CAJ | 학술논문

目的观察粒细胞集落刺激因子(G-CSF)是否可以有效动员骨髓干细胞进入外周血并向缺血-再灌注损伤肾脏归巢,提高肾脏修复的效能.方法 6周龄全身表达绿色荧光蛋白(GFP)的C57BL/6J转基因小鼠提供骨髓,6～8周龄同种无荧光标记的C57BL/6J小鼠20只作为骨髓受体.骨髓移植前,受体小鼠接受致死剂量的γ放射线137Cs照射,骨髓重建情况经流式细胞仪检测确认.骨髓重建完毕后所有小鼠均接受单侧肾脏缺血-再灌注损伤.实验动物分组:(1)对照组(n=10),术前3d至术后4d,皮下注射生理盐水,0.2 ml/d.(2)动员组(n=10),术前3d至术后4d,皮下注射人重组粒细胞集落刺激因子200μg/(kg·d).干细胞动员效果及向肾脏归巢情况经流式细胞仪检测鉴定.损伤1周后取肾脏标本行HE染色,评估肾小管损伤程度.损伤4周后通过组织切片免疫荧光组织化学方法观察并计数血管内皮细胞数.实验数据中计量资料以均数±标准差((x-)±s)表示,两组计量资料之间比较采用独立样本t检验.结果 G-CSF动员后,分别为Sca-1、c-Kit、CD29、CD34阳性的四群干细胞占外周血非红系细胞的比例均高于对照组(P＜0.05).损伤1周后,G-CSF动员组的缺血侧肾脏中,骨髓来源并且分别表达Sca-1、c-Kit、CD34的干细胞的比例均高于对照组(P＜0.05)；肾小管损伤程度评分分值低于对照组(P＜0.05).损伤4周后动员组的肾脏中CD31阳性的内皮细胞数目高于对照组(P＜0.05).结论 (1)G-CSF可以有效动员骨髓干细胞进入外周血并向缺血肾脏归巢；(2) G-CSF动员可以提高肾脏修复的效能,减轻肾脏组织病理学损伤程度.
목적 관찰립세포집락자격인자(G-CSF)시부가이유효동원골수간세포진입외주혈병향결혈-재관주손상신장귀소,제고신장수복적효능.방법 6주령전신표체록색형광단백(GFP)적C57BL/6J전기인소서제공골수,6～8주령동충무형광표기적C57BL/6J소서20지작위골수수체.골수이식전,수체소서접수치사제량적γ방사선137Cs조사,골수중건정황경류식세포의검측학인.골수중건완필후소유소서균접수단측신장결혈-재관주손상.실험동물분조:(1)대조조(n=10),술전3d지술후4d,피하주사생리염수,0.2 ml/d.(2)동원조(n=10),술전3d지술후4d,피하주사인중조립세포집락자격인자200μg/(kg·d).간세포동원효과급향신장귀소정황경류식세포의검측감정.손상1주후취신장표본행HE염색,평고신소관손상정도.손상4주후통과조직절편면역형광조직화학방법관찰병계수혈관내피세포수.실험수거중계량자료이균수±표준차((x-)±s)표시,량조계량자료지간비교채용독립양본t검험.결과 G-CSF동원후,분별위Sca-1、c-Kit、CD29、CD34양성적사군간세포점외주혈비홍계세포적비례균고우대조조(P＜0.05).손상1주후,G-CSF동원조적결혈측신장중,골수래원병차분별표체Sca-1、c-Kit、CD34적간세포적비례균고우대조조(P＜0.05)；신소관손상정도평분분치저우대조조(P＜0.05).손상4주후동원조적신장중CD31양성적내피세포수목고우대조조(P＜0.05).결론 (1)G-CSF가이유효동원골수간세포진입외주혈병향결혈신장귀소；(2) G-CSF동원가이제고신장수복적효능,감경신장조직병이학손상정도.
Objective To investigate mobilization of the bone-marrow-derived stem cell (BMSC) into peripheral blood by granulocyte-colony stimulating factor (G-CSF) to accelerate the renal regeneration.Methods Six-week-old transgenic C57BL/6J mice labeled with green fluorescent protein (GFP) as bone marrow donors and C57BL/6 mice without fluorescence label as recipients ( n =20 ) of bone marrow transplantation were used.All recipients received lethal dose of 8.5 Gy total body γ-ray irradiation with 137 Cs before bone marrow transplantation,and the transplantation of bone marrow mononuclear cells 2 × 105 by retrobulbar injection was done two hours later after irradiation. Bone marrow reconstruction after transplantation was proved by flow cytometry five weeks after transplantation.Six weeks after the bone marrow reconstruction completed,left renal pedicles of all mice were cross-clasped for 30 minutes followed by reperfusion to establish the animal model of ischemia-reperfusion injury.Mice were divided into two groups:( 1 ) Saline control group ( n =10),saline 0.2 ml/day was injected subcutaneously into chimeric mice from 3 days before to 4 days after operation ; (2) G-CSF mobilization group (n =10),chimeric mice were injected subcutanously with recombinant human G-CSF,200μg/kg/day,once a day from three days before surgery for a week.On the 1st day after mobilization,the percentage of stem cell in non-erythroid cells of peripheral blood was detected by using flow cytometry.One week after ischemia,the homing of BMSC to kidney was identified by flow cytometory.Renal tissue sections were stained with Hemotoxylin and Eosin staining method for pathological study,and the degree of renal tubular injury was analyzed by semiquantitative method of Vyacheslav.Four weeks after ischemia,the differences in degree of renal regeneration between the two groups by analysis the numbers of vascular endothelial cells in the kidney.Results After G-CSF mobilization,the percentage of stem cells with Sca-1 +,c-Kit +,CD29 and CD34 + antigen in peripheral blood in G-CSF mobilization group were higher than those in control group.One week after ischemia,mice of mobilization group showed higher percentage of Sca-1 +,c-Kit + and CD34 + bone marrow derived stem cells in tbe kidney compared to control group (P ＜0.05).One week after ischemia,the tubular epithelial damage score of mobilization group was lower significantly than that of the control group (P ＜ 0.05 ) studied by Hemotoxylin and Eosin staining. Four weeks after ischemia,mice of G-CSF mobilization group showed more CD31 positive cells in the kidney compared to control group (P ＜ 0.05 ).Conclusions G-CSF can effectively mediate the mobilization of bone marrow derived stem cells to peripheral blood and homing to kidney.G-CSF mobilization can accelerate renal regeneration and alleviate the degree of renal histopathological changes after ischemia.