Human amnion epithelial cell transplantation provides a local repair of liver damage induced by carbon tetrachloride in mice

doi:10.3969/j.issn.2095-4344.0438

Abstract

Abstract:

BACKGROUND: Human amnion epithelial cells (hAECs) have many merits that embryonic stem cells and adult stem cells do not have, such as no tumorigenicity, rich sources, easy to obtain, low immunogenicity and no medical ethics limit. Therefore, hAECs are expected to be important seed cells for clinical transplantation.
OBJECTIVE: To observe the therapeutic effect of hAECs transplantation labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) in a mouse model of liver damage induced by carbon tetrachloride.
METHODS: hAECs from the human amniotic membrane were collected using enzymatic digestion, and morphology of cells was observed. Expression of keratin 19 in hAECs was detected by immunocytochemistry. Model of liver damage was made in mice by intraperitoneal injection of carbon tetrachloride. Then, CFSE-labeled hAECs were injected into the liver damage mice via tail vein. Histopathological changes and liver function in mice were observed at 7 and 30 days after transplantation, respectively.
RESULTS AND CONCLUSION: The high-purity hAECs were successfully isolated, which expressed keratin 19 shown by immunocytochemical staining. Frozen sections of immunoflrorescence showed that hAECs could be moved to the damaged liver, and exhibited remarkable repair effects on the liver function and histopathology in mice. These findings indicate that hAECs can be used for xenotransplantation and function to promote physiological recovery from liver injury, thereby providing experimental evidence for liver repair with cell transplantation.

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: Tissue Engineering, Stem Cell Transplantation, Immunohistochemistry

CLC Number:

R394.2

Liu Mo-ning, Zhang Yu-kun, Li Yan, Cao Gui-fang, Bai Li-heng. Human amnion epithelial cell transplantation provides a local repair of liver damage induced by carbon tetrachloride in mice[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(5): 698-703.

Figures/Tables 2

2.1 实验动物数量分析纳入小鼠60只均进入结果分析，造模过程中发现死亡和感染后及时补充，无脱失值。 2.2 人羊膜上皮细胞的形态特征原代培养接种24 h后，可见大部分细胞仍悬浮在培养基中，但已可见少量形态呈圆形、三角形、多边形，形态不规则的贴壁细胞，大部分细胞仍悬浮于培养基中(图1A)。培养72 h后换液去除未贴壁细胞，可见细胞多数呈卵圆形，折光性强，细胞形态大小均一(图1B)。第2代细胞呈铺路石样排列呈单片状，上皮细胞形态显著(图1C)。第7代细胞退化严重，折光性减弱，细胞边界模糊，形态多样性，生长缓慢(图1D)。 2.3 各组小鼠肝组织角蛋白19(CK19)的表达爬片完成后的第2代人羊膜上皮细胞，经免疫细胞化学法检测。结果显示人羊膜上皮细胞细胞核经苏木精染色呈蓝色，细胞质可见棕黄色着色，表明CK19在人羊膜上皮细胞的细胞质中呈阳性表达，用DPBS代替一抗孵育的对照组未见棕黄色着色为阴性(图2)。 2.4 小鼠肝组织CFSE标记人羊膜上皮细胞形态使用第2代人羊膜上皮细胞，CFSE染色标记人羊膜上皮细胞后，用荧光显微镜观察，结果显示经过处理的人羊膜上皮细胞的细胞核、细胞质及细胞膜均被CFSE染色标记，发出绿色荧光，荧光呈强阳性(图3)。 2.5 检测CFSE标记的人羊膜上皮细胞在小鼠肝脏内定植情况取移植人羊膜上皮细胞7 d的治疗组小鼠和尾静脉注射生理盐水对照组小鼠肝脏组织，进行冰冻切片制作，DAPI染色后，激光共聚焦显微镜观察见图4，实验组经CFSE标记的人羊膜上皮细胞细胞分布在小鼠肝小叶周围(图4A)，对照组小鼠肝脏组织未见绿色荧光细胞(图4C)。 2.6 人羊膜上皮细胞移植前后小鼠肝功变化与模型组相比，治疗组小鼠的天门冬氨酸转移酶及丙氨酸氨基转移酶表达量明显降低，并接近正常组小鼠肝功水平。结果表明移植人羊膜上皮细胞对小鼠肝功能有恢复作用(表1)。 2.7 移植人羊膜上皮细胞小鼠肝脏病理学变化苏木精-伊红染色结果显示，注射CCl4模型组的肝脏组织表面变性明显，肝脏细胞广泛坏死，有水肿现象，并有明显坏死灶(图5A)。移植人羊膜上皮细胞的治疗组小鼠肝脏组织切片染色后，结果显示小鼠肝脏中变性肝细胞减少，水肿现象有效缓解(图5B)。尾静脉注射生理盐水的对照组苏木精-伊红染色结果与模型组无显著差异(图5C)。"

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