菲立磁标记猪骨髓间充质干细胞自体肝内移植的MR成像

目的:探索标记细胞肝内移植后磁共振威像技术.方法:获取猪自体骨髓间充质干细胞,分离、培养.应用菲立磁(Feridex)标记细胞,普鲁士蓝染色鉴定,标记细胞组n=6)和未标记细胞组(n=4)行经门静脉行肝内移植,分别于移植前,移植后6h、3 d、7d行磁共振T1WI,T2WI,T2*WI序列成像,同时行组织切片普鲁士蓝染色结果:普鲁士蓝染色表明MSCs的标记率达接近100%,磁标记MSCs肝内移植后行磁共振T2*WI序列呈明显低信号改变,并持续至细胞移植后7 d,组织切片普鲁士蓝染色显示7 d后肝内仍有移植的磁标记细胞存在于肝实质及肝血窦中.结论:利用Feridex可以在体外成功标记猪骨髓间充质干细胞,肝脏移植后行磁共振可以对标记细胞进行活体成像.     

细胞因子微球加强骨髓间充质干细胞移植对猪心肌梗死的疗效

目的 通过观察血管内皮生长因子(VEGF)缓释明胶微球与自体骨髓间充质干细胞(MSC)联合移植于猪心肌梗死模型梗死周边区3周后MSC的生存情况,结合磁共振成像(MRI),阐明改善局部微环境对自体MSC移植于缺血心肌后转归的影响,并直观评价MRI对移植MSC在体示踪及半定量分析的可靠性.方法 以乳化冷凝法制备VEGF缓释明胶微球并评价其缓释性能.成年中华小型猪12头,抽取髂骨骨髓并制备自体MSC,以顺磁性氧化铁颗粒(SPIO)和4',6-二脒基-2-苯基吲哚(4',6-Diamidino-2-phenylindole dihydrochloride,DAPI)标记细胞.按照随机化表将动物分为MSC移植组及MSC-VEGF缓释微球联合移植组.开胸结扎冠状动脉左前降支制备小型猪心肌梗死模型后第14天,直视下经心外膜于MSC组动物心肌梗死周边区注射MSC(9×107),于MSC-VEGF缓释微球组注射MSC(9×107)及VEGF缓释明胶微球(含9 μg人重组VEGF165).细胞和(或)微球移植后24 h及21 d,磁共振FGRE序列T2*像检测干细胞移植点低信号区的面积及信号强度.病理组织学检测细胞移植区域心肌组织毛细血管密度及干细胞存活情况.结果 明胶微球平均粒径为(104.0±22.6)μm,体内、外缓释VEGF均可达到10 d以上.小型猪心肌梗死模型建立后第14天,梗死区面积为33.6%±8.9%.细胞移植后24 h,MSC注射点于MRI显示为边界清晰的卵圆形T2*低信号区,细胞移植3周后,两组注射点T2*低信号区面积均减小(P<0.05),其与正常心肌组织间的信号对比度均减低(P<0.05).MSC-VEGF缓释微球组移植点毛细血管密度高于MSC组[(15.2±5.4)个/高倍视野比(10.2±5.0)个/高倍视野,t=2.43,P<0.05].MSC-VEGF缓释微球组MSC注射点移植细胞数多于MSC组[(354±83)个/高倍视野比(278±97)个/高倍视野,t=3.14,P<0.05],而MSC凋亡率则小于MSC-VEGF缓释微球组[(6.4±4.1)%比(11.9±4.8)%,t=2.97,P<0.05].结论 VEGF缓释明胶微球可以改善移植于缺血心肌区3周后MSC的生存情况.移植干细胞所生存的微环境是影响其转归的重要因素.MRI对于移植干细胞位置的在体示踪是可靠的,但不能作为对移植细胞定量或半定量分析的依据.     

联系方式：周涛 chowtao@sina.com. Tel:13985024647

目的 探讨浅低温心脏不停跳心内直视手术对体外循环瓣膜置换患者线粒体耦联因子6（CF6）表达的影响.方法 选择2010年1月至2011年11月广西医科大学第一附属医院择期行人工机械瓣膜置换手术患者50例,使用随机数字表法分为心脏停跳组（停跳组,在中度低温心脏停跳下完成心脏瓣膜置换手术）和浅低温心脏不停跳组（不停跳组,在浅低温心脏跳动下完成手术）,每组25例,分别于体外循环转机前（T1）,转机30 min（T2）,开放升主动脉时（T3）,停体外循环后6 h（T4）、24 h（T5）、72 h（T6）、120 h（T7）7个时间点取静脉血,使用放射免疫分析法测定CF6、6-酮-前列环素F1a（6-Keto-PGF1a）的表达.结果 在T2～T5时间点,停跳组CF6浓度（pg/ml）依次为574.3±103.7、855.3±175.8、665.1±95.6、398.9±74.5,较T1时浓度244.5±52.6升高（P＜0.05）;不停跳组CF6浓度（pg/ml）依次为317.1±93.3、673.9±115.1、452.6±81.2、296.2±61.4,较T1时浓度238.4±49.3升高（P＜0.05）;停跳组变化更明显（P＜0.05）.两组均于T6恢复至T1水平.停跳组T2 ～T4时6-Keto-PGF1a浓度（pg/ml）依次为330.7±67.9、435.6±75.8、235.7±35.0,较T1浓度64.3±18.4升高（P＜0.05）;不停跳组T2～T4时浓度（pg/ml）依次为467.4±43.5、573.9±33.1、356.2±41.9,较T1浓度68.3±19.3升高（P＜0.05）;不停跳组升高明显（P＜0.05）.两组均于T5恢复至T1水平.结论 心脏不停跳手术对心肌包括内皮细胞损伤较小是体外循环手术CF6表达上调相对较小的重要原因.CF6的变化可以影响机体前列环素的表达.     

连续灌注不停跳心脏移植对供心的保护作用

目的 通过建立大鼠同种异位供心停跳心脏移植模型与连续血液灌注供心不停跳心脏移植模型,探讨连续灌注不停跳心脏移植对供心的影响.方法 建立大鼠同种异位连续灌注心脏不停跳心脏移植模型（不停跳移植组）和改良Heron法建立大鼠异位心脏移植模型（停跳移植组）.移植成功后2h检测受体外周血肌酸激酶同工酶（CK-MB）和心肌肌钙蛋白I（cTnI）含量,测定供心心肌脂质过氧化终产物丙二醛（MDA）、过氧化物歧化酶（SOD）含量,电镜观察心肌结构变化,并观察心肌凋亡情况.结果 连续灌注心脏不停跳移植组受体外周血CK-MB及cTnI含量低于停跳移植组,差异有统计学意义（P＜0.05）.与停跳移植组比较,不停跳移植组心肌MDA含量相对较少,SOD含量相对较多,心肌细胞凋亡指数较小,差异均有统计学意义（P＜0.05）.不停跳移植组心肌线粒体等结构损伤相对较轻.结论 心脏不停跳技术能有效减轻心脏移植供心的脂质过氧化反应,保护心肌线粒体,抑制心肌细胞凋亡,可减轻心脏移植供心的再灌注损伤.     

同种异体骨髓间充质干细胞移植对扩张型心肌病心功能衰竭大鼠左心室功能的影响

目的探讨同种异体大鼠骨髓间充质干细胞移植在阿霉素诱导的扩张型心肌病心功能衰竭大鼠心脏内存活、分化的情况及对左心室功能的影响。方法雌性Wistar大鼠55只,随机分成正常对照组(n=10)、模型组(n=15)、诱导前移植组(n=15)和诱导后移植组(n=15)。体外分离培养雄性大鼠骨髓间充质干细胞,传至一代后用10μmol/L5-氮胞苷诱导4周,DAPI标记后,诱导前移植组移植诱导前的骨髓间充质干细胞,诱导后移植组移植诱导后的骨髓间充质干细胞;4周后检测左心室功能及移植细胞存活、分化情况。结果异体骨髓间充质干细胞移植4周后可存活并分化成心肌细胞,表达心肌细胞特异性蛋白;与模型组比较,细胞移植组左心室功能明显改善,且两细胞移植组间差异无显著性。结论同种异体骨髓间充质干细胞移植4周后可存活并分化成心肌细胞,对扩张型心肌病心功能衰竭大鼠的左心室功能有保护作用。     

磁共振成像R2*map示踪超顺磁性氧化铁标记的内皮祖细胞

目的 探讨R2*map在超顺磁性氧化铁(SPIO)标记内皮祖细胞(EPCs)定量检测中的价值.方法 分离和培养Balb/c小鼠后肢骨髓来源的EPCs,培养7 d,用细胞膜红色荧光探针标记的乙酰低密度脂蛋白和异硫氰酸荧光素标记的荆豆凝集素-1双阳性染色法,以及异硫氰酸荧光素标记干细胞抗原1和藻红素标记的血管内皮细胞生长因子受体2的方法上流式细胞仪进行鉴定.用50 μg/ml SPIO及6 μl/ml lipofectamine 2000与EPCs共孵育进行标记后透射电子显微镜观察;制成不同细胞浓度(0～2.0×106/ml)标记及未标记SPIO的细胞琼脂糖模型,进行3.0T磁共振扫描,包括T2*map和T2map扫描,得到R2*map和R2map图像.结果 培养7d后细胞呈现内皮祖细胞特征.SPIO标记的EPCs细胞结构与未标记细胞相比较,无明显改变.R2*和R2值与标记SPIO的细胞浓度呈线性相关(r值分别为0.955,0.922,P值均<0.05);R2*效应明显高于R2效应(t=23.23,P<0.05).结论 磁共振成像R2*map扫描可准确定量示踪SPIO标记的EPCs.     

体外循环心内直视手术对心肌细胞炎症的影响

探讨体外循环(CPB)心脏手术对患者心肌细胞炎症的影响。方法 择期行二尖瓣置换的风心病患者40例，随机分为停跳组和不停跳组，停跳组在心脏停跳下完成瓣膜置换手术，不停跳组在浅低温心脏跳动中完成手术。分别于CPB转机前( T1 )、CPB转机30min( T2)、术中关闭右房时(T3) 、CPB结束后24h(T4)、72h(T5)、120h(T6)共6时间点采集静脉血，ELISA法测定肌钙蛋白I (cTnI )浓度，全自动生化分析仪检测肌酸激酶同工酶MB (CK-MB)浓度。分别于打开和关闭右心房时取右心房组织标本，ELISA法检测心肌匀浆肿瘤坏死因子-α（TNF-α）、白细胞介素( IL)-6、IL-8和IL-10含量。结果 不停跳组静脉血cTnI、CK-MB浓度在T3~T5低于停跳组，差异有统计学意义(P<0.05)。停跳组TNF-α、IL-6、IL-8上升较不停跳组明显(P<0.05)，而IL-10上升幅度较不停跳组小(P<0.05)。结论 CPB心内直视手术时存在心肌炎症，心脏不停跳手术在一定程度上减轻心肌炎症反应，可能是该手术方式减轻心肌损伤的重要机制。     

静脉移植骨髓间充质干细胞对心衰大鼠心肌细胞cx-43表达的影响

目的探讨骨髓间充质干细胞移植对阿霉素诱导的心衰大鼠模型心功能及细胞缝隙连接蛋白-43(cx-43)的影响。方法选取雌性Wistar大鼠(n=38),先随机选取8只作为正常对照组,其余30只通过腹腔注射阿霉素(2.5mg/kg,每周1次,连续6周)建立心衰模型。6周后存活大鼠(n=21)再随机分为细胞移植组(n=11)和移植对照组(n=10)。正常对照组大鼠腹腔注射等量的生理盐水。进行体外分离,纯化,增殖骨髓间充质干细胞(MSCs)并用含有12%胎牛血清的DMEM培养基培养,采用流式细胞仪对体外培养的MSCs表型CD44进行鉴定。取第三代MSCs进行移植,移植前用5-溴-2’脱氧尿苷(BrdU)进行标记。末次阿霉素注射后1周,将5×106个MSCs通过鼠尾静脉注射的方法移植到心衰细胞移植组大鼠。移植对照组大鼠注射等量培养基。细胞移植后4周,采用超声测量心功能及心室重塑,心脏切片行病理免疫组织化学了解移植细胞在受体心脏的存活及cx-43表达情况。结果体外培养的第三代MSCs流式细胞仪鉴定结果显示:CD44阳性细胞数占(99.7±0.9)%,心衰细胞移植组心肌组织中可以见5-溴-2’脱氧尿苷标记的骨髓间充质干细胞(MSCs)归巢并表达cx-43阳性;左室射血分数细胞移植心衰组、对照组与正常对照组分别为(89.36±3.74)%、(72.93±3.08)%和(97.54±2.30)%(P<0.05),心衰细胞移植相对于心衰对照组左室心功能有统计学意义(P<0.05)。结论经静脉移植骨髓间充质干细胞可以归巢心衰大鼠心肌组织而且改善心功能并影响受体心肌组织细胞表达cx-43。     

骨髓间充质干细胞治疗心肌梗死后心功能衰竭的远期疗效

目的采用2’脱氧5-氮杂胞苷诱导的骨髓间充质干细胞移植于心肌梗死后心功能衰竭大鼠心肌中,评价其存活、分化及对心功能的保护作用,并研究其慢性演变。方法体外培养的Wistar大鼠骨髓间充质干细胞,用0.3μmol/L的2’脱氧5-氮杂胞苷体外两次诱导第二代骨髓间充质干细胞,并用溴氮胞苷标记后植入心肌梗死10天心功能衰竭大鼠的心肌疤痕中,同时以注射无血清培养基的实验动物为对照。移植前及移植后1、2、3个月通过心脏射血分数等指标评价其对心功能的保护作用,同时通过双重免疫组织化学及电镜观察对移植细胞的存活和分化进行3个月的慢性动态观察。结果移植后1个月,骨髓间充质干细胞移植组大鼠射血分数(83.3%±8.1%,n=13)高于无血清培养基移植组(47.2%±11.8%,n=12)(P<0.01);并且细胞移植组移植后较移植前心功能(64.3%±10.4%)也有改善(P<0.01)。细胞移植组移植后第2个月(射血分数为83.1%±14.4%,n=7)、第3个月(射血分数为86.3%±3.7%,n=6)心功能改善情况继续保持,与无血清培养基移植组第2个月(射血分数为51.6%±11.2%,n=6)、第3个月(射血分数为49.1%±7.7%,n=6)差异均有显著性(P<0.01)。移植的骨髓间充质干细胞在心肌及疤痕中存活,双重免疫组织化学证实其溴氮胞苷及肌钙蛋白T均阳性。电镜观察提示移植后1个月其在疤痕中具心肌样细胞部分特点,但分化细胞核大浆少,未形成明显肌小节及Z带;移植后3个月,可见明显Z带样结构。结论经2’脱氧5-氮杂胞苷诱导的骨髓间充质干细胞可改善心肌梗死后心功能衰竭大鼠的心功能,并能长期保持(观察3个月);其能在心肌梗死后心功能衰竭大鼠心肌及疤痕中存活,并能逐渐向心肌细胞分化。     

细胞磁共振成像不能区分磁标细胞的存活状态

目的探讨不同状态下磁标记间充质干细胞(MSCs)的体内外磁共振(MR)成像特点,明确MR对细胞存活状态是否有鉴别诊断价值。方法分离培养猪骨髓MSCs,用超顺磁性铁纳米颗粒(SPIO)标记细胞24h。对未标记MSCs、存活的SPIO-MSCs、死亡的SPIO-MSCs、单纯SPIO进行体外凝胶内1.5TMR成像;开胸结扎前降支建立猪心肌梗死模型,于梗死交界区心肌内直接注射未标记MSCs、存活的SPIO-MSCs、死亡的SPIO-MSCs和单纯SPIO,然后进行体内T2*WI-flash序列MR成像。结果存活的SPIO-MSCs、死亡的SPIO-MSCs和单纯SPIO在体内外T2*WI-flash序列MR图像上均显示为低信号区,单凭图像无法区分。结论细胞MR成像无法区分组织磁标细胞、游离铁和含铁病变,因此对长期细胞示踪结果的解读应该慎重。     

Aortic arch repair: let it beat!

Objective aortic arch repair (AAR) on the beating heart may reduce cross-clamping times and offer improved postoperative cardiac function.Methods A single-center review of all patients (n = 24) who underwent surgical AAR during biventricular repair between 01/2006 and 01/2008 was done. All patients were operated on under cardiopulmonary bypass (CPB) with antegrade cerebral perfusion (ACP). During AAR, 13 patients (group 1) received cardioplegic arrest, and were compared to 11 patients (group 2) who underwent a beating-heart modification with selective myocardial perfusion. Seventeen patients had additional intracardiac lesions and underwent simultaneous correction during the procedure.Results Durations of CPB, AAR and ACP did not differ statistically between groups. Cardioplegic arrest time was significantly lower in group 1 (34 ± 13 vs. 76 ± 11 min, p = 0.02) and resulted in a subsequent reduction of myocardial ischemic damage as borne out by lower postoperative levels of troponin T and CK-MB (2.5 ± 0.7 vs. 7.1 ± 1.4 ng/mL, p = 0.02; 68.7 ± 11.5 vs. 149.1 ± 27.2 U/l, p = 0.03). We observed an enhanced patient recovery with shorter inotropic and ventilatory support times (p < 0.05).Conclusion Pediatric aortic arch correction on a CPB beating heart with selective myocardial perfusion is technically feasible and safe. The reduction of the myocardial ischemic time is effective and results in less myocardial damage.     

Cardiac iron removal and functional cardiac improvement by different iron chelation regimens in thalassemia major patients

Heart failure due to myocardial iron overload remains the leading cause of morbidity and mortality in adult thalassemia major (TM) patients. We evaluated the removal of cardiac iron and the changes of cardiac function by different iron chelation in TM patients by T2* cardiac magnetic resonance (CMR). Sixty-seven TM patients (27 males/40 females; mean age, 35 ± 6 years) on different chelation regimens underwent T2* CMR at baseline (t (0)), after 6-14 months (t (1)) and after 32 ± 7 months (t (2)). Patients were divided in four groups according to chelation treatment: group A (deferasirox), group B (deferoxamine), group C (combined treatment, deferoxamine plus deferiprone) and group D (deferiprone alone). Myocardial T2* at t (0) was <10 ms in 8 patients, between 10 and 20 ms in 22 patients and ≥ 20 ms in 37 patients. Progressive changes in T2* were observed at t (1) and t (2). Ten patients (10/36, 27.8 %) in group A, three patients (3/15, 20 %) in group B and three patients (3/12, 25 %) in group C moved from an abnormal T2* to normal values. We observed an improvement of left ventricular ejection fraction and a reduction of end-systolic and end-diastolic left ventricular volumes only in patients in group A with baseline cardiac T2* between 10 and 20 ms. Rigorous compliance to any chelation therapy at proper doses significantly improve myocardial T2*. Treatment with deferasirox significantly improves left ventricular function. Combination therapy seems to ameliorate cardiac T2* in a shorter period of time in severe siderosis.     

艾司洛尔对不停跳心内直视手术心肌的保护作用

目的探讨β1受体阻滞剂艾司洛尔在不停跳心内直视手术中对心肌的保护作用。方法选择22例择期行房间隔修补手术患者,随机分为两组,每组11例,两组均在常温体外循环心脏不停跳心内直视下进行房间隔修补手术。实验组在体外循环开始前于预冲液中加入艾司洛尔1 mg/kg,转机过程中再以300μg.kg-1.min-1的速度从静脉持续输注艾司洛尔,直到手术完成。对照组给予等量的生理盐水代替艾司洛尔,其余和实验组相同。监测两组病例在各个时间点的血流动力学、血气分析、心肌损伤标志物及围手术期各项临床指标,并进行对比。结果在转机过程中,实验组心率(56±8)次/min显著低于对照组(64±9)次/min(P<0.05),实验组cTnI和CK-MB在术后6、12、24 h各个时间点均显著低于对照组(均为P<0.001),两组病例在手术开始前、转机开始前、转机过程中、停机后和手术结束后各个时间点的血气分析指标差异无统计学意义(均为P>0.05)。结论在常温体外循环心脏不停跳心内直视手术中应用艾司洛尔可以显著保护心肌并改善手术操作条件。     

心脏停跳与不停跳先心病矫治术患者围术期cTnⅠ及心肌酶水平变化

目的探讨心脏停跳与不停跳先天性心脏病（先心病）矫治术对心肌的损伤程度,并寻找敏感性评价指标。方法将40例同期拟行先心病矫治术的患者随机分为对照组和观察组各20例,分别于心脏停跳与不停跳下手术,两组麻醉及体外循环（CPB）方法相同。分别于围术期检测血清心肌肌钙蛋白Ⅰ（cTnⅠ）、肌酸激酶同工酶（CK-MB）、乳酸脱氢酶（LDH）、肌酸激酶（CK）、AST、α-羟丁酸脱氢酶（HBDH）,同时观察心电图及心脏超声指标改变。结果两组围术期心电图及心脏超声指标均无明显异常,但术中、术后血清cTnⅠ及CK、CK—MB、LDH水平均明显升高,对照组显著高于观察组;其中血清cTnⅠ水平升高出现早、恢复慢（术后72h时仍显著高于术前水平）。结论心脏不停跳先心病矫治术对心肌的损伤小于心脏停跳手术;cTnⅠ是评价心脏手术围术期心肌损伤敏感的特异指标．     

心脏不停跳二尖瓣置换术对血浆氨基末端脑钠肽前体的影响及其研究

目的 研究氨基末端脑钠肽前体（NT-proBNP）在浅低温体外循环下心脏不停跳二尖瓣置换术围手术期的变化,探讨心脏不停跳二尖瓣置换术的心肌保护效果.方法 40例风湿性心脏病二尖瓣病变患者随机分为试验组（浅低温不停跳组）与对照组（中度低温停跳组）,每组20例,于6个时点采取静脉血,测定静脉血浆NT-proBNP水平.结果 两组NT-proBNP水平在术后2 h无明显升高（P＞0.05）,在术后6 h明显升高,并在术后24 h达到峰值.在术后6 h、24 h、48 h、72 h,停跳组NT-proBNP水平均低于不停跳组（P＜0.05）.结论 浅低温心脏不停跳二尖瓣置换术能减轻缺血缺氧及再灌注损伤引起的心肌损伤,有较好的心肌保护效果.     

Intraportal mesenchymal stem cell transplantation prevents acute liver failure through promoting cell proliferation and inhibiting apoptosis

BACKGROUND: Transplantation of mesenchymal stem cells(MSCs) has been regarded as a potential treatment for acute liver failure(ALF), but the optimal route was unknown. The present study aimed to explore the most effective MSCs transplantation route in a swine ALF model.METHODS: The swine ALF model induced by intravenous injection of D-Gal was treated by the transplantation of swine MSCs through four routes including intraportal injection(In P group), hepatic intra-arterial injection(AH group), peripheral intravenous injection(PV group) and intrahepatic injection(IH group). The living conditions and survival time were recorded. Blood samples before and after MSCs transplantation were collected for the analysis of hepatic function. The histology of liver injury was interpreted and scored in terminal samples. Hepatic apoptosis was detected by TUNEL assay. Apoptosis and proliferation related protein expressions including cleaved caspase-3, survivin, AKT, phospho-AKT(Ser473), ERK and phospho-ERK(Tyr204) were analyzed by Western blotting.RESULTS: The average survival time of each group was 10.7 ±1.6 days(In P), 6.0±0.9 days(AH), 4.7±1.4 days(PV), 4.3±0.8 days(IH), respectively, when compared with the average survival time of 3.8±0.8 days in the D-Gal group. The survival rates between the In P group and D-Gal group revealed a statistically significant difference(P0.01). Pathological and biochemical analysis showed that liver damage was the worst in the D-Gal group, while less injury in the In P group. Histopathological scores revealed a significant decrease in the In P group(3.17±1.04, P0.01) and AH group(8.17±0.76, P0.05) as compared with that in the D-Gal group(11.50±1.32). The apoptosis rate in the In P group(25.0%±3.4%, P0.01) and AH group(40.5%±1.0%, P0.05) was lower than that in the D-Gal group(70.6%±8.5%). The expression of active caspase-3 was inhibited, while the expression of survivin, AKT, phosphoAKT(Ser473), ERK and phospho-ERK(Tyr204) was elevated in the In P group.CONCLUSIONS: Intraportal injection was superior to other pathways for MSC transplantation. Intraportal MSC transplantation could improve liver function, inhibit apoptosis and prolong the survival time of swine with ALF. The transplanted MSCs may participate in liver regeneration via promoting cell proliferation and suppressing apoptosis during the initial stage of ALF.     

Magnetic resonance evaluation of transplanted mesenchymal stem cells after myocardial infarction in swine

Objectives To trace and evaluate intracoronary transplanted mesenchymal stem cells(MSCs) labeled with superparamagnetic iron oxide(SPIO) by using magnetic resonance imaging(MRI) in a swine model of myocardial infarction (MI).Methods MSCs were transfected with a lentiviral vector carrying the gene encoding green fluorescent protein (GFP) and labeled in vitro with SPIO.Two weeks after MI, swine were randomized to intracoronary transplantation of dual -labeled MSCs(n = 10),MSCs-GFP(n = 10) and saline(n = 5).MRI examination was performed with a 1.5T clinical scanner at 24 hours,3 weeks and 8 weeks after cells transplantation. Signal intensity(SI) changes,cardiac function and MI size were measured using MRI.Correlation between MR findings and histomorphologic findings was also investigated. Results The labeling efficiency at a combination of 25μg Fe/ml SPIO and 0.8 pi/ml Lipofectamine 2000 reached 100%.SPIO labeling did not affect GFP fluorescence and dual-labeling did not affect cell proliferation(P>0.05). Multipotentiality was not affected especially for cardiomyocyte-like cells differentiation.Cardiac cell marker of a-MHC and actinin were positively expressed by immunofluorescence staining after induction.SI on T2 * WI decreased substantial- ly in the interventricular septum 24 hours after injection of MSCs.The intensity of hypo-intense signals appeared to increase throughout the later time points.Changes in SI at 24 hours,3 weeks and 8 weeks were 52.98%±10.74%,21.53%±5.40%and 6.23%±2.01%,respectively(P<0.01).DE-MRI demonstrated both dual-labeled MSCSs and MSCs-GFP could dramatically reduce the size of MI and improve cardiac function. Histological data revealed that prussian blue stain-positive cells were found mainly in the border zone which also showed green fluorescence but negative for macrophage marker(CD68).Gross pathologic examination revealed that engrafted MSCs dramatically reduce the extent of necrotic myocardium and promote the regeneration of new,contractile myocardium along the subendocardia     

生长分化因子-11促进小鼠诱导性多能干细胞向心肌细胞定向分化的研究

目的 明确生长分化因子（GDF）-11对小鼠诱导多能干细胞（miPSCs）向心肌细胞定向分化的促进作用,为心肌再生的细胞生物学治疗提供种子细胞和实验依据。方法 常规培养小鼠miPSCs,分为对照组和GDF-11组。GDF-11组在普通分化培养基中加用10 ng/ml的GDF-11。悬滴法诱导培养形成拟胚体（EBs）,每日观察搏动拟胚体数目。采用实时荧光定量PCR检测多能干细胞标志物Oct-4、心脏中胚层标志物Flk-1、心脏祖细胞标志物Nkx2.5、和心肌特异性标志物cTnT的表达变化。用免疫荧光染色观察心肌结构蛋白cTnI的表达水平。结果 GDF-11组的Oct-4表达水平在诱导分化的第3、7天分别为同期对照组的（0.55±0.31）倍（P<0.01）和（0.41±0.57）倍（P<0.05）;诱导分化的第10天,GDF-11组的Flk-1和Nkx2.5表达相分别为对照组的（2.09±0.8）倍（P<0.05）和（2.47±0.22）倍（P<0.01）;cTnT的表达在分化后第10天和第14天分别为对照组的（1.81±0.19）倍（P<0.01）和（1.61±0.20）倍（P<0.01）;两组均出现了心肌样搏动细胞团,GDF-11组搏动EBs百分比要显著高于对照组（P<0.01）。免疫荧光染色显示,GDF-11组的cTnI阳性率要显著高于对照组（P<0.01）。结论 GDF-1能够显著促进miPSCs的心肌定向分化。     

乳鼠心肌细胞联合人工基质移植改善大鼠心肌梗死后心功能的实验研究

目的探讨体外构建液态组织工程学心肌(engineering heart tissue,EHT)的方法,以及其移植于大鼠心肌梗死区域后是否能存活并改善心脏功能。方法结扎雌性Sprague-Dawley(SD)大鼠左冠状动脉,制作心肌梗死模型。分离、标记及培养新生1～3天SD乳鼠心肌细胞。心肌梗死后合格的动物模型随机分成EHT组(n=12)、单纯心肌细胞组(n=12)、单纯基质组(n=12)、对照组(n=11)。移植术后4周,用超声心动图及离体心脏灌流2种手段评价心功能,标本取材做病理学检查及Y染色体多聚酶链检测(polymerase chain reaction,PCR)。结果PCR检测证实EHT组和单纯细胞组移植细胞存活,但病理学检查表明EHT组的移植细胞排列更紧密,细胞间有连接蛋白形成。超声心动图显示EHT组的左室缩短分数最高(22.82±3.44)%,高于细胞组(20.55±4.11)%、人工基质组(17.05±4.57)%和对照组(19.80±3.98)%,P=0.012。心脏灌流模型提示在左心室球囊容积为0.06、0.08、0.10ml时,左心室发展压及dp/dt差异有统计学意义,EHT组最佳(P<0.05)。结论体外成功构建液态EHT后,可以通过注射方法移植于大鼠心肌梗死区域。液态EHT移植后较单纯细胞移植组织形态更佳、更能改善梗死心脏的功能。