| Hepatocellular carcinoma (HCC) is one of the most common liver tumor, which mostly caused by chronic inflammation after HBV, HCV infection or cirrhosis. HCC ranked the No.2 killer in our nation's cancer sort, and the number of deaths related to HCC each year accounts for about 53%in worldwide total deaths. The apoptosis regulation gene, especially B Cell lymphoma/leukemia-2 (Bcl-2) family were deeply researched as the regulatory mechanisms of apoptosis was very complex. Myeloid cell leukemia-1 (Mcl-1) is the most important anti-apoptotic member of Bcl-2 family and the vital anti-apoptotic factor of HCC. It is important to study the Mcl-1 gene abnormal expression in hepatocellular carcinoma to understanding the possible mechanism of tumor occurrence and pathway of anti-tumor therapy. Mcl-1isa new target in anti-tumor therapeutic strategy in the future.RNA interference (RNAi) can be induced by small interference RNA (siRNA), which is new developed technology in recent years. Plasmid or virus carrier that express short hairpin RNA (shRNA). The role of gene silencing of RNAi is specific and effective, which has some merits such as powerful inhibitory effect, good stability and easily absorbed by cells. RNAi provide a new and potent method for gene therapy of tumor and has becoming a widely used tool for gene silencing in current gene therapy research. SiRNA selectively inhibiting gene express makes gene therapy achieving maximal specificity. But puny introduction capability into cell, definite blood stability and non-specific immunogenicity hinder siRNA development for gene therapy. Researchers both in domestic and abroad have not efficiently solved the problem of low transfection efficiency low of siRNA.Gene transfection need suitable carrier. An ideal gene transport system should have high gene transfection rate, favourable target and biocompatibility, fine stability and biological degradation. The viral vector and non-viral vector have different defect to some extent at present. So it is very critical to find an ideal gene carrier. The research of non-viral nanoparticles (NPs) for gene carrier is an important advancing front topic in the fields of nano-biology and tumor gene therapy in internation. It is proved that the binding between NPs and DNA was accomplished by electrostatic attraction. The negatively charge of phosphate backbone of DNA could be binded to the positively charged of vector. Therefore, modification of the NPs surface properties with biomolecules make its surface earring positive charge which can not only protect the condensation of NPs, but also be avaliable to the gene delivery. The magnetic Fe3O4 NPs show the characteristics of magnetic targeting and passive targeting. Amino-function to the surface of NPs could enhance its stability and efficiency of gene transfection. Based on the background and achievement above, we detected Mcl-1 protein expression in HCC tissues and in adjacent tissues by immunohistochemical Envision method, and investigate the relationship between imbalance of apoptotic mechanism and the occurrence of HCC and study the significance of clinical pathology. As a result, there is significant enhancement of Mcl-1 protein expression in poorly differentiated, proliferative HCC tissues than in well differentiated HCC tissues. Mcl-1 positive expression in total 62 HCC tissues (64.5%) is higher than in the adjacent cancer tissues. It were considered to be related to Edmonson grade classification, tumor size, intrahepatic metastasis (P <0.05), and it provided the experimental base for the next gene therapy.We designed and synthesized Mcl-1 siRNA, and Mcl-1 therapeutic shRNA recombinant plasmid through link with psiRNA-Hhlneo vector. Mcl-1 shRNA were tested correct by enzyme electrophoresis and DNA sequencing and were optimized selected through transfection to HepG2 cells to examine Mcl-1 mRNA decreased levels. As a result, we successfully synthesized therapeutic eukaryotic expression vector of Mcl-1 shRNA. There were obviously decrease in Mcl-1 mRNA expression in gene therapy group than in control groups after gene transfection (P<0.01). PsiRNA3 provided the highest inhibition of Mcl-1 mRNA expression, so that we used it to study the therapeutic effect of gene interference targeted to Mcl-1 in HCC. We used improved coprecipitation method to prepare PLL modified Fe3O4 magnetic NPs. TEM, laser particle size analyzer were applied to analysis morphology, particle size, Zeta potential of DMNP. The cytotoxicity of DMNP was investigated by MTT method and its gene transfection efficiency was tested by pEGFP-C1 expression in fluorescence microscope. As a result, the prepared dextran coated Fe3O4 NPs have uniform size and well dispersed. After amino-function modified by PLL, DMNP showed moderate gene protection with its size was (32±4) nm, Zeta potential was +18.23mV. Therefore, we choosed the DMNP as the gene vector in the next experiment.The last part is the experimental study of DNMP mediated Mcl-1 shRNA in the treatment of HCC in vitro and in vivo. At first HepG2 cell was cultivated, then the DNA-NP was transfected into it with or without external magnetic field, after 72h detected inhibition rate of Mcl-1 mRNA and protein by RT-PCR and western blot, detected apoptosis by flow cytometry and fluorescence microscope to inspect gene therapy efficacy in vitro. Established HCC transplantation tumor bearing nude mice model, injected DNA-NP into tumor multiple, drew the growth, curve of tumor, took out the tumor to detect volume inhibition ratio and weight inhibition ratio after 24d. Results indicated that the inhibition ratio of Mcl-1 mRNA of test group were about 54.3% and reached 72.3% after external magnetic field, which increased remarkably than spurious therapy groups (both P<0.01). In test group tumor grows low remarkably, the mean volume inhibition ratio and weight inhibition ratio were also increased significantly than control groups (P<0.01). Which indicated that the DNMP NPs vector mediated Mcl-1 shRNA has very good therapeutic efficacy to HCC which Mcl-1 express is high.
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