Isolation And Screening Of Colorectal Cancer Stem Cells And The Study Of Their Mitochondrial Energy Metabolism Characteristics

Tumors are composed of heterogeneous cells.Cancer stem cells are a small group of cells which are pluripotent and able to self-renew.They play important roles in the occurrence,progression and formation of tumors.Mitochondria are the energy factory in cells,which play a fundamental role in cell proliferation,apoptosis,calcium homeostasis,and other physiological and biochemical activities.The structural and functional abnormalities of mitochondria may be closely related to the occurrence and progression of malignant tumors.However,the detailed mechanism remains to be elucidated.Disclosing properties of mitochondrial energy metabolism and measuring its membrane potential in cancer stem cells will facilitate us to understand the occurrence,progression and recurrence of malignant tumors.At present,it is still technically difficult to accurately determine the mitochondrial membrane potential(??).In this thesis,we isolated and cultured the colon cancer stem cells using CD44 as the colorectal cancer stem cell-specific marker from HT29 cell line.Combining laser scanning confocal microscopy,flow cytometry and immune fluorescence staining technique,we investigated the mitochondrial amount,spatial distribution,respiratory chain activity and mitochondrial movement in colorectal cancer stem cells and non-stem cells.In addition,a new method was established for the detection of mitochondrial membrane potential using Mito Tracker Red CMXRos flow cytometry.The results revealed that mitochondria in the HT29 cancer stem cells mainly surround the nuclei whereas they distribute aggregatively in the cytoplasm in the non-stem cells and the HT29 cancer stem cells contain a significantly higher amount of mitochondria than the non-stem cells.The JC-1 flow cytometry and laser scanning confocal microscopy showed that mitochondria in the HT29 stem cells had higher membrane potentials than those in the non-cancer stem cells,which was further verified by the Mito Tracker Red CMXRos flow cytometry detection.Furthermore,the protein level of an important mitochondrial respiratory chain component,Complex IV,was found significantly higher in the HT29 cancer stem cells than in the non-cancer cells.In summary,these findings disclosed distinct patterns of mitochondrial energy metabolic characteristics in the colorectal cancer HT29 stem cells from non-stem cell.