基于免疫磁珠法的自动化粪便脱落细胞提取仪的研制与应用

Objective To design a device based on immunobead method which enables extraction of exfoliated colonocytes from human feces to become an automatic concentrating process. Methods The automatic device for extraction of exfoliated colonocytes from human feces was designed based on the composition and physiochemical features of the feces as well as the distribution of exfoliated colonocytes, so as to integrate its functions in sample collection, pretreatment, exfoliated colonocyte-immunobead binding,and exfoliated colonocyte- immunobead complex washing. The immunobeads were prepared by coating magnetic beads with monoclonal antibody of epethelial cell adhesion molecule (EpCAM) Ber-EP4 and used to concentrate colorectal cancer cells from cell culture. The binding of colorectal cells to immunobeads was observed under microscopy. Cancer tissues of 10 colorectal cancer patients (or tissues around cancer) were collected. The automatic device for extraction of exfoliated colonocytes from human feces was used to get exfoliated cells from stool specimens of the patients. QRT-PCR was used to detect gene expression of c-myc、cox-2、CD44v6 in tissues and cells collected. Rate of gene positive expression of different original specimens was compared. Results The automatic device for extraction of colonocytes from human feces was designed and manufactured. Extracting exfoliated coloncytes from feces sample was successfully completed by the automatic device and self-prepared immunobeads with coating special monoclonal antibody. The recovery rate of cells was 27%. Rate of c-myc, cox-2 and CD44v6 gene positive expression in cancer tissues or tissues around cancer was 20%, 10% and 20% respectively, and 100% in exfoliated cells from stool specimens. The difference of positive rate among specimens was significant (all P＜0.01). Exfoliated cells from stool specimens collected by the automatic device for extraction of exfoliated colonocytes from human feces were cancer cells. Conclusions As one of the latest-developed approaches, immunobead-based concentration of exfoliated colonocytes appears practical. An automatic device thereby developed may be used for early detection of colorectal cancers.     

基于免疫磁珠法的自动化粪便脱落细胞提取仪的研制与应用

目的 根据免疫磁珠法的原理设计研制自动化粪便脱落细胞提取仪,实现大肠癌脱落细胞的自动化富集.方法 根据粪便组分、理化特性以及粪便脱落细胞分布特点,设计制造包括粪便样本采集装置、粪便样本预处理器、脱落细胞-免疫磁珠结合装置和脱落细胞-免疫磁珠复合物洗涤装置的自动化粪便脱落细胞提取仪.采用抗大肠癌特异表面抗原表皮细胞黏附分子(EpCAM)的单抗Ber-EP4包被至磁珠表面制备免疫磁珠,用于从细胞培养物中富集大肠癌细胞,并在显微镜下观察免疫磁珠与大肠癌细胞的结合效果.收集10例大肠癌患者的癌组织(或癌旁组织),并应用自动化粪便脱落细胞仪提取患者粪便样本的脱落细胞,应用实时荧光定量PCR检测癌组织(或癌旁组织)以及粪便样本的脱落细胞肿瘤相关基因c-myc、cox-2和CD44v6的表达,并对比两种不同来源的样本检出的3种基因的阳性表达率.结果 设计制作了自动化粪便脱落细胞提取仪样机,并且结合自行制备包被特异抗体的免疫磁珠成功提取到直肠癌细胞,细胞回收率达27%.在10例癌组织或癌旁组织中,c-myc、cox-2、CD44v6基因的阳性表达率分别为20%、10%、20%,而在粪便脱落细胞中均为100%,差异有统计学意义(均P＜0.01).自动粪便脱落细胞仪提取的患者粪便样本的脱落细胞为肿瘤细胞.结论 利用免疫磁珠富集大肠癌脱落细胞是可行的,以此为依据制作的自动化脱落细胞提取仪可以用于临床大肠癌的早期筛查.