本文選題：毛囊干細(xì)胞　切入點(diǎn)：分離培養(yǎng)　出處：《新疆醫(yī)科大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：眾所周知,干細(xì)胞是指未成熟細(xì)胞,具有自我更新和分化能力,可分化為胚胎干細(xì)胞和成體干細(xì)胞、其他細(xì)胞、組織和器官。毛囊的周期性生長(zhǎng)提示毛囊存存在著一群具有強(qiáng)增殖能力的細(xì)胞稱(chēng)之為毛囊干細(xì)胞。毛囊干細(xì)胞來(lái)源廣泛且取材較容易；細(xì)胞在體外顯示出很強(qiáng)的生長(zhǎng)能力和集落形成能力,單個(gè)細(xì)胞可分裂增殖至1.7×1038個(gè)；不涉及倫理和道德問(wèn)題的限制；能分化成表皮、皮脂腺和毛囊以及神經(jīng)元、脂肪細(xì)胞和成骨細(xì)胞；因而成為組織工工工程理想的種子細(xì)胞。目前,毛囊干細(xì)胞的篩選純化主要有兩類(lèi)方法：差速貼壁法和免疫磁珠法。其中,免疫磁珠法花費(fèi)昂貴,操作復(fù)雜,篩選過(guò)程中可能會(huì)導(dǎo)致細(xì)胞活性受損,國(guó)內(nèi)僅有少數(shù)有條件的科研單位能夠完成,因而并非理想的篩選純化方法。我們通過(guò)查閱國(guó)內(nèi)外文獻(xiàn),利用我�，F(xiàn)有的實(shí)驗(yàn)設(shè)備和技術(shù)平臺(tái),通過(guò)反復(fù)試驗(yàn)探索,聯(lián)合使用顯微分離技術(shù)、二步酶法以及差速貼壁法,亦可獲得足量高純度、活性好的毛囊干細(xì)胞。方法：經(jīng)倫理學(xué)審查同意后,在實(shí)驗(yàn)過(guò)程中,選用清潔級(jí)SD大鼠的8-9天乳鼠作為研究動(dòng)物。經(jīng)過(guò)嚴(yán)格消毒大鼠乳鼠觸須部組織后,手術(shù)取下含有毛囊的觸須部組織。然后在超凈臺(tái)中,體式顯微鏡下,分離出大鼠觸須部的毛囊組織,切取毛囊外根鞘部位；再分別使用中性蛋白酶和胰酶在自動(dòng)搖床中消化；利用Ⅳ型膠原包被的培養(yǎng)瓶(即差速貼壁法)篩選出貼壁較快的細(xì)胞用10%胎牛血清的角朊細(xì)胞無(wú)血清培養(yǎng)基進(jìn)行培養(yǎng)。所獲細(xì)胞進(jìn)行傳代、消化以及凍存等技術(shù)處理。通過(guò)倒置顯微鏡定期觀(guān)察照相、電子顯微鏡照相、姬姆薩染色、免疫熒光染色、繪制生長(zhǎng)曲線(xiàn)以及流式細(xì)胞術(shù)檢測(cè)等進(jìn)行鑒定。結(jié)果：篩選后的細(xì)胞在倒置相差顯微鏡下觀(guān)察形態(tài)均勻一致,折光性強(qiáng),呈典型的“鋪路石狀”。透射電鏡拍照顯示細(xì)胞體積小,核漿比例大,細(xì)胞器發(fā)育不成熟,處于原始狀態(tài)。繪制生長(zhǎng)曲線(xiàn)顯示,傳代至P3-P6細(xì)胞具有較強(qiáng)的增值能力。流式細(xì)胞術(shù)所獲得的數(shù)據(jù)通過(guò)統(tǒng)計(jì)學(xué)處理表明,所獲細(xì)胞純度可達(dá)90%以上。因此,我們的體會(huì)是聯(lián)用顯微分離技術(shù)、二步酶法以及差速貼壁法可獲得純度高、活性好的毛囊干細(xì)胞,聯(lián)用CD34和β1整合素是較理想的鑒定方法。為進(jìn)一步的誘導(dǎo)分化實(shí)驗(yàn)打下了良好的基礎(chǔ)。
[Abstract]:Objective: as we all know, stem cells are immature cells that have the ability to renew and differentiate themselves into embryonic stem cells and adult stem cells, other cells, Tissue and organ. The periodic growth of hair follicle suggests that there exists a group of hair follicle stem cells with strong proliferative ability. Cells show strong growth and colony formation in vitro, with individual cells dividing and proliferating to 1.7 脳 1038; without ethical and moral limitations; differentiating into epidermis, sebaceous glands, hair follicles, and neurons. Adipocytes and osteoblasts; thus become ideal seed cells for tissue engineering. At present, there are two main methods for screening and purification of hair follicle stem cells: differential adherence and immunomagnetic bead, in which immunomagnetic beads are expensive. The operation is complicated, the screening process may lead to the damage of cell activity, only a small number of qualified scientific research units in China can complete, so it is not an ideal method of screening and purification. Using the existing experimental equipment and technology platform, through repeated experiments and exploration, combined use of microscopic separation technology, two-step enzymatic method and differential adhesion method, we can also obtain sufficient quantity and high purity. Methods: healthy hair follicle stem cells were obtained. Methods: the SD rats of clean grade were selected as the study animals for 8-9 days after the ethical review. After strict disinfection of the tentacles of the rats, the hair follicle stem cells of the SD rats were disinfected strictly, and the hair follicle stem cells of the SD rats were disinfected strictly. The tentacles containing hair follicles were removed by surgery. The hair follicle tissue of rat tentacles was isolated under a pose microscope and then digested in a shaker with neutral protease and trypsin, respectively, and the outer root sheath of the hair follicle was removed. By using the culture bottle coated with type 鈪，

本文編號(hào)：1641231