目的 探讨移植转染肾上腺髓质素(adrenomedullin,ADM)基因的骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)对改善心衰大鼠心脏功能的作用.方法 Wistar大鼠10只,20日龄,体质量30～ 50 g.分离大鼠股骨、胫骨取BMSCs,体外传代培养.将携带ADM基因并标记绿色荧光蛋白(GFP)的腺病毒转染体外培养的第3代BMSCs,在移植前用4’,6-二脒基-2-苯基吲哚(DAPI)对BMSCs进行染色.雄性Wistar大鼠80只,体质量180 ～ 200 g.按体质量将大鼠随机分为2组,对照组10只,皮下注射生理盐水;模型组70只,按170 mg/kg连续4d皮下注射异丙基肾上腺素(isoproterenol,ISO),建立弥漫性心肌损伤的大鼠心衰模型.ISO末次给药后4周,大鼠进行超声心动图检查,将心衰组左室射血分数(LVEF)＜70％的39只大鼠按射血分数高低随机分为3组:未转染组、转染组、生理盐水组,每组13只.将DAPI标记的未转染BMSCs悬液、转染ADM基因BMSC悬液各150 μl(3×106个)、等量生理盐水按上述分组分别分4个点注射到大鼠左心室前壁.对照组只开胸,不做注射.移植后4周行超声心动图检查,取左心室心肌组织,采用Massons染色观察心肌组织纤维化程度,荧光显微镜观察移植细胞,Western blotting检测心肌组织基质金属蛋白酶-2(MMP-2)蛋白表达.结果 大鼠BMSCs体外培养第3天,细胞开始贴壁生长,约第10天细胞趋于融合,呈梭形,走行一致.移植后4周,超声心动图显示,大鼠心脏左心室收缩末径(LVDs)、LVEF、左心室短轴缩短率(LVFS)组间比较差异有统计学意义(F=5.838、32.983、51.714,P＜0.05或P＜0.01).与对照组[(86.50±1.54)％,(50.66±1.87)％]比较,生理盐水组[(56.67±6.86)％,(26.27±4.01)％],转染组[(79.40±1.70)％,(43.48±2.15)％]、未转染组[(69.24±7.30)％,(34.59±5.13)％]LVEF和LVFS明显降低(P均＜0.05)；与生理盐水组比较,转染组和未转染组明显增加(P均＜ 0.05)；与未转染组比较,转染组明显增加(P＜0.05).与对照组[(3.16±0.22)mm]比较,生理盐水组[(5.35±1.57)mm] LVDs明显增加(P＜0.01)；与生理盐水组比较,转染组和未转染组[(3.95±0.55)、(4.24±0.92)mm] LVDs明显降低(P均＜0.05)；对照组、转染组和未转染组组间比较差异无统计学意义(P均＞ 0.05).荧光显微镜下,心肌组织细胞移植区可见GFP和DAPI标记的移植细胞.大鼠心肌纤维化面积及心肌组织MMP-2蛋白表达,组间比较差异有统计学意义(F值分别为533.750、32.777,P均＜0.01).其中生理盐水组[(15.200±0.356)％,0.584±0.013]、转染组[(8.530±0.573)％,0.386±0.017]、未转染组[(10.670±0.369)％,0.438±0.015]心肌纤维化面积比及MMP-2蛋白表达明显高于对照组[(1.070±0.113)％、0.319±0.013,P均＜0.01]；转染组和未转染组低于生理盐水组(P均＜0.05)；转染组低于未转染组(P＜0.05).结论 移植转染ADM基因的BMSCs,可改善心衰大鼠的心脏功能,减轻心肌纤维化.
目的 探討移植轉染腎上腺髓質素(adrenomedullin,ADM)基因的骨髓間充質榦細胞(bone mesenchymal stem cells,BMSCs)對改善心衰大鼠心髒功能的作用.方法 Wistar大鼠10隻,20日齡,體質量30～ 50 g.分離大鼠股骨、脛骨取BMSCs,體外傳代培養.將攜帶ADM基因併標記綠色熒光蛋白(GFP)的腺病毒轉染體外培養的第3代BMSCs,在移植前用4’,6-二脒基-2-苯基吲哚(DAPI)對BMSCs進行染色.雄性Wistar大鼠80隻,體質量180 ～ 200 g.按體質量將大鼠隨機分為2組,對照組10隻,皮下註射生理鹽水;模型組70隻,按170 mg/kg連續4d皮下註射異丙基腎上腺素(isoproterenol,ISO),建立瀰漫性心肌損傷的大鼠心衰模型.ISO末次給藥後4週,大鼠進行超聲心動圖檢查,將心衰組左室射血分數(LVEF)＜70％的39隻大鼠按射血分數高低隨機分為3組:未轉染組、轉染組、生理鹽水組,每組13隻.將DAPI標記的未轉染BMSCs懸液、轉染ADM基因BMSC懸液各150 μl(3×106箇)、等量生理鹽水按上述分組分彆分4箇點註射到大鼠左心室前壁.對照組隻開胸,不做註射.移植後4週行超聲心動圖檢查,取左心室心肌組織,採用Massons染色觀察心肌組織纖維化程度,熒光顯微鏡觀察移植細胞,Western blotting檢測心肌組織基質金屬蛋白酶-2(MMP-2)蛋白錶達.結果 大鼠BMSCs體外培養第3天,細胞開始貼壁生長,約第10天細胞趨于融閤,呈梭形,走行一緻.移植後4週,超聲心動圖顯示,大鼠心髒左心室收縮末徑(LVDs)、LVEF、左心室短軸縮短率(LVFS)組間比較差異有統計學意義(F=5.838、32.983、51.714,P＜0.05或P＜0.01).與對照組[(86.50±1.54)％,(50.66±1.87)％]比較,生理鹽水組[(56.67±6.86)％,(26.27±4.01)％],轉染組[(79.40±1.70)％,(43.48±2.15)％]、未轉染組[(69.24±7.30)％,(34.59±5.13)％]LVEF和LVFS明顯降低(P均＜0.05)；與生理鹽水組比較,轉染組和未轉染組明顯增加(P均＜ 0.05)；與未轉染組比較,轉染組明顯增加(P＜0.05).與對照組[(3.16±0.22)mm]比較,生理鹽水組[(5.35±1.57)mm] LVDs明顯增加(P＜0.01)；與生理鹽水組比較,轉染組和未轉染組[(3.95±0.55)、(4.24±0.92)mm] LVDs明顯降低(P均＜0.05)；對照組、轉染組和未轉染組組間比較差異無統計學意義(P均＞ 0.05).熒光顯微鏡下,心肌組織細胞移植區可見GFP和DAPI標記的移植細胞.大鼠心肌纖維化麵積及心肌組織MMP-2蛋白錶達,組間比較差異有統計學意義(F值分彆為533.750、32.777,P均＜0.01).其中生理鹽水組[(15.200±0.356)％,0.584±0.013]、轉染組[(8.530±0.573)％,0.386±0.017]、未轉染組[(10.670±0.369)％,0.438±0.015]心肌纖維化麵積比及MMP-2蛋白錶達明顯高于對照組[(1.070±0.113)％、0.319±0.013,P均＜0.01]；轉染組和未轉染組低于生理鹽水組(P均＜0.05)；轉染組低于未轉染組(P＜0.05).結論 移植轉染ADM基因的BMSCs,可改善心衰大鼠的心髒功能,減輕心肌纖維化.
목적 탐토이식전염신상선수질소(adrenomedullin,ADM)기인적골수간충질간세포(bone mesenchymal stem cells,BMSCs)대개선심쇠대서심장공능적작용.방법 Wistar대서10지,20일령,체질량30～ 50 g.분리대서고골、경골취BMSCs,체외전대배양.장휴대ADM기인병표기록색형광단백(GFP)적선병독전염체외배양적제3대BMSCs,재이식전용4’,6-이미기-2-분기신타(DAPI)대BMSCs진행염색.웅성Wistar대서80지,체질량180 ～ 200 g.안체질량장대서수궤분위2조,대조조10지,피하주사생리염수;모형조70지,안170 mg/kg련속4d피하주사이병기신상선소(isoproterenol,ISO),건립미만성심기손상적대서심쇠모형.ISO말차급약후4주,대서진행초성심동도검사,장심쇠조좌실사혈분수(LVEF)＜70％적39지대서안사혈분수고저수궤분위3조:미전염조、전염조、생리염수조,매조13지.장DAPI표기적미전염BMSCs현액、전염ADM기인BMSC현액각150 μl(3×106개)、등량생리염수안상술분조분별분4개점주사도대서좌심실전벽.대조조지개흉,불주주사.이식후4주행초성심동도검사,취좌심실심기조직,채용Massons염색관찰심기조직섬유화정도,형광현미경관찰이식세포,Western blotting검측심기조직기질금속단백매-2(MMP-2)단백표체.결과 대서BMSCs체외배양제3천,세포개시첩벽생장,약제10천세포추우융합,정사형,주행일치.이식후4주,초성심동도현시,대서심장좌심실수축말경(LVDs)、LVEF、좌심실단축축단솔(LVFS)조간비교차이유통계학의의(F=5.838、32.983、51.714,P＜0.05혹P＜0.01).여대조조[(86.50±1.54)％,(50.66±1.87)％]비교,생리염수조[(56.67±6.86)％,(26.27±4.01)％],전염조[(79.40±1.70)％,(43.48±2.15)％]、미전염조[(69.24±7.30)％,(34.59±5.13)％]LVEF화LVFS명현강저(P균＜0.05)；여생리염수조비교,전염조화미전염조명현증가(P균＜ 0.05)；여미전염조비교,전염조명현증가(P＜0.05).여대조조[(3.16±0.22)mm]비교,생리염수조[(5.35±1.57)mm] LVDs명현증가(P＜0.01)；여생리염수조비교,전염조화미전염조[(3.95±0.55)、(4.24±0.92)mm] LVDs명현강저(P균＜0.05)；대조조、전염조화미전염조조간비교차이무통계학의의(P균＞ 0.05).형광현미경하,심기조직세포이식구가견GFP화DAPI표기적이식세포.대서심기섬유화면적급심기조직MMP-2단백표체,조간비교차이유통계학의의(F치분별위533.750、32.777,P균＜0.01).기중생리염수조[(15.200±0.356)％,0.584±0.013]、전염조[(8.530±0.573)％,0.386±0.017]、미전염조[(10.670±0.369)％,0.438±0.015]심기섬유화면적비급MMP-2단백표체명현고우대조조[(1.070±0.113)％、0.319±0.013,P균＜0.01]；전염조화미전염조저우생리염수조(P균＜0.05)；전염조저우미전염조(P＜0.05).결론 이식전염ADM기인적BMSCs,가개선심쇠대서적심장공능,감경심기섬유화.
Objective To investigate the effects of transplantation of bone marrow mesenchymal stem cells (BMSCs) transfected with adrenomedullin (ADM) on cardiac function in heart failure rats and the mechanism.Methods BMSCs were isolated from femur and tibia marrow of 10 rats,20 days old,body weight 30-50 g,and in vitro cultured.The third passage of BMSCs were tuansfected with adenovirus containing ADM and labeled with green fluorescent protein(GFP).Before transplantation,BMSCs were labeled with 4',6-diamidino-2-phenylindole (DAPI).Eighty healthy male Wistar rats weighted 180-200 g were randomly divided into 2 groups according to body weight:control group (n =10) was injected with normal saline (NS); diffuse myocardial injury heart failure rat model(n =70) was established by subcutaneous injection of isoproterenol (ISO,170 mg/kg) every day for 4 consecutive days.Four weeks after administration of ISO,heart function was assessed by echocardiography,the 39 rats with left ventricle ejection fraction(LVEF) ＜ 70％ of global heart failure model were randomly divided into three groups in accordance with the level of heart function:untransfected group,transfected group and NS group.DAPI labeled untransfected BMSCs suspension,ADM gene transfected BMSC suspensions (3 × 106/150 μl) and equal volume of NS were injected into the left ventricular anterior wall in 4 places in each goup.Control group received thoracotomy only.Four weeks after transplantation,rats were examined by ultrasound echocardiography,then were sacrificed and left ventricular were dissected.The myocardium was stained with Massons trichrome to analyze myocardial tissue fibrosis.The transplanted cells were observed by fluorescence microscopy and matrix metalloproteinase-2 (MMP-2) expression of myocardial tissue was detected in each group by Western blotting.Results After in vitro culture for three days,the BMSCs began to grow adherently,tended to be fused about 10 days,in the fusiform shape.Four weeks after transplantation,ultrasound echocardiography results showed that rat cardiac left ventricular end systolic diameter (LVDs),LVEF,and left ventricular cardiac fractional shortening (LVFS) were different between groups,and the difference were statistically significant(F =5.838,32.983,51.714,P ＜ 0.05 or P ＜ 0.01).Compared with the control group[(86.50 ± 1.54)％,(50.66 ± 1.87)％],the LVEF and LVFS of NS groups[(56.67 ± 6.86)％,(26.27 ± 4.01)％],the transfected group[(79.40 ± 1.70)％,(43.48 ±2.15)％] and untransfected group[(69.24 ± 7.30)％,(34.59 ± 5.13)％] were significantly lower(all P ＜ 0.05);compared with the NS group,the LVEF and LVFS of the transfected groups and the untransfected group were significantly increased(all P ＜ 0.05) ; compared with the untransfected group,the LVEF and LVFS of the transfected group were increased (all P ＜ 0.05).Compared with the control group [(3.16 ± 0.22)mm],the LVDs of the NS group[(5.35 ± 1.57)mm] was significantly increased (P ＜ 0.01); compared with the NS group,the LVDs of the transfected group and the untransfected group[(3.95 ± 0.55),(4.24 ± 0.92)mm] were significantly decreased (all P ＜ 0.05).There was no statistically significant difference between the control group,the transfected group and the untransfected group in LVDs (P ＞ 0.05).It can clearly be seen that there was GFP and DAPI labeled transplanted cells under a fluorescence microscope in the myocardial tissue transplanted area.There was significant difference in myocardial fibrosis area and the myocardial tissue protein expression of MMP-2 between groups(F =533.75,32.777,all P ＜ 0.01).The area ratio of the NS group[(15.200 ± 0.356)％,0.584 ± 0.013],the transfected group[(8.530 ± 0.573)％,0.386 ± 0.017] and the untransfected group [(10.670 ± 0.369)％,0.438 ± 0.015] and the MMP-2 protein expression were significantly higher than that of the control group[(1.070 ± 0.113)％,0.319 ±0.013,all P ＜ 0.01)]; compared with the NS group,the two index of the transfected group and the untransfected group were decreased (all P ＜ 0.05).Compared with the untransfected group,the two index of the transfected group was decreased (all P ＜ 0.05).Conclusion Transplantation of ADM gene transfected BMSCs can improve heart function of rats with heart failure significantly and reduce myocardial fibrosis.
