海马神经干细胞增生分化及影响因素

发布时间：2018-01-16 14:13

本文关键词：海马神经干细胞增生分化及影响因素　出处：《郑州大学》2010年博士论文　论文类型：学位论文

【摘要】：脑损伤包括有围产期窒息所致的缺氧缺血性脑病是造成人类死亡和伤残的主要疾病之一,给家庭和社会带来了巨大的负担。既往一直认为中枢神经元无法再生,因此,对脑损伤研究的重点是神经元死亡机制以及抑制神经细胞死亡的措施,但是在众多有神经保护作用的措施中,除早期的亚低温干预、促红细胞生成素治疗被证明有临床疗效以及组织纤溶酶原激活物对早期缺血性脑中风有疗效外,其它药物或治疗措施尚未被广泛认可。自从1998年,瑞典科学家Eriksson等应用5-溴脱氧尿苷(BrdU)标记分裂细胞的方法发现成人脑中存在神经元再生现象以来,神经干细胞因具有增生分化的特点和用来进行移植治疗脑损伤的潜在优势迅速成为国内外研究的热点。研究发现,哺乳动物包括人类内源性的神经干细胞存在于海马齿状回颗粒层下和室管膜下区,在生理刺激以及脑损伤等病理因素的刺激下,内源性的神经干细胞可以大量增生并持续数周,新生的细胞可分化成神经元并向脑损伤的区域迁移替代受损的神经元,如果抑制神经干细胞的增生将影响脑损伤后认知功能的恢复,提示内源性神经细胞再生对脑损伤后功能的恢复有重要影响。为了促进脑损伤后神经功能的恢复与重建,近年来国内外对神经干细胞移植治疗脑损伤进行了初步的探讨,国内报道应用人胚胎神经干细胞移植治疗缺氧缺血性脑损伤病人取得了一定的疗效,但争议很大,关于神经干细胞移植目前仍有很多问题尚待解决,特别是神经干细胞的定向分化,迁移和功能连接以及控制神经干细胞的过度增生导致肿瘤的问题需要深入探讨。但是如果能够刺激或者诱导内源性神经干细胞增生分化并替代因脑损伤受损的神经元将可以避免神经干细胞移植存在的潜在技术、伦理等问题,为脑损伤的治疗开辟新的途径。但是由于影响神经干细胞增生、分化的因素比较多,特别是一些临床相关的诊疗措施对神经干细胞的增生是否有影响尚不完全清楚,因此,为了避免不当的临床诊疗措施影响神经干细胞的增生,影响脑损伤后神经功能的修复与重建,需要对可能影响内源性神经干细胞的增生的诊疗措施进一步探讨。方法与结果现代麻醉医学的进展使得新生儿期甚至胎儿期进行手术成为可能,其中异氟醚是临床应用最广泛的麻醉药,既往研究提示异氟醚麻醉是安全的并且有神经保护作用。但是新近的临床研究认为,儿童期多次暴露于麻醉剂导致学习障碍的发生率提高,提示儿科病人用麻醉药应该更加慎重。为了明确异氟醚是否对发育期脑有影响,我们给予不同年龄的大鼠进行异氟醚吸入,以评价对学习记忆以及神经干细胞增生的影响。结果发现14日龄大鼠经异氟醚吸入麻醉后认知功能出现损害,麻醉结束后4周大鼠认知功能指数(26.3%±10.1%)显著低于对照组(56.4%±9.4%),并随麻醉后时间的延长而认知功能损害进一步加重,但是60日龄的成年大鼠异氟醚吸入麻醉后并未出现明显的认知功能损害。为进一步确定异氟醚吸入麻醉对神经行为的影响,采用14日龄小鼠经异氟醚吸入麻醉后应用无偏差的IntelliCage评价学习与记忆功能,结果显示异氟醚吸入的麻醉组与对照组动物学习功能无明显差别,但是记忆功能明显低于对照组。对其机制进一步研究发现,短时间的异氟醚吸入麻醉对动物的血压及体温无明显影响,对海马的神经元以及新生细胞死亡也无明显影响,但是海马齿状回颗粒细胞层的细胞数量在幼年动物麻醉组随麻醉后恢复时间的延长显著低于对照组,但是成年动物麻醉后海马齿状回颗粒细胞层的细胞数量与对照组相比无明显差别。进一步通过检测BrdU和磷酸化组蛋白H3标记的阳性细胞对神经细胞增生进行研究发现,14日龄大鼠在麻醉后24h细胞增生明显降低(21%),随麻醉后时间的延长,细胞增生抑制效应更加明显,麻醉后4周细胞增生减少了71%,而60日龄大鼠细胞增生没有明显减少。新生的BrdU阳性细胞在麻醉后4周通过BrdU-NeuN-GFAP进行表型鉴定,结果发现14日龄大鼠异氟醚吸入麻醉后分化为神经元的数目显著低于对照组,而分化为星形胶质的数目显著高于对照组,进一步通过SOX-2和GFAP双染色检测神经干细胞池发现异氟醚吸入降低了未成熟脑神经干细胞的数量,而对成熟脑的影响则不太明显。头颅放射线照射是治疗或者预防儿童颅脑肿瘤的有效措施之一,但是放射线照射后引起的神经系统的长期副作用已经逐渐引起人们的重视。发育期大脑接受放射线照射后对成年缺血性损伤的影响如何尚未见报道,本研究采用10日龄小鼠给予8Gy照射左侧大脑半球,在照射后50天制作缺氧缺血脑损伤模型,并在脑损伤后30天对脑损伤的程度,神经干细胞的增生分化以及炎症反应进行分析。结果发现在幼年期接受照射的动物在成年期发生缺血性脑损伤的梗塞体积(9.8±1.2mm3)较单纯成年缺血小鼠脑梗死体积(5.1±1.6mm3)几乎增加一倍,脑组织丢失体积分别是32.0±3.5mm3和18.2±5.8mm3也显著增加,对缺氧缺血脑损伤各个部位进行分析发现,放射线照射可加重海马、皮质和纹状体对缺氧缺血脑损伤的敏感性。在幼年期接受射线照射显著降低神经元发生区的体积以及海马齿状回的细胞增生与存活以及神经元再生。BrdU阳性细胞的数量在缺氧缺血脑损伤后明显增加并且与脑损伤的程度相关,但是新生的细胞主要为小胶质细胞。补阳还五汤由黄芪、归尾、川芎、桃仁、红花、芍药和地龙等药物组成,主要用于治疗缺血性脑卒中及其后遗症,其机制被认为主要是改善脑血流及血液流变,拮抗兴奋性氨基酸毒性,但是对神经细胞的再生是否有影响尚未见详细报道。我们采用大鼠MCAO脑缺血(缺血3h再灌注12d)动物模型,观察补阳还五汤(大、中、小剂量)对脑缺血再灌注大鼠神经症状积分、脑组织含水量、病理改变、海马齿状回细胞增殖、分化及其变化的影响。结果发现补阳还五汤可抑制脑缺血再灌注大鼠脑组织含水量增加,降低神经功能障碍评分,明显减轻脑缺血再灌注损伤。电镜检查显示补阳还五汤可以减轻细胞器的水肿,尤其以中剂量组作用较显著。补阳还五汤能够增加BrdU阳性细胞、BrdU/NeuN双标阳性细胞数,刺激神经干细胞的增生及分化成神经元。针灸刺激在脑损伤临床康复促进方面作用已经得到世界卫生组织的认可,并认为针灸可以镇痛维持内稳态,改善脑循环以及对中枢神经系统重要调节等功能,有研究报道,针灸可刺激神经干细胞再生,但对幼年动物的影响如何报道较少。本研究采用14目龄的大鼠,观察电针穴位(曲池,外关,环跳和足三里)刺激,每次30min连续7天,对海马齿状回细胞增殖分化的影响。结果发现末次针刺后4周,用BrdU标记的细胞数目显著高于空白对照组(P=0.0281),90%以上的新增殖细胞分化为神经元。PHH-3阳性细胞数目观察显示,电针刺激穴位对细胞增殖有着长时程的影响。结论 1.异氟醚吸入麻醉对认知与记忆功能的影响与年龄有关,对于发育期未成熟的大脑可引起记忆和认知功能损害,其机制与异氟醚抑制神经干细胞增生和向神经元分化有关。 2.未成熟大脑受到射线照射后,脑内神经干细胞的再生与分化持续低下,对成年缺血性脑损伤有增敏作用。 3.补阳还五汤能够拮抗脑缺血再灌注损伤并能促进海马齿状回神经干细胞增殖和分化。 4.电针穴位刺激能够促进未成熟大鼠海马齿状回干细胞持续的增殖和分化成神经元。
[Abstract]:Brain injury including hypoxic ischemic encephalopathy caused by perinatal asphyxia is one of the main diseases causing human death and disability, brought huge burden to the family and society. It has been considered that the central neurons cannot regenerate, therefore, focus on the study of brain injury is the neuronal death mechanisms and measures to inhibit neuronal cell death, but in there are many neuroprotective measures, in addition to the effects of mild hypothermia on early, erythropoietin therapy proven clinical efficacy and tissue plasminogen activator on the early ischemic stroke have curative effect, other drugs or therapeutic measures have not been widely recognized. Since 1998, Swedish scientists Eriksson bromodeoxyuridine (5- BrdU) since the marker in cell division method found neuron regeneration phenomenon of adult brain, neural stem cell proliferation and differentiation because of its characteristics and For the potential advantages of transplantation for the treatment of brain injury are rapidly becoming a hot research at home and abroad. The study found that mammals including human endogenous neural stem cells and subventricular zone Yu Hai dentate gyrus granular layer, the physiological stimulation and brain damage and other pathological factors under the stimulation of endogenous neural stem cells can proliferate and for weeks, the new cells can differentiate into neurons and brain damage to the regional migration to replace damaged neurons, if the inhibition of neural stem cells proliferation will affect cognitive function after brain injury recovery, suggesting endogenous neural cells to function after brain injury recovery has important effect on regeneration. In order to promote the restoration and reconstruction of nerve function after brain injury, at home and abroad in recent years on neural stem cell transplantation for treating brain injury were discussed, the domestic report of the application of human embryonic neural stem fine Cell transplantation for the treatment of ischemic brain injury patients with hypoxia achieved a certain effect, but the controversy about the transplantation of neural stem cells at present there are still many problems to be solved, especially the differentiation of neural stem cells, migration and function connection and control of neural stem cell proliferation in tumor issues need to be discussed. But if you can stimulation or induction of endogenous neural stem cell proliferation and differentiation of brain damage due to replacement of damaged neurons can avoid neural stem cell transplantation has the potential of technology, ethics and other issues, provides a new way for the treatment of brain injury. But the effects of neural stem cell proliferation and differentiation factors, especially some clinically relevant diagnosis and treatment of neural stem cell proliferation is affected is not clear, therefore, in order to avoid the influence of clinical diagnosis and treatment of inappropriate stem by God Cell proliferation affects the repair and reconstruction of neurological function after brain injury. Further exploration is needed for the diagnosis and treatment of endogenous neural stem cell proliferation.
Methods and results
Modern medical advances in anesthesia during the neonatal period and even fetal surgery is possible, which is the most widely used isoflurane anesthesia, previous studies suggest that are safe and have a neuroprotective effect of isoflurane anesthesia. But recent clinical studies believe that children exposed to lead to the occurrence of many anesthetic to improve the rate of learning disabilities, suggests that pediatric patients with anesthetic should be more careful. In order to clear whether isoflurane has effect on the brain, we give different age rat isoflurane inhalation, to evaluate the effect on learning and memory and neural stem cell proliferation. The results showed that 14 day old rats after isoflurane inhalation cognitive impairment occurred after anesthesia, anesthesia after 4 weeks the cognitive function of the rats index (26.3% + 10.1%) was significantly lower than the control group (56.4% + 9.4%), and with the extension of time after anesthesia and the cognitive function. And further, but the 60 day old adult rats after isoflurane anesthesia did not show significant cognitive impairment. To further determine the impact of isoflurane anesthesia on nerve behavior, using IntelliCage learning and memory function evaluation of 14 day old mice after isoflurane anesthesia after application of no deviation, the results showed that isoflurane inhalation anesthesia group there was no significant difference between control group and animal learning function, but the memory function was significantly lower than the control group. The further study of its mechanism, the short time of isoflurane anesthesia on blood pressure and body temperature of the animal has no obvious effect on hippocampal neurons, and newborn cell death has no significant effect, prolong the recovery time but the number of cells in the hippocampal dentate granule cell layer in juvenile animal with anesthesia group after anesthesia was significantly lower than the control group, but the adult animal anesthesia Houhai dentate Back to the granule cell layer of the cell number compared with the control group no significant difference. By further detection of BrdU positive cells and phosphorylation of histone H3 markers to study the proliferation of neural cells found in 24h cell hyperplasia after anesthesia 14 days old rats decreased significantly (21%), with prolonged time after anesthesia, cell proliferation inhibition the effect is more obvious, anesthesia after 4 weeks of cell proliferation and cell proliferation was decreased by 71%, 60 day old rats were not significantly reduced. BrdU positive cells in the neonatal anesthesia after 4 weeks by BrdU-NeuN-GFAP phenotypic identification, found that isoflurane in rats 14 days after inhalation anesthesia to differentiate into neuron number was significantly lower than the control group, and the number of differentiated into astrocytes was significantly higher than the control group, further through SOX-2 and GFAP double staining to detect the neural stem cell pool found that isoflurane inhalation reduces the immature brain neural stem cell number, But the impact on the mature brain is not obvious.
Cranial irradiation is one of the effective measures for treatment or prevention of pediatric brain tumors, has gradually caused long-term side effects caused by nervous system but after irradiation the people's attention. During the development of the brain to accept the radiation effect on how adult ischemic injury has not been reported, this study used 10 day old mice treated with 8Gy irradiation of the left cerebral hemisphere in 50 days after irradiation, making the model of hypoxia ischemic brain injury, and brain injury in 30 days after the degree of brain injury, neural stem cell proliferation and differentiation and inflammatory reaction were analyzed. Results it infancy irradiated animal ischemic brain damage occurred in adulthood (infarct volume 9.8 + 1.2mm3) compared with adult ischemic mice cerebral infarction volume (5.1 + 1.6mm3) almost doubled, the brain volume loss were 32 + 3.5mm3 and 18.2 + 5.8mm3 also significantly increased, The location of each injury in hypoxic-ischemic brain analysis found that radiation can increase the sensitivity of hippocampus, striatum and cortex of hypoxic ischemic brain damage in infancy. Accept irradiation significantly reduced the occurrence of neurons in the hippocampal dentate gyrus volume and cell proliferation and survival and regeneration of neuronal number of.BrdU positive cells in the hypoxic ischemic brain damage and obviously increased with the extent of brain damage, but the new cells called microglia.
Yang also five soup made from Radix Astragali, Rhizoma Chuanxiong, peach kernel, to the end, safflower, composed of peony and Lumbricus and other drugs, mainly used for the treatment of ischemic stroke and its sequelae, the mechanism is thought to be mainly to improve cerebral blood flow and blood rheology, the antagonist of excitatory amino acid toxicity, but the effect on nerve cell regeneration if there is no detailed reported. We used MCAO in rats with cerebral ischemia (ischemia 3H reperfusion 12D) animal model, to observe the positive also five soup (large, small dose) on cerebral ischemia reperfusion in rats with neurological symptoms score, brain water content, pathological changes of hippocampal dentate gyrus cell proliferation, differentiation and effects of change. Results the positive also five soup can inhibit the cerebral ischemia reperfusion of rat brain tissue water content increased, decreased the score of nerve function, reduce cerebral ischemia reperfusion injury. Electron microscopy showed five Buyanghuanwu decoction can reduce the organelle edema, Especially in the middle dose group, the effect of Buyang Five Decoction can increase the number of BrdU positive cells, the number of BrdU/NeuN double labeled cells, stimulate the proliferation and differentiation of neural stem cells into neurons.
Acupuncture stimulation in brain injury clinical rehabilitation promotion effect has been recognized by the WHO, and believed that acupuncture can maintain homeostasis analgesia, improve cerebral circulation and the central nervous system important regulation function, studies have reported that acupuncture can stimulate neural stem cell regeneration, but the effects on how the juvenile animal reported less.
This study used 14 old rats, to observe the effect of electroacupuncture (acupoint Quchi, Waiguan, Huantiao and Zusanli) stimulation, 30min each time for 7 days, the effect of dentate gyrus cell proliferation and differentiation. The results showed that 4 weeks after the last acupuncture, with the number of cells labeled with BrdU were significantly higher than the control group (P=0.0281), more than 90% of new cell proliferation and differentiation into neurons and.PHH-3 positive cells showed the effect of electroacupuncture has a long range effect on cell proliferation.
conclusion
1., the effect of isoflurane inhalation anesthesia on cognition and memory function is related to age. Immature brain can cause memory and cognitive impairment, and its mechanism is related to isoflurane inhibiting the proliferation of neural stem cells and differentiating into neurons.
After radiation of 2. immature brain, the regeneration and differentiation of neural stem cells in the brain continued to be low, which sensitized the adult ischemic brain injury.
3. tonifying yang and Five Decoction can antagonize the injury of cerebral ischemia reperfusion and promote the proliferation and differentiation of the neural stem cells of the dentate gyrus of the hippocampus.
4. acupoint stimulation of electroacupuncture can promote the continuous proliferation and differentiation of hippocampal gyrus in immature rats.

【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

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