VEGF基因修饰人骨髓间充质干细胞对脑出血大鼠早期脑水肿的影响

VEGF-transfected hBMSCs Aggravate Early Brain Edema in Cerebral Hemorrhage Rats

目的探究血管内皮生长因子(vascular endothelial growth factor, VEGF)基因修饰的人骨髓间充质干细胞(human bone marrow mesenchymal stem cells, hBMSCs)对脑出血大鼠早期脑水肿的影响。方法体外培养hBMSCs和经体外构建的VEGF基因慢病毒载体稳定转染的hBMSCs(hBMSCs/VEGF)细胞。采用尾状核注射Ⅰ型胶原酶和肝素方式,制备脑出血动物模型。术后2 h对大鼠进行改良神经功能缺损(mNSS)评分,鉴定造模是否成功。将SD大鼠随机分为假手术组(仅进针不注射造模剂)、脑出血模型组、生理盐水组、hBMSCs组和hBMSCs/VEGF组。造模后第3天,hBMSCs组和hBMSCs/VEGF组大鼠分别按分组右侧尾壳核内注射细胞悬液(2×106个细胞/kg体质量)进行细胞移植,生理盐水组相同部位注射等量生理盐水,假手术组、模型组大鼠不进行干预。干预后3 d、7 d分别进行mNSS评分,处死大鼠,分离脑右侧尾壳核区脑组织标本,干湿重法检测脑组织标本含水量,HE染色观察脑组织标本病理改变,免疫荧光和Western blot检测VEGF、基质金属蛋白酶-9(matrix metalloproteinase, MMP-9)和水通道蛋白-4(aquaporin 4, AQP-4)表达。结果造模术后2 h,与假手术组大鼠比较,各组大鼠mNSS评分增高,均>8分,提示造模成功;在干预后3 d、7 d,模型组和NS组mNSS评分,脑组织含水量,MMP-9、AQP-4、VEGF蛋白表达高于假手术组(P<0.05),干预后相同时间点,hBMSCs组上述指标较NS组和模型组减少(P<0.001),hBMSCs/VEGF组上述指标高于hBMSCs组(P<0.05);光镜下模型组和NS组大鼠脑组织出现明显出血灶和梗死灶,结构紊乱疏松,水肿改变明显,hBMSCs组出血灶和梗死灶减小,而hBMSCs/VEGF组相较hBMSCs组,脑组织结构疏松,呈空泡状,神经元出现多数核固缩等凋亡改变。结论 hBMSCs移植可改善脑出血大鼠神经功能缺损,减轻脑水肿,但hBMSCs经VEGF基因修饰后移植将增加血管通透性,加重脑出血大鼠的早期脑水肿。

Objective To investigate the effects of human bone marrow mesenchymal stem cells/vascular endothelial growth factor(hBMSCs/VEGF) transplantation on early brain edema in rats with cerebral hemorrhage.Methods Cultured the hBMSCs in vitro, transducted the cells with VEGF recombinant lentivirus vector to abtain the hBMSCs/VEGF cells. Intracerebral hemorrhage(ICH) rat model was established by injection of type Ⅰ collagenase and heparin into the caudate putamen. 2 h after the operation, the rats were evaluated with modified nerve function defect(mNSS) score to ensure whether the model was successfully established. At the third day after the injection, SD rats were randomly divided into sham group(only injected with empty needle), cerebral hemorrhage group, saline group, hBMSCs group and hBMSCs/VEGF group. Saline group, hBMSCs group and hBMSCs/VEGF group mice were injected with normal saline, hBMSCs(2×106 cells/kg body mass) and hBMSCs/VEGF(2×106 cells/kg body mass) respectively;sham group and model group did not perform intervention. On day 3 and 7 after injection, the rats were evaluated with modified neurological function score(mNSS). Then rats were sacrificed, and brain tissue specimens from the right caudate putamen area were separated. The wet and dry weighing method was used to measure the water content, and HE staining was used to evaluate pathological and functional changes. The expressions of VEGF, matrix metalloproteinase 9(MMP-9) and aquaporin 4(AQP-4) proteins were detected by immunofluorescence and Western blot. Results 2 h after injection, compared with rats in sham operation group, mNSS scores of rats in model group were increased, indicating that the models have successfully established. 3 d and 7 d after intervention, the mNSS score, the content of brain water,the expression level of VEGF, MMP-9 and AQP-4 proteins in model group and NS group were significantly higher than those of sham group(P<0.05), while the above values in hBMSCs group were significantly lower than those in saline group and model group(P<0.001), and the above values in hBMSCs/VEGF group were significantly higher than those of hBMSCs group(P<0.05). There were obvious hemorrhage and infarction in the brain tissue in the model group and NS group rats observed under the light microscope. Besides, the brain tissue developed loose structure and edema change.However, the bleeding and infarction in the brain tissue of hBMSCs group mice were reduced. Compared with the hBMSCs group mice, the brain tissue of hBMSCs/VEGF group mice was looser, more vacuoles occurred, and many neurons showed apoptosis changes such as nuclear deflation. Conclusion hBMSCs transplantation could improve neurological function and relieve brain edema. But hBMSCs/VEGF will increase the vascular permeability and then aggravate the early cerebral edema in rats with cerebral hemorrhage.