摘要
目的研究大鼠骨髓间充质干细胞（MSC）对高氧引致新生大鼠肺组织损伤的干预作用。方法采用贴壁选择法分离、培养、扩增大鼠骨髓MSC，并以5-溴脱氧尿嘧啶核苷（BrdU）进行标记，注射前以磷酸盐缓冲液（PBS）稀释；32只3日龄SD大鼠随机分为4组：A、C组高氧（95％）暴露7d后，腹腔分别注射含和不含5×10^4MSC的PBS各50μl；B、D组空气暴露7d后，腹腔分别注射含和不含5×10^4MSC的PBS各50μl。于注射后72h（13日龄）腹腔注射10％水合氯醛后放血处死，肺组织病理学检测辐射状肺泡计数（RAC）；支气管肺泡灌洗液（BALF）细胞计数检测白细胞及其中性粒细胞；ELAsA法检测肺组织匀浆肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β含量。免疫组化双染法检测BrdU和肺泡表面活性物质结合蛋白a（SP—A）表达情况。结果（1）RAC（个）：4组比较差异有统计学意义（11．0±1．0、13．9±1．1、9．6±0．7、14．0±1．1，P=0．000）；A、C组均明显低于D组（均P〈0．05），A组高于C组（P〈0．05）。（2）BALF细胞计数：4组白细胞（10^5／ml：0．85±0．21、0．18±0．10、1．44±0．69、0．21±0．06）和中性粒细胞（10^5／ml：0．32±0．12、0．06±0．02、0．73±0．35、0．07±0．02）计数比较，差异均有统计学意义（均P=0．000）；A、C组均明显高于D组（均P〈0．05）；A组均低于C组（均P〈0．05）。（3）肺组织匀浆TNF-α及IL-1β含量：4组肺组织匀浆TNF-α（pg／ml：173±20、34±4、224±42、35±4）及IL-1β含量（pg／ml：530±74、210±33、948±82、216±30）比较，差异均有统计学意义（均P=0．000）；A、C组均明显高于D组（均P〈0．05），A组低于C组（均P〈0.05）。（4）免疫组化：A、B组均有BrdU及SP-A阳性细胞，A组还可见免疫双染阳性细胞；C、D组仅见SP—A阳性细胞。结论腹腔注射MSC对高氧暴露新生大鼠的肺损伤起保护作用，其保护作用涉及多种机制。
Objective To investigate the influence of marrow-derived mesenchymal stem cells （MSCs） on the lung injury caused by exposure to high oxygen. Methods MSCs were extracted from the femurs of Sprague-Dawley （SD） rat, cultured, amplified, and labeled with 5-bromo-2＇-deoxy-uridinel （BrdU）. Thirty-two 3-day-old SD rats were randomly divided into 4 equal groups： Group A, exposed to high oxygen （95%） for 7 days and then injected intra-peritoneally with 50 μl phosphate buffered solution （PBS） containing 5 ×10^4 MSCs; Group B, exposed to ordinary air for 7 days and then injected intra-peritoneally with PBS containing 5 × 10^4 MSCs; Group C, exposed to high oxygen （95%） for 7 days and then injected intra-peritoneally with PBS without MSC ; and Group D, exposed to ordinary air for 7 days and then injected intra-peritoneally with PBS without MSC. 72 h later, when the rats were 13 days old they were killed Bronchoalveolar lavage fluid （BALF） was obtained to count the numbers of white blood cells and neutrophils. Lung homogenate was made. ELISA was used to examine the contents of tumor necrosis factor （TNF）- α and interleukin （IL）-1β. Radial alveolar count （RAC） of the lungs was conducted under light microscope. Double-staining immunocytochemisty was used to detect the expression of surfactant protein A （SP-A） and BrdU. Results （ 1 ） The RAC values of Groups A and C were （ 11.0 ± 1.0 ） and （9.6 ± 0. 7 ） cells/10 fields, both significantly lower than those of Group B and D [ （ 13.9 ± 1.1 ） and （ 14.0 ± 1.1 ） cells/10 fields respectively, F = 40.41, P = 0. 000 ]. The RAC of Group A was significantly higher than that of Group C （P 〈 0.05 ）. （2） The white blood cell numbers in BALF of Groups A and C were （0.85 ± 0.21 ） and （1.44 ±0.69 ） × 10^5/ml respectively, both significantly higher than those of Groups B and D [ （0.18 ± 0. 10） and （0.21 ± 0.06） × 10^5/ml respectively, F = 21.52, P = 0. 000]. The neutrophil numbers of Groups A and C were （0.32 ±0. 12） and （0.73 ±0.35） × 10^5/ml respectively, both significantly higher than those of Groups B and D [（0. 06 ±0.02） and （0. 07 ±0. 02） × 10^5/ml respectively,F =22.91, P =0. 000]. （3） The TNF-α levels in the homogenate of Groups A and C were （ 173 ± 20） and （224 ± 42） pg/m respectively, both significantly higher than those of Group B and D [ （34 ±4） and （35 ±4） pg/ml respectively, F = 138.1, P =0. 0001. The IL-1β levels in the homogenate of Groups A and C were （530 ± 74） and （948 ± 82） pg/ml respectively, both significantly higher than those of Group B and D [ （210 ±33） and （216 ±30） pg/ml respectively, F =247.4, P =0.000]. （4） BrdU- positive cells and SP-A-positive cells were seen in both Groups A and B, a few double-stained cells were found in Group A, but only SP-A-positive cells, and none BrdU positive cells and double-stained cells were seen in Groups C and D. Conclusion MSC administration has protective effect on the ling injury caused by exposure to high oxygen via different mechanisms.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2008年第38期2715-2718,共4页
National Medical Journal of China
基金
国家自然科学基金资助项目（30772036）
关键词
间质干细胞
肺
婴儿
新生
高氧
Mesenchymal stem cells
Lung
Infant, newborn
Hyperoxia
