人类在体细胞周期核心机制多样性

发布时间：2018-08-20 17:06

【摘要】： 目的：将正常的胃、肠及口腔黏膜上皮组织制备成高质量的单细胞悬液,可供流式细胞仪检测；并建立人类在体细胞的细胞增殖周期研究模型。方法：应用不同浓度的胃蛋白酶和Dispase尝试去除胃、肠、口腔粘液和分离获得粘膜层后,再使用机械剪碎法制备成单细胞悬液并用滤网过滤细胞；另外部分标本采用棉签直接刮取正常人群的口腔黏膜上皮细胞,并用滤网过滤之。然后用流式DNA直方图来验证获得的单细胞的各周期比例,进一步采用Ki67/DNA双参数法检测细胞的增殖能力。对照组是单用机械剪碎法和单用酶消化法以及人类外周血淋巴细胞。结果：我们发现0.05-0.1%的胃蛋白酶浓度和1.2-2.4 u/ml的Dispase浓度能够较好的去除胃、肠、口腔粘液而获得粘膜层,然后剪碎并吹打,制备成胃、肠、口腔粘膜上皮细胞的单细胞悬液；用棉签刮取的口腔黏膜上皮组织滤过之后直接成为单细胞悬液,显微镜下观察发现细胞形态较完整,细胞数量大于1×106,细胞存活率大于90%,从而都能够采用流式技术进行单细胞分析。流式DNA含量法检测胃、肠、口腔黏膜上皮细胞G1期约占80%-82%,S期约占11%-12%, Ki67/DNA双参数法检测胃、肠、口腔黏膜上皮组织的增殖率分别为11.67%、27.79%、23.48%,均低于体外培养的淋巴细胞。结论：利用胃蛋白酶和Dispase能够去除胃、肠、口腔粘液,较容易获得高质量的胃、肠、口腔粘膜上皮细胞的单细胞悬液,样本完全适合FCM分析,结果满意。胃、肠、口腔粘膜上皮细胞具有明显的细胞分裂周期,适合成为人类在体细胞的细胞增殖周期研究模型。目的：以胃、肠和口腔黏膜上皮细胞为研究对象,研究人类正常在体细胞的细胞周期核心调控因子的表达情况。方法：采用Western blot分析各种细胞周期调控因子Cyclins (A, B1, D1, E), CDKs (CDK1,2,4,6)、CKIs (P27, P21, P19, P16)和Rb的表达；并与正常人外周血淋巴细胞(PBL)和体内骨髓单个核细胞(MNC)作为对比分析。结果：Western blot分析结果显示：口腔黏膜上皮细胞(OME)中cyclin D1明显表达,cyclin A、cyclin B1和cyclin E无表达或微弱表达；胃、肠黏膜上皮细胞(GME和IME)中cyclin A、cyclin B1、cyclin D3和cyclin E均无表达或微弱表达。口腔黏膜上皮细胞中CDK2、CDK4和CDK6明显表达,而CDK1无表达或微弱表达；胃肠黏膜上皮细胞中CDK2明显表达,并且胃黏膜上皮细胞中CDK2的表达比肠黏膜上皮细胞的表达要低,而CDK1、CDK4和CDK6无表达或微弱表达。口腔黏膜上皮细胞中p19、p21和p27明显表达,而p16无表达或微弱表达；胃肠黏膜上皮细胞中p19明显表达,而p16、p21和p27无表达或微弱表达。胃、肠、口腔黏膜上皮细胞中Rb无表达或微弱表达。结论：细胞周期调控因子体内细胞与体外培养细胞中的表达以及体内细胞与细胞之间的表达并不完成相同,提示人类在体细胞周期核心机制可能存在多样性。目的：探讨人类在体细胞(胃、肠、口腔黏膜上皮细胞)的细胞周期核心调控机制,即Cyclins时相性起伏及CDKs序列激活规律。方法：Cyclins时相性表达规律：Post-sorting Western blot(分选后蛋白电泳技术)检测四种Cyclins在细胞周期不同时相中的表达。CDKs序列激活规律：Post-sorting Western blot(分选后免疫共沉淀技术)分析Cyclins和CDKs的相互结合规律。阳性对照采用正常人外周血PHA刺激培养的淋巴细胞(PBL)和体内骨髓单个核细胞(MNC)。结果：Cyclins时相性表达规律：体内OME的表达规律是：Cyclin D1在G1早期合成,S期和G2/M期开始下降；Cyclin E、Cyclin A和Cyclin B1无表达或在G1期有微弱表达。体内GME和IME的表达规律是：Cyclin D1、Cyclin E、Cyclin A和CyclinB1无表达或在G1期有微弱表达。CDKs序列激活规律：在OME中,在G1、S.G2/M期,Cyclin D1均与CDK2、CDK4、CDK6结合；在G1期,cyclin D1/CDK4/CDK6结合最多；在G2/M期,cyclin D1/CDK2结合最多。在GME和IME中,在G1、S、G2/M期,Cyclin D1、CyclinE、Cyclin A和Cyclin B1均与CDK2仅有微量的结合。结论：人类在体增殖细胞的细胞周期核心调控机制——Cyclins周期时相性起伏及CDKs序列激活规律,在体内细胞与细胞之间以及体内细胞与体外细胞之间均存在重要的差别,人类在体细胞的细胞周期核心机制存在多样性。
[Abstract]:AIM: To prepare high quality single cell suspension from normal gastric, intestinal and oral mucosal epithelial tissues for flow cytometry detection and to establish a model of human cell proliferation cycle in vivo.
Methods: Different concentrations of pepsin and Dispase were used to remove gastric, intestinal, oral mucus and isolate mucosal layer, then single cell suspension was prepared by mechanical shearing method and the cells were filtered by filter mesh. Other specimens were directly scraped with cotton swabs from normal people's oral mucosal epithelial cells and filtered by filter mesh. The proportions of each cell cycle were verified by flow cytometry, and the proliferation of the cells was detected by Ki67/DNA double parameter assay. The control group was treated by mechanical shredding, enzymatic digestion and human peripheral blood lymphocytes.
RESULTS: We found that 0.05-0.1% pepsin and 1.2-2.4 u/ml Dispase could remove gastric, intestinal and oral mucus to obtain mucosal layer, and then cut and blow to prepare single cell suspension of gastric, intestinal and oral mucosal epithelial cells. Cell suspension and microscopic observation showed that the cell morphology was complete, the number of cells was more than 1 *106, and the cell survival rate was more than 90%. Flow cytometry was used to detect the G1 phase of gastric, intestinal and oral mucosal epithelial cells in 80% - 82%, S phase was about 11% - 12%, and Ki67 / DNA dual parameter method was used to detect gastric, intestinal and oral mucosa. The proliferative rates of membrane epithelium were 11.67%, 27.79% and 23.48%, respectively, which were lower than those of lymphocytes cultured in vitro.
CONCLUSION: Pepsin and Dispase can remove gastric, intestinal and oral mucus. High quality single cell suspension of gastric, intestinal and oral mucosal epithelial cells can be obtained easily. The samples are completely suitable for FCM analysis. The results are satisfactory. A reproductive cycle research model.
AIM: To study the expression of core regulatory factors in human normal cell cycle in gastric, intestinal and oral mucosal epithelial cells.
Methods: Western blot was used to analyze the expression of Cyclins (A, B1, D1, E), CDKs (CDK1, 2, 4, 6), CKIs (P27, P21, P19, P16) and Rb, and compared with normal human peripheral blood lymphocytes (PBL) and bone marrow mononuclear cells (MNC).
Results: Western blot analysis showed that cyclin D1 was significantly expressed in oral mucosal epithelial cells (OME), but cyclin A, cyclin B1 and cyclin E were not or slightly expressed; cyclin A, cyclin B1, cyclin D3 and cyclin E were not or slightly expressed in gastric and intestinal epithelial cells (GME and IME). The expression of CDK2 in gastrointestinal epithelial cells was lower than that in intestinal epithelial cells, but there was no or weak expression of CDK1, CDK4 and CDK6. There was no or weak expression of p19, p16, p21 and p27 in gastrointestinal epithelial cells, and no or weak expression of Rb in gastrointestinal, intestinal and oral epithelial cells.
CONCLUSION: The expression of cell cycle regulators in vivo is not the same as that in vitro, suggesting that there may be diversity in the core mechanism of human cell cycle.
AIM: To investigate the core regulatory mechanism of human cell cycle in vivo (gastric, intestinal and oral mucosal epithelial cells), i.e. Cyclins phase fluctuation and CDKs sequence activation.
METHODS: The expression of four Cyclins in different phases of cell cycle was detected by post-sorting Western blot (post-sorting protein electrophoresis). Activation of CDKs sequence: Post-sorting Western blot (post-sorting immunoprecipitation technique) was used to analyze the interaction between Cyclins and CDKs. Normal peripheral blood PHA stimulates cultured lymphocytes (PBL) and bone marrow mononuclear cells (MNC) in vivo.
Results: Cyclin D1 was synthesized in early G1 phase and decreased in S phase and G2/M phase. Cyclin E, Cyclin A and Cyclin B1 were not expressed or weakly expressed in G1 phase. In OME, Cyclin D1 binds to CDK2, CDK4 and CDK6 at G1, S.G2/M, cyclin D1 / CDK4 / CDK6 at G1, and cyclin D1 / CDK2 at G2/M. In GME and IME, Cyclin D1, Cyclin D1, Cyclin E, Cyclin A and Cyclin B1 bind to CDK2 only slightly.
CONCLUSION: There are important differences between cells in vivo and between cells in vivo, as well as between cells in vivo and in vitro. There are diversity in the core mechanism of human cell cycle.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

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