脊髓损伤的脐带间充质干细胞治疗的实验研究及临床不同治疗方案的疗效对比研究

发布时间：2018-01-07 18:43

  本文关键词：脊髓损伤的脐带间充质干细胞治疗的实验研究及临床不同治疗方案的疗效对比研究　出处：《河北医科大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 颈脊髓损伤 脐带间充质干细胞 小鼠 无骨折脱位 前路减压 后路减压 疗效比较

【摘要】：第一部分脐带间充质干细胞分化为神经细胞并修复脊髓损伤的研究目的:脊髓损伤(spinal cord injury,SCI)是由外伤诱发的一种脊柱外科创伤性疾病,表现为在损伤节段以下出现感觉、运动和自主神经功能障碍。国外流行病学调查显示,每年全球有13万新发脊髓损伤患者,且有超过250万患者正饱受着不同程度的脊髓损伤后遗症困扰,而这些SCI患者每年的医疗支出将超过60亿美元,给家庭及社会造成沉重负担。目前临床上用于治疗SCI的方法主要局限在药物、手术及各种物理干预,SCI的干细胞治疗仍停留在动物相关的基础研究层面,如骨髓基质干细胞移植、嗅鞘细胞移植、脐带血间充质干细胞移植、脐带华通胶来源间充质干细胞(mesenchymal stem cells,MSCs)移植、Nogo A疫苗移植等。由于脐带间充质干细胞具有来源广泛、易扩增、无伦理学方面的限制,已成为基础实验中用于治疗脊髓损伤的新热点和新方向。脐带血间充质干细胞在特定的环境诱导下可以分化为具有不同功能的组织细胞,并促进保护性因子的产生,且具有整合、调节、迁移和分泌等作用。当分化为神经细胞后可以促进神经组织的修复,改善脊髓损伤大鼠的神经功能。国内有学者采用移植人脐带血干细胞的方法来治疗大鼠脊髓损伤,虽获得了满意的疗效,但存在干细胞分化效率较低等问题。本研究旨在提高脐带间充质干细胞体外诱导分化为神经细胞的效率,并将其移植到脊髓损伤小鼠动物模型,为脊髓损伤这一难治性疾病提供了理论基础。方法:采集人脐带并采用植块法、胶原酶消化法及胶原酶与胰酶联合消化法分离人脐带间充质干细胞(Human umbilical cord mesenchymal stem cells,h UCMSCs),培养、扩增后,将Nurr-1转染到脐带间充质干细胞,并联合鸡尾酒因子(ATRA,GGF-2,b FGF,PDGF and forskolin)将脐带间充质干细胞诱导分化为神经细胞,利用免疫荧光技术检测神经细胞标志物表达情况。取昆明系小白鼠进行脊髓损伤模型的建立,并将分化后的细胞移植到脊髓损伤小鼠体内,免疫组化检测细胞移植后的定植情况,判断其治疗效果。结果:人脐带间充质干细胞经过诱导后,细胞开始收缩变圆,进而变得拉伸后成为多角样,在持续增长的过程中,细胞的胞体开始拉丝样出现神经细胞的突触样结构。诱导2周后,借助免疫荧光检测法可以检测出神经细胞特有标志物MAP-2和MBP存在,且经Nurr-1过表达的脐带间充质干细胞分化为神经细胞的效率最高。造模小鼠的脊髓损伤段结构遭到破坏,灰质内出现较多的神经元死亡,前角部分神经元尚存,其中部分神经元可见核固缩。h UCMSCs移植后细胞在损伤部位大量增殖,荧光显微镜下可检测到抗人细胞核阳性细胞,术后小鼠BBB评分逐渐提高,后肢运动功能逐渐改善。结论:经Nurr-1基因转染的脐带间充质干细胞在鸡尾酒因子的诱导下可以获得最好的分化及增殖,移植到受伤脊髓处可以在损伤部位增殖,同时小鼠的后肢功能获得明显改善,表明治疗脊髓损伤疗效确切。第二部分无骨折脱位型脊髓损伤手术与非手术治疗的临床疗效对比研究目的:无骨折脱位型脊髓损伤(cervical spinal cord injury without fracture or dislocation,CSCIWFD)是一类特殊类型的脊髓损伤,临床较多间,通常指颈部受到“较轻微”外力损伤后经过影像学检查,如X线、CT扫描等均未发现明显的颈椎骨折或脱位,但脊髓损伤症状相对较重的一类骨科疾病,大约占到颈脊髓损伤总数的23%。查阅文献发现,CSCIWFD的治疗方法主要分为两种,一种是采用大剂量糖皮质激素冲击、神经营养药物静点及高压氧治疗的保守治疗(非手术治疗),另一类是积极采用外科手段对脊髓进行充分减压的手术治疗。每种治疗方法都可以促使CSCIWFD患者获得一定程度的神经功能恢复,但手术干预作为一种有创性操作,其总体疗效是否优于非手术治疗呢?针对此问题,我们进行了较深入的临床对比研究。方法:回顾性分析2013年10月~2015年9月在门诊就诊和住院治疗的110例CSCIWFD患者的临床资料,其中92例获得了超过18个月的临床随访,根据患者所接受的治疗方法分为两组:A组(非手术组),38例,其中男23例,女15例,年龄19~63岁,平均42.1岁,均接受非手术治疗;B组(手术组),54例,其中男35例,女19例,年龄17~62岁,平均40.8岁,均接受手术治疗。记录手术治疗组患者的手术时间、术中出血量及并发症发生情况;两组分别在治疗前、治疗后1周、治疗后1个月及末次随访时采用日本骨科协会(Japanese Orthopaedic Association,JOA)脊髓损害评分表(17分法)评估治疗效果;末次随访时根据ASIA脊髓损伤分级标准对两组患者进行比较分析。结果:B组所有患者均顺利接受手术,其中10例患者接受颈前路椎间盘切除植骨内固定术,17例患者接受颈前路椎体次全切除内固定术,16例患者接受颈后路单开门椎管扩大成形钛板固定术,11例患者接受颈后路全椎板减压侧块螺钉固定术。平均手术时间175.2 min,平均出血量为210.6 ml。A组JOA评分由治疗前的7.0±1.8分提高至术后1周时的7.9±2.0分、1个月时的9.1±2.2分、3个月时的9.9±2.3分、6个月时的10.5±2.4分和末次随访时的11.2±2.9分,治疗前后比较差异具有显著性(P0.05),神经功能改善率为41.3%±9.6%;B组JOA评分由术前的6.7±1.6分提高至术后1周时的8.9±2.2分、1个月时的10.6±2.5分、3个月时的12.0±2.8分、6个月时的13.9±3.1分和末次随访时的14.5±3.6分,手术前后比较具有显著统计学意义(P0.05),神经功能改善率为70.2%±10.3%。末次随访时根据ASIA分级:A组获A级3例,B级7例,C级10例,D级14例,E级4例;B组获A级3例,B级6例,C级16例,D级17例,E级12例,两组与治疗前比较均具有显著性差异(P0.05)。A组发生消化道出血1例,肺部感染2例,褥疮2例,下肢深静脉血栓1例;B组发生伤口感染1例,硬膜外血肿2例,褥疮1例,硬膜撕裂导致脑脊液漏1例,两组并发症发生率比较差异无统计学意义(P0.05)。结论:与非手术方法相比,脊髓减压内固定术可更快、更好的促进无骨折脱位型脊髓损伤患者神经功能的恢复,且手术治疗不会增加围手术期并发症的发生率。第三部分前、后路减压内固定术治疗无骨折脱位型脊髓损伤疗效对比分析目的:目前国内外文献通过不同方法和侧面比较均已证实无骨折脱位型脊髓损伤(CSCIWFD)手术治疗效果要明显优于各种非手术疗法。因此,临床上一旦遇到CSCIWFD患者出现不同程度的神经功能受损时,就需要采取积极的、合适的手术入路及减压方法来重建或恢复椎管的通畅性。用于治疗CSCIWFD的手术方法很多,常见的有颈椎前路椎间盘摘除植骨内固定术、颈前路椎体次全切除植骨内固定术、颈后路单开门椎管扩大成形内固定术、颈后路全椎板减压侧块螺钉内固定术等。根据手术入路划分,上述术式可分为经前路和后路脊髓减压术,那么前、后路手术在总体疗效上是否存在差异呢,本研究针对这一问题进行了分组比较。方法:回顾性分析2013年09月~2015年12月在我院接受手术治疗的82例CSCIWFD患者的临床资料,根据患者接受的是前路减压还是后路减压将其分为两组:A组(前路组),45例,其中男26例,女19例,年龄22~62岁,平均41.6岁,均接受颈前路减压内固定术治疗;B组(后路组),37例,其中男24例,女13例,年龄19~64岁,平均42.9岁,均接受颈后路减压内固定术治疗。分别记录两组患者的手术时间、术中出血量及并发症发生情况;术前及随访过程中以日本骨科协会(Japanese Orthopaedic Association,JOA)脊髓损害评分表(17分法)评估治疗效果;末次随访时根据ASIA脊髓损伤分级标准对两组患者神经功能恢复恢复情况进行比较分析。结果:两组患者均顺利接受手术,A组平均手术时间(62.6±13.7)min,平均出血量(105.1±16.3)ml;B组手术时间(117.9±22.6)min,术中出血量(240.8±46.3)ml,组间比较差异均有统计学意义(P0.05)。A组JOA评分由治疗前的6.8±1.5分提高至术后2周时9.5±1.9分、3个月时的11.6±2.6分及末次随访时的13.5±3.1分,治疗前后比较差异具有显著性(P0.05),神经功能改善率为65.6%±8.3%,手术前后比较差异具有显著性(P0.05);B组JOA评分由术前的6.6±1.4分提高至术后2周时8.6±1.7分、术后3个月时的10.9±2.5分及末次随访时的13.3±3.4分,手术前后比较具有显著统计学意义(P0.05),神经功能改善率为64.5%±8.5%。术后2周时两组JOA评分比较具有显著性差异(P0.05)。但术后3个月及末次随访时两组比较,差异无统计学意义(P0.05)。末次随访时根据ASIA分级:A组获A级1例,B级6例,C级11例,D级18例,E级9例;B组获A级1例,B级4例,C级11例,D级13例,E级8例,与治疗前比较均具有显著性差异(P0.05),术后组间比较差异无统计学意义(P0.05)。A组发生脑脊液漏2例,喉返神经牵拉损伤1例;钛网下沉1例;B组发生伤口感染1例,液化坏死1例,硬膜外血肿2例,两组并发症发生率比较差异无统计学意义(P0.05)。结论:经前路和后路脊髓减压内固定术均可以有效促进患者神经功能的恢复,围手术期并发症的发生率基本相同,但颈前路手术创伤小,手术时间短,术中出血少,术前应根据患者脊髓受压节段、压迫源及髓内信号长度来进行科学、合理的术式选择。
[Abstract]:The first part of umbilical cord mesenchymal stem cells to differentiate into neural cells and Study on repair of spinal cord injury to spinal cord injury (spinal cord, injury, SCI) is a spinal injury induced by traumatic disease, appears to be felt in the following segmental injury, motor and autonomic dysfunction. Foreign epidemiological survey, every year around the world there are 13 million new cases of patients with spinal cord injury, and has more than 2 million 500 thousand patient is suffering from varying degrees of sequelae of spinal cord injury problems, and these patients with SCI annual medical expenditures will exceed $6 billion, resulting in a heavy burden to family and society. The current clinical treatment for SCI mainly confined to medicine, surgery and physical intervention. Stem cells for the treatment of SCI is still in the animal related basic research level, such as bone marrow stromal stem cell transplantation, transplantation of olfactory ensheathing cells, umbilical cord blood mesenchymal stem cells Transplantation of umbilical cord derived mesenchymal stem cells (mesenchymal stem cells, MSCs) Nogo transplantation, A vaccine transplantation. Because human umbilical cord mesenchymal stem cells have a wide range of sources, easy amplification, no ethical restrictions, has become a new focus and new direction for the treatment of spinal cord injury in experiments. Umbilical cord blood mesenchymal stem cells in specific environment induction can differentiate into tissue cells with different functions, and promote the protection of factors, and with the integration, regulation, migration and secretion function. When the differentiation of neural cells can promote repair of nerve tissue, improve the neurological function of rats with spinal cord injury. Some scholars in China by transplantation of human umbilical cord blood stem cells for treatment of spinal cord injury in rats, although obtained satisfactory curative effect, but the presence of stem cell differentiation efficiency is low. The purpose of this study is to improve the umbilical cord mesenchymal stem In vitro differentiation efficiency of nerve cells, and transplanted into the spinal cord injury animal model for spinal cord injury in mice, the refractory disease provides a theoretical basis. Methods: collecting and using human umbilical cord explant, collagenase and collagenase and trypsin digestion method combined with the separation of human umbilical cord mesenchymal stem (Human umbilical cord mesenchymal stem cell cells, H UCMSCs), cultured, amplified, Nurr-1 was transfected into human umbilical cord mesenchymal stem cells, and combined with cocktail (ATRA, GGF-2, B factor FGF, PDGF and forskolin) of umbilical cord mesenchymal stem cells to differentiate into neural cells, neural cell marker expression detection by using immunofluorescence technique. The establishment of the model of spinal cord injury in Kunming mice, and differentiated cells were transplanted into spinal cord injury in mice in vivo, immunohistochemical detection of colonization after cell transplantation, judgment The effect of the treatment. Results: human umbilical cord mesenchymal stem cells after induction, cells began to shrink and turned round, and then become stretched into angle, in the process of sustained growth, cells began to appear like drawing synapse like structure of nerve cells. After 2 weeks of induction, by immunofluorescence method can detect the existence of MAP-2 and MBP markers specific nerve cells, and the expression of Nurr-1 of human umbilical cord mesenchymal stem cells differentiation into neural cells. The highest efficiency mice model of spinal cord injury while the destruction of the structure in the gray matter more neuron death, anterior horn neurons surviving, some neurons in nuclei pyknotic.H after transplantation of UCMSCs cells in the injury site proliferated under fluorescence microscope to detect anti human nuclear positive cells, postoperative BBB score of mice increased gradually, hindlimb function gradually improved. Conclusion: Nu RR-1 gene transfection of human umbilical cord mesenchymal stem cells can be obtained in the induction of differentiation and proliferation of the best cocktail factor, can be transplanted into the injured spinal cord injury and proliferation in mice, the hind limb function was significantly improved, showed that the curative effect in the treatment of spinal cord injury without fracture exactly. The second part to a comparative study of the clinical effect of surgical treatment for spinal cord injury dislocation and non operation: spinal cord injury without radiographic abnormality (cervical spinal cord injury without fracture or dislocation, CSCIWFD) is a special type of spinal cord injury, in clinic, usually refers to the neck by minor damage after imaging, such as X-ray, cervical fracture or dislocation were obvious no CT scan, but a kind of Department of orthopedics disease symptoms of spinal cord injury is relatively heavy, accounting for about 23%. of the total number of cervical spinal cord injury consulting literature, CSC The treatment of IWFD is mainly divided into two types, one is the use of glucocorticoid, conservative treatment of neurotrophic drugs and intravenous hyperbaric oxygen treatment (non-surgical), another kind is the surgical treatment of active decompression of spinal cord by surgical means. Each treatment method can promote CSCIWFD patients recover a certain degree of nerve function, but surgical intervention as an invasive operation, the overall effect is better than that of non surgical treatment? To solve this problem, we conducted a clinical comparative study deeply. Methods: a retrospective analysis of October 2013 September ~2015 year in outpatient and inpatient treatment of 110 cases of CSCIWFD patients the clinical data of 92 cases received more than 18 months of clinical follow-up, according to the treatment methods, patients were divided into two groups: A group (non operation group), 38 cases, male 23 cases, female 15 cases, age 19~63 Years old, average 42.1 years old, underwent non operative treatment; group B (operation group), 54 cases, male 35 cases, female 19 cases, age 17~62 years old, average 40.8 years old, underwent surgical treatment. The operation time records of the surgical treatment of patients, intraoperative blood loss and complications in two groups; respectively before treatment, 1 weeks after treatment, 1 months after treatment and at the end of the follow-up by the Japanese Association for Department of orthopedics (Japanese Orthopaedic Association, JOA) spinal cord injury score (17 points) to evaluate the therapeutic effect; at the last follow-up according to the two groups of patients with ASIA spinal cord injury classification results: comparative analysis. All of the patients in group B underwent surgery, including 10 patients underwent anterior cervical discectomy and internal fixation, 17 patients underwent anterior cervical corpectomy and internal fixation, 16 patients underwent posterior open-door laminoplasty with titanium plate fixation, 11 cases of patients undergoing carotid 鍚庤矾鍏ㄦ�庢澘鍑忓帇渚у潡铻洪拤鍥哄畾鏈�.骞冲潎鎵嬫湳鏃堕棿175.2 min,骞冲潎鍑鸿��閲忎负210.6 ml.A缁凧OA璇勫垎鐢辨不鐤楀墠鐨，

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