本文選題：喉癌 + 腫瘤干細胞��； 參考：《吉林大學》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)病率有明顯增長的趨勢。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結(jié)合的綜合治療,但病人的生存率并沒有得到太大改善,而且更無法解決腫瘤對放化療抵抗力強及復發(fā)轉(zhuǎn)移的問題。 目前有關(guān)腫瘤干細胞的研究已經(jīng)取得了很大進展,已在白血病及多種實體腫瘤分離出腫瘤干細胞。腫瘤干細胞具有自我更新能力,多向分化潛能,高致瘤性及耐藥性。越來越多的研究發(fā)現(xiàn)腫瘤干細胞對化療具有極強的抵抗性,治療后腫瘤干細胞常常存活下來,又因為其高自我更新能力及增殖潛能,使腫瘤復發(fā),治療失敗。Bmi-1對腫瘤干細胞的自我更新有重要作用,Bmi-1基因失活小鼠的白血病細胞移植入免疫缺陷小鼠后不能再產(chǎn)生白血病細胞。因此分析腫瘤干細胞的化療抵抗性,以及從腫瘤干細胞的角度來探索腫瘤治療,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)于喉癌中腫瘤干細胞的研究已經(jīng)取得了一定進展。CD133是人喉癌Hep-2細胞中腫瘤干細胞標志物,并且CD133+腫瘤干細胞比其他細胞亞群的體外分化和增殖能力強,致瘤性高。這為進一步以腫瘤干細胞的角度研究喉癌的化療抵抗性,以及靶向腫瘤干細胞治療,提供了前提和基礎(chǔ)。 本研究擬通過體內(nèi)外實驗分析喉癌化療后CD133+細胞比例的變化及CD133+細胞對化療藥物的敏感性,探討人喉癌Hep-2細胞中腫瘤干細胞化療抵抗的特性,并通過沉默CD133+腫瘤干細胞胞中的Bmi-1基因,抑制CD133+腫瘤干細胞自我更新能力,抑制其增殖,促進凋亡,增強化療敏感性,為以腫瘤干細胞為靶點治療喉癌進行初步探索。 第一部分：喉癌Hep-2細胞中腫瘤干細胞與化療抵抗關(guān)系的初步研究 目的：探討喉癌Hep-2細胞中CD133+腫瘤干細胞的化療抵抗性,分析Bmi-1在CD133+腫瘤干細胞及CD133-細胞中的表達。方法：①體外培養(yǎng)人喉癌細胞系Hep-2細胞,應用化療藥物(順鉑,5-FU,紫杉醇)作用后,流式細胞儀檢測CD133+細胞比例的變化。②建立裸鼠喉癌移植瘤模型,實驗組以5-FU進行治療,治療結(jié)束后,分離腫瘤組織,通過免疫組織化學染色和流式細胞儀檢測CD133表達情況。③流式細胞儀分選Hep-2細胞系CD133+腫瘤干細胞,CCK-8法檢測其化療敏感性。軟瓊脂克隆形成實驗檢測5-FU作用后CD133+腫瘤干細胞克隆形成率,動物成瘤實驗檢測5-FU作用后CD133+腫瘤干細胞致瘤能力變化。④RT-PCR和Western-blot檢測CD133+腫瘤干細胞和CD133-細胞中Bmi-1的表達。結(jié)果：化療藥物(順鉑,5-FU,紫杉醇)作用48h后CD133+細胞富集2-4倍。CD133+表達率由原來的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表達率為6.7%士1.6%較對照組2.6%士0.96%升高了約3倍。流式細胞分選技術(shù)成功分離純化CD133+腫瘤干細胞,發(fā)現(xiàn)其較CD133-細胞具有明顯的化療抵抗。CD133+腫瘤干細胞經(jīng)5-FU作用后克隆形成能力無明顯降低,成瘤時間延長,最終致瘤能力無明顯下降。CD133+腫瘤干細胞中的Bmi-1 mRNA和蛋白的表達量明顯高于CD133-細胞。結(jié)論：CD133+腫瘤干細胞的存在是喉癌化療抵抗的原因之一,CD133+腫瘤干細胞高表達自我更新關(guān)鍵因子Bmi-1。 第二部分RNAi沉默Bmi-1基因?qū)戆〩ep-2細胞中腫瘤干細胞生物學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,開辟新思路。方法：將前期構(gòu)建的pFIV-H1/U6 Bmi-1 siRNA質(zhì)粒和陰性對照質(zhì)粒轉(zhuǎn)染人喉癌Hep-2細胞系CD133+腫瘤干細胞,RT-PCR、Western-blot檢測轉(zhuǎn)染細胞Bmi-1的表達,選擇抑制效果最好的質(zhì)粒進行后續(xù)實驗。實驗分成3組：空白對照組(Hep-2組),陰性對照組(siRNA-scramble組),實驗組(Bmi-1 siRNA質(zhì)粒轉(zhuǎn)染組),RT-PCR和Western-blot檢測Bmi-1下游基因P16的表達,CCK-8法繪制細胞增殖曲線,軟瓊脂克隆形成能力檢測細胞增殖能力,DAPI染色法及流式細胞儀檢測細胞凋亡,Transwell小室體外侵襲實驗檢測細胞體外侵襲能力,CCK-8法檢測化療敏感性。結(jié)果：RT-PCR和Western-blot發(fā)現(xiàn)實驗組P16基因相對表達水平增高(P0.01)；細胞生長曲線及克隆形成能力顯示,實驗組細胞的增殖受到抑制,與空白組和陰性對照組比較,差異有統(tǒng)計學意義(P0.01);DAPI法和流式細胞儀檢測顯示實驗組細胞凋亡率明顯高于空白組和陰性對照組,差異有統(tǒng)計學意義(P0.01);Transwell小體外侵襲實驗顯示細胞體外侵襲能力降低,實驗組穿膜細胞與陰性對照組及空白對照組相比明顯減少(P0.05)；實驗組CD133+腫瘤干細胞對5-FU的敏感性增加。結(jié)論：應用RNAi技術(shù)沉默Bmi-1基因,抑制CD133+腫瘤干細胞自我更新及增殖,可抑制CD133+腫瘤干細胞的增殖及侵襲能力,促進凋亡,增強對化療的敏感性。
[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.

【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R739.65

【引證文獻】

相關(guān)期刊論文 前1條

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

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本文編號：1875350