Transplantation of endothelial progenitor cells with RNA interference targeting transforming growth factor beta1 inhibits pulmonary fibrosis in rats

doi:10.3969/j.issn.2095-4344.1529

Abstract

Abstract:

BACKGROUND: Abundant animal experiments have shown that transforming growth factor β1 (TGF- β1) gene is a hotspot of pulmonary fibrosis and an important target for drugs.
OBJECTIVE: To explore the therapeutic effect of the transplantation of endothelial progenitor cells with RNA interference targeting TGF-β1 in the rats with pulmonary fibrosis.
METHODS: (1) The siRNA plasmid directed to TGF-β1 was constructed and transfected into endothelial progenitor cells. It was divided into blank group, control group and TGF-β1-siRNA transfection group. The expression of TGF-β1 at 3 and 28 days after transfection was detected by western blot assay. (2) Eighty Wistar rats provided by Beijing Vital River Laboratory Animal Technology Co. Ltd. were divided into control, model, endothelial progenitor cell, and TGF-β1 silent groups. The rat trachea in the latter three groups was injected with 0.3 mL of bleomycin (5 mg/kg) to establish the rat model of pulmonary fibrosis. At 24 hours after modeling, the control and model groups received the injection of 20 μL of normal saline via caudal vein, and the endothelial progenitor cell, and TGF-β1 silent groups subjected to the injection of 20 μL 3×10⁶/L endothelial progenitor cell suspension and TGF-β1 silent endothelial progenitor cell suspension, respectively. (3) After 28 days of transplantation, the levels of interleukin-10, interleukin-6, and tumor necrosis factor-α in the rat bronchoalveolar lavage fluid were detected by ELISA. The pathological changes of lung tissue were observed by hematoxylin-eosin staining. Expression levels of TGF-β1, Smad-2, and Smad-7 were detected by RT-PCR, and western blot assay.
RESULTS AND CONCLUSION: (1) The protein expression of TGF-β1 in the TGF-β1-siRNA transfection group was significantly lower than that in the blank and control groups at 3 and 28 days after transfection (P < 0.05). (2) Compared with the model group, the pulmonary fibrosis was relieved in the endothelial progenitor cell group with reduced alveolar interval thickness and further attenuated in the TGF-β1 silent group. (3) The levels of interleukin-10, interleukin-6, and tumor necrosis factor-α in the endothelial progenitor cell group were significantly lower than those in the model group (P < 0.05). These factor levels in the TGF-β1 silent group were lower than those in the endothelial progenitor cell group (P > 0.05). (4) Compared with the model group, the mRNA and protein expression levels of TGF-β1 and Smad-2 in the endothelial progenitor cell and TGF-β1 silent groups were significantly decreased, the levels of Smad-7 were significantly increased (both P < 0.05). All above levels in the TGF-β1 silent group were significantly superior to those in the endothelial progenitor cell group (P < 0.05). (5) To conclude, TGF-β1 silent endothelial progenitor cell transplantation has a protective effect against pulmonary fibrosis in rats and probably inhibits pulmonary fibrosis by reducing Smad-2 and enhancing Smad-7 expression.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Pulmonary Fibrosis, Endothelial Cells, Gene Silencing, Transforming Growth Factor beta1, Smad2 Protein, Smad7 Protein, Tissue Engineering

CLC Number:

Peng Qiufeng, Gao Jingzhen, Ye Kui, Xing Aimin. Transplantation of endothelial progenitor cells with RNA interference targeting transforming growth factor beta1 inhibits pulmonary fibrosis in rats[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(1): 90-95.

Figures/Tables 6

References

[1] 魏燕华,刘桂桃.肺纤维化的治疗进展[J].临床肺科杂志, 2014,19(2): 336-339.

[2] Kumagai S, Marumo S, Yamanashi K, et al. Prognostic significance of combined pulmonary fibrosis and emphysema in patients with resected non-small-cell lung cancer: a retrospective cohort study. Eur J Cardiothorac Surg. 2014;46(6):e113-119.

[3] 何爱明,金美芳,叶瑞洪.肺成纤维细胞在博来霉素致肺纤维化中的作用机制[J].心肺血管病杂志,2015,34(11):863-866.

[4] Pan Y, Fu H, Kong Q, et al. Prevention of pulmonary fibrosis with salvianolic acid a by inducing fibroblast cell cycle arrest and promoting apoptosis. J Ethnopharmacol. 2014;155(3):1589-1596.

[5] 肖梦.姜黄素对肺纤维化大鼠内皮祖细胞的影响[D]. 重庆:重庆医科大学, 2015.

[6] 陈玉凤.骨髓间充质干细胞对百草枯中毒导致肺纤维化的影响及机制[D]. 福州:福建医科大学,2015.

[7] 刘哲,张晓梅,尹婷,等.肺纤方提取物干预肺纤维化模型大鼠肺微血管内皮细胞新生血管形成的体外研究[J].北京中医药大学学报, 2015,38(4):233-235.

[8] 刘哲,张晓梅,秦慧慧,等.肺纤方提取物干预肺纤维化模型大鼠微血管内皮细胞黏附及游走迁移作用研究[J].中华中医药杂志, 2015,30(11):4042-4044.

[9] Mahmoudi T, Fathi F, Rezaei MJ, et al. Transgenic Mice Bone Marrow-Derived Mesenchymal Stem Cells Attenuate Bleomycin-Induced Pulmonary Fibrosis. International Congress of Immunology & Allergy of Iran, 2016.

[10] Zhou MI, Chen DL, Jiang T, et al. Effects of bone marrow-derived mesenchymal stem cells transfected with survivin on pulmonary fibrosis in mice. Exp Ther Med. 2015;10(5):1857-1864.

[11] Fikry EM, Safar MM, Hasan WA, et al. Bone Marrow and Adipose-Derived Mesenchymal Stem Cells Alleviate Methotrexate-Induced Pulmonary Fibrosis in Rat: Comparison with Dexamethasone. J Biochem Mol Toxicol. 2015;29(7):321-329.

[12] 龚如.内皮祖细胞在缺血性脑血管疾病治疗中的研究进展[J].国际神经病学神经外科学杂志,2014,41(2):130-133.

[13] 李文华,张群辉,戎浩,等.内皮祖细胞与高血压病的应用研究进展[J].中国组织工程研究,2016,20(15):2273-2280.

[14] 毛梅,王健瑜,汪丽,等.内皮祖细胞对急性损伤肺组织的抗炎作用[J].第三军医大学学报,2015,37(24):2444-2447.

[15] Li C, Wilson PB, Levine E, et al. TGF-beta1 levels in pre-treatment plasma identify breast cancer patients at risk of developing post-radiotherapy fibrosis. Int J Cancer. 1999;84(2):155-159.

[16] Lu C, Zhang J, Zhang D, et al. EPCs in vascular repair: how can we clear the hurdles between bench and bedside. Front Biosci (Landmark Ed). 2014;19:34-48.

[17] Rurali E, Bassetti B, Perrucci GL, et al. BM ageing: Implication for cell therapy with EPCs. Mech Ageing Dev. 2016;159:4-13.

[18] Chong MS, Ng WK, Chan JK. Concise Review: Endothelial Progenitor Cells in Regenerative Medicine: Applications and Challenges. Stem Cells Transl Med. 2016;5(4):530-538.

[19] Sun K, Zhou Z, Ju X, et al. Combined transplantation of mesenchymal stem cells and endothelial progenitor cells for tissue engineering: a systematic review and meta-analysis. Stem Cell Res Ther. 2016;7(1):151.

[20] Salmina AB, Morgun AV, Kuvacheva NV, et al. Endothelial progenitor cells in cerebral endothelium development and repair (review). Sovremennye Tehnologii. 2015;6(4):213-221.

[21] Ikutomi M, Sahara M, Nakajima T, et al. Diverse contribution of bone marrow-derived late-outgrowth endothelial progenitor cells to vascular repair under pulmonary arterial hypertension and arterial neointimal formation. J Mol Cell Cardiol. 2015;86:121-135.

[22] Doyle MF, Tracy RP, Parikh MA, et al. Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema. PLoS One. 2017;12(3):e0173446.

[23] Xu BJ, Chen J, Chen X, et al. High shear stress-induced pulmonary hypertension alleviated by endothelial progenitor cells independent of autophagy. World J Pediatr. 2015;11(2):171-176.

[24] 孙宏,刘显东,吕迪宇,等.转化生长因子-β/Smad信号通路对急性肺损伤小鼠免疫平衡的影响[J].中华危重病急救医学, 2016,28(11):967-972.

[25] Ji H, Li Y, Jiang F, et al. Inhibition of transforming growth factor beta/SMAD signal by MiR-155 is involved in arsenic trioxide-induced anti-angiogenesis in prostate cancer. Cancer Sci. 2014;105(12): 1541-1549.

[26] 顾刘宝,卞茸文,涂玥,等.虫草素调控eIF2α/TGF-β/Smad信号通路改善肾间质纤维化的机制[J].中国中药杂志, 2014,39(21):4096-4101.

[27] 邵笑,祝骥,赵峻峰,等.丹参酮ⅡA对大鼠肺纤维化细胞TGF-β1/Smads信号通路的影响[J].浙江中西医结合杂志, 2016,26(5):414-417.