γ-干扰素转基因表达对不同时期博莱霉素致小鼠肺间质纤维化的作用

Effects of murine interferon-γ transgene expression on bleomycin-induced pulmonary fibrosis in mice at different stages

目的 探讨小鼠γ -干扰素 (mIFN -γ)在气道上皮细胞局部转基因高效表达对不同时期博莱霉素致小鼠肺间质纤维化的治疗作用。方法 选择 4周龄的雄性ICR小鼠 (2 5g左右 ) ,使用博莱霉素诱导小鼠肺间质纤维化。将小鼠分为 5组 ,阳性对照组 (PC)为吸入博莱霉素组 ,阴性对照组 (NC)为经鼻吸入生理盐水组 ,基因治疗A、B、C组分别于博莱霉素刺激后 7、14和 2 1d给予小鼠滴入含有小鼠γ -干扰素cDNA片段的重组复制缺陷型腺病毒 (AdCMVmIFN -γ) 5× 10 8空斑形成单位(pfu/mL) ,在给予腺病毒后 2周处死小鼠 ,收集小鼠肺组织及肺泡灌洗液 (BALF) ,右肺经HE和Mas son染色 ,左肺匀浆后检测羟脯氨酸含量。结果 经气道给予AdCMVmIFN γ处理的转基因组小鼠BALF中均可测得高浓度mIFN -γ表达 ,其水平在基因治疗A组 (GTA)、基因治疗B组 (GTB)和基因治疗C组 (GTC)分别为 (895 4 . 6 8± 85 3 .88)pg/mL、(9312. 6 4± 36 1 2 .8)pg/mL和 (96 4 3. 32± 6. 12 74 )pg/mL。博莱霉素刺激后第 7d时给予小鼠mIFN γ转基因处理 (GTA组 ) ,小鼠肺间质纤维化程度和羟脯氨酸含量为 (12 6 7. 0 9± 37 .0 7) μg ,与阳性对照组的 (12 4 4 . 86± 6 2. 4 6 ) μg相近 ,而于14d(GTB组 )和 2 1d(GTC组 )后给予小鼠mIFN- γ转基因?

Objective To investigate the effects of murine interferon-gamma (mIFN-γ) transgene expression via adenoviral vector on bleomycin-induced pulmonary fibrosis in mice at different pathological stages.Methods Specific pathogen-free male ICR mice of 4 week age with an average weight of 25 g were administrated with bleomycin to induce pulmonary interstitial fibrosis.The mice were randomly divided into five groups.Positive control (PC) group was treated with bleomycin only.Negative control (NC) group was treated with saline.Gene therapy groups (GTA, GTB and GTC) were administrated with adenoviral vector carrying murine IFN-γ cDNA (AdCMV mIFN-γ) 5×10 8 plague forming unit (pfu/mL) via nostril instillation at the day 7,14,and 21 after bleomycin administration, respectively.Mice were sacrificed 2 weeks after treated with AdCMV mIFN-γ.The bronchial alveolar lavage fluid (BALF) was recovered and lungs were sampled.The right lung was stained with either hematoxylin-eosin or Masson trichrome.Left lung was weighed and its hydroxyproline (HYP) content was assayed by HCl acid hydrolysis.mIFN-γ concentration in BALF was assayed by enzyme-linked immunosorbent assay (ELISA).Results The concentrations of mIFN-γ in BALF in GTA,GTB and GTC treated by AdCMV mIFN-γ were ( 8 954.68±853.88)pg/mL, ( 9 312.64±361.28)pg/mL and ( 9 643.32±612.74)pg/mL,respectively,which were significantly higher than those in PC and NC groups.The mice treated with AdCMV mIFN-γ at day 7 (GTA) exhibited the similar degree of pulmonary fibrosis and HYP content [( 1 267.09±37.07)μg/g] to those of PC group [HYP was (1 244.86±62.46)μg/g].The pulmonary fibrosis was milder and HYP contents [(477.24±30.79)μg and (734.29±53.12)μg,respectively]were lower in GTB and GTC groups significantly than those in PC group.The pulmonary fibrosis was ameliorated and HYP content was reduced in GTB group significantly compared with those in PC,GTA,and GTC groups (P<0.01) which is similar to those in NC group [(325.24±11.77)μg/g].Conclusions (1)mIFN-γ could be transgenically overexpressed in murine airway and alveolar epithelial cells via AdCMV mIFN-γ nostril administration;(2)At the stage of acute inflammatory response induced by bleomycin, mIFN-γ transgene expression could not alleviate the lung fibrosis at end point;(3)mIFN-γ transgene expression could inhibit proliferation of fibroblast and deposition of collagen at the fibrosing stage;(4)mIFN-γ transgene expression could not reverse the formed pulmonary fibrosis.