| Background: Bile duct carcinoma is a common neoplasm in hepatobiliary system. Recently, the number of patients with bile duct carcinoma has been increasing. The therapeutic efficacy of the cholangiocarcinoma is unsatisfactory,because this tumor shows discontinuous growth and matastasis along the bile ducts, invation in blood vessels, lymphatic vessels, perineural spaces, and loose connective tissue, it results in the poor prognosis of patients with cholangiocarcinoma after complete surgical resection. Additionally there is no distinct advantage for patients receiving radiotherapy, chemotherapy, or liver transplantation.Recently it had been maken obviously progress in the gene therapy of carcinoma. Some laboratory researches of gene therapy have been applied in clinic successfully, however due to lack of an high effective gene of interest, low specificity and efficacy of gene transfer,it is difficult to replace the traditional therapy.Furthermore, carcinogenesis is a multistep process in which a number of genetic alterations occurs, it is difficult to get clinical benefits through identifying one or two genetic alterations.At the same time, reducing the toxicity and side effect is difficult, therefore people start to look for new extracellular substance to conquer the cancer. Recently a novel protein-Apoptin, derived from the chick anemia virus, may provide a mean to overcoming these obstacles. The apoptin can cause depletion of thymocytes and erythroblastoid cells via apoptosis. Interestingly, it can also induce apoptosis in some human malignant/transformed cell lines, and long-term expression of apoptin in normal human cells made it clear that Apoptin has no toxic or transforming activities. Therefore we choose apoptin as a candidate of gene therapy for cholangiocarcinoma.Objective: To find out whether the apoptin is a promising gene for the treatment of cholangiocarcinoma, we constructed a replication deficient recombinant adenovirus vectors containing the apoptin gene and investigated the effects of apoptin on the bile duct carcinoma by transfection in vivo or in vitro.Methods and results:1. Reconstruct the PGEM-T Easy-VP3 containing apoptin gene and Padtrack-CMV-VP3 containing apoptin and green fluorescent protein gene.A poptin gene was amplified by polymerase chain reaction(PCR)and then inserted into PGEM-T Easy and Padtrack-CMV containing green fluorescent protein gene.finally identified the reconstruct plasmids containing the apoptin gene by enzyme cutting and sequences examination.2. Reconstruct a replication deficient recombinant adenoviral vector containing the apoptin geneThe BJ5183 cells, which contains most of the adenoviral genome in supercoiled form, were transformed with Linearize padtrack-cmv-VP3 vector. A recombination of adenoviral vector was taken place in the bacterial cells. By identifying a big fragment which was obtained by Lnearizing Ad-VP3 with Pad, we transferred it into 293 cells, an adenovirus package cell. Base on appearance of green fluorescent protein, the recombinant adenovirus was harvested on the 7th day from 293 cell lines. Finally, the cell suspension was collected to get Ad-VP3 by four rounds of freeze/thaw. Then Ad-VP3 was purified by cesium choride density centrifuge. The titles of Ad-VP3 were 6.56 X 109PFU/ml3. Study on the effect of QBC939, the cell line of bile duct carcinoma, by Ad-VP3 mediated transfection.The most useful MOI (Multiplicity of infection) of QBC939 was determinated by Ad-VP3 mediated transfection, it was 65. Apoptin can induce apoptosis in QBC939 by TUNEL technology, the apoptosis index(AI) on the 7th day, was 61.9% . This is enough to control the growth of bile duct cacinoma cells QBC939.4. Study on Bcl-2 protein expression of bile duct carcinoma by transfection of adenoviral vector containing the apoptin gene.Western Blot was used to deterfiinate the expression of Bcl-2 protein after infection with Ad-VP3 on the 7th day. The results showed that apoptin reduce the expression of Bcl-2 protein obviously. These results suggest that apoptin gene enhance the apoptosis through downregulation of expression of Bcl-2 protein in later phase.5. Study on inhibition effect on cholangiocarcinoma in vivoNude mice were injected subcutaneously with 2X106 QBC939 cells and the tumorswere found after three weeks. Intratumoral injection of Ad-VP3 was conducted only once for the dose of 109PFU. Afterward the tumor size was measured with calipers every three day. Twelve days after treatment, the mice were sacrificed and the tumors were weighted for determinating the ratio of antitumor. The results showed that the volume of QBC939 xenografts in the mice was obviously reduced by apoptin.The ratio of antitumor was significantly higher in Ad-VP3 group than those in control group.6.Study on possible side effects of Ad-VP3During the animal experiment, decreasing in weight gain and weakness were not be observed, this indicates that transfection of apoptin gene is a safe tool for cholangiocarcinoma therapy.Conclusion: Apoptin may induce the apoptosis in bile duct carcinoma cell, and can decrease the expression of Bcl-2 protein in the later phase. A single intratumoral injection of Ad-VP3 virus into a xenogene bile duct carcinoma resulted in significant reduction of tumor growth. No obvious side effect was observed. Taken together we demonstrated that adenovirus vectors expressed the apoptin gene may be a powerful tool for the treatment of cholangiocarcinoma~... |
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