CAJ | 학술논문

背景:骨骼肌细胞移植于损伤心肌可以改善心脏功能,但大部分细胞在移植早期因损伤心肌的缺血及炎性氧化应激环境而发生死亡.研究证实通过细胞预处理或联合基因治疗可以改善移植细胞的生存.目的:探讨人生长激素基因修饰的成肌细胞移植对心肌梗死大鼠心肌细胞凋亡和心功能的影响.设计、时间及地点:细胞学体内实验,于2007-05/2008 12在山西省长治医学院和华中科技大学同济医学院附属协和医院完成.材料:SD大鼠30只,随机分为3组:人生长激素组、绿色荧光蛋白组、模型对照组,10只,组.另取1-3 d龄SD乳鼠用于骨骼肌成肌细胞培养.pLghGHSN质粒、pLgGFPSN质粒由本实验室提供.方法:分别取携带人生长激素基因的pLghGHSN质粒和携带绿色荧光蛋白基因的对照质粒pLgGFPSN,用二步转染法转染E86和PT67包装细胞,G418筛选并扩增阳性克隆.各组大鼠均结扎左冠状动脉前降支制作心肌梗死模型,2周后人生长激素组将转染pLghGHSN的成肌细胞悬液150 μ L(含6×106个成肌细胞)分3点注射于心肌梗死区边缘,绿色荧光蛋白组同法注射等量转染pLgGFPSN的成肌细胞悬液,模型对照组沣射等体积无血清培养基.主要观察指标:细胞移植4周后,超声心动图和血流动力学检查各组心功能变化:苏木精一伊红染色检测心肌梗死面积,天狼星红染色检测胶原纤维容积分数:western blot法检测心肌组织Bax,Bcl-2,人生长激素和caspase3蛋白的表达.结果:与模型对照组比较,人生长激素组左室腔变小,左室游离壁厚度增加,左室舒张末期内径和左室收缩末期内径均显著减小(P<0.05),短轴缩短率显著增加(P<0.05).与绿色荧光蛋白组、模型对照组比较,人生长激素组大鼠左室压力最大上升速率、左室压力最大下降速率绝对值、左室收缩末压均显著升高(P<0.05),左室舒张末压显著降低(P<0.05):血清中类胰岛素样生长因子1质量浓度显著升高(P<0.05);心肌梗死面积明显减小(P<0.05);bax,caspase3蛋白表达水平降低,Bcl-2蛋白表达水平增加,人生长激素蛋白呈阳性表达.结论:人生长激素基因修饰的成肌细胞移植可以抑制心肌细胞凋亡,与单独成肌细胞移植相比能够更有效地缩小心肌梗死面积,更好地改善心肌梗死大鼠的心功能. 揹景:骨骼肌細胞移植于損傷心肌可以改善心髒功能,但大部分細胞在移植早期因損傷心肌的缺血及炎性氧化應激環境而髮生死亡.研究證實通過細胞預處理或聯閤基因治療可以改善移植細胞的生存.目的:探討人生長激素基因脩飾的成肌細胞移植對心肌梗死大鼠心肌細胞凋亡和心功能的影響.設計、時間及地點:細胞學體內實驗,于2007-05/2008 12在山西省長治醫學院和華中科技大學同濟醫學院附屬協和醫院完成.材料:SD大鼠30隻,隨機分為3組:人生長激素組、綠色熒光蛋白組、模型對照組,10隻,組.另取1-3 d齡SD乳鼠用于骨骼肌成肌細胞培養.pLghGHSN質粒、pLgGFPSN質粒由本實驗室提供.方法:分彆取攜帶人生長激素基因的pLghGHSN質粒和攜帶綠色熒光蛋白基因的對照質粒pLgGFPSN,用二步轉染法轉染E86和PT67包裝細胞,G418篩選併擴增暘性剋隆.各組大鼠均結扎左冠狀動脈前降支製作心肌梗死模型,2週後人生長激素組將轉染pLghGHSN的成肌細胞懸液150 μ L(含6×106箇成肌細胞)分3點註射于心肌梗死區邊緣,綠色熒光蛋白組同法註射等量轉染pLgGFPSN的成肌細胞懸液,模型對照組灃射等體積無血清培養基.主要觀察指標:細胞移植4週後,超聲心動圖和血流動力學檢查各組心功能變化:囌木精一伊紅染色檢測心肌梗死麵積,天狼星紅染色檢測膠原纖維容積分數:western blot法檢測心肌組織Bax,Bcl-2,人生長激素和caspase3蛋白的錶達.結果:與模型對照組比較,人生長激素組左室腔變小,左室遊離壁厚度增加,左室舒張末期內徑和左室收縮末期內徑均顯著減小(P<0.05),短軸縮短率顯著增加(P<0.05).與綠色熒光蛋白組、模型對照組比較,人生長激素組大鼠左室壓力最大上升速率、左室壓力最大下降速率絕對值、左室收縮末壓均顯著升高(P<0.05),左室舒張末壓顯著降低(P<0.05):血清中類胰島素樣生長因子1質量濃度顯著升高(P<0.05);心肌梗死麵積明顯減小(P<0.05);bax,caspase3蛋白錶達水平降低,Bcl-2蛋白錶達水平增加,人生長激素蛋白呈暘性錶達.結論:人生長激素基因脩飾的成肌細胞移植可以抑製心肌細胞凋亡,與單獨成肌細胞移植相比能夠更有效地縮小心肌梗死麵積,更好地改善心肌梗死大鼠的心功能.
배경:골격기세포이식우손상심기가이개선심장공능,단대부분세포재이식조기인손상심기적결혈급염성양화응격배경이발생사망.연구증실통과세포예처리혹연합기인치료가이개선이식세포적생존.목적:탐토인생장격소기인수식적성기세포이식대심기경사대서심기세포조망화심공능적영향.설계、시간급지점:세포학체내실험,우2007-05/2008 12재산서성장치의학원화화중과기대학동제의학원부속협화의원완성.재료:SD대서30지,수궤분위3조:인생장격소조、록색형광단백조、모형대조조,10지,조.령취1-3 d령SD유서용우골격기성기세포배양.pLghGHSN질립、pLgGFPSN질립유본실험실제공.방법:분별취휴대인생장격소기인적pLghGHSN질립화휴대록색형광단백기인적대조질립pLgGFPSN,용이보전염법전염E86화PT67포장세포,G418사선병확증양성극륭.각조대서균결찰좌관상동맥전강지제작심기경사모형,2주후인생장격소조장전염pLghGHSN적성기세포현액150 μ L(함6×106개성기세포)분3점주사우심기경사구변연,록색형광단백조동법주사등량전염pLgGFPSN적성기세포현액,모형대조조풍사등체적무혈청배양기.주요관찰지표:세포이식4주후,초성심동도화혈류동역학검사각조심공능변화:소목정일이홍염색검측심기경사면적,천랑성홍염색검측효원섬유용적분수:western blot법검측심기조직Bax,Bcl-2,인생장격소화caspase3단백적표체.결과:여모형대조조비교,인생장격소조좌실강변소,좌실유리벽후도증가,좌실서장말기내경화좌실수축말기내경균현저감소(P<0.05),단축축단솔현저증가(P<0.05).여록색형광단백조、모형대조조비교,인생장격소조대서좌실압력최대상승속솔、좌실압력최대하강속솔절대치、좌실수축말압균현저승고(P<0.05),좌실서장말압현저강저(P<0.05):혈청중류이도소양생장인자1질량농도현저승고(P<0.05);심기경사면적명현감소(P<0.05);bax,caspase3단백표체수평강저,Bcl-2단백표체수평증가,인생장격소단백정양성표체.결론:인생장격소기인수식적성기세포이식가이억제심기세포조망,여단독성기세포이식상비능구경유효지축소심기경사면적,경호지개선심기경사대서적심공능.
BACKGROUND: Skeletal myoblast transplantation can improve cardiac function, but most of cells exhibit apoptosis in the early stage of transplantation due to myocardial ischemia and inflammatory oxidative stress environment. Cell pretreatment or combined gene therapy has been shown to improve the survival of transplanted calls.OBJECTIVE: To investigate the effects of human growth hormone (hGH)-modified myoblast transplantation on myocardial Cellular apoptosis and cardiac function in rats with myocardial infarction.DESIGN, TIME AND SETTING: A cytological experiment in vivo was performed in the Heping Hospital Affiliated to Changzhi Medical College & Union Hospital Affiliated to Tongji Medical Collage, Huazhong University of Science and Technology between May 2007 and December 2008.MATERIALS: Thirty Sprague-Dawley (SD) rats were randomly divided into 3 groups (n = 10): hGH, green fluorescent protein (GFP), and control. An additional number of SD neonate rats aged 1-3 days ware included for culture of myoblasts. Plasmids pLghGHSN and pLgGFPSN were provided by our laboratories.METHODS: Plasmid pLghGHSN carrying hGH gene and plasmid pLgGFPSN carrying GFP were used to respectively transfect E86 and PT67 packaging cells by a two-step transfection protocol, followed by G418 screening and positive clone amplification.Rat models of myocardial infarction were established in each group by ligating the left anterior descending branch coronary artery. Two weeks after myocardial infarction model induction, 150 p L pLghGHSN-transfected myoblast suspension (6×106 myoblasts),pLgGFPSN-transfected myoblast suspension, or serum-free culture medium was injected into the peripheral infarcted regions through 3 points in the hGH, GFP, and control groups, respectively.MAIN OUTCOME MEASURES: At 4 weeks after cell transplantation, cardiac function was examined by echocardiogrephy and hemodynamics, myocardial infarct area was determined by hematoxylin-eosin staining, collagen volume fraction was detected by Sirius red staining, and expression levels of bax, bcl-2, hGH and caspase-3 protein in the myocardial tissue were detected by western blot method.RESULTS: Compared with control group, left ventricular cavity became small, left ventricular free wall was thickened, left ventricular end diastolic diameter and left ventricular end systotic diameter were significantly reduced, and shortening fraction was significantly increased in the hGH group. The maximal change rate of left ventricular pressure rise or decline (±dp/dt <,max>)and left ventricular end-systolic pressure were significantly increased (P < 0.05), left ventricular end-diastolic pressure was significantly decreased (P<0.05), serum level of insulin-like growth factor 1 was significantly increased (P<0.05), myocardial infarct area was significantly reduced (P<0.05), protein levels of bax and caspase-3 were significantly decreased, but protein level of bcl-2 was significantly increased in the hGH group than in the GFP and control groups. In addition, hGH protein-positive expression was found in the hGH group.CONCLUSION: hGH gene-modified myoblast transplantation can inhibit myocardial cell apoptosis and it can more effectively reduce infarct area and better improve the cardiac function of rats with myocardial infarction than simple myoblast transplantation.