本文關(guān)鍵詞： 子宮平滑肌肉瘤 腫瘤干細(xì)胞 無血清懸浮培養(yǎng)　出處：《中南大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：從人子宮平滑肌肉瘤SK-UT-1細(xì)胞(SK-UT-1cells)中分離和培養(yǎng)子宮平滑肌肉瘤干細(xì)胞樣細(xì)胞(Uterine leiomyosarcoma stem cell like cells, ULSSCLCs),并對(duì)其干細(xì)胞特性進(jìn)行鑒定,以揭示腫瘤干細(xì)胞(cancer stem cells, CSCs)在子宮平滑肌肉瘤發(fā)生、發(fā)展中存在重要作用。 方法：用含有生長因子的無血清培養(yǎng)基(Serum free medium,SFM)懸浮培養(yǎng)SK-UT-1cells,篩選出懸浮球細(xì)胞,并將其命名為子宮平滑肌肉瘤干細(xì)胞樣細(xì)胞ULSSCLCs);采用流式細(xì)胞技術(shù)、有限稀釋法、交替在含血清培養(yǎng)基(SSM)和SFM培養(yǎng)基中培養(yǎng)、臺(tái)盼藍(lán)染色、Transwell小室、流式細(xì)胞技術(shù)分別檢測SK-UT-1cells和ULSSCLCs干細(xì)胞表面標(biāo)志物CD133的表達(dá)差異、ULSSCLCs單個(gè)細(xì)胞成球能力、ULSSCLCs的分化能力、SK-UT-1cells及ULSSCLCs的增殖能力的差異、SK-UT-1cells及ULSSCLCs的體外遷移、侵襲能力以及對(duì)不同濃度順鉑作用下的耐藥能力的差異。 結(jié)果：1、SK-UT-1cells能在SFM中形成可以穩(wěn)定傳代的ULSSCLCs,目前已傳到第7代。2、在CD133的表達(dá)中,ULSSCLCs高于SK-UT-1cells,差異有統(tǒng)計(jì)學(xué)意義(P0.05),并且隨ULSSCLCs代數(shù)增加其表達(dá)量增高。3、有限稀釋法可以在顯微鏡下觀察到單個(gè)細(xì)胞成球全過程。4、將SFM中的ULSSCLCs接種于SSM中后可重新貼壁分化,分化后細(xì)胞與SK-UT-1cells形態(tài)無明顯差異。5、在增殖試驗(yàn)中,ULSSCLCs增殖能力強(qiáng)于SK-UT-1cells,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。6、ULSSCLCs遷移(215+14個(gè))、侵襲(161±15個(gè))能力顯著高于SK-UT-1cells(106±11個(gè)).(61±7個(gè)),差異有統(tǒng)計(jì)學(xué)意義(P0.05)。7、SK-UT-1cells和ULSSCLCs經(jīng)化療藥物順鉑誘導(dǎo)凋亡48h后,DDP濃度為40μmol/L、80μmol/L、150μmol/L時(shí),SK-UT-1cells凋亡率高于ULSSCLCs,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。SK-UT-1cells和ULSSCLCs的周期分布在順鉑處理前無明顯差異(P0.05),兩組細(xì)胞經(jīng)順鉑處理48小時(shí)之后：處于S期的SK-UT-1cells細(xì)胞比例較處理前明顯減少(P0.05), ULSSCLCs處于G2期的細(xì)胞比例較處理前明顯升高(P0.05)。 結(jié)論：1、成功從人子宮平滑肌肉瘤SK-UT-1cells中篩選出ULSSCLCs。 2、ULSSCLCs高表達(dá)腫瘤干細(xì)胞表面標(biāo)記物CD133、并具有更強(qiáng)的自我更新、多向分化、增殖、遷移、侵襲及耐藥能力。
[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 鹵15). The ability of invasion was significantly higher than that of SK-UT-1cells(106 鹵11 cells. 61 鹵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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33

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