摘要
目的研究胸腺素β4基因对人肾小管上皮细胞株(HKC)上皮-肌成纤维细胞转分化(EMT)的影响。方法采用RT-PCR和T-A克隆技术,从SW480细胞中扩增胸腺素β4基因全长cDNA,克隆到T载体中,再亚克隆到pIRES2-EGFP质粒载体,得到表达胸腺素β4基因的真核表达载体和阴性对照载体,转染HKC,得到胸腺素β4表达增强的HKC-Tβ4和胸腺素β4表达未受影响的HKC-EGFP。分别采用RT-PCR和Western blot检测HKC、HKC-Tβ4和HKC-EGFP 3组细胞胸腺素β4、α-平滑肌肌动蛋白(-αSMA)和E-钙黏素表达。结果与HKC组及HKC-EGFP组相比,HKC-Tβ4组胸腺素β4和-αSMA表达显著增高,E-钙黏素表达显著降低,差异有统计学意义(P<0.01)。结论胸腺素β4基因转染能诱导HKC发生EMT,提示胸腺素β4是诱导EMT的重要分子,可能成为肾纤维化治疗的靶目标。
Objective To investigate the induced effects of eukaryotic expression vector of human thymosin beta 4 （thymosin β4 ） gene on human tubular epithelial-myofibroblast transdifferentiation （EMT） in vitro. Methods RT-PCR and T-A cloning technique was used to clone the thymosin β4 full length cDNA from SW480 cells, then eukaryotic express vector pIRES2-EGFP containing thymosin β4 cDNA or blank vector was constructed and infected into human renal tubular epithelial cells line （HKC）. HKC-Tβ4 cells expressing thymosin β4 or HKC-EGFP cells was selected. Cells cultured in vitro were divided into three groups： HKC, HKC- Tβ4, HKC-EGFP. Thymosin β4, alpha-smooth muscle actin（α-SMA） and E-cadherin expression were examined by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. Results （1） HKC group：thymosin β4 expression was weakly positive and α-SMA expression was negative, but E-cadherin expression was strong positive. （2）HKC-Tβ4 group： both thymosin β4 and a-SMA expression were strong positive, and E-cadherin expression was weaker than that in HKC group（P〈0.01）. （3）HKC-EGFP group：all of thymosin β4 ,α-SMA and E-cadherin expression were similar to that in HKC group. Conclusion Human renal tubular epithelial-myofibroblast transdifferentiation may be induced by eukaryotic expression vector of human thymosin β4 gene transfection. These results suggest that thymosin β4 might play crucial roles in human renal tubular epithelial-myofibroblast transdifferentiation and might be a promising target for treatment of renal fibrosis.
出处
《重庆医学》
CAS
CSCD
北大核心
2009年第17期2184-2186,2188,共4页
Chongqing medicine
基金
重庆市科委自然基金资助项目(CSTC
2006BB5096)
关键词
胸腺素Β4
人肾小管上皮细胞
肌成纤维细胞
真核表达载体
转分化
thymosin β4
human renal tubular epithelial cells
myofibroblast
eukaryotic expression vector
transdifferentiation
