The Study Of Combine Effect Of SiRN Aeukaryotic Expression Vector Target Rb And TM5-FuNC On Human Cholangiocarcinoma Cell

Background & Objective: Cholangiocarcinoma is mainly named as the extrahepatic bile duct malignant tumor. The prognosis of it isn't idealization. It's scarce in epidemiology and is about 0.01%~0.5% in biopsy, about 3% in the total count of the malignant tumor. It's about 0.07%~0.3% in domestic biopsy. The incidence rate of Cholangiocarcinoma is about 2.32% in all disease of biliary tract. The rate of female and male is 1.46:1. The incident rate is increasing progressively in recent years. The treatments of cholangiocarcinoma were given priority to operation or combine chemotherapy with radiation treatment. But the position of cholangiocarcinoma very deep and concealment in anatomy, it's hard to detect cholangiocarcinoma in earlier period. As a result the effect of operation was discontentment, and the survival rate was very low. The chemiotherapy drug inhibit or kill tumor cells by act on different links of cell breeding, which can treat tumor. This is become a important method for clinical tumor treatment now. But the effect of chemiotherapy drug can not reach the aim of satisfactory. The report of the effect of 5-Fu is only reach 14%-18% regardless of simplex or combine medication.Targeting treatment to malignant tumor is a new therapeutic method which as a result of conjunct nanotechology with chemotherapeutic drug rising in recent years. The principle is that the carrier including various kinds species group affinity carry particles and magnetic targeting particles take drug which could targeted reach to the locus of medication in order to treat disease. This will afford experctation for cure malignant tumor.Our topic group lead by professor zoushengquan succeed get a great topic supported by the grant from The National Hi-Tech Research and Development Program of China (863 Program)(No. 2002AA214061). The core content of this project is that a magnetize cradle which be sacked by metal can reinforcement the TM5-FuNC targeting power while the cradle keeping bile duct drain smoothly. As a result that this will to achieve the real high selective and regional treatment. This will realizated break and new idea of Cholangiocarci- noma treatment, Furthermore, our group of the topic group has investigated deeply on the cholangiocarcinoma bionomics before. We concluded that there are complex network feed back mechanism between the oncogene and the anti-oncogene which is exist in the process of occurrence and develop of cholangiocarcinoma. pRb signal pathway were close importa- nt to the complex network feedback system of cholangiocarcinoma. It's a great break in the research of the molecular cytogenetics in the cancer of extrahepatic biliary duct.We based on the prophase research, put our emphasis on the pRb signal transduction pathway, and make some investigate on the apoptosis of the cholangiocarcinoma and the Fuction of the sensitization of the chemotherapy. We association the siRNA eukaryotic expression vector Rb with TM5-FuNC act on human cholangiocarcinoma cell in vivo and vitro, which will provid us a test base and realization pathway for the union opoerate of nanotechnology and gene therapy. Restricted to the time, we are limited and superficialis level, and we will make the Further step in the Future.1. Methods and results: Eukaryotic expression vector of siRNA Rb were constructed, and used to transiently transfect to QBC939 mediated with LipofectamineTM 2000. The growth of QBC939 cell has been inhibited in some degree compare to the control group form 48h to 72h after transfection. Rb slience caused cell cycle arrest at G0/G1. Apoptotic cell index also increased in some degree. It show certain time dependency in 72h.Rb mRNA and protein under expression significantly compare to the control after transfection.p16 mRNA and protein expression inversed to Rb. E2F1 have identical change tendency with Rb. No animal dead in Acute toxicity test. The most tolerance dosage of TM5-FuNC is 1250mg/kg, which is 5 times dosage of clinical administration. High and middle dosage groups of TM5-FuNC have difference significantly compare to the control group (P <0.05) at 35 after treatment.The high and middle dosage groups of TM5-FuNC have discovered cells apoptosis under electron microscope. The ratio of growth inhibiting of TM5-FuNC and 5-Fu increased significantly after transfection. The tumor volume of TM5-FuNC group and psiRNARb-1+TM5-FuNC group have greatly difference compare with those of the control group and 5-Fu group (P<0.001). The tumor volume of TM5-FuNC group compare to tumor volume of TM5-FuNC group also have some difference (P=0.44).Conclusions:1 We are succeed constructed the eukaryotic expression vector of siRNA Rb which can specifically inhibit Rb expression There are certain inhibited growth effects of QBC939 cell line after transfecting psiRNARb-1 into cell. And Rb slience caused cell cycle arrest at G0/G1. Apoptotic cell index also increased in some degree, which may correlation with the Feedback adjust mechanism of Rb,p16,E2F1,P27kip1 protein . It can be used for later test.2 We get TM5-FuNC the maximum tolerance dosage by acute toxicity experiment which is provided base for medication.3 The TM5-FuNC can inhibit the tumor growth of human cholangiocarcinoma xeno- graft on nude mice. It show that our TM5-FuNC and design ideal of infra-magnetic field targeting get confirmation by test. The Ni-Ti alloy cradle who can be magnetized can high targeting led TM5-FuNC collecting in tumor tissue for treatment effectually and lastly when it keeping bile duct drain smoothly.4 TM5-FuNC + psiRNARb-1 have better inhibit effect for cholangiocarcinoma compare to simplex TM5-FuNC. It would establish a test background for Further inosculatio TM5-FuNC with gene therapy.