本文選題：膠質瘤　切入點：腫瘤干細胞　出處：《濟寧醫(yī)學院》2017年碩士論文

【摘要】：目的:分離、培養(yǎng)人腦膠質瘤原代細胞及其干細胞,為研究膠質瘤生物學特性提供體外試驗模型。方法:選取2016年4月至2016年10月濟寧醫(yī)學院附屬醫(yī)院神經外科住院患者的20例新鮮膠質瘤標本,利用酶消化法分離得到人腦膠質瘤原代細胞單細胞懸液,進行原代細胞培養(yǎng),通過倒置相差顯微鏡觀察腫瘤細胞的形態(tài)學特點,采用免疫組織化學法檢測GFAP、Ki-67表達,并通過CCK-8法繪制細胞生長曲線研究體外培養(yǎng)細胞的增殖情況;同時,通過免疫磁珠法對細胞懸液進行CD133~+細胞分選,利用神經干細胞培養(yǎng)基培養(yǎng)細胞成球,免疫熒光化學法檢測干細胞標志物nestin,行誘導分化實驗后檢測GFAP和細胞表面分化標志物β-Tubulin表達情況。結果:1.人腦膠質瘤原代細胞培養(yǎng)成功17例,失敗3例;成功培養(yǎng)的原代細胞狀態(tài)良好,呈貼壁生長,形態(tài)各異,存在明顯的對數生長期,并可連續(xù)穩(wěn)定傳代培養(yǎng)。其中,WHOⅣ級膠質瘤細胞的對數生長期約出現在第4天,WHOⅡ級膠質瘤細胞的對數生長期約在第10天,WHOⅢ級膠質瘤細胞的對數生長期位于兩者之間。2.細胞增殖實驗顯示所培養(yǎng)細胞在體外增殖活躍,不同病理級別膠質瘤原代細胞的吸光度值差異有統計學意義(P0.05),腫瘤的惡性程度越高,細胞增殖能力越強。通過石蠟切片HE染色和免疫組織化學染色證明所培養(yǎng)的細胞為膠質瘤細胞,且細胞免疫熒光檢測顯示GFAP表達陽性。3.經免疫磁珠法分選得到CD133~+細胞,呈懸浮生長狀態(tài)且能聚集成球,細胞球增殖能力較強,nestin免疫熒光染色檢測結果呈陽性;加入胎牛血清誘導后細胞球貼壁分化,免疫組化法染色檢測分化后的細胞顯示GFAP、β-Tubulin表達陽性,表現為典型的干細胞特征。20例人腦膠質瘤標本中成功培養(yǎng)出膠質瘤干細胞15例,培養(yǎng)成功率為75%。4.人腦膠質瘤組織中存在腫瘤干細胞,腫瘤病理級別越高,細胞球所形成的時間越短,生長速度越快且成球數量越多,干細胞增殖能力越強且所占的比例越高。結論:在體外利用酶消化法從人腦膠質瘤組織中成功進行膠質瘤原代細胞培養(yǎng),且培養(yǎng)成功率較高,適合建立腦膠質瘤體外培養(yǎng)模型;利用免疫磁珠法從人腦膠質瘤組織中分選出一小部分具有干細胞屬性的CD133~+膠質瘤細胞,進一步表明了膠質瘤干細胞的存在,并且膠質瘤病理級別越高,膠質瘤干細胞所占比例越高,增殖能力越強。
[Abstract]:Aim: to isolate and culture primary human glioma cells and stem cells, and to provide an in vitro model for studying the biological characteristics of gliomas.Methods: from April 2016 to October 2016, 20 samples of human glioma were collected from neurosurgical department of the affiliated Hospital of Jining Medical College. The single cell suspension of primary human glioma cells was isolated by enzyme digestion, and the primary cells were cultured.The morphological characteristics of tumor cells were observed by inverted phase contrast microscope, the expression of Ki-67 was detected by immunohistochemical method, and the proliferation of cultured cells in vitro was studied by CCK-8 method.CD133- cells were separated by immunomagnetic beads and cultured in neural stem cell culture medium.The expression of GFAP and 尾 -Tubulin on the surface of stem cells were detected by immunofluorescence assay.The result is 1: 1.The primary cells of human glioma were successfully cultured in 17 cases and failed in 3 cases.The logarithmic growth phase of WHO grade 鈪，

本文編號：1703593