功能化基于线的靶细胞分离:一种从加标外周血中分离癌细胞的离体技术

上皮细胞粘附分子或 EpCAM 是在前列腺癌快速增殖的上皮细胞中过度表达的表面跨膜糖蛋白。要分离表达 EpCAM 的细胞，请从带有功能化镀金尖端的医用级导线开始。功能化层含有共价结合的 EpCAM 特异性抗体的聚合物凝胶。

将导线浸入含有荧光标记癌细胞的外周血悬液中。确保功能化部分保持浸入血液悬浮液中。癌细胞上的 EpCAM 识别并结合在导线上功能化的特异性抗体。

用缓冲液洗涤以去除导丝表面的任何未结合的细胞。弯曲导线并将其安装在含有缓冲液的载玻片上，将功能化部分浸入缓冲液中。使用荧光显微镜检查细胞质和细胞核的明亮荧光，并确认导线上是否存在捕获的细胞。

用释放缓冲液处理电线。缓冲液中的蛋白水解酶裂解聚合物凝胶，将抗体-癌细胞复合物释放到缓冲液中。离心线组件以收集沉淀中释放的抗体细胞复合物。取出导丝和含酶的上清液。将含有目标细胞的沉淀重悬于合适的培养基中。

在 5 毫升外周血中，加入约 500 至 500,000 个细胞，然后倒置管子混合。从储物箱中取出电线后，取下固定电线的橡胶盖，用 1x 磷酸盐缓冲盐水清洗电线，然后将其安装在管盖中。然后，在室温下以每分钟 5 转的速度将试管在倾斜的辊式混合器上孵育 30 分钟。孵育后，在 1x 磷酸盐缓冲盐水中冲洗导丝三次。然后，将电线储存在装有 1x 磷酸盐缓冲盐水的 15 毫升管中，在黑暗中。

使用油笔在载玻片上画一个矩形区域，并在其上加入 500 微升 1x 磷酸盐缓冲盐水。要将导线的功能部分浸入 1x 磷酸盐缓冲盐水中，请弯曲导线的非功能部分并将其放在载玻片上。要计算捕获的细胞数量，请目视检查电线的两侧。检查后，将电线转移回装有 1x 磷酸盐缓冲盐水的 15 毫升管中，并在黑暗中保存。

在 1 毫升 1x 磷酸盐缓冲盐水中，溶解 4 毫克释放缓冲液组分。然后，通过0.2微米无菌过滤器过滤溶液，获得即用型缓冲液。接下来，将释放缓冲液在 37 摄氏度下加热 5 分钟。加热后，转移 1.6 毫升释放缓冲液以完全填充 1.5 毫升反应管。接下来，将导线的功能部分在37摄氏度的释放缓冲液中在水浴中孵育20分钟。

孵育后，将电线以每分钟 500 圈的速度放在摇床上 15 分钟。然后，将金属丝以 300 x g 离心 10 分钟。离心结束后，将导丝从管中移开，盖上盖子，然后再次以 300 x g 离心管 10 分钟。

• Epithelial Cell Adhesion Molecules (EpCAMs) – Overexpressed in rapidly proliferating cells in prostate cancers.
• Free Floating Cancer Cells – Malignant cells in blood that have detached from a primary tumor.
• Cytocentrifugation Method – A procedure used to concentrate cells from a fluid sample.
• Functionalization – Process of equipping the wire with EpCAM-specific antibodies.
• Mccoy's 5a Medium Modified – A commercially available solution for culturing human cells.

• The EpCAMs are surface transmembrane glycoproteins overexpressed in rapidly proliferating epithelial cells (e.g., prostate cancer).
• These molecules bind to specific antibodies bound to a functionalized wire in a fluid suspension (e.g., cytocentrifugation method).
• A washing step removes unattached cells, leaving only the target cells bound to the wire (e.g., free floating cancer cells).
• The immersing and release process allows for the focus on the captured cells on the wire, and their subsequent collection.

• What is an EpCAM and how does it relate to prostate cancer and the isolation process?
• How does cytocentrifugation assist in concentrating the target cells?
• How does the wire's functionalization and release process work in terms of capturing and collecting cells?

• Isolating EpCAM-expressing cells for study can lead to advancements in cancer research (e.g., free floating cancer cells).
• Techniques like cytocentrifugation have numerous applications in cell biology and medical diagnostics (e.g., cytocentrifugation method).
• The process provides greater insights into the nature and behavior of cancers cells, potentially leading to more effective treatments (e.g., EpCAMs).
• Focused studies of such nature often lead to significant scientific advancements in cancer research and treatment.