| Background:The main causes of pathological scar and other fibrosis skin diseases are fibroblast proliferate or metabolic imbalance of collagen. Telomerase,whose key rate-limiting enzyme is TERT,can prolong a shortened telomeres to enhance cell proliferation,whlie telomeres play an important role to keep the stability and cell activity. There were studies which proved elevated expression of fiber cells’ telomerase activity in keloid [3]. In recent years, the reports that thalidomide were used in the treatment of fibrosis skin diseases and other organs’ fibrosis diseases are becoming more and more, it has been confirmed at home and aborad that thalidomide has inhibition against fibrosis skin diseases and other organs’ fibrosis diseases[4]. In the research about thalidomide and telomerase, results have showed that thalidomide can induce multiple myeloma U266 cells apoptosis to suppress cell proliferation by degreeing the values of h TERT ’s m RNA transcription[6]. Being one of stimulation factors for skin fibrosis disease, histamine can promote the proliferation of fibroblasts and the synthesis of collagen [7-8], and is closed to the formation of pathological scar[9].Currently, it hasn’t been reported that thalidomide affected normal dermal fibroblast proliferation and collagen expression directly, and it also hasn’t been reported those on histamine-induced fibroblasts,as well as telomerase.Therefore, our studies explore the effects of thalidomide on histamine-induced cell by testing the changes, including proliferation, telomerase activation and COL-I,IIIm RNA expression. All of those offered the new ideas for researching the potential role of thalidomide against dermal fibrosis, preventing postoperative scarring and the clinical treatment of pathological scar,as well as it’s prognosis.Objective: To explore the effects of thalidomide on histamine-induced fibroblasts, including cell proliferation, the expressions on COL-I,III-m RNA and telomerase m RNAMethods:1 Human dermal fibroblasts were cultured primarily. Cells were identified with detecting the vimentin expression by the dye of immunohistochemistry. Selecting the fourth passages of human dermal fibroblasts for experiments.2 CCK-8 test for normal human dermal fibroblasts proliferationFibroblasts cells were plated at a same density in 96-well plates when they had been digested, and each groups were provided 5 sample sizes. The cells were treated with different concentrations of thalidomide for 24h(0, 10-3,10-4, 10-5 mol/L),and the viability at wave length of 560 nm was analyzed with CCK-8 assay.The data were analyzed by statistical analysis.3 Experimental group and CCK-8 test for histamine-induced human dermal fibroblasts proliferationExperiment was consisted of four groups:control group(K), thalidomide-treated group(S), histamine-treated group(Z), histamine and thalidomide treatde group(Z+S).Fibroblasts cells were plated in 96-well plates at a same density, which concentration is 2×104/ml and each hole was filled whith 200 ul. Group Z+S and Z were treated with 200ul-100 UM histamine while group K and S were treated with only DMEM.After 48 hours culture, the four groups’ nutrient medium were gave up.Group S and Z+S were treated with 10-5 mol/L thalidomide while the other groups were added only DMEM, and each groups were provided 5 sample sizes for another 24 hours culture.Then cell viability was measured using the CCK-8 assay. The data were analyzed by statistical analysis.4 The expressions of COL-I and COL-III m RNA were 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, then COL-I and COL-III m RNA were tested with realtime fluorescence quantitative PCR. Data were analyzed with statistical analysis.5 The expression of h TERTm RNA was tested by realtime fluorescence quantitative PCR(SYBR Green I)Following with the same grouping method to got group Z and Z+S. The expression of h TERTm RNA were tested following with step 4. Data were analyzed with statistical analysis.Results:1 The results of CCK-81.1 The results of CCK-8 in normal human dermal fibroblasts.After beening effected by Concentration 0,10-3,10-4,10-5mol/L of thalidomide, the normal human dermal fibroblasts’ OD were 1.1618±0.0783, 0.898±0.0461, 1.0761±0.0582,1.147±0.050.Four groups’ OD were not all equal(P<0.05), Within the groups, 10-3,10-4mol/L groups showed that cells could be inhibited(P<0.05), however, no significant difference between 10-5mol/L group and the control group was found(P>0.05).1.2 The results of CCK-8 in histamine-induced human dermal fibroblasts proliferation. The OD datas of group K, S, Z, Z+S were 1.1618± 0.0783,1.147±0.050, 1.4182±0.0226, 1.186±0.0515. The four groups’ OD were not all equal(P=0.000). Within the groups, group Z’s OD was higher than group K’s, there were statistically difference(P<0.05), group Z+S’s OD was lower than group Z’s, there were statistically difference(P<0.05). The results suggested that cells not only could be inhibited by 10-3,10-4mol/L thalidomide but also could be activated by 100 UM histamine.Meanwhile, cells could not be inhibited by 10-5mol/L thalidomide, but they could be inhibited after activation by histamine.2 The expressions on Co L-I, Co L-IIIm RNA with effection of thlidomide in histamine-induced human dermal fibroblastsThe Co L-Im RNA RQ datas of group K, S, Z, Z+S were 1.0157± 0.0025, 0.6577±0.0317, 1.7693±0.0358, 0.2557±0.0235. The four groups’ RQ were not all equal(P=0.000). Within the groups, group S’s RQ was lower than group K’s and there were statistically difference(P<0.05). Group Z’s RQ was higher than group K’s and there were statistically difference(P<0.05). Group Z+S’s OD was lower than group Z’s and there were statistically difference(P<0.05). The results suggested that the expression levels of Co L-Im RNA could be activated by 100 UM histamine, as while as they could be inhibited by 10-5mol/L thalidomide, furthermore, they could be inhibited significantly after activation by histamine.The Co L-IIIm RNA RQ data of group K, S, Z, Z+S were 0.988± 0.012, 0.826±0.018, 1.531±0.047, 0.746±0.027. The data of group Z and group K, group S and group K, group Z and group Z+S, which were study need, were statistically analyzed. Results showed that group S’s RQ was lower than group K’s and there were statistically difference(P=0.000). Group Z’s RQ was higher than group K’s and there were statistically difference(P=0.001). Group Z+S’s OD was lower than group Z’s and there were statistically difference(P=0.000). The results suggested that the expression of Co L- IIIm RNA could be activated by 100 UM histamine, as while as they could be inhibited by 10-5mol/L thalidomide. Furthermore, they could be inhibited significantly after activation by histamine.3 The expression of h TERTm RNA with effection of thlidomide in histamine-induced human dermal fibroblastsThe h TERTm RNA RQ data of group Z, Z+S were 1.850±0.054, 0.984 ±0.043. Group Z+S’s RQ was lower than group Z’s and there were statistically difference(P=0.000). It suggested that the expression of h TERTm RNA could be inhibited significantly after activation by histamine.Conclusions:1 Proliferation of fibroblasts and the m RNAexpressions of Co L-I, Co L-III were enhanced by histamine in Concentration of 100 um.2 The m RNAexpressions of Co L-I, Co L-III, h TERT were inhibited by 10-5 mol/L-thlidomide obviously on human dermal fibroblasts enhanced by 100um-histamine.3 The study provided evidences for treatment possibility of thalidomide on Pathological scar and the further study on it. |
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