脐血单个核细胞侧脑室移植对HIBD新生大鼠脑神经元凋亡及Bax、Bcl-2蛋白的影响

Effects of umbilical cord blood mononuclear cells transplantation via lateral ventricle on the neural apoptosis and the expression of Bax and Bcl-2 proteins in neonatal rats with hypoxic-ischemic brain damage

摘要:

目的 探讨脐血单个核细胞移植对缺氧缺血性脑损伤 （HIBD）新生大鼠脑神经元凋亡及Bax、Bcl-2蛋白的影响。方法 7日龄Sprague-Dawley新生大鼠制备缺氧缺血性脑损伤 （HIBD）模型，随机分为正常对照 （N）+生理盐水 （NS）、HIBD+NS、N+脐血单个核细胞 （UCBMC）及HIBD+UCBMC组。N+UCBMC与HIBD+UCBMC组侧脑室注入3×10⁶个UCBMC，N+NS与HIBD+NS组注入等体积NS。移植后7 d采用NeuN/活性Caspase-3免疫荧光双标染色法、TUNEL法观察大脑皮层神经元凋亡，Western blot观察脑组织Bax、Bcl-2蛋白表达。结果 HIBD+NS组的NeuN⁺活性Caspase-3⁺DAPI⁺细胞及TUNEL⁺DAPI⁺细胞均多于N+NS和N+UCBMC组 （P < 0.01）；HIBD+UCBMC组的NeuN⁺活性Caspase-3⁺DAPI⁺细胞及TUNEL⁺DAPI⁺细胞均少于HIBD+NS组 （P < 0.01）。HIBD+NS组的Bax蛋白表达高于N+NS与N+UCBMC组，Bcl-2蛋白低于N+NS与N+UCBMC组 （P < 0.01）；而HIBD+UCBMC组的Bax蛋白较HIBD+NS组降低 （P < 0.01），Bcl-2蛋白则高于HIBD+NS组、N+NS和N+UCBMC组 （P < 0.05）。结论 HIBD新生大鼠侧脑室移植UCBMC可以减轻神经元凋亡，其机制可能与Bcl-2蛋白表达上调，Bax蛋白表达下调有关。

Abstract:

Objective To explore the effects of umbilical cord blood mononuclear cells (UCBMC) transplantation on the neuronal apoptosis and the expression of Bcl-2 and Bax proteins in neonatal rats with hypoxic-ischemic brain damage (HIBD). Methods Seven-day-old Sprague-Dawley neonatal rats were randomly divided into normal control (N)+normal saline (NS), HIBD+NS, N+UCBMC, and HIBD+UCBMC groups. HIBD model was prepared using the classical Rice-Vannucci method. Twenty-four hours after HIBD, UCBMC were transplanted in the N+UCBMC and HIBD+UCBMC groups. Seven days after transplantation, NeuN/Cleaved-Caspase-3 double immunofluorescence staining and TUNEL methods were used to observe neural apoptosis in the cortex. The expression levels of Bax and Bcl-2 proteins were examined by Western blot analysis. Results There were more NeuN⁺ cleaved Caspase-3⁺DAPI⁺ and TUNEL⁺DAPI⁺ cells in the HIBD+NS group compared with the N+NS and N+UCBMC groups (P < 0.01). There were less NeuN⁺ cleaved Caspase-3⁺DAPI⁺ and TUNEL⁺DAPI⁺ cells in the HIBD+UCBMC group compared with the HIBD+NS group (P < 0.01). The concentration of Bax protein was higher and that of Bcl-2 proteins was lower in the HIBD+NS group compared with the N+NS and N+UCBMC groups (P < 0.01). The concentration of Bax protein in HIBD+UCBMC group was lower than that in the HIBD+NS group (P < 0.01). The concentration of Bcl-2 protein was higher compared with the HIBD+NS, N+NS and N+UCBMC groups (P < 0.05). Conclusions UCBMC transplantation via lateral ventricle can upregulate the expression of Bcl-2 protein and down-regulate the expression of Bax protein, thus alleviating brain neural apoptosis in neonatal rats with HIBD.