摘要
目的观察结肠癌HT-29细胞在三维或单层培养条件下细胞形态学、细胞周期、细胞凋亡及对化疗药物5-Fu敏感性的变化。方法分别采用liqu id overlay技术或常规贴壁法对HT-29细胞进行三维或单层培养,通过显微镜观察细胞形态学变化,流式细胞仪测定不同培养方式对细胞周期和凋亡率的影响,并采用rad ial outgrowth测定法或MTT法分别测定三维或单层培养细胞对5-Fu的敏感性。结果三维培养HT-29细胞悬浮生长,形成致密的多细胞球结构,中心出现坏死区,细胞间有较多的微绒毛、紧密连接、桥粒等结构,而单层培养HT-29细胞呈单层贴壁生长,未见紧密连接、桥粒等连接结构;与单层培养细胞相比,三维培养HT-29细胞G1期比例明显升高[(64.95±0.59)%对(36.77±0.51)%,P<0.01]、细胞自发性凋亡率降低[(0.99±0.12)%对(4.79±0.49)%,P<0.01]、对5-Fu产生明显耐药。结论三维培养HT-29细胞在细胞形态学、细胞周期、细胞凋亡等方面与单层培养细胞存在明显差异,可能是导致结肠癌多细胞耐药的重要原因。
Objective To observe the changes of morphology, cell cycle, apoptosis and sensitivity to 5-Fluorouracil （5-Fu） on colon carcinoma HT-29 cells cultured as three-dimensional model or monolayer. Methods HT-29 cells were cultured as three-dimensional model using liquid overlay technique or as monolayer using routine method. The cellular morphology was observed by invert microscope, scanning electron microscope （SEM） or transmission electron microscope （ TEM ）. Cell cycle and apoptotic rate were detected by flow cytometry （ FCM ）. The sensitivity to 5 -Fu on HT-29 spheroids or monolayers was determined by radial outgrowth assay or MTY assay respectively. Results HT-29 cells by three-dimensional culture formed many compact multicellular spheroids （MCS） and floated in the medium. MCS were constituted of hundreds of cells. The centre of HT-29 spheroids formed a necrotic core. Microvilli, tight junction and desmosome could be observed in HT-29 spheroids. When cultured as monolayer, the HT-29 cells were adherent to the bottom of flask and tight junction or desmosome could not be observed. The percentage of G1 phase in HT-29 spheroids was higher than that of monolayer [ （64.95 ±0.59） % vs （36.77 ±0.51 ） %, （P 〈0.01 ） ] and the apoptotic rate in HT-29 spheroids was lower than that of monolayer [ （0.99 _＋0.12） % vs （4.79 ±0.49） %, （P 〈0.01 ） ]. MCS were more resistant to 5 -Fu than monolayer cells. Conclusion There are significant differences in morphology, cell cycle and apoptotic rate between HT-29 spheroids and monolayer, which may be the important causes of the muhicellular resistance of colon carcinoma.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2005年第17期1760-1762,共3页
Journal of Third Military Medical University
基金
中国临床肿瘤学科学基金资助项目(Y-2004-03)
西南医院临床研究专项基金资助项目(SW2004021)~~
关键词
多细胞耐药
细胞周期
凋亡
三维细胞培养
结肠癌
muhicellular resistance
cell cycle
apoptosis
three-dimensional culture
colon carcinoma
