| Preface It is the most important reason making hormone therapy be palliative but not curable for prostate cancer that most malignant cells growth will eventually become hormonal independent. Some studies have discovered that there are two kinds of malignant cells coexisting in prostate cancer; one expresses the AR, while another not. The former makes the activity of AR elevated by sensitization and grow hormonal-independently. The latter is the most important cause that makes the prognosis worse. A treatment aiming at the two kinds of cells is our purpose. Nowadays, there are many gene therapy strategies for prostate cancer. However, because of lacking specificity to prostate cancer cells, most of them are limited for clinical application. Now two prostatic specific promoters, PSA and rPB, which have both two AREs and an ARR, were studied more and more. But the two promoters have activity only in the prostate cancer cells that express AR. On the contrary, they have no activity in those do not express AR. At the same time, some study displayed that all prostate cancer cells had retinoic acid receptor. This experiment was based on these rationales/observations. In the first part of this study, the AREs in the rPB were replaced by RARE. With different construction program, four kinds of modified promoters, rPB-DRs, were constructed successfully. And the rPB-DRs were conjugated with the report genetic carrier. In the second part, the modified promoters, rPB-DRs'activity to priming the report genetic expression in various kinds cells under the regulation of RA was determined. Then the modified promoters'activity to aim at hormonal independent prostate cancer was confirmed. PART ONE:Construction of rPB-DRs and Conjugation with Report Genetic Carrier Objective: To construct the modified promoters, rPB-DRs, which may be capable of aiming at hormonal independent prostate cancer. Materials and Methods: Prostatic specific promoter, rPB was chosen to be reformed. The AREs in the rPB were replaced by RARE. With different construction programs, four kinds of modified promoters, rPB-DRs, were constructed. And the rPB-DRs were conjugated with the report genetic carrier. Results and Discussion: Constructions of recons, the four kinds of rPB-DRs, rPB-luci and the four kinds of rPB-DR-lucis, were confirmed to be successful by electrophoresis and gene sequencing. Four kinds of rPB-DRs were constructed to find the promoter aiming at hormonal independent prostate cancer and having highest degree of specificity. And rPB-DRs were conjugated with the report genetic carrier for the activity determination of the modified promoters. The methods of construction of modified promoters were reported in our study. Some study revealed the sequence representing RARE was "AGGTCA", which was called "half site". Two copies of half site add a basic radical, C or G, could be called DR+1,which had the best activity. DR(3), three direct repeat of DR+1 could respond RA. Therefore, DR(3) was used to replace the ARE, representing RARE. Four different copy conditions were used in the construction of rPB-DRs, including two kinds of replacements in single place, a replacement in two places and a inserting direct. So it is possible to compare the activities between rPB and rPB-DRs, or those between the four kinds of rPB-DRs with different copy conditions. Collating the basic program of gene mutation, we contrived the construction method making use of two steps of PCRs. This method is easy to do and stabilized. It is especially fit for the condition that the mutation bp is much more and the mutation place is situated at the centre of template. Next we will transfect multiple carcinoma cell lines with rPB-luci and rPB-DR-lucis constructed. And the activity of those controlled by RA will be compared.PART TWO:Determination of rPB-DRs'Activity Objectives: To transfect three cancer cell lines with the report genetic carriers conjugated with rPB-DRs and determine the activities of luciferase and the modified promoters indirectly. Materials and Methods: Transfect PC-3 cell lines, LNCaP cell lines and T24 cell lines respectively with rPB-luci and four kinds of rPB-DR-lucis, and then, after adding RA, determine and analyze the activity of luciferase at the best phase point. Results and Discussion: It is necessary to determine the best phase points of luciferase in three cell lines because of the apparent alteration of express activity under stimulus of RA. Through initial experiment, the best phase points of luciferase in three cell lines were determined. Our study showed that, after transfect of PC-3 cell line, the rPB-DRs' activities were much higher than rPB and rPB-DR(6) was the highest. It is confirmed that the modified promoters constructed have activity aiming at hormonal independent prostate cancer not expressing AR. Furthermore, the prostatic specificity of the modified promoters was confirmed by the results of determination after transfect of LNCaP and T24 cell lines. ENDNOTES To sum up, the results of our experiments include: First of all, four different modified promoters, rPB-DR(3A), rPB-DR(3B), rPB-DR(3C) and rPB-DR(6), were constructed successfully. Secondly, the rPB-DRs were conjugated with the report genetic carrier. Through determination the activity of luciferase, the activities of rPB-DRs in three cell lines were determined indirectly. Lastly, by analyzing the results above, we could make the conclusion as followings: The modified promoters, rPB-DRs, can enable the gene therapy aiming at hormonal independent prostate cancer not expressing AR and rPB-DR(6) has the highest activity in the four promoters. At the same time, the modified promoters kept the prostatic specificity. Next, rPB-DR(6) should be chosen to confirm with the suicide gene and construct adv-rPB-DR-tk and experimentalize in vivo or vitro. |
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