结直肠癌HCT116细胞株干样细胞的筛选与鉴定
发布时间：2018-11-15 23:04
【摘要】：目的:本课题通过无血清有限稀释法,从人结直肠癌细胞株HCT116中筛选获得结直肠癌干细胞样克隆球,采用流式细胞术检测克隆球的肿瘤干细胞标志物CD133、CD44的表达、细胞周期分布和软琼脂集落形成实验,鉴定筛选出的克隆球细胞为结直肠肿瘤干样细胞;通过结直肠肿瘤干样细胞耐药性实验,证明筛选出的结直肠肿瘤干样细胞较HCT116细胞具有更强的耐药性。方法:1.运用有限稀释无血清培养法,从结肠癌细胞株HCT116中筛选肿瘤干细胞样克隆球,并扩大培养建立肿瘤干细胞克隆亚系。2.通过流式细胞术检测肿瘤干细胞标志物CD133、CD44和检测细胞周期分布情况,以及软琼脂集落形成实验,鉴定筛选出的克隆球细胞的肿瘤干细胞特性。3.采用CCK-8法研究氟尿嘧啶和槐耳清膏分别对结肠癌细胞生长增殖的抑制作用,比较药物干预后的人结肠癌干样细胞和HCT116细胞表现出的耐药性差异。结果:1、通过无血清有限稀释法筛选出肿瘤干细胞样克隆球14个,细胞株克隆球形成率为28.6%;该群克隆球细胞表现为:较HCT116细胞具有更高的成球率(43.2%),增殖快,可不断传代,予含血清培养基培养可重新分化,表现出肿瘤干细胞不断增殖、自我更新和再分化的特点。2、流式细胞术肿瘤干细胞标志物检测,HCT116中有87.97%的细胞表达CD133+CD44+,克隆球细胞中93.87%的细胞表达CD133+CD44+。流式细胞术检测细胞周期,克隆球中77.18%的细胞处于G1期,HCT116中49.00%的细胞处于G1期,差异有统计学意义。软琼脂集落形成实验中,克隆球1和2的集落形成率分别为51.97%和39.54%,HCT116细胞的集落形成率为5.67%,差异有统计学意义。3、肿瘤干样细胞耐药性实验中,随着用药浓度的升高,氟尿嘧啶和槐耳清膏对肠癌细胞的生长增殖抑制作用均逐渐增强;相同的药物浓度下,肿瘤干样细胞生长增殖被抑制的程度较原株细胞低。结论:1、从人结直肠癌HCT116细胞株中能筛选出肿瘤干细胞样克隆球。2、与原株相比,筛选出的克隆球细胞肿瘤标志物CD133+CD44+阳性表达率和处于G0期的细胞比例更高,具有更高的集落形成率、更强的耐药性,鉴定为肿瘤干样细胞。
[Abstract]:Objective: in this study, a serum-free limited dilution method was used to screen the stem cell like clonal spheres from human colorectal cancer cell line HCT116, and flow cytometry was used to detect the expression of tumor stem cell marker CD133,CD44. The cell cycle distribution and soft Agar colony formation were studied. The cloned spherical cells were identified as colorectal tumor-like cells. The results of drug resistance test showed that the screened stem cells were more resistant than HCT116 cells. Methods: 1. Tumor stem cell like clone spheres were screened from colon cancer cell line HCT116 by using limited dilution serum-free culture method, and tumor stem cell clone sublines were established by expanded culture. 2. Flow cytometry was used to detect the tumor stem cell marker CD133,CD44 and cell cycle distribution, as well as the soft Agar colony forming test to identify the tumor stem cell characteristics of the cloned spherical cells. 3. The inhibitory effects of fluorouracil and Huai er Qing ointment on the growth and proliferation of colon cancer cells were studied by CCK-8 method, and the drug resistance of human colon cancer dry like cells and HCT116 cells were compared. Results: 1. Fourteen tumor stem cell like clones were screened by serum-free limited dilution method. Compared with HCT116 cells, the colony had a higher spheroidization rate (43.2%), rapid proliferation and continuous passage. The cells cultured in serum-containing medium could be redifferentiated and showed the proliferation of tumor stem cells. (2) flow cytometry showed that 87.97% of the HCT116 cells expressed CD133 CD44 and 93.87% of the cloned spheres expressed CD133 CD44. Flow cytometry showed that 77.18% of the cells in the clone sphere were in G1 phase, while 49.00% of the cells in HCT116 were in G1 phase. The difference was statistically significant. In the colony forming test of soft Agar, the colony forming rates of Clone 1 and 2 were 51.97% and 39.54%, respectively. The colony forming rate of HCT116 cells was 5.67. The difference was statistically significant. With the increase of drug concentration, the inhibitory effect of fluorouracil and Huai er Qing ointment on the growth and proliferation of intestinal cancer cells increased gradually. At the same drug concentration, the growth and proliferation of tumor dry-like cells were inhibited to a lesser extent than the original cells. Conclusion: 1. Tumor stem cell like clone spheres can be screened from human colorectal cancer HCT116 cell line. Compared with the original cell line, the positive expression rate of tumor marker CD133 CD44 and the proportion of cells in G0 phase were higher. It has higher colony formation rate and stronger drug resistance, and is identified as a tumor-like cell.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.3
本文编号：2334667
[Abstract]:Objective: in this study, a serum-free limited dilution method was used to screen the stem cell like clonal spheres from human colorectal cancer cell line HCT116, and flow cytometry was used to detect the expression of tumor stem cell marker CD133,CD44. The cell cycle distribution and soft Agar colony formation were studied. The cloned spherical cells were identified as colorectal tumor-like cells. The results of drug resistance test showed that the screened stem cells were more resistant than HCT116 cells. Methods: 1. Tumor stem cell like clone spheres were screened from colon cancer cell line HCT116 by using limited dilution serum-free culture method, and tumor stem cell clone sublines were established by expanded culture. 2. Flow cytometry was used to detect the tumor stem cell marker CD133,CD44 and cell cycle distribution, as well as the soft Agar colony forming test to identify the tumor stem cell characteristics of the cloned spherical cells. 3. The inhibitory effects of fluorouracil and Huai er Qing ointment on the growth and proliferation of colon cancer cells were studied by CCK-8 method, and the drug resistance of human colon cancer dry like cells and HCT116 cells were compared. Results: 1. Fourteen tumor stem cell like clones were screened by serum-free limited dilution method. Compared with HCT116 cells, the colony had a higher spheroidization rate (43.2%), rapid proliferation and continuous passage. The cells cultured in serum-containing medium could be redifferentiated and showed the proliferation of tumor stem cells. (2) flow cytometry showed that 87.97% of the HCT116 cells expressed CD133 CD44 and 93.87% of the cloned spheres expressed CD133 CD44. Flow cytometry showed that 77.18% of the cells in the clone sphere were in G1 phase, while 49.00% of the cells in HCT116 were in G1 phase. The difference was statistically significant. In the colony forming test of soft Agar, the colony forming rates of Clone 1 and 2 were 51.97% and 39.54%, respectively. The colony forming rate of HCT116 cells was 5.67. The difference was statistically significant. With the increase of drug concentration, the inhibitory effect of fluorouracil and Huai er Qing ointment on the growth and proliferation of intestinal cancer cells increased gradually. At the same drug concentration, the growth and proliferation of tumor dry-like cells were inhibited to a lesser extent than the original cells. Conclusion: 1. Tumor stem cell like clone spheres can be screened from human colorectal cancer HCT116 cell line. Compared with the original cell line, the positive expression rate of tumor marker CD133 CD44 and the proportion of cells in G0 phase were higher. It has higher colony formation rate and stronger drug resistance, and is identified as a tumor-like cell.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.3
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