静脉注射骨髓间质干细胞对脊髓损伤修复作用的实验研究

目的探讨静脉注射大鼠骨髓间质干细胞(MSCs)对脊髓损伤后神经修复和功能恢复的影响。方法32只SD大鼠,体重约300g,雌雄不限。体外分离、培养、纯化MSCs,应用流式细胞技术检测MSCs表面细胞标志CD34、CD45、CD29、CD90。根据改良Allen法制备大鼠脊髓损伤模型。暴露T10段脊髓,将一直径为3mm的圆形薄铜垫片置于T10段脊髓表面,以重量为10g的砝码,从5cm高度自由坠落打击该垫片,造成T10段脊髓冲击伤,损伤组24只,假手术组8只。模型建立后24h,损伤组随机分为实验组14只,对照组10只。实验组及假手术组经尾静脉注射Brdu标记的MSCs,对照组经静脉注射PBS。损伤后24h、注射MSCs后1、3、5周评价各组大鼠的神经功能状况,并检测MSCs在体内迁移、存活以及分化情况。结果细胞CD34、CD45阴性表达,CD29、CD90阳性表达。实验组运动功能改善,BBB评分高于对照组(P<0.05)。注射的MSCs在宿主损伤脊髓中聚集并存活,注射MSCs3～5周后部分细胞表达微管相关蛋白2(MAP2)、神经元特异性烯醇化酶(NSE)染色的Brdu阳性细胞。结论MSCs经静脉注射后可向脊髓损伤处聚集并存活,促进神经修复及神经功能的恢复。     

Migration and distribution of bone marrow stromal cells in injured spinal cord with different transplantation techniques

Objective： To study the regularity of migration and distribution of bone marrow stromal cells （BMSCs） in injured spinal cord with intradural space transplantation. Methods： Forty Wistar rats were randomly assigned into 4 groups. The spinal cord injury model was prepared according to the modified Allen method. BMSCs were labeled by CM-Dil. And 5.0x 10^6 cells were transplanted by different channels including intraventricular injection （Group A）,injured spinal cord intrathecally injection （Group B）, remote intrathecally injection at the L3-L4 level （Group C）, and intravenous injection （Group D）. Spinal cord was dissected at 24 hours, 1, 2, 3 and 4 weeks after transplantation. Sections of 4 μm were cut on a cryostat and observed under fluorescence microscopy. Results： No fluorescence was observed 24 hours aftertransplantation in spinal cord injury parenchyma except Group B. One week later, BMSCs in Groups A and C began to migrate to the injured parenchyma; 2-4 weeks later, BMSCs penetrated into the injured parenchyma except Group D. The number of BMSCs decreased at 3-4 weeks after transplantation. The number of cells in Group B decreased faster than that of Groups A and C. Conclusions： BMSCs transplanted through intraventricular injection, injured spinal cord intrathecally injection and remote intrathecal injection could migrate to the injured parenchyma of spinal cord effectively. The number of BMSCs migrated into injured spinal cord parenchyma is rare by intravenous injection.     

骨髓基质细胞移植后在损伤脊髓中的迁移和分布

目的观察骨髓基质细胞（BMSCs）移植后在损伤脊髓中的迁移和分布。方法40只Wistar大鼠随机分成4组，以改良Allen法制备脊髓损伤模型，B、C、D组分别在损伤后立即、24h、7d移植经CM—Dil荧光染料标记的第三代BMSCs，A组注射等量PBS，分别于BMSCs移植后24h、1、2．3、4周取损伤脊髓段作冰冻切片，倒置荧光显微镜下观测移植的BMSCs在损伤脊髓中的迁移和分布。结果移植后1周，各组移植的BMSCs基本位于硬脊膜下，移植后2—4周，BMSCs迁移、聚集在损伤脊髓的中心，D组损伤脊髓内BMSCs数量明显多于B、C组。结论（1）移植的BMSCs迁移聚集在损伤脊髓的中心；（2）在脊髓损伤后7d移植BMSCs较损伤后立即或1d后移植更利于BMSCs在损伤区的聚集。     

骨髓基质细胞经不同移植途径在损伤脊髓中的迁移

目的 观察骨髓基质细胞(BMSCs)经不同移植途径在损伤脊髓中的迁移和分布规律.方法 40只Wistar大鼠随机分成4组,以改良Allen法制备脊髓损伤模型.第3代BMSCs经CM-Dil荧光染料标记后按不同途径移植.A组:脊髓损伤后行第四脑室注射移植;B组:脊髓损伤后行损伤区蛛网膜下注射移植;C组:脊髓损伤后行损伤区远端蛛网膜下注射移植;D组:脊髓损伤后行股静脉注射移植.分别于移植后24 h、1、2、3、4周取损伤脊髓段作冰冻切片,倒置荧光显微镜下观测移植的BMSCs在损伤脊髓中心的聚集情况.结果 移植后24 h:除B组外脊髓损伤区未发现红色荧光;移植后1周,A、C组移植的BMSCs开始向脊髓损伤区迁移;移植后2周,除D组外,各组BMSCs迁移、聚集在损伤脊髓的中心;移植后3-4周,脊髓损伤区BMSCs数量减少,B组减少速度更快.结论 经第4脑室、硬脊膜下移植的BMSCs能迁移聚集在损伤脊髓的中心,经静脉移植的BMSCs极少迁移到损伤脊髓的中心.     

多次移植骨髓基质干细胞有利于脊髓损伤修复

[目的] 研究多次经蛛网膜下腔移植骨髓基质干细胞对Wistar大鼠脊髓损伤的功能修复作用.[方法] 骨髓基质干细胞经体外分离、培养并用Hoecst33342标记.按照Allen方法把60只在T_(10)-T_(12)平面损伤的Wistar大鼠随机分4组,A、B、C、D组(对照组).在损伤平面蛛网膜下腔中段放置一硅胶管,在7 d后,注入1×10~6个骨髓基质干细胞.在2、3、5、7、12周荧光显微镜、免疫组化检测骨髓基质干细胞在损伤段脊髓的存活、分布、分化情况并作计数观察.使用BBB评分观测后肢功能恢复.[结果] 移植后7～14 d骨髓基质干细胞达到高峰,在7 d后表达巢蛋白及神经丝蛋白阳性.随时间的延长,移植在损伤部位的骨髓基质干细胞数量及神经元样细胞均有减少,但移植3次的细胞数减少速度较其他两组慢.移植3次组大鼠的BBB评分较移植1、2次组有明显的提高,P＜0.01,有统计学意义.[结论] 多次移植骨髓基质干细胞更有利于脊髓损伤的恢复.     

骨髓基质细胞体外分化移植治疗大鼠脊髓损伤的初步研究

[目的]探讨大鼠骨髓基质细胞体外分化为神经干细胞后移植治疗大鼠脊髓损伤的可行性。[方法]骨髓基质细胞经培养及定向分化为神经干细胞，后者由5-溴脱氧尿嘧啶核苷法标记，制备大鼠脊髓损伤模型，伤后第9d移植神经干细胞，实验分组：细胞移植组、PBS填充组、正常对照组。应用组化法观察移植细胞是否存活，取材前24h显露坐骨神经，行辣根过氧化物酶逆行示踪法观察脊髓损伤处的修复重建。[结果]骨髓基质细胞在定向分化为神经干细胞后标记并移植于脊髓损伤区，标记的阳性细胞可在受体脊髓内检测到，辣根示踪技术显示细胞移植组较PBS填充组阳性细胞明显增多，差别有统计学意义。[结论]大鼠骨髓基质细胞在体外分化为神经干细胞后移植于脊髓损伤区，移植细胞可以存活，并参与脊髓损伤处神经传导通路的结构重建。     

骨髓间充质干细胞治疗脊髓损伤的研究进展

骨髓间充质干细胞以其良好的增殖和多向分化能力,取材方便,易于分离培养和自体移植无免疫源性等特征而成为细胞移植治疗脊髓损伤研究的重点之一。目前已证实蛛网膜下腔注射是最理想的骨髓间充质干细胞治疗途径。早期临床应用骨髓间充质干细胞移植是安全的,对脊髓损伤的修复作用是肯定的,其作用机制可能与骨髓间充质干细胞的替代作用、神经营养作用、抑制免疫反应及促进轴突再生等有关。     

骨髓间充质干细胞移植治疗脊髓损伤的研究进展

脊髓损伤（spinal cord injury,SCI）会导致损伤平面以下肢体感觉和运动功能障碍。其致残率较高,在给患者本人带来身心伤害的同时还会对其家庭以及社会造成巨大的经济负担,严重影响患者的日常生活及工作能力。流行病学调查显示,全球每年因外伤性SCI而丧失肢体感觉、运动功能的人数高达60多万。目前,临床上治疗SCI的主要方式包括药物（如大剂量类固醇激素冲击）、手术、物理及康复训练等。SCI后神经的自我修复能力有限,依靠药物和外科手术的传统治疗方式多局限在病症的缓解,疗效难以令人满意,无法从根本上解决问题。Barinaga认为SCI后运动功能的丧失源于神经细胞的凋亡。目前已有一些早期临床试验证明了干细胞移植治疗SCI的可行性,使受损神经细胞、组织得到修复或替换成为可能。近年来研究发现骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）可以分化为神经元样细胞,可使轴突再生、突触重建,进而恢复受损脊髓功能。BMSCs移植为SCI的治疗提供了新思路,被认为是创伤性SCI的有效治疗方法。BMSCs移植治疗SCI具有多样性,即BMSCs可以通过多种不同的应用方法,以不同的机制发挥治疗SCI的作用。笔者就BMSCs移植治疗对SCI的研究进展予以综述,以期为临床治疗SCI提供理论参考。     

Analysis of allogeneic and syngeneic bone marrow stromal cell graft survival in the spinal cord

Bone marrow stromal cells (MSC) are attractive candidates for developing cell therapies for central nervous system (CNS) disorders. They can be easily obtained, expanded in culture, and promote modest functional recovery following transplantation into animal models of injured or degenerative CNS. While syngeneic MSC grafts can be used efficiently, achieving long-term survival of allogeneic MSC grafts has been a challenge. We hypothesize that improved graft survival will enhance the functional recovery promoted by MSC. To improve MSC graft survival, we tested two dosages of the immune suppressant cyclosporin A (CsA) in an allogeneic model. Syngeneic transplantation of MSC where cells survive well without immune suppression was used as a control. Sprague-Dawley rats treated with standard dose (n = 12) or high-dose (n = 12) CsA served as allogeneic hosts; Fisher 344 rats (n = 12) served as syngeneic hosts. MSC were derived from transgenic Fisher 344 rats expressing human placental alkaline phosphatase and were grafted into cervical spinal cord. Animals treated with standard dose CsA showed significant decreases in allograft size 4 weeks posttransplantation; high CsA doses yielded significantly better graft survival 4 and 8 weeks posttransplantation compared to standard CsA. As expected, syngeneic MSC transplants showed good graft survival after 4 and 8 weeks. To investigate MSC graft elimination, we analyzed immune cell infiltration and cell death. Macrophage infiltration was high after 1 week in all groups. After 4 weeks, high-dose CsA and syngeneic animals showed significant reductions in macrophages at the graft site. Few T lymphocytes were detected in any group at each time point. Cell death occurred throughout the study; however, little apoptotic activity was detected. Histochemical analysis revealed no evidence of neural differentiation. These results indicate that allogeneic transplantation with appropriate immune suppression permits long-term survival of MSC; thus, both allogeneic and syngeneic strategies could be utilized in devising novel therapies for CNS injury.     

骨髓基质干细胞在损伤脊髓内向少突胶质细胞定向分化的研究

目的 探讨移植骨髓基质干细胞(BMSCs)在损伤脊髓内向少突胶质细胞分化的可能性.方法 应用低温包埋免疫电镜技术观察迁移在损伤脊髓内1、3、5周的移植BMSCs超微结构,应用免疫荧光标记和激光共聚焦技术观察迁移在损伤脊髓内1、3、5周的移植BMSCs表达髓磷脂碱性蛋白(MBP)、髓鞘蛋白前脂蛋白(PLP)的情况.结果 移植1周,迁移在损伤脊髓白质内的BMSCs体积较小,突起少,细胞核较小,核仁清楚,染色质分布尚均匀,线粒体、粗面内质网和核糖体等细胞器发达,具有少突胶质细胞的超微结构特点;移植3周和5周,迁移在损伤脊髓白质内的BMSCs具有成熟少突胶质细胞的超微结构特点,并形成髓鞘样结构.移植1周后,迁移在损伤脊髓白质内的BMSCs开始表达MBP和PLP;移植3周和5周后,迁移在损伤脊髓白质内的BMSCs继续表达MBP和PLP.结论 移植BMSCs在损伤脊髓内可能会部分分化为功能性少突胶质细胞.     

Bone marrow stromal cell transplantation preserves gammaaminobutyric acid receptor function in the injured spinal cord

A surprising shortage of information surrounds the mechanisms by which bone marrow stromal cells (BMSC) restore lost neurologic functions when transplanted into the damaged central nervous system. In the present study, we sought to elucidate whether BMSCs express the neuron-specific gamma-aminobutyric acid (GABA) receptor when transplanted into injured spinal cord. To examine this, we harvested and cultured rat femoral BMSCs. We then subjected Sprague-Dawley rats to thoracic spinal cord injury (SCI) with a pneumatic impact device. Fluorescence-labeled BMSCs (n = 7) were transplanted stereotactically or the vehicle in which these cells were cultured (n = 4) was introduced stereotactically into the rostral site of SCI at 7 days after injury. We evaluated GABA receptor function by measuring the binding potential for 125I-iomazenil (125I-IMZ) through in vitro autoradiography at 4 weeks after BMSC transplantation and simultaneously examined the fate of the transplanted BMSCs by immunocytochemistry. We found that the transplanted BMSC migrated toward the core of the injury and were densely distributed in the marginal region at 4 weeks after transplantation. BMSC transplantation significantly increased the binding potential for 125I-IMZ (p = 0.0376) and increased the number of GABA receptor-positive cells (p = 0.0077) in the marginal region of the injury site. Some of the transplanted BMSCs were positive for microtubule-associated protein-2 and the alpha1 subunit of GABA(A) receptor in the region of injury. These findings suggest that BMSCs have the potential to support the survival of neurons in the marginal region of SCI and can partly differentiate into neurons, regenerating spinal cord tissue at the site of injury.     

骨髓基质细胞经CM-Dil标记后的活性及移植到损伤脊髓的示踪观察

目的 观察骨髓基质细胞(BMSCs)经CM-Dil荧光染料标记后的生物活性及移植后在损伤脊髓中的迁移和分布.方法 取Wistar大鼠的第3代BMSCs消化后分两组,实验组和对照组,实验组以CM.DiI标记,另一组正常细胞不标记,测定生长曲线.以改良Allen法制备脊髓损伤模型,将CM.DiI标记的BMSCs通过蛛网膜下腔注射移植,分别于移植后24 h、1、2、3、4周取损伤脊髓段作冰冻切片,倒置荧光显微镜下观测移植的BMSCs在损伤脊髓中的迁移和分布规律.结果 BMSCs经CM-Dil荧光染料标记成功率100%,实验组和对照组的生长曲线基本吻合;移植后24 h,脊髓损伤区未见标记细胞,移植后1周,标记的BMSCs开始迁移到损伤区硬脊膜下,移植后2～3周.BMSCs迁移、聚集在损伤脊髓的中心,移植后4周,仍有标记细胞聚集在脊髓损伤中心.结论 (1)BMSCs经CM-Dil荧光染料标记后的生物活性保持正常;(2)CM-Dil标记的BMSCs移植后能迁移聚集在损伤脊髓的中心,CM-Dil在活体内至少能维持4周     

骨髓基质干细胞移植联合应用G-CSF对大鼠脊髓损伤修复的影响

[目的]探讨骨髓基质干细胞(bone marrow stromal stem cells,BMSCs)移植联合应用粒细胞集落刺激因子(granulocyt colony stimulating factor,G-CSF)对大鼠脊髓损伤的治疗修复作用。[方法]48只Wistar大鼠采用改良的Allen’s装置在T11水平制成大鼠脊髓损伤模型,随机分成4组(n=12):A组为BMSCs移植联用G-CSF组,B、C组为单纯BMSCs移植组和G-CSF治疗组,D组为损伤对照组。术后1、2、3、4周采用Basso-Beattie-Bresnahan(BBB)评分评价大鼠后肢神经功能恢复情况,术后4周取材HE染色观察,免疫荧光染色检测神经元特异性烯醇化酶(neuron-specific enolase,NSE)、神经胶质纤维酸性蛋白质(glial fibrillary acidic protein,GFAP)和神经丝蛋白200(neurofilament 200,NF-200)的表达变化。[结果]术后1~4周,A组评分均明显高于其他3组,D组最低,差异均有统计学意义(P0.01);B组术后3、4周高于C组(P0.01)。HE与免疫荧光染色显示,BMSCs联合应用G-CSF对脊髓损伤的修复作用最好,D组恢复最差,B、C组介于A、D组之间。A组在脊髓损伤区及周缘NSE、NF 200阳性细胞均较B组多,C组未见明显的NSE、NF 200阳性细胞,但在损伤周缘有大量的GFAP阳性细胞,并向脊髓损伤空腔内延伸;D组损伤区看见大量结构杂乱的GFAP阳性细胞,瘢痕组织形成明显,损伤区未见明显的脊髓再生现象及NSE、NF-200阳性细胞。[结论]BMSCs移植能在脊髓损伤周围存活并分化;移植联用G-CSF更能促进神经修复及功能的恢复;二者联用具有协同作用。     

骨髓间充质干细胞移植治疗脊髓损伤研究

20世纪90年代成功分离出骨髓间充质干细胞(BMSC)并移植用于治疗急性脊髓损伤动物模型,引起了广泛关注。BMSC移植治疗脊髓损伤的实验研究主要有单独移植、联合支架移植、联合细胞移植、联合药物移植及转基因干细胞移植等。该文就BMSC生物学特性、BMSC移植治疗脊髓损伤研究现状及进展作一简要综述。     

Combination of bone marrow stromal cell transplantation with mobilization by granulocyte-colony stimulating factor promotes functional recovery after spinal cord transection

Purpose  

Spinal cord injury (SCI) results in severe neurological deficit. However, the functional recovery following SCI is very poor due to the neural lost and limited axonal regeneration. To date, there was no effective treatment. Recent studies have shown that bone marrow stromal cells (BMSCs) transplantated into the central nervous system (CNS) can survive and differentiate into neuronal-like cells. Additionally, granulocyte colony-stimulating factor (G-CSF) can mobilize hematopoietic stem cells and inhibit neural cell apoptosis. Thus, we aimed to evaluate the combined effect of BMSC transplantation and G-CSF administration on rats with traverse spinal cord injury.