Research On Microchip-based Capillary Electrophoresis With Laser Induced Fluorescence Detection Of Drug-resistant In Single Cell

Microchip-based Capillary Electrophoresis(μCE) has the unique superiority and succes in single cell detection about the quantitative and quantitative analysis, with advantages of small sample size, high separation efficiency and high compatibility. Laser Induced Fluorescence(LIF) is the MCE first used and still most commonly used as detection technique with advantages of high sensitivity, strong specificity and fast response.In this dissertation, multidrug resistant cell line HepG2/ADM was induced by stepwise selection on exposure to increaseing doses. The method of μCE-LIF is applicated to separate and determinate the content of Doxorubicin(DOX), Reduced Glutathione(GSH), Oxidized Glutathione(GSSG) and Reactive Oxygen Species(ROS) in single cell during drug-resistant cell culture and TMP reversing DOX resistance within the 72 h firstly. The main contents of this dissertation are summarized as following :1. Introduce the research status of multi-drug resistance mechanism about cell and the resistance mechanism of DOX(content of intracellular DOX, GSH, GSSG, ROS) and TMP reverse DOX resistance mechanism are summarized.2. Multidrug resistant cell line HepG2/ADM was induced by stepwise selection on exposure to increaseing doses. The inverted microscope and fluorescence microscope was used to observed the cell morphological changes and the gathering of DOX. The content of DOX in single-cell was dectected by μCE-LIF. The human liver multi- resistant cell line HepG2 / ADM was established after 6 months of continuous culture. DOX mainly accumulated in the nucleus at the beginning of culture, and DOX and its metabolites could be detected. In the late induction, DOX could not be detected. This experiment furtherly confirmed that DOX toxicity to the tumor cells was directly related to the content of DOX.3. The content of GSH, GSSG and ROS was detected by μCE-LIF single cell analysis. GSH and GSSG was dynamically labeled by the 2, 3-naphthalene-dicarboxaldehyde( NDA). ROS was derivatized by dihydrorhodamine 123, which can be permitted to enter the cell. GSH, GSSG and ROS could be detected simultaneously. It was found that both GSH and GSSG could be derivatized online by NDA. During the formation of multidrug resistance cells, the content of GSH inside the cell was increased, and the content of GSSG and ROS was decreased gradually as the DOX content in culture medium increased. This research reflects somewhat the resistance mechanism of DOX is related to the toxin immunity of cell that is to enhance and the apoptosis pathway blocking.4. The ability of reversing of DOX resistance about TMP was investigated by μCE-LIF single cell analysis. Fist of all, using phenol blue and MTT experiment to explore the toxicity of TMP on cell, we found that the best concentration of TMP to reverse HepG2/ADM cells resistance is 20 μg/mL. HepG2 / ADM cells are set to a negative control group(without any drugs), the chemotherapy drug group(1.0 μg/m L DOX), ligustrazine group(20 μg/m L TMP), the chemotherapy drug + ligustrazine group(1.0 μg/m L DOX + 20 μg/mL TMP). After drug induced 72 h, GSH, GSSG and DOX can be detected simultaneously by μCE-LIF. The results showed that the content of DOX in cell is increased obviously in TMP+DOX group and nearly seven times more than the DOX group, but haven’t reach prompt drug-resistant cell apoptosis not yet. TMP reverse of DOX resistance preliminary. TMP and DOX are co-adminiatered more time to reverse DOX resistance completely.