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Numb基因过表达的人脐带间充质干细胞移植对胆汁淤积性肝纤维化的干预作用及其机制
DOI: 10.12449/JCH260110
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：慕永平、赵长青、张士豪负责设计论文框架，起草论文；张士豪、杨明艳、邢飞飞、陈高峰负责实验操作，研究过程的实施；张士豪、赵长青负责数据收集，统计学分析，图表绘制；慕永平、张士豪负责论文修改；慕永平、刘平、刘伟、陈佳美负责拟定写作思路，指导撰写文章；慕永平负责最后定稿。张士豪和赵长青对本文贡献相同，同为第一作者。
Effect and mechanism of transplantation of human umbilical cord mesenchymal stem cells with overexpression of the Numb gene in treatment of cholestatic liver fibrosis
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摘要:
目的 探讨Numb基因过表达的人脐带间充质干细胞（hUC-MSC）移植对胆汁淤积性肝纤维化（CLF）的干预作用及其机制。 方法 采用慢病毒转染技术过表达hUC-MSC的Numb基因（hUC-MSCNumb-OE），并以空载慢病毒转染的hUC-MSC（hUC-MSCOE-EV）为阴性对照。采用胆管结扎（BDL）建立CLF大鼠模型，随机分为BDL组、hUC-MSC组、hUC-MSCOE-EV组和hUC-MSCNumb-OE组，同时设有假手术组（Sham组）。各干预组于BDL后经脾脏一次性注射相应细胞，4周末取材。检测血清生化学、肝组织病理、肝组织羟脯氨酸（Hyp）水平、肝星状细胞活化、胆管反应、肝再生及Numb-p53信号轴关键分子的表达水平。计量资料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 与BDL组比较，hUC-MSC组和hUC-MSCOE-EV组血清生化学指标（天冬氨酸氨基转移酶、γ-谷氨酰转移酶、总胆汁酸、总胆红素和直接胆红素）、肝纤维化相关指标（Hyp、α-平滑肌肌动蛋白、肿瘤坏死因子-α和转化生长因子-β1）、胆管反应指标（细胞角蛋白7、细胞角蛋白19）均显著降低，差异有统计学意义（P值均＜0.05）；且hUC-MSCNumb-OE组的上述指标较hUC-MSCOE-EV组进一步改善，差异有统计学意义（P值均＜0.05）。此外，与hUC-MSCOE-EV组比较，hUC-MSCNumb-OE组肝再生相关指标（白蛋白、肝细胞核因子4α）、Numb-p53信号轴相关分子（Numb、pNumb、Mdm2、p53）的表达水平均较hUC-MSCOE-EV组显著改善，差异有统计学意义（P值均＜0.05）。 结论 过表达Numb基因可增强hUC-MSC对CLF的干预效果，其机制可能与激活Numb-PTBL-p53-HNF4α轴，促hUC-MSC肝向分化，进而促进肝再生有关。 -
关键词:
- 肝纤维化 /
- Numb基因 /
- 脐带间充质干细胞 /
- 胆管反应 /
- Numb-PTBL-p53-HNF4α轴
Abstract:Objective To investigate the effect and mechanism of transplantation of human umbilical cord mesenchymal stem cell （hUC-MSC） with overexpression of the Numb gene in the treatment of cholestatic liver fibrosis （CLF）. Methods The technique of lentiviral transfection was used to induce the overexpression of the Numb gene in hUC-MSC （hUC-MSCNumb-OE）， and hUC-MSC transfected with empty vector （hUC-MSCOE-EV） was used as negative control. Bile duct ligation （BDL） was performed to establish a rat model of CLF， and then the rats were randomly divided into BDL group， hUC-MSC group， hUC-MSCOE-EV group， and hUC-MSCNumb-OE group， while a sham-operation group was also established. The rats in the intervention groups were given a single splenic injection of the corresponding cells after BDL， and samples were collected at the end of week 4. Related indicators were measured， including serum biochemistry， liver histopathology， the content of hydroxyproline （Hyp） in the liver， hepatic stellate cell activation， ductular reaction， liver regeneration， and the expression levels of key molecules in the Numb-p53 signaling axis. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results Compared with the BDL group， the hUC-MSC group and the hUC-MSCOE-EV group had significant reductions in the levels of serum biochemical parameters （aspartate aminotransferase， gamma-glutamyl transpeptidase， total bile acid， total bilirubin， and direct bilirubin）， liver fibrosis markers （the content of Hyp and the expression levels of alpha-smooth muscle actin， tumor necrosis factor-α， and transforming growth factor-beta 1）， and ductular reaction markers （the expression levels of CK7 and CK19） （all P <0.05）， and compared with the hUC-MSCOE-EV group， the hUC-MSCNumb-OE group had significantly greater improvements in the above indicators （all P <0.05）. In addition， compared with the hUC-MSCOE-EV group， the hUC-MSCNumb-OE group had significant improvements in the expression levels of liver regeneration-related markers （albumin and hepatocyte nuclear factor 4α） and the molecules associated with the Numb-p53 signaling axis （Numb， pNumb， Mdm2， and p53） （all P <0.05）. Conclusion Overexpression of the Numb gene can enhance the therapeutic effect of hUC-MSC on CLF， possibly by activating the Numb-PTBL-p53-HNF4α axis， promoting the hepatic differentiation of hUC-MSCs and subsequently enhancing liver regeneration. -
注： a，hUC-MSCNumb-OE镜下形态学观察（×50）；b，Numb的mRNA表达水平，**P<0.01；c，hUC-MSCNumb-OE成骨诱导（茜素红染色，×40）；d，hUC-MSCNumb-OE成脂诱导（油红O染色，×400）；e，hUC-MSCNumb-OE流式细胞鉴定。
图 1 hUC-MSCNumb-OE细胞表型和分化潜能检测
Figure 1. Phenotype and differentiation potential detection of hUC-MSCNumb-OE
注： a，苏木素-伊红染色（×100）；b，天狼星红胶原染色（×100）；c，血清生化指标和肝组织Hyp水平。ALT，丙氨酸氨基转移酶；AST，天冬氨酸氨基转移酶；ALP，碱性磷酸酶；GGT，γ-谷氨酰转移酶；TBA，总胆汁酸；TBil，总胆红素； DBil，直接胆红素； IBil，间接胆红素； Hyp，羟脯氨酸。*P<0.05，**P<0.01。
图 2 hUC-MSC Numb-OE移植对肝脏炎症反应及肝纤维化的影响
Figure 2. Effect of hUC-MSC Numb-OE transplantation on hepatic inflammation and fibrosis
注： a，α-SMA免疫组化染色（×200）；b，肝组织TNF-α、TGF-β1和 α-SMA免疫印迹条带；c，TNF-α、TGF-β1和 α-SMA免疫印迹条带灰度积分；d，TNF-α、TGF-β1和α-SMA的mRNA表达水平。TNF-α，肿瘤坏死因子-α；TGF-β1，转化生长因子-β1；α-SMA，α-平滑肌肌动蛋白；GAPDH，甘油醛-3-磷酸脱氢酶。*P<0.05，**P<0.01。
图 3 hUC-MSCNumb-OE移植对HSC活化的影响
Figure 3. Effect of hUC-MSCNumb-OE transplantation on hepatic stellate cell activation
注： a，CK7免疫组化染色（×200）；b，CK19免疫组化染色；c，CK7和CK19免疫印迹条带；d，CK7和CK19免疫印迹条带灰度积分；e，CK7和CK19的mRNA表达水平。CK7，细胞角蛋白7；CK19，细胞角蛋白19；GAPDH，甘油醛-3-磷酸脱氢酶。*P<0.05，**P<0.01。
图 4 hUC-MSCNumb-OE移植对胆管反应的影响
Figure 4. Effect of hUC-MSCNumb-OE transplantation on ductular reaction
注： a，Alb免疫组化染色（×200）；b，HNF4α免疫组化染色（×200）；c，Alb、HNF4α免疫印迹条带；d，Alb、HNF4α免疫印迹条带灰度积分；e，Alb、HNF4α mRNA表达水平；f，Numb1/2、pNumb、Mdm2和p53免疫印迹条带；g，Numb1/2、pNumb、Mdm2和p53免疫印迹条带灰度积分；h，Numb、Mdm2和p53的mRNA表达水平；i，血清Alb水平。Alb，白蛋白；HNF4α，肝细胞核因子4α；GAPDH，甘油醛-3-磷酸脱氢酶。*P<0.05，**P<0.01。
图 5 hUC-MSCNumb-OE移植对肝细胞增殖及Numb1/2-p53轴的影响
Figure 5. hUC-MSCNumb-OE transplantation on liver cell proliferation and Numb1/2-p53 axis
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