粒细胞集落刺激因子动员自体骨髓源性细胞对博来霉素致小鼠肺损伤的治疗作用

目的 观察粒细胞集落刺激因子(G-CSF)动员自体骨髓源性细胞(BMDC)对博来霉素所致小鼠肺损伤的修复作用.方法 采用随机数字表法将60只骨髓重建后小鼠分为重建动员组(博来霉素+G-CSF)和重建对照组(博来霉素+生理盐水),每组30只;75只健康小鼠分为未重建动员组30只(博来霉素+G-CSF)、未重建对照组30只(博来霉素+生理盐水)和空白对照组15只(生理盐水+生理盐水).各组分别在实验第3、第7和第14天处死1/3小鼠取材.通过HE染色、Masson胶原染色、肺通透指数和羟脯氨酸含量评价疗效.免疫组织化学方法检测肺组织中转化生长因子-β_1(TGF-β_1)、γ-干扰素、基质金属蛋白酶_9(MMP-9)和金属蛋白酶组织抑制物-1(TIMP-1)的表达;流式细胞仪和激光共聚焦显微镜检测肺组织内BMDC表达.将20只健康小鼠分为观察动员组(博来霉素+G-CSF)和观察对照组(博来霉素+生理盐水),每组10只,不设终点,观察小鼠存活时间.计量资料比较采用t检验(两组间)和单因素方差分析(多组间),组间两两比较采用LSD法;等级资料比较采用Mann-Whitney μ检验(两组间)和Kruskal-Wallis H检验(多组间);采用Kaplan-Meier法进行生存分析.结果 实验第7天,重建动员组肺泡炎程度的平均秩次为15.3,肺间质纤维化程度的阳性面积(×10~3)为46士8,肺羟脯氨酸含量为(O.44±0.09)μg/mg,TGF-β_1、γ-干扰素和MMP-9表达的积分吸光度值(×10~3)分别为111±23、250±72和59±19,均明显低于重建埘照组[28.0、(73±10)×10~3、(0.52±0.07)μg/mg、(161 ±35)×10~3、(299±31)×10~3和(314±77)×10~3];未重建动员组肺泡炎程度的平均秩次为22.7,肺间质纤维化程度的阳性面积(×10~3)为27±15,肺羟脯氨酸含量为(0.41±0.05)μg/mg,肺通透指数为(43.8 ±9.9)×10~(-3),TGF-β_1、γ-干扰素和MMP-9表达的积分吸光度值(×10~3)分别为132±55、178±23和101±54,均明显低于未重建对照组[33.9、(56±13)×10~3、(0.49±0.08)μg/mg、(53.6±8.7)×10~(-3)、(320±98)×10~3、(409±61)×10~3和(288±75)×10~3].重建动员组在第7大肺内BMDC表达的绿色荧光蛋白阳性细胞比例(0.65±0.13)明显高于重建对照组(0.46±0.11).结论 G-CSF动员BMDC对博来霉素所敛肺损伤有一定的保护作用,本实验方法尚不能明显延长小鼠的存活时间.

The effect of autologous bone marrow-derived cells mobilized by granulocyte colony stimulating factor on bleomycin-induced lung injury in mice

Objective To investigate the contribution of mobilized autologous bone marrow-derived cells ( BMDC) to lung repair after lung injury induced by bleomycin, and the mechanisms of any protective effects conferred by BMDC. Methods Sixty marrow-reconstructed mice were randomly divided into 2 groups: group A [bleomycin + granulocyte colony stimulating factor( G-CSF) ] and group B (bleomycin +saline). Seventy-five normal mice were randomly divided into 3 groups: group C (bleomycin + G-CSF) :group D (bleomycin + saline) and group N (saline only). Each group was further divided into 3 subgroups,which were sacrificed respectively on days 3, 7 and 14. Therapeutic evaluations were made by means of HE stain, Masson' s trichrome stain, hydroxyproline concentration and pulmonary permeability index. The expressions of TGF-β_1, IFN-γ, MMP-9 and TIMP-1 in the lung tissue were detected by immunohistochemistry. Intrapulmonary BMDC was evaluated by flow cytometry and laser scanning confocal microscope. Another 20 mice were randomly divided into 2 groups including group E ( bleomycin + G-CSF) and group F (bleomycin + saline). The survival time of each mouse was observed without end point Results The alveolitis score ( mean rank 15.3 ) , the pulmonary fibrosis score (46 ±8 ) , the hydroxyproline concentrations (0.44 ±0.09) μg/mg, the TGF-β_1, level (111 ±23), the IFN-γ level (250 ±72) and the MMP-9 level (59±19) were significantly decreased in reconstructed treatment group on day 7 as compared to reconstructed control group, which was respectively (mean rank 28.0) , (73 ± 10) , (0. 52 ±0. 07)μg/mg, (161±35) , (299 ±31) and (314±77). Likewise, the alveolitis (mean rank 22. 7) , the pulmonary fibrosis (27±15), the hydroxyproline concentrations (0.41±0.05) μg/mg, the pulmonary permeability index (43.8 4-9.9)× 10~(-3),the TCF-β_1 level(132±55),the IFN-γ level(178±23),and the MMP-9 level ( 101 ±54) in non-reconstructed treatment group on day 7 were significantly lower than those in non-reconstructed control group, (mean rank 33.9), (56 ±13), (0.49±0.08) μg/mg, (54±9)×10~(-3), (320±98), (409 ±×61), (288±75), the differences being statistically significant (P<0.05). The intrapulmonary BMDC level of reconstructed treatment group (0. 65 ±0. 13) was significantly higher than that in reconstructed control group (0.46±0. 11) , P <0. 05. Conclusion Mobilization of BMDC by G-CSF showed a protective effect on lung injury induced by bleomycin in mice, but did not have significant influence on survival time.