| IntroductionHepatoma is one of the most common cancer worldwide. In China its mortality rate is the first or second malignancies. Each year about 45% of new cases are in China and it is tendency to onset in younger. The combined treatment is the main method for liver tumors at present. But hepatoma cells MDR (multidrug resistance, MDR) severely limits the effect of the treatment. Some data indicate that more than 90% of patients with liver cancer and the death of the MDR. How to reverse the MDR is an urgent task to improve the treatment of liver cancer. The ideal method of tumor MDR reversal must be efficient, asepsis and target (be specific to tumor while no damage to normal tissue). But recent researches are far from this standard. Therefore a series of studiesof MDR HCC have being carried out to explore new method which is the key to improving the effect on cancer control, and that is of important practical significance and value at present. The present researches were divided four parts. It would try to find a new low toxicity, efficient and targetted method for MDR reversion of HCC, and try to provide a theoretical basis to improve comprehensive treatment effects, clinical gene therapy and high-dose chemotherapy for liver cancer, which had Built the model of MDR human hepatoma cell and subcutaneous transplant tumor, explored the reversal effects and mechanisms on MDR of human hepatoma cells and transplant tumor through using acoustic microbubbles joint ultrasound taget-mediating for the antisense oligonucleotides transfection of mdr1 and mrp resistance gene. In this study, it had improved the efficiency of the MDR reversal by using acoustic microbubbles and ultrasonic cavitation effect to facilitate the efficient of targeted transfection and expression of gene in tumor cells for antisense oligonucleotide technologies of molecular biology in combintion with acoustics microbubbles and ultrasonic treatment. It is a strategy and technical innovation.Part 1 Establishment of HCC model for MDR cells and biological characterizationSection 1 Establishment of QGY Multidrug-resistant cell line induced by exposure of increasing concentration by degrees and intermission affect approach of CDDP and biological characterization Objective: To establish human hepatoma cell multidrug resistance model QGY and preliminary testing their resistance. Methods : The human hepatoma cell line for the pro-inhibiting cell, the use of chemotherapy drug cisplatin (cisplatin. CDDP), a low concentration increases delivery-resistant cells induced by intermittent role. Light microscopy, electron microscopy observation of morphological and ultrastructural characteristics of the new cell lines. Determination of cell doubling time and the mapping of the growth curve, observation of cell colony formation and flow cytometry (Flow Cytometry, FCM) detection of cell cycle distribution, identification of new biological characteristics. MTT assay of cell drug sensitivity, immunohistochemistry detecion of the expression of cells resistant Protein P-gp, MRP and LRP, apoptotic protein Bcl-2 and Bax. Results: During the 15 weeks, the MDR cell lines QGY/CDDP successful. QGY/CDDP in morphology and ultrastructure with the parental cells QGY is no different. General characterazation: MDR cell population doubling time than the parent cells extended 21-hour, colony formation was significantly lower than the parental cells, the cells in G0/G1 phase increased, the cell ratio in S and G2/M phase decreased, and the difference that with the parental cell QGY was statistically significant (P <0.05). Susceptibility testing revealed: QGY/CDDP cells were highly resistant to CDDP, RI (resistance index, RI) 10.35; of ADM (ADM). 5-FU (5-fluorouracil) and AST (arsenic trioxide) also resistance, RI 5.50, 8.51 and 9.23 respectively. The results of immunohistochemical were that staining positive for cell MRP highest rate of 23.89%, P-gp cells staining positive for 13.26%; the rate of LRP, Bcl-2 and Bax-positive cells was close to the parental cells. Conclusion: The new cell lines have MDR QGY/CDDP characteristics, resistance mechanisms mediating by the MRP and P-gp. MRP-mediating mechanism, while LRP, QGY/CDDP Bcl-2/Bax may not be involved in the formation of resistance.Section 2 Establishment of HePG2 Multidrug-resistant cell line induced by X-radiation exposure approach and biological characterizationObjective: To establish human hepatoma HepG2 cell MDR model and preliminary testing their resistance. Methods: The human hepatoma cell line HepG2 cells for parent was to X-ray irradiation at 6 MV linear accelerator, induce cell resistance. HepG2 cells after irradiation by X-ray accumulation, was inoculated with the 0.4μg/ml concentration of ADM (adriamycin, ADM) was cultured, good stability proliferation and growth of cells, suggesting that cell cytotoxicity against ADM. HepG2/ADM named for the upcoming new cell lines. Light microscopy, electron microscopy observation of morphological and ultrastructural characteristic of the new cell lines. Determination of cell adhesion rate, cell colony formation rate, and FCM detection of cell cycle distribution, observation HepG2/ADM biological characteristics. MTT assay sensitivity in HepG2/ADM cells, immunohistochemistry detecion of the expression of cells resistant Protein P-gp, MRP and LRP, apoptotic protein Bcl-2 and Bax, FCM in cell Rh123 intake and discharge capacity, indirect detection of P-gp and MRP functions. Results: During the 45 days, the cumulative dose of X-ray irradiation 24GY, treated cells in culture medium containing 0.4μg/ml ADM growth, HepG2/ADM MDR cell lines successfully established. HepG2/ADM in morphology and ultrastructure with the parental cells HepG2 was no significant different. General characterazation: HepG2 cells adherent rate, rate of colony formation significantly reduced, the proportion of cells in G0/G1 phase increased, the proportion of cells in S and G2/M phase decreased, that difference with the parental cells was statistically significant (P <0.05). Susceptibility testing revealed: HepG2/ADM cells were highly resistant to ADM, RI to 30.31; meanwhile to CDDP, 5-Fu and AsT also produced resistance, RI 6.12, 6.94 and 9.15. Immunohistochemical staining shows that the highest rate of P-gp positive staining cells to 85.62%, MRP rate of 12.36% positive staining cells, LRP positive staining cells with the parental cells close, Bcl-2 and Bax were 27.35%, 6.25% positive staining cells, Bcl-2/Bax ratio increased, all the differences with the parental HepG2 cells had statistically significance (P <0.05). Conclusion: new cell line HepG2 with MDR characteristics its resistance was combined by P-gp, MRP-mediating and apoptotic protein Bcl-2, the main mechanism mediating by P-gp.Part 2 Effects and mechanisms of hepatoma cells MDR reversed by acoustic microbubbles joint ultrasound target-mediating ASON of drug resistance gene transfectionObjective: To discuss the effect and mechanism of QGY/CDDP and HepG2/ADM human hepatoma cell line MDR reversed by ASON + ultrasound +contrast microbubbles. Methods: Log phase cells to adjust as a single cell suspension were to mdr1-ASON+ microbubbles+ ultrasound and mrp-ASON+ contrast microbubbles + ultrasound treatment, cell growth to be good, detection of the various indicators: In QGY/CDDP cells, MTT assay to detect changes in cell resistance, cell adhesion rate determination, detection of cell cycle distribution (FCM), apoptosis; TUNNEL determination to observe the biological characteristics after cells receiving the experimental treatment; RT-PCR assay to detect the mRNA expression of mdr1and mrp gene; Western-blot assay to detect the expression changes of protein P-gp and MRP; immunohistochemical staining assay to detect the expression of cell apoptotic protein bcl-2 and bax. In HepG2 cells, fluorescence AO/EB determination to detect apoptosis, other detection methods agreed to experimental treatment QGY/CDDP cell line testing. Results: The drug resistance of QGY/CDDP cells to CDDP, ADM, 5-FU and AsT was down to a certain extent, RI reduced: mdr1-ASON processed, compared with the control group the difference was no statistically significant (P> 0.05); mrp-ASON processed, compared with the negative control group the difference was statistically significant (P <0.05), and compared with the control group, the difference was no statistically significant (P> 0.05). mdr1-ASON after transfection, cell cycle distribution, relative expressing amonts of P-gp, MRP protein, and percentage of positive cells of apoptosis protein in the cells were smaller changes, compared with the control group the difference was not statistically significant (P> 0.05); and the rate of cells adherent, TUNNEL detection of apoptosis resulted, relative expression amonts of resistance gene mRNA to a certain extent for statistically significant (p <0.05). mrp-ASON after transfection, the six indicators of cells adherent rate, cell cycle distribution, TUNNEL determination of apoptotic cells, relative expression amonts of resistance gene mRNA, relative expressing amonts of P-gp, MRP protein were certain changes, the differences were statistically significant (P<0.05); immunohistochemical results of apoptotic protein was a small change. HepG2 cells after experimental treatment, the seven indicators of drug sensitivity of cells, cell adhesion rate, cell cycle distribution, apoptosis, mRNA expression of resistance gene, percentage of positive cells of resistance protein and apoptotic protein, showed certain cells changes, and compared with the control group the difference was statistically significant (P <0.05); also seen colony processing, changes of indicators were larger than that after mrp-ASON treatment. Conclusion: mdr1-ASON+ acoustic contrast microbubbles + ultrasound treatment and mrp-ASON+ contrast microbubbles + ultrasound exposure QGY/CDDP could partly reverse human hepatoma HepG2 cells MDR. Combination of ultrasound treatment, Ultrasound contrast microbubbles have a better target-mediating roles for ASON gene transfection than cationic liposomes, which showed a stronger role reversal to MDR of hepatoma cell lines. In QGY/CDDP cells, mrp-ASON transfection had a significantly stronger role in the reversal of MDR colony transfection than mdr1-ASON transfection; In HepG2 cells, mdr1-ASON and mrp-ASON transfection have greater role in the reversal of the MDR, transfection of MDR reversal colony stronger.Part 3 Establishment and characterization MDR model of hepatocellular carcinoma in nude miceObjective: To establish a human hepatoma QGY/CDDP, HepG2/ADM nude mouse model of multi-drug resistance and identified their resistance. Methods: Log phase QGY/CDDP, HepG2 cells to adjust as the single cell suspension, by the experimental groups, were directly injected subcutaneously, so MDR tumor cells were inoculated in nude mice subcutaneously, to observe and record the growth of tumors. When the implanted tumors grew to a certain size, two nude mice were randomly killed in each group, dissecting transplanted tumor tissue, extracting tumor cells to cultivate, when cells were in the logarithmic growth phase for resistance testing. Light microscopy, electron microscopy detection of morphological and ultrastructural characteristic of tumor cells. Observation of transplant tumor doubling time and the mapping of the growth curve, determination of cell colony formation rate and cell cycle distribution, to observe the biological characteristics of transplanted cells. MTT to detect the sensitivity of tumor cells to drug. FCM to detect the expression of P-gp and MRP. Western blot to determine the expression of cell apoptosis protein Bcl-2 and Bax. Results: In QGY/CDDP, HepG2/ADM nude mice, the average survival period was 40±15 days. Growth Statistical analysis of transplanted tumor size: the growth of the two cell tumors was slower than that of the parental cells transplanted tumor, and the difference was statistically significant (P <0.05). MDR tumor cells in morphology and ultrastructure of the cells had no significant difference to pro counterparts. Detection of biological characteristics of the transplanted tumor cells: two strains of cell doubling time were longer (P <0.05), the rate of cell colony formation decreased (P <0.05), the proportion of cells in G0/G1 phase of the cell cycle distribution increased, the proportion of cells in S and G2/M phases declined, which the difference was statistically significant compared with parental implanted tumor cells (P <0.05). Results of MTT assay: Two MDR tumor cells were resistant to ADM, CDDP, 5-FU and AsT, and the RI of QGY/CDDP tumor cells to CDDP was the highest, he RI of HepG2/ADM tumor cells to ADM was the highest, compared with each parental implanted tumor cells, which the difference had statistically significant (P <0.05). The results of detection of resistance protein and apoptosis protein: In implanted tumor cells QGY/CDDP, the rate of positive cells of MRP protein staining was the highest to 23.85% (P <0.05), the rate of positive cells of P-gp cells staining was 7.44% (P <0.05); the rate of positive cells of Bcl-2 and Bax cells staining was close to that of the parental tumor cells. In HepG2/ADM transplanted tumor cells, the rate of positive cells of P-gp cells staining was highest to 84.97%, 10.26% rate of cells staining positive MRP, and relative expressing amonts of Bcl-2 and Bax protein, which the difference was statistically significant (P <0.05) compared with the parental transplanted tumor cells. Conclusion: To successfully establish the human hepatoma QGY/CDDP, HepG2/ADM nude mice model. The main resistant machanism may be of the expression of mdr1, protein P-gp, mrp, protein MRP (P190) and apoptotic proteins Bcl-2/Bax. The implanted model had the high success rate and had repeatable, stable biological characteristics.Part 4 Effect and mechanisms of reversal of hepatocellular carcinoma MDR in nude mice by acoustic microbubbles joint ultrasound target-mediating ASON of drug resistance gene transfectionObjective: To preliminary study the effect and mechanism of reversal human hepatocellular carcinoma MDR in nude mice in vivo by ASON+ ultrasound contrast microbubbles+ ultrasound treatment. Method: The nude mice implanted tumor in vivo, by the lab groups, were given experimental treatment: mrp-ASON+ ultrasound contrast microbubbles+ ultrasound treatment to QGY/CDDP transplanted tumor, mdr1-ASON+ ultrasound contrast microbubbles+ ultrasound treatment to HepG2/ADM transplanted tumor, corresponding ASON+ cationic liposome+ ultrasound as a positive control, corresponding MDR tumor + switch dye injected as a negative control. At the end of death in nude mice in vivo, transplanted tumor was stripped, the corresponding tumor cells were extracted and cultivated. Cells were in the logarithmic growth phase for MTT susceptibility testing, mRNA expression of mdr1 and mrp gene measured by RT-PCR, and expression of P-gp cells and protein MRP was detected by Western blot, expression of apoptosis protein of bcl-2 and bax was determined by immunohistochemical staining, the implanted tumor cell membrane ATPase activity was determined. Results: After in vivo treatment of the human hepatocellular carcinoma xenograft in nude mice, the implanted tumor cells were detected to find: after mrp-ASON+ultrasound contrast microbubbles+ultrasound treatment to QGY/CDDP transplanted tumor, compared with the negative control group, the drug resistance of transplanted tumor cells to CDDP, ADM, 5-FU and AsT significantly reduced (P <0.05); compared with the positive control group, the resistance reduced to a certain extent, but the difference was not statistically significant (p>0.05), RI reduced. RT-PCR detection showed: the expression of mrp gene of transplanted tumor cell was significantly decreased (to mRNA expression of mdr1 gene have a relatively small impact), and compared with the control group, the difference was significant (P <0.05). Western-blot analysis showed: the expression of MRP of transplanted tumor cells declined to a certain (absolute value of MRP greater than that of P-gp), and compared with the control group, the difference was statistically significant (P <0.05). The immunohistochemistry results of apoptotic protein showed: bcl-2 and bax of transplanted tumor cells had changed to some degree, and compared with the control group, the difference was statistically significant (P> 0.05). Determination of cell membrane ATPase activity can be found that: the cell membrane ATPase activity of transplanted tumor cells increased to some degree, however, no significant difference compared with the control group was significant (P <0.05). After Mdr1-ASON+ ultrasound contrast microbubbles + ultrasound treatment to HepG2/ADM transplanted tumor, the drug-resistance of transplanted tumor cells to ADM, CDDP, 5-FU and AsT significantly reduced, and compared with the control group, the difference was statistically significant (P <0.05), RI reduced. RT-PCR detection showed: the mRNA expression of mdr1 gene and mrp gene of transplanted tumor cells decreased significantly higher than that of the control group (P <0.05). Western blot results showed that: the expression of P-gp and MRP gene of transplanted tumor cells was significantly decreased (absolute value of P-gp greater than that of MRP), and compared with the control group, the difference was statistically significant (P <0.05). The immunohistochemistry results of apoptotic protein showed: the bcl-2 and bax of transplanted tumors noticeable changed significantly higher than the control group (P <0.05). Determination of cell membrane ATPase activity can be seen that: the cell membrane ATPase activity of transplanted tumor cells was up to a certain extent, and compared with the control group, the difference was statistically significant (P <0.05). Conclusion: ASON+ ultrasound acoustic microbubbles+ ultrasound treatment in vivo could reverse the human hepatocellular carcinoma MDR xenograft in nude mice, and ultrasound acoustic microbubbles had a better target-mediating roles for ASON gene transfection than cationic liposomes combination of ultrasound, which showed a stronger role reversal to hepatocellular carcinoma MDR in nude mice. Analysis of the possible reasons for this phenomenon: the ultrasonic irradiation led to the breakdown of microbubbles, cavitation effect of micro-bubble breakdown promoted the transfection and expression of ASON in the cells. The formation of MDR of the transplanted hepatocellular carcinoma QGY/CDDP in nude mice is mainly mediating by mrp, little relations with mdr1 mediating mechanisms and anti-apoptotic mechanisms, accordingly the effect to reverse cells MDR was by down the expression of mrp and its encoded protein. The formation of MDR of the transplanted hepatocellular carcinoma HepG2/ADM was of mdr1-mediating mechanisms, mrp-mediating mechanisms and the anti-apoptotic cell mechanisms to enhance the role of a common mechanism, the main mechanism mediating by mdr1, accordingly the effect to reverse cells MDR was by reducing the role of the three mechanisms, in particular, achieved by down the expression of mdr1and its encoded protein P-gp.Summaries1. CDDP delivery using low concentration increases-intermittent role and Linac X-ray irradiation method to establish two MDR cell lines model: QGY/CDDP and HepG2/ADM. The two cell lines have a high level of MDR character . QGY/CDDP to CDDP, ADM, 5-FU and AsT resistance index were 10.35, 5.50, 8.51 and 9.23. HepG2/ADM to CDDP, ADM, 5-FU and AsT, RI were 6.12, 30.31 ,6.94 and 9.15.2. QGY/CDDP's main mechanism of MDR is mediating by the mrp and its encoded protein MRP. There is little mdr1 and cell-mediating anti-apoptotic mechanisms and strengthen mechanisms. HepG2 cells, it's MDR mechanism mediating by mdr1and protein P-gp,mrp and protein MRP and the anti-apoptotic ,But the main mechanism ismdr1 and protein P-gp mediating.3.Irradiation of mdr1, mrp-ASON+acoustic microbubbless+ultrasound can partially reverse the multidrug resistance of QGY/CDDP and HepG2/ADM human hepatoma cells. acoustic microbubbles and ultrasound combined have a better target-mediating roles for MDR gene ASON transfection than cationic liposomes and ultrasound combined, it's showed a stronger reversed effect in MDR of hepatoma cell lines. In the role of QGY/CDDP reversed MDR ,the mrp-ASON transfection significantly stronger than mdr1-ASON transfection. For HepG2/ADM, mdr1-ASON and mrp-ASON transfection have great effect in the reversal of the MDR, mdr1-ASON Transfection more stronger.4. MDR hepatoma cell suspension were inoculated subcutaneously in nude mice directly. we established human hepatoma QGY/CDDP and HepG2/ADM xenograft model in nude mice. The transplanted tumor cells are highly tolerance tumor cells :the RI of QGY/CDDP transplant tumor cells to CDDP,ADM,5-FU and AsT is 9.28, 4.50, 7.52,8.36; the RI of HepG2/ADM transplanted tumor cells to ADM, CDDP, 5-FU and AsT is 6.05, 27.94, 6.73 , 8.96.5. Irradiation of mrp-ASON+ acoustic microbubbles + ultrasound can reverse partial the resistance of MDR QGY/CDDP human hepatoma cells that transplanted in nude mice. mdr1-ASON+ acoustic microbubbless + ultrasound irradiation can reverse partial the resistance of MDR HepG2/ADM human hepatoma cells that transplanted in nude mice. acoustic microbubbles and ultrasound combined have a better target- mediating for MDR gene ASON transfection than cationic liposomes and ultrasound combined,it's showed a stronger reversed effect in MDR of transplanted tumor cells.6. Acoustic microbubbles joint ultrasound target-mediating for MDR gene ASON transfection reversed the resistance of MDR human hepatoma cell lines and transplanted tumor, maybe major mechanisms is closed down or blocking mdr1 mrp and the encoded protein expression, thus lowering the resistance of cells. other mechanisms may include to improve the cell sensitivity of drug , improve cell membrane ATPase activity,promote cell uptake of the drug , increase intracellular drug concentration and changes in the expression of apoptosis-related gene,lowered Bcl-2/Bax ratio and so on. Maybe the reasons that acoustic microbubbles agent and ultrasound combined have a better taget- mediating for MDR gene ASON transfection and reversed effect in MDR than cationic liposomes and ultrasound combined: low-intensity ultrasound exposure may lead to micro-bubble bursting and micro-bubble cavitation effect for the bursting can accelerate ASON transfection and expression in the cells ,then the MDR cell lines and transplanted tumor cells resistance decreased.
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