The Study Of TG2 Expression And Role In Human Gingival Fibroblasts Induced By Nifedipine

Background and purposeDrug-induced gingival overgrowth(DGO) is a kind of fibrotic disease of gingiva which is caused by long-term administration of anticonvulsants, various calcium channel blockers and immunosuppressive agents. The main features of DGO are the number increase of fibroblasts and an accumulation of extracellular matrix. Due to the undefinity of the molecular mechanisms, there is still no effective medicine for DGO therapy.Recent years, transglutaminase 2, an Ca2+ dependent enzyme ubiquitously distributed in cells was considered to play a vital role in the fibrotic disease, Celiac disease, diabetes mellitus, and Alzheimer disease. Previous study has demonstrated that the expression of TG2 increased in the gingiva of rats stimulated by NIF. The present study uses the cultured human gingival fibroblasts in vitro to explore the function that TG2 may be involved in DGO.Methods1. Clinical healthy gingival tissue from 3 systematically healthy subjects and gingival fibroblasts were cultured in vitro by tissue explant and double-enzyme digestion method. Immunohistochemical identification was used to detect the gingival fibroblasts.2. MTT assay was used to acquire the growth curve of the human gingival fibroblasts in order to determine the optimal time. According to the growth curve,3rd passage of gingival fibroblasts was cultured by 0μg/ml、0.6μg/ml、0.8μg/ml、1.0μg/ml、 1.5μg/ml、2.0μg/ml NIF to determine the appropriate concentration of NIF.3. Flow cytometry assay was used to measure intracellular Ca2+ of gingival fibroblasts stimulated by 1.0 u g/ml NIF.4. Microplate method were used to quantify the TG2 activity of gingival fibroblasts stimulated by 1.0μg/ml NIF.5. Human gingival fibroblasts were divided into 6 groups and stimulated 24h by 1.0μg/mL NIF with MDC at different concentrations of 0μmol/L、50μmol/L、 100 μmol/L 150μmol/L、200μmol/L and 250μmol/L to determine the the appropriate concentration of MDC.6. According to above results, human gingival fibroblasts was divided into the following groups: Control group C:DMEM Group A:DMEM+1.0μg/ml NIF Group B:B1:DMEM+1.0 μg/mlNIF+50μmol/L MDCB2:DMEM+1.0μg/ml NIF+100μmol/L MDCB3:DMEM+1.0μg/ml NIF+150μmol/L MDC Human gingival fibroblasts were divided into 5 groups and cultured 24h with different stimulations, then to study the expression of TG2 and a-SMA by Western-Blot. Quantitative real-time PCR technology was used to measure the mRNA expression of TG2 and a-SMA.7.Statistical analysis All values are expressed as mean± standard error (SD). Statistical analysis was carried out by using Student’s t test for comparisons between two groups, or one-way ANOVA test of group comparison, withp values less than 0.05 considered statistically significant.Results1. Cell culture and identification Two days after the culture of gingival tissue, cells started to grow from the tissue. Observed under the microscope, cells were unregularly arranged, sometimes radically, with a spiral-like shape. At high magnification, cell nucleus and cores were round and clear. Immunohistochemistry showed that the fibroblasts were stained negatively for keratin but positively for vimentin.2. Cell growth and NIF administration Cell growth curve showed that the number of fibroblasts proliferated rapidly in the early 24h, and then grew into the plateau with a very low rate of number increase. So we choose 24h as the culture time tolerance for the following experiments. Under the concentration from 0.6-2.0 u g/ml of NIF owned the ability to promote proliferation. There was no significant difference in 0.8,1.0 and 1.2 μg/ml group with 1.0μg/ml arriving at a peak in cell number. Then 1.0 μg/ml NIF was used to stimulate the cultured cells to study the role of TG2 during the NIF administration.3. Detection of Ca2+ concentration in gingival fibroblasts Flow cytometry assay showed that the fluorescence intensity for the the control group was 6815+79.02. After stimulated by 1.0μg/ml NIF, the fluorescence intensity increased significantly to 11926±131.644. TG2 enzyme activity Biotinamidopentylamine (BAPA)was incorporated into the fibroblasts to detect the TG2 enzyme activity by microplate enzyme assay. The optical intensity in the control group and the 1.0ug/mL NIF group were 0.134±0.05 and 0.334±0.12. There was a significant increase of TG2 enzyme activity when the fibroblasts were administered by 1.0ug/mL NIF.5. Determine the the appropriate concentration of MDC MTT colorimetric assay showed MDC at concentration of 50,100,150 u mol/L inhibited the proliferation of fibroblasts when compared to the control group, but there was not significance among these three groups.When the concentration increased to 200μmol/L, the growth of the fibroblasts were greatly affected and number of cells decreased significantly. So we use MDC at concentration of 50,100,150μmol/L in the following experiments.6. TG2 and a-SMA determination The results of the western blot showed both TG2 and a-SMA In NIF group significantly increased when compared to the control group. After adding gradient concentration of MDC in the culture medium, the expression of TG2 and a-SMA protein decreased gradually when compared to the NIF group.7. Gene expression of TG2 and a-SMA RT-PCR demonstrated the gene expression of TG2 in NIF group increased significantly when compared to the control group, while it was inhibited significantly by gradient concentration of MDC. The gene expression of a-SMA in NIF group increased about 5 folds when compared to the control group. MDC at concentration of 50,100,150μmol/l could significantly decrease the expression of a-SMA gene when compared to the NIF group. However, there was no significant difference among these three groups.Conclusions1. Our results showed certain concentration of NIF can promote the proliferation of gingival fibroblasts, which suggests that gingival fibroblasts proliferation related to calcium antagonists may play an important role in the development of DGO.2. NIF can improve the concentration of intracellular Ca2+ in gingival fibroblasts, which implicates that it is the increased Ca2+induced by NIF that initiates a series of cell bioactivity in the development of DGO.3.The expression of Ca2+ dependent TG2 and its enzyme activity increase in the gingival fibroblasts after stimulated by NIF. And the increased TG2 mediates the transformation of fibroblasts into myofibroblasts. So TG2 plays an important role in the pathogenesis of DGO, suggesting that it could be used as a therapeutic target for DGO.