诱导多能性干细胞移植减轻实验性脑出血大鼠急性炎症反应和神经细胞损伤

发布时间：2018-04-29 13:17

本文选题：诱导多能性干细胞 + 脑出血　；参考：《郑州大学》2014年硕士论文

【摘要】：出血性脑卒中（ICH）是一种严重的脑卒中亚型，它具有高发病率、死亡率、致残率和复发率的特点，且目前尚缺乏有效的治疗措施。由脑出血引发的大脑损伤的机制较复杂，而其最初的伤害（原发性损害）主要体现在血肿在脑实质内的形成和扩张；其次继发性的损害是由三种相互交织的级联反应引起的，包括颅内炎症反应，红细胞裂解和凝血酶的产生等，继发性损害可通过脑水肿、神经细胞凋亡和退化引起严重的神经功能障碍，甚至死亡。越来越多的证据表明，炎症在脑出血引起的继发性脑损害中起到极其关键的作用，并提示炎症通路是具有应用前景的脑出血治疗靶点。干细胞治疗在实验性ICH模型的神经修复等研究中得到广泛应用。诱导多能干细胞（iPS细胞/iPSCs）是一种新的干细胞群，该干细胞群的获得是通过导入特定的转录因子将终末分化的体细胞重编程为多能干细胞，并且因为避免了伦理性或免疫原性的问题被视为有应用前景的临床细胞治疗候选。最近的研究表明，，它可减少在缺血性脑卒中和人类疾病的其他动物模型中的炎症性损害。不过，目前还不清楚iPSCs的治疗是否有可能在出血性脑卒中后的急性先天性炎症反应中发挥抗炎作用。在本项研究中，我们在胶原酶诱发的大鼠脑出血性卒中模型中移植iPS细胞，并探讨其对脑急性炎症、神经损伤和恢复的治疗效果。目的: 在胶原酶诱发的大鼠脑出血性卒中模型中，探讨诱导多能干细胞移植对脑急性炎症、神经损伤和恢复的治疗效果。方法: 1.选取80只SD大鼠，20只立体注射无菌生理盐水为假手术组（Sham组）；60只立体定位注射Ⅶ型胶原酶于左侧纹状体，建立脑出血模型；造模后6小时后随机分成两组分别原位注射诱导多能干细胞(5μl PBS中悬浮1×106个iPSCs细胞)和磷酸缓冲液(5μl PBS），即移植组（iPSCs组）与模型组（PBS组）； 2.建模后第48小时，脑血肿周围组织荧光定量PCR检测两组大鼠血肿周围炎症因子水平；第3天，免疫荧光染色检测血肿周围的炎症细胞与神经凋亡细胞，并且采用干湿重法与磁共振成像分析来测量两组大鼠脑含水量；采用改良肢体平衡试验MLPT在建模后第0,1,3,7,14,28以及42天分别对两组大鼠进行行为学功能评价； 3.第42天，尼氏染色观察脑出血后神经元损伤修复效果以及免疫荧光染色观察胶质疤痕厚度和探测移植的干细胞。结果: 1.与模型组相比，移植组促进大鼠神经功能恢复，MLPT评分从第14天起至42天均高于模型组（P0.01）； 2.移植组脑血肿旁组织中促炎性因子白介素1β（IL-1β）、IL-6和肿瘤坏死因子α（TNF-α）显著下降，而抗炎性因子IL-10升高了2倍。出血半球的脑含水量较模型组低（P0.05），非出血半球无差别；中性粒细胞和小胶质细胞较模型组均有不同程度的下降（P0.05），以TUNEL和活化的半胱天冬蛋白酶3（active caspase-3）为标记的坏死与凋亡的神经细胞在移植组中细胞数低于模型组（P0.05）。 3.移植的干细胞存活于血肿周围；与模型组相比，移植组的神经细胞恢复较好（P0.05），胶质疤痕的厚度较低（P0.01）。结论: 移植诱导多能性干细胞可能通过减轻实验性脑出血急性炎症反应和损伤促进神经功能恢复。
[Abstract]:Hemorrhagic stroke (ICH) is a serious subtype of cerebral apoplexy. It has the characteristics of high incidence, mortality, disability and recurrence rate, and it is still lack of effective treatment. The mechanism of brain injury caused by cerebral hemorrhage is complex, and its initial injury (primary damage) is mainly reflected in the formation and formation of hematoma in the brain parenchyma. The secondary damage is caused by three interlaced cascade reactions, including intracranial inflammation, erythrocyte lysis, and the production of thrombin. Secondary damage can be caused by brain edema, nerve cell apoptosis and degeneration, causing severe neurological dysfunction and death. More and more evidence suggests that inflammation is in the brain. It plays an important role in secondary brain damage caused by blood. It also suggests that inflammatory pathway is a promising therapeutic target for intracerebral hemorrhage.
Stem cell therapy is widely used in the research of neural repair of experimental ICH models. Induced pluripotent stem cells (iPS cell /iPSCs) is a new stem cell group. The stem cell group is obtained by reprogramming the end differentiated somatic cells by introducing specific transcription factors into pluripotent stem cells, and because it avoids ethical or exemption. The problem of Phytophthora is considered as a promising candidate for clinical cell therapy. Recent studies have shown that it can reduce inflammatory damage in ischemic stroke and other animal models of human disease. However, it is not clear whether the treatment of iPSCs is likely to occur in acute congenital inflammatory responses after hemorrhagic stroke. In this study, we transplanted iPS cells in the rat model of cerebral hemorrhagic stroke induced by collagenase, and discussed the therapeutic effects on acute brain inflammation, nerve injury and recovery.
Objective:
In the rat model of intracerebral hemorrhagic stroke induced by collagenase, the therapeutic effects of induced pluripotent stem cell transplantation on acute inflammation, nerve injury and recovery were explored.
Method:
1. 80 SD rats were selected and 20 sterile saline were injected into the sham group (group Sham); 60 stereotaxic collagenase was injected into the left striatum, and the cerebral hemorrhage model was established. After 6 hours, the model was randomly divided into two groups to induce the pluripotent stem cells (5 mu L PBS suspended in 1 * 106 iPSCs cells) and the phosphate buffer solution. 5 L PBS), namely the transplant group (group iPSCs) and the model group (group PBS).
2. after forty-eighth hours of modeling, fluorescence quantitative PCR around hematoma was used to detect the level of inflammatory factors around hematoma in two groups of rats; third days, immunofluorescence staining was used to detect the inflammatory cells and apoptotic cells around hematoma, and the water content of the two groups of rats was measured by dry wet weight method and magnetic resonance imaging (MRI), and the improved limb balance was improved. Experiment MLPT evaluated behavioral function of two groups of rats on 0,1,3,7,14,28 and 42 days after modeling respectively.
3. on forty-second days, Nissl staining was used to observe the repair effect of neuron damage after intracerebral hemorrhage, and the thickness of glial scar and the detection of transplanted stem cells were observed by immunofluorescence staining.
Result:
1. compared with the model group, the transplantation group promoted the recovery of neurological function in rats, and the MLPT score increased from fourteenth days to 42 days, which was higher than that of the model group (P0.01).
In 2. transplantation group, the proinflammatory factor (IL-1 beta), IL-6 and tumor necrosis factor alpha (TNF- alpha) in the Para Para tissue were significantly decreased, while the anti inflammatory factor IL-10 increased by 2 times. The water content of the cerebral hemorrhage hemisphere was lower than that of the model group (P0.05), and the non hemorrhagic hemisphere was not different. The neutrophils and microglia were different to the model group. The number of cells with necrosis and apoptosis marked by TUNEL and activated caspase 3 (active caspase-3) was lower in the transplanted group than in the model group (P0.05).
3. the transplanted stem cells survived around the hematoma. Compared with the model group, the nerve cells recovered well in the transplantation group (P0.05), and the thickness of the glial scar was low (P0.01).
Conclusion:
Transplantation induced pluripotent stem cells may promote neurological function recovery by alleviating acute inflammation and injury in experimental 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】：R743.3

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