CAJ | 학술논문

目的通过肥大细胞与肺成纤维细胞接触和非接触共体培养,观察肥大细胞对肺成纤维细胞增殖、转化及功能的影响.方法分离大鼠肺成纤维细胞和腹腔肥大细胞,实验分为3组:接触共育组、非接触共育组、对照组.计数各组肺成纤维细胞数量,通过免疫细胞化学方法检测肌成纤维细胞特异性标志物α-平滑肌肌动蛋白(α-SMA),噻唑蓝(MTT)比色法测定肺成纤维细胞增殖率,通过酶联免疫吸附试验(ELISA)法检测培养液中Ⅰ型胶原蛋白含量.结果各组肺成纤维细胞数目在培养的第5天均达峰值,分别为接触共育组(3.70±0.83)×106个/孔；非接触共育组(1.40 ±0.32)×106个/孔；对照组(1.10 ±0.23)×106个/孔,接触共育组与非接触共育组和对照组比较,差异有统计学意义(P＜0.05)；MTT检测各组肺成纤维细胞吸光度(A)值:接触共育组为0.2520±0.0318,非接触共育组为0.1250±0.0212,对照组为0.1210±0.0124,接触共育组与非接触共育组和对照组比较,差异有统计学意义(P＜0.05)；各组α-SMA染色的平均吸光度(IA)值分别为0.3190±0.0436、0.1840±0.0190、0.1730±0.0226,接触共育组与非接触共育组和对照组比较,差异有统计学意义(P＜0.05)；接触共育组,培养液中Ⅰ型胶原蛋白含量明显高于非接触共育组和对照组[(192.56±2.37)、(143.58±2.07)、(121.43±1.98) μg/L,P＜0.05).结论肥大细胞可能通过紧密接触来促进肺成纤维细胞的增殖、转化及胶原合成.
목적 통과비대세포여폐성섬유세포접촉화비접촉공체배양,관찰비대세포대폐성섬유세포증식、전화급공능적영향.방법 분리대서폐성섬유세포화복강비대세포,실험분위3조:접촉공육조、비접촉공육조、대조조.계수각조폐성섬유세포수량,통과면역세포화학방법검측기성섬유세포특이성표지물α-평활기기동단백(α-SMA),새서람(MTT)비색법측정폐성섬유세포증식솔,통과매련면역흡부시험(ELISA)법검측배양액중Ⅰ형효원단백함량.결과 각조폐성섬유세포수목재배양적제5천균체봉치,분별위접촉공육조(3.70±0.83)×106개/공；비접촉공육조(1.40 ±0.32)×106개/공；대조조(1.10 ±0.23)×106개/공,접촉공육조여비접촉공육조화대조조비교,차이유통계학의의(P＜0.05)；MTT검측각조폐성섬유세포흡광도(A)치:접촉공육조위0.2520±0.0318,비접촉공육조위0.1250±0.0212,대조조위0.1210±0.0124,접촉공육조여비접촉공육조화대조조비교,차이유통계학의의(P＜0.05)；각조α-SMA염색적평균흡광도(IA)치분별위0.3190±0.0436、0.1840±0.0190、0.1730±0.0226,접촉공육조여비접촉공육조화대조조비교,차이유통계학의의(P＜0.05)；접촉공육조,배양액중Ⅰ형효원단백함량명현고우비접촉공육조화대조조[(192.56±2.37)、(143.58±2.07)、(121.43±1.98) μg/L,P＜0.05).결론 비대세포가능통과긴밀접촉래촉진폐성섬유세포적증식、전화급효원합성.
Objective To investigate the possible roles of mast cells on the proliferation and transformation of lung fibroblast cells by contact and non-contact co-culture of the two cell lines.Methods Fibroblast cells were separated from adult rat lung and cultured in vitro with trypsinization.Collect rat peritoneal lavage solution to separate and purify mast cells with mast cell separating medium under asepsis circumstance,then qualificated with aniline blue.Cells were divided into 3 groups,each with three subgroups:control group; contact co-culture group; non-contact co-culture group.Each group were cultured for five days,counting lung fibroblast cells under microscope everyday to set up cell growth curve.After five days,α-smooth muscle actin (α-SMA) was measured by immunocy tochemistry methods in myofibroblasts,lung fibroblast cell hyperplasia rate was measured with methyl thiazol tetrazolium (MTT) colorimetric method and cells count,collagen Ⅰ protein in culture were measured with enzyme linked immunosorbent assay (ELISA) method.Results The difference of Fb amount between contact co-culture group and other two groups were significant [(3.70 ± 0.83) × 106,(1.40 ± 0.32) × 106,(1.10 ± 0.23) × 106 ; P ＜0.05) ; Contact co-culture group enhance the generation of lung fibroblast cells compared with control group and non-contact co-culture group (A:0.2520 ± 0.0318,0.1250 ± 0.0212,0.1210 ± 0.0124; P＜0.05) ; Compared with non-contact co-culture group and control group,the average optical value (IA) of massive α-SMA positive cells in contact co-culture group increased significantly (0.3190 ± 0.0436,0.1840 ±0.0190,0.1730-±0.0226; P ＜0.05) ; Compared with control group and non-contact co-culture group,collagen Ⅰ protein were significantly higher in contact co-culture group [(192.56 ± 2.37),(143.58±2.07),(121.43±1.98) μg/L; P＜0.05].Conclusion Mast cellscan upregulate the synthesis of collagen Ⅰ protein by a compact contact way with lung fibroblast cells ; The proliferation and transformation of lung fibroblast cells is more significant when contact co-culture with mast cells.