中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2013年
10期
768-774
,共7页
刘少鹏%俞小芳%钟一红%方艺%刘红%许嵘%郭佳%蔡洁茹%刘同强
劉少鵬%俞小芳%鐘一紅%方藝%劉紅%許嶸%郭佳%蔡潔茹%劉同彊
류소붕%유소방%종일홍%방예%류홍%허영%곽가%채길여%류동강
干细胞移植%再灌注损伤%细胞增殖%细胞凋亡%急性肾损伤
榦細胞移植%再灌註損傷%細胞增殖%細胞凋亡%急性腎損傷
간세포이식%재관주손상%세포증식%세포조망%급성신손상
Stem cell transplantation%Reperfusion injury%Cell proliferation%Apoptosis%Acute kidney injury
目的 探讨经大鼠左侧肾动脉移植脂肪干细胞(ASCs)对急性缺血性肾损伤(iAKI)的治疗作用,同时观察ASCs的体内分布情况.方法 采用胶原酶消化法分离培养ASCs并鉴定.夹闭SD大鼠双侧肾蒂45 min建立iAKI模型.按随机数字表法分成两组:对照组(再灌注后即刻经左肾动脉注射500μl PBS,n=30)和ASCs治疗组(再灌注后即刻经肾动脉移植5× 105 ASCs,n=30).术后12、24、48、72 h和1周时处死大鼠,测定血清肌酐值(Scr),光学显微镜观察肾脏病理损伤及细胞凋亡、炎性反应及细胞增生情况,荧光显微镜观察ASCs在肾、肺、肝、脾、心脏的停留时间及分布情况.结果 培养的第3代ASCs具有成脂、成骨等多向分化潜能,流式细胞仪检测高表达CD29 (99.35％)和CD90 (92.88％),低表达CD34(0.48％)和CD45 (3.51％).ASCs治疗组Scr水平各时间点均显著低于对照组(均P＜0.05).与对照组相比,治疗组左侧肾小管间质损伤评分在再灌注12、24、48 h时显著降低(均P＜0.05);肾组织TUNEL染色和巨噬细胞浸润染色积分在再灌注12、24、48、72 h和1周时显著降低(均P＜0.05);核增殖抗原在再灌注48 h时显著增加(P＜0.05),72 h和1周时显著减少(均P＜0.05).与治疗组右肾相比,治疗组左侧肾小管间质损伤评分在再灌注24 h时显著降低(P＜0.05);肾组织TUNEL染色阳性细胞数在再灌注1周时减少(P＜0.05);巨噬细胞浸润染色积分在再灌注12、48、72 h和1周时减少(均P＜0.05);核增殖抗原48 h时增加(P＜0.05),72 h和1周时减少(均P＜ 0.05).荧光显微镜观察显示ASCs在肾脏内停留时间超过1周,但移植48 h后明显减少,肺、肝、脾、心脏仅观察到极少量的Dil阳性细胞.结论 ASCs经肾动脉移植可显著改善iAKI大鼠肾功能、减轻肾脏病理损伤和细胞凋亡、减少炎性细胞浸润、促进损伤后修复,这可能与经肾动脉移植可提高ASCs进入损伤肾脏的数量,减少循环过程中的其他器官截留效应有关.
目的 探討經大鼠左側腎動脈移植脂肪榦細胞(ASCs)對急性缺血性腎損傷(iAKI)的治療作用,同時觀察ASCs的體內分佈情況.方法 採用膠原酶消化法分離培養ASCs併鑒定.夾閉SD大鼠雙側腎蒂45 min建立iAKI模型.按隨機數字錶法分成兩組:對照組(再灌註後即刻經左腎動脈註射500μl PBS,n=30)和ASCs治療組(再灌註後即刻經腎動脈移植5× 105 ASCs,n=30).術後12、24、48、72 h和1週時處死大鼠,測定血清肌酐值(Scr),光學顯微鏡觀察腎髒病理損傷及細胞凋亡、炎性反應及細胞增生情況,熒光顯微鏡觀察ASCs在腎、肺、肝、脾、心髒的停留時間及分佈情況.結果 培養的第3代ASCs具有成脂、成骨等多嚮分化潛能,流式細胞儀檢測高錶達CD29 (99.35％)和CD90 (92.88％),低錶達CD34(0.48％)和CD45 (3.51％).ASCs治療組Scr水平各時間點均顯著低于對照組(均P＜0.05).與對照組相比,治療組左側腎小管間質損傷評分在再灌註12、24、48 h時顯著降低(均P＜0.05);腎組織TUNEL染色和巨噬細胞浸潤染色積分在再灌註12、24、48、72 h和1週時顯著降低(均P＜0.05);覈增殖抗原在再灌註48 h時顯著增加(P＜0.05),72 h和1週時顯著減少(均P＜0.05).與治療組右腎相比,治療組左側腎小管間質損傷評分在再灌註24 h時顯著降低(P＜0.05);腎組織TUNEL染色暘性細胞數在再灌註1週時減少(P＜0.05);巨噬細胞浸潤染色積分在再灌註12、48、72 h和1週時減少(均P＜0.05);覈增殖抗原48 h時增加(P＜0.05),72 h和1週時減少(均P＜ 0.05).熒光顯微鏡觀察顯示ASCs在腎髒內停留時間超過1週,但移植48 h後明顯減少,肺、肝、脾、心髒僅觀察到極少量的Dil暘性細胞.結論 ASCs經腎動脈移植可顯著改善iAKI大鼠腎功能、減輕腎髒病理損傷和細胞凋亡、減少炎性細胞浸潤、促進損傷後脩複,這可能與經腎動脈移植可提高ASCs進入損傷腎髒的數量,減少循環過程中的其他器官截留效應有關.
목적 탐토경대서좌측신동맥이식지방간세포(ASCs)대급성결혈성신손상(iAKI)적치료작용,동시관찰ASCs적체내분포정황.방법 채용효원매소화법분리배양ASCs병감정.협폐SD대서쌍측신체45 min건립iAKI모형.안수궤수자표법분성량조:대조조(재관주후즉각경좌신동맥주사500μl PBS,n=30)화ASCs치료조(재관주후즉각경신동맥이식5× 105 ASCs,n=30).술후12、24、48、72 h화1주시처사대서,측정혈청기항치(Scr),광학현미경관찰신장병리손상급세포조망、염성반응급세포증생정황,형광현미경관찰ASCs재신、폐、간、비、심장적정류시간급분포정황.결과 배양적제3대ASCs구유성지、성골등다향분화잠능,류식세포의검측고표체CD29 (99.35％)화CD90 (92.88％),저표체CD34(0.48％)화CD45 (3.51％).ASCs치료조Scr수평각시간점균현저저우대조조(균P＜0.05).여대조조상비,치료조좌측신소관간질손상평분재재관주12、24、48 h시현저강저(균P＜0.05);신조직TUNEL염색화거서세포침윤염색적분재재관주12、24、48、72 h화1주시현저강저(균P＜0.05);핵증식항원재재관주48 h시현저증가(P＜0.05),72 h화1주시현저감소(균P＜0.05).여치료조우신상비,치료조좌측신소관간질손상평분재재관주24 h시현저강저(P＜0.05);신조직TUNEL염색양성세포수재재관주1주시감소(P＜0.05);거서세포침윤염색적분재재관주12、48、72 h화1주시감소(균P＜0.05);핵증식항원48 h시증가(P＜0.05),72 h화1주시감소(균P＜ 0.05).형광현미경관찰현시ASCs재신장내정류시간초과1주,단이식48 h후명현감소,폐、간、비、심장부관찰도겁소량적Dil양성세포.결론 ASCs경신동맥이식가현저개선iAKI대서신공능、감경신장병리손상화세포조망、감소염성세포침윤、촉진손상후수복,저가능여경신동맥이식가제고ASCs진입손상신장적수량,감소순배과정중적기타기관절류효응유관.
Objective To explore the therapeutic effect of adipose derived stem cells (ASCs)transplanted via left renal artery on rat acute ischemia reperfusion kidney injury (iAKI) and the distribution of ASCs in different organs.Methods ASCs were isolated from inguinal subcutaneous adipose tissue of male SD ras.iAKI model was set in male SD rats by clipping bilateral renal pedicles for 45 min (ischemia reperfusion model).The iAKI rats were randomized into two groups (n =30):control group (renal intra-arterial administration of 500 μl PBS) and ASCs transplantation group (renal intra-arterial administration of 5×105 ASCs).Rats were sacrificed at 12,24,48,72 hours and 1 week after reperfusion to measure renal function by serum creatinine (Scr).Renal pathology,cell apoptosis,inflammation and cell proliferation were analyzed by optical microscope.Distributions of ASCs were measured by fluorescent microscopy.Results ASCs at its third passage had the capacities for adipogenic and osteogenic differentiation,postive for CD29,CD90,and negative for CD34,CD45.Compared with control group,Scr in ASCs transplantation group were significantly lower at all time points (P ＜ 0.05); score of left renal tubular interstitial damage degree in ASCs transplantation group was markedly lower at 12 hours,24 hours,48 hours (P ＜ 0.05); TUNEL and macrophage infiltration score in ASCs transplantation group were significantly lower (P ＜ 0.05); proliferating antigen increased at 48 hours and decreased at 72 hours and 1 week (P ＜ 0.05).Meanwhile,comparing with right kidneys in ASCs transplantation group,score of left renal tubular interstitial damage degree was markedly lower at 24 hours(P ＜ 0.05); the number of TUNEL positive cells at 1 week was observably lower (P ＜ 0.05); macrophage infiltration score were dramatically lower at 12 hours,48 hours,72 hours and 1 week; proliferating antigen increased at 48 hours and decreased at 72 hours and 1 week (P ＜ 0.05).Fluorescence microscope observation showed that residence time of ASCs in kidney was more than 7 days,but the number of ASCs significantly reduced after 48 hours,few Dil positive cells could be observed in lung,liver,spleen and heart.Conclusions ASCs transplantation via renal artery can significantly improve renal function and ameliorate pathological damage,relieve apoptosis and macrophage infiltration,and enhance the repair process after iAKI.It may due to renal intra-arterial transplantation increasing the amounts of ASCs migrated into the kidney in iAKI and reducing ASCs distribution in other organs.
