人脑胶质瘤原代细胞及其干细胞的分离、培养和鉴定

发布时间：2018-04-03 04:18

  本文选题：胶质瘤　切入点：肿瘤干细胞　出处：《济宁医学院》2017年硕士论文

【摘要】：目的:分离、培养人脑胶质瘤原代细胞及其干细胞,为研究胶质瘤生物学特性提供体外试验模型。方法:选取2016年4月至2016年10月济宁医学院附属医院神经外科住院患者的20例新鲜胶质瘤标本,利用酶消化法分离得到人脑胶质瘤原代细胞单细胞悬液,进行原代细胞培养,通过倒置相差显微镜观察肿瘤细胞的形态学特点,采用免疫组织化学法检测GFAP、Ki-67表达,并通过CCK-8法绘制细胞生长曲线研究体外培养细胞的增殖情况;同时,通过免疫磁珠法对细胞悬液进行CD133~+细胞分选,利用神经干细胞培养基培养细胞成球,免疫荧光化学法检测干细胞标志物nestin,行诱导分化实验后检测GFAP和细胞表面分化标志物β-Tubulin表达情况。结果:1.人脑胶质瘤原代细胞培养成功17例,失败3例;成功培养的原代细胞状态良好,呈贴壁生长,形态各异,存在明显的对数生长期,并可连续稳定传代培养。其中,WHOⅣ级胶质瘤细胞的对数生长期约出现在第4天,WHOⅡ级胶质瘤细胞的对数生长期约在第10天,WHOⅢ级胶质瘤细胞的对数生长期位于两者之间。2.细胞增殖实验显示所培养细胞在体外增殖活跃,不同病理级别胶质瘤原代细胞的吸光度值差异有统计学意义(P0.05),肿瘤的恶性程度越高,细胞增殖能力越强。通过石蜡切片HE染色和免疫组织化学染色证明所培养的细胞为胶质瘤细胞,且细胞免疫荧光检测显示GFAP表达阳性。3.经免疫磁珠法分选得到CD133~+细胞,呈悬浮生长状态且能聚集成球,细胞球增殖能力较强,nestin免疫荧光染色检测结果呈阳性;加入胎牛血清诱导后细胞球贴壁分化,免疫组化法染色检测分化后的细胞显示GFAP、β-Tubulin表达阳性,表现为典型的干细胞特征。20例人脑胶质瘤标本中成功培养出胶质瘤干细胞15例,培养成功率为75%。4.人脑胶质瘤组织中存在肿瘤干细胞,肿瘤病理级别越高,细胞球所形成的时间越短,生长速度越快且成球数量越多,干细胞增殖能力越强且所占的比例越高。结论:在体外利用酶消化法从人脑胶质瘤组织中成功进行胶质瘤原代细胞培养,且培养成功率较高,适合建立脑胶质瘤体外培养模型;利用免疫磁珠法从人脑胶质瘤组织中分选出一小部分具有干细胞属性的CD133~+胶质瘤细胞,进一步表明了胶质瘤干细胞的存在,并且胶质瘤病理级别越高,胶质瘤干细胞所占比例越高,增殖能力越强。
[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，

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