本文關(guān)鍵詞：人表皮干細(xì)胞的分離和培養(yǎng)的研究　出處：《江西醫(yī)學(xué)院》2005年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 角朊細(xì)胞 表皮干細(xì)胞 細(xì)胞培養(yǎng) 角蛋白19 β_1整合素 無血清培養(yǎng)基

【摘要】：目的研究表皮干細(xì)胞的體外分離、培養(yǎng)及鑒定方法,觀察表皮干細(xì)胞在有血清和無血清培養(yǎng)基中的生長特性,并對其在體外的培養(yǎng)條件進(jìn)行探索,以期在體外獲得人表皮干細(xì)胞的良好增殖。 方法①采用DispaseⅡ酶和胰蛋白酶兩步法分離出角朊細(xì)胞和成纖維細(xì)胞,并以人成纖維細(xì)胞條件培養(yǎng)基為基礎(chǔ)制備表皮干細(xì)胞培養(yǎng)基,用以人表皮干細(xì)胞(Ⅳ型膠原快速粘附法富集分離)的體外培養(yǎng),以角朊細(xì)胞培養(yǎng)為對照組。觀察人表皮干細(xì)胞的細(xì)胞形態(tài)和一般生長狀態(tài),比較人表皮干細(xì)胞和角朊細(xì)胞的克隆形成率和克隆維持時間,同時采用免疫細(xì)胞化學(xué)法檢測β_1整合素和K_(19) 的表達(dá),鑒定表皮干細(xì)胞。②將富集分離出的人表皮干細(xì)胞分兩組培養(yǎng),分別加入同源成纖維細(xì)胞條件培養(yǎng)基配制的人表皮干細(xì)胞有血清培養(yǎng)基和無血清培養(yǎng)基,觀察和比較在這兩種培養(yǎng)基中的細(xì)胞形態(tài)、克隆形成率、生長曲線、β_1整合素和K_(19)的強陽性表達(dá)率。 結(jié)果①在Ⅳ型膠原快速包被的培養(yǎng)皿中快速貼壁細(xì)胞經(jīng)繼續(xù)培養(yǎng),可見細(xì)胞克隆數(shù)增多,兩周后可見大克隆形成。與角朊細(xì)胞相比,表皮干細(xì)胞有更強的克隆形成能力并可形成較大克隆,兩者的形成率分別為17.04±1.01%和8.72±0.73%,兩者比較有顯著性差異(P0.01);人表皮干細(xì)胞β_1整合素和K_(19)免疫細(xì)胞化學(xué)染色呈強陽性。②細(xì)胞形態(tài)學(xué)分析和生長曲線均表明,培養(yǎng)5 天前,無血清培養(yǎng)的人表皮干細(xì)胞增殖細(xì)胞數(shù)較有血清培養(yǎng)的多,第9 天達(dá)到細(xì)胞基本融合狀態(tài)并出現(xiàn)接觸抑制;而在有血清培養(yǎng)基中,人表皮干細(xì)胞則繼續(xù)增殖,第11 天時達(dá)到生長峰值,隨后進(jìn)入平臺期。克隆性分析表明,兩種培養(yǎng)基培養(yǎng)的表皮干細(xì)胞均呈克隆性生長,但比較克隆數(shù)目和大小、克隆形成率和克隆持續(xù)時間,有血清組明顯優(yōu)于無血清組(P0.01)。細(xì)胞周期分析顯示,人表皮干細(xì)胞在有血清培養(yǎng)基中G_0～G_1期細(xì)胞比例明顯
[Abstract]:Objective to study the isolation, culture and identification methods of epidermal stem cells, observe the growth characteristics of epidermal stem cells in serum-free and serum free medium, and explore their culture conditions in vitro, in order to obtain a good proliferation of human epidermal stem cells in vitro.
Methods using the Dispase II enzyme and trypsin two step isolated keratinocytes and fibroblasts, and human fibroblast conditioned medium for preparation of epidermal stem cell culture medium, with human epidermal stem cells (type IV collagen rapid adhesion by enrichment) in vitro culture of keratinocytes training for the control group. Observation of cell morphology and growth status of epidermal stem of human epidermal stem cells and cloning of keratinocytes and clone formation rate of maintenance time, at the same time using immunocytochemical method to detect beta _1 integrin and K_ (19) expression and identification of epidermal stem cells. The enrichment and separation of the the human epidermal stem cells cultured and divided into two groups, respectively adding homologous fibroblast conditioned medium of human epidermal stem cells serum medium and serum free medium, observe and compare the two culture medium on cell morphology, The positive rate of positive expression of clone formation rate, growth curve, beta _1 integrin and K_ (19).
The fast coated culture dishes in the rapidly adherent cells by cultured in collagen, visible cell clones increased after two weeks showed a large colony formation. Compared with keratinocytes, epidermal stem cells have a stronger ability to form clones and formed large clones, the formation rate is respectively 17.04 and 1.01%. 8.72 + 0.73% of the two, there is significant difference between them (P0.01); human epidermal stem cells beta _1 integrin and K_ (19) immunocytochemical staining showed strong positive analysis. The cell morphology and growth of Qu Xianjun culture showed that 5 days ago, serum-free culture of human epidermal stem cell proliferation cell number of serum culture more than ninth days to reach the cells fusion and contact inhibition; and in serum medium, human epidermal stem cells continued to proliferate, achieve the growth peak of eleventh days, then into the platform. Clonal analysis showed that two The cultured cells showed clonal growth of cultured epidermal stem, but the number and size of clones, clone formation rate and clone duration, serum group was significantly better than that of non serum group (P0.01). Cell cycle analysis showed that human epidermal stem cells in serum medium G_0 and the percentage of cells in G_1 phase significantly

【學(xué)位授予單位】：江西醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R329.2

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本文編號：1372698