本文關(guān)鍵詞：天麻素對(duì)海人酸干預(yù)的神經(jīng)干細(xì)胞分化的影響　出處：《鄭州大學(xué)》2009年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 神經(jīng)干細(xì)胞 分化 天麻素 海人酸

【摘要】：目的和背景 自90年代科學(xué)家們分離、培養(yǎng)出神經(jīng)干細(xì)胞(neural stem cells,NSCs)以來(lái),人們相繼從各種動(dòng)物及人的中樞神經(jīng)系統(tǒng)(central neural system,CNS)內(nèi)分離、培養(yǎng)出了神經(jīng)干細(xì)胞,因其具有很強(qiáng)的分裂、增殖及自我更新能力,從而打破了傳統(tǒng)上認(rèn)為神經(jīng)細(xì)胞不能再生的觀念。神經(jīng)干細(xì)胞的發(fā)現(xiàn)對(duì)神經(jīng)損傷、神經(jīng)退行性疾病等的治療以及深入研究動(dòng)物的生長(zhǎng)發(fā)育和分化具有重要的意義,為中樞神經(jīng)系統(tǒng)的結(jié)構(gòu)和功能重建提供了新的手段,具有廣闊的應(yīng)用前景,因而神經(jīng)干細(xì)胞的研究也就成了當(dāng)今生命科學(xué)的研究熱點(diǎn)。從神經(jīng)干細(xì)胞的分離、體外培養(yǎng)及其生物學(xué)特性的研究,到移植治療神經(jīng)系統(tǒng)疾病,都取得了可喜的成績(jī)。但中樞神經(jīng)系統(tǒng)的再生是一個(gè)十分復(fù)雜的課題,神經(jīng)干細(xì)胞在體內(nèi)如何分化也是個(gè)難題,尚存在大量的未知問(wèn)題需要進(jìn)一步深入研究。 缺氧缺血性腦病是臨床常見(jiàn)病、多發(fā)病,如腦血栓、腦梗塞、腦血管痙攣等,其致死和致殘率很高。缺氧缺血性腦病也常發(fā)生于圍產(chǎn)期的胎兒和新生兒,不僅可引起患兒死亡,而且是造成腦癱、癲癇及智力發(fā)育遲緩或智力低下的重要原因。據(jù)統(tǒng)計(jì),國(guó)內(nèi)每年約有75萬(wàn)新生兒發(fā)生窒息,約有25萬(wàn)嬰兒因此成為傷殘兒或低智兒童。近年來(lái)的研究表明:缺血再灌注可導(dǎo)致腦內(nèi)興奮性氨基酸(EAA)的大量釋放,并對(duì)神經(jīng)細(xì)胞產(chǎn)生興奮性毒性作用,興奮性氨基酸的毒性作用可能是通過(guò)以下兩條途徑產(chǎn)生:一是主要由氨基-3-羥基-5-甲基-4-異惡唑丙酸受體(AMPA-R)和海人藻酸受體(KA-R)過(guò)度興奮所介導(dǎo)的神經(jīng)細(xì)胞急性滲透性腫脹,以Na~+內(nèi)流為特征,可在數(shù)小時(shí)內(nèi)發(fā)生;二是主要由N-甲基-D-天(門(mén))冬氨酸受體(NMDA-R)過(guò)度興奮所介導(dǎo)的神經(jīng)細(xì)胞延遲性損傷,以Ca~(2+)內(nèi)流為特征,可在數(shù)小時(shí)至數(shù)日后發(fā)生,細(xì)胞內(nèi)鈣離子超載可能是興奮性氨基酸引起細(xì)胞損傷或死亡的共同病理學(xué)機(jī)制。興奮性氨基酸受體過(guò)度興奮可導(dǎo)致神經(jīng)細(xì)胞急性滲透性腫脹或延遲性損傷,可能導(dǎo)致神經(jīng)功能缺損、智能落后、姿勢(shì)異常等后遺癥。 因此,本研究旨在觀察天麻素在腦缺血后興奮性氨基酸增多時(shí)的作用,研究其在神經(jīng)干細(xì)胞分化中的影響,是否可誘導(dǎo)神經(jīng)干細(xì)胞向神經(jīng)元方向分化,改善腦缺氧后由于神經(jīng)干細(xì)胞向膠質(zhì)細(xì)胞分化導(dǎo)致的認(rèn)知或肢體活動(dòng)障礙,這對(duì)于研究如何誘導(dǎo)神經(jīng)干細(xì)胞在缺血缺氧后向神經(jīng)元分化,研究其在缺血性腦血管病、神經(jīng)系統(tǒng)變性疾病、癲癇、神經(jīng)損傷等疾病中的應(yīng)用具有重要意義。 材料與方法 以新生24h內(nèi)的Wistar大鼠為實(shí)驗(yàn)動(dòng)物,體外分離并培養(yǎng)神經(jīng)干細(xì)胞,傳代培養(yǎng),并進(jìn)行細(xì)胞單克隆實(shí)驗(yàn)。在神經(jīng)干細(xì)胞分化時(shí)加海人酸和天麻素干預(yù),海人酸濃度分別為100μmol/l、500μmol/l、1mmol/l、2mmol/l、5mmol/l,天麻素濃度為0.5mg/l。免疫熒光法和免疫組化SP法檢測(cè)加入海人酸和天麻素干預(yù)后神經(jīng)干細(xì)胞分化后細(xì)胞比例的變化,AO/EB熒光雙染檢測(cè)加入海人酸后部分神經(jīng)干細(xì)胞的凋亡,MTT比色法測(cè)定海人酸對(duì)神經(jīng)干細(xì)胞分化速度的影響。 結(jié)果 1.神經(jīng)干細(xì)胞可以分化為神經(jīng)元和膠質(zhì)細(xì)胞,其比例為23.07%±1.51%和76.93%±1.51%。 2.KA干預(yù)組神經(jīng)元和膠質(zhì)陽(yáng)性細(xì)胞比例分別為13.99%±2.07%和86.01%±2.07%。不同濃度的海人酸干預(yù)的組之間神經(jīng)元和膠質(zhì)細(xì)胞比例無(wú)明顯差異(F=1.43,p＞0.05),正常分化組與KA干預(yù)組所分化細(xì)胞比例有顯著統(tǒng)計(jì)學(xué)差異(F=368.21,p＜0.001),海人酸可以誘導(dǎo)神經(jīng)干細(xì)胞向膠質(zhì)細(xì)胞分化。 3.海人酸和天麻素干預(yù)組神經(jīng)元和膠質(zhì)陽(yáng)性細(xì)胞比例分別約為17.16%±2.14%和82.84%±2.14%,與只加海人酸組相比差異有統(tǒng)計(jì)學(xué)意義(F=115.6,p＜0.01),天麻素可可在一定程度上減少海人酸興奮性的影響,誘導(dǎo)神經(jīng)干細(xì)胞向神經(jīng)元分化。 4.MTT檢測(cè)加入海人酸和天麻素后早期部分神經(jīng)干細(xì)胞死亡,但可促進(jìn)存活的神經(jīng)干細(xì)胞分化。 結(jié)論 1.本實(shí)驗(yàn)所分離細(xì)胞的可分化為神經(jīng)元、少突膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞,是神經(jīng)干細(xì)胞。 2.海人酸可以誘導(dǎo)神經(jīng)干細(xì)胞向膠質(zhì)細(xì)胞分化。 3.天麻素可在一定程度上減少海人酸興奮性的影響,誘導(dǎo)神經(jīng)干細(xì)胞向神經(jīng)元分化。
[Abstract]:Purpose and background
From 90s scientists, culture of neural stem cells (neural stem cells, NSCs), have been from the central nervous system of various animal and human (central neural, system, CNS) in isolation, cultured from neural stem cells, because of its strong division, proliferation and self-renewal capacity, thus breaking the the traditional idea that neural cells cannot regenerate. The discovery of neural stem cells on neural injury, plays an important role in the treatment of neurodegenerative diseases such as growth and differentiation of animal research, provides a new method for central nervous system structure and function reconstruction, so it has broad application prospects. Neural stem cell research has become the research hotspot of life science. From neural stem cell separation, and Study on the biological characteristics of cultured in vitro, to transplantation for the treatment of diseases of the nervous system, are taken Gratifying achievements have been made. However, the regeneration of central nervous system is a very complex subject. How to differentiate neural stem cells in vivo is also a difficult problem. There are still a lot of unknown problems that need further study.
Hypoxic ischemic encephalopathy is a common clinical disease, frequently occurring disease, such as cerebral thrombosis, cerebral infarction, cerebral vasospasm, the mortality and disability rate is very high. Hypoxic ischemic encephalopathy often happen to perinatal fetuses and newborns, can not only cause of death in children, but also is an important reason causing cerebral palsy, epilepsy and mental retardation or low intelligence. According to statistics, about 750 thousand of neonatal asphyxia at home every year, about 250 thousand babies become disabled children or children with low intelligence. Recent studies show that ischemic reperfusion can lead to brain excitatory amino acid (EAA) release, and produce excitatory toxic effect on nerve cells, toxicity excitatory amino acids may be produced through the following two ways: one is mainly composed of -3- amino hydroxy -5- methyl -4- isoxazole propionate receptor (AMPA-R) and kainate receptor (KA-R) mediated by nerve hyperexcitability fine Acute cellular osmotic swelling, with Na~+ internal flow characteristics, can occur within hours; the two is mainly composed of N- methyl -D- aspartate receptor (door) (NMDA-R) delayed injury hyperexcitability mediated by nerve cells in Ca~ (2+) internal flow characteristics, can occur in several the number of hours to days after, intracellular calcium overload may be the common pathological excitatory amino acids induced cell injury or death. The mechanism of excitatory amino acid receptors can lead to neuronal hyperexcitability of acute osmotic swelling or delayed injury may lead to neurological impairment, intelligent backward, abnormal posture and other sequelae.
Therefore, the purpose of this study was to observe the gastrodin in excitatory amino acids after cerebral ischemia increased the role of the research in the effect of neural stem cell differentiation, induce differentiation of neural stem cells into neurons, improve the brain after hypoxia for neural stem cell differentiation cognitive or limb movement disorder leads to glial cells, the for the study of how to induce cell differentiation to neurons in hypoxia ischemia after neural stem on the ischemic cerebrovascular disease, epilepsy, neurodegenerative diseases, nervous system diseases, has important significance in the application of nerve injury.
Materials and methods
In the neonatal Wistar rats within 24h as experimental animal, isolate and culture neural stem cells, cultured, and cell clone experiments. Plus kainic acid and gastrodin in the differentiation of neural stem cells when hormone intervention, kainic acid concentrations were 100 mol/l, 500 mol/l, 1mmol/l, 2mmol/l, 5mmol/l. The change of gastrodin concentration of 0.5mg/l. by immunofluorescence and immunohistochemistry SP method with kainic acid and gastrodin percentage of stem cells after neural stem cell differentiation and prognosis, AO/EB fluorescent staining with cell apoptosis after kainic acid part of neural stem, to determine the effects of kainic acid on the differentiation of neural stem cells speed MTT colorimetric method.
Result
1. neural stem cells can differentiate into neurons and glial cells, the proportion of which is 23.07% + 1.51% and 76.93% + 1.51%.
No significant difference between the proportion of neurons and glial cells 2.KA positive cells proportion of intervention group neurons and glia were 13.99% + 2.07% and 86.01% + 2.07%. different concentrations of kainic acid intervention group (F=1.43, P > 0.05), normal group and KA intervention group the differentiation proportion of differentiated cells with significant difference (F=368.21, P < 0.001), kainic acid can induce neural stem cells to differentiate into glial cells.
3. kainic acid and gastrodin intervention group positive cells were neurons and glia were approximately 17.16% + 2.14% and 82.84% + 2.14%, compared with only kainic acid group had significant difference (F=115.6, P < 0.01), gastrodin kainic acid reduced cocoa excitatory effects induced in a certain extent. Differentiation of neural stem cells into neurons.
4.MTT was used to detect the early death of some neural stem cells after the addition of sea human acid and gastrodin, but it could promote the differentiation of surviving neural stem cells.
conclusion
1. the cells isolated from the experiment can differentiate into neurons, oligodendrocytes and astrocytes, which are neural stem cells.
2. sea human acids can induce neural stem cells to differentiate into glial cells.
3. gastrodin can reduce the effect of the excitability of the sea human acid to some extent and induce the neural stem cells to differentiate into neurons.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R329;R285

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 賈延R，

本文編號(hào)：1401450