【作者】 张永红；

【导师】 龚建平；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【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。更多还原

【Abstract】 Objective:Making high-qualified single cell suspensions out of normal gastic mucous epithelia (GME), intestinal mucous epithelia (IME) and oral mucous epithelia (OME) in order to be smoothly detected by flow cytometry (FCM) and building up a model for studying human in vivo cell-cycle regulatory mechanism.Methods:We used pepsin and Dispase at different concentrations to remove gastic mucus,intestinal mucus and oral mucus and separated mucous membranes, then cut them to pieces, blew gently and finally filtered them to make single cell suspensions; used cotton bud to scrape out normal people’s OME and filter them. Next we used DNA content method of FCM to detect the cell proportion at every phase and furtherly used Ki67/DNA double parameter method to detect cell proliferation ability. Taking scissoring alone and digesting alone and PBL as control.Results:We made use of 0.05-0.1% pepsin and 1.2-2.4 u/ml Dispase to remove mucus successfually from the GME, IME and OME and obtained mucous membranes, then cut them to pieces and blew repeatedly to make high-qualified single cell suspensions; We also made high-qualitied single cell suspensions by scraping and filtering OME. At last we obtained a lot of intact cells, cell quantity>1×106and cell survival rate>90% and the cells can be detected successfually by FCM. The rates of GME, IME and OME were 80%-82% at G1 phase and 11%-12% at S phase. The proliferating rates of GME, IME and OME were 11.67%,27.79% and 23.48% respectively by the Ki67/DNA double parameter method and were lower than those of PBL.Conclusions:We made use of pepsin and Dispase to produce high-qualified single cell suspensions out of GME, IME and OME which were much fit for analysis on FCM and got satisfied results and they have an evident cell division cycle and are competent for a model of studying human in vivo cell-cycle regulatory mechanism. Objective:Study on the expression of cell cycle regulators of human in vivo cells taking gastric mucous epithelia (GME).. intestinal mucous epithelia (IME) and oral mucous epithelia (OME) for examples.Methods:We applied Western blot to detect the expression of cell cycle regulators like Cyclins (A, B1, D1, E), CDKs (CDK1,2,4,6), CKIs (P27, P21, P19, P16) and Rb taking PBL and MNC as positive control.Results:There existed obvious cyclin Dl expression but no or faint cyclin A, cyclin B1 and cyclin E expression in the OME; and there were no or faint cyclin Dl, cyclin A, cyclin B1 and cyclin E expression in the GME and IME. There existed obvious CDK2, CDK4 and CDK6 expression but no or faint CDK1 expression in the OME; and there were obvious CDK2 expression and no or faint CDK1, CDK4 and CDK6 expression in the GME and IME, and CDK2 expression in GME was lower than those in IME. There were obvious p19, p21 and p27 expression but no or faint p16 expression in the OME; and there existed obvious p19 expression but no or faint p16, p21 and p27 expression in the GME and IME. There were no or faint Rb expression in the OME, GME and IME.Conclusions:The significant various expression of core cell cycle regulators between the in-vivo cells and the cultured cells and between the in-vivo cells and another in-vivo cells reveals the diversity of human in-vivo core cell-cycle mechanism. Objective:To study on human in-vivo cell-cycle core mechanism-cyclins schedule and CDKs activation taking GME, IME and OME for examples.Methods:Cyclins schedule:we used post-sorting western blot to dectect at which phase the expression of four cyclins were. CDKs activation:we used post-sorting immuno-precipitation to detect how the four cyclins were associated with the four CDKs. Taking PBL and MNC as positive control.Results:Cyclins expression schedule:In the OME, cyclin E, cyclin A and cyclin B1 are not expressed or only a little; cyclin D1 are expressed distinctly during all the cycle phases. From G1 early phase, S phase to G2/M phase, cyclin D1 expression decrease gradually in the OME. In the GME and IME, cyclin D1, cyclin E, cyclin A and cyclin B1 are not expressed or only a little. CDKs scheduled activation:In the G1, S and G2/M phases of the OME, cyclin D1 can combine with CDK2, CDK4 and CDK6; in G1 phase, cyclin D1 mainly combine with CDK4/CDK6, and in G2/M phase mainly with CDK2. In the G1, S and G2/M phases of the GME and IME, only a weak association existed between CDK2 and Cyclin D1, Cyclin E, cyclin A or cyclin B1.Conclusions:The significant diversity existed between the in-vivo cells and the cultured cells and between the in-vivo cells and another in-vivo cells about human in-vivo cell-cycle core mechanism, namely cyclins schedule and CDKs activation, which revealed the diversity of human in-vivo cell-cycle core mechanism.更多还原