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目的 :分析外源性p16表达对CDK4,CyclinD1及pRb的影响 ,探讨其抑制细胞生长的机制。方法 :绘制生长曲线 ,比较细胞倍增时间 ,分析HNE1,# 4 - 2及 # 3- 2克隆中细胞增殖状况 ;利用流式细胞仪分析上述细胞中细胞周期分布的改变 ;结合WesternBlot方法 ,分析外源性p16表达对CDK4,CyclinD1及pRb的影响。结果 :生长曲线示 ,HNE 1倍增时间为 2 3 4h ,# 3- 2和 # 4 - 2分别为 2 8 8h和 31 2h。流式细胞仪示 ,HNE1和 # 3- 2细胞周期无明显差别 (P >0 .0 5 ) ,# 4 - 2细胞G0 /G1期细胞数明显增多 ,S期的细胞数显著降低 (P <0 .0 1和P <0 .0 5 )。WesernBlot示外源性p16表达对CDK4表达水平影响并不显著 ,但下调 # 4 - 2中CyclinD1的表达 ,# 3- 2中CyclinD1的表达反而增强 ;各细胞系均可检测到低磷酸化的pRb ,但在 # 4 - 2 ,# 3- 2中表达强于HNE1及Hela细胞 ;HNE1还可检获高磷酸化的pRb。结论 :外源性p16表达使细胞停滞在G1期 ,抑制细胞生长 ,但其主要机制并非改变CDK4表达水平 ;而对CyclinD1的影响 ,随p16表达水平而变动 ,最终表现为抑制pRb磷酸化。
OBJECTIVE: To analyze the effect of exogenous p16 expression on CDK4, CyclinD1 and pRb and to explore the mechanism of its inhibition on cell growth. Methods: The growth curves were drawn and the cell doubling time was compared. The cell proliferation status in HNE1, # 4-2 and # 3- 2 clones was analyzed. The cell cycle distribution was analyzed by flow cytometry. Combined with Western Blot analysis, Effect of p16 expression on CDK4, CyclinD1 and pRb. Results: The growth curves showed that the doubling time of HNE was 2334h and that of # 3-2 and # 4-2 were respectively 2 8 8h and 31 2h. Flow Cytometry showed that there was no significant difference between HNE1 and # 3- 2 cell cycle (P> 0.05). The number of cells in G0 / G1 phase of # 4-2 cells was significantly increased and the number of cells in S phase was significantly decreased (P < 0 .0 1 and P <0. 0 5). WesernBlot showed that the expression of exogenous p16 had no significant effect on the expression of CDK4, but downregulated the expression of CyclinD1 in # 4 - 2 and increased the expression of CyclinD1 in # 3-2. However, the expression of pRb in hypoxia was detected in all cell lines , But it was stronger than that of HNE1 and Hela cells in # 4 - 2 and # 3 - 2; HNE1 also could sequester highly phosphorylated pRb. CONCLUSION: Exogenous p16 expression arrests cells in G1 phase and inhibits cell growth. However, the main mechanism is not to change the expression level of CDK4. The effect on CyclinD1 changes with the expression of p16 and eventually inhibits the phosphorylation of pRb.