人肾癌干细胞的培养鉴定及其端粒酶活性的研究
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摘要
目的:通过对人肾癌细胞的无血清培养获取肾癌干细胞,找出人肾癌干细胞的成熟的培养方法;进一步检测肾癌细胞、肾癌干细胞中端粒酶的活性,从而探讨端粒酶活性在肾癌发生、发展及治疗方面的重要意义。
方法:利用含有表皮生长因子(Epidermal Growth Factor,EGF)、碱性成纤维生长因子(basic Fibroblast Growth Factor,bFGF)的DMEM/F12培养基无血清悬浮培养分离肾癌干细胞,并观察其生长特性;通过流式细胞仪检测其CD133、CD34的表达情况及其凋亡率,以正常肾组织细胞及肾癌细胞作为对照,采用端粒重复序列扩增的方法(Telomeric Repeat Amplification Protocol,TRAP)实时定量检测肾癌干细胞端粒酶的活性。
结果:肾癌干细胞接种于DMEM/F12无血清培养基后,细胞呈圆形悬浮于培养液中;2天后有细胞球生成,每个细胞球含3～8个细胞,折光性较强;7天后细胞球明显增多,体积增大,为规则的圆形或卵圆形。无血清培养的肾癌干细胞球CD133~+CD34~-率明显高于含血清培养的肾癌细胞CD133~+CD34~-率,凋亡率较血清培养的肾癌细胞低;正常肾组织细胞未检测出有CD133及CD34的表达,三者间比较有显著意义(F=328.25,P＜0.05)。肾癌干细胞球和肾癌细胞端粒酶活性均高于正常肾组织细胞(F=278.74,P＜0.05),而前二者之间端粒酶活性无明显差异。
结论:与含血清培养的肾癌细胞相比无血清悬浮培养的肾癌干细胞表面标志CD133呈高表达,有着较低的凋亡率,二者端粒酶的活性均高于正常肾组织细胞。
Objective: Method of suspension culture in serum free medium (SFM) was used to enrich renal carcinoma (RCC) stem cells in order to find the mature methods of culturing human renal carcinoma stem cells ; Telomerase activity in renal carcinoma cells and renal carcinoma stem cells was envalued to discuss the significance to telomerase activity for the occurrence , development and treatment of RCC .
Methods:Serum free medium (SFM) containing Epidermal Growth Factor (EGFX basic Fibroblast Growth Factor (bFGF) was used to enrich RCC stem cells while observing their growing characteristic . And flow cytometry was used to detect the expression of CD133 and CD34 and the apoptotic rate of RCC stem cells . Then real-time quantitative TRAP assay was applied to envalue telomerase activity in CD133~+ and CD133~- cells with normal renal cells as negative control.
Result: After incubated in serum-free medium , Renal carcinoma stem cells were round and suspended . Two days later , cell mass generated . Each cell mass contained 3-8 cell , with strong refraction . Seven days later, cell mass became more , presented big body that was regular , round or elliptical . CD133~+CD34~- rate in renal carcinoma stem cell mass was significantly greater , and apoptotic rate lower , in serum-free suspension culture compared with in serum suspension culture . CD133 and CD34 expression was not determined in normal renal tissue . There were significant differences among groups (F=328.25, P<0.05) . Telomerase activity was greater in renal carcinoma stem cells and renal carcinoma cells compared with normal renal cells (F=278.74, P<0.05) . No significant difference was detected between renal carcinoma stem cells and renal carcinoma cells .
Conclusions: Compared with serum cultured renal carcinoma cells, serum-free cultured renal carcinoma cells surface marker CD133 presents higher expression , apoptotic rate lower . Moreover , telomerase activity is higher in renal carcinoma stem cells and renal carcinoma cells compared with normal renal tissue .
方法:利用含有表皮生长因子(Epidermal Growth Factor,EGF)、碱性成纤维生长因子(basic Fibroblast Growth Factor,bFGF)的DMEM/F12培养基无血清悬浮培养分离肾癌干细胞,并观察其生长特性;通过流式细胞仪检测其CD133、CD34的表达情况及其凋亡率,以正常肾组织细胞及肾癌细胞作为对照,采用端粒重复序列扩增的方法(Telomeric Repeat Amplification Protocol,TRAP)实时定量检测肾癌干细胞端粒酶的活性。
结果:肾癌干细胞接种于DMEM/F12无血清培养基后,细胞呈圆形悬浮于培养液中;2天后有细胞球生成,每个细胞球含3～8个细胞,折光性较强;7天后细胞球明显增多,体积增大,为规则的圆形或卵圆形。无血清培养的肾癌干细胞球CD133~+CD34~-率明显高于含血清培养的肾癌细胞CD133~+CD34~-率,凋亡率较血清培养的肾癌细胞低;正常肾组织细胞未检测出有CD133及CD34的表达,三者间比较有显著意义(F=328.25,P＜0.05)。肾癌干细胞球和肾癌细胞端粒酶活性均高于正常肾组织细胞(F=278.74,P＜0.05),而前二者之间端粒酶活性无明显差异。
结论:与含血清培养的肾癌细胞相比无血清悬浮培养的肾癌干细胞表面标志CD133呈高表达,有着较低的凋亡率,二者端粒酶的活性均高于正常肾组织细胞。
Objective: Method of suspension culture in serum free medium (SFM) was used to enrich renal carcinoma (RCC) stem cells in order to find the mature methods of culturing human renal carcinoma stem cells ; Telomerase activity in renal carcinoma cells and renal carcinoma stem cells was envalued to discuss the significance to telomerase activity for the occurrence , development and treatment of RCC .
Methods:Serum free medium (SFM) containing Epidermal Growth Factor (EGFX basic Fibroblast Growth Factor (bFGF) was used to enrich RCC stem cells while observing their growing characteristic . And flow cytometry was used to detect the expression of CD133 and CD34 and the apoptotic rate of RCC stem cells . Then real-time quantitative TRAP assay was applied to envalue telomerase activity in CD133~+ and CD133~- cells with normal renal cells as negative control.
Result: After incubated in serum-free medium , Renal carcinoma stem cells were round and suspended . Two days later , cell mass generated . Each cell mass contained 3-8 cell , with strong refraction . Seven days later, cell mass became more , presented big body that was regular , round or elliptical . CD133~+CD34~- rate in renal carcinoma stem cell mass was significantly greater , and apoptotic rate lower , in serum-free suspension culture compared with in serum suspension culture . CD133 and CD34 expression was not determined in normal renal tissue . There were significant differences among groups (F=328.25, P<0.05) . Telomerase activity was greater in renal carcinoma stem cells and renal carcinoma cells compared with normal renal cells (F=278.74, P<0.05) . No significant difference was detected between renal carcinoma stem cells and renal carcinoma cells .
Conclusions: Compared with serum cultured renal carcinoma cells, serum-free cultured renal carcinoma cells surface marker CD133 presents higher expression , apoptotic rate lower . Moreover , telomerase activity is higher in renal carcinoma stem cells and renal carcinoma cells compared with normal renal tissue .
引文
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