本文選題：喉癌 + 腫瘤干細(xì)胞��； 參考：《吉林大學(xué)》2011年博士論文

【摘要】：喉癌是東北地區(qū)患病率較高的惡性腫瘤,并且其發(fā)病率有明顯增長(zhǎng)的趨勢(shì)。目前主要采用手術(shù)、放化療及生物治療等多種治療手段有機(jī)結(jié)合的綜合治療,但病人的生存率并沒(méi)有得到太大改善,而且更無(wú)法解決腫瘤對(duì)放化療抵抗力強(qiáng)及復(fù)發(fā)轉(zhuǎn)移的問(wèn)題。 目前有關(guān)腫瘤干細(xì)胞的研究已經(jīng)取得了很大進(jìn)展,已在白血病及多種實(shí)體腫瘤分離出腫瘤干細(xì)胞。腫瘤干細(xì)胞具有自我更新能力,多向分化潛能,高致瘤性及耐藥性。越來(lái)越多的研究發(fā)現(xiàn)腫瘤干細(xì)胞對(duì)化療具有極強(qiáng)的抵抗性,治療后腫瘤干細(xì)胞常常存活下來(lái),又因?yàn)槠涓咦晕腋履芰霸鲋碀撃?使腫瘤復(fù)發(fā),治療失敗。Bmi-1對(duì)腫瘤干細(xì)胞的自我更新有重要作用,Bmi-1基因失活小鼠的白血病細(xì)胞移植入免疫缺陷小鼠后不能再產(chǎn)生白血病細(xì)胞。因此分析腫瘤干細(xì)胞的化療抵抗性,以及從腫瘤干細(xì)胞的角度來(lái)探索腫瘤治療,將是克服化療抵抗的理想方法,最終改善腫瘤的治療效果。目前關(guān)于喉癌中腫瘤干細(xì)胞的研究已經(jīng)取得了一定進(jìn)展。CD133是人喉癌Hep-2細(xì)胞中腫瘤干細(xì)胞標(biāo)志物,并且CD133+腫瘤干細(xì)胞比其他細(xì)胞亞群的體外分化和增殖能力強(qiáng),致瘤性高。這為進(jìn)一步以腫瘤干細(xì)胞的角度研究喉癌的化療抵抗性,以及靶向腫瘤干細(xì)胞治療,提供了前提和基礎(chǔ)。 本研究擬通過(guò)體內(nèi)外實(shí)驗(yàn)分析喉癌化療后CD133+細(xì)胞比例的變化及CD133+細(xì)胞對(duì)化療藥物的敏感性,探討人喉癌Hep-2細(xì)胞中腫瘤干細(xì)胞化療抵抗的特性,并通過(guò)沉默CD133+腫瘤干細(xì)胞胞中的Bmi-1基因,抑制CD133+腫瘤干細(xì)胞自我更新能力,抑制其增殖,促進(jìn)凋亡,增強(qiáng)化療敏感性,為以腫瘤干細(xì)胞為靶點(diǎn)治療喉癌進(jìn)行初步探索。 第一部分：喉癌Hep-2細(xì)胞中腫瘤干細(xì)胞與化療抵抗關(guān)系的初步研究 目的：探討喉癌Hep-2細(xì)胞中CD133+腫瘤干細(xì)胞的化療抵抗性,分析Bmi-1在CD133+腫瘤干細(xì)胞及CD133-細(xì)胞中的表達(dá)。方法：①體外培養(yǎng)人喉癌細(xì)胞系Hep-2細(xì)胞,應(yīng)用化療藥物(順鉑,5-FU,紫杉醇)作用后,流式細(xì)胞儀檢測(cè)CD133+細(xì)胞比例的變化。②建立裸鼠喉癌移植瘤模型,實(shí)驗(yàn)組以5-FU進(jìn)行治療,治療結(jié)束后,分離腫瘤組織,通過(guò)免疫組織化學(xué)染色和流式細(xì)胞儀檢測(cè)CD133表達(dá)情況。③流式細(xì)胞儀分選Hep-2細(xì)胞系CD133+腫瘤干細(xì)胞,CCK-8法檢測(cè)其化療敏感性。軟瓊脂克隆形成實(shí)驗(yàn)檢測(cè)5-FU作用后CD133+腫瘤干細(xì)胞克隆形成率,動(dòng)物成瘤實(shí)驗(yàn)檢測(cè)5-FU作用后CD133+腫瘤干細(xì)胞致瘤能力變化。④RT-PCR和Western-blot檢測(cè)CD133+腫瘤干細(xì)胞和CD133-細(xì)胞中Bmi-1的表達(dá)。結(jié)果：化療藥物(順鉑,5-FU,紫杉醇)作用48h后CD133+細(xì)胞富集2-4倍。CD133+表達(dá)率由原來(lái)的1.55%士0.28%增至5.16%士0.86%,4.94%士0.58%,3.66%士0.59%。裸鼠喉癌移植瘤模型中發(fā)現(xiàn)5-FU治療組CD133表達(dá)率為6.7%士1.6%較對(duì)照組2.6%士0.96%升高了約3倍。流式細(xì)胞分選技術(shù)成功分離純化CD133+腫瘤干細(xì)胞,發(fā)現(xiàn)其較CD133-細(xì)胞具有明顯的化療抵抗。CD133+腫瘤干細(xì)胞經(jīng)5-FU作用后克隆形成能力無(wú)明顯降低,成瘤時(shí)間延長(zhǎng),最終致瘤能力無(wú)明顯下降。CD133+腫瘤干細(xì)胞中的Bmi-1 mRNA和蛋白的表達(dá)量明顯高于CD133-細(xì)胞。結(jié)論：CD133+腫瘤干細(xì)胞的存在是喉癌化療抵抗的原因之一,CD133+腫瘤干細(xì)胞高表達(dá)自我更新關(guān)鍵因子Bmi-1。 第二部分RNAi沉默Bmi-1基因?qū)戆〩ep-2細(xì)胞中腫瘤干細(xì)胞生物學(xué)行為的影響及化療增敏作用 目的：探討RNAi沉默Bmi-1基因后喉癌Hep-2中CD133+腫瘤干細(xì)胞增殖、凋亡、侵襲及化療敏感性的變化,為喉癌的治療尋找新的靶點(diǎn),開(kāi)辟新思路。方法：將前期構(gòu)建的pFIV-H1/U6 Bmi-1 siRNA質(zhì)粒和陰性對(duì)照質(zhì)粒轉(zhuǎn)染人喉癌Hep-2細(xì)胞系CD133+腫瘤干細(xì)胞,RT-PCR、Western-blot檢測(cè)轉(zhuǎn)染細(xì)胞Bmi-1的表達(dá),選擇抑制效果最好的質(zhì)粒進(jìn)行后續(xù)實(shí)驗(yàn)。實(shí)驗(yàn)分成3組：空白對(duì)照組(Hep-2組),陰性對(duì)照組(siRNA-scramble組),實(shí)驗(yàn)組(Bmi-1 siRNA質(zhì)粒轉(zhuǎn)染組),RT-PCR和Western-blot檢測(cè)Bmi-1下游基因P16的表達(dá),CCK-8法繪制細(xì)胞增殖曲線,軟瓊脂克隆形成能力檢測(cè)細(xì)胞增殖能力,DAPI染色法及流式細(xì)胞儀檢測(cè)細(xì)胞凋亡,Transwell小室體外侵襲實(shí)驗(yàn)檢測(cè)細(xì)胞體外侵襲能力,CCK-8法檢測(cè)化療敏感性。結(jié)果：RT-PCR和Western-blot發(fā)現(xiàn)實(shí)驗(yàn)組P16基因相對(duì)表達(dá)水平增高(P0.01)；細(xì)胞生長(zhǎng)曲線及克隆形成能力顯示,實(shí)驗(yàn)組細(xì)胞的增殖受到抑制,與空白組和陰性對(duì)照組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.01);DAPI法和流式細(xì)胞儀檢測(cè)顯示實(shí)驗(yàn)組細(xì)胞凋亡率明顯高于空白組和陰性對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.01);Transwell小體外侵襲實(shí)驗(yàn)顯示細(xì)胞體外侵襲能力降低,實(shí)驗(yàn)組穿膜細(xì)胞與陰性對(duì)照組及空白對(duì)照組相比明顯減少(P0.05)；實(shí)驗(yàn)組CD133+腫瘤干細(xì)胞對(duì)5-FU的敏感性增加。結(jié)論：應(yīng)用RNAi技術(shù)沉默Bmi-1基因,抑制CD133+腫瘤干細(xì)胞自我更新及增殖,可抑制CD133+腫瘤干細(xì)胞的增殖及侵襲能力,促進(jìn)凋亡,增強(qiáng)對(duì)化療的敏感性。
[Abstract]:Laryngeal cancer is a malignant tumor with high prevalence in northeastern China, and its incidence has a tendency to increase obviously. At present, the combination of surgery, radiotherapy and chemotherapy and biological therapy is mainly used in the integrated treatment, but the survival rate of the patients has not been greatly improved, and it is more unable to solve the tumor's strong resistance to radiotherapy and chemotherapy. The problem of transfer.
At present, a lot of progress has been made in the research of cancer stem cells. Cancer stem cells have been isolated in leukemia and various solid tumors. The cancer stem cells have the ability to renew themselves, multidirectional differentiation potential, high tumorigenicity and resistance. More and more studies have found that the stem cells of tumor stem are highly resistant to chemotherapy and after treatment. The tumor stem cells often survive and cause the tumor to relapse because of their high self-renewal ability and proliferation potential. The treatment failure.Bmi-1 plays an important role in the self renewal of the tumor stem cells. The leukemia cells of the Bmi-1 gene inactivated mice can not produce the leukemia cells after transplantation into the immune deficient mice. Therefore, the analysis of the tumor stem cells is analyzed. The resistance to therapy and the exploration of cancer treatment from the angle of tumor stem cells will be the ideal method to overcome the resistance of chemotherapy and ultimately improve the therapeutic effect of the tumor. At present, the research on the cancer stem cells in the larynx cancer has made some progress..CD133 is the marker of the swelling of the tumor stem cells in the Hep-2 cells of human larynx cancer and the CD133+ tumor stem cells. The differentiation and proliferation ability and high tumorigenicity of the cells in vitro are higher than those of other subsets. This provides the premise and basis for the further study of the chemotherapeutic resistance of larynx cancer with the angle of tumor stem cells, as well as the target to the treatment of tumor stem cells.
This study is to analyze the changes in the proportion of CD133+ cells after chemotherapy and the sensitivity of CD133+ cells to chemotherapeutic drugs in vitro and in vivo, to explore the characteristics of chemotherapeutic resistance of cancer stem cells in Hep-2 cells of human larynx cancer, and to inhibit the self-renewal capacity of CD133+ tumor stem cells by silencing the Bmi-1 gene in the cell of CD133+ tumor stem cells and inhibiting the self-renewal capacity of the cancer stem cells. Its proliferation, promotion of apoptosis, and enhancement of chemosensitivity are the primary exploration of treating cancer of the larynx with the target of cancer stem cells.
Part one: preliminary study on the relationship between cancer stem cells and chemotherapy resistance in laryngeal carcinoma Hep-2 cells
Objective: To investigate the chemotherapeutic resistance of CD133+ tumor stem cells in Hep-2 cells of larynx cancer, and to analyze the expression of Bmi-1 in CD133+ tumor stem cells and CD133- cells. Methods: (1) the human laryngeal carcinoma cell line Hep-2 cells were cultured in vitro, and the change of the proportion of CD133+ cells was detected by flow cytometry after the action of chemotherapeutic drugs (cisplatin, 5-FU, paclitaxel). The experimental group was treated with 5-FU, and the experimental group was treated with 5-FU. After the treatment, the tumor tissue was separated. The expression of CD133 was detected by immunohistochemistry and flow cytometry. The Hep-2 cell line CD133+ tumor stem cells were selected by flow cytometry, and the sensitivity of chemotherapy was detected by CCK-8. The soft agar clone formation test was used to detect 5-FU. The clone formation rate of CD133+ tumor stem cells after action, and animal tumorigenesis test to detect the tumorigenicity of CD133+ tumor stem cells after 5-FU action. (4) RT-PCR and Western-blot were used to detect the expression of Bmi-1 in CD133+ tumor stem cells and CD133- cells. Results: 2-4 times the.CD133+ table was enriched after the action of chemotherapeutic drugs (cisplatin, 5-FU, paclitaxel) and 48h CD133+ cells. The rate of arrival was increased from 1.55%, 0.28% to 5.16%, 0.86%, 4.94%, 0.58%, and 3.66% 0.59%. nude mouse larynx tumor transplanted tumor model. The expression rate of CD133 in the 5-FU group was 6.7% 1.6% and 2.6% times higher than that of the control group 2.6% 0.96%. The flow cytometry technique successfully separated and purified the CD133+ tumor stem cells, and found that the CD133- cells were more obvious than those of the CD133- cells. The clone formation ability of.CD133+ tumor stem cells after 5-FU action was not significantly reduced, the time of tumor formation was prolonged, and the final tumorigenicity of.CD133+ tumor stem cells was not significantly decreased. The expression of Bmi-1 mRNA and protein in the tumor stem cells was significantly higher than that of CD133- cells. Conclusion: the existence of CD133+ tumor stem cells is one of the reasons for the resistance to chemotherapy in larynx cancer. CD133+ tumor stem cells highly expressed self renewal key factor Bmi-1.
The second part is the effect of RNAi silencing Bmi-1 gene on the biological behavior of cancer stem cells in laryngeal carcinoma Hep-2 cells and the sensitization effect of chemotherapy.
Objective: To explore the proliferation, apoptosis, invasion and chemosensitivity of CD133+ tumor stem cells in Hep-2 of larynx cancer Hep-2 after silencing Bmi-1 gene in Hep-2, and to find new targets for the treatment of larynx cancer, and to open new ideas. Method: transfection of pFIV-H1/U6 Bmi-1 siRNA plasmid and negative control plasmid to CD133+ tumor stem of human larynx cancer cell line. Cell, RT-PCR, and Western-blot were used to detect the expression of Bmi-1 in transfected cells and select the best plasmids for subsequent experiments. The experiments were divided into 3 groups: blank control group (Hep-2 group), negative control group (siRNA-scramble group), experimental group (Bmi-1 siRNA plasmid transfection group), RT-PCR and Western-blot detection of Bmi-1 downstream gene P16 expression, CCK-8 method was plotted. Cell proliferation curve, soft agar clone formation ability test cell proliferation ability, DAPI staining and flow cytometry to detect cell apoptosis, Transwell cell invasion test to detect cell invasion ability in vitro, CCK-8 method to detect chemosensitivity. Results: RT-PCR and Western-blot found that the relative expression level of P16 gene in experimental group was increased (P0.01 The cell growth curve and the clone formation ability showed that the proliferation of the experimental group was inhibited, and the difference was statistically significant compared with the blank group and the negative control group (P0.01). DAPI and flow cytometry showed that the apoptosis rate of the experimental group was significantly higher than that in the blank group and the negative control group (P0.01); Transwell The invasive ability of the cells decreased in vitro, and the membrane cells in the experimental group decreased significantly compared with the negative control group and the blank control group (P0.05), and the sensitivity of the CD133+ tumor stem cells in the experimental group increased to 5-FU. Conclusion: the RNAi technique was used to silence the Bmi-1 gene and inhibit the self renewal and proliferation of the CD133+ tumor stem cells. The proliferation and invasion ability of CD133+ tumor stem cells can promote apoptosis and enhance sensitivity to chemotherapy.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R739.65

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王泉;胡衛(wèi);陳濤;王成雙;詹玲;;Bmi1在腫瘤干細(xì)胞中的研究進(jìn)展[J];廣東醫(yī)學(xué);2012年23期

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本文編號(hào)：1875350