| Hepatocellular carcinoma is among most common malignant tumor internationally. According to the data provided by world health organization, it has new cases of 260,000 annually. It is ranked as the third place among the malignant tumors with high mortality in China. The development of liver cancer is very complicated and not clear yet. It is involved in many steps and triggered by many factors, such as initiation, promotion, and development. This process engrosses numerous gene participation and mutation, and finally leads to formation of liver cancer. To date, it is common accepted that the development of hepatocellular carcinoma is associated with liver cirrhosis, virus hepatitis, aflatoxin, gene mutation and certain geographic locations. Unfortunately, most cases of hepatocellular carcinoma are already in advanced stage when patients get confirmation of their clinical diagnosis.Even though a lot of progress on the treatment methods was been made, such as surgery techniques, radiation therapy, chemotherapy and combination of them, they did not have a profound improved effect on the prognosis of liver cancer. The mortality of liver cancer is still close to the it's incidence. New method to treat this disease still needs to be developed.Currently, new methods named as gene target therapy for hepatocellular carcinoma are gradually applied clinically, which include suicide gene therapy, immune gene therapy, induction gene therapy, multiple kinas therapy, anti-angiogenesis therapy and virus gene therapy. Among them, the virus mediated gene therapy is becoming the research focus.In the early 1970s, Folkman firstly hypothesized that growth and nourishment of malignant tumor based on the neo-angiogenesis. After that, numerous studies proved his assumption. When its diameter reaches 1mm tumor it will go into the dormant stage or aggression if there is no neo-angiogensis to support its growth. However, if there is neo-angiogenesis it will grow out of control. The growth of new blood vessel into the tumor can provide all kinds of nutrition and oxygen, and take all the metabolic products. Therefore, anti-angiogenesis treatment is a reasonable method to control the growth of tumor and cut off the nutrition supply, which is being the spotlight for hepatocellular carcinoma therapy.Recently studies have proved that process of neo-angiogenesis is the formation of new capillary vaginated from the existed blood vessel. In many tissues, most vascular system is relatively static, which means the formation of rare blood vessels, except for the endometrial, placenta and repairing tissue. The local balance of "pro" and "con" factors for blood vessel formation determines the initiation of neo-angiogenesis. The balance plays a very important role in regulating the formation of new blood vessels.Studies demonstrated that all endogenous inhibiting factors for angiogenesis could decrease tumor growth in animal models, which further proved the growth of tumor is based on the neo-angiogenesis. However, there are also concerns about using the inhibiting factors for clinical treatment of malignant tumor, such as 1) Repetitive and long time administration of inhibiting factor is necessary before remission of tumor under the effect of radiation, chemotherapy and surgery.2) Application of high dose of exogenous recombinant protein also carries them with some potential toxic and contagious factors, which could yield harmful immune reactions to body. 3) Cost of the recombinant protein is very high and makes a big economic burden on the patients.Gene therapy is to transfer the certain DNA or RNA into the cell and make the cell express certain protein, which encoded by the inserted DNA or RNA. Application of gene therapy for anti-angiogenesis in the treatment of tumor will overcome the shortcomings of protein therapy mentioned in the previous paragraph. The advantage of gene therapy is 1) it can save a lot of cost by transuding cell with targeted gene and let the cells make the recombinant protein by themselves.2) it will mainly focus on the treatment of local tumor by transuding the tumor cells with the targeted gene. This method can make a local high concentration of therapeutic protein, which could improve the effect of anti-angiogenesis significantly.Nowadays, more than 200 clinical gene therapy trails carried on world widely. About half of them are targeting the treatment of malignant tumor, which is performed by tranducing the tumor cells with targeted gene. Currently, gene therapy by using inhibiting factors of anti-angiogenesis is targeting on the endothelial cells, which is genetic stable and does not have the character of heterogeneity like tumor cells do. Gene therapy also can make the targeted gene express for a while in vivo and save a lot expense when compared to the protein therapy.The most used vector for gene therapy is adenovirus. This vector has high efficiency and expression. It can also induce the immune reaction against the tumor cells by transducing them.Canstatin is newly discovered endogenous inhibiting factor for anti-angiogenesis. It belongs to the collagen type IV and has an a2 chain in its C terminal non-collagen NCI domain. It has been demonstrated to have the inhibiting effect on the immigration of endothelial cell and prevent the tube formation in vitro, as well as inducing apoptosis of endothelial cells. In vivo some studies also showed that canstatin protein could effectively inhibit the growth of human prostrate cancer and renal cancer cell in the SCID model. Its anti-angiogenesis effect is more potent than endostatin. However, to date, no report has been seen about the treatment of human hepatocellular carcinoma by using adenovirus mediated canstatin gene therapy.In the present study, a human canstatin gene was cloned and the adenovirus vector, which carries canstatin, was constructed. The adenovirus was been injected into the marine hepatocellular carcinoma model. The effects of treatment of tumor were been observed by the investigation of the change of volume of tumor, vessel density and expression of caspase-3 and flk-1. The mechanism was also been discussed in the present study.Objective:clone and sequence analysis of human canstatin geneMethods:mRNA was extracted from human placenta tissue. Canstatin gene was amplified by RT-PCR, and then inserted into the plasmid pGEM-T. The plasmid containing canstatin was transferred into E.coli. The positive clone which contains the recombinant plasmid pGEM-T/canstatin was picked out and its'sequence was analyzed.Results:total mRNA was extracted from human placenta tissue. Results of total mRNA gel electrophoresis showed three bands, which is 28s,18s and 5s respectively. The concentration of total mRNA is measured by spectrophotometer, which showed A26o=0.878, A280=0.411 and A260/A280 =2.122. The final concentration of total mRNA is 1.76g/L. The product of RT-PCR showed same length to the target gene canstatin. The product is ligated with plasmid PGEM-T overnight, and then was transfected into E coli DH5α. The transfected DH5a was then transferred onto the LB plate with ampicillin.5 white clones were picked up and digested by Bam HI and HINDⅢ.1 of 5 clones was confirmed to be a positive clone which contains the canstatin gene. The positive clone was sent to the company to get the sequence analysis. The result is 100% identical to the canstatin sequence of gene bank.Conclusion:Canstatin gene successfully cloned from human placenta tissue, which made a good step for the further research. Objective:To construct canstatin prokaryotic expression vector, expressed and purified recombinant human Canstatin protein.Methods:Bam HI and Hind III from the recombinant plasmid pGEM-T/ canstatin on the cut canstatin gene, inserted into expression plasmid pET-22b (+) the corresponding sites and transformed E.coli BL21 after IPTG-induced recombinant protein expression, SDS-PAGE electrophoresis analysis of protein expression. Ultrasonication biomass, Ni-NTA affinity chromatography purification of recombinant protein, PBS for its dialysis repatriation.Results:The recombinant plasmid PGEM-T/canstatin in the Bam HI and HindⅢdouble digestion, the electrophoresis confirmed the cut canstatin cDNA was recovered in the target gene inserted into plasmid pET-22b (+) the corresponding sites, transforming E. coliBL21, pick 5 Bam HI and the colonies to do HindⅢrestriction enzyme digestion, all the colonies were cut out corresponding to the original plasmid with the target gene two specific bands, were confirmed positive clones. After IPTG induction, SDS-PAGE electrophoresis analysis showed that the 24KD protein expression about the emergence of new bands after induction 1h,2h,3h,4h expressed protein accounted for a percentage of the total bacterial proteins were 17.2%, stroke 17.8%,21.0% and 22.4%. Ni-NTA affinity chromatography after 125Mm, 250mM elution purification of the target protein.Conclusion:The successful construction of the canstatin prokaryotic expression vector, expressed and purified recombinant human canstatin protein for further study laid the foundation for its anti-tumor effect. Objective:To verify that recombinant human protein in vivo Canstatin the therapeutic effect of liver cancer.Methods:The SMMC-7721 liver cancer cell line established 30 subcutaneous tumor model of hepatocellular carcinoma in nude mice, when the tumor grew to 2-3mm, the randomly divided into 3 groups, namely PBS control group,5-Fu treatment group, Canstatin treatment group route of administration for the tumor injection. Treatment for 3 weeks with the compass and the cursor on a regular basis the amount of cards ulnar subcutaneous tumor size, the end of treatment, remove the tumor, conventional biopsy, detection of tumor angiogenesis.Results:The treatment of subcutaneous tumor in nude mice results show that the first 3 days after treatment began, canstatin treatment group tumor volume was significantly smaller than the control group, to the first six days, there are very significant difference P<0.01. The end of treatment, although 5-Fu the most significant effect of treatment group, but the drug toxicity evident in mice treated ataxia obvious pathology confirmed severe liver damage.Conclusion:Recombinant human canstatin protein can effectively inhibit the growth of human hepatocellular carcinoma, anti-tumor effect was positively correlated with the dosage, and mechanism of action is to inhibit tumor formation of new blood vessels. Objective:To combine the anti-angiogenesis and gene therapy, and construct the adenovirus containing canstatin gene.Methods:The targeted canstatin gene was expanded by PCR on the plasmid of PGEM-T/canstatin, and then digested by EcoRl and BamHI. The product was inserted the correspondence site of plasmid PCA13; Two plasmids of PCA13-cans and PBGHE3 were co-transfected into 293 package cells. The yielded adenoviruses were confirmed by PCR. Finally, viruses were further purified by the Cscl density gradient centrifuge. The final concentration of virus was tested by TCH150.Results:709bp DNA which contains castatin gene was amplified from the plasmid of PET/canstatin by PCR. It was then inserted into plasmid of PCA13 by the digestion of EcoRl and Bam HI. The constructed plasmid of PCA13-cans was analyzed to be correct by PCR and enzyme digestion. Adenoviruses were yielded by co-transfection of 293 cells with two plasmids. The final concentration of Ad-canstatin is 3.1×109 pfu/ml.Conclusion:Ad-canstatin can be yielded by the co-transfection of 293 cell with plasmids including PCA13-cans and PBGHE3.Objective:Investigation of inhibiting effect of ad-canstatin gene therapy on the hepatocellular carcinoma in mice modelMethods:The human hepatocellular carcinoma model was established by using human liver cancer cell line SMMC-7721 implanted into the SCID mice. Firstly, human liver cancer cell line SMMC-7721 was cultured in vitro. Cultured medium was changed every 3 days.30 SCID mice were divided into three groups, which are ad-canstatin group, GFP group and PBS control group respectively. Each mouse of three groups was received 0.1 ml injection of cancer cell suspension subcutaneously in the axillae. When the tumor volume is about 50mm3, each mouse of ad-canstatin group was received a injection into the tumor with a virus dose of 8×109 pfu, GFP group with a virus dose of 8×109 pfu and PBS 0.1 ml for PBS control group, respectively. All tumors get only two injections and interval between two injections is 72 hours.1. The tumor growth curve is drawn by measure the volume of tumor using function V=π/6×(length xwidth2).2. The inhibition rate of tumor growth is measured by the function R=(Vcontrol-Vad-canstatin)/Vcontrol×100%.3. Histological and immunochemical analysis of tissues:all animals were sacrificed at day 30 after injection. The tissue samples were harvested and fixed by 10% formalin, and then paraffin embedded.5μm sections was cut and stained with H.E. Some sections were also immunostained with Caspase-3 and Flk-1.Results:1. The volume difference of tumor was shown at day 3 after injection between ad-canstatin group and other two groups. Ad-canstatin group displayed significant small tumor size when compared to other two groups (p<0.05). With the time develops, tumor size of three groups are growing. However, tumors of ad-canstatin group grow much slowly in size than the other two groups. The volume of tumor in ad-canstatin group still is significantly small than the other two groups (p<0.05).2. The necrotizing tissue in ad-canstatin group is significantly large than that of other two groups. 3. The expression of Flk-1 in ad-canstatin group is significant low than that of PBS group (χ2=7.778, p<0.01); The expression of ad-canstatin group is significant high than that of GFP groups and PBS groups (χ2=6.563 andχ2=10.50, P<0.01 respectively).Conclusion:Ad-canstatin gene therapy can effectively inhibit the growth of hepatocellular carcinoma. It has a lot of advantage such as easy operation, convinent and less side effects. Conclusions of the project1. Successfully cloned of human canstatin gene.2. Successfully established canstatin prokaryotic expression vector and recombinant protein canstatin.3. The use of recombinant canstatin proteins to inhibit liver cancer in nude mice experiments.4. Successful construction of vector of ad-canstatin.5. Ad-canstatin gene therapy can effectively inhibit the growth of hepatocellular carcinoma.In summary, Ad-canstatin is very promising to become a new therapeutic drug for hepatocellular carcinoma due to its anti-angiogenesis effect, the mechanism includes:1)the expressed canstatin protein can inhibit the growth of tumor blood vessel.2) It can induce the tumor cells to express the apoptosis factor caspase-3 which increase the apoptosis and decrease flk-1 of liver cancer cells. When used Ad-canstatin in gene therapy, it can be applied as a new method to treat the liver cancer and will have a bright future.
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