芒硝对驴皮成纤维细胞增殖、凋亡的影响

doi:10.16431/j.cnki.1671-7236.2020.07.021

摘要/Abstract

摘要： 为研究不同浓度的芒硝对体外培养的驴皮成纤维细胞增殖、凋亡的影响，本试验采用组织块法体外培养驴皮成纤维细胞，并用HE、Masson染色鉴定；再添加高（2 000、1 500 μg/mL）、中（1 000、500、250 μg/mL）和低浓度（100、50、10 μg/mL）芒硝体外培养驴皮成纤维细胞，用MTT和Caspase-3法分别检测成纤维细胞增殖率和凋亡率；最后用实时荧光定量PCR、ELISA法检测不同浓度（1 500、250、10 μg/mL）芒硝培养液中影响驴皮成纤维细胞胶原蛋白合成的相关基因表达量及蛋白浓度。结果显示，培养5 d时，驴皮成纤维细胞开始从组织块边缘爬出，25 d后长满培养皿；HE染色呈典型的梭形，驴皮成纤维细胞中胶原纤维经Masson染色呈蓝色。中浓度芒硝培养液组成纤维细胞的增殖率显著高于高、低浓度组（P<0.05），相应地，中浓度组的凋亡因子Caspase-3活性显著低于其他两组（P<0.05）。相比于24 h，芒硝作用于成纤维细胞48 h能显著提高胶原蛋白合成相关基因的表达量（P<0.05），250 μg/mL浓度的效果最佳。综合上述结果，组织块培养法易于分离培养驴皮成纤维细胞，250 μg/mL浓度的芒硝在48 h能显著提高驴皮成纤维细胞的增殖率及其胶原蛋白合成相关基因的表达量，此结果可为芒硝促进驴皮成纤维细胞的增殖作用以及驴皮伤口愈合机制的研究提供理论依据。

关键词: 驴皮; 成纤维细胞; 芒硝; 胶原蛋白; 基因

Abstract: The effect of different concentrations of mirabilite on the proliferation and apoptosis of donkey skin fibroblasts in vitro was evaluated in this study.The skin fibroblasts were cultured in vitro by the tissue block method,and identified by HE and Masson staining.Then,the fibroblasts were cultured with high (2 000,1 500 μg/mL),medium (1 000,500 and 250 μg/mL) and low concentration (100,50 and 10 μg/mL) of mirabilite.The proliferation rate and apoptosis rate of fibroblasts were measured by the methods of MTT and Caspase-3 respectively.Finally,the expression of genes and proteins that affected the collagen synthesis of donkey skin fibroblasts were detected by Real-time fluorescence quantitative PCR and ELISA in different concentrations of mirabilite media (1 500,250 and 10μg/mL).The results showed that the fibroblasts of donkey skin began to climb out from the edge of the tissue mass at the 5th day of culture,and grew up in the whole culture dish at the 25th day.HE staining showed a typical spindle shape,and the collagen fibers of donkey skin fibroblasts were blue by Masson staining.The proliferation rate of fibroblasts in the medium concentration group was significantly higher than that in the high and low concentration groups (P<0.05),correspondingly,apoptotic factor Caspase-3 activity was significantly lower in the medium concentration group (P<0.05).Compared with 24 h,the mirabilite could significantly increase the expression of genes related to collagen synthesis after 48 h (P<0.05),and the effect was best with 250 μg/mL of mirabilite.In summary,the method of tissue block culture was easy to isolate and culture donkey skin fibroblasts.250 μg/mL of mirabiliten could significantly improve the proliferation rate of donkey skin fibroblasts after 48 h and the expression of genes related to collagen synthesis.This result could provide theoretical basis for the study of the effects of mirabilite on the proliferation of donkey skin fibroblasts and the mechanism of wound healing.

Key words: donkey skin; fibroblast; mirabilite; collagen; genes

中图分类号:

S813.24

张媛, 王艳萍, 刘迥, 苗凯, 臧睿, 曾维斌. 芒硝对驴皮成纤维细胞增殖、凋亡的影响[J]. 中国畜牧兽医, 2020, 47(7): 2159-2170.

ZHANG Yuan, WANG Yanping, LIU Jiong, MIAO Kai, ZANG Rui, ZENG Weibin. Effect of Mirabiliteon on Proliferation and Apoptosis of Donkey Fibroblasts[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(7): 2159-2170.

参考文献

[1] KOURBETI I S,HALKID A,VAKIS A F,et al.Infections in patients undergoing craniotomy:Risk factors associated with post-craniotomy meningitis[J].Journal of Neurosurgery,2015,122(5):1113-1119.
[2] 中国药典委员会:中华人民共和国药典[M].北京,中国医学科学出版社,2010. CHINA PHARMACOPAEIA COMMITTEE:Pharmacopoeia of the People's Republic of China[M].Beijing:China Medical Science Press,2010.
[3] MOSTAFA A,MAHMOUD S,MUSTAFA B,et al.Prospective study of incidence and predictors of peripheral intravenous catheter-induced complications[J].Therapeutics and Clinical Risk Management,2014,12(9):993-1001.
[4] 刘飞.芒硝外用在创面愈合中的作用及机制的研究[D].重庆:西南医科大学,2018. LIU F.Research on the effect and mechanism of mirabilite external use in wound healing[D].Chongqing:Southwest Medical University,2018.(in Chinese)
[5] 刘莎.芒硝溶液对于大鼠机械性皮损作用的实验研究[D].长春:长春中医药大学,2015. LIU S.Research of mirabilite to the mechanical injury of the rats'skin[D].Changchun:Changchun University of Chinese Medicine,2015.(in Chinese)
[6] 王艳萍,高帅,刘迥,等.驴皮成纤维细胞的体外培养研究[J].中国畜牧兽医,2017,44(8):2255-2260. WANG Y P,GAO S,LIU J,et al.Study on in vitro culture of donkey skin fibroblasts[J].China Animal Husbandry & Veterinary Medicine,2017,44(8):2255-2260.(in Chinese)
[7] 张建成,黄宇坚.人心房成纤维细胞体外组织块贴壁培养与鉴定[J].莆田学院学报,2010,17(2):40-42. ZHANG J C,HUANG Y J.Establishment of using tissue block adhering wall method culture human atrial fibroblasts in vitro and their identifying[J].Journal of Putian University,2010,17(2):40-42.(in Chinese)
[8] MASSAGUE J.TGF-β signalling in context[J].Nature Reviews Molecular Cell Biology,2012,13(10):616-630.
[9] YU Y,XIAO C H,TAN L D,et al.Cancer-associated fibroblasts induce epithelial-mesenchymal transition of breast cancer cells through paracrine TGF-β signalling[J].British Journal of Cancer,2014,110(3):724-732.
[10] JAVLE M I,YNTAN D F.Biomarkers of TGF-beta signaling pathway and prognosis of pancreatic cancer[J].PLoS One,2014,9(1):e85924.
[11] LEASK A,ABRAHAM D J.TGF-beta signaling and the fibrotic response[J].The FASEB Journal,2004,18(6):816-827.
[12] MEAD A L,WONG T T,CORDEIRO M F,et al.Evaluation of anti-TGF-beta2 antibody as a new postoperative anti-scarring agent in glaucoma surgery[J].Investigative Opthalmology & Visual Science,2003,44(8):3394-3401.
[13] ZOL B K,MARK S,NAKSHATRA K R,et al.Temporal expression of the transforming growth factor-beta pathway in the rabbit ear model of wound healing and scarring[J].Journal of the American College of Surgeons,2007,205(1):78-88.
[14] QIN Z,XIA W,FISHER GARY J,et al.YAP/TAZ regulates TGF-β/Smad3 signaling by induction of Smad7via AP-1 in human skin dermal fibroblasts[J].Cell Communication and Signaling,2018,16(1):18.
[15] MASSAGUE J.How cells read TGF-β signals[J].Nature Reviews Molecular Cell Biology,2000,1(3):169-178.
[16] CHIN G S,LIU W,PELED Z,et al.Differential expression of transforming growth factor-beta receptors Ⅰ and Ⅱ and activation of Smad3 in keloid fibroblasts[J].Plastic and Reconstructive Surgery,2001,108(2):423-429.
[17] PUROHIT T,HE T Y,QIN Z P,et al.Smad3-dependent regulation of type Ⅰ collagen in human dermal fibroblasts:Impact on human skin connective tissue aging[J].Journal of Dermatological Science,2016,83(1):80-83.
[18] HUANG D,HONG S K,WANG H G.Pressure therapy upregulates matrix metalloproteinase expression and downregulates collagen expression in hypertrophic scar tissue[J].Chinese Medical Journal,2013,126(17):3321-3324.
[19] VERRECCHIA F,MAUVIEL A.Transforming growth factor-beta signaling through the Smad pathway:Role in extracellular matrix gene expression and regulation[J].Journal of Investigative Dermatology,2002,118(2):211-215.
[20] DONG C,ZHU S,WANG T,et al.Deficient Smad7 expression:A putative molecular defect in scleroderma[J].Proceedings of the National Academy of Science of the United States of America,2002,99(6):3908-3913.
[21] MONTOLEONE G,KUMBEROVA A,CROFT N M,et al.Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease[J].Journal of Clinical Investigation,2001,108(4):601-609.
[22] 李震,王英,刘惠莉,等.牛乳腺上皮细胞的分离培养[J].上海农业学报,2000,16(3):30-33. LI Z,WANG Y,LIU H L,et al.Study on isolation and culture of bovine mammary epithelial cells[J].Acta Agriculturae Shanghai,2000,16(3):30-33.(in Chinese)
[23] 林桂娟,王恬,陈才勇.机械破碎法分离奶牛乳腺上皮细胞的体外培养研究[J].畜牧与兽医,2004,7:6-8. LIN G J,WANG T,CHEN C Y.A study on primary culture of cow mammary epithelial cell separated directly[J].Animal Husbandry & Veterinary Medicine,2004,7:6-8.(in Chinese)
[24] 覃梅梅,盘雪莹.中药芒硝外敷对腹部手术后患者胃肠功能恢复的影响[J].护理学报,2009,16(15):53-54. QIN M M,PAN X Y.Effect of external application of mirabilite on gastrointestinal function recovery in patients after abdominal operation[J].Journal of Nursing(China),2009,16(15):53-54.(in Chinese)
[25] LU Y,HAO C,HE W,et al.Experimental research on preventing mechanical phlebitis arising from indwelling needles in intravenous therapy by external application of mirabilite[J].Experimental and Therapeutic Medicine,2017,15(1):276-282.
[26] 王芸,王赟.芒硝外敷治疗腹膜透析患者皮下隧道感染的护理[J].中华护理杂志,2013,48(11):1015-1016. WANG Y,WANG Y.Nursing care of patients with subcutaneous tunnel infection treated with mirabilite external application[J].Chinese Journal of Nursing,2013,48(11):1015-1016.(in Chinese)
[27] GOPI K K,XINGGUI S,SHYAMAL CB,et al.Hydrogen sulfide chemical biology:Pathophysiological roles and detection[J].Nitric Oxide,2013,35(30):5-20.
[28] RUSZCZAK Z.Effect of collagen matrices on dermal wound healing[J].Advanced Drug Delivery Reviews,2003,55(12):1595-1611.
[29] ROMANIC A M,ADACHI E,KADLER K E,et al.Copolymerization of pNcollagen Ⅲ and collagen Ⅰ.pNcollagen Ⅲ decreases the rate of incorporation of collagen Ⅰ into fibrils,the amount of collagen Ⅰ incorporated,and the diameter of the fibrils formed[J].Journal of Biological Chemistry,1991,266(19):12703-12709.
[30] VOLK S W,WANG Y,MAULDIN E A,et al.Dimi-nished type Ⅰ collagen promotes myofibroblast differen-tiation and increases scar deposition in cutaneous wound haeling[J].Cells Tissues Organs,2011,194(1):25-37.
[31] ZHAI XX,TANG ZM,DING JC,et al.Expression of TGF-β1/mTOR signaling pathway in pathological scar fibroblasts[J].Molecular Medicine Reports,2017,15(6):3467-3472.
[32] LIU R M,DESAI L P.Reciprocal regulation of TGF-β and reactive oxygen species:A perverse cycle for fibrosis[J].Redox Biology,2015,9(9):565-577.
[33] BROWN F C,TUTTLE E,WESTERVELD M,et al.Visual memory in post-anterior right temporal lobectomy patients and adult normative data for the brown location test(BLT)[J].Epilepsy Behavior,2010,17(2):215-220.
[34] PELTONEN J,HSIAO L,JAAKKOLA S,et al.Activation of collagen gene expression in keloids:Co-localization of type Ⅰ and Ⅵ collagen and transforming growth factor-beta 1 mRNA[J].The Journal of Investigative Dermatology,1991,97(2):240-248.
[35] VERRCCHIA F,CHU M L,MAUYIEL A.Identification of novel TGF-beta/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach[J].Journal of Biological Chemistry,2001,276(20):17058-17062.
[36] GHOSH A K,MORI Y,DOWLING E,et al.Trichostatin a blocks TGF-beta-induced collagen gene expression in skin fibroblasts:Involvement of Sp1[J].Biochemical and Biophysical Research Communications,2007,354(2):420-426.
[37] 刘波,果磊.SiRNA-Smad3沉默人瘢痕疙瘩成纤维细胞中Smad3对纤维连接蛋白及Ⅰ型胶原表达的影响[J].重庆医科大学学报,2010,35(4):522-526. LIU B,GUO L.Effects of SiRNA-mediated Smad3 gene silence on the expression of fibronectin and collagen Ⅰ in human keloid fibroblasts[J].Journal of Chongqing Medical University,2010,35(4):522-526.(in Chinese)
[38] HU Z C,SHI F,LIU P,et al.TIEG1 Represses Smad7-mediated activation of TGF-β1/Smad signaling in keloid pathogenesis[J].Journal of Investigative Dermatology,2017,137(5):1051-1059.