Capture Of Circulating Tumor Cells Of Breast Cancer And Detection Of Estrogen Receptor In Cells

The main reason that breast cancer can lead to death is the recrudescence and metastasis ofthe cancer, the breast cancer cells easily migrate to the blood and circulate with the blood toform a new metastases after a series of complex mechanisms. Detection of the circulating tumorcells (CTCs) plays an important role on the early detection of cancer metastasis, monitoringrecurrence and assessing the prognosis efficacy. The level of estrogen receptors (ER) is animportant indicator of breast cancer and can be used to determine whether apply endocrinetherapy or not, and the distribution of ER in CTCs may be differed from the initial tumor cells,if the ER does not exist in the initial tumor cells but in the CTCs, the endocrine therapy alsocould benefit the patients.Microfluidic chip was applied to the capture of CTCs and Immunohistochemistry wasapplied to the detection of the ER distribution of the captured cells. The capture and detectionmethods based on microfluidic chip had a number of advantages such as small space occupation,little reagent consumption, high throughput, automated analysis process, quick analysis speed,easy integration and so on. This paper consisted of three parts, as follows:In the chapter1, some relative knowledge including capture and detection method of CTCs,the testing methods and their significance of the ER, and their relationships with the tumor werereviewed, which focused on the detection methods of CTC with microfluidic chip and theirresearch progress.In the chapter2, the microfluidic chip based CTCs’ capture method was established.Anti-epithelial cell adhesion molecule (EpCAM) antibody could specifically identify EpCAM onthe membrane of CTCs, the microfluidic chip was modified with the anti-EpCAM, and the CTCscould be specifically captured by the anti-EpCAM in the chip when they passed through the chip.Antibodies modified conditions, injection velocity and injection methods were optimized, and the capture efficiency of MCF-7using three kinds of chips: the micro-post, curve and spiralstructures were compared, respectively, and the results showed that the capture efficiency of themicro-post structure is the best. Under the condition of antibody coating directly, incubating for3h,10μL/min flowing rate of the syringe pump, the capture efficiency of the micro-poststructure was drawn to be the highest.In the chapter3, the distribution of ER in MCF-7cells captured by microfluidic chip wasdetected using immunoenzyme techniques and immunofluorescence technology. Immunoenzymetechniques: the MCF-7cells firstly eluted from the microfluidic chip with glycine-hydrochloricacid buffer, incubated with anti-ERα antibodies, and then successively added the HRP-labeledantibody and substrate reagent so that the distribution of ER in the cells was analyzedqualitatively, and the results showed that ER were positive, but the specific location of the ERcould not be determined. Immunofluorescence with laser scanning confocal microscopetechnology: the FITC-anti-ERα was firstly composed using the fluorescein isothiocyanate(FITC), then the captured MCF-7cells by the microfluidic chip were fixed by theparaformaldehyde, changed membrane permeability by Trion X-100and sealed by albumin frombovine serum (BSA). The cells were incubated with the fluorescent probe (anti-ERα-FITC), andwhen the FITC-anti-ERα and ERα reacted adequately, the excessive unbound fluorescentantibodies were washed away. The results showed that ER was mainly distributed in the nucleusof MCF-7using the laser scanning confocal microscope technology.