【摘要】：目的:本課題通過(guò)無(wú)血清有限稀釋法,從人結(jié)直腸癌細(xì)胞株HCT116中篩選獲得結(jié)直腸癌干細(xì)胞樣克隆球,采用流式細(xì)胞術(shù)檢測(cè)克隆球的腫瘤干細(xì)胞標(biāo)志物CD133、CD44的表達(dá)、細(xì)胞周期分布和軟瓊脂集落形成實(shí)驗(yàn),鑒定篩選出的克隆球細(xì)胞為結(jié)直腸腫瘤干樣細(xì)胞;通過(guò)結(jié)直腸腫瘤干樣細(xì)胞耐藥性實(shí)驗(yàn),證明篩選出的結(jié)直腸腫瘤干樣細(xì)胞較HCT116細(xì)胞具有更強(qiáng)的耐藥性。方法:1.運(yùn)用有限稀釋無(wú)血清培養(yǎng)法,從結(jié)腸癌細(xì)胞株HCT116中篩選腫瘤干細(xì)胞樣克隆球,并擴(kuò)大培養(yǎng)建立腫瘤干細(xì)胞克隆亞系。2.通過(guò)流式細(xì)胞術(shù)檢測(cè)腫瘤干細(xì)胞標(biāo)志物CD133、CD44和檢測(cè)細(xì)胞周期分布情況,以及軟瓊脂集落形成實(shí)驗(yàn),鑒定篩選出的克隆球細(xì)胞的腫瘤干細(xì)胞特性。3.采用CCK-8法研究氟尿嘧啶和槐耳清膏分別對(duì)結(jié)腸癌細(xì)胞生長(zhǎng)增殖的抑制作用,比較藥物干預(yù)后的人結(jié)腸癌干樣細(xì)胞和HCT116細(xì)胞表現(xiàn)出的耐藥性差異。結(jié)果:1、通過(guò)無(wú)血清有限稀釋法篩選出腫瘤干細(xì)胞樣克隆球14個(gè),細(xì)胞株克隆球形成率為28.6%;該群克隆球細(xì)胞表現(xiàn)為:較HCT116細(xì)胞具有更高的成球率(43.2%),增殖快,可不斷傳代,予含血清培養(yǎng)基培養(yǎng)可重新分化,表現(xiàn)出腫瘤干細(xì)胞不斷增殖、自我更新和再分化的特點(diǎn)。2、流式細(xì)胞術(shù)腫瘤干細(xì)胞標(biāo)志物檢測(cè),HCT116中有87.97%的細(xì)胞表達(dá)CD133+CD44+,克隆球細(xì)胞中93.87%的細(xì)胞表達(dá)CD133+CD44+。流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期,克隆球中77.18%的細(xì)胞處于G1期,HCT116中49.00%的細(xì)胞處于G1期,差異有統(tǒng)計(jì)學(xué)意義。軟瓊脂集落形成實(shí)驗(yàn)中,克隆球1和2的集落形成率分別為51.97%和39.54%,HCT116細(xì)胞的集落形成率為5.67%,差異有統(tǒng)計(jì)學(xué)意義。3、腫瘤干樣細(xì)胞耐藥性實(shí)驗(yàn)中,隨著用藥濃度的升高,氟尿嘧啶和槐耳清膏對(duì)腸癌細(xì)胞的生長(zhǎng)增殖抑制作用均逐漸增強(qiáng);相同的藥物濃度下,腫瘤干樣細(xì)胞生長(zhǎng)增殖被抑制的程度較原株細(xì)胞低。結(jié)論:1、從人結(jié)直腸癌HCT116細(xì)胞株中能篩選出腫瘤干細(xì)胞樣克隆球。2、與原株相比,篩選出的克隆球細(xì)胞腫瘤標(biāo)志物CD133+CD44+陽(yáng)性表達(dá)率和處于G0期的細(xì)胞比例更高,具有更高的集落形成率、更強(qiáng)的耐藥性,鑒定為腫瘤干樣細(xì)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.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.3

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本文編號(hào)：2334667