| Objective: To screen early warning factors of squamous intraepithelial lesion(SIL)in cervical microbial community,and explore the mechanism of Lactobacillus inhibiting cervical cancer cell and cervical precancerous cell,we established a co-culture system of Lactobacillus crimpus with cervical cancer cell line(Si Ha cells)and cervical precancerous cell line(Ect1/E6E7 cells),so as to provide important theoretical basis and experimental basis for lactobacillus to prevent and treat SIL and cervical cancer.Methods: 1.A certain amount of Lactobacillus crimpus was obtained by plate counting method,and co-cultured with cervical cancer cell line(Si Ha cells)and cervical precancerous cell line(Ect1/E6E7 cells)respectively in a series of gradient multiplicity of infection(MOI).The growth of cells after co-culture was observed,and the optimal multiplicity of infection was selected,and then Lactobacillus crimpus was co-cultured with Si Ha cells and Ect1/E6E7 cells to observe Lactobacillus’ sticky effect on cells and cell morphology after adhesion.2.Lactobacillus crimpus was co-cultured with Si Ha cells for 6h,12 h,18h and24 h,and with Ect1/E6E7 cells for 6h,12 h,18h,24 h,30h,36 h,42h and 48 h.CCK-8 kit was used to detect the effect of Lactobacillus crimpus on the proliferation of Si Ha cells and Ect1/E6E7 cells,and the effect of Lactobacillus crimpus on the apoptosis of Si Ha cells and Ect1/E6E7 cells was detected by flow cytometry.Transwell chamber and Matrigel were used to detect the effect of Lactobacillus crimpus on cell migration and invasion.3.ELISA kit was used to detect the expression of IL-2,IL-4,IL-10,IL-12 and IFN-γ in the cell supernatant before and after Lactobacillus crimpus co-culture with Si Ha cells and Ect1/E6E7 cells.4.Total RNA and protein were collected before and after co-culture of Lactobacillus crimpus with Si Ha cells and Ect1/E6E7 cells.The m RNA and protein levels of ABCG2,PCNA,ATM,TDG,LIG1,OGG1 and HMGB1 were detected by real-time PCR and Western-blot.Results: 1.The optimal MOI for co-culture of Lactobacillus crimpus with Si Ha cells and Ect1/E6E7 cells was 200:1.A large number of Lactobacillus crimpus adhered to the membrane of Siha cells.With the extension of co-culture time,the cells gradually became smaller and round,the gap between cells increased,and the growth of cells was significantly inhibited.After co-culture for 12 hours,the floating cells and cell debris increased.After co-culture for 18 hours,the number of round cells with intracellular vacuity and shrunken nuclei gradually increased,and the number of adherent cells further decreased.For Ect1/E6E7 cells,however,Lactobacillus crimpus less adhered to the membrane of cells,and the cell morphology remained unchanged.With the extension of co-culture time,the intercellular space increased slightly,and the floating cells and cell debris increased gradually.Some vacuoles appeared in some cytoplasm after co-culture for 18 h and 24 h.2.Compared with the Si Ha cells blank control group,the proliferation of Siha cells began to be significantly inhibited after affected with Lactobacillus crimpus for 12 h(P < 0.001).Compared with the Ect1/E6E7 cells blank control group,the proliferation of Ect1/E6E7 cells was promoted after Lactobacillus crimpus affected 6 h and 12 h(P ≤ 0.001).With the extension of co-culture time,the cell proliferation was significantly inhibited after 24 h(P =0.019,P = 0.009,P < 0.001,P < 0.001,P < 0.001).3.Compared with the blank control group,the apoptotic rates of Si Ha cells and Ect1/E6E7 cells began to be significantly increased after Lactobacillus crimpus affected 6 h(P ≤ 0.001).4.Compared with the blank control group,the migration and invasion ability of Si Ha cells were significantly decreased after treated with Lactobacillus crimpus for 18 h(P < 0.05),while only the migration ability of Ect1/E6E7 cells was significantly inhibited after treated with Lactobacillus crimpus for 18 h(P ≤0.001).5.In the experiment of Si Ha cells affected with Lactobacillus crimpus,compared with the blank control group,the cytokines IL-2,IL-12 and IFN-γ in the supernatant were significantly increased after affected 6 hours(P < 0.001,P= 0.001,P = 0.013),and the cytokines IL-2,IL-4,IL-12 and IFN-γ were significantly increased after affected 12 hours(P < 0.001,P = 0.001,P = 0.008,P = 0.016),the cytokines IL-4,IL-12 and IFN-γ were significantly increased after 18 hours(P < 0.001,P = 0.016,P = 0.006),and IL-4,IL-10,IL-12 and IFN-γ were significantly increased after 24 hours(P < 0.001,P < 0.001,P =0.045,P = 0.033),while IL-2 was significantly decreased(P = 0.005).In the experiment of Ect1/E6E7 cells affected with Lactobacillus crimpus,compared with the blank control group,the levels of IL-2 and IFN-γ in the supernatant were significantly increased(P = 0.027,P = 0.033)but the levels of IL-4 and IL-10 were significantly decreased(P < 0.001,P = 0.028)after affected 6 hours.The levels of IL-4 and IL-10 were significantly decreased(P < 0.001,P = 0.028)after affected 12 hours.After affected 18 h,the level of IL-4 was significantly decreased(P = 0.002)and IFN-γ was significantly increased(P = 0.033).After affected 24 h,the levels of IL-12 and IFN-γ were significantly increased(P =0.036,P < 0.001)and IL-4 was significantly decreased(P < 0.001).6.The results of PCR showed that PCNA,ATM,LIG1 and HMGB1 in Si Ha cells were significantly higher expressed than in Ect1/E6E7 cells(P = 0.026,P = 0.002,P= 0.005,P = 0.028).While the m RNA of TDG and OGG1 in Si Ha cells was significantly lower than that in Ect1/E6E7 cells(P = 0.004,P = 0.006).In the experiment of Si Ha cells affected with Lactobacillus crimpus,compared with the blank control group,the m RNA levels of PCNA,ATM,LIG1 and HMGB1 in Si Ha cells treated with Lactobacillus crimpus decreased significantly after 12h(P = 0.010,P = 0.036,P = 0.024),and the m RNA levels of PCNA,ATM,LIG1,OGG1 and HMGB1 in Si Ha cells treated with Lactobacillus crimpus decreased significantly after 18 h(P = 0.018,P = 0.016,P = 0.002,P = 0.025,P= 0.015).After co-cultured 24 h,the m RNA levels of PCNA,ATM,LIG1,OGG1 and HMGB1 in Si Ha cells treated with Lactobacillus crimpus decreased significantly(P = 0.009,P = 0.007,P = 0.010,P = 0.011,P = 0.012),while the m RNA levels of ABCG2 and TDG in Si Ha cells treated 24 h were not significantly changed by Lactobacillus.In the experiment of Ect1/E6E7 cells affected with Lactobacillus crimpus,compared with the blank control group,TDG m RNA was significantly inhibited at 18 h and 24 h(P = 0.011,P = 0.004),while the expression of ABCG2,PCNA,ATM,LIG1,OGG1 and HMGB1 were not significantly changed.The results of Western-blot showed that the expressions of PCNA,ATM,LIG1 and HMGB1 in Si Ha cells were significantly higher than those in Ect1/E6E7 cells(P = 0.001,P = 0.007,P < 0.001,P =0.001).The expressions of TDG and OGG1 in Si Ha cells were significantly lower than those in Ect1/E6E7 cells(P < 0.001,P = 0.030).In the experiment of Si Ha cells affected with Lactobacillus crimpus,compared with the blank control group,the protein levels of PCNA,OGG1 and HMGB1 in Si Ha cells treated with Lactobacillus crimpus were significantly decreased after 12 h(P = 0.017,P= 0.010,P = 0.017),and the protein levels of ABCG2,PCNA,ATM,LIG1,OGG1 and HMGB1 were significantly decreased after 18 h(P = 0.026,P =0.011,P = 0.035,P = 0.043,P < 0.001,P = 0.029).The protein levels of PCNA,ATM,LIG1,OGG1 and HMGB1 in Si Ha cells were significantly decreased(P< 0.001,P = 0.008,P = 0.034,P = 0.006,P = 0.003,P = 0.001),while the expression of TDG in Si Ha cells treated with Lactobacillus did not change significantly.In the experiment of Ect1/E6E7 cells affected with Lactobacillus crimpus,compared with the blank control group,ABCG2 protein decreased significantly after 6 h(P = 0.021),ABCG2 and LIG1 protein decreased significantly after 12 h(P = 0.002,P = 0.033),and ABCG2 and LIG1 protein decreased significantly after 18 h(P = 0.003,P = 0.002),while TDG protein increased significantly(P = 0.007).After 24 h of treatment,ABCG2,PCNA,ATM,LIG1,OGG1 and HMGB1 protein levels were significantly decreased(P< 0.001,P = 0.049,P = 0.014,P < 0.001,P = 0.017,P = 0.046),while TDG protein levels were significantly increased(P < 0.001).Conclusion: 1.When the MOI is 200:1,lactobacillus crimpus has the best effect in co-culture with Si Ha cells and Ect1/E6E7 cells,which can be used as the MOI of the co-culture system model in this study.2.Lactobacillus crimpus can significantly inhibit the proliferation of Si Ha cells and Ect1/E6E7 cells,but the inhibition effect on Ect1/E6E7 cells is not as good as that of Si Ha cells.3.Lactobacillus crimpus can significantly promote the apoptosis of Si Ha cells and Ect1/E6E7 cells,but the apoptosis promoting effect of Lactobacillus crimp on Ect1/E6E7 cells is not as good as that of Si Ha cells.4.Lactobacillus crimpus can significantly inhibit the migration and invasion ability of Si Ha cells,but had no significant effect on the migration and invasion of Ect1/E6E7 cells,and the migration and invasion ability of Ect1/E67 cells was significantly lower than that of Si Ha cells.5.Lactobacillus crimpus can significantly promote Si Ha cells and Ect1/E6E7 cells to secrete Th1 cytokines IL-2,IL-12 and IFN – γ,promote Si Ha cells but inhibit Ect1/E6E7 cells to secrete Th2 cytokines IL-4 and IL-10.6.The differentially expressed genes PCNA,ATM,LIG1 and HMGB1 in Ect1/E6E7 cells were significantly lower than those in Si Ha cells,while TDG and OGG1 in Ect1/E6E7 cells were significantly higher than those in Si Ha cells.7.Lactobacillus crimpus could significantly inhibit the expression of ABCG2,PCNA,ATM,LIG1,OGG1 and HMGB1 in Si Ha cells,but had no significant effect on the expression of TDG.9.Lactobacillus crimpus can significantly inhibit the expression of ABCG2,PCNA,ATM,LIG1,OGG1 and HMGB1 at protein level in Ect1/E6E7 cells,and promote the expression of TDG protein. |
PDF Full Text Request