Single Cell RT-PCR Detection Of Exfoliated Cells In Sputum On Chip For Lung Cancer Diagnosis

Lung cancer is one of the most common cancers in the world,and its mortality rate ranks among the highest in malignant tumors.Usually,the onset of lung cancer is hidden,and patients with early lung cancer are easily missed because they have no obvious clinical symptoms.Therefore,the realization of early detection of lung cancer is of great significance for the early diagnosis of lung cancer and the screening of high-risk lung cancer populations.Liquid biopsy based on microfluidic technology is an effective method for tumor detection in recent years.At present,related research and clinical applications of liquid biopsy based on microfluidic technology are mainly focused on the detection of serum tumor markers and circulating tumor cells(CTC)in the blood.Generally,serum tumor marker detection can be used to assist diagnosis and monitor tumor progression and recurrence,but there is currently no specific lung cancer serum tumor markers,which are prone to false positives and false negatives.CTC in blood can usually be detected as a biomarker for cancer diagnosis and treatment assessment,but CTC detection faces the problem of false negatives caused by large total peripheral blood,less CTC,and lack of tumor markers on the cell membrane surface.Sputum cytology is a unique diagnostic method that distinguishes respiratory diseases from other tissue and organ diseases.Its detection has the advantages of non-invasive,simple,and repeatable operation.Studies have shown from imaging that cancer cells can be detected in sputum before solid tumors form.Based on this,in this paper,we use sputum as the analysis object of microfluidic liquid biopsy to perform efficient sorting and cytological analysis of exfoliated tumor cells in sputum.This provides a new method for early diagnosis of lung cancer and screening of high-risk populations of lung cancer.Different from the liquid biopsy method that targets blood,sputum as the detection object faces unique problems: First,The sputum environment is different from the blood environment.The main interfering cells in the sputum separation process are granulocytes and non-tumor epithelial cells,among which granulocytes are mainly neutrophils,and non-tumor epithelial cells are mainly columnar epithelial cells and squamous epithelial cells.Second,the distinction between tumor cells and white blood cells is mainly in the blood environment,and the key to confirming tumor cells in the sputum environment is the distinction between tumor cells and non-tumor epithelial cells.The research content of this thesis will focus on solving these problems.The first chapter is preface.First,it summarizes the current research progress of the application of microfluidic technology in liquid biopsy for tumor diagnosis.It describes the common tumor detection methods including liquid biopsy and advantages of microfluidics in liquid biopsy.Subsequently,the research status of microfluidic technology in liquid biopsy is introduced,and the advantages and disadvantages of various liquid biopsy marker molecules are summarized.Finally,the significance and application of chip single-cell RT-PCR are introduced.The second chapter,single cell RT-PCR detection of exfoliated tumor cells in sputum for lung cancer diagnosis.In this chapter,we draw on the sorting and capture strategies of circulating tumor cells in peripheral blood.By designing high-throughput spiral chips and microwell array chips,sorting and single-cell capture of exfoliated tumor cells in a complex environment unique to sputum.Aiming at the problem of distinguishing tumor cells from epithelial cells in the sputum environment,we proposed and implemented single-cell RT-PCR detection using tumor cell function-related markers TERT and HK-2.Distinguish tumor cells from normal epithelial cells in the sputum environment,and provide a feasible solution for the identification of tumor cells in sputum.From this,we have achieved lung cancer detection by single-cell RT-PCR detection of tumor cell function-related markers in sputum.