| Prostate cancer(PC)is one of the most common carcinomas in males. In recent years, prostate cancer has continuously increased in our country. Despite the initial effectiveness of hormone therapy, many patients with metastatic disease eventually progress to an androgen-resistant state. Various new treatment modalities are currently being developed, but none has yet shown a survival benefit in patients with hormone-refractory prostate cancer. Thus, in a long future, the invasion and metastasis of tumor cells will be one of the momentous tasks for preclinical and clinical researches.As we all known, the metastasis of tumor cell is a very complex and multistep process, but chick embryo model systems offer a number of unique advantages to study it. Since the chick embryo serves as a naturally immunodeficiency host capable of sustaining grafted tissues and cells without species-specific restrictions. Different chick embryo model systems allow for comprehensive analysis of specific stages and aspects of cancer cell dissemination. Chorioallantoic membrane (CAM) provides a uniquely supportive environment for primary tumor formation and a source of angiogenesis. In addition, the CAM blood vessel network provides conduits for tumor cell intravasation, dissemination, and vascular arrest and finally, a repository where arrested cells extravasate to form micro metastatic foci.In early study, we have applied phage random 7 peptides library to perform differentiated selection, and obtained PC high metastasis associated short peptides specifically combining human prostate cell line PC3M, and consequently, systematically identified the influences on PC3M biological behaviors. We found that some of the peptides inhibited PC3M cell spread, proliferation, and had inhibitory influence on migration and invasion of PC3M cell. In order to study the role of these peptides in the metastasis of prostatic cancer cells, we made the tumor spontaneous metastasis model in chick embryo chorioallantoic membrane system that could quantitatively detect the metastasis of human prostatic carcinoma cells, i.e. opened a little window in the center of long circumference, and the tumor cells were inoculated on the dropped CAM, after a period of time, the lower CAM were harvested for assay. As for human-specific Alu sequences widespread in human genome, its quantity can represent the number of human prostate cancer cells PC3M, so we have extracted genomic DNA to estimated numbers of human prostatic cancer cells within each lower CAM followed by sensitive real-time quantification PCR amplifying human specific Alu sequence.The results are as following:1. Establishment of human prostate cancer metastasis CAM modelUsing the side window method, exposured CAM, after inoculation of human prostate cancer cell PC3M isolated chick lower CAM, By immunohistochemical staining CD44 molecules in tumor cells, metastasis of tumor cells are usually found in the chorioallantoic membrane of the ectoderm, and formatted metastases foci here clearly. There are a number of tumor cells in the interstitial mesoderm formatting metastasis foci, and closed to the vessel of mesoderm and beneath the ectoderm.The results confirmed that prostate cancer cells PC3M in human prostate cancer metastasis CAM model transferred to the lower CAM successfully.2. Quantitative detection of the metastasis of human prostate cancer cells in the chicken chorioallantoic membrane model(1) The sensitive, accurate and repeatable standard curve of Alu sequence-specific real-time fluorescence quantitative PCR were established: Y=-3.463Log10X+28.760, amplification efficiency E = 94.536%, the best linear interval are 102 ~ 105, the lowest detectible magnitude is 100.(2) Quantitative analysis of metastasis of tumor cells in the chick embryo chorioallantoic membrane model: 1×106 PC3M cells were inoculated on the CAM at the age of 10d , 48 hours after inoculation, extracted genomic DNA of lower CAM. human-specific Alu sequences were amplified by real time quantitative PCR , by standard curve we obtained the number of PC3M cells in the lower CAM :1754.60±182.504. 220bp specific band were obtained by gel electrophoresis analysis of PCR products.3. Inhibition of human prostate cancer cell binding peptides in metastasis of prostate cancer cell PC3M1) The spontaneous metastasis of PC3M characterized by time-dependence and concentration-dependence in this model : It was found in prostate cancer metastasis model quantity of migrated tumor cells are increased with time and vaccination levels increasing. Inoculation of 5×105/25μl tumor cells, after 24 hours got the quantity of tumor cells by real-time PCR: 166.35±11.363, which closed to the minimum detectable amount of the transfer level of 100 cells. Peptide inhibition experiments carried out at this time will be more sensitive results.(2) The inhibition effect of peptides in prostate cancer metastasis and modes of action: The research of peptide inhibition were carried through different delivery methods, including peptides binding to PC3M before the cells were incubation , peptides and PC3M were mixed when inoculation, were dropped onto CAM in 12 hours after inoculation. We found that it is that peptides were inoculated with PC3M cells prior grafting onto CAM could insure the inhibitory action, and the inhibition rate is 23.41%. In statistical student t analysis, P<0.05. Result indicated that the inhibit of prostate cancer cells metastasis by prostate cancer metastasis associated peptides with the way of combination.In sum, we have made a tumor spontaneous metastasis model with chick chorioallantoic membrane that could quantitatively detected the metastatic ability of human prostate cancer cells, and on this basis, we detected the inhibitory effect of short peptides concerning the metastasis of human prostatic cancer in tumor metastasis. This research provides the clues to study the mechanism of invasion and metastasis of prostate cancer, and the experimental basis for targeted therapy of prostate carcinoma, which supplies hope for finding a new clinical treatment way.
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