Role of mature liver cells and oval cells in liver regeneration after liver injury

doi:10.3969/j.issn.2095-4344.0401

Abstract

Abstract:

BACKGROUND: Retrorsine (RS) is a chemical agent for the long-term inhibition of mature liver cell division and proliferation.
OBJECTIVE: To establish a rat model of liver injury by combined use of RS and 1/3 partial hepatectomy, to observe the proliferation of liver cells and oval cells in rats after liver injury, and to discuss the relationship between liver regeneration and mature liver cells and oval cells after liver injury.
METHODS: Thirty male Sprague-Dawely rats were randomized into two groups (n=15 per group): RS group and control group. Rats in the RS group were subjected to intraperitoneal injection of RS, 30 mg/kg, twice in total, with 2 weeks in between; and rats in the control group were injected physiological saline instead of RS. Four weeks after the last injection, the 1/3 partial hepatectomy was performed in two groups of rats. Liver pathological and morphological changes as well as cell proliferation were observed, and CK19 and C-kit immunohistochemical detections of the rat liver in the two groups were conducted at different time points after operation.
RESULTS AND CONCLUSION: At 20 days after operation, the body mass of the RS group rats was still lower than the baseline, and the liver increase was obviously less than that in the control group; there was cell body swelling shown by hematoxylin-eosin staining, loose cytoplasm, extensive vacuoles degeneration of liver cells, and clustered or scattered oval cells around the portal area of small bile duct and in the hepatic lobule. However, in the control group, the body mass was close to the baseline, liver damage was lighter than that in the RS group, a large number of mature liver cells proliferated under BrdU Immunofluorescence at 20 days after operation; liver oval cells proliferated and distributed in the liver cell line at 14 days after operation, with morphology and immunohistochemical markers consistent with oval cells in the RS group. These findings indicate that the rat model of acute liver injury is successfully established by combining retrorsine with 1/3 partial hepatectomy, liver poisoning and the proliferation of liver stem cells and mature liver cells after poisoning can be seen in the experiment, which firmly confirm that liver cell renewal and regeneration after injury is accredited to the combined action of liver stem cells in liver basin and mature liver cells.

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: Hepatectomy, Hepatocytes, Tissue Engineering

CLC Number:

R394.2

Wang Ya-ping, Kong Xiang-ping, Zhuo Li, Tang Xiao-ping, Gao Hong-bo, Tong Ming-hua. Role of mature liver cells and oval cells in liver regeneration after liver injury[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(1): 40-45.

Figures/Tables 2

2.3 苏木精-伊红染色结果肝脏1/3切除后第3天，倒千里光碱/肝脏1/3切除组大鼠肝脏可见胞体肿大、胞浆疏松、肝细胞广泛空泡变性，散在肝组织小灶凝固性坏死(图3A)；术后第7天可见肝细胞小灶状坏死、汇管区小胆管周围和肝小叶内可见成簇或散在分布的卵圆细胞增生 (图3B)；第14天这种有增生倾向的卵圆细胞减少，部分细胞呈团束状、细胞间无管腔或有围管现象(图3C)。肝脏1/3切除组仅见肝细胞和胆管细胞轻微固缩、部分轻度水变性和少数细胞轻微空泡变性，第14天5例中2例损伤较重的大鼠汇管区可见炎细胞浸润，伴少量卵圆细胞出现并向小叶内延伸(图3D)。直到术后第20天仍能观察到卵圆细胞的增生。 2.4 BrdU免疫荧光化学检测结果见图4。肝脏1/3切除后第3天，倒千里光碱/肝脏1/3切除组大鼠肝组织内可见散在数个BrdU阳性的肝卵圆细胞，细胞核小，仅正常肝细胞核的1/4左右，多出现在细胆管壁和基膜外侧及少部分肝细胞索中(图4A)。肝脏1/3切除组大鼠肝组织内可见较多的BrdU阳性成熟肝细胞。核体积较大，圆形，与附近肝细胞核体积相仿，只出现在肝板内(图4D)。其中，倒千里光碱/肝脏1/3切除组在各时间点上均看到少量成熟肝细胞的增殖。两组大鼠肝脏BrdU阳性细胞在术后第7天增殖最活跃，直到第14天仍维持在一定高的水平上，其中倒千里光碱/肝脏1/3切除组出现数十个至数百个BrdU阳性卵圆细胞团(图4B)。同一时间点，对照组未见肝细胞团出现(图4E)，BrdU阳性细胞数与实验组接近，稍低于实验组，但组间差异无显著性意义(P > 0.05)。随着时间的增加，增殖细胞的数量逐渐减少，20 d左右基本消失(图4C、F)。值得注意的是：①倒千里光碱/肝脏1/3切除组的各时间点上均可见少量成熟肝细胞的增殖；②肝脏1/3切除组术后14 d切片中可见BrdU阳性的肝卵圆细胞，多出现在肝细胞索中，形态与倒千里光碱/肝脏1/3切除组卵圆细胞一致。 2.5 CK19和C-kit免疫组织化学染色结果阳性细胞表现为细胞浆呈棕黄色，均匀弥漫性。倒千里光碱/肝脏1/3切除组大鼠术后第3天在肝脏汇管区附近可见核呈卵圆形，胞质较少的CK19阳性细胞，多出现在细胆管壁和基膜外侧(图5A)；第7天阳性细胞增加，并逐渐向肝小叶内穿插生长(图5B)；之后阳性细胞数逐渐减少，20 d左右仅见少量阳性细胞(图5C)。C-Kit染色结果基本和CK19相同(图5D-F)。肝脏1/3切除组术后3，7 d在切片中均未见CK19阳性细胞(图5G)，术后第14天作者在其中2例大鼠切片中发现CK19阳性肝卵圆细胞，多出现在肝细胞索中，形态和免疫组织化学标记与倒千里光碱/肝脏1/3切除组卵圆细胞一致(图5H)。2例大鼠直到术后第20天仍能观察到卵圆细胞的增生(图5I)。"

References

[1] Chen S, Xu L, Lin N,et al.Activation of Notch1 signaling by marrow-derived mesenchymal stem cells through cell-cell contact inhibits proliferation of hepatic stellate cells. Life Sci. 2011;89(25-26):975-981.

[2] 张伟,陈孝平.人肝脏祖细胞研究进展[J].中华肝脏病杂志, 2006, 14(11): 875-877.

[3] Zhang Y, Bai XF, Huang CX. Hepatic stem cells: existence and origin. World J Gastroenterol.2003;9(2):201-204.

[4] 刘君,薛玲.肝卵圆细胞分化调控机制的研究进展[J].中华肝脏病杂志, 2005, 13(12): 951-953.

[5] Walkup MH, Gerber DA.Hepatic stem cells: in search of. Stem Cells.2006;24(8):1833-1840.

[6] Shupe T, Petersen BE. Potential applications for cell regulatory factors in liver progenitor cell therapy. Int J Biochem Cell Biol;2011;43(2): 214-221.

[7] Burra P, Bizzaro D, Ciccocioppo R,et al.Therapeutic application of stem cells in gastroenterology: an up-date. World J Gastroenterol.2011;17(34):3870-3880.

[8] Best DH, Butz GM, Coleman WB. Cytokine-dependent activation of small hepatocyte-like progenitor cells in retrorsine-induced rat liver injury. Exp Mol Pathol.2010;88(1): 7-14.

[9] Gordon GJ, Coleman WB, Hixson DC, et al.Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response. Am J Pathol.2000;156(2): 607-619.

[10] Kohneh-Shahri N, Regimbeau JM, Terris B,et al.Liver repopulation trial using bone marrow cells in a retrorsine-induced chronic hepatocellular injury model. Gastroenterol Clin Biol.2006;30(3): 453-459.

[11] Laconi E, Oren R, Mukhopadhyay DK,et al.Long-term, near-total liver replacement by transplantation of isolated hepatocytes in rats treated with retrorsine. Am J Pathol. 1998;153(1): 319-329.

[12] Toma W, Trigo JR, de Paula AC,et al.Preventive activity of pyrrolizidine alkaloids from Seneciobrasiliensis (Asteraceae) on gastric and duodenal induced ulcer on mice and rats. J Ethnopharmacol.2004;95(2-3):345-351.

[13] Small AC, Kelly WR, Seawright AA, et al.Pyrrolizidine alkaloidosis in a two month old foal. Zentralbl Veterinarmed A.1993;40(3): 213-218.

[14] Picard C, Lambotte L, Starkel P, et al.Retrorsine: a kinetic study of its influence on rat liver regeneration in the portal branch ligation model.J Hepatol.2003;39(1):99-105.

[15] 马明,盛哲津,张梦杰,等.肝脏大部分切除实验在小鼠肝再生研究中的应用[J].中国细胞生物学学报, 2011, 33(12): 112-118.

[16] Mroczek T, Glowniak K, Wlaszczyk A. Simultaneous determination of N-oxides and free bases of pyrrolizidine alkaloids by cation-exchange solid-phase extraction and ion-pair high-performance liquid chromatography. J Chromatogr A.2002;949(1-2): 249-262.

[17] 余宏宇,李文林,谢东甫,等.小鼠单纯肝切除后再生肝组织内大小核分裂相和卵圆细胞的观察[J].第二军医大学学报, 2005,26(3): 251-256.

[18] 廖芝玲,陈加玲,邝晓聪,等.倒千里光碱对肝大部分切除小鼠肝脏损伤后再生修复的影响[J].中国组织工程研究与临床康复，2010, 14(6):1023-1026.

[19] Kimura M, Watanabe M, Ishibashi N, et al.Acyclic retinoid NIK-333 accelerates liver regeneration and lowers serum transaminase activities in 70% partially hepatectomized rats, in vivo. Eur J Pharmacol.2010;643(2-3): 267-273.

[20] Gomez EV, Perez YM, Sanchez HV,et al.Antioxidant and immunomodulatory effects of Viusid in patients with chronic hepatitis C. World J Gastroenterol.2010;16(21):2638-2647.

[21] 李劲,张建,刘光泽,等. D-氨基半乳糖与脂多糖对小鼠肝脏损伤后再生修复的影响[J].南方医科大学学报,2012, 32(1): 50-54.

[22] Copper RA, Bowers RJ, Beckham CJ, et al.Preparative separation of pyrrolizidine alkaloids by high-speed counter-current chromatography. J Chromatogr A.1996; 732(1): 43-50.

[23] Sieders E, De Somer F, Bouchez S,et al.Haemostasis monitoring during sequential aortic valve replacement and liver transplantation. Acta Gastroenterol Belg.2010;73(1): 65-68.

[24] Ichinohe N, Kon J, Sasaki K,et al.Growth ability and repopulation efficiency of transplanted hepatic stem cells, progenitor cells, and mature hepatocytes in retrorsine-treated rat livers. Cell Transplant.2012;21(1):11-22.