【作者】 宫晨；

【导师】 郭风劲；

【作者基本信息】 华中科技大学 ， 外科学， 2013， 博士

【摘要】 目的：1.研究无血清培养法和免疫磁珠分选法用于人骨肉瘤类肿瘤干细胞的分离培养,探讨两者作为分选方法的可行性。2.研究Nanog在人骨肉瘤及其类肿瘤干细胞的中的表达及意义。3.研究LY294002对人骨肉瘤类肿瘤干细胞增殖和细胞周期的影响,并初步探讨其抑制增殖作用可能的机制。4.研究LY294002对人骨肉瘤类肿瘤干细胞凋亡的影响,并初步探讨其诱导凋亡作用可能的机制。方法：1.无血清培养法分离人骨肉瘤类肿瘤干细胞；免疫磁珠分选CD133+CD44+人骨肉瘤细胞；免疫组织化学法鉴定人骨肉瘤类肿瘤干细胞CD44和CD133的表达情况；蛋白印迹法检测骨肉瘤及其类肿瘤干细胞Nanog的表达并进行半定量分析。2.实验分组：LY294002在培养基中的浓度分别为0μmol/L(对照组)、5μmol/L、15μmol/L和45μmol/L。3.CCK-8法检测人骨肉瘤类肿瘤干细胞在不同LY294002浓度下的增殖情况；碘化丙啶PI染色流式细胞术检测人骨肉瘤类肿瘤干细胞在不同LY294002浓度下细胞周期的变化；蛋白印迹法法检测人骨肉瘤类肿瘤干细胞在不同LY294002浓度下总AKT、磷酸化AKT和β-肌动蛋白的表达情况。4. Annexin V/PI双染法流式细胞术检测人骨肉瘤类肿瘤干细胞在不同LY294002浓度下的凋亡情况；蛋白印迹法检测半胱天冬酶-3、活化型半胱天冬酶-3、半胱天冬酶-9、活化型聚腺苷二磷酸核糖聚合酶和β-肌动蛋白的表达情况。结果：1.采用无血清培养法可以从人骨肉瘤MG63细胞中分离出成“细胞球”生长的人骨肉瘤类肿瘤干细胞；采用免疫磁珠法可以从人骨肉瘤MG63细胞中分离出CD133+CD44+人骨肉瘤细胞,该细胞亦可在含表皮生长因子和成纤维生长因子的无血清培养基中形成“细胞球”；将上述骨肉瘤类肿瘤干细胞球传代于无血清培养基仍可形成相似的人骨肉瘤类肿瘤干细胞球；人骨肉瘤类肿瘤干细胞CD44和CD133均呈阳性表达；人骨肉瘤类肿瘤干细胞Nanog表达水平强于人骨肉瘤MG63细胞(P<0.05)。2.与对照组相比较,无论何种浓度的LY294002均对人骨肉瘤类肿瘤干细胞的增殖有一定的抑制作用,且其抑制作用与剂量有一定的相关性；各干预组与对照组比较人骨肉瘤类肿瘤干细胞中Go/G1期的细胞比例明显增多(P<0.01),增殖指数明显减低(P<0.01)；随着LY294002浓度的不断增加,人骨肉瘤类肿瘤干细胞中的pAKT表达量逐渐下降。3.随着LY294002浓度的逐渐增加,各干预组活细胞有所下降,而凋亡有所增加,这种对凋亡的促进作用同样表达出一定的剂量依赖性,其中45μmol/L组最为明显(P<0.01)；在对照组和干预组中均可以检测到半胱天冬酶-3的表达,但是活化型半胱天冬酶-3只有在干预组中才能观察到明显的表达,且活化型半胱天冬酶-3和聚腺苷二磷酸核糖聚合酶的表达量随LY294002浓度的增高也呈现上升趋势。结论：1.无血清培养法可以用于人骨肉瘤类肿瘤干细胞的分离和培养；Nanog作为维持细胞多能性的关键蛋白,可能成为分离和鉴定入骨肉瘤类肿瘤干细胞的标志之一。2.LY294002对人骨肉瘤类肿瘤干细胞表现出对增殖的抑制作用,该作用可能是通过其抑制PI3K而导致其下游AKT活性减弱,细胞发生G0/G1期细胞周期阻滞所介导的。3.LY294002对人骨肉瘤类肿瘤干细胞表现出对凋亡的促进作用,该作用可能是通过其抑制PI3K导致其下游AKT活性减弱,从而激活半胱天冬酶-9、半胱天冬酶-3和聚腺苷二磷酸核糖聚合酶等参与的凋亡通路所介导的。更多还原

【Abstract】 Objective1. To isolate and culture human osteosarcoma cancer stem-like cells with serum-free cell culture method and magnetic activated cell sorting method, and to explore the feasibility of both as sorting method.2. To investigate the expression and significance of Nanog in human osteosarcoma and its cancer stem-like cells.3. To investigate the effect of LY294002on the proliferation and cell cycle of human osteosarcoma cancer stem-like cells, and to explore the possible mechanism in the inhibitory effect on proliferation.4. To investigate the effect of LY294002on the apoptosis of human osteosarcoma cancer stem-like cells, and to explore the possible mechanism to enhance apoptosis.Methods1. Human osteosarcoma cancer stem-like cells were isolated with serum-free media. CD133+CD44+human osteosarcoma cancer stem-like cells were isolated with magnetic activated cell sorting method. The expression levels of CD44and CD133in human osteosarcoma cancer stem-like cells were detected by using immunohistochemistry. The expression of Nanog in human osteosarcoma and its cancer stem-like cells were detected by western blot and analyzed by semi-quantitative method.2. Experimental groups:the concentration of LY294002in culture medium was Oumol/L (control group),5μmol/L,15μmol/L and45μmol/L respectively.3. The proliferation of human osteosarcoma cancer stem-like cells under different LY294002concentrations was detected by CCK-8method. The cell cycle disturbutions of human osteosarcoma cancer stem-like cells under different LY294002concentrations was detected by Propyl iodide staining andflow cytometry. Western blotting was employed to detect the expression levels of total Akt, p-Akt and β-actin in human osteosarcoma cancer stem-like cells in different LY294002concentrations.4. Annexin V/PI double staining and flow cytometry was employed to detect the apoptosis of human osteosarcoma cancer stem-like cells under different LY294002concentrations. Western blotting was employed to detect the expression levels of Caspase-3cleaved Caspase-3、Caspase-9、cleaved poly ADP-ribose polymerase.Results1. Human osteosarcoma cancer stem-like cells coule be isolated from human osteosarcoma MG63cells with serum-free media method, and coule grow as cell spheres. The CD133+CD44+human osteosarcoma cells could be isolated from human osteosarcoma MG63cells with magnetic activated cell sorting method, and cells could form cell spheres in serum-free media contained EGF and bFGF. The human osteosarcoma cancer stem-like cells sphere as previously described could be passaged in serum-free media and form similar human osteosarcoma stem-like cells spheres. The osteosarcoma cancer stem-like cells show positive expressions of CD44and CD133. The Nanog was higher expressed in osteosarcoma cancer stem-like cells than human osteosarcoma MG63cells (P<0.05).2. Compared with the control group, the proliferation of human osteosarcoma cancer stem-like cells could be significantly inhibited by LY294002regardless of concentration, and the inhibitory effect of LY294002on osteosarcoma cancer stem-like cells maybe dose-dependent. Compared with the control group, LY294002increase the number of osteosarcoma cancer stem-like cells in G0/G1phase (P<0.01) and a corresponding decrease in proliferation index (P<0.01) after treatment with LY294002. LY294002led to a decrease phosphorylation of AKT, and the expressions of phosphorylated AKT was corresponding decreased as the increasing LY294002concentration.3. The decrease of viable cells was corresponding to a increase in apoptotic cells with different concentrations of LY294002. The effect of apoptotic enhancement also maybe dose-dependent, and in45μmol/L group was significant (P<0.01). Caspase-3expression could be detected in both control group and the intervention groups, but cleaved caspases-3expression could only be obviously observed in the intervention groups, and cleaved caspases-3and poly ADP-ribose polymerase expression increased with concentration of LY294002.Conclusion1. Human osteosarcoma cancer stem-like cells could be isolated and cultured in serum-free medium; Nanog, a key protein in the maintenance of cell pluripotency, may serve as a marker for prostate cancer stem cells for isolation and identification of human osteosarcoma cancer stem-like cells.2. LY294002could inhibit the proliferation of human osteosarcoma cancer stem-like cells, and this effect may be relative to by inhibition of PI3K and its downstream AKT activity, and induction of G0/G1cell cycle arrest.3. LY294002could induce the apoptosis of human osteosarcoma cancer stem-like cells, and the effect may be related to by AKT activity reduction caused by inhibition of PI3K, and activation of Caspase-9, Caspaser3and poly ADP-ribose polymerase.更多还原