骨髓干细胞移植后Western blot免疫印迹分析鼠抗肌萎缩蛋白表达的变化

Immunoblot changes of dystrophin expression in mice after bone marrow stem cells transplantation

目的:Western blot免疫印迹分析骨髓干细胞移植后mdx鼠抗肌萎缩蛋白表达的变化。方法:实验于2004-09/12在中山大学附属第一医院神经科实验室完成。①选取7～8周龄mdx鼠25只,随机数字表法分为骨髓移植4,8,12,16周组、空白对照组,5只/组。另选取4～6周龄C57鼠20只作为供体鼠,10周龄C57鼠5只作为阳性对照组。②各组在移植前均给予照射剂量为7Gy的放疗预处理。放疗完毕后,骨髓移植4,8,12,16周组均进行尾静脉细胞移植,移植细胞数为2×107/只,0.3～0.5mL;空白对照组、阳性对照组尾静脉输入0.3mL磷酸盐缓冲液。③骨髓移植4,8,12,16周组分别于对应时间点处死,取其腓肠肌,制备抗肌萎缩蛋白样品,进行抗肌萎缩蛋白Western blot免疫印迹分析,以GAPDH作为内参。结果:25只7～8周龄mdx鼠、5只10周龄C57鼠全部进入结果分析。骨髓干细胞移植后抗肌萎缩蛋白Western Blot免疫印迹分析结果:空白对照组无抗肌萎缩蛋白的表达;骨髓移植4周组仅可见抗肌萎缩蛋白的微弱表达,dystrophin/GAPDH灰度比值约为0.095±0.267;骨髓移植12周组约为0.218±0.338;随骨髓移植时间的延长抗肌萎缩蛋白表达量逐渐增加,移植后16周的表达量较移植后8周明显增加(0.393±0.385,0.173±0.284;t=6.062,P<0.05),但仍明显低于阳性对照组1.172±0.328(t=3.14,P<0.05)。结论:骨髓干细胞移植mdx鼠后应用WesternBlot免疫印迹分析可于不同时间检测到抗肌萎缩蛋白的表达,且随移植时间延长表达量增多,提示骨髓干细胞移植可长久持续参与受损骨骼肌的修复与再生。

AIM： To investigate the immunoblot changeS of dystrophin expression in mdx mice after b.one marrow stem cells transplantation. METHODS： The experiment was conducted in the laboratory of Department of Neurology, the First Affiliated Hospital, Sun Yat-sen University from September to December 2004. （1）Twenty-five mdx mice of 7-8 weeks old were selected and randomly divided into transplantation 4, 8, 12 and 16 weeks groups and blank control group with 5 mice in each group. Meanwhile, 20 C57 mice of 4 to 6 weeks old were selected as donor mice, and 5 C57 mice aged 10 weeks served as positive control mice. （2）All mice were preconditioned ,with 7 Gy γ-ray. After radiotherapy, 2×10^7 marrow stem cells per mouse （about 0.3-0.5 mL） were-injected into the vena caudalis of mice; 0.3 mL PBS was injected into the blank control group and positive control group. （3）The mice in 4 transplantation groups were killed at each time point, and the gastrocnemius was harvested to prepare dystrophin samples. The amount of dystrophin expression was detected by Westem blot with GAPDH as control. RESULTS： All 25 mdx mice and 5 C57 mice were involved in the result analysis. Western Blot analysis of dystrophin after transplantation： No dystrophin was detected in the blank control group; in the 4 weeks after transplantation group, only few dystrophin expressions were detected, and the dystrophin/GAPDH was about 0.095±0.267; in the 12 weeks after transplantation group, dystrophin/GAPDH was about 0.218±0.338; dystrophin expressions were increased with time, at 16 weeks after transplantation, the expressions were much more than those at 8 weeks （dystrophin/GAPDH： 0.393± 0.385, 0.173±0.284, t =6.062, P 〈 0.05）, but still much less than the positive control group （1.172±0.328, t =3.14, P 〈 0.05）. CONCLUSION： The bone marrow stem cells after transplantation can express dystrophin detected by Western Blot and the expression increases with time, which indicates that bone marrow stem cells can participate in the repair and regeneration of injured somatic muscles permanently and constantly.