骨髓单个核细胞移植治疗大鼠脑出血后脑损伤的实验研究

发布时间：2018-03-17 06:06

本文选题：大鼠　切入点：骨髓单个核细胞　出处：《郑州大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景与目的脑出血（intracerebral hemorrhage,ICH）其定义是原发性的非外伤性的脑实质内出血，是一种常见的急性脑血管疾病，它的特点是发病率、病死率和致残率都很高。然而目前尚缺乏有效的治疗手段。脑出血后脑损伤机制主要包括血肿压迫、脑水肿、炎症和细胞凋亡等多个方面，，其中炎症在脑出血的继发性脑损伤中起关键作用。因此，防治脑出血后炎症反应引起的继发性脑损伤，对于减少后遗神经功能损害，提高患者生存质量，具有非常重要的临床意义。近年来干细胞移植已成为神经科疾病治疗的热点。骨髓来源的单个核细胞(Bonemarrow mononuclear cells,BMMNCs)是由多种细胞组成的异质群，包含造血干细胞、间充质干细胞、内皮祖细胞等多种干、祖细胞成分。BMMNCs用于脑血管疾病治疗有许多优势，如获取分离方便，避免了免疫排斥反应，无需培养、可自体异体移植、无道德伦理学争议；已较多应用于心脏、下肢缺血性疾病方面上的动物实验和Ⅰ期临床研究且已证实其有效性和安全性。骨髓单个核细胞对脑出血是否有神经保护作用，到底发挥怎样的作用，目前未见相关报道。本研究将观察评估骨髓单个核细胞移植后脑出血大鼠的神经行为功能的恢复情况、脑水肿程度及神经元细胞凋亡的情况，并检测移植后在出血灶侧周围炎症细胞即小胶质细胞活化和中性粒细胞浸润情况；进一步探讨骨髓单个核细胞对脑出血后继发性脑损伤治疗的效果，及其在继发性脑损伤炎症反应中可能治疗机制。方法应用立体定位技术将Ⅳ型胶原酶注入大鼠尾状核纹状体内制作实验性脑出血模型，假手术组在同等条件下注射等量生理盐水。体重250-300g的雄性SD大鼠建模后按随机化原则分为假手术组、ICH组、PBS组、BMMNCs移植组；对用于提取细胞的大鼠连续腹腔注射Brdu,14天后收集大鼠骨髓细胞，用Fi-coll密度梯度离心法提取、分离、纯化大鼠骨髓单个核细胞；Brdu标记BMMNCs用以体内示踪。于ICH6h后经股静脉输注3×107BMMNCs或等量PBS。采用改良神经功能损伤评分（modi-fied neurological severity score,mNSS）评估脑出血后1d、3d、7d、14d神经行为功能改善情况；干湿重法测定血肿周围脑组织含水量；FJC(Fluoro-JadeC)染色法检测出血灶周围神经元细胞凋亡情况；免疫荧光染色法检测脑出血侧小胶质细胞（IBA-1+）活化及中性粒细胞浸润(MPO+)情况；同时对标记的Brdu行免疫荧光染色观察BMMNCs脑内的分布。结果 1.假手术组在术后1d、3d、7d、14d各个时间点均未见明显神经功能缺损症状，各时间点mNSS评分均为0分，ICH组和PBS组神经功能缺损症状在术后1d时达高峰，7d时均可见较明显的恢复，14d时有进一步的恢复。BMMNCs组神经功能评分在7d、14d各个时间点较ICH组和PBS组明显降低，差异有统计学意义(P0.05)；在脑出血后3d，BMMNCs移植组的大鼠脑组织含水量明显降低，及神经元凋亡数目也显著降低，与ICH组及PBS组比较，差异有统计学意义(P0.05)； 2.在脑出血后1d，BMMNCs移植组可见Brdu阳性细胞散在分布于出血灶周围，3d时达高峰，7d时呈下降趋势；ICH组及PBS组在出血灶周围无Brdu阳性细胞。脑出血后3d，BMMNCs移植组出血侧活化增殖的小胶质细胞数和浸润的中性粒细胞数均较ICH组与PBS组明显减少，差异有统计学意义(P0.01)。结论 1．移植的骨髓单个核细胞能够迁移、归巢至脑出血灶周围区域并存活，可显著减轻出血后脑水肿程度，减少神经元细胞凋亡，显著促进神经行为功能的恢复。 2．骨髓单个核细胞移植可减轻出血侧小胶质细胞的增殖、活化和中性粒细胞的浸润。 3．骨髓单个核细胞对实验性大鼠脑出血的治疗作用，其机制可能与其抑制脑出血后继发性脑损伤中相关的炎症反应有关。
[Abstract]:Background and purpose
Cerebral hemorrhage (intracerebral hemorrhage ICH) is defined as the primary non traumatic intracerebral hemorrhage is a common acute cerebrovascular disease, which is characteristic of the incidence, fatality rate and mutilation rate are very high. However, there is still lack of effective treatment. The cerebral blood brain mechanism damage including hematoma oppression, brain edema, inflammation and apoptosis in many aspects, including inflammation plays a key role in the secondary brain injury of intracerebral hemorrhage. Therefore, the secondary brain injury caused by inflammatory reaction after cerebral hemorrhage prevention, to reduce the sequela of nervous function damage, improve the quality of life of patients, with clinical significance very important. In recent years, stem cell transplantation has become a hot topic in treatment of neurological diseases. Mononuclear cells derived from bone marrow (Bonemarrow mononuclear cells, BMMNCs) is composed of a variety of cells including hematopoietic stem cell heterogeneity group. , a variety of mesenchymal stem cells, endothelial progenitor cells stem progenitor cells, components of.BMMNCs for the treatment of cerebral vascular disease has many advantages, such as convenient for separation, avoid immunological rejection, without culture, autologous transplantation, no moral and ethical disputes; has more should be used for heart, hand on the ischemic disease of lower extremity animal experiment and clinical study of stage I and confirmed its effectiveness and safety. Bone marrow mononuclear cells have neuroprotective effect on cerebral hemorrhage, in the end how to play the role of, at present has not been reported. This study will evaluate the neurobehavioral function of bone marrow mononuclear cells transplantation cerebral hemorrhage rat brain recover. The degree of edema and neuronal apoptosis, and detected after transplantation in inflammatory cells around the hemorrhage side that microglia activation and neutrophil infiltration; further study of bone The effect of medullary mononuclear cells on secondary brain injury after intracerebral hemorrhage and the possible mechanism of treatment in secondary brain injury.
Method
The application of stereo positioning technology of type IV collagenase injected into rat caudate nucleus striatum produced in experimental intracerebral hemorrhage model, sham operation group injected under the same conditions of saline. Male SD rats weighing 250-300g modeling after randomly divided into sham operation group, ICH group, PBS group, BMMNCs group of transplantation; for the extraction of cells in rats by intraperitoneal injection of Brdu, collected 14 days after rat bone marrow cells by Fi-coll density gradient centrifugation extraction, separation, purification of rat bone marrow mononuclear cells; Brdu marker for BMMNCs. After ICH6h in vivo via femoral vein infusion of 3 x 107BMMNCs or equivalent PBS. with improved neural function the injury score (modi-fied neurological severity score, mNSS) assessment of cerebral hemorrhage after 1D, 3D, 7d, 14d neurobehavioral function improvement; Determination of perihematomal brain tissue water content of dry wet weight method; FJC (Fluoro-JadeC) staining. Neuronal apoptosis was detected around the hemorrhagic foci. Microglia (IBA-1+) activation and neutrophil infiltration (MPO+) were detected by immunofluorescence staining. Meanwhile, labeled Brdu was used to detect the distribution of BMMNCs brain in immunofluorescence staining.
Result
1. in the sham surgery group after operation, 1D, 3D, 7d, 14d each time point showed no obvious symptoms of neurological deficits, each time point mNSS score was 0 points, ICH group and PBS group of neurological symptoms in the postoperative 1D reached the peak, 7d showed obvious recovery, when 14d recovery.BMMNCs group further neurological score in 7d, 14d each time point were significantly lower than that in ICH group and PBS group decreased, the difference was statistically significant (P0.05); at 3D after cerebral hemorrhage, brain tissue of rats with BMMNCs transplantation group water content decreased significantly, and the number of apoptotic neurons were significantly decreased, compared with group ICH and in PBS group, the difference was statistically significant (P0.05);
In 2. 1D after ICH, BMMNCs transplantation group showed Brdu positive cells scattered in the bleeding around, reached a peak at 3D, 7d decreased; ICH group and PBS group of Brdu positive cells in the cerebral hemorrhage bleeding around. After 3D, the number of neutrophil number of microglial cell transplantation group the activation and proliferation and infiltration of BMMNCs were lower than ICH group and PBS group were significantly reduced, the difference was statistically significant (P0.01).
conclusion
1. transplantation of bone marrow mononuclear cells can migrate and migrate to the surrounding areas of cerebral hemorrhage and survive. It can significantly reduce the degree of cerebral edema after hemorrhage, reduce neuronal apoptosis, and significantly promote the recovery of neurobehavioral function.
2. transplantation of bone marrow mononuclear cells can reduce the proliferation, activation and infiltration of neutrophils in the haemorrhagic microglia.
3. the therapeutic effect of bone marrow mononuclear cells on experimental intracerebral hemorrhage in rats may be related to inhibiting inflammatory reaction related to secondary brain injury after intracerebral hemorrhage.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.34

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