【摘要】： 目的：將正常的胃、腸及口腔黏膜上皮組織制備成高質(zhì)量的單細(xì)胞懸液,可供流式細(xì)胞儀檢測(cè)；并建立人類在體細(xì)胞的細(xì)胞增殖周期研究模型。 方法：應(yīng)用不同濃度的胃蛋白酶和Dispase嘗試去除胃、腸、口腔粘液和分離獲得粘膜層后,再使用機(jī)械剪碎法制備成單細(xì)胞懸液并用濾網(wǎng)過(guò)濾細(xì)胞；另外部分標(biāo)本采用棉簽直接刮取正常人群的口腔黏膜上皮細(xì)胞,并用濾網(wǎng)過(guò)濾之。然后用流式DNA直方圖來(lái)驗(yàn)證獲得的單細(xì)胞的各周期比例,進(jìn)一步采用Ki67/DNA雙參數(shù)法檢測(cè)細(xì)胞的增殖能力。對(duì)照組是單用機(jī)械剪碎法和單用酶消化法以及人類外周血淋巴細(xì)胞。 結(jié)果：我們發(fā)現(xiàn)0.05-0.1%的胃蛋白酶濃度和1.2-2.4 u/ml的Dispase濃度能夠較好的去除胃、腸、口腔粘液而獲得粘膜層,然后剪碎并吹打,制備成胃、腸、口腔粘膜上皮細(xì)胞的單細(xì)胞懸液；用棉簽刮取的口腔黏膜上皮組織濾過(guò)之后直接成為單細(xì)胞懸液,顯微鏡下觀察發(fā)現(xiàn)細(xì)胞形態(tài)較完整,細(xì)胞數(shù)量大于1×106,細(xì)胞存活率大于90%,從而都能夠采用流式技術(shù)進(jìn)行單細(xì)胞分析。流式DNA含量法檢測(cè)胃、腸、口腔黏膜上皮細(xì)胞G1期約占80%-82%,S期約占11%-12%, Ki67/DNA雙參數(shù)法檢測(cè)胃、腸、口腔黏膜上皮組織的增殖率分別為11.67%、27.79%、23.48%,均低于體外培養(yǎng)的淋巴細(xì)胞。 結(jié)論：利用胃蛋白酶和Dispase能夠去除胃、腸、口腔粘液,較容易獲得高質(zhì)量的胃、腸、口腔粘膜上皮細(xì)胞的單細(xì)胞懸液,樣本完全適合FCM分析,結(jié)果滿意。胃、腸、口腔粘膜上皮細(xì)胞具有明顯的細(xì)胞分裂周期,適合成為人類在體細(xì)胞的細(xì)胞增殖周期研究模型。 目的：以胃、腸和口腔黏膜上皮細(xì)胞為研究對(duì)象,研究人類正常在體細(xì)胞的細(xì)胞周期核心調(diào)控因子的表達(dá)情況。 方法：采用Western blot分析各種細(xì)胞周期調(diào)控因子Cyclins (A, B1, D1, E), CDKs (CDK1,2,4,6)、CKIs (P27, P21, P19, P16)和Rb的表達(dá)；并與正常人外周血淋巴細(xì)胞(PBL)和體內(nèi)骨髓單個(gè)核細(xì)胞(MNC)作為對(duì)比分析。 結(jié)果：Western blot分析結(jié)果顯示：口腔黏膜上皮細(xì)胞(OME)中cyclin D1明顯表達(dá),cyclin A、cyclin B1和cyclin E無(wú)表達(dá)或微弱表達(dá)；胃、腸黏膜上皮細(xì)胞(GME和IME)中cyclin A、cyclin B1、cyclin D3和cyclin E均無(wú)表達(dá)或微弱表達(dá)�？谇火つど掀ぜ�(xì)胞中CDK2、CDK4和CDK6明顯表達(dá),而CDK1無(wú)表達(dá)或微弱表達(dá)；胃腸黏膜上皮細(xì)胞中CDK2明顯表達(dá),并且胃黏膜上皮細(xì)胞中CDK2的表達(dá)比腸黏膜上皮細(xì)胞的表達(dá)要低,而CDK1、CDK4和CDK6無(wú)表達(dá)或微弱表達(dá)�？谇火つど掀ぜ�(xì)胞中p19、p21和p27明顯表達(dá),而p16無(wú)表達(dá)或微弱表達(dá)；胃腸黏膜上皮細(xì)胞中p19明顯表達(dá),而p16、p21和p27無(wú)表達(dá)或微弱表達(dá)。胃、腸、口腔黏膜上皮細(xì)胞中Rb無(wú)表達(dá)或微弱表達(dá)。 結(jié)論：細(xì)胞周期調(diào)控因子體內(nèi)細(xì)胞與體外培養(yǎng)細(xì)胞中的表達(dá)以及體內(nèi)細(xì)胞與細(xì)胞之間的表達(dá)并不完成相同,提示人類在體細(xì)胞周期核心機(jī)制可能存在多樣性。 目的：探討人類在體細(xì)胞(胃、腸、口腔黏膜上皮細(xì)胞)的細(xì)胞周期核心調(diào)控機(jī)制,即Cyclins時(shí)相性起伏及CDKs序列激活規(guī)律。 方法：Cyclins時(shí)相性表達(dá)規(guī)律：Post-sorting Western blot(分選后蛋白電泳技術(shù))檢測(cè)四種Cyclins在細(xì)胞周期不同時(shí)相中的表達(dá)。CDKs序列激活規(guī)律：Post-sorting Western blot(分選后免疫共沉淀技術(shù))分析Cyclins和CDKs的相互結(jié)合規(guī)律。陽(yáng)性對(duì)照采用正常人外周血PHA刺激培養(yǎng)的淋巴細(xì)胞(PBL)和體內(nèi)骨髓單個(gè)核細(xì)胞(MNC)。 結(jié)果：Cyclins時(shí)相性表達(dá)規(guī)律：體內(nèi)OME的表達(dá)規(guī)律是：Cyclin D1在G1早期合成,S期和G2/M期開(kāi)始下降；Cyclin E、Cyclin A和Cyclin B1無(wú)表達(dá)或在G1期有微弱表達(dá)。體內(nèi)GME和IME的表達(dá)規(guī)律是：Cyclin D1、Cyclin E、Cyclin A和CyclinB1無(wú)表達(dá)或在G1期有微弱表達(dá)。CDKs序列激活規(guī)律：在OME中,在G1、S.G2/M期,Cyclin D1均與CDK2、CDK4、CDK6結(jié)合；在G1期,cyclin D1/CDK4/CDK6結(jié)合最多；在G2/M期,cyclin D1/CDK2結(jié)合最多。在GME和IME中,在G1、S、G2/M期,Cyclin D1、CyclinE、Cyclin A和Cyclin B1均與CDK2僅有微量的結(jié)合。 結(jié)論：人類在體增殖細(xì)胞的細(xì)胞周期核心調(diào)控機(jī)制——Cyclins周期時(shí)相性起伏及CDKs序列激活規(guī)律,在體內(nèi)細(xì)胞與細(xì)胞之間以及體內(nèi)細(xì)胞與體外細(xì)胞之間均存在重要的差別,人類在體細(xì)胞的細(xì)胞周期核心機(jī)制存在多樣性。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

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