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中华医学超声杂志（电子版）

中华医学超声杂志（电子版） ›› 2015, Vol. 12 ›› Issue (08) : 652 -656. doi: 10.3877/cma.j.issn.1672-6448.2015.08.014

所属专题： 文献；

基础研究

超声辐照微泡促骨髓间充质干细胞修复大鼠急性肾损伤的实验研究
唐海林1, 王志刚2,†(), 李巧2, 李攀2   
  1. 1. 310012　杭州，浙江省立同德医院超声科
    2. 400010　重庆医科大学附属第二医院超声科　重庆医科大学超声影像学研究所
  • 收稿日期:2014-11-09 出版日期:2015-08-01
  • 通信作者: 王志刚
  • 基金资助:
    浙江省自然科学基金(No.Y2080071)

Targeted delivery of bone mesenchymal stem cells by ultrasound destruction of microbubbles promotes kidney recovery in acute kidney injury

Hailin Tang1, Zhigang Wang2,†(), Qiao Li2, Pan Li2   

  1. 1. Department of Ultrasound, Tongde Hospital of Zhejiang Province, Hangzhou 310012, China
    2. Institute of Ultrasound Imaging, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China
  • Received:2014-11-09 Published:2015-08-01
  • Corresponding author: Zhigang Wang
  • About author:
    Corresponding author: Wang Zhigang, Email:
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引用本文：

唐海林, 王志刚, 李巧, 李攀. 超声辐照微泡促骨髓间充质干细胞修复大鼠急性肾损伤的实验研究[J]. 中华医学超声杂志（电子版）, 2015, 12(08): 652-656.

Hailin Tang, Zhigang Wang, Qiao Li, Pan Li. Targeted delivery of bone mesenchymal stem cells by ultrasound destruction of microbubbles promotes kidney recovery in acute kidney injury[J]. Chinese Journal of Medical Ultrasound (Electronic Edition), 2015, 12(08): 652-656.

目的

探讨超声辐照靶向破坏微泡促骨髓间充质干细胞（MSCs）修复大鼠急性肾小管坏死的作用。

方法

选择40只SD大鼠构建急性肾小管坏死模型。将制备成功的40只急性肾小管坏死大鼠随机分为4组：单纯模型组、1.0 W/cm2超声辐照+微泡组（1.0 US+MB组）、干细胞组（MSCs组）、1.0 W/cm2超声辐照+微泡+干细胞组（1.0 US+MB+MSCs组），每组各10只。1.0 US+MB组、1.0 US+MB+MSCs组大鼠均以强度为1.0 W/cm2及频率为1 MHz的超声辐照肾脏组织，并尾静脉输入0.5 ml造影剂，密度约为1.0×108个/ml，辐照5 s，停5 s，共1 min。在急性肾小管坏死模型建立后第1天及第3天分别辐照1次。1.0 US+MB+MSCs组大鼠最后一次超声辐照完毕后1 min内经股静脉注入MSCs1 ml（密度为2.0×106个/ml）。同时MSCs组大鼠经股静脉注入MSCs1 ml（密度为2.0×106个/ml）。单纯模型组不做任何处理。MSCs移植7 d后将各组大鼠处死并取材，采用Western印迹法测定各组大鼠肾组织肝细胞生长因子（HGF）及表皮生长因子（EGF）蛋白表达并进行定量分析，采用HE染色观察各组大鼠肾小管坏死情况并进行评分。采用单因素方差分析比较单纯模型组、1.0 US+MB组、MSCs组、1.0 US+MB+MSCs组大鼠HGF蛋白表达水平、EGF蛋白表达水平、肾小管上皮细胞坏死评分差异，进一步组间两两比较采用SNK-q检验。

结果

Western印迹结果显示，HGF在各组相对分子质量为84 000处均有特异性条带，EGF在各组相对分子质量为6 000处均有特异性条带，其中1.0 US+MB+MSCs组的条带最为明显。单纯模型组、1.0 US+MB组、MSCs组、1.0 US+MB+MSCs组大鼠HGF蛋白表达水平分别为0.16±0.30、0.35±0.50、0.37±0.50、0.70±0.60，EGF蛋白表达水平分别为0.19±0.40、0.41±0.50、0.43±0.50、0.82±0.70，肾小管上皮细胞坏死评分分别为（4.1±0.8）、（3.6±0.6）、（1.8±0.4）、（1.0±0.3）分。1.0 US+MB+MSCs组大鼠HGF蛋白、EGF蛋白表达水平均高于单纯模型组、1.0 US+MB组及MSCs组大鼠，且差异均有统计学意义（HGF蛋白：q值分别为23.6、18.7、9.6；EGF蛋白：q值分别为21.8、17.1、9.3；均P<0.05)；1.0 US+MB+MSCs组大鼠肾小管坏死评分低于单纯模型组、1.0 US+MB组及MSCs组大鼠，且差异均有统计学意义（q值分别为20.1、16.7、6.9，均P<0.05)，提示1.0US+MB+MSCs组大鼠肾小管修复情况优于其他各组大鼠。

结论

1.0 W/cm2超声辐照微泡促进MSCs归巢并修复急性肾小管坏死，可为急性肾小管坏死提供一种新型的干细胞移植治疗方法。

关键词: 超声检查, 微泡, 造影剂, 骨髓间充质干细胞, 急性肾小管坏死
Objective

To explore whether ultrasound microbubble destruction promotes recovery of the kidney in acute tubular necrosis (ATN) in rats.

Methods

ATN model was induced in forty Sprague-Dawley (SD) rats. Forty SD rats were randomly divided into the following groups after the establishment of animal models of ATN injury (n=10): (1) Model group alone (control group); (2) 1.0 W/cm2 US+MB (US/MB group), Bubble (0.5 ml)+ 1.0 W/cm2 US; (3) MSCs group ; (4) 1.0 W/cm2 US+MB + MSCs group (US/MB + MSCs group). 0.5 ml infusion of microbubbles via the contralateral femoral vein were infused at days 1 and 3 after ATN. Simultaneous insonation was started at the lower back region of rats, using an ultrasound gene transfection treatment meter with the following settings: insonate for 5 s at 1 MHz and 1.0 W/cm2 with a 5 s pause, totaling 60 s in US/MB group and US/MB + MSCs group. US/MB + MSCs group received an infusion of rat MSCs (2×106 in 1 ml saline) via the contralateral femoral vein at 1 minute after US/MB at 3 days after ATN. Simultaneousin MSCs group, a concentration of 2×106 in 1 ml saline were infused via the contralateral femoral vein at 3 days after ATN. Control group received no treatment. The rats were killed and their kidneys were excised at 7 days after MSCs transplantation. Western blotting was applied to determine hepatocyte growth factor (HGF) and epidermal growth factor (EGF). Histologicalexamination wasperformed to evaluate the extent of tubular necrosis. Statistical significances of the level of HGF and EGF expression and the extent of tubular necrosis were determined by one-way ANOVA followed by Student-Newman-Keuls-q for the comparison between two groups.

Results

The special band of 83 kilodalton (KD) was found in all groups. A prominent band consistent with HGF was seen in the US/MB +MSCs group. Special band of 6 KD was found in all groups. A prominent band consistent with EGF was seen in the US/MB +MSCs group. Western blotting results showed that the levels of HGF protein expression were (0.16±0.30), (0.35±0.50), (0.37±0.50) and (0.70±0.60), respectively. The levels of EGF protein expression were (0.19±0.40), (0.41±0.50), (0.43±0.50) and (0.82±0.70), respectively. The extent of tubular necrosis and dilation were (4.1±0.8), (3.6±0.6), (1.8±0.4) and (1.0±0.3), respectively. The levels of HGF protein were higher in the US/MB +MSCs group compared with those of the control group, US/MB group and MSCs group, respectively (q=23.6, q=18.7 and q=9.6, P<0.05). The levels of EGF were higher in the US/MB +MSCs group compared with those of the control group, US/MB group and MSCs group, respectively (q=21.8, q=17.1 and q=9.3, P<0.05). The extent of tubular necrosis and dilation was milder in the US/MB +MSCs group compared with those of the control group, US/MB group and MSCs group, respectively (q=20.1, q=16.7 and q=6.9, P<0.05). The extent of tubular necrosis and dilation was significantly milder in the US/MB + MSCs group than those of all other groups.

Conclusion

Microbubble destruction by 1.0 W/cm2 ultrasound can promote both the homing of mesenchymal stem cells to kidney tissue and the recovery of the kidney in ATN in rats, which provide a novel and efficient approach for cellular therapy.

Key words: Ultrasonography, Microbubble, Contrast media, Mesenchymal stem cell, Acute tubular necrosis
图1 Western印迹法检测各组大鼠HGF蛋白的表达。HGF蛋白在各组相对分子质量为84 000处均有特异性条带，其中1.0 US+MB+MSCs组的条带最为明显
图2 Western印迹法检测各组大鼠EGF蛋白的表达。EGF蛋白在各组相对分子质量为6 000处均有特异性条带，其中1.0 US+MB+MSCs组的条带最为明显
表1 各组小鼠HGF蛋白、EGF蛋白表达水平及肾小管上皮细胞坏死评分比较（+s）
组别 鼠数 HGF蛋白表达水平 EGF蛋白表达水平 肾小管上皮细胞坏死评分（分）
单纯模型组 10 0.16±0.30a 0.19±0.40c 4.1±0.80e
1.0 US+MB组 10 0.35±0.50 0.41±0.50 3.6±0.60
MSCs组 10 0.37±0.50 0.43±0.50 1.8±0.40
1.0 US+MB+MSCs组 10 0.70±0.60b 0.82±0.70d 1.00±0.30f
F值 ? 47.68 45.72 37.83
P值 ? <0.05 <0.05 <0.05
图3～6 各组大鼠近端小管上皮细胞肿胀及坏死情况（HE，×400）。图3　为单纯模型组；图4　为1.0 W/cm2超声辐照+微泡组；图5　为干细胞组；图6　为1.0 W/cm2超声辐照+微泡+干细胞组。HE染色结果显示，1.0US+MB+MSCs组大鼠肾小管上皮细胞变性或坏死的数目、肾小管上皮细胞空泡数目及肾小管上皮细胞肿胀数目较其他各组大鼠减少
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