动员骨髓干细胞对大鼠缺血再灌注肾损伤缺氧诱导因子～1α和表皮生长因子表达的影响

Effect of mobilization bone marrow stem cells on hypoxia inducible factor-1a and epidermal growth factor in rats with renal injury induced by ischemic reperfusion

目的 观察干细胞因子（SCF）和粒细胞集落刺激因子（G-CSF）动员自体骨髓干细胞对急性肾小管坏死大鼠缺氧诱导因子-1α（HIF-1α）及表皮生长因子（EGF）表达的影响,探讨SCF和G-CSF治疗大鼠缺血再灌注肾损伤的可能机制.方法 160只8 ～ 10周龄雄性SD大鼠随机分4组：对照组（A组）、缺血再灌注组（B组）、SCF＋ G-CSF治疗组（C组）、SCF＋ G-CSF对照组（D组）.免疫组织化学法（SP法）检测肾组织HIF-1α和链霉素亲和素-生物素-过氧化物酶（SABC）法检测肾组织CD34＋细胞、EGF的表达；RT-PCR检测肾组织中EGF mRNA的表达.结果 1.术后B组、C组肾小管上皮细胞变性、坏死、脱落,随时间推移,肾小管逐渐修复,但C组修复程度明显好于B组.各时间点C组大鼠病理学评分均低于B组,差异有统计学意义（P＜0.05）.2.A组、D组CD34＋细胞表达差异无统计学意义；术后5 d,B组、C组肾组织中CD34＋细胞表达明显增高,与A组、D组相比,差异有统计学意义（P＜0.05）,C组与B组比较增高明显（P＜0.05）,随时间推移逐渐下降.3.B组、C组术后5 d最高,而后逐渐降低,C组各时相与A组、B组、D组比较差异有统计学意义（P＜0.05）.4.EGF蛋白表达：B组在第17天达高峰,显著高于A组（P＜0.05）；C组在第10天达高峰,显著高于A组、B组和D组（P＜0.05）.B组和C组表达量达到高峰后随时间推移逐渐降低.5.肾组织EGF mRNA检测：A组、D组可见少许EGF表达.B组、C组于术后5 d增强,C组显著高于B组,差异有统计学意义（P＜0.05）.术后10 d,C组表达最明显,显著高于A组、B组和D组（P＜0.05）.术后17 d,B组表达达到高峰,高于A组和D组（P＜0.05）.术后24 d,B组、C组表达均降低；术后31d,B组、C组表达降至接近正常,各组间差异无统计学意义（P＞0.05）.6.HIF-1α表达与下游基因产物EGF呈正相关（r=0.815,P＜0.01）.结论 SCF联合G-CSF动员足量的骨髓干细胞“归巢”至损伤部位是促进肾小管损伤修复的关键因素；SCF和G-CSF动员骨髓干细胞能通过增加肾组织HIF-1α及EGF的表达,发挥抗损伤及促修复作用.

Objective To investigate the mobilization of granulocyte-colony stimulating factor（G-CSF） with stem cell factor（SCF） in bone marrow stem cells（BMSC）,and to observe the therapeutic effects of BMSC mobilization on repairing ischemia/reperfusion（I/R） renal injury induced by hypoxia inducible factor-1α （HIF-1α） and epidermal growth factor（EGF）,and to investigate the mechanism for BMSC mobilization for repairing I/R renal injury.MethodsOne hundred and sixty male Sprague-Dawley rats（age：8-10 weeks） were randomly allocated into 4 groups（40 rats in each group）：control group （group A）,I/R group（group B）,SCF ＋ G-CSF treatment group （group C） and SCF ＋ G-CSF control group（group D）.The expressions of CD34 ＋ and EGF were measured by immunohistochemistry technique,and the expression of EGF mRNA was measured by reverse transcriptase-polymerase chain reaction.Results 1.After I/R injury,the renal tubular epithelial cells were in a state of degeneration,necrosis,even exfoliation.As the time prolonged,the necrotic renal tubules were repaired gradually,but the repair in group C was not good compared with that in group B.The level of group C was obviously lower than that of group B on the 5th,10th,17th and 24th day（P ＜ 0.05）.2.There was no significant difference in the percentage of CD34 ＋ cells between group A and D（P ＞ 0.05）.On 5 d postoperative,the level of CD34 ＋ cells of group B and group C was conspicuous higher than group A and D （P ＜ 0.05）,and that of group C was conspicuous higher than that of group B （P ＜ 0.05）.They descended gradually from the 5 th day with the time prolonged.3.The expressions of HIF-1c of group B and group C had a highly positive reaction on the 5th day postoperatively and the level of HIF-1α of group B and group C decended to the normal level with the time prolonged,and at each time,the expression of HIF-1α of group C was significantly higher than that of other groups （P ＜ 0.05）.4.On the 5th day postoperative,the expression of EGF of group B and group C showed a higher positive reaction on the 5th day postoperatively and the level of EGF in group B and group C descended to the normal level with the time prolonged,and at each time the expression of EGF of group C was significantly higher than that of the other groups （P ＜0.05）.5.The expressions of EGF mRNA of group B and C showed higher positive reaction on the 5th day postoperatively（P ＜ 0.05）.The level of EGF mRNA of group B was higher significantly than that of group A on the 17th day postoperatively（P ＜ 0.05）.The level of EGF mRNA of group C was a peak and higher than that of other groups on the 10th day postoperatively（P ＜ 0.05）.The level of EGF of group B and group C descended to a normal level with the time prolonged.6.There was a positive correlation between the expressions of HIF-1α and EGF （r =0.815,P ＜ 0.01）.Conclusions Combination of SCF with the mobilization of G-CSF sufficiently homed BMSC to injured renal tissue was an important factor for the recovery of injured renal tubules.Combination of SCF with G-CSF may promote the expression of HIF-lα and EGF in the kidney may be one of the mechanisms of why combination of SCF with G-CSF can promote recovery from renal ischemic reperfusion injury.