The Diversity Of Human In Vivo Core Cell-cycle Mechanism

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.