全身亚低温联合人脐带血间充质干细胞移植改善新生大鼠缺氧缺血性脑损伤后认知障碍和能量衰竭的机制研究

Mechanism of mild whole-body hypothermia combined with human umbilical cord blood-derived mesenchymal stem cell trans-plantation in ameliorating cognitive impairment and energy failure after hypoxic-ischemic brain damage in neonatal rats

摘要:

目的 探究全身亚低温联合人脐带血间充质干细胞（hCMNCs）移植改善新生大鼠缺氧缺血性脑损伤（HIBD）后认知障碍和能量衰竭的机制。方法 7日龄新生SD乳鼠330只。随机分为11组，每组30只。空白对照组（A组）、HIBD常温组（B组）、HIBD亚低温持续72 h组（C组）、HIBD亚低温24 h干细胞移植组（D组）、HIBD亚低温48 h干细胞移植组（E组）、HIBD亚低温72 h移植组（F组）、HIBD亚低温24 h后持续3 d移植干细胞（G组）、HIBD 24 h干细胞移植组（H组）、HIBD 48 h干细胞移植组（I组）、HIBD 72 h干细胞移植组（J组）、HIBD 24 h、48 h、72 h干细胞移植组（K组）。乳鼠生长的第10天、第21天和第35天对其进行称重；通过Morris水迷宫实验检测乳鼠学习和记忆能力，超微电镜观察分析乳鼠脑组织神经元细胞及突触超微结构的变化，苏木精-伊红（HE）染色观察脑组织病理学变化，酶联免疫吸附试验检测乳鼠脑组织炎症因子和干细胞分化标记蛋白含量，免疫荧光染色检测脐带CD24和CD29阳性表达率，荧光显微镜测量线粒体膜电位。结果 A、B、C、D、E、F、G、H、I、J、K组乳鼠生长第10天、第21天和第35天体重比较，采用重复测量设计的方差分析，结果 ①不同时间点乳鼠体重有差异（F =13.285，P =0.000），各组乳鼠第21天与第35天体重比较，差异有统计学意义（P <0.05）；②各组乳鼠体重有差异（F =20.097，P =0.000），B组乳鼠体重较A组降低（P <0.05），C～K组乳鼠体重较B组升高（P <0.05），G、I组乳鼠体重较B、C、D、E、F、H、J、K组均升高（P <0.05），且G组与I组乳鼠体重比较，差异无统计学意义（P >0.05）；③各组乳鼠体重变化趋势有差异（F =28.712，P =0.000）。C～F组3种反射（悬崖调转反射、阴性趋地性、步态反射）时间较A组延长（P <0.05），但较B组缩短（P <0.05）；C组3种反射时间较A、D～J组延长（P <0.05）；G～K组3种反射时间与A组比较，差异无统计学意义（P >0.05）；C～F组3种反射时间较G～K组延长（P <0.05）。HE染色结果显示，与A组比较，B～K组乳鼠HIBD模型复制成功后第3天，脑间质水肿，小血管间隙增宽，左侧大脑出现大片坏死，细胞溶解、破坏、消失；皮质海马神经元排列紊乱，核固缩、崩解，细胞浓染，并见小胶质细胞。第21天，B～K组与A组有差异，B～K组脑组织见不同程度的损伤，其中B组可见大的梗塞灶及液化空洞形成。C～K组表现为部分炎症细胞；B组与C～K组比较，脑损伤程度较大。其中G、J、K组脑损伤及炎症程度较C～F、H、I组小。B～K组逃避潜伏期较A组延长（P <0.05），G、J、K组逃避潜伏期较B～F、H组缩短（P <0.05）。B～K组神经元致密区长度较A组变短（P <0.05），厚度较A组变薄（P <0.05），而突触间隙宽度较A组变厚（P <0.05）。C～K组乳鼠神经元致密区长度较B组变长（P <0.05），厚度较B组变厚（P <0.05），突触间隙宽度较B组变薄（P <0.05）。G～J组乳鼠神经元致密区长度较D～F、K组变长（P <0.05），厚度较D～F、K组变厚（P <0.05），突触间隙宽度较D～F、K组变薄（P <0.05）。A、B、C、D、E、F、G、H、I、J、K组乳鼠HIBD模型复制成功后第3天、第8天、第12天的TNF-α、IL-6、Nestin、TUBB、MBP比较，采用重复测量设计的方差分析，结果 ①不同时间点TNF-α、IL-6、Nestin、TUBB、MBP有差异（F =39.451、19.754、36.957、16.794和16.958，均P =0.000）；②各组乳鼠TNF-α、IL-6、Nestin、TUBB、MBP有差异（F =10.719、10.159、43.271、5.947和11.217，P =0.000、0.000、0.000、0.012和0.000）；B～K组TNF-α、IL-6在HIBD模型复制成功第3天、第8天和第12天较A组升高（P <0.05），C～K组TNF-α、IL-6较B组降低（P <0.05）。C～K组Nestin、TUBB和MBP较B组升高（P <0.05）。③各组乳鼠TNF-α、IL-6、Nestin、TUBB、MBP变化趋势有差异（F =22.678、25.483、6.597、20.159和20.154，P =0.000、0.000、0.003、0.000和0.000）。A～C组CD24和CD29阳性表达率较E～K组降低（P <0.05），G、I组较H、J、K组CD24和CD29阳性表达率升高（P <0.05）。B～K组线粒体膜电位较A组降低（P <0.05），C～K组线粒体膜电位较B组升高（P <0.05），G～J组线粒体膜电位较C～F组升高（P <0.05）。结论 亚低温联合UCMSCs能改善HIBD乳鼠认知障碍和能量衰竭，对脑神经有保护作用。

Abstract:

Objective To explore the mechanism of mild whole-body hypothermia combined with human umbilical cord blood-derived mesenchymal stem cell transplantation in ameliorating cognitive impairment and energy failure after hypoxic-ischemic brain damage (HIBD) in neonatal rats.Methods A total of 330 7-day-old neonatal SD rats were randomly divided into 11 groups, including blank control group (A), HIBD + normal temperature group (B), HIBD + 72-hour mild hypothermia group (C), HIBD + 24-hour mild hypothermia + stem cell transplantation group (D), HIBD + 48-hour mild hypothermia + stem cell transplantation group (E), HIBD + 72-hour mild hypothermia + stem cell transplantation group (F), HIBD + 24-hour mild hypothermia + 3-day stem cell transplantation group (G), HIBD + 24-hour stem cell transplantation group (H), HIBD + 48-hour stem cell transplantation group (I), HIBD + 72-hour stem cell transplantation group (J), and HIBD + 24-hour, 48-hour, 72-hour stem cell transplantation group (K), with 30 animals in each group. The rats were weighed and compared in the 10th, 21st and 35th days of the growth. The learning and memory abilities of rats were detected by Morris water maze test. The ultrastructural changes of neurons and synapses in rat brain tissues were observed and analyzed by electron microscopy. The histopathological changes in the rat brain tissues were observed with H&E staining. Serum levels of inflammatory factors and stem cell differentiation markers were detected by enzyme-linked immunosorbent assay. The positive expression rates of CD24 and CD29 in the umbilical cord were detected by immunofluorescence. The mitochondrial membrane potential was detected by a fluorescence microscope.Results The rats in all the groups were weighed and compared on the 10th, 21st and 35th days of growth. The repeated measures ANOVA showed that the body weight of rats at different time points was different (F = 13.285, P = 0.000), and that the body weight of rats at 21 d and 35 d in each group was significantly different (P < 0.05). The body weight of rats was also different among the groups (F = 20.097, P = 0.000), where the body weight of rats in group B was lower than that in group A (P < 0.05), the body weight of rats in groups C to K was higher than that in group B (P < 0.05), and the body weight of rats in group G and I was higher than that in groups B to K (P < 0.05). There was no significant difference in body weight of rats between group I and group G (P > 0.05). The change trend of body weight of rats in each group was different (F = 28.712，P = 0.000). The response time of cliff avoidance reaction, negative geotaxis and gait reflex in group C, D, E and F was longer than that in group A (P < 0.05), but was shorter than that in group B (P < 0.05). The response time of cliff avoidance reaction, negative geotaxis and gait reflex in group C was longer than that in group A, D, E, F, G, H, I and J (P < 0.05), while that was not different among group A and group G, H, I, J and K (P < 0.05). The response time of cliff avoidance reaction, negative geotaxis and gait reflex in group C, D, E and F was longer than that in group G, H, I, J and K (P < 0.05). The results of H&E staining on the 3rd day after the successful establishment of HIBD model showed interstitial cerebral edema, dilated perivascular spaces, large areas of necrosis in the left brain, cytolysis, cytoclasis, cell loss, irregular arrangement of cortical and hippocampal neurons, karyopyknosis, karyorrhexis, darkly stained cells, and the presence of microglia in rats of group B to K compared with group A. On the 21st day, the injury in group B to K was different from than in group A, and various degrees of injury was observed in groups B to K, where large infarcts and cavities due to liquefactive necrosis were observed in group B while some inflammatory cells were observed in group C to K. The degree of brain damage was greater in group B than that in group C to K, while the degrees of brain injury and inflammation in G, J and K groups were lower than those in C to F, H and I groups. The escape latency of rats in group B to K was longer than that in group A (P < 0.05), and the escape latency of groups G, J and K was shorter than that of groups B to F and H (P < 0.05). The postsynaptic density was shorter and thinner, and the synaptic cleft was wider in rats of groups B to K than those in group A (P < 0.05). Compared with group B, the postsynaptic density was longer and thicker, and the synaptic cleft was narrower in groups C to K (P < 0.05). Compared with groups D to F and K, the postsynaptic density was longer and thicker, and the synaptic cleft was narrower in groups G to J (P < 0.05). The levels of TNF-α, IL-6, Nestin, TUBB and MBP were compared in groups A to K on the 3rd, 8th and 12th day after modeling, and the results were analyzed by repeated measures ANOVA. The levels of TNF-α, IL-6, Nestin, TUBB and MBP were significantly different at different time points (F = 39.451, 19.754, 36.957, 16.794 and 16.958, all P =0.000) and among groups (F = 10.719, 10.159, 43.271, 5.947 and 11.217, P = 0.000, 0.000, 0.000, 0.012 and 0.000). The serum levels of TNF-α and IL-6 in groups B to K were significantly higher than those in group A, while the serum levels of TNF-α and IL-6 in groups C to K were significantly lower than those in group B on the 3rd, 8th and 12th day after modeling (P < 0.05). Compared with group B, the serum levels of Nestin, TUBB and MBP in groups C to K were significantly higher (P < 0.05). The change trends of the serum levels of TNF-α, IL-6, Nestin, TUBB and MBP were also different among the groups (F = 22.678, 25.483, 6.597, 20.159 and 20.154, P = 0.000, 0.000, 0.003, 0.000 and 0.000). The positive expression rates of CD24 and CD29 in groups A to C were lower than those in groups E to K (P < 0.05), and the positive expression rates of CD24 and CD29 in groups G and I were higher than those in groups H, J and K (P < 0.05). The mitochondrial membrane potential of groups B to K was lower than that of group A (P < 0.05), the mitochondrial membrane potential of groups C to K was higher than that of group B (P < 0.05), and the mitochondrial membrane potential of groups G to J was higher than that of groups C to F (P < 0.05).Conclusions Mild hypothermia combined with human umbilical cord blood-derived mesenchymal stem cell transplantation can improve the cognitive impairment and energy failure of HIBD neonatal rats and exhibits a neuroprotective effect.