SELDI-TOF-MS技术对大肠癌裸鼠动物模型早期蛋白组学研究
发布时间：2018-08-18 15:43
【摘要】：目的：利用结肠癌细胞悬液皮下接种BALB/C裸小鼠,构建HCT-116人结肠癌裸鼠动物模型,观测时间与移植瘤大小变化,检测分析接种后不同时间裸鼠体内微量蛋白谱的变化,寻找肿瘤相关的差异蛋白,探索大肠癌的早期诊断指标。 方法：复苏培养HCT-116大肠癌细胞,收集第3-5代对数生长期细胞,将细胞悬液调至密度1×107/ml,皮下接种于10只3-4周龄裸鼠背部皮下,接种剂量为0.1ml/只,每隔15天测量瘤体大小并记录.对照组同时用无血清培养基接种(0.1ml/只)。实验组和对照组裸鼠分别在接种后20d、40d和60d取血,前两次采用断尾法取血0.1ml/只,最后一次采用摘眼球取血0.5ml/只后处死裸鼠并剥离瘤体,测量瘤体体积,剥离的瘤体立即送病理科进行病理切片检查。所取血液标本立即4℃3000rpm离心5min,将分离的血清分装3管,30μL/管并进行编号,利用SELDI-TOF-MS技术对裸鼠血清进行微量蛋白分析,将获得的微量蛋白质谱图用Ciphergen ProteinChip3.0和Ciphergen Biomaker Wizard3.1软件进行数据的采集、校正和分析,筛选与大肠癌相关的微量差异蛋白,并对筛选出的差异蛋白在蛋白数据库中进行比对。 结果：裸鼠皮下接种后15天长出肿瘤,成瘤率100%。筛选出与肿瘤相关的具有规律性变化的5个差异蛋白(p0.01),其分子量分别为2357Da、2424Da、2637Da、8182Da、10133Da。其中2637Da、8182Da、10133Da三种微量蛋白在接种后裸鼠体内表达逐渐增强,2357Da,2424Da两种微量蛋白在接种后裸鼠体内表达逐渐减弱,并在Swiss-Prot蛋白库中进行数据检索,初步鉴定为Reactive oxygen species modulator1、 Leukocyte-specific transcript1protein、Brevinin-2R、Prosalusin、Calcitonin。在肿瘤细胞的培养基里面筛选出了一个差异蛋白,其分子量为4160Da,鉴定为WW domain-containing oxidoreductase或者Peptide YY。 结论：1裸鼠皮下接种HCT-116细胞能成功构建移植瘤动物模型,此模型易于操作,成功率高,模型稳定,可为研究大肠癌提供帮助。2SELDI-TOF-MS技术对结肠癌裸鼠移植瘤模型差异蛋白的检测是有效的,能发现早期大肠癌的标志蛋白。3癌细胞体外培养发现的新物质证明了癌细胞的疾病进程并不是孤立的而是和外界紧密关联的。
[Abstract]:Objective: to establish a nude mouse model of HCT-116 human colon cancer by subcutaneously inoculating colon cancer cell suspension into BALB/C nude mice, observe the changes of time and size of transplanted tumor, and analyze the changes of microprotein spectrum in nude mice at different time after inoculation. To search for tumor related differential proteins and to explore the early diagnostic criteria of colorectal cancer. Methods: HCT-116 colorectal cancer cells were cultured after resuscitation. The cells of logarithmic growth phase were collected and the cell suspension was adjusted to 1 脳 107 / ml. The cells were subcutaneously inoculated subcutaneously on the back of 10 nude mice aged 3-4 weeks. The tumor size was measured and recorded every 15 days. The control group was inoculated with serum-free medium (0.1ml/ only). The nude mice in the experimental group and the control group were collected blood at 20 days after inoculation for 40 days and 60 days after inoculation respectively. The first two blood samples were taken from 0.1ml/ by severed tail method, the last one was taken out of eyeball to get blood 0.5ml/, and then the nude mice were killed and stripped off the tumor body to measure the volume of the tumor. The dissected tumor was immediately sent to the pathology department for pathological section examination. The blood samples were immediately centrifuged at 4 鈩，
本文编号：2189928
[Abstract]:Objective: to establish a nude mouse model of HCT-116 human colon cancer by subcutaneously inoculating colon cancer cell suspension into BALB/C nude mice, observe the changes of time and size of transplanted tumor, and analyze the changes of microprotein spectrum in nude mice at different time after inoculation. To search for tumor related differential proteins and to explore the early diagnostic criteria of colorectal cancer. Methods: HCT-116 colorectal cancer cells were cultured after resuscitation. The cells of logarithmic growth phase were collected and the cell suspension was adjusted to 1 脳 107 / ml. The cells were subcutaneously inoculated subcutaneously on the back of 10 nude mice aged 3-4 weeks. The tumor size was measured and recorded every 15 days. The control group was inoculated with serum-free medium (0.1ml/ only). The nude mice in the experimental group and the control group were collected blood at 20 days after inoculation for 40 days and 60 days after inoculation respectively. The first two blood samples were taken from 0.1ml/ by severed tail method, the last one was taken out of eyeball to get blood 0.5ml/, and then the nude mice were killed and stripped off the tumor body to measure the volume of the tumor. The dissected tumor was immediately sent to the pathology department for pathological section examination. The blood samples were immediately centrifuged at 4 鈩，
本文编号：2189928
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