野生型对照鼠高。结论RunX3基因缺失对骨髓造血干细胞的自我更新没有影响,但其可能参与了骨髓造血干细胞的分化过程。     

肝细胞生长因子对骨髓内皮祖细胞的动员作用

目的: 分析肝细胞生长因子(HGF)能否动员骨髓内皮祖细胞,以及动员的内皮祖细胞能否参与创伤修复时的血管新生和内皮修复.方法: 将腺病毒HGF载体(adenovirus vector encoding HGF gene, Ad-HGF)经尾静脉注射到Balb/c小鼠体内,用ELISA方法检测血浆HGF水平的变化;用流式细胞术检测外周血CD34 细胞含量变化;对外周血单个核细胞进行分离、培养,并对生长的细胞克隆进行内皮细胞表面标志Tie-2、vW因子的免疫组化检测.建立雌性小鼠CCl4肝损伤模型,静脉移植HGF处理后雄性小鼠外周血单个核细胞到其体内,4 W后利用原位杂交技术检测新生肝组织中是否存在雄性细胞.结果: 注射Ad-HGF能明显提高小鼠血浆的HGF水平,并使外周血中以CD34、Tie-2和vW因子等为标志的内皮祖细胞的数量显著增多.这些细胞参与肝损伤修复时的血管新生.结论: HGF对骨髓内皮祖细胞具有明显的动员作用.     

骨髓间充质干细胞和内皮祖细胞移植促进血流重建的比较

目的:比较骨髓间充质细胞(Bone Marrow Mesenchymal Stem Cells,BM/MSC)和骨髓源内皮祖细胞(Bone Marrow Endothelialprogenitor cells,BM/EPC)移植促进血流重建的效果,为进一步优化骨髓干细胞移植治疗肢体缺血提供理论基础。方法:获取Lewis大鼠骨髓单个核细胞,在体外培养分化为MSC和EPC。采用Lewis大鼠建立单侧后肢缺血模型。在模型建立后3天,将0.8mlD-Hanks液注入大鼠缺血侧后肢,为对照组(n=6);将8×106个骨髓MSC植入大鼠缺血侧后肢,为MSC组(n=6);将体外培养的8×106个EPC植入大鼠缺血侧后肢,为EPC组(n=6)。细胞移植后3周行缺血大鼠后肢动脉造影,检测缺血侧后肢侧支血管数;获取缺血侧后肢腓肠肌,分别行CD31和α-SMA免疫组化染色,计算毛细血管密度和小动脉密度。结果:MSC组与EPC组侧支血管数无显著性差异,二者均高于对照组;EPC组毛细血管密度明显高于MSC组,二者均高于对照组;MSC组与EPC组小动脉密度无显著性差异,二者均高于对照组。结论:骨髓间充质干细胞移植和内皮祖细胞移植均能够明显促进血流重建,而且骨髓间充质干细胞在治疗肢体缺血性疾病中的优势应该受到重视。     

Transplantation of bone marrow-derived mononuclear cells improves mechanical hyperalgesia, cold allodynia and nerve function in diabetic neuropathy

Relief from painful diabetic neuropathy is an important clinical issue. We have previously shown that the transplantation of cultured endothelial progenitor cells or mesenchymal stem cells ameliorated diabetic neuropathy in rats. In this study, we investigated whether transplantation of freshly isolated bone marrow-derived mononuclear cells (BM-MNCs) alleviates neuropathic pain in the early stage of streptozotocin-induced diabetic rats. Two weeks after STZ injection, BM-MNCs or vehicle saline were injected into the unilateral hind limb muscles. Mechanical hyperalgesia and cold allodynia in SD rats were measured as the number of foot withdrawals to von Frey hair stimulation and acetone application, respectively. Two weeks after the BM-MNC transplantation, sciatic motor nerve conduction velocity (MNCV), sensory nerve conduction velocity (SNCV), sciatic nerve blood flow (SNBF), mRNA expressions and histology were assessed. The BM-MNC transplantation significantly ameliorated mechanical hyperalgesia and cold allodynia in the BM-MNC-injected side. Furthermore, the slowed MNCV/SNCV and decreased SNBF in diabetic rats were improved in the BM-MNC-injected side. BM-MNC transplantation improved the decreased mRNA expression of NT-3 and number of microvessels in the hind limb muscles. There was no distinct effect of BM-MNC transplantation on the intraepidermal nerve fiber density. These results suggest that autologous transplantation of BM-MNCs could be a novel strategy for the treatment of painful diabetic neuropathy.     

Early endothelial progenitor cells in bone marrow are a biomarker of cell therapy success in patients with critical limb ischemia

Background aimsEndothelial progenitor cells (EPC) have been proposed for autologous angiogenic therapy. The objectives of this study were to quantify EPC in the peripheral blood and bone marrow mononuclear cells (BM-MNC) of patients with critical limb ischemia that had received BM-MNC as a cell therapy product, and to study the putative relationship between the presence of EPC and the process of neovascularization in toe or transmetatarsal amputation specimens.MethodsEarly and late endothelial progenitor cells (CFU-EC and ECFC) were cultivated and quantified according to published methods in peripheral blood and BM-MNC from patients with critical limb ischemia (CLI; n = 11) enrolled in the OPTIPEC trial (http://clinicaltrials.gov/ct2/show/NCT00377897) to receive BM-MNC as a cell therapy product.ResultsEight out of the 11 patients had undergone amputations. Three of the patients displayed a neoangiogenic process that was associated with a higher number of CFU-EC in BM-MNC, while CD3⁺, CFU-GM and CD34⁺ in BM-MNC, and EPC in peripheral blood, did not correlate with the appearance of newly formed vessels. As expected, circulating CFU-EC and ECFC counts were significantly lower in CLI patients compared with age-matched controls.ConclusionsIn patients with critical limb ischemia, EPC in peripheral blood were decreased compared with healthy individuals. However, in BM-MNC we found that relative numbers of CFU-EC could be used as an indicator to discriminate patients with neoangiogenic processes. These results need to be confirmed in a randomized study.     

Autologous bone marrow mononuclear cell infusion and hyperbaric oxygen therapy in type 2 diabetes mellitus: an open-label,randomized controlled clinical trial

Background aimsThe use of bone marrow mononuclear cells (BM-MNCs) has achieved great outcomes in clinical practice. We aim to evaluate the efficacy and safety of autologous BM-MNC infusion and hyperbaric oxygen therapy (HOT) in type 2 diabetes mellitus.MethodsThis single-center, randomized, open-label, controlled clinical trial with a factorial design included two phases. The patients received standard medical therapy in the run-in phase; in the treatment phase, patients with glycated hemoglobin of 7.5–9.5% were randomly assigned into four groups and underwent BM-MNC infusion along with HOT (BM-MNC+HOT group), BM-MNC infusion (BM-MNC group), HOT (HOT group) and standard medical therapy (control group), respectively. The area under the curve of C-peptide was recorded as a primary end point. Our research is registered at ClinicalTrials.gov (NCT00767260).ResultsA total of 80 patients completed the follow-up. At 12 months after treatment, the area under the curve of C-peptide (ng/mL per 180 min) of the BM-MNC+HOT group and the BM-MNC group were significantly improved (34.0% and 43.8% from the baseline, respectively). The changes were both significant compared with that in the control group, but no remarkable change was observed in the HOT group. Treatment-related adverse events were mild, including transient abdominal pain (n = 5) and punctual hemorrhage (n = 3).ConclusionsBM-MNC infusion for type 2 diabetes mellitus improves islet function and metabolic control, with mild adverse effects. HOT does not synergize with BM-MNC infusion.     

Long-term effects of bone marrow mononuclear cell transplantation on left ventricular function and remodeling in rats

This study was performed to evaluate the long-term effect on left ventricular function and remodeling in a rat model of bone marrow cell transplantation (BMT) into acute infarcted myocardium. After myocardial infarction was induced in inbred Lewis rats by left anterior descending artery ligation, the ischemic area was directly injected with saline, peripheral blood mononuclear cells (PB-MNCs) or bone marrow mononuclear cells (BM-MNCs). Cardiac function and structure were evaluated by echocardiography before the operation, and on day 1 and 2 months post-infarct. The collagen content, the number of vessels and the vasculogenesis were examined by histology and immunohistochemistry. We found at 2 months post-infarct, BMT significantly improved cardiac systolic function and recovered diastolic function. Transplantation of BM-MNCs, but not PB-MNCs, reversed remodeling and reduced collagen density. Vessel counts showed greater angiogenesis occurred in the animals transplanted with BM-MNCs. Furthermore, a vascular endothelial cell-specific marker was detected in the transplanted bone marrow cells. Our data suggest that BM-MNC transplantation results in long-term improvement in left ventricular function-especially diastolic function- and remodeling, possibly related with the reduction of the amount of the collagen and enhancement of neovascularization.     

Autologous bone marrow mononuclear cell therapy improves symptoms in patients with end-stage peripheral arterial disease and reduces inflammation-associated parameters

Background aimsThe purpose of this study was to evaluate the effect of autologous bone marrow mononuclear cells (BM-MNCs) on symptoms and perfusion indices in severely symptomatic patients with peripheral arterial disease (PAD) without further option for endovascular or surgical revascularization.MethodsOnly patients with severe symptomatic PAD (Fontaine class IIb-IV, Rutherford category 3–6) not amenable for revascularization were treated. Bone marrow from both cristae iliacae was harvested; MNCs were isolated by the Ficoll density-gradient method and transplanted by means of intra-arterial and intramuscular injection in the index limb. Functional (pain score, ulcer healing, maximum walking distance) and perfusion indices such as ankle-brachial-index and transcutaneous oxygen pressure were documented before and after BM-MNC therapy. Additionally, serum concentration of C-reactive protein and interleukin-6 were measured as markers of inflammation before and after BM-MNC treatment.ResultsSixteen consecutive patients (four women; mean age, 63.0 ± 13 years) were treated with a mean dose of 4.2 ± 2.2 × 10⁸ BM-MNCs. At 6 months' follow-up, ankle-brachial-index, transcutaneous oxygen pressure and maximum walking distance significantly increased, whereas C-reactive protein and interleukin-6 conversely decreased (P < 0.01 versus baseline values), resulting in 88% limb salvage, 75% pain reduction and 71% complete wound healing and/or reduction of ulcer size. One major and one minor amputation were performed, both in patients with Rutherford category 6.ConclusionsAutologous BM-MNC therapy in patients with end-stage PAD improves tissue perfusion indices and decreases markers of inflammation. If our observations could be confirmed by large-scale, randomized controlled trials, BM-MNC transplantation could become an alternative therapeutic option for patients with end-stage PAD.     

脂肪干细胞在周围神经损伤修复中的作用

周围神经损伤的修复是临床外科中的一个难题。尽管周围神经系统在损伤后具有内在的自我修复能力,但一般很难达到完全功能恢复,特别是近端的损伤或者大段的神经缺损。近年来,基于干细胞的细胞治疗为周围神经再生带来了曙光。大量研究表明干细胞可促进周围神经损伤的再生,然而其作用机制还不明确。为此,本文将对脂肪干细胞在周围神经损伤修复中作用包括向雪旺细胞分化、神经营养、血管形成、神经元保护、靶器官保护和免疫调节等作用进行归纳,并进一步探讨其潜在的作用机制。     

Peripheral Nerve Injuries Treatment: a Systematic Review

Patients with peripheral nerve injuries, especially severe injury, often face poor nerve regeneration and incompletely functional recovery, even after surgical nerve repair. Current researches have extensively focused on the new approaches for the treatment of peripheral nerve injuries. This review summarizes treatments of peripheral nerve injures, from conventional suturing method, to conduit coaptation with stem cell and growth factor, and review the developments of research and clinical application of these therapies.     

Current progress in use of adipose derived stem cells in peripheral nerve regeneration

Unlike central nervous system neurons; those in the peripheral nervous system have the potential for full regeneration after injury. Following injury, recovery is controlled by schwann cells which replicate and modulate the subsequent immune response. The level of nerve recovery is strongly linked to the severity of the initial injury despite the significant advancements in imaging and surgical techniques. Multiple experimental model shave been used with varying successes to augment the natural regenerative processes which occur following nerve injury. Stem cell therapy in peripheral nerve injury may be an important future intervention to improve the best attainable clinical results. In particular adipose derived stem cells(ADSCs) are multipotent mesenchymal stem cells similar to bone marrow derived stem cells, which are thought to have neurotrophic properties and the ability to differentiate into multiple lineages. They are ubiquitous within adipose tissue; they can form many structures resembling the mature adult peripheral nervous system. Following early in vitro work; multiple small and large animal in vivo models have been used in conjunction with conduits, autografts and allografts to successfully bridge the peripheral nerve gap. Some of the ADSC related neuroprotective and regenerative properties have been elucidated however much work remains before a model can be used successfully in human peripheral nerve injury(PNI). This review aims to provide a detailed overview of progress made in the use of ADSC in PNI, with discussion on the role of a tissue engineered approach for PNI repair.     

A Silk Fibroin/Collagen Nerve Scaffold Seeded with a Co-Culture of Schwann Cells and Adipose-Derived Stem Cells for Sciatic Nerve Regeneration

As a promising alternative to autologous nerve grafts, tissue-engineered nerve grafts have been extensively studied as a way to bridge peripheral nerve defects and guide nerve regeneration. The main difference between autogenous nerve grafts and tissue-engineered nerve grafts is the regenerative microenvironment formed by the grafts. If an appropriate regenerative microenvironment is provided, the repair of a peripheral nerve is feasible. In this study, to mimic the body’s natural regenerative microenvironment closely, we co-cultured Schwann cells (SCs) and adipose-derived stem cells (ADSCs) as seed cells and introduced them into a silk fibroin (SF)/collagen scaffold to construct a tissue-engineered nerve conduit (TENC). Twelve weeks after the three different grafts (plain SF/collagen scaffold, TENC, and autograft) were transplanted to bridge 1-cm long sciatic nerve defects in rats, a series of electrophysiological examinations and morphological analyses were performed to evaluate the effect of the tissue-engineered nerve grafts on peripheral nerve regeneration. The regenerative outcomes showed that the effect of treatment with TENCs was similar to that with autologous nerve grafts but superior to that with plain SF/collagen scaffolds. Meanwhile, no experimental animals had inflammation around the grafts. Based on this evidence, our findings suggest that the TENC we developed could improve the regenerative microenvironment and accelerate nerve regeneration compared to plain SF/collagen and may serve as a promising strategy for peripheral nerve repair.     

Supernatant of bone marrow mesenchymal stromal cells induces peripheral blood mononuclear cells possessing mesenchymal features

Increasing evidence shows that some cells from peripheral blood fibroblast-like mononuclear cells have the capacity to differentiate into mesenchymal lineages. However, the insufficiency of these cells in the circulation challenges the cell isolation and subsequently limits the clinical application of these cells. In the present study, the peripheral blood mononuclear cells (pbMNCs) were isolated from wound animals and treated with the supernatant of bone marrow mesenchymal stromal cells (bmMSCs). Results showed these pbMNCs were fibroblast-like, had stromal morphology, were negative for CD34 and CD45, but positive for Vimentin and Collagen I, and had the multipotency to differentiate into adipocytes and osteoblasts. We named these induced peripheral blood-derived mesenchymal stromal cells (ipbMSCs). Skin grafts in combination with ipbMSCs and collagen I were applied for wound healing, and results revealed ipbMSC exhibited similar potency and effectiveness in the promotion of wound healing to the bmMSCs. Hereafter, we speculate that the mixture of growth factors and chemokines secreted by bmMSCs may play an important roles in the induction of the proliferation and mesenchymal differentiation of mononuclear cells. Our results are clinically relevant because it provide a new method for the acquisition of MSCs which can be used as a candidate for the wound repair.