CAJ | 학술논문

目的探讨脂肪间充质干细胞(ADSC)对烧伤脓毒症小鼠肾脏损伤的作用及相关机制. 方法 (1)取5只C57 BL/6J小鼠双侧腹股沟脂肪组织,采用酶消化法、密度梯度离心法及贴壁法分离培养纯化小鼠ADSC,取第3代细胞行形态学观察、测定细胞生长曲线,流式细胞仪检测细胞表面分子标记物,并行成骨及成脂肪细胞诱导分化鉴定.(2)另取37只C57BL/6J小鼠,按随机数字表法分为正常对照组、生理盐水组、ADSC组.正常对照组5只,其余2组各16只.生理盐水组及ADSC组小鼠造成背部15％ TBSAⅢ度烫伤后,创面中心注射铜绿假单胞菌菌液制成烧伤脓毒症小鼠模型,之后分别经尾静脉注射生理盐水和ADSC,均于伤后12、24、48、72 h观察肾脏组织病理学变化、检测血肌酐和尿素氮水平,并采用实时荧光定量PCR法检测肾脏组织TNF-α、IL-12、IL-10以及环氧合酶2(COX2) mRNA表达.正常对照组小鼠不行任何处理,但均行上述检测.对数据行多因素方差分析及LSD-t检验. 结果 (1)分离培养的细胞传至第3代时,形态均一与Fb相似,排列规则；CD90+、CD105+、CD34-、CD45-细胞百分比均在90％以上；细胞可向成脂和成骨细胞分化；经鉴定,细胞为ADSC.(2)伤后12～72 h,生理盐水组小鼠肾小管和肾小球中中性粒细胞浸润逐渐增多,管腔受损、肾小球结构被破坏.ADSC组小鼠伤后各时相点肾组织病理学损伤程度均轻于生理盐水组.生理盐水组各时相点血肌酐、尿素氮水平均明显高于正常对照组(P值均小于0.01)；伤后12～72 h,与生理盐水组比较,ADSC组血肌酐、尿素氮水平均明显下降(P值均小于0.01).与正常对照组比较,伤后24 h生理盐水组及ADSC组TNF-α、IL-12 mRNA表达量均升高(P值均小于0.05).伤后24 h,ADSC组TNF-α mRNA水平(1.58±0.19)明显低于生理盐水组的3.36±0.30(P＜0.05).伤后24 h,ADSC组IL-10、COX2 mRNA水平分别为2.89±0.47、4.90±0.59,均高于正常对照组的1.00±0.15、1.00±0.27和生理盐水组的1.32±0.38、1.57±0.38(P值均小于0.05). 结论 ADSC可降低血肌酐和尿素氮水平,促进抗炎因子IL-10、COX2生成,抑制促炎因子TNF-α、IL-12释放,对烧伤脓毒症小鼠肾脏损伤有保护作用. 目的探討脂肪間充質榦細胞(ADSC)對燒傷膿毒癥小鼠腎髒損傷的作用及相關機製. 方法 (1)取5隻C57 BL/6J小鼠雙側腹股溝脂肪組織,採用酶消化法、密度梯度離心法及貼壁法分離培養純化小鼠ADSC,取第3代細胞行形態學觀察、測定細胞生長麯線,流式細胞儀檢測細胞錶麵分子標記物,併行成骨及成脂肪細胞誘導分化鑒定.(2)另取37隻C57BL/6J小鼠,按隨機數字錶法分為正常對照組、生理鹽水組、ADSC組.正常對照組5隻,其餘2組各16隻.生理鹽水組及ADSC組小鼠造成揹部15％ TBSAⅢ度燙傷後,創麵中心註射銅綠假單胞菌菌液製成燒傷膿毒癥小鼠模型,之後分彆經尾靜脈註射生理鹽水和ADSC,均于傷後12、24、48、72 h觀察腎髒組織病理學變化、檢測血肌酐和尿素氮水平,併採用實時熒光定量PCR法檢測腎髒組織TNF-α、IL-12、IL-10以及環氧閤酶2(COX2) mRNA錶達.正常對照組小鼠不行任何處理,但均行上述檢測.對數據行多因素方差分析及LSD-t檢驗. 結果 (1)分離培養的細胞傳至第3代時,形態均一與Fb相似,排列規則；CD90+、CD105+、CD34-、CD45-細胞百分比均在90％以上；細胞可嚮成脂和成骨細胞分化；經鑒定,細胞為ADSC.(2)傷後12～72 h,生理鹽水組小鼠腎小管和腎小毬中中性粒細胞浸潤逐漸增多,管腔受損、腎小毬結構被破壞.ADSC組小鼠傷後各時相點腎組織病理學損傷程度均輕于生理鹽水組.生理鹽水組各時相點血肌酐、尿素氮水平均明顯高于正常對照組(P值均小于0.01)；傷後12～72 h,與生理鹽水組比較,ADSC組血肌酐、尿素氮水平均明顯下降(P值均小于0.01).與正常對照組比較,傷後24 h生理鹽水組及ADSC組TNF-α、IL-12 mRNA錶達量均升高(P值均小于0.05).傷後24 h,ADSC組TNF-α mRNA水平(1.58±0.19)明顯低于生理鹽水組的3.36±0.30(P＜0.05).傷後24 h,ADSC組IL-10、COX2 mRNA水平分彆為2.89±0.47、4.90±0.59,均高于正常對照組的1.00±0.15、1.00±0.27和生理鹽水組的1.32±0.38、1.57±0.38(P值均小于0.05). 結論 ADSC可降低血肌酐和尿素氮水平,促進抗炎因子IL-10、COX2生成,抑製促炎因子TNF-α、IL-12釋放,對燒傷膿毒癥小鼠腎髒損傷有保護作用.
목적 탐토지방간충질간세포(ADSC)대소상농독증소서신장손상적작용급상관궤제. 방법 (1)취5지C57 BL/6J소서쌍측복고구지방조직,채용매소화법、밀도제도리심법급첩벽법분리배양순화소서ADSC,취제3대세포행형태학관찰、측정세포생장곡선,류식세포의검측세포표면분자표기물,병행성골급성지방세포유도분화감정.(2)령취37지C57BL/6J소서,안수궤수자표법분위정상대조조、생리염수조、ADSC조.정상대조조5지,기여2조각16지.생리염수조급ADSC조소서조성배부15％ TBSAⅢ도탕상후,창면중심주사동록가단포균균액제성소상농독증소서모형,지후분별경미정맥주사생리염수화ADSC,균우상후12、24、48、72 h관찰신장조직병이학변화、검측혈기항화뇨소담수평,병채용실시형광정량PCR법검측신장조직TNF-α、IL-12、IL-10이급배양합매2(COX2) mRNA표체.정상대조조소서불행임하처리,단균행상술검측.대수거행다인소방차분석급LSD-t검험. 결과 (1)분리배양적세포전지제3대시,형태균일여Fb상사,배렬규칙；CD90+、CD105+、CD34-、CD45-세포백분비균재90％이상；세포가향성지화성골세포분화；경감정,세포위ADSC.(2)상후12～72 h,생리염수조소서신소관화신소구중중성립세포침윤축점증다,관강수손、신소구결구피파배.ADSC조소서상후각시상점신조직병이학손상정도균경우생리염수조.생리염수조각시상점혈기항、뇨소담수평균명현고우정상대조조(P치균소우0.01)；상후12～72 h,여생리염수조비교,ADSC조혈기항、뇨소담수평균명현하강(P치균소우0.01).여정상대조조비교,상후24 h생리염수조급ADSC조TNF-α、IL-12 mRNA표체량균승고(P치균소우0.05).상후24 h,ADSC조TNF-α mRNA수평(1.58±0.19)명현저우생리염수조적3.36±0.30(P＜0.05).상후24 h,ADSC조IL-10、COX2 mRNA수평분별위2.89±0.47、4.90±0.59,균고우정상대조조적1.00±0.15、1.00±0.27화생리염수조적1.32±0.38、1.57±0.38(P치균소우0.05). 결론 ADSC가강저혈기항화뇨소담수평,촉진항염인자IL-10、COX2생성,억제촉염인자TNF-α、IL-12석방,대소상농독증소서신장손상유보호작용.
Objective To investigate the effect of adipose-derived stem cells (ADSC) on renal injury in mice with burn injury and sepsis and its underlying mechanism.Methods (1) Adipose tissue was collected from both inguinal regions of 5 C57BL/6J mice to isolate,culture and purify ADSC through enzyme digestion,density gradient centrifugation,and adherence method.Cells of the third passage were used in the experiment.The morphologic change in cells was observed and the growth curve of cells was determined.The expression of cell surface antigen phenotype was analyzed by flow cytometry,and the cells were identified by adipogenic and osteogenic differentiation.(2) Another 37 C57BL/6J mice were divided into normal control group (n =5),saline group (n =16),and group ADSC (n =16) according to the random number table.The mice in saline group and group ADSC were injected with Pseudomonas aeruginosa after being subjected to 15％ TBSA full-thickness burn on the back to reproduce septic burn model.Then the mice were injected with saline and ADSC through tail vein respectively.At post burn hour (PBH) 12,24,48,and 72,the pathological change in kidney tissue was observed,tie levels of blood urea nitrogen and serum creatinine were determined,and the levels of TNF-α,IL-12,IL-10,and cyclooxygenase-2 (COX2) mRNA were determined with real-time fluorescence quantitative PCR in both groups.Above-mentioned indexes were also examined in the normal control group (without burn).Data were processed with multifactor analysis of variance and LSD-t test.Results (1) Cells in the third passage were orderly arranged with the shape similarto fibroblasts.The percentages of CD90 +,CD105 +,CD34-,and CD45-cells were all above 90％.The cells could differentiate into osteoblasts and adipocytes.The cells were identified to be ADSC.(2) From PBH 12 to PBH 72,the neutrophil infiltration gradually increased,and the structure of kidney tubules and glomeruli were deranged in saline group.The pathological change in kidney tissue in group ADSC was less serious than that of normal control group at each time point.From PBH 12 to PBH 72,the levels of blood urea nitrogen and serum creatinine in saline group were significantly higher than those of normal control group and group ADSC (P values all below 0.01).Compared with those of the normal control group,the levels of TNF-α and IL-12 mRNA were higher in group ADSC and saline group at PBH 24 (P values all below 0.05).At PBH 24,the level of TNF-α mRNA in group ADSC (1.58±0.19) was lower than that of saline group (3.36 ±0.30,P ＜0.05).At PBH 24,the levels of IL-10 and COX2 mRNA in group ADSC (2.89 ±0.47,4.90 ±0.59) were higher than those in normal control group (1.00 ±0.15,1.00 ±0.27)and saline group (1.32 ± 0.38,1.57 ± 0.38,P values all below 0.05).Conclusions ADSC can decrease the levels of blood urea nitrogen and serum creatinine,promote the production of anti-inflammatory cytokines IL-10 and COX2,and reduce the release of the pro-inflammatory cytokines TNF-α and IL-12 to offer protective effects against renal injury in burn mice with sepsis.