Differentiation of bone marrow mesenchymal stem cells into hepatocytes under induction by interleukin-1 beta combined with hepatocyte growth factor in vitro

doi:10.3969/j.issn.2095-4344.1831

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells have multi-lineage differentiation potential and can differentiate into a variety of cell lineages. It is currently considered that bone marrow mesenchymal stem cells are the most promising seed cells for the treatment of various end-stage liver diseases including liver failure. How to induce cell differentiation is the focus of research.
OBJECTIVE: To explore the ability of hepatocyte growth factor combined with interleukin-1β to induce bone marrow mesenchymal stem cells to differentiate into hepatocytes, and to find a suitable induction method.
METHODS: Passage 3 bone marrow mesenchymal stem cells were divided into control group, hepatocyte growth factor group, and low-, middle- and high-dose combined induction group (20 μg/L hepatocyte growth factor+5, 10, 20 μg/L interleukin-1β respectively). Morphological changes of the cells in each group were observed under inverted microscope. Immunohistochemical methods were used to detect expression of cytokeratin 18 and albumin at 7, 14, and 21 days of induction.
RESULTS AND CONCLUSION: There was no morphological change in the control group, whereas in the other four groups, the cells gradually developed from fusiform and prismatic to polygonal and round, similar to the morphology of hepatocytes. No cytokeratin 18 and albumin expression was observed in the control group, while cytokeratin 18 and albumin positively expressed in the other four groups, and the expression levels were gradually increased with the extension of induction time (P < 0.05). At each time point, the expression of albumin and CK18 upregulated with the increase of interleukin-1β concentration (P < 0.05). Therefore, combined use of hepatocyte growth factor and interleukin-1β has better induction effect than hepatocyte growth factor used alone. Moreover, the induction ability of the combined use is strengthened with the increase of interleukin 1β concentration between 0-20 μg/L.

Key words: liver failure, bone marrow mesenchymal stem cells, interleukin 1β, hepatocyte growth factor, induced differentiation, hepatocytes

CLC Number:

Pan Lijuan, Wang Rongli. Differentiation of bone marrow mesenchymal stem cells into hepatocytes under induction by interleukin-1 beta combined with hepatocyte growth factor in vitro[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(33): 5328-5333.

Figures/Tables 2

2.1 骨髓间充质干细胞的原代、传代培养及鉴定结果刚接种时细胞呈圆形，4 h可贴壁，1 d后可见大量透明且不规则的贴壁细胞，杂质细胞多。24 h首次换液后杂质细胞较前减少，三四天可见较多集落形成。刚开始时细胞呈梭形，中央可见细胞核。1周后可见细胞密集呈漩涡状生长，10 d左右细胞呈成纤维细胞样生长。待细胞融合至80%-90%时将细胞按1∶2比例传代接种于新的培养瓶中。待传至第3代后将较为纯化的骨髓间充质干细胞用于诱导培养肝细胞样细胞。传代后杂质细胞减少，细胞生长速度增快，3-5 d即可传代1次，传代后细胞形态见图1。 2.2 骨髓间充质干细胞的鉴定结果第3代骨髓间充质干细胞用于流式细胞检测，CD29、CD90为阳性，造血系干细胞表面标记CD45为阴性，见图2。 2.3 诱导分化后细胞形态空白对照组细胞形态未发生改变，呈长梭形。加入肝细胞生长因子和不同质量浓度白细胞介素1β后，倒置相差显微镜下观察各组细胞形态学变化。随着时间的延长，骨髓间充质干细胞分裂增殖停止，而且细胞发生形态学改变，在第6天至第7天时，细胞逐渐由双极成纤维细胞形态转变为上皮样细胞形态，随后细胞质进一步收缩，最终变为圆形且有双细胞核的细胞。第21天时细胞形态以类圆形为主，细胞呈肝细胞样，见图3。 2.4 免疫组化染色检测细胞中白蛋白表达空白对照组白蛋白始终表达阴性，未见阳性着色。加入细胞因子各组细胞白蛋白表达均为阳性，证明能够诱导分化为肝细胞样细胞，见图4，并且随着诱导时间延长白蛋白表达呈增加趋势(P均< 0.05)；随着白细胞介素1β质量浓度的增加白蛋白表达增加(P均< 0.05)，见表2。 2.5 免疫组化染色检测细胞中细胞角蛋白18表达空白对照组细胞角蛋白18始终表达阴性，未见阳性着色。加入细胞因子各组细胞角蛋白18表达均为阳性，胞浆出现棕黄色颗粒，证明能够诱导分化为肝细胞样细胞，见图5，并且随着诱导时间延长细胞角蛋白18表达呈增加趋势(P均< 0.05)；随着白细胞介素1β质量浓度的增加细胞角蛋白18表达增加(P均<0.05)，见表3。"

References

[1]Sun L, Fan X, Zhang L, et al. Bone mesenchymal stem cell transplantation via four routes for the treatment of acute liver failure in rats. Int J Mol Med. 2014;34(4):987-996.

[2]Yuan S, Jiang T, Sun L, et al. The role of bone marrow mesenchymal stem cells in the treatment of acute liver failure. Biomed Res Int. 2013;2013:251846.

[3]Zhang H, Fang J, Wu Y, et al. Mesenchymal stem cells protect against neonatal rat hyperoxic lung injury. Expert Opin Biol Ther. 2013;13(6):817-829.

[4]荣为为,韩明子,金世柱,等.骨髓间充质干细胞在组织工程研究中的应用新进展[J].现代生物医学进展, 2017,17(5):982-984.

[5]He Q, He X, Wang Y, et al. Transplantation of bone marrow-derived mesenchymal stem cells (BMSCs) improves brain ischemia-induced pulmonary injury in rats associated to TNF-α expression. Behav Brain Funct. 2016; 12: 9.

[6]Moore JK, Stutchfield BM, Forbes SJ. Systematic review: the effects of autologous stem cell therapy for patients with liver disease. Aliment Pharmacol Ther. 2014;39(7):673-685.

[7]Levine P, McDaniel K, Francis H, et al. Molecular mechanisms of stem cell therapy in alcoholic liver disease. Dig Liver Dis. 2014; 46(5):391-397.

[8]Salomone F, Barbagallo I, Puzzo L, et al. Efficacy of adipose tissue-mesenchymal stem cell transplantation in rats with acetaminophen liver injury. Stem Cell Res. 2013;11(3):1037-1044.

[9]Scott TR, Kronsten VT, Hughes RD, et al. Pathophysiology of cerebral oedema in acute liver failure. World J Gastroenterol. 2013;19(48):9240-9255.

[10]郑盛,尤丽英.间充质干细胞分化为功能性肝细胞的研究进展[J].中华肝胆外科杂志,2013,19(5):396-400.

[11]Bernal W, Hyyrylainen A, Gera A, et al. Lessons from look-back in acute liver failure? A single centre experience of 3300 patients. J Hepatol. 2013;59(1):74-80.

[12]Podoll AS, DeGolovine A, Finkel KW. Liver support systems--a review. ASAIO J. 2012;58(5):443-449.

[13]谢树才,张剑权,蒋锡丽,等.骨髓间充质干细胞诱导分化为肝细胞的方法及机制研究与进展[J].中国组织工程研究,2016, 20(50): 7586-7593.

[14]李伟,汪林,曾杰,等.瘀胆血清诱导骨髓间充质干细胞向肝细胞的分化[J].中国组织工程研究,2016,20(6):771-776.

[15]孙红,李继尧,于吉人. IL-1β对离体培养的醋氨酚损伤的小鼠肝细胞的保护作用及其机制的初步探讨[J].胃肠病学和肝病学杂志,1999, (1):13-15.

[16]de Girolamo L, Lucarelli E, Alessandri G, et al. Mesenchymal stem/stromal cells: a new ''cells as drugs'' paradigm. Efficacy and critical aspects in cell therapy. Curr Pharm Des. 2013;19(13): 2459-2473.

[17]Sun L, Fan X, Zhang L, et al. Bone mesenchymal stem cell transplantation via four routes for the treatment of acute liver failure in rats. Int J Mol Med. 2014;34(4):987-996.

[18]潘兴南,魏开鹏.肝细胞生长因子的浓度和诱导时间对人骨髓间充质干细胞向肝细胞分化的影响[J].肝脏,2013,18(6):382-385.

[19]袁仙丽,李明才,李燕,等.白细胞介素-38及其相关细胞因子在炎症中的作用[J].中国细胞生物学学报,2013,35(8):1232-1237.

[20]张东锋,王煜姝,滕军放.骨髓间充质干细胞对肝豆状核变性合并肝纤维化肝功能的临床研究[J].中国免疫学杂志,2016,32(2):193-196.

[21]罗显克,陆正峰,姜海行,等.肿瘤坏死因子-α刺激骨髓间充质干细胞后对肝星状细胞凋亡的促进作用[J].世界华人消化杂志,2012,20(19): 1713-1719.

[22]霍大浪,刘代顺,宁家辉,等. SD大鼠骨髓间充质干细胞-肝细胞共培养上清液细胞因子分析[J].重庆医学,2014,43(28):3766-3768.

[23]Lin L, Lin H, Bai S, et al. Bone marrow mesenchymal stem cells (BMSCs) improved functional recovery of spinal cord injury partly by promoting axonal regeneration. Neurochem Int. 2018;115: 80-84.

[24]陈鹏飞,魏文斌,谭远忠,等.生长因子体外诱导骨髓间充质干细胞向肝样细胞的分化[J].中国组织工程研究,2012,16(49):9214-9220.

[25]张美华,余卫,王巧瑜,等.骨髓间充质干细胞治疗大鼠白毒伞中毒致急性肝衰竭的效果[J].广东医学,2016,37(13):1917-1919.

[26]荣为为,韩明子,金世柱,等.骨髓间充质干细胞在组织工程研究中的应用新进展[J].现代生物医学进展,2017,17(5):982-984.

[27]王晓东,许烂漫,王丽萍,等.骨髓间充质干细胞免疫调节作用对急性肝功能衰竭大鼠肝再生的影响[J].中华传染病杂志,2016,34(2):97.

[28]潘兴南,魏开鹏.肝细胞生长因子的浓度和诱导时间对人骨髓间充质干细胞向肝细胞分化的影响[J].肝脏,2013,18(6):382-385.

[29]Xu TB, Li L, Luo XD, et al. BMSCs protect against liver injury via suppressing hepatocyte apoptosis and activating TGF-β1/Bax singling pathway. Biomed Pharmacother. 2017;96:1395-1402.

[30]徐土炳,李莉,罗兴迪,等.骨髓间充质干细胞在肝细胞损伤恢复中的作用[J].实用医学杂志,2017,33(5):687-692.

[31]焦栓林,赵晓蕊,欧阳洪,等.自体骨髓间充质干细胞联合促肝细胞生长素治疗失代偿期酒精性肝硬化疗效分析[J].实用肝脏病杂志, 2017,20(6):773-774.

[32]符真,张剑权,李志,等. BMSCs对肝癌肝切除后肝细胞再生的作用[J].海南医学,2017,28(16):2665-2668.

[33]徐土炳,李莉,罗兴迪,等.骨髓间充质干细胞在肝细胞损伤恢复中的作用[J].实用医学杂志,2017,33(5):687-692.

[34]夏金华,夏建川,林万飞.骨髓源间充质干细胞注射疗法对肝硬化大鼠模型免疫系统的调节作用及其相关机制研究[J].免疫学杂志,2017, 33(7):623-628.