人皮肤鳞状细胞癌A431、Colon16细胞系侧群细胞分选及其生物学特性的初步鉴定

发布时间：2018-04-18 15:31

本文选题：鳞状细胞癌 + A431细胞系　；参考：《北京协和医学院》2012年博士论文

【摘要】：肿瘤干细胞(Cancer Stem Cell, CSC)是存在于肿瘤组织中的一小部分具有干细胞性质的细胞群体,具有自我更新能力和不定向分化潜能。CSC是肿瘤形成及其不断生长的根源之一。Goodell等利用DNA特异性染料Hoechst33342和流式细胞术发现了侧群细胞(side population cells, SP cells)。SP细胞具有类似干细胞的自我更新和分化潜能,与干细胞有很多共性。目前绝大多数研究把分离出的SP细胞作为干细胞,利用流式细胞术,建立了SP细胞分选法。这一方法可用于未知表面标志物的正常干细胞和CSC分离,至今已在很多肿瘤组织中分离到了SP细胞。皮肤鳞状细胞癌是常见的皮肤科肿瘤,具有一定的侵袭力和转移力,鳞状细胞癌的CSC研究目前还处于初级阶段,其生物学特性还不明朗,其表面标志物尚未明确。本研究目的在于探讨A431细胞系、colon16细胞系及皮肤鳞癌组织中CSC是否存在,如存在,进一步对A431细胞系中CSC生长特性、干细胞标志分子的表达等部分生物学功能进行探讨,为研究有效治疗鳞状细胞癌药物奠定理论和实验基础。研究方法和内容 1、采用SP法分选人鳞状细胞癌A431,Colon16细胞系SP和非SP细胞,明确其中是否存在SP细胞及其比例。 2、分选并培养人A431细胞系中SP细胞,采用细胞增殖实验MTT法、克隆形成实验,比较SP和非SP细胞的增殖能力和克隆形成能力；RT-PCR法检测干细胞标志ABCG2分子在两者中的表达。 3、分选并培养人A431细胞系中的水平和非SP细胞,检测其维A酸受体RARα、 RARβ、RARγ、RXRα、RXRβ、RXR γ等的表达差异。 4、利用免疫组织化学方法观察日光性角化病、Bowen病和皮肤鳞状细胞癌组织中干细胞相关标记分子ABCG2、CK19和P63表达和定位,并结合图像分析技术对三者的表达进行分析。结果 1、人皮肤鳞状细胞癌A431细胞系中存在SP细胞,约占总细胞数的1.1%。人皮肤鳞状细胞癌Colon16细胞系存在SP细胞,约占总细胞数的1%。 2、人皮肤鳞状细胞癌A431细胞系中SP细胞较非SP细胞有更强的增殖能力和克隆形成能力,SP细胞组和非SP细胞组克隆形成数分别为114.8+4.95和44.5+3.67(P0.05),SP细胞干细胞标志分子ABCG2mRNA表达量较明显高于非侧群细胞(P0.05)。 3、人皮肤鳞状细胞癌A431细胞系中SP细胞较非SP细胞RARα、RARβ、RAR γ mRNA水平明显增高(P0.05)；而RXRa、RXRβ、RXR γ mRNA水平无明显差异(P0.05)。 4、ABCG2、CK19阳性反应物质定位于细胞膜和胞浆,皮肤鳞状细胞癌中ABCG2、CK19阳性细胞数显著高于日光性角化病和Bowen病(p0.01),而后两者之间无统计学差异；日光性角化病、Bowen病和皮肤鳞状细胞癌中ABCG2和CK19阳性病例比例逐渐增加,分别为ABCG2:40%,71.4%,84.6%和CK19：40%、50%、92.3%。P63阳性反应物质定位于细胞核,阳性反应细胞数在三者之间无统计学差异,所有病例均有P63阳性细胞。结论 1、人皮肤鳞状细胞癌细胞系A431、Colon16中存在SP细胞,采用流式细胞侧群分选技术可以成功分离、培养并进行细胞生物学实验。 2、A431细胞系中SP细胞具有干细胞特性,可培养出全克隆细胞集落。其增殖活性明显优于非SP细胞。SP细胞高表达干细胞标志分子ABCG2。 3、皮肤鳞状细胞癌组织中可检测到干细胞相关标志分子ABCG2、CK19、P63阳性细胞,这些分子在日光性角化病、Bowen病及皮肤鳞状细胞癌中的表达存在差异,这些分子可能与皮肤鳞状细胞癌的病理过程或预后相关。 4、干细胞标志分子ABCG2和维A酸受体RAR α、RAR β、RAR γ在A431细胞系SP和非SP细胞中的表达水平存在差异,这可能与肿瘤耐药相关,可成为皮肤鳞状细胞癌药物治疗的突破口,为且.维A酸辅助治疗皮肤鳞状细胞癌提供依据。
[Abstract]:Cancer stem cells (Cancer Stem Cell, CSC) is a small part of the tumor tissue with stem cell properties of the cell population, have the ability to self renew and differentiation of.CSC tumor formation and growth of root.Goodell using DNA specific dye Hoechst33342 and flow cytometry found side group cells (side population cells, SP cells) in.SP cells with similar stem cell self-renewal and differentiation potential, there are a lot of similarities with stem cells. Most of the isolated SP cells as stem cells using flow cytometry, established SP cell sorting method. This method can be used for the unknown surface markers of normal stem cells and CSC separation, has been in many tumor tissues were isolated from SP cells. Skin squamous cell carcinoma is a common tumor of Department of Dermatology, has certain invasiveness and metastasis CSC, study of squamous cell carcinoma is still in its infancy, its biological characteristics is not clear, the surface marker is not yet clear. The purpose of this study is to investigate the A431 cell line, the existence of CSC, colon16 cell lines and tissues such as skin squamous cell carcinoma, further growth of CSC characteristics of A431 cells, stem cell marker part of the biological function of molecule expression were discussed, lay a theoretical and experimental basis for the research of effective treatment of squamous cell carcinoma of the drug.
Research methods and contents
1, A431, Colon16 cell line SP and non SP cells were selected by SP method to determine whether there were SP cells and their proportion.
2, sorting and culturing SP cells in human A431 cell line, using cell proliferation test MTT method, clone formation experiment, comparing SP and non SP cell proliferation ability and colony forming ability; RT-PCR method to detect stem cell marker ABCG2 molecule expression in both.
3, the level and non SP cells in the human A431 cell line were selected and cultured, and the differences in the expression of the A acid receptor RAR alpha, RAR beta, RAR gamma, RXR a, RXR beta and RXR gamma were detected.
4, immunohistochemical staining was used to observe the expression and location of stem cell related markers ABCG2, CK19 and P63 in keratosis of Bowen, CK19 and P63.
Result
1, there are SP cells in the A431 cell line of human skin squamous cell carcinoma, which accounts for about 1.1%. of the total number of 1.1%. cells. There are SP cells in the skin squamous cell carcinoma Colon16 cell line, accounting for about 1%. of the total cell number.
2, human skin squamous cell carcinoma A431 cell line SP cells than non proliferation and SP cell clones have stronger ability of forming cells in SP group and non SP group cell clone number were 114.8+4.95 and 44.5+3.67 (P0.05), cell marker ABCG2mRNA expression was significantly higher than that of non SP cells SP stem cell (P0.05).
3, SP cell in human skin squamous cell carcinoma A431 cell line is significantly higher than that in non SP cell RAR alpha, RAR beta, RAR mRNA level (P0.05), while RXRa, RXR beta and RXR gamma level have no significant difference.
4, ABCG2, CK19 positioning the positive material in cell membrane and cytoplasm, ABCG2 in squamous cell carcinoma of the skin, the number of CK19 positive cells was significantly higher than that of actinic keratosis and Bowen disease (P0.01), and no significant difference between the two; actinic keratosis, Bowen disease and skin squamous cell carcinoma ABCG2 and CK19 positive cases proportion gradually increase, respectively ABCG2:40%, 71.4%, 84.6% and CK19:40%, 50%, 92.3%.P63 positive reaction substance located in the nucleus, there was no significant difference in the number of positive cells between the three, all cases had P63 positive cells.
conclusion
1, there are SP cells in human skin squamous cell carcinoma cell line A431 and Colon16, which can be successfully isolated, cultured and carried out in cell biology experiments by flow cytometry side group sorting technology.
2, SP cells in A431 cell line have the characteristics of stem cells, which can cultivate colony of whole clone cells. Its proliferative activity is better than that of non SP cells,.SP cells express high expression of stem cell marker ABCG2..
3, detection of stem cell related markers ABCG2, can be in skin squamous cell carcinoma CK19, P63 positive cells, these molecules in actinic keratosis, differences in expression of Bowen's disease and squamous cell carcinoma, these molecules may be the pathological and cutaneous squamous cell carcinoma related process or prognosis.
4, stem cell markers ABCG2 and A retinoic acid receptor RAR alpha, RAR beta, there are differences in the expression level of RAR gamma A431 SP cells and non SP cells, which may be associated with tumor resistance, can become a breakthrough, skin squamous cell carcinoma and drug treatment. Vitamin A acid in the treatment of skin squamous cancer cells provide the basis.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.5

【参考文献】