The Effects Of Thalidomide On TGF-?1 MRNA?MMP-1mRNA?TIMP-1mRNA In Normal And Histamine-induced Human Dermal Fibroblasts

Backgroud:Fibroblast is one of the most important cells in skin dermis, attached to the collagen fibers and able to synthesize and secrete large amounts of collagen, which plays a significant role in maintaining skin elasticity and toughness. Many kinds of diseases have obvious relation to metabolic disorder of collagen. Studies have shown that systemic scleroderma and pathological scar are caused by increased proliferative activity of fibroblast and excess deposition of collagen-based extracellular matrix. Transforming growth factor-?1?TGF-?1? can promote the proliferation of fibroblasts, stimulate collagen synthesis and collagenase production. TGF-?1 is considered to be the most important cytokine in scar formation,which is able to strongly promote the deposition of extracellular matrix. In normal circumstances, the synthesis and degradation of extracellular matrix can keep dynamic equilibrium, in order to maintain the structure and function of the tissues. Matrix metalloproteinases?MMPs? and its inhibitor of matrix metalloproteinase?TIMPs? play crucial roles in this balance. MMP-1 is a vital member of MMPs and it is the key enzyme in the course of collagen degradation which plays a key role in starting collagen degradation in wound repair. TIMP-1 is the most important inhibitory factor of MMP-1 which can form a complex with MMP-1 in a 1:1 ratio to inhibit activity of the enzyme. As thalidomide has achieved good results on treatment of diseases such as leprosy and erythema nodosum, the researchers have found that thalidomide has the effects of anti-inflammatory, antineoplastic and immunomodulatory. It has been currently proved that THD can fight the process of pulmonary fibrosis by inhibiting TGF-? and TNF-? factors. A large number of experiments have proved that histamine secreted by mast cells can stimulate fibroblast proliferation and collagen synthesis, so fibrosis model of pathological cell in this experiment is induced with histamine as the stimulating factor of fibroblast. This topic has studied the effects of THD on TGF-?1, MMP-1 and TIMP-1 secreted by histamine-induced human fibroblasts by culturing human fibroblasts in vitro.Objective:To explore the effects of thalidomide on expressions of TGF-?1 mRNA?MMP-1mRNA and TIMP-1mRNA in normal and histamine-induced human dermal fibroblasts.Methods:1 Human dermal fibroblasts were cultured primarily in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum using tissue-digestion mixing method.2 Cells were identified with detecting the vimentin expression by immunohistochemical staining.3 Experimental groupExperiment was consisted of four groups:control group?normal fibroblasts?, thalidomide-treated group, histamine-treated group, histamine and thalidomide treatde group.4 There was no effect on cell proliferation by 10^-5mol/L thalidomide at previous experiment test. The concentrations of histamine was 100 UM in this study after consulting documents.5 The expressions of TGF-?1 mRNA was tested by realtime fluorescence quantitative PCR?SYBR Green I?.Following with the same grouping method above, 3 samples were provided by each groups. All samples were cultivated in 25 ml culture flasks respectively. The total RNA was extracted from cells after 24 hours, then TGF-?1 mRNA were tested with realtime fluorescence quantitative PCR. Data were analyzed with statistical analysis.6 The expressions of MMP-1 mRNA and TIMP-1 mRNA were tested by realtime fluorescence quantitative PCR?SYBR Green I?.The expressions of MMP-1 mRNA and TIMP-1 mRNA were tested following with step 5. Datum were analyzed with statistical analysis.Results:1 The effect of thalidomide on the expression of TGF-?1 mRNA of histamine-induced human dermal fibroblasts.The TGF-?1 mRNA RQ datas of the four group were 0.9837�0.0125?0.849�0.0276?2.5653�0.0935?0.648�0.0321. The RQ datas of the four groups were not all equal?P<0.05?.Comparison of the two two groups were statistically significant?P<0.05?. The results suggested that the expression of TGF-?1 mRNA could be increased by 100 UM histamine. But they could be inhibited by thalidomide, even significantly after activating by histamine.2 The effect of thalidomide on the expression of MMP-1 mRNA of histamine-induced human dermal fibroblasts.The MMP-1 mRNA RQ datas of the four group were 0.9973�0.0074?1.2773�0.0351?1.071�0.0137?1.7523�0.0334. The RQ datas of the four groups were not all equal?P<0.05?.Comparison of the two two groups were statistically significant?P<0.05?. The results suggested that histamine and thalidomide could promote the expressions of MMP-1 m RNA.And the effect of thalidomide on MMP-1 mRNA was more significant after activating by histamine.3 The effect of thalidomide on the expression of TIMP-1 mRNA of histamine-induced human dermal fibroblasts.The TIMP-1 mRNA RQ datas of the four group were 0.997�0.007?0.1687�0.0015?0.9563�0.0312?0.1173�0.005. The variance of four groups were irregular. There was no significant difference between control group and histamine group. Compared with the control group, the expressions of TIMP-1 mRNA of thalidomide group was decreased?P<0.05?. The expressions of TIMP-1 mRNA of histamine and thalidomide group was decreased compared with histamine group?P<0.05?. The expressions of TIMP-1 mRNA of histamine and thalidomide group was decreased compared with thalidomide group?P<0.05?.The results suggested that the expression of TIMP-1 mRNA could be inhibited by 10^-5mol/L thalidomide. Furthermore, they could be inhibited more significantly after activating by histamine.Conclusions:1 The expression of TGF-?1 mRNA could be increased by histamine in concentration of 100 UM.2 10^-5mol/L thalidomide could promote the expressions of MMP-1 mRNA of normal fibroblasts. And the effect of thalidomide on MMP-1 mRNA was more significant after activating by 100 UM histamine.3 The expressions of TGF-?1 mRNA and TIMP-1mRNA of normal fibroblasts could be inhibited by 10^-5mol/L thalidomide. And the effects of thalidomide on TGF-?1 mRNA and TIMP-1mRNA were more significant after activating by histamine.