本文選題：Hep-2細胞系 + 腫瘤干細胞��； 參考：《復(fù)旦大學(xué)》2010年博士論文

【摘要】： 第一部分人喉癌細胞系Hep-2和AMC-HN-8中側(cè)群細胞的檢測和分選 目的探討人喉癌細胞系中是否存在腫瘤側(cè)群細胞及建立可靠的喉癌細胞系側(cè)群細胞(SP細胞)檢測及分選的操作規(guī)程。 方法以人喉癌細胞系Hep-2和AMC-HN-8為研究對象,5μg/mL Hoechst 33342, 150μmol/L維拉帕米,1μg/mL碘化丙啶,應(yīng)用流式細胞技術(shù)檢測細胞系中SP細胞的比例并對Hep-2中SP細胞進行分選并檢測其純度。以含血清培養(yǎng)基培養(yǎng)SP及non-SP細胞及細胞爬片,觀察細胞活性及培養(yǎng)后細胞形態(tài)。 結(jié)果Hep-2細胞SP比例為17.1±2.0%,AMC-HN-8中SP的比例為11.8±1.7%。經(jīng)維拉帕米處理后比例分別降為0%及0.3±0.1%。流式細胞術(shù)分選Hep-2細胞SP的純度可達99.2±0.2%,non-SP細胞的純度達98.5±0.5%；在含血清培養(yǎng)基中SP及non-SP細胞均貼壁生長,死細胞少,爬片檢查提示均為典型的鱗癌細胞表現(xiàn)。 結(jié)論喉癌細胞系中存在SP細胞,能被維拉帕米所抑制,流式細胞技術(shù)可有效的分選SP細胞。 第二部分人喉癌Hep-2細胞系中腫瘤側(cè)群細胞生物學(xué)特性 目的通過與non-SP相比較,研究喉癌Hep-2細胞系腫瘤SP細胞的生物學(xué)特性。 方法分選的1×104SP細胞及non-SP細胞以0.2ml無血清培養(yǎng)基種植于同一96孔板上,在1、3、5、7d觀察生長狀態(tài)并用CCK-8法測定細胞的增殖狀態(tài),繪制細胞生長曲線,比較兩組細胞的增殖速度；分選后細胞在含血清培養(yǎng)基中培養(yǎng)的0、4、8、12d用流式細胞儀動態(tài)測試SP細胞在培養(yǎng)體系中的百分比,以評估其分化能力；分選的1×104SP細胞及non-SP細胞種植于同一96孔板上,培養(yǎng)1天后以2Gy的劑量射線照射,2d后用CCK-8法測定細胞的增殖狀態(tài),計算射線對細胞的抑制率,比較兩組細胞對放射線的耐受性；將1×105,5×104及2×104的SP及non-SP細胞分別注射于8只NOD/SCID鼠的腋窩皮下,8周后觀察成瘤情況,比較兩組細胞的致瘤性。 結(jié)果在無血清環(huán)境下SP細胞及non-SP呈不貼壁、球形、半透明狀,培養(yǎng)后SP細胞漸成簇生長,而non-SP細胞不能形成細胞球；在第1、3、5、7d時SP細胞的吸光度分別為0.665±0.017,1.086±0.069,1.387±1.107,1.675±0.07,而non-SP細胞為0.694±0.053 0.951±0.031 1.049±0.092,1.008±0.086,除第1d外均具有顯著性差異；分選后SP細胞在含血清培養(yǎng)基中培養(yǎng)的4、8、12d時SP的比例為47.8±1.1%,27.8±3.6%和17.3±1.9%,而non-SP培養(yǎng)后為2.2±0.1%,4.7±0.4%和4.9±0.3%；經(jīng)射線照射后SP細胞與non-SP細胞的抑制率分別為6.7±3.5%和29.9±4.9%(t=14.295,p=.000)；細胞數(shù)為5×104,SP與non-SP的成瘤數(shù)分別為7和2(p=0.41),細胞數(shù)為2×104時成瘤數(shù)SP細胞成瘤數(shù)為6,而non-SP不能成瘤(p=0.007)。 結(jié)論Hep-2細胞系中的SP細胞具有很強的增殖、分化、成瘤能力,對射線具有耐受性,證明該SP細胞具有干細胞的相關(guān)特性,它富含腫瘤起始細胞。但同時也說明SP細胞也是不均質(zhì)的,我們不能將SP細胞認為是腫瘤干細胞。 第三部分 人喉癌Hep-2細胞系腫瘤側(cè)群細胞中干細胞相關(guān)基因的表達分析 目的探討干細胞相關(guān)基因CD133、ABCG2、Bmi1、Notch2及PTEN在SP細胞中的表達。 方法以Realtime PCR檢測SP細胞與non-SP細胞CD133、ABCG2、Bmi1、Notch2及PTEN在SP細胞中的表達的差異。 結(jié)果CD133、ABCG2、Bmi1及NOTCH 2在SP細胞中高表達,而PTEN在SP細胞與non-SP細胞中表達無明顯差異 結(jié)論干細胞相關(guān)基因在SP細胞的形成及維持中可能起重要作用,ABCG2在SP細胞的形成中起重要作用,這些基因可能成為喉癌治療的潛在靶點。
[Abstract]:Part one detection and sorting of side population cells in human laryngeal carcinoma cell lines Hep-2 and AMC-HN-8
Objective to investigate whether there are tumor side population cells in human laryngeal cancer cell lines and establish a reliable procedure for the detection and sorting of laryngeal cancer cell line side population cells (SP cells).
Methods the human larynx cell line Hep-2 and AMC-HN-8 were used as the study object, 5 mu g/mL Hoechst 33342, 150 mu mol/L verapamil and 1 g/mL iodide iodide. The proportion of SP cells in the cell lines was detected by flow cytometry and the SP cells in Hep-2 were selected and the purity was detected. SP and non-SP cells and cell crawling tablets were cultured with blood containing culture medium. The activity of the cells and the morphology of the cells were observed.
Results the proportion of SP in Hep-2 cells was 17.1 + 2%, and the proportion of SP in AMC-HN-8 was 11.8 + 1.7%. after treatment by 0% and 0.3 + 0.1%. flow cytometry. The purity of SP of Hep-2 cells was 99.2 + 0.2%, and the purity of non-SP cells was 98.5 + 0.5%. The SP and non-SP cells in the serum containing medium were all adhered to the wall, and the dead cells were few. The results showed that all of them were typical squamous cell carcinoma.
Conclusion there are SP cells in laryngeal cancer cell lines, which can be inhibited by Vera Pammy. Flow cytometry can effectively separate SP cells.
Biological characteristics of tumor side population cells in second human laryngeal cancer Hep-2 cell lines
Objective to study the biological characteristics of tumor SP cells in Hep-2 cell line of laryngocarcinoma by comparing with non-SP.
Methods the 1 x 104SP cells and non-SP cells were planted on the same 96 pore plate with 0.2ml serum-free medium. The growth state of the cells was observed in 1,3,5,7d and the proliferation of cells was measured by CCK-8. The proliferation rate of the two groups of cells was compared. The cells cultured in the medium containing serum were fined by flow cytometry after the separation. Cytosmeter dynamically measured the percentage of SP cells in the culture system in order to evaluate their differentiation ability. 1 x 104SP cells and non-SP cells were planted on the same 96 orifice plates. After 1 days of culture, the cells were irradiated with 2Gy dose rays. After 2D, the proliferation of cells was measured by CCK-8 method, and the inhibition rate of rays on the cells was calculated. The two groups of cells were compared to the radiation. Tolerance, 1 x 105,5 x 104 and 2 * 104 SP and non-SP cells were injected subcutaneously in the armpit of 8 NOD/SCID mice, and the tumor formation was observed after 8 weeks, and the tumorigenicity of the two groups of cells was compared.
Results in serum-free environment, SP cells and non-SP were not adherent, spherical and translucent, and SP cells grew gradually after culture, while non-SP cells could not form cell spheres; the absorbance of SP cells at the time of 1,3,5,7d was 0.665 + 0.017,1.086 + 0.069,1.387 + 0.07 respectively, while non-SP cells were 0.694 + 0.053 0.951 + 0.031 1.049 + 0.. 092,1.008 + 0.086, except for 1D, had significant differences. The proportion of SP in SP cells cultured in serum medium after separation was 47.8 + 1.1%, 27.8 + 3.6% and 17.3 + 1.9%, while non-SP culture was 2.2 + 0.1%, 4.7 + 0.4% and 4.9 + 0.3%, and the inhibition rates of SP cells and non-SP cells after irradiation were respectively 4.9% (t=14.295, p=.000); the number of cells was 5 x 104, the number of SP and non-SP was 7 and 2 (p=0.41), and the number of cells in the number of cells was 2 x 104, and the number of tumor cells was 6, while non-SP could not become a tumor (p=0.007).
Conclusion SP cells in Hep-2 cell line have strong proliferation, differentiation, tumorigenicity and tolerance to radiation. It is proved that the SP cells have the related characteristics of stem cells, which are rich in tumor starting cells. But it also indicates that SP cells are also heterogeneous, and we can not consider SP cells to be cancer stem cells.
The third part
Expression of stem cell related genes in tumor side population cells of human laryngeal carcinoma Hep-2 cell line
Objective to investigate the expression of stem cell related genes CD133, ABCG2, Bmi1, Notch2 and PTEN in SP cells.
Methods the expressions of CD133, ABCG2, Bmi1, Notch2 and PTEN in SP cells were detected by Realtime PCR. The differences between SP cells and CD133 cells were observed.
Results CD133, ABCG2, Bmi1 and NOTCH 2 were highly expressed in SP cells, while PTEN showed no significant difference between SP cells and non-SP cells.
Conclusion stem cell related genes may play an important role in the formation and maintenance of SP cells, and ABCG2 plays an important role in the formation of SP cells. These genes may be potential targets for the treatment of larynx cancer.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R739.65

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本文編號：2085214