The Preliminary Study Of Paracarcinoma Tissue Biomarkers Using In Diagnosing Prostate Cancer

ObjectiveTo determine the pathological pattern of the prostate tissues which got from radical prostatectomy and cystoprostatectomy.Then isolate and purify the luminal epithelial cells and basal cells of the prostate tissues after primary culture,finally obtain five kinds of cell groups including luminal epithelial cells and basal cells of normal benign prostate tissues,luminal epithelial cells and basal cells of paracarcinoma tissues,luminal epithelial cells of prostate cancer tissues.Comparing the gene expression profile of different cell groups by Transcriptome sequencing to explore the potential diagnostic biomarkers of paracarcinoma tissue.MethodObtain the prostate tissues from radical prostatectomy and cystoprostatectomy.Then isolate the cancer tissues and paracarcinoma tissues by experienced pathologists.The pathological pattern of the prostate tissues were tested by HE staining and immunohistochemistry used two markers AMACR and 34?E12.Wash the prostate tissues and cut them into pieces within 4 hours.Then the tissues were immersed into type I collagenase for 18 hours,afterthat the residues should be treated with trypsin for 15 min.The digested tissues were then filtered and planted into a 75cm2 culture flask filled with DMEMF12 medium containing 10%FBS for primary culture.When the cells were fusion over 90%,isolate them from the flask sidewall and stain them with corresponding antibody.The paracarcinoma tissues cells and normal benign prostate tissues cells were stained by Trop2 and CD49 f,while the cancer tissues cells were stained only by Trop2.In the forward two kinds of tissues,the luminal epithelial cells and basal cells were isolated and purified by FACS(Fluorescence-activated cell sorting).For the cancer tissues cells,we just need to isolated the luminals because there were no basals in the malignant prostate tumours.After the FACS,above-mentioned five kinds of cell groups could be harvested.Then Transcriptome sequencing was performed and the gene expression profiles of the different cell groups were assayed to explore the potential diagnosticmarkers of paracarcinoma tissue which may used in diagnosing prostate cancer.Results(1)Histopathological examination of prostate cancer tissue under the microscope can be seen the structure of prostate acinar disorders,basal layer disappeared,showing a trend of interstitial invasive growth,while the cell atypia significantly.The immunohistochemistry suggested that AMACR +,34?E12-.There was no significant difference between the paracarcinoma tissue and the nomal prostatic tissue in HE staining,and the prostate gland bubble structure was regular and the basal cell layer existed.No abnormal nuclear changes were observed and the immunohistochemistry shown AMACR-,34?E12 +.(2)The fresh prostate tissue has been completely digested by a variety of digestive enzymes.Small pieces of prostate epithelial colony formation can be seen in a part of samples after 72 hours,about 10 days after inoculation the cell fusion up to 90% above in most of the samples.There were no significant differences between the paracarcinoma tissue and the nomal prostatic tissue cells in the flow cytometry after stained by CD49 f and Trop2 antibody togethere.The Trop2 + cells could be divided into two subgroups with Trop2 +CD49fhigh and Trop2 + CD49 flow.After stained by Trop2 antibody,the cancer cells can be divided into two subgroups with Trop2+ and Trop2-.Flow cytometry was used to determine the percentage of target cells to make sure the percentage was above85% in all the samples used for Transcriptome sequencing.(3)Paracarcinoma tissues luminal epithelial cells were similar to cancer tissues but completely different from nomal prostate tissues on the expression level of some genes.The basal epithelial cells of paracancerous tissue also have different expression profiles for some genes with normal prostate basal cells.ConclusionThe prostate cancer tissues luminal epithelial cells are similar to cancer tissues luminal epithelial cells and are different from normal prostate luminal epithelial cells at the expression level of some genes.The prostate cancer tissue basal cells and normal prostate tissue basal cells also have completely different gene expression profiles.There may exist potential specific biomarkers which could distinguish paracarcinoma tissues from nomal prostate tissues.And the high degree ofenrichment of cancer-related signaling pathways are also likely to play an important role in the early stages of cancer.