本文選題：鱗狀細(xì)胞癌 + A431細(xì)胞系　； 參考：《北京協(xié)和醫(yī)學(xué)院》2012年博士論文

【摘要】：腫瘤干細(xì)胞(Cancer Stem Cell, CSC)是存在于腫瘤組織中的一小部分具有干細(xì)胞性質(zhì)的細(xì)胞群體,具有自我更新能力和不定向分化潛能。CSC是腫瘤形成及其不斷生長的根源之一。Goodell等利用DNA特異性染料Hoechst33342和流式細(xì)胞術(shù)發(fā)現(xiàn)了側(cè)群細(xì)胞(side population cells, SP cells)。SP細(xì)胞具有類似干細(xì)胞的自我更新和分化潛能,與干細(xì)胞有很多共性。目前絕大多數(shù)研究把分離出的SP細(xì)胞作為干細(xì)胞,利用流式細(xì)胞術(shù),建立了SP細(xì)胞分選法。這一方法可用于未知表面標(biāo)志物的正常干細(xì)胞和CSC分離,至今已在很多腫瘤組織中分離到了SP細(xì)胞。皮膚鱗狀細(xì)胞癌是常見的皮膚科腫瘤,具有一定的侵襲力和轉(zhuǎn)移力,鱗狀細(xì)胞癌的CSC研究目前還處于初級(jí)階段,其生物學(xué)特性還不明朗,其表面標(biāo)志物尚未明確。本研究目的在于探討A431細(xì)胞系、colon16細(xì)胞系及皮膚鱗癌組織中CSC是否存在,如存在,進(jìn)一步對(duì)A431細(xì)胞系中CSC生長特性、干細(xì)胞標(biāo)志分子的表達(dá)等部分生物學(xué)功能進(jìn)行探討,為研究有效治療鱗狀細(xì)胞癌藥物奠定理論和實(shí)驗(yàn)基礎(chǔ)。 研究方法和內(nèi)容 1、采用SP法分選人鱗狀細(xì)胞癌A431,Colon16細(xì)胞系SP和非SP細(xì)胞,明確其中是否存在SP細(xì)胞及其比例。 2、分選并培養(yǎng)人A431細(xì)胞系中SP細(xì)胞,采用細(xì)胞增殖實(shí)驗(yàn)MTT法、克隆形成實(shí)驗(yàn),比較SP和非SP細(xì)胞的增殖能力和克隆形成能力；RT-PCR法檢測干細(xì)胞標(biāo)志ABCG2分子在兩者中的表達(dá)。 3、分選并培養(yǎng)人A431細(xì)胞系中的水平和非SP細(xì)胞,檢測其維A酸受體RARα、 RARβ、RARγ、RXRα、RXRβ、RXR γ等的表達(dá)差異。 4、利用免疫組織化學(xué)方法觀察日光性角化病、Bowen病和皮膚鱗狀細(xì)胞癌組織中干細(xì)胞相關(guān)標(biāo)記分子ABCG2、CK19和P63表達(dá)和定位,并結(jié)合圖像分析技術(shù)對(duì)三者的表達(dá)進(jìn)行分析。 結(jié)果 1、人皮膚鱗狀細(xì)胞癌A431細(xì)胞系中存在SP細(xì)胞,約占總細(xì)胞數(shù)的1.1%。人皮膚鱗狀細(xì)胞癌Colon16細(xì)胞系存在SP細(xì)胞,約占總細(xì)胞數(shù)的1%。 2、人皮膚鱗狀細(xì)胞癌A431細(xì)胞系中SP細(xì)胞較非SP細(xì)胞有更強(qiáng)的增殖能力和克隆形成能力,SP細(xì)胞組和非SP細(xì)胞組克隆形成數(shù)分別為114.8+4.95和44.5+3.67(P0.05),SP細(xì)胞干細(xì)胞標(biāo)志分子ABCG2mRNA表達(dá)量較明顯高于非側(cè)群細(xì)胞(P0.05)。 3、人皮膚鱗狀細(xì)胞癌A431細(xì)胞系中SP細(xì)胞較非SP細(xì)胞RARα、RARβ、RAR γ mRNA水平明顯增高(P0.05)；而RXRa、RXRβ、RXR γ mRNA水平無明顯差異(P0.05)。 4、ABCG2、CK19陽性反應(yīng)物質(zhì)定位于細(xì)胞膜和胞漿,皮膚鱗狀細(xì)胞癌中ABCG2、CK19陽性細(xì)胞數(shù)顯著高于日光性角化病和Bowen病(p0.01),而后兩者之間無統(tǒng)計(jì)學(xué)差異；日光性角化病、Bowen病和皮膚鱗狀細(xì)胞癌中ABCG2和CK19陽性病例比例逐漸增加,分別為ABCG2:40%,71.4%,84.6%和CK19：40%、50%、92.3%。P63陽性反應(yīng)物質(zhì)定位于細(xì)胞核,陽性反應(yīng)細(xì)胞數(shù)在三者之間無統(tǒng)計(jì)學(xué)差異,所有病例均有P63陽性細(xì)胞。 結(jié)論 1、人皮膚鱗狀細(xì)胞癌細(xì)胞系A(chǔ)431、Colon16中存在SP細(xì)胞,采用流式細(xì)胞側(cè)群分選技術(shù)可以成功分離、培養(yǎng)并進(jìn)行細(xì)胞生物學(xué)實(shí)驗(yàn)。 2、A431細(xì)胞系中SP細(xì)胞具有干細(xì)胞特性,可培養(yǎng)出全克隆細(xì)胞集落。其增殖活性明顯優(yōu)于非SP細(xì)胞。SP細(xì)胞高表達(dá)干細(xì)胞標(biāo)志分子ABCG2。 3、皮膚鱗狀細(xì)胞癌組織中可檢測到干細(xì)胞相關(guān)標(biāo)志分子ABCG2、CK19、P63陽性細(xì)胞,這些分子在日光性角化病、Bowen病及皮膚鱗狀細(xì)胞癌中的表達(dá)存在差異,這些分子可能與皮膚鱗狀細(xì)胞癌的病理過程或預(yù)后相關(guān)。 4、干細(xì)胞標(biāo)志分子ABCG2和維A酸受體RAR α、RAR β、RAR γ在A431細(xì)胞系SP和非SP細(xì)胞中的表達(dá)水平存在差異,這可能與腫瘤耐藥相關(guān),可成為皮膚鱗狀細(xì)胞癌藥物治療的突破口,為且.維A酸輔助治療皮膚鱗狀細(xì)胞癌提供依據(jù)。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.5

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相關(guān)期刊論文 前2條

1 林旭勇;劉樹立;劉楠;楊小n\;徐洪濤;王恩華;;干細(xì)胞標(biāo)記物CK19、Notch3、CD133、P75NTR、STRO-1及ABCG2在肺鱗癌中的表達(dá)及意義[J];中國肺癌雜志;2009年04期

2 楊麗娟;耿松梅;;全反式維A酸對(duì)A431細(xì)胞中BMI-1表達(dá)的影響[J];中國皮膚性病學(xué)雜志;2009年10期

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本文編號(hào)：1768967