| Objects Hepatocellular carcinoma(HCC) is one of the most common malignant tumors in our country, which is gravely threatening people's health because of its high malignant degree and bad prognosis.Exairesis is still the first-line and most effective treatment to HCC nowadays. Chemotherapy, as an important adjunctive treatment to HCC, has to settle the two tough difficulties of cytotoxicity to normal cells and drug resistance of tumor cells. It is researched that Survivin is the most powerful inhibiting apoptosis factor ever been found with the dual function of inhibiting apoptosis and regulating cellcycle, and it has a close relationship with chemoresistance. Nanoparticle gene transporter, an non-viral gene transporter ,is well developed in recent years because of its safety, efficacy and fine property. In this paper: 1. to prepare polylactic-chitosan(PLA-CS)modified nanoparticles and analyze its properties and vitro transfection efficiency; 2. to test directly using molecular beacon which is a new living cell detection technique and prove that Survivin is high expressed in Be17402; 3.to use Survivin antisense oligodeoxynucleotide (ASODN) loaded PLA-CS nanoparticles to enhance the apoposis of HCC induced by adriamycin(ADM).Methods 1.to prepare PLA-CS NP with small particle diameter and uniform distribution using solvent emulsionizated volatilixation method and with optimized conditions of technique; to detect Zeta electric potentials and particle diameter distributions of PLA-CS NP and PLA-CS/DNA using Malvern Zetasizer 3000 E; to detect particle diameter and appearance of PLA-CS NP by Atomic force microscope (AFM). Then to observe the combination of PLA-CS NP and DNA and the PLA-CS's protection to DNA by electrophoresis; to detect the transfection efficiency and cytotoxicity of PLA-CS NP in vitro by MTT assay and Flow cytometer (FCM); 2. to transfect molecular beacon designed according to Survivin into HL7702 and Be17402 via PLA-CS NP, then to detect the changes of Survivin transcripts, and to detect the influence of cell growth caused by molecular beacon using Typan blue staining; to confirm the accuracy of molecular beacon detection using RT-PCR;3. to transfect the Survivin ASODN loaded PLA-CS nanoparticles into hepatoma carcinoma cells, then to detect the change of Survivin by methods of RT-PCR and Western blot, to detect cytostasis using MTT assay, and to detect apoptosis rate of HCC using FCM. After acting with ADM, to detect cytostasis using MTT assay and to observe the change of cell apoptotic index using FCM and AO/EB staining.Results 1.Detection results of the particle diameter and distribution using laser particle size analysor Malvem Zetasizer 3000E showed that prepared PLA-CS NP had small mean diameter of 87 nm and the particle diameter had well Gaussian distribution. It was observed by AFM that NPs were global ones with smooth and complete surface and without adhering and agglomeration. Zeta electric potential was+10mV±1.5mV when PLA-CS NP coupled with DNA. It was analyzed from electrophoresis results that NP and plasmid DNA can be combined together effectively. MTT results showed inhibition ratio of cell growth was less than 10% as the concentration of PLA-CS NP was below 3μg/μl.The transfection efficiency can reach 43.5% detected by FCM. 2. After transfecting molecular beacon loaded PLA-CS NP into HL7702 and Be17402, it was observed that there was hardly any fluorescence in HL7702 when strong green fluorescence signal was found in Be17402. Trypan blue staining essay showed cell vigor was uninfluenced basically. RT-PCR confirmed that the Survivin mRNA in Be17402 was higher expressed than that in HL7702. 3.24 hours after all the groups having been transfected by Survivin ASODN, the mRNA and expression of proteins of them all decreased, especially the one of the group with the concentration of 800ng/ml of Survivin ASODN, and there was significant difference with the control group(P＜0.05); MTT assay results showed that all cells group's proliferation activity degraded disparity after having been transfected by survivn ASODN. FCM detection results showed that 24 hours after the efficient of ASODN the apoptosis rates were 7.88+0.15%, 14.16+0.35%, 32.73+0.78% respectively in the Be17402 groups with concentration of 400ng/ml, 600ng/ml and 800ng/ml, and there was significant difference contrasting to blank group with apoptosis rates of 0.50+0.06% and SODN transfected group with apoptosis rates of 0.57+0.09%(P＜0.05). After the acting of ADM, apoptosis rate of Be17402 was 40.34+0.78% in the group with concentration of 600ng/ml,and there was significant difference contrasting to the apoptosis rates of 16.94+0.54% or 15.07+0.35% respectively in groups using ADM or ASODN transfected only. AO/EB staining results showed more cells in ADM+ASODN group had the change of nucleus morphous including karyopycnosis, thick stain and disruption of nucleus. MTT assay results showed that the cytostasis degree had obvious difference (P＜0.05) between blank control group with ADM group or ASODN group 24 hours after having been transfected.,and there was significant difference between ADM+ASODN group with each of other three groups (P＜0.05).Conclusions 1.PLA-CS NP with mean diameter of 87 nm and even distribution had been prepared successfully. The NP whose surface had been modified by CS to enable it have positive charge can combine with plasmid DNA reversibly, then PLA-CS NP gene transport system with low cytotoxicity and high transfection efficiency was assembled. 2. That a method of detecting genetic transcript in living monoplast cells by using molecular beacon can be expected to be well developed as a method of detecting tumor markers in the early stage. 3. The transfection induced by Survivin ASODN which has efficient function of blocking loaded PLA-CS NP gene carrier which is safe and efficient has high transfection efficiency. Using PLA-CS NP gene carrier to induce the transfection of Survivin ASODN, it can be more efficient to block survivin and down regulate the expressions of protein and mRNA of surviving which can promote the apoptosis of Be17402 finally. Using ASODN to block expression of survivin can inhibit the growth of HCC efficiently and increase the apoptosis of cell. Also, that can improve the chemosensibility of ADM and the effect of routine chemotherapy. All these make it potential in clinical application.
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