| Backgroud and AimsProstate cancer(PC) is one of common male urological malignant tumors, which is the second leading cause of male cancerous death in USA. In recent years, the incidence of PC has been rising dramatically in China. The androgen deprivation therapy(ADT) is a important clinical treatment on PC and its metastatic carcinoma at present, but almost all of the patients accepting ADT lead to castration-resistant prostate cancer(CRPC), which is insensitive to ADT and has a bad prognosis.Prostate apoptosis response-4gene(par-4) is a kind of promotive apoptosis genes found by Sells. The unique core domain of par-4sufficient for selective apoptosis induction in cancer cells(SAC) can induce apoptosis of both CRPC and ADPC cells, but harmless to normal cells. The adeno-associated virus(AAV) has received attention in research of vectors in gene therapy because of its low immunogenicity, high security, stable expression of object gene, etc. Chick embryo chorioallantoic membrane(CAM) is formed between days4and5by partial fusion of chorion and allantois. Between days6and7of incubation,the CAM and its blood vessels cover the entire surface of yolk sac, and capillary become extremely rich along with the embryo age increase. The CAM is a kind of natural immunocompromised host and can tolerate certain temperature changes which is appropriate for the growth of xenograft tumor and detection of biology behaviour. So the CAM is usually used as a xenograft tumor model in biological experiments.We have successfully constructed the rAAV-NT4-SAC-HA2-TAT which carried the neurotrophin-4(NT4) and transactivating protein(TAT) based on the apoptosis inducing mechanisms of SAC and application of adeno-associated viral vector. In this research we aim to construct the CAM xenograft tumor model of PC and observe the inhibition of expression product SAC to xenograft tumor after rAAV transfected.Materials and methods:1. Recover and cultivate the human prostate cancer cell line PC-3, collect cells in logarithmic phage and centrifuge, then adjust the cell concentration to5～8×106/20u L with PBS. In the clean bench, open a window at the air chamber of the9th day’s chick embryo after the egg shell disinfected with alcohol, rive the air chamber membrane and expose the CAM. Then PC-3cells were inoculated on it. Closed the window with transparent adhesive tape and continue to incubate. Then we observe the growth, angiogenesis and morphology of the implanted tumors.2.3days later, we choose the embryos emerging implanted tumors (dia.≥3mm)and divided into3groups randomly,8eggs in each group. Different treatments were given as below. PBS group:PBS,100μL, AAV group:AAV,1×109cfu/100μL, rAAV group:rAAV-NT4-SAC-HA2-TAT,1×109cfu/100μL. The growth of xenograft tumors were observed all the time and the size were measured4days after treatment. Then the tumors were detached completely, fixed with formalin, embeded by paraffin and cut into slices. The expression of object gene was detected with immunofluorescence test. The effects of SAC on implanted tumors was confirmed by comparing tumor volumes and hematoxylin-eosin staining of rAAV group to those of another groups.Results:1. The cancer cells distributed scatteredly after inoculation. The CAM of inoculation area became thicken and rough with a clear border24hours later. We could see clear white tumor group2days later, but the capillary gathering was not apparent.3days later the tumor continued to increase and capillary gathering began to be observed. 2. Tumors of PBS group and AAV group continued to increase after treatment. We could observe the rich capillaries distributing radially around tumor. Volumes of tumor in rAAV group were smaller than other two groups generally. The statistic results showed that there were statistical differences between rAAV group and PBS group or AAV group(P<0.01). The expression of SAC was confirmed by immunofluorescence test. Microscopically, the growth of tumors was similar in PBS group and AAV group. The infiltrating tumor cells extends irregularly through the CAM as cords with new vascular formation. The invasiveness of cancer cells in rAAV group was inhibited so that the tumor cells groups were limited under the microscope.Conclusions:1. The xenograft tumor model of human PCa on CAM was constructed successfully.2. The fusion peptide SAC expressed by rAAV which was transfected into xenograft tumor induced apoptosis of PC-3cells and growth of the tumors was inhibited, which laid a foundation for the application of SAC to the gene therapy to PCa, especially CRPC. |
PDF Full Text Request