中华手外科杂志
中華手外科雜誌
중화수외과잡지
CHINESE JOURNAL OF HAND SURGERY
2009年
2期
117-120
,共4页
冯勇%张高孟%梁炳生%马学晓%李继峰%顾玉东
馮勇%張高孟%樑炳生%馬學曉%李繼峰%顧玉東
풍용%장고맹%량병생%마학효%리계봉%고옥동
肌萎缩%去神经支配%心脏营养素-1
肌萎縮%去神經支配%心髒營養素-1
기위축%거신경지배%심장영양소-1
]Muscular atrophy%Denervation%Cardiotrophin-1
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Lenti-GFP溶液20μl(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、Western 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)μm2,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)μm2(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)μm2,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)μm2(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.
目的 探討重組慢病毒介導的心髒營養素-1(cardiotrophin-1,CT-1)基因轉染延緩小鼠失神經骨骼肌萎縮的療效.方法 將36隻Swiss小鼠,隨機分為實驗組和對照組,每組18隻,切斷脛神經,建立右下失神經支配肌萎縮模型.于實驗組和對照組失神經腓腸肌分彆轉染重組慢病毒載體Lenti-GFP-CT-1和Lenti-GFP溶液20μl(108TU/ml),轉染後2、4週,每箇時間點分彆取3隻小鼠,于熒光顯微鏡下觀察綠色熒光蛋白(GFP)的錶達、Western blot檢測CT-1的錶達;取6隻小鼠行單次收縮力、彊直收縮力的檢測,測定肌濕重、肌纖維橫截麵積,併觀察肌纖維超微結構的變化.結果 慢病毒轉染2,4週,兩組腓腸肌中均可見大量GFP錶達.Western blot檢測顯示實驗組失神經腓腸肌中有明顯的CT-1錶達(P均<0.01).轉染2週,實驗組肌肉單次收縮力恢複率、彊直收縮力恢複率、肌濕重維持率和肌纖維橫截麵積分彆為[(47.61±6.25)%,x-±s,下同]、(56.08±5.47)%、(63.02±5.23)%、(1372.42±149.73)μm2,均明顯高于對照組,後者分彆為(27.23±5.06)%、(30.78±4.67)%、(52.41±4.98)%、(1147.28±128.67)μm2(P均<0.01);4週時,實驗組上述各項指標分彆為(33.13±4.76)%、(36.59±5.67)%、(51.46±5.36)%、(1209.12±142.57)μm2,仍明顯高于對照組,後者分彆為(16.40±5.48)%、(15.35±4.08)%、(39.15±6.12)%、(989.45±136.12)μm2(P均<0.01).此外,實驗組肌漿網的擴張程度明顯減輕.結論 慢病毒介導的基因治療有較高的轉染效率,其介導的CT-1基因轉染能有效延緩小鼠失神經骨骼肌的萎縮,其療效至少可以維持4週.
목적 탐토중조만병독개도적심장영양소-1(cardiotrophin-1,CT-1)기인전염연완소서실신경골격기위축적료효.방법 장36지Swiss소서,수궤분위실험조화대조조,매조18지,절단경신경,건립우하실신경지배기위축모형.우실험조화대조조실신경비장기분별전염중조만병독재체Lenti-GFP-CT-1화Lenti-GFP용액20μl(108TU/ml),전염후2、4주,매개시간점분별취3지소서,우형광현미경하관찰록색형광단백(GFP)적표체、Western blot검측CT-1적표체;취6지소서행단차수축력、강직수축력적검측,측정기습중、기섬유횡절면적,병관찰기섬유초미결구적변화.결과 만병독전염2,4주,량조비장기중균가견대량GFP표체.Western blot검측현시실험조실신경비장기중유명현적CT-1표체(P균<0.01).전염2주,실험조기육단차수축력회복솔、강직수축력회복솔、기습중유지솔화기섬유횡절면적분별위[(47.61±6.25)%,x-±s,하동]、(56.08±5.47)%、(63.02±5.23)%、(1372.42±149.73)μm2,균명현고우대조조,후자분별위(27.23±5.06)%、(30.78±4.67)%、(52.41±4.98)%、(1147.28±128.67)μm2(P균<0.01);4주시,실험조상술각항지표분별위(33.13±4.76)%、(36.59±5.67)%、(51.46±5.36)%、(1209.12±142.57)μm2,잉명현고우대조조,후자분별위(16.40±5.48)%、(15.35±4.08)%、(39.15±6.12)%、(989.45±136.12)μm2(P균<0.01).차외,실험조기장망적확장정도명현감경.결론 만병독개도적기인치료유교고적전염효솔,기개도적CT-1기인전염능유효연완소서실신경골격기적위축,기료효지소가이유지4주.
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. Deaervated gastrocnemius muscles of the experimental group and control group were administered with 20 μl ( 108 TU/ml) of recombinant lentivirus vector carrying CT-1 eDNA and with 20 μl of Lenti-GFP solution respectively. Two and 4 weeks following the transfection, 3 mice each were checked for transection efficacy by observing expression of green fluorescent protein (GFP) under fluorescent microscope. Westem blot assays were done to validate the expression of CT-1.Twitch tension and tetanie tension, muzzle wet weight preservation rate, muscle fiber cross-sectional area and ultrastruture 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 transfeetion. Westem blot showed significantly inereaticl expression of CT-1 in the experimental group compared with the control group ( P < 0.01 ). Two weeks after transfection,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)μm2 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)μm2 respectively(all P <0.01).At 4 weeks.the above-mendoned parameters were(33.13±4.76)%,(36.59 4-5.67)%.(51.46-±5.36)%,and(1209.12±142.57)μm2 respectively in the experimental group,being significantly greater than these in the control group which were(16.40±5.48)%,(15.35±4.08)%,(39.15±6.12)%and (989.45 4-136.12)μm2 respectively(all P<0.01).Dilation of the sareoplasmic refieulum of denervated muscles in the experimental group wss markedly alleviated.Conclusion Lentivirus mediated gene therapy can aehieve high transection efficacy.CT-1 gene tmnsfeefion call produce llighly egieient and sustained CT-1 secretion in vivo and delay denervation indueed skeletal muscle atrophy for at least 4 weeks.
