Effect of different transplantation approaches of human umbilical cord menchymal stem cells on repairing hepatic ischemia-reperfusion injury in rat models
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摘要:
目的 探讨不同途径移植人脐带间充质干细胞（HU-MSCs）对大鼠肝脏缺血-再灌注损伤（HIRI）的修复效果。 方法 将40只雄性SD大鼠随机分为A组（对照组）、B组（模型组）、C组（门静脉移植干细胞组）、D组（尾静脉移植干细胞组），每组10只，B、C、D组采用Pringle法建立HIRI模型，其中B组不予进行干细胞移植，C组和D组分别经门静脉、尾静脉移植氯甲基苯甲酰氨（CM-DiL）标记的HU-MSCs 106个。分别于术前、移植后第1、3、5日抽血检测丙氨酸转氨酶（ALT）、肿瘤坏死因子（TNF）-α水平；移植后第5日处死各组大鼠，肝脏取材行苏木素-伊红（HE）及dUTP缺口末端标记（TUNEL）染色观察肝脏损伤情况，在激光共聚焦显微镜下观察HU-MSCs在各组大鼠肝脏分布情况。 结果 干细胞移植后C、D组ALT水平较B组降低，且C组ALT水平低于D组（均为P < 0.05）；C、D组炎症因子TNF-α水平明显低于B组，且与D组相比，C组下降更显著（均为P < 0.05）。肝组织HE染色提示B组肝脏损伤最严重，C组肝脏损伤轻于D组。肝组织TUNEL染色结果显示，C、D组的凋亡指数（AI）低于B组，且C组较D组低，差异均有统计学意义（均为P < 0.05）。激光共聚焦显微镜下可见，移植后C组与D组均可见CM-DiL标记的HU-MSCs，且C组的HU-MSCs数量高于D组（P < 0.05）。 结论 移植HU-MSCs能减轻HIRI，且门静脉移植效果优于尾静脉移植效果。 Abstract:Objective To investigate the effect of different transplantation approaches of human umbilical cord menchymal stem cells (HU-MSCs) on repairing hepatic ischemia-reperfusion injury (HIRI) in rat models. Methods Forty male SD rats were randomly divided into the A (control group), B (model group), C (portal vein transplantation of MSCs group) and D (tail vein transplantation of MSCs group) groups (n=10 for each group). In the B, C and D groups, HIRI rat models were established by Pringle maneuver. MSC transplantation was not delivered in the B group, and chloromethyl-benzamidodialkylcarbocyanine (CM-DiL) labeled-HU-MSCs (106) were transplanted via portal vein or tail vein in the C or D groups. Blood samples were collected for detection of the expression levels of alanine aminotransferase(ALT) and tumor necrosis factor (TNF)-α before and 1, 3 and 5 d after transplantation. At 5 d post-transplantation, all rats in each group were sacrificed. The liver tissues were prepared for hematoxylin-eosin (HE) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) staining for evaluation of liver injury. The distribution of HU-MSCs in the rat liver in each group was observed under laser confocal microscope. Results After HU-MSCs transplantation, the expression levels of ALT in the C and D groups were significantly down-regulated compared with that in the B group, and the level of ALT in the C group was significantly lower than that in the D group (all P < 0.05). The expression levels of inflammatory cytokine TNF-α in the C and D groups were significantly lower than that in the B group and the TNF-α level in the C group was considerably lower than that in the D group (all P < 0.05). HE staining of the liver tissue prompted that the liver injury was the most severe in the B group, and the liver injury in the C group was less severe compared with that in the D group. TUNEL staining of the liver tissue revealed that the apoptosis index (AI) in the C and D groups was significantly lower than that in the B group, and the AI in the C group was significantly lower compared with that in the D group (all P < 0.05). Under laser confocal microscope, CM-DiL-labeled HU-MSCs were noted in the C and D groups after transplantation, and the quantity of HU-MSCs in the C group was significantly higher than that in the D group (all P < 0.05). Conclusion Transplantation of HU-MSCs can mitigate the severity of HIRI. And transplantation via portal vein transplantation yields higher clinical efficacy compared with the tail vein route. -
图 2 荧光显微镜下CM-DiL标记第3代HU-MSCs的表现
Figure 2. The third generation of HU-MSCs labeled with CM-DiL by fluorescence microscope
图 3 各组大鼠术后不同时间ALT和TNF-α水平的比较
Figure 3. Comparison of the levels of ALT and TNF-α in rats among each group at different times
图 4 HU-MSCs移植后第5日各组大鼠肝脏病理学表现（HE，×400）
A图示A组肝索及肝窦排列规则，细胞膜完整；B图示B组肝组织结构紊乱，大量炎症细胞聚集、浸润，肝小叶中央静脉和肝血窦淤血明显，肝窦狭窄，肝索排列不规则，肝脏细胞不同程度的肿胀及气球样变性，偶有点状坏死；C图示C组肝小叶结构尚可，少量肝细胞呈气泡样改变，较B组轻；D图示D组肝细胞肿胀、变性，肝窦狭窄，损伤程度重于C组
Figure 4. Pathological changes of liver in rats among each group at 5th d after HU-MSCs transplantation
图 5 HU-MSCs移植后第5日各组大鼠肝脏细胞凋亡结果（TUNEL，×200）
A~D图分别为A~D组的TUNEL染色图片
Figure 5. The results of liver cell apoptosis in rats among each group at 5th d after HU-MSCs transplantation
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