组蛋白脱乙酰酶6抑制剂Tubastatin A对人皮肤成纤维细胞增殖和运动的影响及其分子机制

张灿; 张琼; 张均辉; 王凡; 张家平

doi:10.3760/cma.j.cn501120-20200519-00274

组蛋白脱乙酰酶6抑制剂Tubastatin A对人皮肤成纤维细胞增殖和运动的影响及其分子机制

doi: 10.3760/cma.j.cn501120-20200519-00274

张灿¹,
张琼²,
张均辉³,
王凡¹,
张家平^1, ,

1.
陆军军医大学（第三军医大学）第一附属医院整形外科，重庆　　400038
2.
陆军军医大学（第三军医大学）第一附属医院全军烧伤研究所，创伤、烧伤与复合伤国家重点实验室，重庆　　400038
3.
陆军军医大学（第三军医大学）第一附属医院内分泌科，重庆　　400038

基金项目:

国家自然科学基金面上项目 81873936

详细信息

通讯作者:
张家平，Email：japzhang@aliyun.com

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出版历程
- 收稿日期: 2020-05-19

Effects and molecular mechanism of histone deacetylase 6 inhibitor Tubastatin A on the prolifera- tion and movement of human skin fibroblasts

1.
Department of Plastic Surgery, the First Affiliated Hospital of Army Medical University (the Third Military Medical University), Chongqing 400038, China
2.
State Key Laboratory of Trauma, Burns, and Combined Injury, Institute of Burn Research, the First Affiliated Hospital of Army Medical University (the Third Military Medical University), Chongqing 400038, China
3.
Department of Endocrinology, the First Affiliated Hospital of Army Medical University (the Third Military Medical University), Chongqing 400038, China

Funds:

General Program of National Natural Science Foundation of China 81873936

More Information

Corresponding author: Zhang Jiaping, Email: japzhang@aliyun.com

摘要

摘要: 目的探讨组蛋白脱乙酰酶6（HDAC6）抑制剂Tubastatin A对人皮肤成纤维细胞（HSF）增殖及运动性的影响及其可能的分子机制。方法采用实验研究方法。取对数生长期HSF，按随机数字表法分为阴性对照组及1 μmol/L Tubastatin A组、5 μmol/L Tubastatin A组、10 μmol/L Tubastatin A组。阴性对照组加入含终体积分数0.1%二甲基亚砜的DMEM培养液（以下简称完全培养液），其余3组分别加入含相应终物质的量浓度Tubastatin A的完全培养液。常规培养24 h后，采用细胞计数试剂盒8（CCK-8）法和5-乙炔基-2'-脱氧尿嘧啶核苷（EdU）染色检测细胞增殖活力；在活细胞工作站下观察细胞3 h内运动范围，计算细胞曲线运动速度；采用蛋白质印迹法检测胞外信号调节激酶1/2（ERK1/2）及磷酸化ERK1/2（p-ERK1/2）的蛋白表达量，并计算p-ERK1/2与ERK1/2比值，以此表示ERK1/2活性。CCK-8法行细胞增殖活力检测样本数为6，其余实验样本数为3。对数据行单因素方差分析及LSD检验。结果培养24 h后，CCK-8法和EdU染色显示，与阴性对照组比较，1 μmol/L Tubastatin A组、5 μmol/L Tubastatin A组、10 μmol/L Tubastatin A组细胞增殖活力均显著下降（P<0.01）。培养24 h后，CCK-8法显示，与1 μmol/L Tubastatin A组比较，10 μmol/L Tubastatin A组细胞增殖活力显著下降（P<0.05）；EdU染色显示，与1 μmol/L Tubastatin A组比较，5 μmol/L Tubastatin A组、10 μmol/L Tubastatin A组细胞增殖活力显著下降（P<0.05或P<0.01）。观察3 h内，1 μmol/L Tubastatin A组、5 μmol/L Tubastatin A组、10 μmol/L Tubastatin A组细胞运动范围较阴性对照组明显缩小。观察3 h内，阴性对照组细胞曲线运动速度为（0.780±0.028）μm/min，明显快于1 μmol/L Tubastatin A组、5 μmol/L Tubastatin A组、10 μmol/L Tubastatin A组细胞的（0.594±0.023）、（0.469±0.028）、（0.391±0.021）μm/min（P<0.01）；1 μmol/L Tubastatin A组细胞曲线运动速度明显快于5 μmol/L Tubastatin A组和10 μmol/L Tubastatin A组（P<0.01）；5 μmol/L Tubastatin A组细胞曲线运动速度明显快于10 μmol/L Tubastatin A组（P<0.05）。培养24 h后，与阴性对照组比较，1 μmol/L Tubastatin A组、5 μmol/L Tubastatin A组、10 μmol/L Tubastatin A组细胞ERK1/2的活性显著下降（P<0.01）；与1 μmol/L Tubastatin A组比，5 μmol/L Tubastatin A组和10 μmol/L Tubastatin A组细胞ERK1/2的活性显著下降（P<0.01）；与5 μmol/L Tubastatin A组比较，10 μmol/L Tubastatin A组细胞ERK1/2的活性显著下降（P<0.05）。结论 HDAC6抑制剂Tubastatin A可能通过抑制ERK1/2活性，从而抑制HSF增殖及运动。
- 细胞增殖 /
- 细胞运动 /
- 组蛋白脱乙酰酶6 /
- Tubastatin A /
- 人皮肤成纤维细胞 /
- 胞外信号调节激酶1/2
Abstract: Objective To explore the effects and possible molecular mechanism of histone deacetylase 6 (HDAC6) inhibitor Tubastatin A on the proliferation and movement of human skin fibroblasts (HSFs). Methods The experimental research method was used. HSFs in logarithmic growth phase were taken and divided into negative control group, 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group according to the random number table. The HSFs in negative control group were added with Dulbecco′s modified eagle medium with the final volume fraction of 0.1% dimethyl sulfoxide (hereinafter referred to as the complete medium), and the other three groups were added with the complete medium with the corresponding final molarity of Tubastatin A. After 24 h of conventional culture, the cell proliferation activity was detected using cell counting kit 8 (CCK-8) method and 5-ethynyl-2'-deoxyuridine (EdU) staining; the range of motion of cells within 3 h was observed under the living cell workstation, and the curve movement velocity of the cells was calculated. The protein expressions of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2) were detected by Western blotting, and the ratio of p-ERK1/2 to ERK1/2 was calculated to represent the activity of ERK1/2. The sample number in cell proliferation activity detection with CCK-8 method was 6, while the sample numbers in other experiments were 3. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results After 24 h of culture, CCK-8 method and EdU staining showed that compared with negative control group, the cell proliferation activities in 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group were significantly decreased (P<0.01). After 24 h of culture, CCK-8 method showed that compared with 1 μmol/L Tubastatin A group, the cell proliferation activity in 10 μmol/L Tubastatin A group was significantly decreased (P<0.05); EdU staining showed that compared with 1 μmol/L Tubastatin A group, the cell proliferation activities in 5 μmol/L Tubastatin A group and 10 μmol/L Tubastatin A group were significantly decreased (P<0.05 or P<0.01). Within 3 h of observation, the ranges of cell motion in 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group were obviously reduced compared with that in negative control group. Within 3 h of observation, the curve movement velocity of cells in negative control group was (0.780±0.028) μm/min, which was obviously faster than (0.594±0.023), (0.469±0.028), and (0.391±0.021) μm/min of 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group (P<0.01); the curve movement velocity of cells in 1 μmol/L Tubastatin A group was obviously faster than those in 5 μmol/L Tubastatin A group and 10 μmol/L Tubastatin A group (P<0.01); the curve movement velocity of cells in 5 μmol/L Tubastatin A group was obviously faster than that in 10 μmol/L Tubastatin A group (P<0.05). After 24 h of culture, compared with negative control group, the activities of ERK1/2 of cells in 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group were decreased significantly (P<0.01); compared with 1 μmol/L Tubastatin A group, the activities of ERK1/2 of cells in 5 μmol/L Tubastatin A group and 10 μmol/L Tubastatin A group were decreased significantly (P<0.01); compared with 5 μmol/L Tubastatin A group, the activity of ERK1/2 of cells in 10 μmol/L Tubastatin A group was decreased significantly (P<0.05). Conclusions HDAC6 inhibitor Tubastatin A may mediate the inhibitory effect on proliferation and movement of HSFs by inhibiting the activity of ERK1/2.
- Cell proliferation /
- Cell movement /
- Histone deacetylase 6 /
- Tubastatin A /
- Human skin fibroblast /
- Extracellular signal-regulated kinase 1/2
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1 EdU染色法检测阴性对照组及Tubastatin A处理3组HSF增殖活力 EdU-DAPI×630，图中标尺为25 μm。1A、1B、1C.分别为阴性对照组细胞EdU染色、细胞核染色、细胞EdU染色与细胞核染色重叠图片，细胞核完整，EdU阳性染色细胞较多；1D、1E、1F.分别为1 μmol/L Tubastatin A组细胞EdU染色、细胞核染色、细胞EdU染色与细胞核染色重叠图片，细胞核完整，EdU阳性染色细胞较阴性对照组明显减少；1G、1H、1I及1J、1K、1L.分别为5 μmol/L Tubastatin A组、10 μmol/L Tubastatin A组细胞EdU染色、细胞核染色、细胞EdU染色与细胞核染色重叠图片，EdU阳性染色细胞均较1 μmol/L Tubastatin A组减少

注：HSF为人皮肤成纤维细胞，EdU为5-乙炔基-2'-脱氧尿嘧啶核苷，DAPI为4'，6-二脒基-2-苯基吲哚；细胞EdU阳性染色为绿色，细胞核阳性染色为蓝色，绿色+蓝色双荧光染色为增殖的HSF

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2 活细胞工作站观察阴性对照组及Tubastatin A处理3组人皮肤成纤维细胞3 h内的运动范围倒置相差显微镜×100。2A.阴性对照组细胞运动范围较大；2B、2C、2D.分别为1 μmol/L Tubastatin A组、5 μmol/L Tubastatin A组、10 μmol/L Tubastatin A组细胞运动范围，均较图2A明显缩小

注：细胞运动起点均为坐标（0，0），运动终点为位于4个象限中的圆点，连接两者之间的曲线为细胞运动轨迹，数据前“-”表示相应轴线上的方向

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3 蛋白质印迹法检测阴性对照组及Tubastatin A处理3组人皮肤成纤维细胞中ERK1/2及p-ERK1/2的蛋白表达水平

注：ERK1/2为胞外信号调节激酶1/2，p-ERK1/2为磷酸化ERK1/2，GAPDH为3-磷酸甘油醛脱氢酶；1.阴性对照组，2、3、4.分别为1 μmol/L Tubastatin A组、5 μmol/L Tubastatin A组、10 μmol/L Tubastatin A组

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