The Effects Of Nordy On HPV16 Subgenes-Immortalized Human Endocervical Cells

PART 1 EFFECTS OF NORDY ON PROLIFERATION AND DIFFERENTIATION AND APOPTOSIS OF HPV16 SUBGENES IMMORTALIZED HUMAN END- -OCERVICAL CELLSObjective: To investigate the effects of Nordy on proliferation and differentiation and apoptosis of HPV16 subgenes-immortalized human endocervical cells(H8 cells)in vitro。Methods: HPV16 subgenes-immortalized human endocervical cells (H8 cells) were cultured in vitro. After treated with Nordy, the proliferation of H8 was tested with MTT assay; The effect of Nordy on cell cycle and apoptosis of H8 cells was analyzed by flow cytometry (FCM) and Annexin V-FITC method; The expression of mcm5 in H8 cells was detected by immunocytochemistry. Morphological changes were observed by light and electron microscopy.Results:â‘ 10umol/L, 50umol/L, 75 umol/L and 100umol/L Nordy could inhibit proliferation of H8 cells significantly in a time and dose-dependent manner.â‘¡Expression of mcm5 decreased significantly after H8 cell was treated with 100umol/L Nordy for 72h.â‘¢According to FCM, H8 cells accumulated in G₀/G₁ phase, and the percentages of S and G₂/M phase cells decreased after treated with 10umol/L,50umol/L, 75 umol/L ,100umol/L Nordy for 72h in a dose-dependent manner. The result of Annexin V-FITC detection showed the apoptosis rate of experimental group cells was higher than control group cells obviously (p<0.05).â‘£In the control group, H8 cells displayed marked pleomorphism, with large and hyperchromatic nucleus. Nuclear mitoses could be observed easily. After treated with Nordy, the cells became regular and smaller, mitoses figures decreased, nuclei of H8 cells became smaller and regular. By electron microscopy, the mature organelles such as mitochondrias and endocytoplasmic reticulums increased obviously and the apoptosis bodys could be observed easily in the cells of the test group.Conclusions: Nordy could inhibit proliferation and promote apoptosis of H8 cells,Nordy also could induce the differentiation of H8 cell, which might be achieved by inhibiting synthesis of cell DNA , blocking cell cycle in G0/G1 phase and down-regulating the activity of mcm5 protein . PART 2 INHIBITION EFFECT OF NORDY ON HPV16 E6 GENE IN HUMAN IMMORTALIZED ENDOCERVICAL CELLSObjective: To study the inhibition effect of Nordy on HPV16 E6 gene in human immortalized endocervical cells.Methods: The expressions of E6 and P53 proteins of H8 cells were detected by immunocytochemistry, the expression of HPV16E6 mRNA of H8 cells was detected by RT-PCR method.Results:â‘ According to immunocytochemistry, expressions of E6 and P53 proteins of H8 cells after treated with 100umol/L Nordy for 72h were weaker than control group obviously.â‘¡Treated with 50umol/L, 100umol/L, 200umol/L Nordy for 72h, The HPV16E6 mRNA expressions of H8 cells were lower than the control group significantly(p<0.05).Conclusion: Nordy could inhibit the expression of HPV16E6 gene mRNA in H8 cells to influence transcription of viral gene and decrease expressions of E6 and P53 proteins.