中华结核和呼吸杂志
中華結覈和呼吸雜誌
중화결핵화호흡잡지
Chinese Journal of Tuberculosis and Respiratory Diseases
2009年
12期
902-907
,共6页
姜寒水%李惠萍%胡洋%褚建新%陈岗%张容轩%郑卉
薑寒水%李惠萍%鬍洋%褚建新%陳崗%張容軒%鄭卉
강한수%리혜평%호양%저건신%진강%장용헌%정훼
肺纤维化%博来霉索%骨髓细胞%粒细胞集落刺激因子
肺纖維化%博來黴索%骨髓細胞%粒細胞集落刺激因子
폐섬유화%박래매색%골수세포%립세포집락자격인자
Pulmonary fibrosis%Bleomycin%Bone marrow cells%Granulocyte colony-stimulating factor
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.
目的 觀察粒細胞集落刺激因子(G-CSF)動員自體骨髓源性細胞(BMDC)對博來黴素所緻小鼠肺損傷的脩複作用.方法 採用隨機數字錶法將60隻骨髓重建後小鼠分為重建動員組(博來黴素+G-CSF)和重建對照組(博來黴素+生理鹽水),每組30隻;75隻健康小鼠分為未重建動員組30隻(博來黴素+G-CSF)、未重建對照組30隻(博來黴素+生理鹽水)和空白對照組15隻(生理鹽水+生理鹽水).各組分彆在實驗第3、第7和第14天處死1/3小鼠取材.通過HE染色、Masson膠原染色、肺通透指數和羥脯氨痠含量評價療效.免疫組織化學方法檢測肺組織中轉化生長因子-β_1(TGF-β_1)、γ-榦擾素、基質金屬蛋白酶_9(MMP-9)和金屬蛋白酶組織抑製物-1(TIMP-1)的錶達;流式細胞儀和激光共聚焦顯微鏡檢測肺組織內BMDC錶達.將20隻健康小鼠分為觀察動員組(博來黴素+G-CSF)和觀察對照組(博來黴素+生理鹽水),每組10隻,不設終點,觀察小鼠存活時間.計量資料比較採用t檢驗(兩組間)和單因素方差分析(多組間),組間兩兩比較採用LSD法;等級資料比較採用Mann-Whitney μ檢驗(兩組間)和Kruskal-Wallis H檢驗(多組間);採用Kaplan-Meier法進行生存分析.結果 實驗第7天,重建動員組肺泡炎程度的平均秩次為15.3,肺間質纖維化程度的暘性麵積(×10~3)為46士8,肺羥脯氨痠含量為(O.44±0.09)μg/mg,TGF-β_1、γ-榦擾素和MMP-9錶達的積分吸光度值(×10~3)分彆為111±23、250±72和59±19,均明顯低于重建塒照組[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];未重建動員組肺泡炎程度的平均秩次為22.7,肺間質纖維化程度的暘性麵積(×10~3)為27±15,肺羥脯氨痠含量為(0.41±0.05)μg/mg,肺通透指數為(43.8 ±9.9)×10~(-3),TGF-β_1、γ-榦擾素和MMP-9錶達的積分吸光度值(×10~3)分彆為132±55、178±23和101±54,均明顯低于未重建對照組[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].重建動員組在第7大肺內BMDC錶達的綠色熒光蛋白暘性細胞比例(0.65±0.13)明顯高于重建對照組(0.46±0.11).結論 G-CSF動員BMDC對博來黴素所斂肺損傷有一定的保護作用,本實驗方法尚不能明顯延長小鼠的存活時間.
목적 관찰립세포집락자격인자(G-CSF)동원자체골수원성세포(BMDC)대박래매소소치소서폐손상적수복작용.방법 채용수궤수자표법장60지골수중건후소서분위중건동원조(박래매소+G-CSF)화중건대조조(박래매소+생리염수),매조30지;75지건강소서분위미중건동원조30지(박래매소+G-CSF)、미중건대조조30지(박래매소+생리염수)화공백대조조15지(생리염수+생리염수).각조분별재실험제3、제7화제14천처사1/3소서취재.통과HE염색、Masson효원염색、폐통투지수화간포안산함량평개료효.면역조직화학방법검측폐조직중전화생장인자-β_1(TGF-β_1)、γ-간우소、기질금속단백매_9(MMP-9)화금속단백매조직억제물-1(TIMP-1)적표체;류식세포의화격광공취초현미경검측폐조직내BMDC표체.장20지건강소서분위관찰동원조(박래매소+G-CSF)화관찰대조조(박래매소+생리염수),매조10지,불설종점,관찰소서존활시간.계량자료비교채용t검험(량조간)화단인소방차분석(다조간),조간량량비교채용LSD법;등급자료비교채용Mann-Whitney μ검험(량조간)화Kruskal-Wallis H검험(다조간);채용Kaplan-Meier법진행생존분석.결과 실험제7천,중건동원조폐포염정도적평균질차위15.3,폐간질섬유화정도적양성면적(×10~3)위46사8,폐간포안산함량위(O.44±0.09)μg/mg,TGF-β_1、γ-간우소화MMP-9표체적적분흡광도치(×10~3)분별위111±23、250±72화59±19,균명현저우중건시조조[28.0、(73±10)×10~3、(0.52±0.07)μg/mg、(161 ±35)×10~3、(299±31)×10~3화(314±77)×10~3];미중건동원조폐포염정도적평균질차위22.7,폐간질섬유화정도적양성면적(×10~3)위27±15,폐간포안산함량위(0.41±0.05)μg/mg,폐통투지수위(43.8 ±9.9)×10~(-3),TGF-β_1、γ-간우소화MMP-9표체적적분흡광도치(×10~3)분별위132±55、178±23화101±54,균명현저우미중건대조조[33.9、(56±13)×10~3、(0.49±0.08)μg/mg、(53.6±8.7)×10~(-3)、(320±98)×10~3、(409±61)×10~3화(288±75)×10~3].중건동원조재제7대폐내BMDC표체적록색형광단백양성세포비례(0.65±0.13)명현고우중건대조조(0.46±0.11).결론 G-CSF동원BMDC대박래매소소렴폐손상유일정적보호작용,본실험방법상불능명현연장소서적존활시간.
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.
