癌旁组织标志物用于早期诊断前列腺癌的初步研究

发布时间：2018-03-04 06:05

本文选题：前列腺癌　切入点：癌旁组织　出处：《天津医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:获取前列腺癌组织、癌旁组织及正常前列腺组织标本并验证其病理学类型。分离纯化经过原代培养后的各细胞株分别获得前列腺癌管腔上皮细胞、癌旁组织管腔上皮细胞、癌旁组织基底细胞、良性前列腺组织管腔上皮细胞及良性前列腺基底上皮细胞等5种细胞亚群。对比分析癌旁组织、良性前列腺组织及前列腺癌组织管腔上皮细胞的基因表达谱,同时对比分析癌旁组织和良性前列腺组织基底上皮细胞的基因表达谱,初步探索在癌旁组织中是否可能存在能够应用于前列腺癌临床诊断的特异性标记物,为临床早期诊断前列腺癌及制定后续诊疗计划提供一个新的思路。方法:直接从前列腺癌根治术及膀胱前列腺切除术获取相应的前列腺组织标本。对来源于前列腺癌根治术的组织标本由经验丰富的病理科医师分离其中的恶性癌组织及良性癌旁组织。对最终得到的前列腺癌组织、癌旁组织及来源于膀胱前列腺切除术的正常前列腺组织分别进行HE染色及AMACR和34βE12抗体的免疫组化染色以确定所获得的组织标本的可靠性。对获取的新鲜前列腺组织在4h以内进行洗涤,剪碎,之后加入一型胶原酶混合消化液消化处理18h并对未完全消化的残渣使用胰蛋白酶处理约15min;将消化后得到的细胞接种到75cm2细胞培养瓶中并使用含10%FBS的DMEM/F12培养基进行原代前列腺细胞培养,细胞增殖到一定程度后对癌旁组织细胞和良性前列腺组织细胞均同时染色Trop2和CD49f抗体,通过流式细胞分选分别获得此2种细胞株的高纯度基底细(Trop2+CD49fhigh)和管腔上皮细胞(Trop2+CD49flow)亚群。由于前列腺癌组织缺乏基底细胞,对前列腺癌组织细胞染色Trop2抗体,通过流式细胞分选获得其管腔上皮细胞亚群(Trop2+)。对以上五种细胞亚群进行二代转录组测序,分析癌旁组织、正常前列腺组织及前列腺癌组织管腔上皮细胞的基因表达谱,同时分析癌旁组织和正常前列腺组织基底上皮细胞的基因表达谱。结果:(1)所获取的前列腺癌组织HE染色切片在显微镜下可见前列腺腺泡结构紊乱,基底层消失,呈现出间质侵袭性生长的趋势,同时细胞异型性明显,免疫组化提示AMACR+,34βE12-;所获取的前列腺癌旁组织及正常前列腺组织HE染色切片在光镜下无明显区别,腺泡结构规整,基底细胞层存在,未见细胞核异常改变,免疫组化均为AMACR-,34βE12+。(2)由手术室直接获取的新鲜前列腺组织(包括癌、癌旁及正常前列腺组织)先后经过多种消化酶联合作用后基本消化完全,接种72h后可见小片前列腺上皮集落形成,接种后10天左右大多数样品细胞细胞融合达90%以上。使用Trop2和CD49f抗体双染的癌旁和正常前列腺组织细胞在流式细胞分析中的细胞分群情况相似,且Trop2+的细胞群中根据CD49f的表达情况大致可以分为Trop2+CD49fhigh和Trop2+CD49flow两个亚群。经过原代培养后的癌组织细胞进行TROP2抗体的染色后,流式细胞分析可见细胞主要可分为TROP2+和TROP2-两个细胞亚群。流式分选获得的用于转录组测序的各细胞样本通过流式细胞分析测得其目标细胞比例均在85%以上。(3)通过转录组测序及对不同细胞群基因表达谱的分析,我们发现癌旁组织管腔上皮细胞在部分基因的表达水平上与癌组织相似,而且不同于正常前列腺管腔上皮;癌旁组织的基底上皮细胞在部分基因的表达上与正常前列腺基底细胞也存在不同的基因表达谱。结论:前列腺癌旁组织管腔上皮细胞在部分基因的表达水平上与前列腺癌相似而且不同于正常前列腺管腔上皮细胞,前列腺癌旁组织基底细胞和正常前列腺组织基底细胞也存在完全不同的基因表达谱,其中的差异表达基因如PSG5、RNA5-8S5等基因都可能成为潜在的能够将癌旁组织与正常前列腺组织区分开来的特异性标志物,而差异表达基因中富集程度较高的与癌症相关的信号通路也很有可能在癌症发病早期即发挥着重要的作用。
[Abstract]:Objective: to get prostate cancer, paracancerous tissue and normal prostate tissue samples and verify its pathological types. After separation and purification of the cells were cultured respectively after prostate cancer luminal epithelial cells, tissue adjacent to cancer luminal epithelial cells, basal cell carcinoma, 5 benign prostate epithelial cells and the lumen benign prostate epithelial cells and basal cell subsets. Comparative analysis of expression profiles of tumor adjacent tissues and benign prostate tissue and prostate cancer luminal epithelial cell genes, compared with basal epithelial cell carcinoma and benign prostate tissue gene expression profiling, explore specific markers can be used in clinical diagnosis of prostate cancer whether there may exist in noncancerous tissue, for the early clinical diagnosis of prostate cancer and provide a new idea to develop follow-up plan. Methods: from Resection of prostate tissue specimens were obtained and radical prostatectomy for prostate cancer. Bladder cancer originates in malignant prostate cancer tissue samples by experienced pathologist and the separation of the benign tissue adjacent to carcinoma of prostate cancer tissue. The normal prostate tissue adjacent tissues and from the bladder and prostate excision were stained by HE and AMACR and 34 beta E12 antibody immunohistochemical staining to determine the reliability of the obtained samples. The fresh prostate tissue obtained by washing, within 4H and cut into pieces, then add a mixture of collagenase digestion and trypsin digestion of 18h treatment on the undigested the residue of about 15min; after digestion of the cultured cells were inoculated into 75cm2 cells and the bottle containing 10%FBS DMEM/F12 medium primary prostate cell culture A, cell proliferation to a certain degree of carcinoma cells and benign prostate tissue cells were also stained Trop2 and CD49f antibody by flow cytometry respectively the 2 cell lines of high purity fine substrate (Trop2+CD49fhigh) and luminal epithelial cells (Trop2+ CD49flow). A subgroup of prostate cancer due to lack of basal cell the staining of Trop2 antibody, prostate cancer cells by flow cytometry to obtain the luminal epithelial cell subsets (Trop2+). The two generation transcriptome sequencing for these five cell subsets analysis, paracancerous tissue, expression of normal prostate tissue and prostate cancer in luminal epithelial cells of basal epithelial gene, and analysis cell carcinoma and normal prostate tissue gene expression. Results: (1) the prostate cancer tissue HE staining in the microscope prostate gland structure disorder, The basal layer disappeared, showing interstitial invasive growth trend, at the same time, cell atypia, immunohistochemical staining showed that AMACR+ 34 beta E12-; the prostate cancer adjacent tissues and normal prostate tissue HE staining had no significant difference in the light microscope, acinar regular structure and basal cell layer exists, abnormal change neclei, immunohistochemistry for AMACR- 34 beta, E12+. (2) fresh prostate tissue obtained directly by the operation room (including cancer, prostate cancer and normal tissues) after combined effects of a variety of digestive enzymes after complete digestion, colony formation showed the small prostate epithelial in 72h after inoculation, about 10 days after inoculation of most samples cell fusion was more than 90%. Double staining using Trop2 and CD49f antibodies in cancer and adjacent normal prostate tissue were similar in flow cytometric analysis of cell segmentation, cell group and Trop2+ according to CD49f The expression of Trop2+CD49fhigh and Trop2+CD49flow can be roughly divided into two subsets. After staining after primary culture cancer cells with TROP2 antibody, flow cytometry analysis showed that cells can be divided into TROP2+ and TROP2- two cells. Flow cytometry was used for each cell sample by transcriptome sequencing flow cytometry analysis was used to test the target cell ratio was above 85%. (3) by transcriptome sequencing of different cell populations and gene expression analysis, we found that the adjacent tissues in luminal epithelial cells and carcinoma tissue in the expression level of some genes are similar, but different from normal prostate luminal; carcinoma the basal epithelial cells of basal and expression of genes in normal prostate cells have different gene expression profiles of prostate cancer tissues. Conclusion: luminal epithelial cells in gene The expression level of prostate cancer and similar and different from normal prostate luminal epithelial cells, prostate carcinoma basal cells and normal prostate tissue basal cell also exist different gene expression profiles, the differentially expressed genes such as PSG5, RNA5-8S5 genes may be potential to specific markers to distinguish cancerous tissue with normal prostate tissue, and cancer related signaling pathways differentially expressed genes in high degree of enrichment is also very likely that play an important role in the pathogenesis of early cancer.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

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