發(fā)布時(shí)間：2018-01-23 09:11

  本文關(guān)鍵詞： 原子力顯微鏡 淫羊霍苷 人臍帶間充質(zhì)干細(xì)胞 成骨細(xì)胞 紫杉醇 鼻咽癌細(xì)胞 細(xì)胞骨架　出處：《暨南大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 本論文主要分為兩部分：(1)利用原子力顯微鏡(AFM)研究淫羊霍苷(ICA)誘導(dǎo)人臍帶間充質(zhì)干細(xì)胞(hUCMSCs)成骨分化的作用機(jī)制。(2)利用AFM研究紫杉醇誘導(dǎo)鼻咽癌細(xì)胞(CNE-2)凋亡。 本文第一部分：通過形態(tài)學(xué),細(xì)胞表面標(biāo)志物以及多向分化能力三個(gè)方法鑒定hUCMSCs。使用改良鈣鈷法對(duì)加入ICA誘導(dǎo)后的細(xì)胞進(jìn)行ALP染色,確定ICA是否具有誘導(dǎo)hUCMSCs分化為成骨細(xì)胞的作用。通過ALP陽性細(xì)胞計(jì)數(shù),確定最佳促進(jìn)成骨分化的ICA添加濃度,使用相同濃度的ICA誘導(dǎo)hUCMSCs向成骨細(xì)胞分化.結(jié)果表明：(1)光學(xué)顯微鏡圖像顯示,培養(yǎng)了7天的細(xì)胞為長(zhǎng)梭形,是典型的成纖維細(xì)胞形態(tài)；培養(yǎng)了10天的細(xì)胞極性排列,集落呈漩渦狀。(2)流式細(xì)胞儀(FCAS)檢測(cè)分離到的hUCMSCs表面抗原CD29、CD44、CD 105陽性表達(dá),CD34、CD45和人白細(xì)胞抗原HLA-DR陰性表達(dá)。(3)ICA能增加間充質(zhì)干細(xì)胞的ALP的活性,促進(jìn)礦化結(jié)節(jié)的形成(P0.05)。ICA最佳促進(jìn)成骨分化濃度為1×10-6mol／L。(4)AFM的圖像表明淫羊霍苷在蓋玻片上呈分散狀分布(粒徑分布在70±25nm左右),加入細(xì)胞共培養(yǎng)5天時(shí)在細(xì)胞表面上聚集并呈微米域分布(粒徑分布在96±21nm左右)。培養(yǎng)時(shí)間為10天時(shí)在細(xì)胞表面出現(xiàn)200-300 nm的微米孔,培養(yǎng)時(shí)間為15天時(shí)細(xì)胞表面微米孔的深度變小,細(xì)胞膜開始愈合,表現(xiàn)出細(xì)胞胞吞作用的現(xiàn)象。與分化前相比較,hUCMSCs的細(xì)胞形態(tài)由長(zhǎng)梭形變?yōu)榉叫?細(xì)胞膜的粗糙度增大,細(xì)胞表面有明顯的小突觸,是因?yàn)槌晒欠只蠹?xì)胞內(nèi)形成鈣結(jié)節(jié)。此結(jié)果為研究淫羊霍苷誘導(dǎo)間充質(zhì)干細(xì)胞的成骨分化的作用機(jī)制提供了一種直觀的方法。 本文第二部分：應(yīng)用AFM對(duì)紫杉醇誘導(dǎo)鼻咽癌細(xì)胞(CNE-2)凋亡的研究。(1)首先,采用MTT法檢測(cè)紫杉醇對(duì)鼻咽癌細(xì)胞株(CNE-2)的生長(zhǎng)抑制性,結(jié)果表明,紫杉醇作用后,細(xì)胞的存活率顯著下降,且呈現(xiàn)顯著的劑量依賴性。(2)采用不同濃度的TritonX-100處理CNE-2細(xì)胞,并用AFM對(duì)其進(jìn)行形貌性質(zhì)的研究,結(jié)果顯示1ml／L的TritonX-100可以有效地制備出細(xì)胞骨架,為下一步研究紫杉醇作用后細(xì)胞骨架的變化提供了一個(gè)實(shí)驗(yàn)依據(jù)。(3)用AFM對(duì)紫杉醇誘導(dǎo)12小時(shí),24小時(shí)和對(duì)照組CNE-2細(xì)胞的形貌,超微結(jié)構(gòu)和粗糙度進(jìn)行測(cè)量。與對(duì)照組細(xì)胞比較,發(fā)現(xiàn)誘導(dǎo)12小時(shí)后,細(xì)胞的體積開始皺縮,細(xì)胞絨毛減少；誘導(dǎo)24小時(shí)后,細(xì)胞發(fā)生明顯的皺縮,細(xì)胞絨毛消失。并且隨著誘導(dǎo)時(shí)間增加,細(xì)胞膜粗糙度增大。(4)用AFM對(duì)1ml／L濃度TritonX-100處理100μg／ml紫杉醇誘導(dǎo)12小時(shí)的CNE-2細(xì)胞進(jìn)行成像；結(jié)果顯示,紫杉醇可以破壞CNE-2細(xì)胞的細(xì)胞骨架。原子力顯微鏡是研究細(xì)胞骨架的一種可視化的工具。
[Abstract]:This thesis is divided into two parts: 1) the mechanism of osteogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) induced by ICA (atomic force microscope AFM) was studied. (. 2) AFM was used to study the apoptosis of nasopharyngeal carcinoma cell line CNE 2 induced by paclitaxel. In the first part of this paper, we used morphology, cell surface markers and multidirectional differentiation ability to identify the hUCMSCs.The modified calcium cobalt method was used to detect the ALP staining of the cells induced by ICA. To determine whether ICA can induce the differentiation of hUCMSCs into osteoblasts. By counting the positive cells of ALP, the best concentration of ICA to promote osteogenic differentiation is determined. The differentiation of hUCMSCs into osteoblasts was induced by the same concentration of ICA. The results showed that the cells cultured for 7 days were fusiform and typical fibroblasts. After 10 days of culture, the hUCMSCs surface antigen CD29 and CD44 were detected by flow cytometry (FCAS). The positive expression of CD105 and the negative expression of CD34-CD45 and human leukocyte antigen HLA-DR could increase the ALP activity of mesenchymal stem cells. Promoting the formation of mineralized nodules (P0.05A. ICA) the best concentration of osteogenic differentiation was 1 脳 10 ~ (-6) mol / L 路L 路L ~ (4) (AFM). The particle size distribution is about 70 鹵25 nm. After 5 days of co-culture, the cells were clustered on the cell surface and distributed in micron domain (particle size distribution was about 96 鹵21 nm). When the culture time was 10 days, the micropores of 200-300 nm were found on the surface of the cells. When the culture time was 15 days, the depth of the micropore on the cell surface became smaller, the cell membrane began to heal, showing the phenomenon of cell endocytosis, which was compared with that before differentiation. The cell morphology of hUCMSCs changed from long spindle to square, the roughness of cell membrane increased, and the surface of the cell had obvious small synapses. The results provide an intuitive method for the study of the mechanism of osteogenic differentiation of mesenchymal stem cells induced by aridonin. In the second part of this paper, AFM was used to study the apoptosis of nasopharyngeal carcinoma cell line CNE-2 induced by paclitaxel. The growth inhibition of paclitaxel on nasopharyngeal carcinoma cell line CNE-2 was detected by MTT assay. The results showed that the survival rate of CNE-2 cells decreased significantly after paclitaxel treatment. In a dose-dependent manner, CNE-2 cells were treated with different concentrations of TritonX-100, and their morphology and properties were studied by AFM. The results showed that 1 ml / L TritonX-100 could effectively produce cytoskeleton. It provides an experimental basis for the further study of cytoskeleton changes after paclitaxel treatment. (3) the morphology of CNE-2 cells induced by paclitaxel for 12 hours and 24 hours with AFM. The ultrastructure and roughness were measured. Compared with the control group, it was found that after 12 hours of induction, the volume of the cells began to shrink and the villi of the cells decreased. After 24 hours of induction, the cells shrank obviously and the villi disappeared, and increased with the induction time. AFM was used to image the CNE-2 cells treated with 100 渭 g / ml paclitaxel at the concentration of 1 ml / L TritonX-100 for 12 hours. The results show that paclitaxel can destroy the cytoskeleton of CNE-2 cells. Atomic force microscopy is a visual tool for studying cytoskeleton.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R739.63

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