Therapeutic effect of autologous source induced pluripotent stem cell transplantation on chronic hepatitis B-induced liver injury mice

doi:10.3969/j.issn.2095-4344.1551

Abstract

Abstract:

BACKGROUND: Autologous source induced pluripotent stem cell transplantation to improve liver tissue structure and function is a new research direction in the treatment of liver injury caused by chronic hepatitis B.
OBJECTIVE: To observe the effect of autologous skin-derived induced pluripotent stem cell transplantation on chronic hepatic injury in HBV transgenic mice.
METHODS: Achronic hepatic injury model of HBV transgenic mice (provided by the Beijing Vitalstar Biotechnology Co., Ltd. in China) was established with intraperitoneal injection of carbon tetrachloride. At 1 week after injection of carbon tetrachloride, skin fibroblasts from the model mice were reprogrammed into induced pluripotent stem cells and identified in vitro. Induced pluripotent stem cells (0.1 mL) were transplanted into the liver of model mice through the portal vein pathway as experimental group, and PBS (0.1 mL) was injected into the other mice as control group. Colonization of induced pluripotent stem cells was observed by frozen section at 7 and 14 days of transplantation. Liver function of the mice was detected by ELISA, and liver histopathological changes were observed by hematoxylin-eosin staining of paraffin section.
RESULTS AND CONCLUSION: Skin fibroblasts from model mice were successfully reprogrammed into induced pluripotent stem cells in vitro that had multipotent differentiation potential. At 7 days after transplantation, fluorescent labeled induced pluripotent stem cells were found in the mouse liver of the experimental group. The levels of serum alanine aminotransferase, aspartate aminotransferase, glutamyl transpeptidase, and total bilirubin in the experimental group were lower than those in the control group, and the level of albumin was higher than that in the control group (P < 0.05). Liver cell degeneration, necrosis and inflammatory cell infiltration in the mouse liver of the experimental group were significantly improved as compared with the control group. These results suggest that portal vein transplantation of autologous source induced pluripotent stem cells can treat chronic hepatitis B-induced liver injury in mice.

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: Hepatitis B, Induced Pluripotent Stem Cells, Stem Cell Transplantation, Tissue Engineering

CLC Number:

Pan Lijuan, Wang Rongli. Therapeutic effect of autologous source induced pluripotent stem cell transplantation on chronic hepatitis B-induced liver injury mice[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(5): 773-778.

Figures/Tables 1

2.1 诱导性多能干细胞的形态与鉴定传至第3代的诱导性多能干细胞呈克隆样生长，椭圆形细胞克隆边界清晰，克隆中细胞体积小，细胞核大，核仁明显，细胞形态一致，折光性强，排列紧密，见图1A；免疫荧光染色结果显示，细胞中多能性标志OCT4、NANOG和SSEA-1呈阳性表达，提示细胞具有多能性，见图1B；体外分化能力检测结果显示，将细胞悬浮培养10 d后形成拟胚体，提示细胞具有体外分化增殖能力，见图1C。上述结果表明转染后获得的细胞具有诱导性多能干细胞特征。 2.2 参与研究小鼠一般情况及数量分析 24只HBV转基因小鼠在造模后饮食次数减少、行动略迟缓、体质量减轻，对照组造模6周内死亡1只，PBS注射后死亡1只；实验组造模6周内死亡2只，诱导性多能干细胞移植后10只小鼠无死亡；两组均有10只小鼠的实验数据计入结果分析。 2.3 诱导性多能干细胞在小鼠肝脏中的定植为了细胞移植后示踪，门静脉注射前用荧光染料CFSE标记诱导性多能干细胞，CFSE与细胞膜、细胞质及细胞核上的蛋白结合，呈现绿色荧光，与细胞核的结合最多，见图2A，经CFSE染料标记的悬浮诱导性多能干细胞在荧光显微镜下观察可见绿色荧光，细胞核部位荧光强度较胞质高，说明诱导性多能干细胞活性较好；移植7 d后，实验组冰冻切片在荧光显微镜下观察显示小鼠肝脏内有点状散在分布的绿色荧光，强弱不等，说明荧光标记的诱导性多能干细胞在肝脏内定植，由于一旦细胞分裂，标记的荧光将减弱，提示移植的诱导性多能干细胞可能存在不同程度的分化，见图2B；移植后14 d，实验组小鼠肝脏冰冻切片在荧光显微镜下无明显绿色荧光出现，提示随着诱导性多能干细胞的分裂增殖，荧光逐渐减弱，见图2C。 2.4 诱导性多能干细胞移植对小鼠肝功能的影响为了明确移植诱导性多能干细胞对小鼠肝功能的影响，分别检测了移植7，14 d后血清中谷丙转氨酶、谷草转氨酶、谷氨酰转肽酶、白蛋白、总胆红素水平，见图3。对照组与正常小鼠肝功能比较，血清谷丙转氨酶、谷草转氨酶、谷氨酰转肽酶、总胆红素水平均升高，白蛋白水平降低，差异有显著性意义(P < 0.05)；与对照组比较，移植后7 d和14 d实验组血清谷丙转氨酶、谷草转氨酶、谷氨酰转肽酶、总胆红素水平均不同程度的降低，白蛋白水平则升高，差异有显著性意义(P < 0.05)；与移植后7 d比较，实验组移植14 d后血清谷丙转氨酶、谷草转氨酶、谷氨酰转肽酶、总胆红素水平均降低，白蛋白水平升高，差异有显著性意义(P < 0.05)，提示诱导性多能干细胞移植后，随着时间的延长，肝功能得到改善。 2.5 诱导性多能干细胞移植对损伤肝脏组织结构的影响为了深入研究移植诱导性多能干细胞对肝脏损伤的治疗作用，对损伤肝脏的组织病理学特征进行观察。移植7 d后，对照组小鼠肝脏中肝小叶结构紊乱，可见坏死的肝细胞，较多肝细胞内含有大小不一的脂滴呈脂肪变性，炎症细胞浸润明显，见图4A；移植14 d后，对照组小鼠肝脏中变性、坏死的细胞增多，中央静脉位于偏位，大量炎症细胞浸润，见图4B。移植7 d后，实验组小鼠肝脏的单个显微视野中坏死和脂肪变性的肝细胞数量减少，浸润的炎症细胞减少，门管区的小叶间静脉中可见上皮样细胞，见图4C；移植14 d后，实验组小鼠肝脏的单个显微视野中肝细胞脂肪变性、坏死及炎症细胞浸润改善明显，假小叶减少，可见肝小叶重建，见图4D。"

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