自体骨髓间充质干细胞移植治疗兔肾缺血再灌注损伤

Autologous bone marrow mesenchymal stem cell transplantation for the treatment of rabbit renal ischemia/reperfusion injury

目的:骨髓间充质干细胞可分化为肾小管上皮细胞,有效促进损伤肾脏功能的修复,但以往研究多以大鼠作为实验对象采用同种异体移植。实验采用自体骨髓间充质干细胞行兔左肾动脉灌注治疗,以观察移植细胞对缺血再灌注损伤后肾组织结构和功能修复的影响。方法:实验于2006-03/12在解放军成都军区昆明总医院完成。①实验材料:健康2-3月龄日本大耳白兔24只,雌雄不限,体质量(2.6±0.7)kg,由解放军昆明总医院实验动物中心提供,实验过程中对动物处置符合动物伦理学标准。将24只兔随机均分为移植组和对照组。②实验方法:以红细胞破碎法结合贴壁培养法分离培养兔骨髓间充质干细胞,免疫细胞化学染色鉴定细胞表面标记物CD34、CD105、CD106的表达。采用右肾切除联合左肾动脉缺血85min再灌注的方法,制备兔肾缺血再灌注损伤模型。造模后移植组经右股动脉插管行自体骨髓间充质干细胞左肾动脉灌注治疗,对照组注入等量生理盐水。③实验评估:分别在移植前及移植后3,7,14,21,28d行肾功检测和肾组织病理学观察。结果:①刚接种的细胞主要为圆形,第7天左右细胞呈现克隆性生长,呈骨髓间充质干细胞特征性的旋涡状生长,细胞形态多呈梭形,传代细胞生长速度明显增快,细胞体积明显增大。P3代细胞CD34染色均呈阴性反应,而CD105、CD106染色阳性细胞在超过98%。②造模后第3天两组的血清肌酐、尿素氮均达最高水平,自第7天起移植组血清肌酐、尿素氮水平逐渐低于对照组,第21天两组间血清肌酐和尿素氮差异显著(P<0.05)。③缺血再灌注损伤后两组肾小管上皮细胞变性、坏死甚至脱落,随着时间延长,坏死肾小管逐渐修复,但移植组修复程度明显好于对照组。结论:移植的自体骨髓间充质干细胞可在一定程度上加速缺血再灌注损伤后肾组织结构和功能的恢复。

AIM：Bone marrow mesenchymal stem cells （BMSCs） can differentiate into tubular epithelial cells for the functional reparation of injured renal tissue. But, many of these were implemented by rat allogenic transplantation. This study aimed to observe the effect of autologous BMSCs transplantation via the renal artery on the impaired renal structural and functional recovery following renal ischemia/reperfusion injury in rabbits. METHODS： Experiments were performed from March to December 2006 at the Kunming General Hospital of Chinese PLA. Twenty-four healthy Japanese flap-eared rabbits aged 2-3 months old of either gender, （2.6±0.7） kg, were provided by Laboratory Animal Center of Kunming General Hospital of Chinese PLA. The protocol was conducted in accordance with animal ethical standards. These animals were equally randomly divided into a transplantation group and a control group. BMSCs were isolated and cultivated by red blood cells disintegration and adherent culture. The cells were identified by the immunohistoehemical analysis of CD34, CD105, CD106 staining. Rabbit models of ischemia/reperfusion injury were established by ablating the right kidney and clamping the artery of the left kidney for 85 minutes followed by reperfusion. BMSCs and saline were injected into the kidney via the renal artery in the transplantation group and the control group respectively. Before transplantation and 3, 7, 14, 21 and 28 days after transplantation, renal function was examined and the renal tissues were sampled for pathological observation. RESULTS： The cells of just segregation were round. The cell clones began to form after 7 days, and whirlpool growth appeared in fusiform. Passaged cells grew rapidly and became bigger. Third passage of cells was negative for CD34. 98% cells were positive for CD105 and CD106. Three days later, serum creatinine and urea nitrogen levels reached a peak in both groups. Seven days later, serum creatinine and urea nitrogen levels were lower in the transplantation group compared to the control group. Significant differences in serum creatinine and urea nitrogen, levels were detected between the two groups at the 21st day （P 〈 0.05）. After ischemia/reperfusion injury, the renal tubular epithelial cells were in a status of degeneration, necrosis, even exfoliation. With the time prolonged, the necrotic renal tubular was repaired gradually, but it was repaired better in the transplantation group compared to the control group. CONCLUSION：The transplanted autologous BMSCs contribute to the textural and functional reparation for injured renal tissues.