眼睑基底细胞癌相关成纤维细胞的分离培养及生物学特性研究

发布时间：2018-01-04 16:11

  本文关键词：眼睑基底细胞癌相关成纤维细胞的分离培养及生物学特性研究　出处：《川北医学院》2016年硕士论文　论文类型：学位论文

  更多相关文章： 眼睑基底细胞癌 癌相关成纤维细胞 正常成纤维细胞 分离 培养 生物学特性

【摘要】：目的:对眼睑基底细胞癌相关成纤维细胞(CAFs)及眼睑正常成纤维细胞(NFs)进行分离、培养、纯化、鉴定,初步研究CAFs与NFs的细胞形态、生长增殖及骨膜蛋白(PN或POSTN)表达等生物学特性的差异。方法:(1)用组织块贴壁法培养获得原代眼睑基底细胞癌CAFs和眼睑正常NFs,通过胰酶差速消化法和反复传代培养法进行细胞的分离、纯化;(2)通过倒置相差显微镜观察CAFs和NFs细胞形态,应用免疫细胞化学染色法检测细胞表达的特异性蛋白,对CAFs和NFs进行鉴定;(3)采用MTT比色法对连续培养7天的细胞数量进行检测并绘制生长曲线,通过酶联免疫吸附试验(ELISA)法检测CAFs与NFs细胞培养上清液中PN的含量,应用免疫荧光细胞化学染色方法检测CAFs与NFs中PN的表达。结果:(1)成功分离出了纯化的眼睑CAFs和NFs;(2)眼睑CAFs呈长梭形,胞质突减少,胞浆内可见较多颗粒,细胞大小不一,生长较快,排列紊乱,有重叠生长现象;眼睑CAFs和NFs的细胞免疫化学染色,前者波形蛋白(VIM)、α平滑肌肌动蛋白(α-SMA)、成纤维细胞活化蛋白(FAP)呈阳性,细胞角蛋白(CK)呈阴性,后者仅VIM呈阳性,CK、α-SMA、FAP均呈阴性。(3)与NFs相比,眼睑CAFs生长增殖较快,倍增时间较短;ELISA法测定CAFs与NFs细胞培养上清液中PN的含量,分别为((9.26±2.35)ng/ml)和((2.57±0.41)ng/ml);细胞免疫荧光化学染色检测眼睑CAFs中PN表达强于NFs,差异具有统计学意义(P0.05)。结论:与NFs相比,眼睑基底细胞癌中CAFs在形态结构、生长增殖、骨膜蛋白表达等生物学特征存在显著差异,提示眼睑基底细胞癌细胞微环境的变化导致NFs生物学特性和功能的改变而形成了CAFs,同时与眼睑基底细胞癌细胞相互作用,促进或抑制眼睑基底细胞癌的发生发展。
[Abstract]:Objective: to isolate, culture, purify and identify the basal cell carcinoma associated fibroblasts (CAFs) and normal eyelid fibroblasts (NFS). The cell morphology of CAFs and NFs was studied. Methods CAFs and normal eyelid NFs were obtained from primary eyelid basal cell carcinoma (BCC) and normal eyelid (NFs). The cells were separated and purified by trypsin differential digestion and repeated passage culture. (2) the morphology of CAFs and NFs cells were observed by inverted phase contrast microscope, and the specific proteins expressed by the cells were detected by immunocytochemical staining. CAFs and NFs were identified. MTT colorimetry was used to detect the number of cells cultured for 7 days and to draw the growth curve. The content of PN in the supernatant of CAFs and NFs cells was detected by Elisa. The expression of PN in CAFs and NFs was detected by immunofluorescence cytochemical staining. (2) CAFs of eyelid is fusiform, cytoplasmic process is decreased, more granules can be seen in the cytoplasm, cell size is different, growth is faster, arrangement is disordered, and there is overlapping growth phenomenon; The vimentin vimentin (VIMA) and 伪 smooth muscle actin (伪 -SMAA) were positive in eyelid CAFs and NFs. Cytokeratin (CK) was negative, only VIM was positive, 伪 -SMA-FAP was negative. (3) compared with NFs, CAFs of eyelid grew and proliferated faster and the doubling time was shorter. The content of PN in supernatant of CAFs and NFs cells was determined by ELISA method. They were 9.26 卤2.35 ng / ml and 2.57 卤0.41 ng / ml, respectively. The expression of PN in eyelid CAFs was stronger than that in NFS by immunofluorescence staining, and the difference was statistically significant (P 0.05). Conclusion: compared with NFs. There were significant differences in the biological characteristics of CAFs in eyelid basal cell carcinoma (BCC), such as morphology, growth and proliferation, and expression of periosteal protein. It is suggested that the changes of microenvironment of eyelid basal cell carcinoma cells lead to the change of biological characteristics and function of NFs, which results in the formation of CAFs, and the interaction with BCC cells. Promote or inhibit the development of basal cell carcinoma of eyelid.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R739.71

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