人脐带间充质干细胞经不同移植途径治疗肝硬化大鼠模型的效果比较

邱英麒; 王宏伟; 朱宏岩; 於洪亮; 谢凡; 姜翠宝

doi:10.3969/j.issn.1001-5256.2023.12.016

人脐带间充质干细胞经不同移植途径治疗肝硬化大鼠模型的效果比较

DOI: 10.3969/j.issn.1001-5256.2023.12.016

1.
海口市人民医院临床研究中心，海口 570208
2.
上海泉生生物工程有限公司，上海 201401

基金项目:

国家卫健委干细胞临床研究项目 (MR-46-21-014648)

伦理学声明：本研究方案于2018年11月12日经由上海交通大学实验动物伦理委员会审批，批号：20181112，符合实验室动物管理与使用准则。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：邱英麒负责课题研究和设计；姜翠宝负责研究数据收集分析，论文撰写；谢凡、朱宏岩参与研究数据的获取分析解释过程；王宏伟提供指导意见，以及论文修改与定稿；於洪亮负责文章校核与修订。

详细信息

通信作者:
姜翠宝， jiangcuibao@xibaozhiliao.cn （ORCID： 0000-0001-6213-0829）

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出版历程
- 收稿日期: 2022-11-21
- 录用日期: 2023-02-03
- 出版日期: 2023-12-12

Efficacy of human umbilical cord mesenchymal stem cells via different transplantation approaches in treatment of rats with liver cirrhosis

1.
Clinical Research Center of Haikou People’s Hospital，Haikou 570208，China
2.
Shanghai Quansheng Bioengineering Co.，Ltd.，Shanghai 201401，China

Research funding:

National Health Commission Project： Stem Cell Clinical Research (MR-46-21-014648)

More Information

Corresponding author: JIANG Cuibao， jiangcuibao@xibaozhiliao.cn （ORCID： 0000-0001-6213-0829）

摘要

摘要: 目的探讨人脐带间充质干细胞（hUC-MSC）制剂（冷冻细胞制剂和新鲜细胞制剂）通过两种给药途径（门静脉/尾静脉）移植对肝硬化模型大鼠的治疗效果。方法 70只SPF级健康雄性SD大鼠随机分为正常组（n=13，予以普通自来水及普通鼠粮喂养）和肝硬化模型组（n=57，给予CCl₄/橄榄油溶液混合液，皮下多点注射造模）。第8周观察两组大鼠的生长状况，取正常组及肝硬化模型组小鼠各3只行组织病理学检查，明确肝硬化形成。选取50只肝硬化模型组大鼠按随机数字表法分为模型组、门静脉（新鲜细胞制剂）组、门静脉（冷冻细胞制剂）组、尾静脉（新鲜细胞制剂）组、尾静脉（冷冻细胞制剂）组，每组10只。分别取新鲜或冷冻hUC-MSC制剂通过门静脉或尾静脉途径移植。给药4周后，比较各组大鼠肝功能指标、肝纤维化程度变化。计量资料两组间比较采用成组t检验；多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。结果造模第8周时，肝硬化模型组大鼠肝脏形成多个大小不等的假小叶，符合肝硬化诊断标准，ALT、AST、TBil、ALP水平相较于正常组均明显升高（P值均<0.001），肝硬化大鼠造模成功。第12周时5组大鼠ALT、AST、TBil、ALP水平比较差异均有统计学意义（F值分别为232.00、177.10、112.30、121.70，P值均<0.001）。进一步两两比较结果显示，模型组ALT、AST、TBil、ALP水平均显著高于正常组（P值均<0.01）；门静脉（新鲜细胞制剂）组、门静脉（冷冻细胞制剂）组、尾静脉（新鲜细胞制剂）组、尾静脉（冷冻细胞制剂）组ALT、AST、TBil、ALP水平均显著低于模型组（P值均<0.01）。结论 hUC-MSC移植4周可改善肝硬化模型大鼠肝功能和肝纤维化程度，细胞制剂的不同、给药途径的不同对治疗结果无明显影响。
- 肝硬化，实验性 /
- 间质干细胞移植 /
- 大鼠， Sprague-Dawley
Abstract: Objective To investigate the therapeutic effect of the frozen and fresh preparations of human umbilical cord mesenchymal stem cells （hUC-MSC） on a rat model of liver cirrhosis after transplantation via the portal vein or the caudal vein. Methods A total of 70 specific pathogen-free healthy male Sprague-Dawley rats were randomly divided into normal group （13 rats fed with ordinary tap water and rat food） and liver cirrhosis model group （57 rats given subcutaneous multi-point injection of mixed carbon tetrachloride/olive oil solution）. At week 8， the growth of rats was observed for both groups， and 3 rats were selected from each group for histopathological examination to confirm the formation of liver cirrhosis. A total of 50 rats were selected from the liver cirrhosis model and were divided into model group， portal vein group+fresh cell preparation group， portal vein+frozen cell preparation group， caudal vein+fresh cell preparation group， and caudal vein+frozen cell preparation group using a random number table， with 10 rats in each group. Fresh or frozen hUC-MSC were transplanted via the portal vein or the caudal vein， and after 4 weeks of administration， the different groups were compared in terms of the changes in liver function parameters and liver fibrosis degree. Continuous data were expressed as mean±standard deviation， and the independent-samples t test was used for comparison between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results At week 8 of modeling， the model group showed the formation of pseudolobules of different sizes in the liver and met the diagnostic criteria for liver cirrhosis， with significant increases in the levels of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， total bilirubin （TBil）， and alkaline phosphatase （ALP） compared with the normal group （all P<0.001）， suggesting that the rat model of liver cirrhosis was established successfully. There were significant differences in the levels of ALT， AST， TBil， and ALP between the five groups （F=232.00， 177.10， 112.30， 121.70， all P<0.001）. Further comparison between two groups showed that the model group had significantly higher levels of ALT， AST， TBil， and ALP than the normal group （all P<0.01）， and the portal vein group+fresh cell preparation group， the portal vein+frozen cell preparation group， the caudal vein+fresh cell preparation group， and the caudal vein+frozen cell preparation group had significantly lower levels of ALT， AST， TBil， and ALP than the model group （all P<0.01）. Conclusion There are significant improvements in liver function and liver fibrosis degree in a rat model of liver cirrhosis at week 4 after the transplantation of hUC-MSC， and frozen or fresh cell preparation and different transplantation approaches have no significant influence on treatment outcome.
- Liver Cirrhosis， Experimental /
- Mesenchymal Stem Cell Transplantation /
- Rats， Sprague-Dawley

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图 1 各组大鼠体质量变化趋势

Figure 1. Body weight change trend of rats in each group

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图 2 模型组和正常组的HE、Masson、天狼星红染色结果

注： a，正常组（HE染色，×100）；b，造模组（HE染色，×100）；c，正常组（Masson染色，×100）；d，造模组（Masson染色，×100）；e，正常组（天狼星红染色，×100）；f，造模组（天狼星红染色，×100）。

Figure 2. H&E，Masson and Sirus red staining results of model group and normal group

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图 3 第12周终点各组肝功能指标结果比较

注：与正常组比较，*P<0.01；与模型组比较，^#P<0.01。

Figure 3. Comparison the indicators of liver function among groups at the 12^th week

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图 4 第12周终点各组染色结果（×100）

Figure 4. H&E， Masson， Sirus red staining results among groups at the 12^th week （×100）

下载: 全尺寸图片幻灯片

表 1 第8周大鼠肝功能检测结果比较

Table 1. Comparison of liver function test results of rats at the 8^th week

组别	动物数（只）	ALT（U/L）	AST（U/L）	TBil（μmol/L）	ALP（U/L）
正常组	10	50.04±3.96	197.00±5.95	0.55±0.08	114.00±7.75
肝硬化模型组	50	102.61±7.25	316.63±12.05	1.20±0.18	197.60±11.00
t值		32.48	47.13	18.11	28.80
P值		<0.001	<0.001	<0.001	<0.001

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表 2 第12周大鼠肝功能检测结果比较

Table 2. Comparison of liver function test results of rats at the 12^th week

组别	动物数（只）	ALT（U/L）	AST（U/L）	TBil（μmol/L）	ALP（U/L）
正常组	10	62.68±7.25	203.00±12.12	0.53±0.10	134.00±8.94
模型组	10	162.38±8.61^1）	348.55±12.12^1）	2.00±0.21^1）	273.50±18.43^1）
门静脉（新鲜细胞制剂）组	10	86.60±7.40 ²^）	241.95±11.35 ²^）	0.56±0.11 ²^）	148.88±19.92 ²^）
门静脉（冷冻细胞制剂）组	10	59.61±7.49 ²^）	141.89±15.40 ²^）	0.95±0.16 ²^）	177.88±8.85 ²^）
尾静脉（新鲜细胞制剂）组	10	68.80±5.62 ²^）	230.04±12.22 ²^）	0.45±0.14 ²^）	136.75±7.13 ²^）
尾静脉（冷冻细胞制剂）组	10	88.71±5.92 ²^）	242.08±22.59 ²^）	1.06±0.18 ²^）	190.38±11.81 ²^）
F值		232.00	177.10	112.30	121.70
P值		<0.001	<0.001	<0.001	<0.001
注：与正常组比较，1）P<0.01；与模型组比较，2）P<0.01。

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