DKK3基因过表达对人瘢痕疙瘩成纤维细胞增殖和凋亡的影响
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摘要
目的:探究DKK3基因过表达对人瘢痕成纤维细胞增殖和凋亡的影响。方法:取瘢痕疙瘩组织21例和正常皮肤组织20例,采用qRT-PCR检测其中DKK3 mRNA的表达水平。构建真核表达质粒pc DNA3. 0-DKK3,通过脂质体介导的方法将其转入瘢痕成纤维细胞中(DKK3组),未转染细胞为对照组,转染pc DNA3. 0空质粒的细胞为pc DNA组。转染48 h后,qRT-PCR和Western blot检测转染效率; CCK-8法检测成纤维细胞的增殖情况;双染法检测成纤维细胞的凋亡情况。结果:DKK3 mRNA在瘢痕疙瘩组织中表达下调(P <0. 05)。3组瘢痕疙瘩成纤维细胞中DKK3 mRNA和蛋白的表达差异有统计学意义(P <0. 05),DKK3组高于其他两组(P <0. 05)。3组细胞凋亡和增殖情况差异有统计学意义(P <0. 05),DKK3组增殖减少,凋亡增加(P <0. 05)。结论:DKK3基因过表达可抑制瘢痕疙瘩成纤维细胞的增殖并诱导其发生凋亡。
Aim: To investigate the effects of overexpression of DKK3 gene on proliferation and apoptosis of human keloid fibroblasts. Methods: A total of 21 cases of keloid tissue and 20 cases of normal skin tissue were taken. qRT-PCR was used to detect the expression level of DKK3 mRNA. The eukaryotic expression plasmid pc DNA3. 0-DKK3 was constructed and transferred into scar fibroblasts by liposome-mediated method(DKK3 group),untransfected cells were used as control group,and empty plasmid pc DNA3. 0 was transfected as negative control(pc DNA group). After transfection for 48 hours,qRT-PCR and Western blot were used to detect the transfection efficiency; the proliferation of fibroblasts was detected by CCK-8 method; the apoptosis of fibroblasts was detected by double staining method. Results: The level of DKK3 mRNA was down-regulated in keloid tissue(P < 0. 05). The levels of DKK3 mRNA and protein in keloid fibroblasts of the three groups were significantly different(P < 0. 05),which was higher in the DKK3 group than those in the other two groups(P <0. 05). The apoptosis and proliferation among the 3 groups were statistically different(P < 0. 05),the proliferation of keloid fibroblasts in DKK3 group was decreased(P < 0. 05),and the apoptosis was increased(P < 0. 05). Conclusion: Overexpression of DKK3 gene could inhibit proliferation and induce apoptosis of keloid fibroblasts.
Aim: To investigate the effects of overexpression of DKK3 gene on proliferation and apoptosis of human keloid fibroblasts. Methods: A total of 21 cases of keloid tissue and 20 cases of normal skin tissue were taken. qRT-PCR was used to detect the expression level of DKK3 mRNA. The eukaryotic expression plasmid pc DNA3. 0-DKK3 was constructed and transferred into scar fibroblasts by liposome-mediated method(DKK3 group),untransfected cells were used as control group,and empty plasmid pc DNA3. 0 was transfected as negative control(pc DNA group). After transfection for 48 hours,qRT-PCR and Western blot were used to detect the transfection efficiency; the proliferation of fibroblasts was detected by CCK-8 method; the apoptosis of fibroblasts was detected by double staining method. Results: The level of DKK3 mRNA was down-regulated in keloid tissue(P < 0. 05). The levels of DKK3 mRNA and protein in keloid fibroblasts of the three groups were significantly different(P < 0. 05),which was higher in the DKK3 group than those in the other two groups(P <0. 05). The apoptosis and proliferation among the 3 groups were statistically different(P < 0. 05),the proliferation of keloid fibroblasts in DKK3 group was decreased(P < 0. 05),and the apoptosis was increased(P < 0. 05). Conclusion: Overexpression of DKK3 gene could inhibit proliferation and induce apoptosis of keloid fibroblasts.
引文
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[18]UCHIDA D,SHIRAHA H,KATO H,et al.Potential of adenovirus-mediated REIC/Dkk-3 gene therapy for use in the treatment of pancreatic cancer[J].J Gastroenterol Hepatol,2014,29(5):973
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[2]SLEMP AE,KIRSCHNER RE.Keloids and scars:a review of keloids and scars,their pathogenesis,risk factors,and management[J].Curr Opin Pediatr,2006,18(4):396
[3]LEDON JA,SAVAS J,FRANCA K,et al.Intralesional treatment for keloids and hypertrophic scars:a review[J].Dermatol Surg,2013,39(12):1745
[4]ZHU HY,BAI WD,WANG HT,et al.Peroxisome proliferator-activated receptor-gamma agonist inhibits collagen synthesis in human keloid fibroblasts by suppression of early growth response-1 expression through upregulation of miR-543 expression[J].Am J Cancer Res,2016,6(6):1358
[5]BUTLER PD,LONGAKER MT,YANG GP.Current progress in keloid research and treatment[J].J Am Coll Surg,2008,206(4):731
[6]周毕峰,常宏,石定,等.瘢痕疙瘩的治疗新进展[J].现代生物医学进展,2016,16(7):1394
[7]RANG Z,WANG ZY,PANG QY,et al.MiR-181a Targets phlpp2 to augment akt signaling and regulate proliferation and apoptosis in human keloid fibroblasts[J].Cell Physiol Biochem,2016,40(3/4):796
[8]HSIEH SY,HSIEH PS,CHIU CT,et al.Dickkopf-3/REICfunctions as a suppressor gene of tumor growth[J].Oncogene,2004,23(57):9183
[9]YIN DT,WU W,LI M,et al.DKK3 is a potential tumor suppressor gene in papillary thyroid carcinoma[J].Endocr Relat Cancer,2013,20(4):507
[10]GUO CC,ZHANG XL,YANG B,et al.Decreased expression of Dkk1 and Dkk3 in human clear cell renal cell carcinoma[J].Mol Med Rep,2014,9(6):2367
[11]SLEMP AE,KIRSCHNER RE.Keloids and scars:a review of keloids and scars,their pathogenesis,risk factors,and management[J].Curr Opin Pediatr,2006,18(4):396
[12]RHETT JM,GHATNEKAR GS,PALATINUS JA,et al.Novel therapies for scar reduction and regenerative healing of skin wounds[J].Trends Biotechnol,2008,26(4):173
[13]HAHN JM,GLASER K,MCFARLAND KL,et al.Keloidderived keratinocytes exhibit an abnormal gene expression profile consistent with a distinct causal role in keloid pathology[J].Wound Repair Regen,2013,21(4):530
[14]MAO B,WU W,DAVIDSON G,et al.Kremen proteins are dickkopf receptors that regulate Wnt/beta-catenin signalling[J].Nature,2002,417(6889):664
[15]CADIGAN KM.Wnt/beta-catenin signaling:turning the switch[J].Dev Cell,2008,14(3):322
[16]XING Y,CLEMENTS WK,KIMELMAN D,et al.Crystal structure of aβ-catenin/Axin complex suggests a mechanism for theβ-catenin destruction complex[J].Genes Dev,2003(22):2753
[17]WANG Z,MA LJ,KANG Y,et al.Dickkopf-3(Dkk3)induces apoptosis in cisplatin-resistant lung adenocarcinoma cells via the Wnt/beta-catenin pathway[J].Oncol Rep,2015,33(3):1097
[18]UCHIDA D,SHIRAHA H,KATO H,et al.Potential of adenovirus-mediated REIC/Dkk-3 gene therapy for use in the treatment of pancreatic cancer[J].J Gastroenterol Hepatol,2014,29(5):973
[19]YANG ZR,DONG WG,LEI XF,et al.Overexpression of Dickkopf-3 induces apoptosis through mitochondrial pathway in human colon cancer[J].World J Gastroenterol,2012,18(14):1590