摘要
目的探讨miR-9能否通过靶向PI3K抑制增生性瘢痕成纤维细胞的生长。方法自2020年9月至2021年9月,北部战区总医院烧伤整形科选取12只健康成年的新西兰长耳兔随机分成对照组(6只)和瘢痕组(6只),对瘢痕组进行造模。HE染色检测两组组织形态。实时荧光定量PCR检测组织中miR-9和PI3K的m RNA表达情况,对miR-9和PI3K的表达用Pearson相关系数进行相关性分析。取人增生性瘢痕成纤维细胞(hypertrophic scar fibroblast,HSFb),分成psi CHECK-WT-PI3K+miR-9对照组、psiCHECK-WT-PI3K+miR-9模拟物组、psiCHECK-MUT-PI3K+miR-9对照组、psiCHECKMUT-PI3K+miR-9模拟物组,分别转染相应的序列,转染后48 h采用荧光素酶报告基因检测试剂盒检测荧光素酶和肾荧光素酶的表达。取HSFb,分为miR-9对照组和miR-9模拟物组,转染后0、12、24、36和48 h,采用噻唑蓝法检测实验检测细胞增殖活性,处理后24 h,采用Annexin V-FITC/PI试剂盒测定细胞凋亡情况,蛋白免疫印迹法检测PI3K、Caspase 3和Bcl-2蛋白表达情况。细胞实验中样本数均为3。对数据行t检验及Pearson相关系数分析。结果HE染色结果证明瘢痕组的组织中成纤维细胞过度增殖、炎症细胞浸润,模型构建成功。实时荧光定量PCR实验检测结果,与对照组相比瘢痕组miR-9显著下调(t=11.020,P<0.05),与对照组相比瘢痕组PI3K表达上调明显(t=10.840,P<0.05)。Pearson相关系数进行相关性分析在所有组织中,miR-9和PI3K的表达呈负相关。转染后48 h,psiCHECK-WT-PI3K+miR-9模拟物组PI3K活性明显低于psi CHECK-WT-PI3K+miR-9对照组的PI3K活性(t=8.648,P=0.001);psiCHECK-MUT-PI3K+miR-9对照组和psiCHECK-MUTPI3K+miR-9模拟物组PI3K活性相近(t=2.053,P=0.109)。miR-9模拟物组转染后12、24、36和48 h时,细胞的增殖能力明显低于miR-9对照组(P<0.05)。miR-9模拟物组转染24 h后,HSFb早期凋亡比例高于miR-9对照组(t=11.210,P=0.001)。miR-9能够下调PI3K的表达,并且能够抑制Bcl-2的表达,上调Caspase 3的含量。结论miR-9在增生性瘢痕组织中表达下调,可以通过打靶PI3K抑制HSFb的生长。
Objective To explore whether miR-9 can inhibit the growth of hypertrophic scar fibroblasts by targeting PI3K.Methods From September 2020 to September 2021,12 healthy adult New Zealand long-eared rabbits were randomly divided into control group(6 rabbits)and scar group(6 rabbits).The histological morphology of both groups was detected by HE staining.The m RNA expression of miR-9 and PI3K in tissues was detected by real-time fluorescent quantitative PCR,and the expression of miR-9 and PI3K was analyzed by Pearson’s correlation coefficient.The human hypertrophic scar fibroblasts(HSFb)were divided into psiCHECK-WT-PI3K+miR-9 control group,psiCHECK-WT-PI3K+miR-9 mimic group,psiCHECK-MUT-PI3K+miR-9 control group and psi CHECK-MUT-PI3K+miR-9 mimic group.The corresponding sequences were transfected respectively.The expression of luciferase and renilla luciferase were detected by luciferase reporter gene assay at 48 h after transfection.The HSFb was divided into miR-9control group and miR-9 mimic group.At 0,12,24,36 and 48 h after transfection,the cell proliferation was detected by MTT.At 24 h after treatment,the cell apoptosis was detected by Annexin V-FITC/PI kit,and the expression of PI3K,caspase 3 and Bcl-2 protein was detected by protein immunoblotting.The number of samples in cell experiment was 3.The data were analyzed by t-test and Pearson’s correlation coefficient.Results In HE staining,the excessive proliferation of fibroblasts and inflammatory cell infiltration were found in scar tissue.The model was successfully constructed.Compared with the control group,the miR-9 expression was significantly down-regulated(t=11.020,P<0.05)and PI3K expression was significantly up-regulated in the scar group(t=10.840,P<0.05).Pearson’s correlation analysis showed that the expression of miR-9 and PI3K was negatively correlated in all tissues.At 48 h after transfection,the PI3K activity in the psi CHECK-WT-PI3K+miR-9 mimic group was significantly lower than that in the psiCHECK-WT-PI3K+miR-9 control group(t=8.648,P=0.001).The PI3K activity in the psiCHECK-MUT-PI3K+miR-9 control group was similar to that in the psiCHECKMUT-PI3K+miR-9 mimic group(t=2.053,P=0.109).At 12,24,36 and 48 h after transfection,the proliferation ability of miR-9 mimic group was significantly lower than that of the miR-9 control group(P<0.05).At 24 h after transfection,the early apoptotic rate of HSFb in the miR-9 mimic group was higher than that in the miR-9 control group(t=11.210,P=0.001).Mi R-9 can down regulate the expression of PI3K,inhibit the expression of Bcl-2 and up regulate the content of caspase 3.Conclusion Mi R-9 is down-regulated in the hypertrophic scar tissue,which can inhibit the growth of HSFb by targeting PI3K.
作者
林枫
郭冰玉
白泽明
陶凯
王洪一
LIN Feng;GUO Bingyu;BAI Zeming;TAO Kai;WANG Hongyi(Department of Burn and Plastic Surgery,General Hospital of Northern Theater Command,Shenyang 110016,China)
出处
《中国美容整形外科杂志》
CAS
2022年第11期663-667,共5页
Chinese Journal of Aesthetic and Plastic Surgery
基金
国家自然科学基金青年科学基金(82002047)
国家自然科学基金面上项目(81971845)。
