Effect Of TRPM7 Channel On Proliferation And Migration Of Human Buccal Fibroblasts

Objective: To investigate whether the transient receptor potential ion channel TRPM7(Transient receptor potential melastatin 7)is expressed in human buccal mucosal fibroblasts and the effect of inhibiting TRPM7 ion channels on the proliferation and migration of human buccal mucosal fibroblasts.Methods: The tissues used in this experiment were derived from the Oral and Maxillofacial Surgery Operating Room of the Affiliated Hospital of Southwest Medical University.Human buccal mucosal fibroblasts were cultured in vitro using the tissue block method.1.After isolation and purification to identify the fibroblasts by immunofluorescence staining;2.The expression of TRPM7 ion channels in human buccal mucosal fibroblasts was detected by reverse transcription polymerase chain reaction(RT-PCR)and immunofluorescence.The whole-cell patch-clamp technique records the whole-cell current of the human oral buccal mucosal fibroblasts TRPM7 ion channel;3.Set the cultured cells into 3 groups: control group,TRPM7 shRNA group and 2-APB group.The control group was normal cultured human buccal mucosal fibroblasts without special treatment.The TRPM7 shRNA group was transfected with human buccal mucosal fibroblasts using TRPM7 shRNA virus.After 48 h,the transfection effect was observed under an inverted fluorescence microscope.The expression of TRPM7 ion channel was again detected by RT-PCR to verify whether the TRPM7 ion channel was successfully inhibited.Whole-cell patch-clamp records the changes in whole-cell currents of human buccal mucosal fibroblasts in the TRPM7 shRNA group.In the 2-APB group,500 ?mol / L 2-aminoethylbiphenylborate(2-APB)was applied to human buccal mucosal fibroblasts to inhibit the expression of TRPM7 ion channels.After 48 h,recording was performed using whole-cell patch clamp.Whole-cell current changes of human buccal mucosal fibroblasts in the 2-APB group;4.After the TRPM7 shRNA group and the 2-APB group successfully inhibited the TRPM7 ion channel,they were cultured for 24 hours with the control group.MTT cell proliferation experiments and cell planning were used.The trace test was used to detect the proliferation and migration of the three groups of cells,and to compare the effects of inhibition of TRPM7 ion channels on the proliferation and migration of human buccal mucosal fibroblasts;5.Statistical methods: SPSS17 and GraphPad Prism7 The software was used for statistical analysis and histogram drawing.The difference was statistically significant with p <0.05.Results: 1.The human oral buccal mucosal fibroblast cells were successfully cultured by tissue block method and passed to the third generation.After isolation and purification,they were identified by immunofluorescence staining.The results were in line with the biological characteristics of fibroblasts,and the cultured cells were confirmed to be human buccal mucosal fibroblasts;2.Using RT-PCR and immunofluorescence to confirm that human buccal mucosal fibroblasts functionally express TRPM7 ion channels,and the whole-cell patch-clamp technique records typical TRPM7 ion channel whole-cell currents;3.TRPM7 shRNA was successfully transfected into human buccal mucosal fibroblasts after 48 h.The cells showed specific green fluorescence under an inverted fluorescence microscope.RT-PCR results showed that TRPM7 ion channels in TRPM7 shRNA group cells were not expressed,and whole-cell patch clamp The experimental results showed that the TRPM7 ion channel whole cell current of human buccal mucosal fibroblasts in the TRPM7 shRNA group was significantly weakened.After 500 ?mol / L 2-APB was applied to human buccal mucosal fibroblasts for 48 h,patch-clamp technique was used to record the whole-cell current in the 2-APB group.In the absence of Mg2 + in the electrode solution,a typical TRPM7 ion channel was recorded.The whole cell current was blocked.The above experimental results show that both the TRPM7 shRNA group and the 2-APB group can successfully inhibit the expression of TRPM7 ion channels in human buccal mucosal cells after 48 h of treatment;4.MTT cell proliferation experiments were used to test the proliferation of the three groups of cells.The results showed that compared with the control group,the cell proliferation rate of the 2-APB group decreased by about 25% and the cell proliferation rate of the TRPM7 shRNA group decreased by about 35%,The differences were statistically significant(p <0.05).Cell scratch test was used to detect the migration ability of the three groups of cells.The results showed that compared with the control group,the scratch healing rate of the 2-APB group decreased by about 67% and that of the TRPM7 shRNA group by about 53%.%,The differences were statistically significant(p <0.05).The above results indicate that inhibition of TRPM7 ion channels significantly inhibited the proliferation and migration of human buccal mucosal fibroblasts.Conclusion: Human buccal mucosal fibroblasts functionally express TRPM7 ion channels,and TRPM7 ion channels are involved in the regulation of human buccal mucosal fibroblast proliferation and migration capabilities.