人脐带间充质干细胞对感染状态下人肺泡Ⅱ型上皮细胞的影响

摘要

[目的]探讨人脐带间充质干细胞(hUCMSC)对感染铜绿假单胞菌(PA)状态下人肺泡Ⅱ型上皮细胞(A549)的影响.[方法]人肺泡Ⅱ型上皮细胞A549(1×105/mL)2 mL与PA(3×104 CFU/mL)2 mL共培育6 h后,加入hUCMSC(1×106/mL)2 mL为实验组,加入等量磷酸缓冲液(PBS)为感染组,A549与PBS及培养基共培育为对照组.比较组间A549细胞形态学改变(透射电镜),A549细胞存活率(CCK-8新型细胞增殖检测试剂盒),A549细胞凋亡率(Annexin V-FITC/PI双染色流式细胞仪)以及A549肺表面活性物质A(SP-A)表达量(Western Blot).[结果]电镜细胞形态学观察显示,感染组A549细胞受损明显,胞质出现空泡变性,染色质高度凝集,可见凋亡小体;实验组细胞包膜结构完整,核膜完整,核仁明显,核染色质电子密度低,染色质均一,未见凋亡小体;对照组A549细胞结构完整,细胞膜表面微绒毛丰富,核膜完整,核周间隙结构正常,染色质均一;感染组与对照组相比,感染组A549细胞细胞存活率显著降低[(70.35±2.89)%与(97.37±2.07)%,n=3,P<0.01],凋亡率显著增加[(8.63%±0.16)%与(2.55±0.11)%,n=3,P<0.01],SP-A表达显著降低[(0.105±0.01)与(0.232±0.015),n=3,P<0.01];实验组与感染组相比,实验组细胞存活率显著增加[(85.69±3.07)%与(70.35±2.89)%,n=3,P<0.01],凋亡率显著下降[(6.34±0.12)%与(8.63±0.16)%,n=3,P<0.01],感染组中,PA可能损伤A549细胞,使其SP-A表达量著减少(n=5,P<0.05),实验组中,hUCMSC可能对感染后A549细胞的保护作用,其SP-A表达量增加(n=5,P<0.05).[结论]hUCMSC能抑制感染状态下的A549细胞凋亡,保护A549细胞分泌SP-A.%[Objective]To investigate the effect of human umbilical cord mesenchymal stem cells on infected state of human alve?olar type Ⅱ epithelial cells.[Methods]Human alveolar type Ⅱ epithelial cells A549(1×105/mL)2 mL and PA(3×104 CFU/mL)2 mL has grown after 6 hours,add hUCMSC(1 × 106/mL)2 mL as the experimental group,add equal amounts of phosphate buffer (PBS)for infection,A549 and PBS and the medium has grown as the control group. A549 cells morphological changes between the compared groups(Transmission electron microscope,TEM),A549 cell viability(new CCK-8 cell proliferation assay Kit),A549 cells apoptosis(Annexin V-FITC/PI double staining flow cytometry)and the expression of A549 pulmonary surfactant A(SP-A) (Western blot).[Results]Transmission electron microscope cell morphology observation displayed ,infection group A549 cell dam?aged obviously,cell quality appeared empty bubble degeneration,chromatin height agglutination,visible apoptosis bodies;experi?ment group cell package film structure full,nuclear film full,nucleolus obviously,nuclear chromatin electronic density low,chroma? tin uniform,no apoptotic bodies;control group A549 cell structure full,membrane surface micro-fluff rich,nuclear film full,nucle?ar week clearance structure normal,chromatin uniform;infection group and control group compared,Infection group A549 cell sur?vival significantly reduced[(70.35±2.89)% and(97.37±2.07)%,n=3,P<0.01],apoptosis rate significantly increased[(8.63%± 0.16)%and(2.55±0.11)%,n=3,P<0.01],In the infected group,PA could damage A549 cells and decrease the amount of SP-A ex?pression(n=5,P<0.05). In the experiment group,the protective effect of hUCMSC on the A549 cells after infection may increase the expression of SP-A(n=5,P<0.05);[Conclusions]HUCMSC inhibits the infection of A549 cells apoptosis and protection of A549 cells secrete SP-A.