The Effect Of Ultrasound Irradiating Docetaxel-loaded Microbubbles On Apoptosis In Human Liver Cancer Cells MHCC97-H And The Expression Of PCNA

Part ? The Effect and mechanism of Ultrasound irradiating Docetaxel-Loaded Microbubbles on apoptosis in Human Liver Cancer Cells MHCC97-HObjective To study the effects and its probable mechanism of ultrasound-irradiation docetaxel-loaded microbubbles on apoptosis in human liver cancer cells MHCC97-H.Methods The MHCC97-H cells were randomly divided into 4 groups: control group(CON), Docetaxel-loaded Microbubbles group(DM), Ultrasound group(US) and Ultrasound irradiating Docetaxel-loaded Microbubbles group(DM+US). The livability of MHCC97-H cells was detected by MTT test and the morphological changes of cells were observed through inverted microscope. The apoptosis rate of cells were detected with flow cytometry(FCM) and the charges of apoptosis protein were detected using apoptosis assay kit. The expression of PCNA protein was detected using cell immunofluorescence and flow cytometry, and the expression of PCNA protein and MAPK pathway members were detected by western blot of each group, respectively.Results After 24 h, MTT test showed that the livability of MHCC97-H cells of DM group, US group and DM+US group were(76.0 ± 3.0)%?(73.0 ± 5.0)% and(52.0 ± 6.0)% respectively, which was significantly declined comparing with control group. The morphological of tumor cells of control group remain normal size and grow eugenic, while the cells of DM group, US group and DM+US group decreased, the shape was irregular, the intercellular space increased, and cells were apoptosis in the medium,the DM+US group has changed significantly. FCM pictured the apoptosis rate of DM+US group was(26.81 ± 2.82)%, which was declined clearly compared with(2.90 ± 0.99) % of the control group(P<0.05). The changes of various apoptosis protein detected by apoptosis assay kit in DM+US group, including Caspase-3, c IAP-1, SMAC, and HSP70 etc. The expression of PCNA protein showed down-regulation detected by cell immunofluorescence and flow cytometry in DM+US group. Western-blot showed that DM+US group down-regulated the expression of PCNA and p-ERK1/2 protein, and meanwhile up-regulated that of p-p 38 protein in human liver cancer cells MHCC97-H.Conclusion The method of Ultrasound irradiating docetaxel-loaded microbubbles can notability induce human liver cancer cells MHCC97-H apoptosis by regulating the expression of PCNA and related protein.Part ? The Effect of Ultrasound-irradiation Docetaxel-loaded Microbubbles on the Tumorigenesis of MHCC97-H cells inoculated into Nude Mice and the Expression of PCNAObjective This study attempt to explore the effect of ultrasound-irradiation docetaxel-loaded microbubbles on the tumorigenesis of human liver cancer cells MHCC97-H inoculated into nude mice and the expression of PCNA, in order to provide a new method for ultrasound triggered drug-loaded microbubbles for the therapy of liver cancer.Methods MHCC97-H cells were subcutaneously inoculated into the right flank of nude mice to establish and treat the transplanted model of human liver cancer, and observed the growth of nude mice. The nude mice were euthanized after 30 days, the body weight of all nude mice was analyzed and toke out the tumor tissue. The morphology changes of tumor cells were observed by HE staining and transmission electron microscope. The expression of PCNA protein was detected by immunohistochemistry and the optical density can be determined by image analysis.Results Successfully established the model of human liver cancer cell carcinoma xenografts in nude mice, the tumor rate in nude mice model injected with MHCC97-H was 100%. The tumor tissue was confirmed to be liver cancer by pathological observation HE staining, the experimental nude mice survived in good condition, no obvious abnormal activities. The changes of nude mice body weight between DM+US group and CON group, the difference was statistically significant; the cell morphology and structure observed by HE staining and transmission electron microscopy(SEM) showed that DM+US group of tumor tissue cells density declined, and cells obviously apoptosis. The result of immunohistochemical of the nude mice tumor tissue determined the expression of PCNA protein in DM+US group(69.42 ± 16.72) declined dramatically, while the CON group of optical density value was(109.76 ± 15.37), the difference between them was statistically significant.Conclusion The method of ultrasound irradiating docetaxel-loaded microbubbles on the tumorigenesis of MHCC97-H cells inoculated into nude mice and the expression of PCNA protein had obvious inhibitory effect.