本文關(guān)鍵詞：海馬神經(jīng)干細(xì)胞增生分化及影響因素　出處：《鄭州大學(xué)》2010年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 麻醉 異氟謎 放射線照射治療 補(bǔ)陽還五湯 電針 缺血缺氧 神經(jīng)元再生 海馬

【摘要】：腦損傷包括有圍產(chǎn)期窒息所致的缺氧缺血性腦病是造成人類死亡和傷殘的主要疾病之一,給家庭和社會帶來了巨大的負(fù)擔(dān)。既往一直認(rèn)為中樞神經(jīng)元無法再生,因此,對腦損傷研究的重點是神經(jīng)元死亡機(jī)制以及抑制神經(jīng)細(xì)胞死亡的措施,但是在眾多有神經(jīng)保護(hù)作用的措施中,除早期的亞低溫干預(yù)、促紅細(xì)胞生成素治療被證明有臨床療效以及組織纖溶酶原激活物對早期缺血性腦中風(fēng)有療效外,其它藥物或治療措施尚未被廣泛認(rèn)可。自從1998年,瑞典科學(xué)家Eriksson等應(yīng)用5-溴脫氧尿苷(BrdU)標(biāo)記分裂細(xì)胞的方法發(fā)現(xiàn)成人腦中存在神經(jīng)元再生現(xiàn)象以來,神經(jīng)干細(xì)胞因具有增生分化的特點和用來進(jìn)行移植治療腦損傷的潛在優(yōu)勢迅速成為國內(nèi)外研究的熱點。研究發(fā)現(xiàn),哺乳動物包括人類內(nèi)源性的神經(jīng)干細(xì)胞存在于海馬齒狀回顆粒層下和室管膜下區(qū),在生理刺激以及腦損傷等病理因素的刺激下,內(nèi)源性的神經(jīng)干細(xì)胞可以大量增生并持續(xù)數(shù)周,新生的細(xì)胞可分化成神經(jīng)元并向腦損傷的區(qū)域遷移替代受損的神經(jīng)元,如果抑制神經(jīng)干細(xì)胞的增生將影響腦損傷后認(rèn)知功能的恢復(fù),提示內(nèi)源性神經(jīng)細(xì)胞再生對腦損傷后功能的恢復(fù)有重要影響。為了促進(jìn)腦損傷后神經(jīng)功能的恢復(fù)與重建,近年來國內(nèi)外對神經(jīng)干細(xì)胞移植治療腦損傷進(jìn)行了初步的探討,國內(nèi)報道應(yīng)用人胚胎神經(jīng)干細(xì)胞移植治療缺氧缺血性腦損傷病人取得了一定的療效,但爭議很大,關(guān)于神經(jīng)干細(xì)胞移植目前仍有很多問題尚待解決,特別是神經(jīng)干細(xì)胞的定向分化,遷移和功能連接以及控制神經(jīng)干細(xì)胞的過度增生導(dǎo)致腫瘤的問題需要深入探討。但是如果能夠刺激或者誘導(dǎo)內(nèi)源性神經(jīng)干細(xì)胞增生分化并替代因腦損傷受損的神經(jīng)元將可以避免神經(jīng)干細(xì)胞移植存在的潛在技術(shù)、倫理等問題,為腦損傷的治療開辟新的途徑。但是由于影響神經(jīng)干細(xì)胞增生、分化的因素比較多,特別是一些臨床相關(guān)的診療措施對神經(jīng)干細(xì)胞的增生是否有影響尚不完全清楚,因此,為了避免不當(dāng)?shù)呐R床診療措施影響神經(jīng)干細(xì)胞的增生,影響腦損傷后神經(jīng)功能的修復(fù)與重建,需要對可能影響內(nèi)源性神經(jīng)干細(xì)胞的增生的診療措施進(jìn)一步探討。 方法與結(jié)果 現(xiàn)代麻醉醫(yī)學(xué)的進(jìn)展使得新生兒期甚至胎兒期進(jìn)行手術(shù)成為可能,其中異氟醚是臨床應(yīng)用最廣泛的麻醉藥,既往研究提示異氟醚麻醉是安全的并且有神經(jīng)保護(hù)作用。但是新近的臨床研究認(rèn)為,兒童期多次暴露于麻醉劑導(dǎo)致學(xué)習(xí)障礙的發(fā)生率提高,提示兒科病人用麻醉藥應(yīng)該更加慎重。為了明確異氟醚是否對發(fā)育期腦有影響,我們給予不同年齡的大鼠進(jìn)行異氟醚吸入,以評價對學(xué)習(xí)記憶以及神經(jīng)干細(xì)胞增生的影響。結(jié)果發(fā)現(xiàn)14日齡大鼠經(jīng)異氟醚吸入麻醉后認(rèn)知功能出現(xiàn)損害,麻醉結(jié)束后4周大鼠認(rèn)知功能指數(shù)(26.3%±10.1%)顯著低于對照組(56.4%±9.4%),并隨麻醉后時間的延長而認(rèn)知功能損害進(jìn)一步加重,但是60日齡的成年大鼠異氟醚吸入麻醉后并未出現(xiàn)明顯的認(rèn)知功能損害。為進(jìn)一步確定異氟醚吸入麻醉對神經(jīng)行為的影響,采用14日齡小鼠經(jīng)異氟醚吸入麻醉后應(yīng)用無偏差的IntelliCage評價學(xué)習(xí)與記憶功能,結(jié)果顯示異氟醚吸入的麻醉組與對照組動物學(xué)習(xí)功能無明顯差別,但是記憶功能明顯低于對照組。對其機(jī)制進(jìn)一步研究發(fā)現(xiàn),短時間的異氟醚吸入麻醉對動物的血壓及體溫?zé)o明顯影響,對海馬的神經(jīng)元以及新生細(xì)胞死亡也無明顯影響,但是海馬齒狀回顆粒細(xì)胞層的細(xì)胞數(shù)量在幼年動物麻醉組隨麻醉后恢復(fù)時間的延長顯著低于對照組,但是成年動物麻醉后海馬齒狀回顆粒細(xì)胞層的細(xì)胞數(shù)量與對照組相比無明顯差別。進(jìn)一步通過檢測BrdU和磷酸化組蛋白H3標(biāo)記的陽性細(xì)胞對神經(jīng)細(xì)胞增生進(jìn)行研究發(fā)現(xiàn),14日齡大鼠在麻醉后24h細(xì)胞增生明顯降低(21%),隨麻醉后時間的延長,細(xì)胞增生抑制效應(yīng)更加明顯,麻醉后4周細(xì)胞增生減少了71%,而60日齡大鼠細(xì)胞增生沒有明顯減少。新生的BrdU陽性細(xì)胞在麻醉后4周通過BrdU-NeuN-GFAP進(jìn)行表型鑒定,結(jié)果發(fā)現(xiàn)14日齡大鼠異氟醚吸入麻醉后分化為神經(jīng)元的數(shù)目顯著低于對照組,而分化為星形膠質(zhì)的數(shù)目顯著高于對照組,進(jìn)一步通過SOX-2和GFAP雙染色檢測神經(jīng)干細(xì)胞池發(fā)現(xiàn)異氟醚吸入降低了未成熟腦神經(jīng)干細(xì)胞的數(shù)量,而對成熟腦的影響則不太明顯。 頭顱放射線照射是治療或者預(yù)防兒童顱腦腫瘤的有效措施之一,但是放射線照射后引起的神經(jīng)系統(tǒng)的長期副作用已經(jīng)逐漸引起人們的重視。發(fā)育期大腦接受放射線照射后對成年缺血性損傷的影響如何尚未見報道,本研究采用10日齡小鼠給予8Gy照射左側(cè)大腦半球,在照射后50天制作缺氧缺血腦損傷模型,并在腦損傷后30天對腦損傷的程度,神經(jīng)干細(xì)胞的增生分化以及炎癥反應(yīng)進(jìn)行分析。結(jié)果發(fā)現(xiàn)在幼年期接受照射的動物在成年期發(fā)生缺血性腦損傷的梗塞體積(9.8±1.2mm3)較單純成年缺血小鼠腦梗死體積(5.1±1.6mm3)幾乎增加一倍,腦組織丟失體積分別是32.0±3.5mm3和18.2±5.8mm3也顯著增加,對缺氧缺血腦損傷各個部位進(jìn)行分析發(fā)現(xiàn),放射線照射可加重海馬、皮質(zhì)和紋狀體對缺氧缺血腦損傷的敏感性。在幼年期接受射線照射顯著降低神經(jīng)元發(fā)生區(qū)的體積以及海馬齒狀回的細(xì)胞增生與存活以及神經(jīng)元再生。BrdU陽性細(xì)胞的數(shù)量在缺氧缺血腦損傷后明顯增加并且與腦損傷的程度相關(guān),但是新生的細(xì)胞主要為小膠質(zhì)細(xì)胞。 補(bǔ)陽還五湯由黃芪、歸尾、川芎、桃仁、紅花、芍藥和地龍等藥物組成,主要用于治療缺血性腦卒中及其后遺癥,其機(jī)制被認(rèn)為主要是改善腦血流及血液流變,拮抗興奮性氨基酸毒性,但是對神經(jīng)細(xì)胞的再生是否有影響尚未見詳細(xì)報道。我們采用大鼠MCAO腦缺血(缺血3h再灌注12d)動物模型,觀察補(bǔ)陽還五湯(大、中、小劑量)對腦缺血再灌注大鼠神經(jīng)癥狀積分、腦組織含水量、病理改變、海馬齒狀回細(xì)胞增殖、分化及其變化的影響。結(jié)果發(fā)現(xiàn)補(bǔ)陽還五湯可抑制腦缺血再灌注大鼠腦組織含水量增加,降低神經(jīng)功能障礙評分,明顯減輕腦缺血再灌注損傷。電鏡檢查顯示補(bǔ)陽還五湯可以減輕細(xì)胞器的水腫,尤其以中劑量組作用較顯著。補(bǔ)陽還五湯能夠增加BrdU陽性細(xì)胞、BrdU/NeuN雙標(biāo)陽性細(xì)胞數(shù),刺激神經(jīng)干細(xì)胞的增生及分化成神經(jīng)元。 針灸刺激在腦損傷臨床康復(fù)促進(jìn)方面作用已經(jīng)得到世界衛(wèi)生組織的認(rèn)可,并認(rèn)為針灸可以鎮(zhèn)痛維持內(nèi)穩(wěn)態(tài),改善腦循環(huán)以及對中樞神經(jīng)系統(tǒng)重要調(diào)節(jié)等功能,有研究報道,針灸可刺激神經(jīng)干細(xì)胞再生,但對幼年動物的影響如何報道較少。 本研究采用14目齡的大鼠,觀察電針穴位(曲池,外關(guān),環(huán)跳和足三里)刺激,每次30min連續(xù)7天,對海馬齒狀回細(xì)胞增殖分化的影響。結(jié)果發(fā)現(xiàn)末次針刺后4周,用BrdU標(biāo)記的細(xì)胞數(shù)目顯著高于空白對照組(P=0.0281),90%以上的新增殖細(xì)胞分化為神經(jīng)元。PHH-3陽性細(xì)胞數(shù)目觀察顯示,電針刺激穴位對細(xì)胞增殖有著長時程的影響。 結(jié)論 1.異氟醚吸入麻醉對認(rèn)知與記憶功能的影響與年齡有關(guān),對于發(fā)育期未成熟的大腦可引起記憶和認(rèn)知功能損害,其機(jī)制與異氟醚抑制神經(jīng)干細(xì)胞增生和向神經(jīng)元分化有關(guān)。 2.未成熟大腦受到射線照射后,腦內(nèi)神經(jīng)干細(xì)胞的再生與分化持續(xù)低下,對成年缺血性腦損傷有增敏作用。 3.補(bǔ)陽還五湯能夠拮抗腦缺血再灌注損傷并能促進(jìn)海馬齒狀回神經(jīng)干細(xì)胞增殖和分化。 4.電針穴位刺激能夠促進(jìn)未成熟大鼠海馬齒狀回干細(xì)胞持續(xù)的增殖和分化成神經(jīng)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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R329

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