不同剂量人脐带间充质干细胞移植对新生大鼠脑白质损伤的修复作用

doi:10.7499/j.issn.1008-8830.2310081

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摘要目的比较不同剂量人脐带间充质干细胞（human umbilical cord mesenchymal stem cells, hUC-MSCs）对新生大鼠脑白质损伤（white matter injury, WMI）的修复作用。方法将2日龄Sprague-Dawley新生大鼠随机分为5组：假手术组、WMI组和hUC-MSCs组（低剂量组、中剂量组、高剂量组），每组24只。成功制备新生大鼠WMI模型24 h后，WMI组经侧脑室注射无菌PBS，hUC-MSCs组注射不同剂量hUC-MSCs。造模后14 d、21 d，采用苏木精-伊红染色法观察各组侧脑室周围组织病理变化，实时荧光定量聚合酶链式反应法检测各组脑组织中髓鞘碱性蛋白（myelin basic protein, MBP）、胶质纤维酸性蛋白（glial fibrillary acidic protein, GFAP）mRNA表达水平，免疫组化法观察GFAP、神经元核蛋白（neuron-specific nuclear protein, NeuN）在侧脑室周围组织表达水平；TUNEL染色观察各组侧脑室周围组织细胞凋亡情况。造模后21 d，采用Morris水迷宫实验观察各组新生大鼠的空间学习记忆能力。结果造模后14 d、21 d，WMI组和低剂量组侧脑室周围组织均可见大量细胞核固缩、核破裂，神经纤维排列紊乱，与WMI组比较，中、高剂量组上述病理改变均减轻；与高剂量组比较，中剂量组神经纤维排列相对整齐。与WMI组比较，低剂量组MBP、GFAP mRNA表达水平差异无统计学意义（P>0.05），中、高剂量组MBP mRNA表达水平升高，GFAP mRNA表达水平降低；中剂量组MBP mRNA表达水平高于高剂量组，中剂量组GFAP mRNA表达水平低于高剂量组（P<0.05）。与WMI组比较，低剂量组GFAP、NeuN阳性表达差异无统计学意义（P>0.05），中、高剂量组NeuN阳性表达升高，GFAP阳性表达降低（P<0.05）；中剂量组NeuN阳性表达高于高剂量组，GFAP阳性表达低于高剂量组（P<0.05）。与WMI组比较，低剂量组凋亡细胞数差异无统计学意义（P>0.05），中、高剂量组凋亡细胞数少于WMI组（P<0.05）；中剂量组凋亡细胞数少于高剂量组（P<0.05）。与WMI组比较，低剂量组逃避潜伏期时间差异无统计学意义（P>0.05）；自潜伏期第3天开始，中剂量组逃避潜伏期时间少于WMI组（P<0.05）；中、高剂量组穿越平台次数多于WMI组（P<0.05）。结论低剂量hUC-MSCs对新生大鼠WMI修复效果欠佳，中、高剂量hUC-MSCs均有修复作用，中剂量效果更优。

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关键词 ：脑白质损伤, 人脐带间充质干细胞, 剂量, 新生大鼠

Abstract：Objective To compare the repair effects of different doses of human umbilical cord mesenchymal stem cells (hUC-MSCs) on white matter injury (WMI) in neonatal rats. Methods Two-day-old Sprague-Dawley neonatal rats were randomly divided into five groups: sham operation group, WMI group, and hUC-MSCs groups (low dose, medium dose, and high dose), with 24 rats in each group. Twenty-four hours after successful establishment of the neonatal rat white matter injury model, the WMI group was injected with sterile PBS via the lateral ventricle, while the hUC-MSCs groups received injections of hUC-MSCs at different doses. At 14 and 21 days post-modeling, hematoxylin and eosin staining was used to observe pathological changes in the tissues around the lateral ventricles. Real-time quantitative polymerase chain reaction was used to detect the quantitative expression of myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) mRNA in the brain tissue. Immunohistochemistry was employed to observe the expression levels of GFAP and neuron-specific nuclear protein (NeuN) in the tissues around the lateral ventricles. TUNEL staining was used to observe cell apoptosis in the tissues around the lateral ventricles. At 21 days post-modeling, the Morris water maze test was used to observe the spatial learning and memory capabilities of the neonatal rats. Results At 14 and 21 days post-modeling, numerous cells with nuclear shrinkage and rupture, as well as disordered arrangement of nerve fibers, were observed in the tissues around the lateral ventricles of the WMI group and the low dose group. Compared with the WMI group, the medium and high dose groups showed alleviated pathological changes; the arrangement of nerve fibers in the medium dose group was relatively more orderly compared with the high dose group. Compared with the WMI group, there was no significant difference in the expression levels of MBP and GFAP mRNA in the low dose group (P>0.05), while the expression levels of MBP mRNA increased and GFAP mRNA decreased in the medium and high dose groups. The expression level of MBP mRNA in the medium dose group was higher than that in the high dose group, and the expression level of GFAP mRNA in the medium dose group was lower than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the protein expression of GFAP and NeuN in the low dose group (P>0.05), while the expression of NeuN protein increased and GFAP protein decreased in the medium and high dose groups. The expression of NeuN protein in the medium dose group was higher than that in the high dose group, and the expression of GFAP protein in the medium dose group was lower than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the number of apoptotic cells in the low dose group (P>0.05), while the number of apoptotic cells in the medium and high dose groups was less than that in the WMI group, and the number of apoptotic cells in the medium dose group was less than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the escape latency time in the low dose group (P>0.05); starting from the third day of the latency period, the escape latency time in the medium dose group was less than that in the WMI group (P<0.05). The medium and high dose groups crossed the platform more times than the WMI group (P<0.05). Conclusions Low dose hUC-MSCs may yield unsatisfactory repair effects on WMI in neonatal rats, while medium and high doses of hUC-MSCs have significant repair effects, with the medium dose demonstrating superior efficacy.

Key words： White matter injury Human umbilical cord mesenchymal stem cell Dose Neonatal rat

收稿日期: 2023-10-19

基金资助:国家自然科学基金（地区科学基金项目82060288）。

通讯作者: 朱艳萍，女，教授。Email：1119788145@qq.com。 E-mail: 1119788145@qq.com

作者简介: 张军，男，硕士研究生；

引用本文:

张军,李明霞,王超等. 不同剂量人脐带间充质干细胞移植对新生大鼠脑白质损伤的修复作用[J]. 中国当代儿科杂志, 2024, 26(4): 394-402.

ZHANG Jun,LI Ming-Xia,WANG Chao et al. Repair effect of different doses of human umbilical cord mesenchymal stem cells on white matter injury in neonatal rats[J]. CJCP, 2024, 26(4): 394-402.

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