人瘢痕疙瘩成纤维细胞相关基因表达及青蒿琥酯的作用
인반흔흘탑성섬유세포상관기인표체급청호호지적작용
Expressions of the related genes in fibroblasts of human keloid and the effect of artesunate
  • 万方数据
    中华烧伤杂志 중화소상잡지
    16
    2013年 2期 185-190 ,共6页

    肖亮%贺光照%余泽浩%唐小清%任玉涵

    초량%하광조%여택호%당소청%임옥함

    瘢痕疙瘩%成纤维细胞%青蒿素类%钙/钙调蛋白依赖性丝氨酸蛋白激酶%分化抑制因子1 瘢痕疙瘩%成纖維細胞%青蒿素類%鈣/鈣調蛋白依賴性絲氨痠蛋白激酶%分化抑製因子1
    반흔흘탑%성섬유세포%청호소류%개/개조단백의뢰성사안산단백격매%분화억제인자1
    Keloid%Fibroblasts%Artemisinins%Calcium/calmodulin dependent serine protein kinase%Inhibitors of differentiation 1
    目的 验证瘢痕疙瘩Fb中是否存在钙/钙调蛋白依赖性丝氨酸蛋白激酶(CASK)和分化抑制因子1(ID1)的异常表达,观察青蒿琥酯对二者的影响. 方法 收集笔者单位患者手术后废弃的瘢痕疙瘩样本15个和正常皮肤组织样本12个,采用组织微粒贴壁法行Fb原代培养,取第3～8代细胞用于实验.免疫荧光染色观察2种Fb中CASK和ID1的表达,瘢痕疙瘩Fb用不同浓度青蒿琥酯作用不同时间,通过噻唑蓝比色法确定药物的半数抑制浓度(IC50)并作为后续实验药物干预浓度.选取正常皮肤Fb设为正常对照组(添加培养液处理),收集瘢痕疙瘩Fb分为瘢痕对照组(添加培养液处理)及瘢痕给药组(添加含IC50青蒿琥酯的培养液处理),流式细胞术检测各组Fb细胞周期和凋亡的变化,RT-PCR、蛋白质印迹法检测各组Fb中CASK和ID1的基因与蛋白表达情况.对数据行单因素方差分析及LSD-t检验. 结果 瘢痕疙瘩和正常皮肤Fb中均存在CASK和ID1表达,选择75 mg/L青蒿琥酯为后续实验干预浓度.(1)G0/G1期和G2/M期的细胞百分比3组之间比较,差异有统计学意义(F值分别为118.064、163.840,P值均小于0.01).其中瘢痕给药组G0/G1期为(91.4±1.4)％,明显高于瘢痕对照组的(80.7±0.3)％和正常对照组的(82.4±0.6)％(t值分别为12.740、9.872,P值均小于0.05)；G2/M期为(6.9±0.3)％,明显低于瘢痕对照组的(13.7±0.3)％和正常对照组的(12.7±0.8)％(t值分别为43.702、12.276,P值均小于0.05).(2)早晚期细胞凋亡率3组之间比较,差异均有统计学意义(F值分别为61.879、4710.862,P值均小于0.01).其中瘢痕给药组早期凋亡率为(7.1±1.0)％,明显高于瘢痕对照组的(2.6±0.4)％和正常对照组的(2.7±0.3)％(t值分别为7.974、7.767,P值均小于0.05)；晚期凋亡率为(14.9±0.3)％,明显高于瘢痕对照组的(2.3±0.3)％和正常对照组的(2.5±0.4)％(t值分别为72.882、69.792,P值均小于0.05).(3)瘢痕对照组CASK基因表达量为0.658±0.024,低于正常对照组的1.076±0.008(t =28.997,P＜0.01)；瘢痕给药组的基因表达量为0.855±0.008,较瘢痕对照组上升(t=13.549,P＜0.01).瘢痕对照组CASK蛋白表达量为0.067±0.007,低于正常对照组的0.179±0.015(t=12.042,P＜0.01)；瘢痕给药组为0.132±0.010,较瘢痕对照组上升(t=9.498,P＜0.01).(4)瘢痕对照组ID1基因表达量为0.416±0.006,高于正常对照组的0.317±0.020(t=8.299,P＜0.01)；瘢痕给药组为0.217±0.009,较瘢痕对照组下降(t=32.417,P＜0.01).瘢痕对照组ID1蛋白的表达量为0.789±0.034,高于正常对照组的0.366±0.029(t=16.341,P＜0.01)；瘢痕给药组为0.114±0.006,较瘢痕对照组下降(t=33.978,P＜0.01). 结论 推测CASK和ID1参与了瘢痕疙瘩Fb的增殖过程,青蒿琥酯抑制瘢痕疙瘩Fb增殖的机制可能与上调CASK和下调ID1表达有关.
    목적 험증반흔흘탑Fb중시부존재개/개조단백의뢰성사안산단백격매(CASK)화분화억제인자1(ID1)적이상표체,관찰청호호지대이자적영향. 방법 수집필자단위환자수술후폐기적반흔흘탑양본15개화정상피부조직양본12개,채용조직미립첩벽법행Fb원대배양,취제3～8대세포용우실험.면역형광염색관찰2충Fb중CASK화ID1적표체,반흔흘탑Fb용불동농도청호호지작용불동시간,통과새서람비색법학정약물적반수억제농도(IC50)병작위후속실험약물간예농도.선취정상피부Fb설위정상대조조(첨가배양액처리),수집반흔흘탑Fb분위반흔대조조(첨가배양액처리)급반흔급약조(첨가함IC50청호호지적배양액처리),류식세포술검측각조Fb세포주기화조망적변화,RT-PCR、단백질인적법검측각조Fb중CASK화ID1적기인여단백표체정황.대수거행단인소방차분석급LSD-t검험. 결과 반흔흘탑화정상피부Fb중균존재CASK화ID1표체,선택75 mg/L청호호지위후속실험간예농도.(1)G0/G1기화G2/M기적세포백분비3조지간비교,차이유통계학의의(F치분별위118.064、163.840,P치균소우0.01).기중반흔급약조G0/G1기위(91.4±1.4)％,명현고우반흔대조조적(80.7±0.3)％화정상대조조적(82.4±0.6)％(t치분별위12.740、9.872,P치균소우0.05)；G2/M기위(6.9±0.3)％,명현저우반흔대조조적(13.7±0.3)％화정상대조조적(12.7±0.8)％(t치분별위43.702、12.276,P치균소우0.05).(2)조만기세포조망솔3조지간비교,차이균유통계학의의(F치분별위61.879、4710.862,P치균소우0.01).기중반흔급약조조기조망솔위(7.1±1.0)％,명현고우반흔대조조적(2.6±0.4)％화정상대조조적(2.7±0.3)％(t치분별위7.974、7.767,P치균소우0.05)；만기조망솔위(14.9±0.3)％,명현고우반흔대조조적(2.3±0.3)％화정상대조조적(2.5±0.4)％(t치분별위72.882、69.792,P치균소우0.05).(3)반흔대조조CASK기인표체량위0.658±0.024,저우정상대조조적1.076±0.008(t =28.997,P＜0.01)；반흔급약조적기인표체량위0.855±0.008,교반흔대조조상승(t=13.549,P＜0.01).반흔대조조CASK단백표체량위0.067±0.007,저우정상대조조적0.179±0.015(t=12.042,P＜0.01)；반흔급약조위0.132±0.010,교반흔대조조상승(t=9.498,P＜0.01).(4)반흔대조조ID1기인표체량위0.416±0.006,고우정상대조조적0.317±0.020(t=8.299,P＜0.01)；반흔급약조위0.217±0.009,교반흔대조조하강(t=32.417,P＜0.01).반흔대조조ID1단백적표체량위0.789±0.034,고우정상대조조적0.366±0.029(t=16.341,P＜0.01)；반흔급약조위0.114±0.006,교반흔대조조하강(t=33.978,P＜0.01). 결론 추측CASK화ID1삼여료반흔흘탑Fb적증식과정,청호호지억제반흔흘탑Fb증식적궤제가능여상조CASK화하조ID1표체유관.
    Objective To verify whether abnormal expression of calcium/calmodulin dependent serine protein kinase (CASK) and inhibitors of differentiation 1 (ID1) exist in Fb of keloid,and to observe the effect of artesunate on two genes.Methods Fifteen samples of keloid and 12 samples of normal skin tissue (discarded) excised from patients admitted to our hospital were collected.Tissue particle adherent method was used in the primary culture of Fb,and cells from the third to the eighth passage were used for test.Expressions of CASK and ID1 in Fb harvested from both sources were observed with immunofluorescence staining.Fb of keloid were stimulated with artesunate in various concentration for different time,and the median inhibitory concentration (IC50) was determined with the MTT colorimetric assay,which served as the intervention concentration of artesunate.Fb of normal skin were set as normal control group (NC,treated with medium solution).Fb of keloid were divided into scar control group (SC,treated with medium solution)and scar administration group (SA,treated with artesunate in IC50).The cycle and apoptosis of Fb were detected with flow cytometric assay,and the nucleic acid and protein expressions of CASK and ID1 of Fb in each group were determined with RT-PCR and Western blotting.Data were processed with one-way analysis of variance and LSD-t test.Results Expressions of CASK and ID1 were detected in two kinds of Fb.The concentration of 75 mg/L was selected as the intervention concentration of artesunate.(1) There were statistically significant differences among the three groups in the percentages of cells in G0/G1 phase and G2/M phase (with F values respectively 118.064 and 163.840,P values all below 0.01).The percentage of cells in G0/G1 phase of group SA was (91.4 ± 1.4) ％,which was significantly higher than that of group SC and group NC [respectively (80.7 ± 0.3) ％ and (82.4 ± 0.6) ％,with t values respectively 12.740 and 9.872,P values all below 0.05].The percentage of cells in G2/M phase of group SA was (6.9 ± 0.3) ％,which was significantly lower than that of group SC and group NC [respectively (13.7 ± 0.3) ％ and (12.7 ± 0.8) ％,with t values respectively 43.702 and 12.276,P values all below 0.05].(2) There were statistically significant differences among the three groups in the early and late apoptotic rates (with F values respectively 61.879 and 4710.862,P values all below 0.01).The early and late apoptotic rates of group SA were respectively (7.1 ± 1.0) ％ and (14.9 ± 0.3) ％,which were significantly higher than those of group SC and group NC [with early apoptotic rate respectively (2.6 ± 0.4) ％ and (2.7 ± 0.3) ％,t values respectively 7.974 and 7.767,P values all below 0.05 ; with late apoptotic rate respectively (2.3 ± 0.3) ％and (2.5 ± 0.4) ％,t values respectively 72.882 and 69.792,P values all below 0.05].(3) The mRNA expression of CASK in group SC was 0.658 ± 0.024,and it was lower than that of group NC (1.076 ±0.008,t =28.997,P ＜0.01) and group SA (0.855 ±0.008,t =13.549,P ＜0.01).The protein expression of CASK in group SC was 0.067 ± 0.007,and it was lower than that of group NC (0.179 ± 0.015,t =12.042,P ＜0.01) and groupSA (0.132±0.010,t =9.498,P ＜0.01).(4) The mRNA expression ofID1 in group SC was 0.416 ±0.006,which was higher than that of group NC (0.317 ±0.020,t =8.299,P ＜0.01) and groupSA (0.217±0.009,t =32.417,P ＜0.01).The protein expression of ID1 in groupSC was0.789±0.034,and it was higher than that of group NC (0.366 ±0.029,t =16.341,P ＜0.01) and groupSA (0.114±0.006,t =33.978,P ＜0.01).Conclusions It is speculated that CASK and ID1 participate in the proliferation of Fb in keloid.The mechanism of artesunate in inhibiting the proliferation of Fb in keloid may be related to the up-regulation of CASK and down-regulation of ID1.
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