喉癌Hep-2细胞中肿瘤干细胞化疗抵抗及靶向封闭Bmi-1化疗增敏的研究

发布时间：2018-05-11 19:42

本文选题：喉癌 + 肿瘤干细胞　；参考：《吉林大学》2011年博士论文

【摘要】：喉癌是东北地区患病率较高的恶性肿瘤,并且其发病率有明显增长的趋势。目前主要采用手术、放化疗及生物治疗等多种治疗手段有机结合的综合治疗,但病人的生存率并没有得到太大改善,而且更无法解决肿瘤对放化疗抵抗力强及复发转移的问题。目前有关肿瘤干细胞的研究已经取得了很大进展,已在白血病及多种实体肿瘤分离出肿瘤干细胞。肿瘤干细胞具有自我更新能力,多向分化潜能,高致瘤性及耐药性。越来越多的研究发现肿瘤干细胞对化疗具有极强的抵抗性,治疗后肿瘤干细胞常常存活下来,又因为其高自我更新能力及增殖潜能,使肿瘤复发,治疗失败。Bmi-1对肿瘤干细胞的自我更新有重要作用,Bmi-1基因失活小鼠的白血病细胞移植入免疫缺陷小鼠后不能再产生白血病细胞。因此分析肿瘤干细胞的化疗抵抗性,以及从肿瘤干细胞的角度来探索肿瘤治疗,将是克服化疗抵抗的理想方法,最终改善肿瘤的治疗效果。目前关于喉癌中肿瘤干细胞的研究已经取得了一定进展。CD133是人喉癌Hep-2细胞中肿瘤干细胞标志物,并且CD133+肿瘤干细胞比其他细胞亚群的体外分化和增殖能力强,致瘤性高。这为进一步以肿瘤干细胞的角度研究喉癌的化疗抵抗性,以及靶向肿瘤干细胞治疗,提供了前提和基础。本研究拟通过体内外实验分析喉癌化疗后CD133+细胞比例的变化及CD133+细胞对化疗药物的敏感性,探讨人喉癌Hep-2细胞中肿瘤干细胞化疗抵抗的特性,并通过沉默CD133+肿瘤干细胞胞中的Bmi-1基因,抑制CD133+肿瘤干细胞自我更新能力,抑制其增殖,促进凋亡,增强化疗敏感性,为以肿瘤干细胞为靶点治疗喉癌进行初步探索。第一部分：喉癌Hep-2细胞中肿瘤干细胞与化疗抵抗关系的初步研究目的：探讨喉癌Hep-2细胞中CD133+肿瘤干细胞的化疗抵抗性,分析Bmi-1在CD133+肿瘤干细胞及CD133-细胞中的表达。方法：①体外培养人喉癌细胞系Hep-2细胞,应用化疗药物(顺铂,5-FU,紫杉醇)作用后,流式细胞仪检测CD133+细胞比例的变化。②建立裸鼠喉癌移植瘤模型,实验组以5-FU进行治疗,治疗结束后,分离肿瘤组织,通过免疫组织化学染色和流式细胞仪检测CD133表达情况。③流式细胞仪分选Hep-2细胞系CD133+肿瘤干细胞,CCK-8法检测其化疗敏感性。软琼脂克隆形成实验检测5-FU作用后CD133+肿瘤干细胞克隆形成率,动物成瘤实验检测5-FU作用后CD133+肿瘤干细胞致瘤能力变化。④RT-PCR和Western-blot检测CD133+肿瘤干细胞和CD133-细胞中Bmi-1的表达。结果：化疗药物(顺铂,5-FU,紫杉醇)作用48h后CD133+细胞富集2-4倍。CD133+表达率由原来的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发现5-FU治疗组CD133表达率为6.7%士1.6%较对照组2.6%士0.96%升高了约3倍。流式细胞分选技术成功分离纯化CD133+肿瘤干细胞,发现其较CD133-细胞具有明显的化疗抵抗。CD133+肿瘤干细胞经5-FU作用后克隆形成能力无明显降低,成瘤时间延长,最终致瘤能力无明显下降。CD133+肿瘤干细胞中的Bmi-1 mRNA和蛋白的表达量明显高于CD133-细胞。结论：CD133+肿瘤干细胞的存在是喉癌化疗抵抗的原因之一,CD133+肿瘤干细胞高表达自我更新关键因子Bmi-1。第二部分RNAi沉默Bmi-1基因对喉癌Hep-2细胞中肿瘤干细胞生物学行为的影响及化疗增敏作用目的：探讨RNAi沉默Bmi-1基因后喉癌Hep-2中CD133+肿瘤干细胞增殖、凋亡、侵袭及化疗敏感性的变化,为喉癌的治疗寻找新的靶点,开辟新思路。方法：将前期构建的pFIV-H1/U6 Bmi-1 siRNA质粒和阴性对照质粒转染人喉癌Hep-2细胞系CD133+肿瘤干细胞,RT-PCR、Western-blot检测转染细胞Bmi-1的表达,选择抑制效果最好的质粒进行后续实验。实验分成3组：空白对照组(Hep-2组),阴性对照组(siRNA-scramble组),实验组(Bmi-1 siRNA质粒转染组),RT-PCR和Western-blot检测Bmi-1下游基因P16的表达,CCK-8法绘制细胞增殖曲线,软琼脂克隆形成能力检测细胞增殖能力,DAPI染色法及流式细胞仪检测细胞凋亡,Transwell小室体外侵袭实验检测细胞体外侵袭能力,CCK-8法检测化疗敏感性。结果：RT-PCR和Western-blot发现实验组P16基因相对表达水平增高(P0.01)；细胞生长曲线及克隆形成能力显示,实验组细胞的增殖受到抑制,与空白组和阴性对照组比较,差异有统计学意义(P0.01);DAPI法和流式细胞仪检测显示实验组细胞凋亡率明显高于空白组和阴性对照组,差异有统计学意义(P0.01);Transwell小体外侵袭实验显示细胞体外侵袭能力降低,实验组穿膜细胞与阴性对照组及空白对照组相比明显减少(P0.05)；实验组CD133+肿瘤干细胞对5-FU的敏感性增加。结论：应用RNAi技术沉默Bmi-1基因,抑制CD133+肿瘤干细胞自我更新及增殖,可抑制CD133+肿瘤干细胞的增殖及侵袭能力,促进凋亡,增强对化疗的敏感性。
[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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R739.65

【引证文献】