| Objective The failure of chemiotherapy for malignant tumor mainly attribute to the tolerance of tumor cells to anti-tumor drug. Few research had been carried into execution on tolerance of HepG2 cell growing under the selective pressure of cisplatin. This study is aim at the selective results made by HepG2 cells which were cultured under the selective pressure of cisplatin and to observe the internal environment change of survival HepG2 cells, the possible initiation site of HepG2 cell signal conduction and the selective response of HepG2 cells to the cisplatin cytotoxicity in development. The overall objective of this study is to understand in detail that how the HepG2 cells tolerance to cisplatin and lay the groundwork in seeking of key position of tolerance to cisplatin in HepG2 cell and provide guidance forclinical chemiotherapy.Methods:Chapter 1 Establishment of HepG2 cell strain toleranced to cisplatin: HepG2 cell strain HepG2 /CDDP toleranced to cisplatin in two concentrations were established by continuously adding cisplatin with the manner of double increase at earlier period and increase progressively at anaphase. MTT technique was used to test the tolerance of HepG2 cell and HepG2 /CDDP to the cytotoxicity of cisplatin. Immune histochemisty technique and RT-PCR were used to detect the expression of MDR (LRP, MRP) which reflecting the tolerance to anti-tumor drugs of HepG2 cell and HepG2 /CDDP. ultrastructural change and different between HepG2 and HepG2 /CDDP were distinguished by transmission electron microscope.Chapter 2 Observation on the change of ROS in cells and its influence on mitochondrion function in the process of HepG2 cell toleranced to cisplatin: Confocal microscopy was used to detect the ROS level and its change in cells real time in situ by taking advantage of the propert that DCFH-DA (2',7'-dichloro fluorescein) could generate luminescence fluorescein evoked directly by ROS. The change of mitochondrion membrane potential in the process of HepG2 cell tolerant to cisplatin was detected with fluorescentintensity which was observed under confocal microscopy by using fluorescent probe Rhodamine-123. The changes of mRNAs of cytochrome C, NF-κB and a and b subunit of ATP enzyme which reflecting the change of cell internal environment and mitochondrion function were tested by RT-PCR.Chapter 3 Change of apoptosis related genes in the process of HepG2 cell toleranced to cisplatin: The expression of Bd-2/Bax, PKC, PTr, TGF-βRII, c-myc and P53 were detected respectively by immune histochemisty technique.Results:Chapter 1:1. Two HepG2 /CDDP cell strains were established successfully: HepG2/CDDP0.4 was tolerant to cisplatin in low degree and HepG2/CDDP2.0 was tolerant to cisplatin in middle degree. The drug resistance index was 1.89 and 7.36 respectively.2. LRP expression of Cells in low and middle degree drug resistance were both positive,and MRP expression of middle degree drug resistance strain was negative. Maternal cell and middle degree drug resistance strain had different ultrastructures : HepG2/CDDP2.0 had more abundance Golgi complex3. and rough endoplasmic reticulum then maternal cells, but no significant change of mitochondrion was seen in the two strains. Chapter 2 : 1. In the process of low degree drug resistance formation to cisplatin, it was found that ROS in HepG2/CDDP0.4cells increased significantly at 24h after adding cisplatin and decreased at 48h and there was no significant difference between HepG2/CDDP0.4cells and maternal cells. Mitochondrion membrane potential decreased significantly at 24h after adding cisplatin and gradually got recovery after 48h. Mitochondrion membrane potential had no significant difference between HepG2/CDDP0.4cells and maternal cells after 5 days 2. Cytochrome C, a and b subunit of ATP enzyme had no significant change.Chapter 3:1. Bcl-2 increased in fluctuation manner with adding cisplatin. Bax decreased in paranet to the time. Relative ratio of Bcl-2/Bax s...
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