人子宫平滑肌肉瘤干细胞样细胞的分离及其特性的初步鉴定

发布时间：2018-02-23 17:29

本文关键词： 子宫平滑肌肉瘤肿瘤干细胞无血清悬浮培养　出处：《中南大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：从人子宫平滑肌肉瘤SK-UT-1细胞(SK-UT-1cells)中分离和培养子宫平滑肌肉瘤干细胞样细胞(Uterine leiomyosarcoma stem cell like cells, ULSSCLCs),并对其干细胞特性进行鉴定,以揭示肿瘤干细胞(cancer stem cells, CSCs)在子宫平滑肌肉瘤发生、发展中存在重要作用。方法：用含有生长因子的无血清培养基(Serum free medium,SFM)悬浮培养SK-UT-1cells,筛选出悬浮球细胞,并将其命名为子宫平滑肌肉瘤干细胞样细胞ULSSCLCs);采用流式细胞技术、有限稀释法、交替在含血清培养基(SSM)和SFM培养基中培养、台盼蓝染色、Transwell小室、流式细胞技术分别检测SK-UT-1cells和ULSSCLCs干细胞表面标志物CD133的表达差异、ULSSCLCs单个细胞成球能力、ULSSCLCs的分化能力、SK-UT-1cells及ULSSCLCs的增殖能力的差异、SK-UT-1cells及ULSSCLCs的体外迁移、侵袭能力以及对不同浓度顺铂作用下的耐药能力的差异。结果：1、SK-UT-1cells能在SFM中形成可以稳定传代的ULSSCLCs,目前已传到第7代。2、在CD133的表达中,ULSSCLCs高于SK-UT-1cells,差异有统计学意义(P0.05),并且随ULSSCLCs代数增加其表达量增高。3、有限稀释法可以在显微镜下观察到单个细胞成球全过程。4、将SFM中的ULSSCLCs接种于SSM中后可重新贴壁分化,分化后细胞与SK-UT-1cells形态无明显差异。5、在增殖试验中,ULSSCLCs增殖能力强于SK-UT-1cells,差异有统计学意义(P0.05)。6、ULSSCLCs迁移(215+14个)、侵袭(161±15个)能力显著高于SK-UT-1cells(106±11个).(61±7个),差异有统计学意义(P0.05)。7、SK-UT-1cells和ULSSCLCs经化疗药物顺铂诱导凋亡48h后,DDP浓度为40μmol/L、80μmol/L、150μmol/L时,SK-UT-1cells凋亡率高于ULSSCLCs,差异有统计学意义(P0.05)。SK-UT-1cells和ULSSCLCs的周期分布在顺铂处理前无明显差异(P0.05),两组细胞经顺铂处理48小时之后：处于S期的SK-UT-1cells细胞比例较处理前明显减少(P0.05), ULSSCLCs处于G2期的细胞比例较处理前明显升高(P0.05)。结论：1、成功从人子宫平滑肌肉瘤SK-UT-1cells中筛选出ULSSCLCs。 2、ULSSCLCs高表达肿瘤干细胞表面标记物CD133、并具有更强的自我更新、多向分化、增殖、迁移、侵袭及耐药能力。
[Abstract]:Objective: to isolate and culture Uterine leiomyosarcoma stem cell like cells from human leiomyosarcoma SK-UT-1 cell line SK-UT-1 cells, and to identify the stem cell characteristics of Uterine leiomyosarcoma stem cell like cells. To reveal that cancer stem cells play an important role in the development and development of uterine leiomyosarcoma. Methods: SK-UT-1 cells were cultured in Serum free medium SFM, a serum-free medium containing growth factor, and the suspension ball cells were selected and named as ULSSCSCsLCsL cells. The cells were cultured alternately in serum-containing medium (SSM) and SFM medium, and Trypan blue staining was performed in Transwell chamber. The difference of CD133 expression between SK-UT-1cells and ULSSCLCs stem cells was detected by flow cytometry. The differentiation ability of ULSSCLCs and the proliferation ability of SK-UT-1 cells and ULSSCLCs were compared with the migration of SK-UT-1 cells and ULSSCLCs in vitro. The difference in invasiveness and resistance to different concentrations of cisplatin. Results: SK-UT-1 cells could form stable ULSSCLC cells in SFM, which had been transferred to the 7th generation. The expression of ULSSCLCs in CD133 was higher than that of SK-UT-1 cells. The difference was statistically significant (P0.05), and the expression of ULSSCLC cells increased with the increase of ULSSCLCs algebra. Microscopically, the whole process of single cell spheroidization was observed. After inoculating ULSSCLCs in SFM into SSM, the adherent differentiation could be reobserved. There was no significant difference in morphology between the cells after differentiation and that of SK-UT-1cells. The proliferative ability of LCs was stronger than that of SK-UT-1 cells in proliferative test. The difference was statistically significant (P 0.05U. 6UT-1cells, 161 卤15). The ability of invasion was significantly higher than that of SK-UT-1cells(106 卤11 cells. 61 卤7. The difference was statistically significant (P 0.05. SK-UT-1cells and SK-UT-1cells). After 48 hours of apoptosis induced by cisplatin, the apoptosis rate of SK-UT-1 cells in ULSSCLCs was higher than that of ULSSCLCs at the concentration of 40 渭 mol / L ~ 80 渭 mol / L ~ (150 渭 mol / L). There was no significant difference in cell cycle distribution between P0.05 SK-UT-1 cells and ULSSCLCs before and after cisplatin treatment, and there was no significant difference between the two groups after 48 hours of cisplatin treatment. The percentage of SK-UT-1cells cells in S phase was significantly lower than that before treatment, while the proportion of ULSSCLCs cells in G2 phase was significantly higher than that before treatment. Conclusion: 1, ULSSCLCswere successfully screened from SK-UT-1cells of human uterine leiomyosarcoma. 2ULSSCLCs expressed CD133on tumor stem cells, and had stronger ability of self-renewal, multi-differentiation, proliferation, migration, invasion and drug resistance.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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