蛋白激酶C在肾上腺素抑制人正常皮肤及增生性瘢痕成纤维细胞增殖中作用的实验研究

An experimental study on the role of protein kinase C in the down-regulation of fibroblast proliferation in normal skin and hyperplastic scar by adrenaline

目的观察蛋白激酶C(PKC)在肾上腺素诱导人正常皮肤成纤维细胞(NFb)和增生性瘢痕成纤维细胞(SFb)增殖抑制中的作用。方法体外培养人NFb和SFb,均加入酚妥拉明(终浓度为0×10-6、3×10-6μmol/L)培养1h,再加入肾上腺素(终浓度0.00、0.05、0.10、0.20μmol/L)培养24h.用噻唑蓝(MTT)法检测NFb和SFb的增殖活力,计算细胞存活率。收集细胞培养上清液,检测乳酸脱氢酶(LDH)活性。用蛋白印迹(Western blot)法检测两种细胞磷酸化PKC的表达水平并进行半定量分析。结果(1)与未刺激时(酚妥拉明0×10-6μmol/L+肾上腺素0.00μmol/L)比较,单独用0.05、0.10、0.20μmol/L肾上腺素刺激以及3×10-6μmol/L酚妥拉明与0.20μmol/L肾上腺素联用时,NFb、SFb增殖活力及存活率均明显下降(P<0.05或0.01)。(2)与未刺激时比较,单独应用0·20μmol/L肾上腺素或3×10-6μmol/L酚妥拉明与各种剂量肾上腺素联用后,SFb、NFb培养上清液中LDH活性差异均无统计学意义(P>0.05).(3)单独应用0.05、0.10、0.20μmol/L肾上腺素时,NFb、SFb磷酸化PKC表达水平均明显高于未刺激时(P<0.01).单用3×10-6μmol/L酚妥拉明时,NFb磷酸化PKC表达水平为123±5,高于未刺激时(80±5,P<0.01),而SFb磷酸化PKC表达水平与未刺激时接近(P>0.05).3×10-6μmol/L酚妥拉明分别与0.05、0.10、0·20μmol/L肾上腺素联用时,两种细胞的磷酸化PKC表达水平低于3种剂量肾上腺素单独作用时(P<0.01).结论肾上腺素通过与α受体结合激活PKC,从而抑制NFb和SFb增殖。

Objective To investigate the role of protein kinase C （PKC） in the down-regulation of fibroblast proliferation in normal skin （NFb） and hyperplastic scar （SFb） by adrenaline. Methods Human NFb and SFb cells were cuhured in vitro. Phentolamine （ in final concentrations of 0 and 3×10^-6μmol/L） was added to the culture medium. One hour later, adrenaline in different final concentrations （0.00, 0.05, 0.10, 0.20μmol/L） was added to the culture medium and incubated for 24 hours. The cellular proliferation activity and cell viability rate were determined with MTT. The cell culture supernatant was harvested for the determination of LDH activity to assess the toxicity of phentolamine and adrenaline. The phosph-PKC activity was determined with Western-blotting and was semiquantitatively analyzed. Results （ 1 ） After stimulation with adrenaline alone, or combined 0. 20μmol/L adrenaline with 3×10^-6μmol/L phentolamine, the cell viability of both NFb and SFb decreased significantly（ P 〈0.05 or 0.01 ）. （2） There was no difference in the LDH activity between the cells either stimulated by adrenaline in all concentrations or by combination of adrenaline and phentolamine （ P 〉 0. 05 ）. （ 3 ） The phosphorylation of PKC in NFb and SFb cells stimulated by 0.05, 0. 10, 0. 20μmol/L adrenaline was obviously higher than that before stimulation（ P 〈 0. 01 ）. When phentolamine in the concentration of 3×10^-6μmol/L was used alone for stimulation, the phosphoryla- tion of PKC in NFb cells （ 123±5） was alsoevidently higher than that before stimulation （80±5, P 〈0.01 ）. But there was no such effect on SFb cells （ P 〉 0. 05 ）. When adrenline in the concentration of O. 05,0.10 or 0. 20μmol/L was separately added together with phentolamine in the dose of 3×10^6μmol/L for the stimulation, the iJhosphorylation of PKC in NFb and SFb cells was evidently lower than that when 3 different concentrations of adrenaline was used alone for stimulation（ P 〈0.01 ）. Conclusion Adrenaline can inhibit the proliferation of NFh and SFb by activating PKC through binding ct adrenaline receptor.