Combined use of RT-PCR and gel electrophoresis to detect expression of transforming growth factor beta1 in mouse lung fibroblasts in vitro

doi:10.3969/j.issn.2095-4344.2017.08.021

Abstract

Abstract:

BACKGROUND: As a combination of reverse transcription (RT) and polymerase chain reaction (PCR), RT-PCR has been used to detect gene expression levels in cells and tissues, RNA virus contents in cells and specific gene cloned cDNA sequences.
OBJECTIVE: To detect the inhibitory effcet of Stealth siRNAs on the expression of transforming growth factor β1 (TGF-β1).
METHODS: There were blank control, empty vector transfection, stealth_48, stealth_166, and stealth_594 groups. Three stealth siRNAs aimed at different sequences in TGF-β1 mRNA were made, and were then transfected into BALB/c mouse lung fibroblasts in vitro. The expressions of TGF-β1 and connective tissue growth factor were detected by RT-PCR.
RESULTS AND CONCLUSION: In different time periods, the TGF-β1 expression was differentially depressed by three stealth siRNAs, especially stealth_166. The inhibitory effects varied with time, which could be detective at 48 hours, reached the peak at 72 hours and then began to attenuate at 96 hours. Our findings show that the inhibitory effect of stealth siRNAs on the TGF-β1 expression in mouse lung fibroblasts can be detected by RT-PCR.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: RNA, Small Interfering, Transforming Growth Factor beta1, Fibroblasts, Tissue Engineering

CLC Number:

R318

Yu Wei-tao, Wang Dong-jian, Ai Ling-yan. Combined use of RT-PCR and gel electrophoresis to detect expression of transforming growth factor beta1 in mouse lung fibroblasts in vitro[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(8): 1268-1272.

Figures/Tables 3

References

[1] Selman M,King TE Jr,Pardo A.Idiopathic Pulmonary Fibrosis:progress in understanding its pathogenesis and implications for thrapy.Ann Intern Med.2001;134:136-151.

[2] Scaffidi AK, Petrovic N, Moodley YP, et al. alpha(v)beta(3) Integrin interacts with the transforming growth factor-beta (TGF-beta) type II receptor to potentiate the proliferative effects of TGF-beta1 in living human lung fibroblasts.J Biol Chem. 2004;279(36): 37726-37733.

[3] 余维涛,崔社怀,杨雪梅.特异性stealth siRNA抑制小鼠肺成纤维细胞TGF-β1表达的实验研究[J].第三军医大学学报,2007, 29(21):2038-2040.

[4] 孟翠萍,罗武,陈灵.ACEI联合激素治疗肾病综合征疗效及TGF-β1检测的临床意义[J].实用医学杂志,2012,28(2):297-299.

[5] 厉琴,刘畅,樊荣等.消化性溃疡患者血清IL-2、IL-6和TGF-β1检测的临床意义[J].放射免疫学杂志,2013,26(2):143-144.

[6] 董志珍,姚登福,沈预程等.肝细胞肝癌组织TGF-β和IGF-Ⅱ异常表达与HBV复制[J].江苏医药,2008,34(7):649-651.

[7] 叶军,薛冬英,张洁等.慢性肝炎肝纤维化患者肝组织和血清中TGF-β1、TIMP-1检测及其意义[J].肝脏,2007,12(4):294-295.

[8] Huang Y,Zou Q,Wang S P,et al.The discovery approaches and detection methods of microRNAs.Mol Biol Rep.2011; 38(6):463-472.

[9] 党裔武,陈罡,容敏华.实时定量RT-PCR检测肝癌miRNA表达中内参的选择[J].临床与实验病理学杂志,2012,28(10):1102-1106.

[10] 王珂,周长满,于恩华. 成年大鼠嗅球和鼻腔嗅粘膜成鞘细胞的分离、培养与鉴定[J]. 解剖学报,2002, 33(5):488-491.

[11] Qi W,Chen X,Poronnik P,et al.Transforming growth factor-beta/connective tissue grouth factor axis in the kidney. Int J Biochen Cell Biol.2008;40(1):9-13.

[12] 杨云华，艾维.慢性肾脏病与肾脏纤维化研究进展[J].实用医学杂志，2009,25(20)：3357-3359.

[13] Li HH, Cai Q, Wang YP,et al. The role of transforming growth factor-β<sub>1</sub>/connective tissue growth factor signaling pathway in paraquat-induced pulmonary fibrosis. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2016; 34(7):484-488.

[14] Yao R, Cao Y, He YR, et al.Adiponectin attenuates lung fibroblasts activation and pulmonary fibrosis induced by paraquat.PLoS One. 2015;10(5):e0125169.

[15] Hao S, Wang W, Yu Z, et al.[Adrenomedullin inhibits TGF-β1-induced procollagen expression in cultured human fetal lung fibroblasts via Smad2/3 pathway].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014;30(11):1125-1128.

[16] Estany S, Vicens-Zygmunt V, Llatjós R, et al.Lung fibrotic tenascin-C upregulation is associated with other extracellular matrix proteins and induced by TGFβ1.BMC Pulm Med. 2014;14:120.

[17] Zhang M, Cao SR, Zhang R, et al. The inhibitory effect of salvianolic acid B on TGF-β1-induced proliferation and differentiation in lung fibroblasts.Exp Lung Res. 2014;40(4): 172-185.

[18] Xu X, Wan X, Geng J, et al.Kinase inhibitors fail to induce mesenchymal-epithelial transition in fibroblasts from fibrotic lung tissue.Int J Mol Med. 2013;32(2):430-438.

[19] Mirzamani MS, Nourani MR, Imani Fooladi AA,et al. Increased expression of transforming growth factor-β and receptors in primary human airway fibroblasts from chemical inhalation patients. Iran J Allergy Asthma Immunol. 2013; 12(2):144-152.

[20] Wójcik KA, Skoda M, Koczurkiewicz P, et al.Apigenin inhibits TGF-β1 induced fibroblast-to-myofibroblast transition in human lung fibroblast populations.Pharmacol Rep. 2013; 65(1):164-172.

[21] 高晓方,崔社怀.干扰素γ对实验大鼠肺纤维化的治疗作用及机制的初步研究[J].第三军医大学学报, 2005, 27(2): 143-145.

[22] Ruvkun G. Molecular biology: Glimpses of a tiny RNA world. Science. 2001,294:797-799.

[23] Dillon CP, Sandy P, Nencioni A, et al. RNAi as an experimental and therapeutic tool to studyand regulate physiological and disease processes. Annu Rev Physiol. 2005;67: 147-173.

[24] Baarsma HA, Engelbertink LH, van Hees LJ, et al.Glycogen synthase kinase-3 (GSK-3) regulates TGF-β?-induced differentiation of pulmonary fibroblasts.Br J Pharmacol. 2013;169(3):590-603.

[25] Shi Y, Dong Y, Duan Y,et al.Substrate stiffness influences TGF-β1-induced differentiation of bronchial fibroblasts into myofibroblasts in airway remodeling.Mol Med Rep.2013 ; 7(2):419-424.

[26] Robinson PM, Blalock TD, Yuan R, et al.Hammerhead ribozyme-mediated knockdown of mRNA for fibrotic growth factors: transforming growth factor-beta 1 and connective tissue growth factor.Methods Mol Biol. 2012;820:117-132.

[27] Ren DQ, Liu M, Guo YZ,et al. [Liver X receptor agonist T0901317 inhibits TGF-β1-induced α-SMA expression in normal human lung fibroblasts].Nan Fang Yi Ke Da Xue Xue Bao. 2011;31(5):744-748.

[28] Zhang J, Wu L, Feng MX, et al.Pulmonary fibroblasts from COPD patients show an impaired response of elastin synthesis to TGF-β1.Respir Physiol Neurobiol. 2011;177(3): 236-240.

[29] Fu JH, Yang HP, Pan L, et al. [Effects of TGF-β1 on gene expression of connective tissue growth factor in lung fibroblasts].Zhongguo Dang Dai Er Ke Za Zhi. 2011;13(1): 36-39.

[30] Ding H, Zhou FQ, Cai HR, et al. Expression of caveolin-1 and extracellular matrix induced by transforming growth factor beta1 in human fetal lung fibroblasts. Zhonghua Jie He He Hu Xi Za Zhi. 2010;33(4):280-283.

[31] Pegorier S, Campbell GA, Kay AB, et al. Bone morphogenetic protein (BMP)-4 and BMP-7 regulate differentially transforming growth factor (TGF)-beta1 in normal human lung fibroblasts (NHLF).Respir Res. 2010;11:85.

[32] Sullivan DE, Ferris M, Nguyen H, et al.TNF-alpha induces TGF-beta1 expression in lung fibroblasts at the transcriptional level via AP-1 activation.J Cell Mol Med. 2009;13(8B): 1866-1876.

[33] Shi JQ, Xie M, Wang YJ, et al. [The effect or rosiglitazone on signal pathways of transforming growth factor-beta1 in human lung fibroblast].Sichuan Da Xue Xue Bao Yi Xue Ban. 2009; 40(4):598-603.

[34] 应方微,黎明,李志.角膜营养不良家系患病角膜细胞角蛋白及b-FGF与TGF-b1表达的研究[J].中国实用眼科杂志,2011,29(7): 731-734