本文選題：神經(jīng)干細(xì)胞　切入點(diǎn)：神經(jīng)球　出處：《大連理工大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：神經(jīng)干細(xì)胞因其移植后不具有致癌性等優(yōu)點(diǎn),已成為治療神經(jīng)系統(tǒng)退行性疾病和神經(jīng)系統(tǒng)損傷的最優(yōu)選擇。為了更有效地利用神經(jīng)干細(xì)胞,神經(jīng)干細(xì)胞的低溫保存技術(shù)成為一個(gè)重要的課題。目前,低溫保存神經(jīng)干細(xì)胞主要采用的是慢速凍存,玻璃化法作為一種新型的,簡便的冷凍保存方法可以有效地克服慢速凍存的缺點(diǎn)。本文一方面對(duì)神經(jīng)干細(xì)胞慢速凍存過程中神經(jīng)干細(xì)胞球的直徑尺寸與冷凍保護(hù)劑的種類、濃度進(jìn)行了實(shí)驗(yàn)。另一方面對(duì)玻璃化溶液的種類,導(dǎo)入過程等方面的問題進(jìn)行初步的研究。 首先對(duì)神經(jīng)干細(xì)胞的慢速凍存進(jìn)行了研究。通過神經(jīng)干細(xì)胞生長曲線的測定,確定了低溫保存神經(jīng)干細(xì)胞的最佳時(shí)間及狀態(tài)。用處于最佳時(shí)間狀態(tài)的單細(xì)胞懸液、不同尺寸的細(xì)胞球(直徑30～50μm球,直徑80～100μm球)分別進(jìn)行了七個(gè)不同濃度3%,5%,7%,8%,10%,15%,20%DMSO的凍存比較。從而確定了神經(jīng)干細(xì)胞處于對(duì)數(shù)生長期后期,神經(jīng)球直徑約80μm～100μm,DMSO濃度為8%時(shí)凍存效果最佳,其復(fù)蘇后細(xì)胞活率約83%。不同直徑的神經(jīng)干細(xì)胞球?qū)鋬霰Ｗo(hù)劑的濃度需求是不同的。且神經(jīng)干細(xì)胞間親密的聯(lián)系和皺縮信號(hào)與直徑尺寸共同作用,影響著神經(jīng)干細(xì)胞的凍存復(fù)蘇。 其次,對(duì)神經(jīng)干細(xì)胞玻璃化保護(hù)劑進(jìn)行了實(shí)驗(yàn)比較。通過在冷臺(tái)上動(dòng)態(tài)直觀地觀察以及神經(jīng)干細(xì)胞復(fù)蘇率的比較,優(yōu)選出了適合神經(jīng)干細(xì)胞玻璃化凍存的冷凍保護(hù)劑20%(v/v)二甲基亞砜(DMSO)+20%(v/v)乙二醇(EG)+10%(w/v)乙酰胺(Acetamide)+0.3M蔗糖(Sucrose)。然后研究了所配制的玻璃化保護(hù)劑導(dǎo)入過程中操作溫度、導(dǎo)入時(shí)間對(duì)細(xì)胞活率的影響。實(shí)驗(yàn)溫度分別選擇了室溫(20℃-26℃)和冰水混合物(0℃-4℃)的溫度下30s、60s、90s、120s和150s不同時(shí)間導(dǎo)入對(duì)神經(jīng)干細(xì)胞活率的影響。最后確定了在室溫下,60s連續(xù)導(dǎo)入神經(jīng)干細(xì)胞的活率最高。
[Abstract]:Neural stem cells (NSCs) have become the best choice for the treatment of neurodegenerative diseases and nervous system injuries due to their lack of carcinogenicity after transplantation. Cryopreservation of neural stem cells has become an important subject. At present, cryopreservation of neural stem cells is mainly by slow and quick freezing, and vitrification is a new type of cryopreservation. The simple method of cryopreservation can effectively overcome the shortcoming of slow freezing. On the one hand, the diameter of neural stem cell ball and the types of cryopreservation protectants during the slow freezing of neural stem cells are studied in this paper. On the other hand, the kinds of glass solution and the process of introducing glass solution were studied. In this paper, we first studied the cryopreservation of neural stem cells. By measuring the growth curve of neural stem cells, we determined the best time and state of cryopreservation of neural stem cells. Seven cell spheres of different sizes (30 ~ 50 渭 m in diameter and 80 ~ 100 渭 m in diameter) were used to compare the cryopreservation of seven different concentrations (30 渭 m, 50 渭 m and 80 渭 m DMSO). The best cryopreservation effect was obtained when the diameter of neural stem cells was about 80 渭 m, 100 渭 m DMSO was 8, and the diameter of neural stem cells was about 80 渭 m and 100 渭 m DMSO was 8. The cell viability after resuscitation is about 83%. The different diameter of neural stem cell balls require different concentrations of cryoprotectants. And the close connection between neural stem cells and the interaction between shrinkage signal and diameter size, It affects the cryopreservation and resuscitation of neural stem cells. Secondly, the experimental comparison of neural stem cell vitrification protectants was carried out. The dynamic and intuitive observation on the cold stage and the comparison of neural stem cell resuscitation rate were carried out. The chilled protectant 20v / v) dimethyl sulfoxide (DMSOO) 20g / v) ethylene glycol (EG10) and acetamide (Acetamide) 0.3M sucrose (0.3M) were selected for cryopreservation of neural stem cells (NSCs). Then the operating temperature of the prepared glass protectant during the introduction process was studied. The effect of the time of introduction on the viability of neural stem cells was studied. The experimental temperature of 20 鈩，

本文編號(hào)：1566262