人骨髓来源间充质干细胞对新生大鼠高氧肺损伤干预作用的研究

目的 研究人骨髓来源间充质干细胞对新生大鼠高氧肺损伤的干预作用.方法 采用贴壁选择法分离、培养、扩增hMSCs,并予BrdU进行标记;32只3日龄SD大鼠随机分为A、B、、C、D4组,每组8只;A组:高氧暴露+hMSCs注射组,B组:空气暴露+hMSCs注射组,C组:高氧暴露对照组,D组:空气暴露对照组.A、C组:高氧(95%)暴露后7 d,腹腔分别注射含5×105 MSC的磷酸盐缓冲液(PBS)、单纯的PBS 50 μl,B、D组:空气暴露后7 d,腹腔分别注射含5×105 hMSCs的PBS、单纯的PBS 50 μl.注射后3 d处死全部动物取肺组织,ELASA法检测肺组织匀浆TNFα、TGFβ1水平;免疫组织化学方法检查肺组织BrdU积分情况,HE染色观察肺组织学形态学结构,做辐射状肺泡计数(RAC);RT-PCR方法检测各组肺组织Alu序列表达情况.结果 (1)A、B、C、D 4组TNFα水平分别为142.933±24.017,79.033±11.573,224.088±41.915,76.500±10.373(F=59.970,P=0.000);而TGFβ1水平分别为1726.484±91.086,1530.359±173.441,2047.717±152.057,1515.777±131.049(F=24.977,P=0.000).(2)RAC值分别为11.145±1.331,13.941±0.985,9.595±0.672,14.819±1.080(F=43.234,P=0.000).(3)RT-PCR检测显示A、B组均有Alu序列表达,而C、D组均未见Alu序列表达.免疫组织化学显示A、B组均有BrdU阳性染色细胞,BrdU积分分别为0.230±0.026,0.190±0.015,t=3.769,P=0.002;C、D组均未见阳性染色细胞.结论 新生大鼠腹腔注射hMSC后肺组织有hMSCs定植,且与暴露的条件相关;hMSCs可改善新生大鼠因高氧引致的肺组织损伤.

Influence of human bone marrow-derived mesenchymal stem cells on the lung of newborn rats damaged by hyperoxia

OBJECTIVE: To evaluate whether human mesenchymal stem cells (hMSCs) administration alter the clinical course of hyperoxia-induced lung injury. METHODS: hMSCs were obtained from bone marrow aspirates from healthy donors after informed consent was signed, hMSCs were separated, cultured, amplified, identified and labeled with BrdU. For BrdU labeling, a sterile stock solution was added to the culture medium 48 h before the end of culture, at a final concentration of 10 micromol/L. Thirty-two 3-day old SD rats from four litters were randomly divided into four groups, as hyperoxia exposed + hMSC group (A), air-exposed + hMSC group (B), hyperoxia exposed group (C), and air-exposed group (D). The rats from the group A and the group C were placed in a sealed Plexiglas chamber with a minimal in- and outflow, providing six to seven exchanges per hour of the chamber volume and maintaining O2 levels above 95%, while the rats in the group B and the group D were only exposed to room air. Seven days later, all of them were taken out of the chamber, rats in the group A and B were injected intraperitoneally with hMSCs (1 x 10(5) in 50 microl of PBS) immediately, while the rats in the group C and D were only treated with 50 microl of PBS 3 days later. All the animals were sacrificed by an injection of sodium pentobarbital (120 mg/kg), perfused with cold 0.9% NaCl, and the left lungs were removed, the upper lobes of which were ground as tissue homogenates and used for ELISA, while the inferior lobes were stored at -70 degrees C until use for RT-PCR. The right lungs were fixed in situ for 2 h by the intratracheal instillation with 10% neutral formalin and then postfixed for 24 h. Sagittal sections (4-microm) of paraffin-embedded middle lobe and upper lobe of the right lung were used for immunohistochemistry and histology, respectively. RESULTS: (1) There was a significant difference in the value of RAC (raditive alveoli coant) among the 4 groups (11.145 +/- 1.331, 13.941 +/- 0.985, 9.595 +/- 0.672, 14.819 +/- 1.080, F = 43.234, P = 0.000). RAC in group A and C were significantly reduced compared with subjects in group D (P < 0.05, P < 0.05); and there was also a significant difference between group A and group C (P < 0.05), but not between group B and D subjects (P > 0.05). (2) There were significant differences in the levels of both TNFalpha and TGFbeta(1) in the homogenate of lungs among the 4 groups (142.933 +/- 24.017, 79.033 +/- 11.573, 224.088 +/- 41.915, 76.500 +/- 10.373, F = 59.970, P = 0.000; 1726.484 +/- 91.086, 1530.359 +/- 173.441, 2047.717 +/- 152.057, 1515.777 +/- 131.049, F = 24.977, P = 0.000). The levels of TNFalpha and TGFbeta1 were significantly elevated in both group A and group C when compared with subjects in group D (P < 0.05 for both). Concentrations of TNFalpha and TGFbeta1 were both significantly decreased in group A versus group C (P < 0.05 for both). There was no significant difference between group B and D subjects in the fields of TNFalpha and TGFbeta(1) (P > 0.05 for both). (3) BrdU-labelled cells were observed at alveolar wall and bronchioles in both group A and group B, and there was a significant difference in BrdU-labeled cells between two groups (0.230 +/- 0.026, 0.190 +/- 0.015; t = 3.769, P = 0.002), but none was found in group C and group D. Electrophoresis of the PCR products showed a 224 bp band, specific for Alu mRNA, in 7 of 8 rats of group A and 5 of 8 rats of group B, respectively, but no such band was found in group C and group D. CONCLUSION: hMSCs administered by intraperitoneal injection could be implanted in the lungs of newborn rats, and they could effectively protect the rats against damage to the lungs caused by hyperoxia.