摘要
目的探讨重组慢病毒介导的心脏营养素-1(cardiotrophin-1,CT-1)基因转染延缓小鼠失神经骨骼肌萎缩的疗效。方法将36只Swiss小鼠,随机分为实验组和对照组,每组18只,切断胫神经,建立右下失神经支配肌萎缩模型。于实验组和对照组失神经腓肠肌分别转染重组慢病毒载体Lenti-GFP-CT-1和Lend—GFP溶液20越(108TU/ml),转染后2,4周,每个时间点分别取3只小鼠,于荧光显微镜下观察绿色荧光蛋白(GFP)的表达、Westem blot检测CT-1的表达;取6只小鼠行单次收缩力、强直收缩力的检测,测定肌湿重、肌纤维横截面积,并观察肌纤维超微结构的变化。结果慢病毒转染2、4周,两组腓肠肌中均可见大量GFP表达。Western blot检测显示实验组失神经腓肠肌中有明显的CT-1表达(P均〈0.01)。转染2周,实验组肌肉单次收缩力恢复率、强直收缩力恢复率、肌湿重维持率和肌纤维横截面积分别为[(47.61±6.25)%,^-x±s,下同]、(56.08±5.47)%、(63.02±5.23)%、(1372.42±149.73)μm^2,均明显高于对照组,后者分别为(27.23±5.06)%、(30.78±4.67)%、(52.41±4.98)%、(1147.28±128.67)μm^2(P均〈0.01);4周时,实验组上述各项指标分别为(33.13±4.76)%、(36.59±5.67)%、(51.46±5.36)%、(1209.12±142.57)μm^2,仍明显高于对照组,后者分别为(16.40±5.48)%、(15.35±4.08)%、(39.15±6.12)%、(989.45±136.12)时(P均〈0.01)。此外,实验组肌浆网的扩张程度明显减轻。结论慢病毒介导的基因治疗有较高的转染效率,其介导的CT-1基因转染能有效延缓小鼠失神经骨骼肌的萎缩,其疗效至少可以维持4周。
Objective To explore the effect of delaying atrophy of mice denervated skeletal muscles by lentivirus mediated gene transfer of cardiotrophin-1. Methods Thirty-six Swiss mice were randomly divided into experimental group and control group, with 18 each. A model of gastrocnemius muscle denervation atrophy was established at the right lower limb by transecting the tibial nerve. Denervated gastrocnemius muscles of the experimental group and control group were administered with 20μl ( 108 TU/ml) of recombinant lentivirus vector carrying CT-1 cDNA and with 20μl of Lenti-GFP solution respectively. Two and 4 weeks following the transfection, 3 mice each were checked for transfecfion efficacy by observing expression of green fluorescent protein (GFP) under fluorescent microscope. Western blot assays were done to validate the expression of CT-1. Twitch tension and tetanic tension, muscle wet weight preservation rate, muscle fiber cross-sectional area and ultrastructure of the denervated muscles were measured and observed in 6 mice at each timepoint to evaluate the extent of muscle atrophy. Results Strong GFP expression was observed in the gastrocnemius muscles of both groups at 2 and 4 weeks following lentivirus transfection. Western blot showed significantly increased expression of CT-1 in the experimental group compared with the control group (P 〈 0.01 ). Two weeks after transfecfion, twitch tension and tetanic tension, muscle wet weight preservation rate and muscle fiber cross-sectional area of the denervated muscles in the experimental group were (47.61 ± 6.25)%, (56.08± 5.47)%, (63.02 ± 5.23)% and (1372.42 ± 149.73)μm^2 respectively, being significantly higher than those in the control group, which were (27.23±5.06)%, (30.78±4.67)%, (52.41 ±4.98)% and (1147.28± 128.67)μm^2 respectively (all P 〈 0.01 ). At 4 weeks, the above-mentioned parameters were ( 33.13 ± 4.76) %, ( 36.59 ± 5.67) %, (51.46 ± 5.36)%, and (1209.12 ± 142.57)μm^2 respectively in the experimental group, being significantly greater than those in the control group which were ( 16.40 ± 5.48) %, ( 15.35 ± 4.08 ) %, ( 39.15 ± 6.12) % and (989.45 ± 136.12)μm^2 respectively (all P 〈 0.01 ). Dilation of the sarcoplasmic reticulum of denervated muscles in the experimental group was markedly alleviated. Conclusion Lentivirus mediated gene therapy can achieve high transfection efficaey. CT-1 gene transfection can produce highly efficient and sustained CT-1 secretion in vivo and delay denervation induced skeletal muscle atrophy for at least 4 weeks.
出处
《中华手外科杂志》
CSCD
北大核心
2009年第2期117-120,共4页
Chinese Journal of Hand Surgery
基金
国家自然科学基金资助项目(30170964,30571918)