本文選題：前列腺癌　切入點：癌旁組織　出處：《天津醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:獲取前列腺癌組織、癌旁組織及正常前列腺組織標本并驗證其病理學(xué)類型。分離純化經(jīng)過原代培養(yǎng)后的各細胞株分別獲得前列腺癌管腔上皮細胞、癌旁組織管腔上皮細胞、癌旁組織基底細胞、良性前列腺組織管腔上皮細胞及良性前列腺基底上皮細胞等5種細胞亞群。對比分析癌旁組織、良性前列腺組織及前列腺癌組織管腔上皮細胞的基因表達譜,同時對比分析癌旁組織和良性前列腺組織基底上皮細胞的基因表達譜,初步探索在癌旁組織中是否可能存在能夠應(yīng)用于前列腺癌臨床診斷的特異性標記物,為臨床早期診斷前列腺癌及制定后續(xù)診療計劃提供一個新的思路。方法:直接從前列腺癌根治術(shù)及膀胱前列腺切除術(shù)獲取相應(yīng)的前列腺組織標本。對來源于前列腺癌根治術(shù)的組織標本由經(jīng)驗豐富的病理科醫(yī)師分離其中的惡性癌組織及良性癌旁組織。對最終得到的前列腺癌組織、癌旁組織及來源于膀胱前列腺切除術(shù)的正常前列腺組織分別進行HE染色及AMACR和34βE12抗體的免疫組化染色以確定所獲得的組織標本的可靠性。對獲取的新鮮前列腺組織在4h以內(nèi)進行洗滌,剪碎,之后加入一型膠原酶混合消化液消化處理18h并對未完全消化的殘渣使用胰蛋白酶處理約15min;將消化后得到的細胞接種到75cm2細胞培養(yǎng)瓶中并使用含10%FBS的DMEM/F12培養(yǎng)基進行原代前列腺細胞培養(yǎng),細胞增殖到一定程度后對癌旁組織細胞和良性前列腺組織細胞均同時染色Trop2和CD49f抗體,通過流式細胞分選分別獲得此2種細胞株的高純度基底細(Trop2+CD49fhigh)和管腔上皮細胞(Trop2+CD49flow)亞群。由于前列腺癌組織缺乏基底細胞,對前列腺癌組織細胞染色Trop2抗體,通過流式細胞分選獲得其管腔上皮細胞亞群(Trop2+)。對以上五種細胞亞群進行二代轉(zhuǎn)錄組測序,分析癌旁組織、正常前列腺組織及前列腺癌組織管腔上皮細胞的基因表達譜,同時分析癌旁組織和正常前列腺組織基底上皮細胞的基因表達譜。結(jié)果:(1)所獲取的前列腺癌組織HE染色切片在顯微鏡下可見前列腺腺泡結(jié)構(gòu)紊亂,基底層消失,呈現(xiàn)出間質(zhì)侵襲性生長的趨勢,同時細胞異型性明顯,免疫組化提示AMACR+,34βE12-;所獲取的前列腺癌旁組織及正常前列腺組織HE染色切片在光鏡下無明顯區(qū)別,腺泡結(jié)構(gòu)規(guī)整,基底細胞層存在,未見細胞核異常改變,免疫組化均為AMACR-,34βE12+。(2)由手術(shù)室直接獲取的新鮮前列腺組織(包括癌、癌旁及正常前列腺組織)先后經(jīng)過多種消化酶聯(lián)合作用后基本消化完全,接種72h后可見小片前列腺上皮集落形成,接種后10天左右大多數(shù)樣品細胞細胞融合達90%以上。使用Trop2和CD49f抗體雙染的癌旁和正常前列腺組織細胞在流式細胞分析中的細胞分群情況相似,且Trop2+的細胞群中根據(jù)CD49f的表達情況大致可以分為Trop2+CD49fhigh和Trop2+CD49flow兩個亞群。經(jīng)過原代培養(yǎng)后的癌組織細胞進行TROP2抗體的染色后,流式細胞分析可見細胞主要可分為TROP2+和TROP2-兩個細胞亞群。流式分選獲得的用于轉(zhuǎn)錄組測序的各細胞樣本通過流式細胞分析測得其目標細胞比例均在85%以上。(3)通過轉(zhuǎn)錄組測序及對不同細胞群基因表達譜的分析,我們發(fā)現(xiàn)癌旁組織管腔上皮細胞在部分基因的表達水平上與癌組織相似,而且不同于正常前列腺管腔上皮;癌旁組織的基底上皮細胞在部分基因的表達上與正常前列腺基底細胞也存在不同的基因表達譜。結(jié)論:前列腺癌旁組織管腔上皮細胞在部分基因的表達水平上與前列腺癌相似而且不同于正常前列腺管腔上皮細胞,前列腺癌旁組織基底細胞和正常前列腺組織基底細胞也存在完全不同的基因表達譜,其中的差異表達基因如PSG5、RNA5-8S5等基因都可能成為潛在的能夠?qū)┡越M織與正常前列腺組織區(qū)分開來的特異性標志物,而差異表達基因中富集程度較高的與癌癥相關(guān)的信號通路也很有可能在癌癥發(fā)病早期即發(fā)揮著重要的作用。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.25

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