Neuroprotective effect and mechanism of human umbilical cord blood mesenchymal stem cells on hypoxic-ischemic brain injury in neonatal rats

doi:10.3969/j.issn.2095-4344.1648

Abstract

Abstract:

BACKGROUND: Although it has been confirmed that human umbilical cord blood mesenchymal stem cells transplantation can promote the recovery of neural function, its mechanism is unclear.
OBJECTIVE: To observe the therapeutic effect of human umbilical cord blood mesenchymal stem cells transplantation on hypoxic-ischemic brain injury in neonatal rats and to analyze its possible mechanism.
METHODS: Sixty 10-day-old neonatal Sprague-Dawley rats (provided by the Experimental Animal Center of North Sichuan Medical College in China) were placed in a simulated hypobaric environment at 6 000-meter altitude to live and exercise. The animal model of ischemic-hypoxic brain injury at high altitude was established by exercising 60 min/d for 15 days in the swimming tank of the cabin. At 24 hours after modeling, 60 rats were randomly divided into transplantation group and model group. In the transplantation group, DAPI labeled human umbilical cord blood mesenchymal stem cells were injected into the rat hippocampus, while in the model group, the same volume of PBS was injected into the rat hippocampus. Another 30 rats were taken as blank control group, with no modeling and treatment. At 48 hours after cell transplantation, apoptosis of neurons was detected by TUNEL method, and western blot assay was used to detect the expression of Caspase-3 and p-Akt protein in brain tissue. At 21 days after cell transplantation, the learning and memory abilities of rats were measured by Morris water maze test. At 28 days after cell transplantation, the expression of inflammatory factors in rat brain tissue was detected by ELISA and pathological changes of the rat brain tissue were observed histologically.
RESULTS AND CONCLUSION: (1) The results of TUNEL staining showed that only a small number of apoptotic cells were observed in the blank control group, and a large number of apoptotic cells were observed in the model group. The number of apoptotic cells in the transplantation group was higher than that in the blank control group but lower than that in the model group. There was a significant difference in the number of apoptotic cells among the three groups. (2) The expression of Caspase-3 protein in the rat brain tissue was significantly lower in the transplantation group than the model group (P < 0.05), while the expression of p-Akt protein in the transplantation group was significantly higher than that in the model group (P < 0.05). (3) In the Morris water maze test, the escape latency and swimming distance in the transplantation group were significantly shorter than those in the model group (P < 0.05). (4) Compared with the model group, the levels of tumor necrosis factor α, interleukin 1β and interleukin 10 were significantly reduced in the transplantation group (P < 0.05), while the level of interleukin 8 was significantly increased in the transplantation group (P < 0.05). (5) In the model group, the rats appeared to have neuronal edema in the hippocampus, with unclear chromatin structure and vacuoles, and severe ependymal cell edema. In the transplantation group, there was milder neuronal edema in the rat hippocampus compared with the model group, and the chromatin structure was still unclear but with no formation of vacuoles. To conclude, human umbilical cord blood mesenchymal stem cells can improve the learning and memory abilities of neonatal rats with hypoxic-ischemic brain injury by regulating the expression of inflammatory factors and inhibiting neuronal apoptosis. PI3K/Akt signaling pathway may play a role in inhibiting apoptosis.

Key words: Cord Blood Stem Cell Transplantation, Hypoxia-Ischemia, Brain, Caspase 3, Tissue Engineering

CLC Number:

Zhou Yu, Mo Guoliang, Zhao Jing, Fang Ping. Neuroprotective effect and mechanism of human umbilical cord blood mesenchymal stem cells on hypoxic-ischemic brain injury in neonatal rats[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(13): 2035-2041.

Figures/Tables 1

2.1 实验动物数量分析参加实验120只大鼠，30只大鼠造模失败，其余90只大鼠均进入结果分析。 2.2 人脐血间充质干细胞的培养与标记细胞接种后72 h开始贴壁，多呈梭形，7 d后梭形细胞逐渐增多，细胞排列具有一定的方向性，呈成纤维样细胞形态；第3代细胞增殖迅速，可见大量成纤维细胞样集落，细胞呈梭形，方向一致，见图1A，B。取第3代人脐血间充质干细胞进行DAPI标记，荧光显微镜观察细胞核呈蓝色强荧光，DAPI标记阳性率在98%以上，见图1C。 2.3 各组脑组织细胞凋亡情况细胞移植48 h后，TUNEL染色可见空白对照组仅见极少量的凋亡细胞，模型组可见大量细胞凋亡，移植组细胞凋亡介于空白对照组与模型组之间，见图2。空白对照组、模型组、移植组凋亡细胞数分别为(3.2±0.6)，(59.8±9.5)，(22.8±6.4)个，模型组、移植组凋亡细胞数均高于空白对照组(P < 0.05)，移植组凋亡细胞数低于模型组(P < 0.05)。 2.4 各组Western blot检测结果细胞移植48 h后，模型组脑组织Caspase-3蛋白表达明显高于空白对照组，p-Akt蛋白表达明显低于空白对照组(P < 0.05)；移植组脑组织Caspase-3蛋白表达明显低于模型组，p-Akt蛋白表达明显高于模型组(P < 0.05)；移植组脑组织Caspase-3蛋白表达明显高于空白对照组，p-Akt蛋白表达明显低于空白对照组(P < 0.05)，见图3。 2.5 各组大鼠学习记忆能力检测 Morris水迷宫实验检测结果显示，与空白对照组比较，模型组大鼠逃避潜伏期与游泳距离均明显延长(P < 0.05)；与模型组比较，移植组大鼠逃避潜伏期与游泳距离均明显缩短(P < 0.05)，但移植组大鼠逃避潜伏期与游泳距离仍显著长于空白对照组(P < 0.05)，见表1。 2.6 各组脑组织匀浆炎症因子检测与空白对照组比较，模型组肿瘤坏死因子α、白细胞介素1β、白细胞介素10质量浓度明显升高(P < 0.05)，白细胞介素8水平明显降低(P < 0.05)；与模型组比较，移植组肿瘤坏死因子α、白细胞介素1β、白细胞介素10质量浓度明显降低(P < 0.05)，白细胞介素8水平明显升高(P < 0.05)；与空白对照组比较，移植组肿瘤坏死因子α、白细胞介素1β、白细胞介素8质量浓度明显升高(P < 0.05)，白细胞介素10水平明显降低(P < 0.05)，见表2。 2.7 各组脑组织病理观察结果空白对照组大鼠脑组织海马区内神经元细胞包膜完整，核大而圆，细胞间隙正常，细胞突起清晰；模型组大鼠海马区神经元排列稀疏、散乱，核染色质结构不清，有空泡形成，室管膜细胞重度水肿；移植组大鼠海马区神经元水肿程度轻于模型组，神经元排列稍紊乱，可见少量细胞丢失，见图4。 2.8 人脐血间充质干细胞的存活与分布观察细胞移植28 d后，移植组大鼠左侧脑组织海马内可见大量DAPI阳性细胞，多分布于齿状回，见图5。"

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