喉癌细胞系肿瘤侧群细胞的研究

发布时间：2018-06-30 05:57

本文选题：Hep-2细胞系 + 肿瘤干细胞　；参考：《复旦大学》2010年博士论文

【摘要】： 第一部分人喉癌细胞系Hep-2和AMC-HN-8中侧群细胞的检测和分选目的探讨人喉癌细胞系中是否存在肿瘤侧群细胞及建立可靠的喉癌细胞系侧群细胞(SP细胞)检测及分选的操作规程。方法以人喉癌细胞系Hep-2和AMC-HN-8为研究对象,5μg/mL Hoechst 33342, 150μmol/L维拉帕米,1μg/mL碘化丙啶,应用流式细胞技术检测细胞系中SP细胞的比例并对Hep-2中SP细胞进行分选并检测其纯度。以含血清培养基培养SP及non-SP细胞及细胞爬片,观察细胞活性及培养后细胞形态。结果Hep-2细胞SP比例为17.1±2.0%,AMC-HN-8中SP的比例为11.8±1.7%。经维拉帕米处理后比例分别降为0%及0.3±0.1%。流式细胞术分选Hep-2细胞SP的纯度可达99.2±0.2%,non-SP细胞的纯度达98.5±0.5%；在含血清培养基中SP及non-SP细胞均贴壁生长,死细胞少,爬片检查提示均为典型的鳞癌细胞表现。结论喉癌细胞系中存在SP细胞,能被维拉帕米所抑制,流式细胞技术可有效的分选SP细胞。第二部分人喉癌Hep-2细胞系中肿瘤侧群细胞生物学特性目的通过与non-SP相比较,研究喉癌Hep-2细胞系肿瘤SP细胞的生物学特性。方法分选的1×104SP细胞及non-SP细胞以0.2ml无血清培养基种植于同一96孔板上,在1、3、5、7d观察生长状态并用CCK-8法测定细胞的增殖状态,绘制细胞生长曲线,比较两组细胞的增殖速度；分选后细胞在含血清培养基中培养的0、4、8、12d用流式细胞仪动态测试SP细胞在培养体系中的百分比,以评估其分化能力；分选的1×104SP细胞及non-SP细胞种植于同一96孔板上,培养1天后以2Gy的剂量射线照射,2d后用CCK-8法测定细胞的增殖状态,计算射线对细胞的抑制率,比较两组细胞对放射线的耐受性；将1×105,5×104及2×104的SP及non-SP细胞分别注射于8只NOD/SCID鼠的腋窝皮下,8周后观察成瘤情况,比较两组细胞的致瘤性。结果在无血清环境下SP细胞及non-SP呈不贴壁、球形、半透明状,培养后SP细胞渐成簇生长,而non-SP细胞不能形成细胞球；在第1、3、5、7d时SP细胞的吸光度分别为0.665±0.017,1.086±0.069,1.387±1.107,1.675±0.07,而non-SP细胞为0.694±0.053 0.951±0.031 1.049±0.092,1.008±0.086,除第1d外均具有显著性差异；分选后SP细胞在含血清培养基中培养的4、8、12d时SP的比例为47.8±1.1%,27.8±3.6%和17.3±1.9%,而non-SP培养后为2.2±0.1%,4.7±0.4%和4.9±0.3%；经射线照射后SP细胞与non-SP细胞的抑制率分别为6.7±3.5%和29.9±4.9%(t=14.295,p=.000)；细胞数为5×104,SP与non-SP的成瘤数分别为7和2(p=0.41),细胞数为2×104时成瘤数SP细胞成瘤数为6,而non-SP不能成瘤(p=0.007)。结论Hep-2细胞系中的SP细胞具有很强的增殖、分化、成瘤能力,对射线具有耐受性,证明该SP细胞具有干细胞的相关特性,它富含肿瘤起始细胞。但同时也说明SP细胞也是不均质的,我们不能将SP细胞认为是肿瘤干细胞。第三部分人喉癌Hep-2细胞系肿瘤侧群细胞中干细胞相关基因的表达分析目的探讨干细胞相关基因CD133、ABCG2、Bmi1、Notch2及PTEN在SP细胞中的表达。方法以Realtime PCR检测SP细胞与non-SP细胞CD133、ABCG2、Bmi1、Notch2及PTEN在SP细胞中的表达的差异。结果CD133、ABCG2、Bmi1及NOTCH 2在SP细胞中高表达,而PTEN在SP细胞与non-SP细胞中表达无明显差异结论干细胞相关基因在SP细胞的形成及维持中可能起重要作用,ABCG2在SP细胞的形成中起重要作用,这些基因可能成为喉癌治疗的潜在靶点。
[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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.65

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