抗Ⅳ型胶原酶单链抗体对博莱霉素诱导小鼠肺纤维化的防治作用

Anti-type Ⅳ collagenase single-chain antibody ameliorates bleomycin-induced experimental pulmonary fibrosis in mice

目的研究抗Ⅳ型胶原酶单链抗体scFv（3G11）对博莱霉素（BLM）诱导的小鼠肺纤维化的防治作用。方法C57BL／6小鼠分为空白对照组、盐水对照组、BLM组和低、中、高剂量scFv（3G11）组。BLM组气管内注射BLM400ng／g；低、中、高剂量scFv（3G11）组注射BLM当天记为0d，第1～7天每天腹腔注射scFv（3G11）15、30、45μg／g。第21天处死盐水对照组、BLM组和中剂量scFv（3G11）组小鼠各6只，取左肺行组织病理学观察，并测定肺泡隔面积比和有核细胞计数；另处死空白对照组、盐水对照组、BLM组和低、中、高剂量scFv（3G11）组小鼠各6只，取肺组织测定羟脯氨酸含量以衡量肺胶原沉积状况。取小鼠腹腔巨噬细胞进行原代培养，采用明胶酶谱法观察不同浓度scFv（3G11）（0、15、30、45、60Ixmol／L）对巨噬细胞分泌基质金属蛋白酶2（MMP-2）和MMP-9的影响。结果组织病理学观察显示scFv（3G11）组小鼠肺纤维化程度明显轻于BLM组，肺泡隔面积比（45．3％±3．2％）和有核细胞计数（451±47）均明显低于BLM组（59．0％±3．0％，599±42，均P〈0．01）。中、高剂量scFv（3G11）组小鼠肺组织羟脯氨酸含量[分别为（0．82±0．05）和（0．80±0．03）μg／mg]明显低于BLM组[（0．92±0．07）μg／mg，P〈0．05，P〈0．01]。明胶酶谱分析显示，scFv（3G11）浓度为15μmol／L时即可抑制巨噬细胞分泌的MMP-2和MMP-9的活性。结论抗Ⅳ型胶原酶单链抗体scFv（3G11）能抑制BLM诱导的小鼠肺纤维化。

Objective To study the effect of anti-type 1V collagenase single-chain antibody scFv （3G11 ） on bleomycin （BLM）-induced pulmonary fibrosis. Methods C57BL/6 mice were divided into six groups （ blank, saline control, BLM, low-dose treatment, intermediate-dose treatment, and high-dose treatment）, the animal model of pulmonary fibrosis was induced with 400 ng/g intratracheal BLM. The day that treatment groups were injected with BLM was marked Day 0. Then at Days 1 -7, scFv（3Gll ） was injected once intraperitoneally each day with three dosage [ low-dose treatment （15 μg/g）, intermediatedose treatment （30 μg/g） and high-dose treatment （45μg/g） ]. At Day 21, 6 mice of each group （saline control, BLM, intermediate-dose treatment） were sacrificed and pathomorphological changes of left lungs were evaluated with HE stained sections. Meanwhile, 6 mice of each group （blank, saline control, BLM, low-dose treatment, intermediate-dose treatment, and high-dose treatment） were sacrificed, the content of hydroproline was detected to measure the degree of collagen deposition. Macrophages were extracted from murine abdominal cavity and primarily cultured. And the inhibition of matrix metalloproteinase （ MMP）-2 and MMP-9 excretion by different concentrations of scFv （3Gll） （O, 15, 30, 45, and 60 p, mol/L）was determined by gelatin zymography. Results By image analysis, the degree of fibrosis in the lungs of treated group was lighter than that of the BLM group. The percentages of the area of interalveolar septum （45.3% ± 3.2%） and the number of nucleated cells （451 ± 47） of treated group were remarkably reduced as compared with the BLM group（59. 0% ± 3.0%, 599 ± 42, both P 〈0. 01）. The content of hydroproline of the intermediate-dose treatment group [ （0. 82 ±0. 05） μg,/mg] and high-dose treatment group [ （0. 80 ± 0. 03 ） μg/mg ] was lower than that of the BLM group [ （0. 92 ± 0.07 ） μg/mg, P 〈 0. 05, P 〈 0. 01 ]. The result of gelatin zymography demonstrated that the excretion of MMP-2 and MMP-9 by macrophage was inhibited by scFv （3G11 ） at the concentration of 15μmol/L Conclusion Single-chain antibody directed against type 1V collagenase might inhibit the development of bleomycin-induced pulmonary fibrosis.