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目的观察脐带间充质干细胞(UCMSCs)植入HIE大鼠模型仔鼠后存活、增殖及分化情况。方法取人脐带组织,经胶原酶和胰蛋白酶消化后,分离UCMSCs,采用5-溴脱氧尿苷(BrdU)标记。将孕鼠随机分为实验组(n=6)和对照组(n=1)。实验组结扎孕鼠子宫动脉15 min,建立宫内全脑缺氧缺血损伤模型,幼鼠出生20 d(P20)随机分为干细胞组(n=24)和PBS组(n=19)。干细胞组仔鼠小脑延髓池穿刺注射UCMSCs,PBS组注射PBS。移植后1、2、3、4周,采用免疫组织化学方法检测其BrdU、巢蛋白(Nes-tin)、神经元特异性烯醇化酶(NSE)、胶原纤维酸性蛋白(GFAP)表达,以硫堇染色观察其神经元形态。对照组正常分娩,同样检测作同期对照。结果干细胞组于移植后1周海马齿状回即出现BrdU、Nestin、NSE和GFAP阳性细胞;BrdU和Nestin阳性细胞于移植1～3周逐渐增高,组间两两比较具有统计学意义(Pa<0.01),移植3～4周阳性细胞数目无明显差异(P>0.05);NSE和GFAP阳性细胞于1～4周均渐增加,组间两两比较具有统计学意义(Pa<0.01)。硫堇染色观察其海马细胞形态,对照组神经细胞排列整齐,神经元呈多形性,数目多;PBS组神经细胞排列紊乱,神经元数目减少;干细胞组细胞排列较整齐,神经元数目较多。结论UCMSCs经小脑延髓池移植至HIE大鼠模型能存活,并分化为神经干细胞、神经元及星形胶质细胞。
Objective To observe the survival, proliferation and differentiation of umbilical cord mesenchymal stem cells (UCMSCs) implanted in HIE rat model. Methods Human umbilical cord tissue was harvested and UCMSCs were isolated after digestion with collagenase and trypsin and labeled with BrdU. Pregnant mice were randomly divided into experimental group (n = 6) and control group (n = 1). The experimental group was ligated with uterine artery of pregnant rats for 15 min to establish the model of intrauterine hypoxic-ischemic injury. The 20-day-old pups were randomly divided into three groups: the stem cell group (n = 24) and the PBS group (n = 19). UCMSCs were injected into the cisterna magna of stem cells in vitro and PBS in PBS group. The expression of BrdU, Nes-tin, neuron-specific enolase (NSE) and collagen fiber acidic protein (GFAP) were detected by immunohistochemical staining at 1, Pansy staining to observe its neuronal morphology. Control group normal delivery, the same test for the same period control. Results BrdU, Nestin, NSE and GFAP positive cells appeared in the hippocampal dentate gyrus of the stem cell group one week after transplantation. BrdU and Nestin positive cells gradually increased from 1 to 3 weeks after transplantation, and there was statistical significance between groups (Pa < 0.01). There was no significant difference in the number of positive cells between 3 and 4 weeks after transplantation (P> 0.05). The NSE and GFAP positive cells increased gradually from 1 to 4 weeks after operation, with statistical significance (P <0.01). Thionine staining was used to observe the morphological changes of hippocampal cells. The neurons in the control group were arranged neatly, and the number of neurons was plentiful. The number of neurons in the PBS group was disordered and the number of neurons was decreased. The cells in the stem cells were arranged more regularly with more neurons . Conclusions UCMSCs can be transplanted into the HIE rat model via the cisterna magna and survive in vitro and differentiate into neural stem cells, neurons and astrocytes.