| Multi-drug resistance (MDR) remains one of the major obstacles to successful chemotherapy. More than 90% of tumor patients died of MDR. Consequently, there is still an urgent need of a viable strategy to overcome MDR. An ideal reversal approach should meet the following standards: efficient, nontoxic and most importantly specifically targeting only the tumor tissues. The commonly used MDR reversal agents are far from the above standards. Chemical agents, for example, usually exhibit considerable reversal effect in vitro, while the serious side effects prevent their clinical applications. Gene therapy may play a role when the MDR is mediated by a single established mechanism. However most MDRs are caused by vast factors, and furthermore many of the factors remain undiscovered.Ultrasound refers to sound waves with frequencies above 20kHz. It is generated from mechanical vibrations. As high frequency and short wave length, ultrasound has several advantages as follows: ①transmit in a definite direction; ② it can be focused; ③ beam can spread through the tissues; ④ have a variety of bio-effects on cells or tissues; ⑤ultrasound regimes are convenient and acoustic dosage can be exactly managed. Although several research groups have documented ultrasound effects on the MDR reversal, the published data are confused because the ultrasound parameters differs greatly from different laboratory. In this study, we aimed to explore the possibility of using ultrasound for the purpose of MDR reversal, to select the optimized parameters of therapeutic ultrasound, and to investigate possible mechanism of ultrasound-induced MDR reversal. PART ONEEstablishment and Characterization of Multi-drug Resistant Tumor Cell linesObjective To establish multi-drug resistant tumor cell lines and to assess their resistant characteristics. Methods Human hepatoma cell line HepG2 was exposed to adriamycin (ADM) in the culture medium for the establishment of drug resistant cell lines in two ways as follows: concentrations of ADM was stepwise increased for long exposure; a single high concentration was used for short exposure. Morphologic studies were performed with optical microscopy and transmission electron microscopy. Drug sensitivities were determined by MTT assay, and P-gp activity was evaluated by Rh123 influx and efflux assay. Immunocytochemistry staining was employed to assess the expression of MDR1, MRP and LRP, and proteins of apoptosis genes including Bcl-2 and Bax. Results Three months were taken to establish drug resistant cell lines HepG2/ADMs and HepG2/ADMi. HepG2/ADMs was the result of pulsed exposure to ADM and HepG2/ADMi was the result of stepwise continuous exposure. No obvious morphologic changes were observed between resistant and parental cell. Resistance indices (RI) of HepG2/ADMs and HepG2/ADMi to ADM were 30.5 and 51 respectively. Meanwhile, HepG2/ADMs and HepG2/ADMi were also resistant to VCR and VP-16 and the RI were 40.16, 57.06 and 11.65, 15.53 respectively. Either decreased Rh123 influx or increased efflux was detected. Positive expressions of P-pg, MRP, Bcl-2 and Bax were seen. Conclusion Both cell lines HepG2/ADMs and HepG2/ADMi were multi-drug resistant. HepG2/ADMi was more resistant to drugs than HepG2/ADMs. P-gp and MRP played a major role in the development of MDR, and both Bcl-2 and Bax were partly involved. PART TWOEffect of Ultrasound on Cell Viability and Determination of Sublethal Ultrasound DosageObjective To observe the effect of ultrasound exposure on the cell viability and to determine the sublethal ultrasound dosage. Methods Cell viability was evaluated by the trypan blue exclusion test after HepG2 and HepG2/ADMi cells were exposed to various dosages of ultrasound. Proliferative activities were assessed by MTT assay, and colony-forming abilities were determined in soft agar. Cell cycle analysis was performed by flow cytometry (FCM). Results Ultrasound sublethal dosage was determined by ultrasound parameters and cell types. Sublethal dosage of 0.8M...
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