Experimental Study On The Protective Effects Of Umbilical Cord Mesenchymal Stem Cells Combined With Simvastatin On RF/6A Cells Cultured In High Glucose

Objective: To observe the effects of simvastatin on the biological characteristics of RF/6A cells cultured in high glucose; to investigate the protective effects of h UCMSCs combined with simvastatin on RF/6A cells cultured in high glucose, the changes of TLR4 inflammatory signaling pathway and its mechanism.Method:(1) The RF/6A cells were divided into the following six groups: normal control group(NG group), high glucose group(HG group), simvastatin 0.1?mol/L group(SIM 0.1 group), simvastatin 1?mol/L group(SIM 1 group), simvastatin 5?mol/L group(SIM 5 group), simvastatin 10?mol/L group(SIM 10 group). When RF/6A cells grew to 60%~70%, it was switched to high glucose culture medium for 48 h, examined after adding different concentrations of simvastatin for 24 h. MTT is to detect the proliferation ability; adhesion assay test is to detect the adhesion ability; Transwell chambers are used to detect the migration ability; tube test is to detect the angiogenic ability.(2) The RF/6A cells were divided into the following five groups: normal control group(NG group), high glucose group(HG group), simvastatin 1?mol/L group(SIM group), h UCMSCs 1:1 RF/6A group(MSCs group), simvastatin 1?mol/L joint h UCMSCs 1:1 RF/6A group(SIM+MSCs group). MSCs group uses the Transwell chamber co-culture system in which h UCMSCs was added to the upper chamber, and RF/6A cells were added to the lower one. SIM+MSCs group co-culture system was added 1?mol/L simvastatin in culture medium. MTT is to detect the proliferation ability; adhesion assay test is to detect the adhesion ability; Transwell chambers are used to detect the migration ability; tube assay is to detect the angiogenic ability; immunofluorescence staining is used to detect the expression of TLR4?NF-?B?TNF-??IL-1? in RF/6A cells; RT-PCR is to detect the m RNA level of TLR4?NF-?B?TNF-??IL-1? in RF/6A cells.Results:(1) The ability of proliferation, adhesion, migration and tube formation of RF/6A cells of HG group was lower than NG group, and the differences were statistically significant(P<0.05). The proliferation ability of SIM 0.1 group, SIM 1 group and SIM 5 group(0.32±0.02?0.36±0.01?0.27±0.02) was better than HG group(0.15±0.01)(P<0.05), but the proliferation ability of SIM 10 group(0.14±0.03) was lower than HG group,the differences were no statistically significant(P>0.05); the percentage area occupied by the adherent cells of SIM 0.1 group, SIM 1 group and SIM 5 group [(34.08±1.89)%,(46.23±1.02)%,(26.05±2.12)%/200 magnification] was higher than HG group [(16.17 ± 2.84)%/200 magnification](P<0.05), but SIM 10 group [(10.46±1.27)%/200 magnification] was lower than HG group(P>0.05); the percentage area occupied by the migrating cells of SIM 0.1 group, SIM 1 group, SIM 5 group and SIM 10 group [(23.28±2.38)%,(33.15±2.91)%,(17.06±1.03)%,(13.49±0.57)%/40 magnification] was higher than HG group [(10.26±2.05)%/40 magnification]( P<0.05); the bureaucratic forming ability of SIM 0.1 group, SIM 1 group, SIM 5 group and SIM 10 group [(15.40±1.51),(19.80±0.84),(11.20±1.30),(8.00±1.00) cells/40 magnification] was higher than HG group[(5.40±1.14) cells/40 magnification](P<0.05).(2) The proliferation, adhesion, migration and tube formation ability of SIM group, MSCs group, and SIM+MSCs group were better than HG group, the differences were statistically significant(P<0.05). The immunofluorescence staining results showed that the expression of TLR4, NF-?B, TNF-?, IL-1? of HG group was higher than NG group; the expression of TLR4, NF-?B, TNF-?, IL-1? of SIM+MSCs group was lower than HG group. RT-PCR results showed that the m RNA level of TLR4, NF-?B, TNF-?, IL-1? increased in HG group compared with NG group(P<0.05); the m RNA level of TLR4, NF-?B, TNF-?, IL-1? decreased in SIM+MSCs group compared with HG group(P<0.05); compared with HG group, the m RNA level of NF-?B, TNF-?, IL-1? in SIM group and TLR4 in MSCs group was no obvious differences(P>0.05).Conclusion:(1) The biological characteristics of RF/6A cells cultured in high glucose decreased than the normal cells; the biological function of RF/6A cells cultured in simvastatin 1?mol/L is improved significantly.(2) HUCMSCs and simvastatin can inhibit the expression of TLR4 pathway inflammatory factors, improve the repairing effect of RF/6A cells, and provide the theoretical basis for the clinical application of h UCMSCs on DR therapy.