细胞因子缓释微球联合骨髓间充质干细胞移植治疗缺血性心脏病疗效评价及MRI在体示踪的实验研究
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摘要
第一部分
血管内皮生长因子缓释明胶微球制备及性能评价
目的:制备血管内皮生长因子明胶微球并评价其缓释性能。方法:以乳化冷凝法制备VEGF缓释明胶微球。20%的明胶溶液滴加于橄榄油中搅拌至形成乳浊液,丙酮固化,异丙醇悬浮后以不同孔径筛网筛分。离心干燥后,戊二醛交联,清洗后冷冻干燥,得淡黄色明胶微球。钴60照射灭菌。将微球浸泡于磷酸盐缓冲液中24小时,微球充分溶胀后,以显微镜自带软件测量微球粒径。取灭菌冻干明胶微球9 mg,平均分成3组,将1.5μCi~(125)Ⅰ-VEGF溶于30μl PBS中,平均滴加于3组明胶微球,4℃过夜。PBS离心洗涤2次。将微球重悬于2ml PBS中,于Γ-计数器检测总放射性强度。之后将重悬的微球置于37℃恒温摇床中,每24 h全量更换溶出液。以Γ-计数器检测各时点微球中的剩余放射性强度,绘制释放曲线。昆明小鼠42只,随机分为14组,每组3只。取明胶微球冻干粉126 mg,平均分为42份,将21μCi~(125)Ⅰ-VEGF溶于420μl PBS中,平均滴加于每份明胶微球,4℃过夜。将每份微球重悬于100μl PBS中,注射于小鼠左后肢皮下组织中。每24 h处死一组小鼠,剪取左下肢,以Γ-计数器检测组织中的剩余放射性强度。以每组放射性强度的平均值绘制~(125)Ⅰ-VEGF释放曲线。
结果:溶胀后的明胶微球平均粒径为(202.0±44.7)μm。微球体外缓释性能测试实验中,明胶微球在前4天释放VEGF速度较快,在第96小时,已由微球释放于溶出液中的VEGF的累积量占微球中初始VEGF总含量的48.73%。之后释放速度渐减慢,于第144小时微球中~(125)Ⅰ-VEGF的剩余量为初始量的46.98%。此后微球中VEGF的剩余量达到平台期。微球体内缓释性能测试实验中,微球中~(125)Ⅰ-VEGF剩余量占初始总含量的百分比呈逐渐下降的趋势。在第96小时,小鼠后肢~(125)Ⅰ-VEGF的剩余量为51.9%,于第144小时,小鼠后肢~(125)Ⅰ-VEGF的剩余量为41.89%,在第288小时,~(125)Ⅰ-VEGF的剩余量为9.98%。
结论:明胶微球包载VEGF后能够实现VEGF的缓慢释放。血管内皮生长因子缓释微球对缺血性心脏病治疗作用的实验研究
目的:探讨血管内皮生长因子(vascular endothelial growth factor,VEGF)缓释明胶微球能否诱导缺血心肌血管新生。
方法:以乳化冷凝法制备VEGF缓释明胶微球,并以1,1'-双十八烷-3,3,3',3'-四甲基-吲哚-羧花青-高氯酸盐(DiI)荧光标记。中华小型猪18头,按照随机化表将动物分为VEGF缓释微球移植组、空载微球移植组及对照组。开胸结扎冠状动脉左前降支制备小型猪心梗模型。心肌梗死模型建立后14 d磁共振检查后行二次开胸,暴露左室前壁近心尖部,于VEGF缓释微球移植组动物梗死周边区心肌注射包载VEGF的缓释微球,每只动物注射3点,每点注射量3 mg(含VEGF 3μg);于空载微球组梗死周边区心肌注射等量未包裹VEGF的缓释微球,每只动物注射3个点。对照组注射含等量VEGF的磷酸盐缓冲液。微球移植后24 h(基线)及21 d(终点),电影MRI和增强MRI采集动物心功能参数并测量心梗面积。终点指标检测结束后处死动物进行组织学检测,免疫组化法检测微球移植局部缺血心肌的纤维化程度、毛细血管密度及微球降解情况。
结果小型猪左前降支结扎后第14天,延迟增强磁共振成像显示左室前壁、心尖部及室间隔均有延迟增强灶。在微球移植后24h及与移植后21d,各组间心肌梗死面积及心功能参数均无统计学差异。组织学结果提示在微球移植于缺血心肌3周后,VEGF缓释微球移植组微球移植点处心肌组织纤维化程度低于空载微球移植组及对照组。VEGF缓释微球移植组微球注射点处心肌毛细血管密度高于空载微球移植组及对照组(9.8±1.5/HPF vs 4.1±0.9 and 3.7±1.1/HPF,P<0.05)。明胶微球移植于缺血心肌3周后,组织间隙中可见明胶微球降解碎片。
结论血管内皮生长因子缓释明胶微球能够诱导缺血心肌区新生血管形成。细胞因子缓释微球加强骨髓间充质干细胞移植对心肌梗死疗效的研究
目的本研究通过观察血管内皮生长因子(vascular endothelial growth factor,VEGF)缓释明胶微球与自体骨髓间充质干细胞(mesenchymal stem cells,MSCs)联合移植于猪心肌梗死模型梗死周边区3周后MSCs的生存情况,结合磁共振成像(magneticresonance imaging,MRI),阐明改善局部微环境对自体MSCs移植于缺血心肌后转归的影响。
方法以乳化冷凝法制备VEGF缓释明胶微球。中华小型猪18头。抽取髂骨骨髓并制备自体MSCs,以注射用顺磁性氧化铁颗粒(superparamagnetic iron oxide particles,SPIO)、4',6-二脒基-2-苯基吲哚(4',6-Diamidino-2-phenylindole dihydrochloride,DAPI)标记细胞并进行标记率检测。按照随机化表将动物分为对照组、MSCs移植组(MSCs组)及MSCs-VEGF缓释微球联合移植组(MSCs-VEGF组)。开胸结扎冠状动脉左前降支制备小型猪心肌梗死模型。模型建立后第14天,磁共振延迟增强成像(delatedenhancement MRI,DE-MRI)检测梗死面积后行二次开胸,直视经心外膜于MSCs组动物心肌梗死周边区注射MSCs 3个点(3×10~7/点),于MSCs-VEGF缓释微球组每只动物注射3个点,每点注射MSCs 3×10~7及VEGF缓释明胶微球3mg(含人重组VEGF165 3μg),于对照组动物心梗周边区相应部位注射培养基DMEM。微球移植后24 h(基线)及21 d(终点),电影MRI和增强MRI采集动物心功能参数包括射血分数、左室舒张末期容积、左室收缩末期容积、左室前壁室壁增厚率,并测量心梗面积。终点指标检测结束后处死动物进行组织学检测,免疫组化法检测微球移植局部缺血心肌的纤维化程度、毛细血管密度及干细胞存活情况。
结果DAPI对MSCs的标记率达100%。心梗模型建立后第14天,小型猪心肌梗死面积为33.6%±8.9%。细胞移植后24 h,三组间心梗面积及心功能参数均无统计学差异。细胞移植3周后,MSCs组及MSCs-VEGF组左室前壁室壁增厚率高于对照组(-5.23±3.82和-1.52±4.81比-15.53±3.42,P<0.05),MSCs-VEGF组左室收缩末期容积小于对照组(31.20±3.56 vs 37.05±6.26,P<0.05)。MSCs-VEGF组细胞注射点心肌毛细血管密度高于MSCs组及对照组(18.18±6.6比10±3.58和12.81±4.26/HPF;P<0:05)。MSCs-VEGF缓释微球组MSCs注射点存活的移植干细胞数多于MSCs组(383.07±96.33/高倍视野比285.17±73.97/高倍视野,P<0.0001),而MSCs凋亡率则低于MSCs组5.24±5.44%vs 10.24±5.04%,P<0.05]。
结论VEGF缓释明胶微球可以提高移植于缺血心肌区3周MSCs的生存率。移植干细胞所生存的微环境是影响其转归的重要因素。磁共振在体示踪氧化铁纳米颗粒标记移植干细胞的量化分析及可靠性评价
目的通过观察移植于猪心肌梗死模型梗死周边区及正常心肌区顺磁性氧化铁颗粒标记的自体骨髓间充质干细胞(mesenchymal stem cells,MSCs)所形成低信号区的信号变化情况,结合组织学检查结果,评价MRI对干细胞移植于缺血心肌后数量演变的监测价值。
方法中华小型猪6头,抽取髂骨骨髓并制备自体骨髓间质干细胞,以注射用顺磁性氧化铁颗粒(superparamagnetic iron oxide particles,SPIO)和4',6-二脒基-2-苯基吲哚(4',6-Diamidino-2-phenylindole dihydrochloride,DAPI)标记细胞并进行标记率检测。开胸结扎冠状动脉左前降支制备小型猪心肌梗死模型。模型建立后14 d,行二次开胸,直视经心外膜于每只动物心肌梗死周边区和正常心肌区各注射标记MSCs 2个点,每点注射MSCs 3×10~7。同时分别于各区注射培养基2个点作为对照点。细胞移植后24 h及21 d,快速梯度回波序列(FGRE序列)T_2~*像检测干细胞移植点低信号区的面积及信号强度。T_2~*信号减低区范围的测量由FGRE面积法实现,其信号减低的程度以正常心肌区和该区的T_2~*值之差与正常心肌区T_2~*值的百分比表示。病理组织学检查心肌细胞形态、瘢痕形成、毛细血管密度及干细胞存活情况。不同时点MSCs注射点低信号区面积及T_2~*信号强度的比较采用重复测量方差分析及配对t检验。干细胞注射点与对照点毛细血管密度的比较、梗死周边区及正常心肌区MSCs注射点信号衰减幅度的比较采用成组资料t检验。
结果DAPI及SPIO对MSCs的标记率均达100%。细胞移植后24 h,SPIO标记MSCs注射点于MRI显示为边界清晰的卵圆形T_2~*低信号区,MSCs注射后24 h,梗死周边区与正常心肌区MSCs注射点T_2~*低信号区的信号强度[(67±5.48)%vs(61.92±7.76)%t=1.65 P=0.1158)]及面积[(0.56±0.24)cm~2 vs(0.52±0.25)cm~2,t=0.39,P=0.7044)]均无统计学差异。移植3周后,T_2~*低信号区与正常心肌区的对比程度均较前下降,梗死周边区降低至(40.12±5.93)%(t=9.53,P<0.0001);正常心肌区降低至(46.92±6.25)%(t=11.03,P<0.0001)。且梗死周边区MSCs注射点低信号区T_2~*减低的程度大于正常心肌区MSCs注射点且两组间信号强度减弱的幅度具有显著性差异(26.88±7.27 vs 15±4.51,F=20.08,P=0.0003),此时梗死周边区MSCs注射点T_2~*低信号区的信号对比度低于正常心肌区MSCs注射点(t=-2.48,P=0.0234)。同时,T_2~*低信号区的面积亦均较前降小,但低信号区面积减小的程度在梗死周边区及正常心肌区无显著性差异(F=1.15,P=0.2982)。正常心肌区MSCs注射点组织中荧光标记的细胞核分布密度高于梗死边缘区MSCs注射点(106±25/HPF vs143±31/HPF,t=-2.47,P=0.0293)。梗死边缘区MSCs注射点组织中毛细血管分布密度高于该区对照点(13.4±4.0/HPF vs 9.4±3.1/HPF,t=2.49,P=0.0229)。
结论MRI可在一定时间内实现对移植干细胞的示踪并反映移植MSCs在心肌局部的数量变化趋势。移植干细胞所生存的微环境是影响其生存时间的重要因素。氧化铁颗粒标记的间充质干细胞排除铁颗粒机制的初步探讨
目的本研究从细胞学实验的角度探讨在间充质干细胞分裂增殖过程中标记于干细胞胞浆中的超顺磁性氧化铁颗粒(superparamagnetic iron oxide,SPIO)标记率的演变,以明确干细胞分裂增殖对SPIO标记率变化的影响。
方法抽取猪髂骨骨髓,密度梯度离心后获取单个核细胞层,以差时贴壁的方法纯化培养骨髓间充质干细胞(MSCs)。第一代细胞达到80%融合时,以1:3比例接种于两组六孔板中。以含有10%胎牛血清的高糖DMEM培养。在细胞达60-70%融合时,换以含铁25 ng/L及多聚赖氨酸375 pg/L的培养基为细胞换液,并培养细胞48 h。之后,细胞达到80%融合。将另一六孔板中的细胞以1:3比例传代至新的3块六孔板中,其中两块板的六个孔底部放置灭菌盖玻片。其中一组细胞换以含15%胎牛血清的高糖DMEM培养,而另一组细胞换以含5%胎牛血清的低糖DMEM培养。每隔24小时取出相应组别的细胞爬片进行固定、染色、检测。其余细胞分别以两种不同的培养基继续培养传代。将制备好的细胞爬片进行普鲁士蓝染色并估测细胞氧化铁颗粒标记率。
结果:SPIO标记干细胞48小时后,细胞爬片普鲁士蓝染色可见所有细胞中均含有蓝染颗粒,位于细胞核周围。将SPIO标记的细胞以不同条件的培养基继续培养,两组细胞SPIO标记率均逐渐减低,细胞中铁颗粒的量逐渐减少。以含15%胎牛血清的高糖DMEM继续培养的细胞组,标记后72小时细胞达到80%融合,进行第一次传代,传代后24小时,SPIO标记率较前略有降低(98±4.39)%(P=0.0409),培养72小时后,第二次传代,传代后24小时,细胞SPIO标记率降低为(88.67±3.93)%,与第一次传代后相比有统计学差异(P=0.0004)。继续培养72小时后,第三次传代,传代后24小时可见SPIO标记率进一步降低至(44.5±7.74)%,与第二次传代后24小时相比有统计学差异(P<0.0001)。第四次传代后24小时,SPIO标记率为(6.17±2.64)%。以含5%FBS的低糖DMEM继续培养的细胞组,标记后96小时细胞达到80%融合,进行第一次传代,传代后24小时,SPIO标记率较前略有降低(97±0.86)%(P=0.0172),培养120小时后,第二次传代,传代后24小时,细胞SPIO标记率降低为(87±4.29)%,与第一次传代后相比有统计学差异(P=0.0004)。继续培养144小时后,第三次传代,传代后24小时可见SPIO标记率进一步降低至(46.7±7.06)%,与第二次传代后24小时相比有统计学差异(P<0.0001)。第四次传代后24小时,SPIO标记率为第四次传代后24小时,SPIO标记率为(5.17±3.06)%。在两组细胞,均在标记以后第四次传代后24小时,SPIO的标记率下降至10%以下,在高糖DMEM组历时9天,而在低糖DMEM组历时16天。在标记后第四次传代后24小时,两组细胞SPIO标记率无统计学差异[(5.17±3.06)%比(6.17±2.64)%,P=0.558]。
结论SPIO标记的间充质干细胞排除SPIO的机制主要为细胞的分裂增殖。细胞分裂增殖越快,细胞清除SPIO的速率也越快。
Part One
The study of the sustained release performance of gelatin microspheres containing vascular endothelial growth factor
Objective The present study was aimed to investigate whether gelatin microspheres containing VEGF could release VEGF persistently.
Methods Gelatin microspheres were prepared through glutaraldehyde crosslinking of gelatin in aqueous solution dispersed in an oil phase.The gelatin microspheres were sterilized by ~(60)Co radiation.The mean diameter of these microspheres was measured after swelling in phosphate buffer saline for 24h.The in vitro release test of ~(125)Ⅰ-VEGF from ~(125)Ⅰ-VEGF-impregnated gelatin microspheres was carred out on a shaker at 37℃. The VEGF-impregnated microspheres were placed in phosphate buffer solution and the buffer was changed periodically.The amount of ~(125)Ⅰ-VEGF was measured by a gamma counter.The release character of ~(125)Ⅰ-VEGF-incorporating gelatin microspheres was also assessed in vivo.Gelatin microspheres were incorporating ~(125)Ⅰ-labeled VEGF was subcutaneously injected into the mouse hind limb.As a control,the same amount of ~(125)Ⅰ-VEGF in aqueous solution was subcutaneously.The mice were killed at intervals, and the limbs with the microspheres injection sites were cut and measured on a gamma counter to evaluate the time profile of in vivo degradation of the gelatin microspheres.
Results The diameter of the gelatin microspheres was 202.2±44.7μm.The remaining radioactivity of microspheres decreased with time in vitro and in vivo.In vitro, ~(125)Ⅰ-VEGF was released from the gelatin microspheres within 96 hours up to 48.73%. The residual radioactivity of ~(125)Ⅰ-VEGF at 144 hours remained 46.98%.From then on, the remaining radioactivity of ~(125)Ⅰ-VEGF did nearly not decrease with time.The remaining radioactivity of ~(125)Ⅰ-VEGF in vivo decreased with time.At 96 hours,144 hours,and 288 hours after gelatin microspheres transplantation,the residual radioactivity of ~(125)Ⅰ-VEGF remained 51.9%,41.89,and 9.98%,respectively.
Conclusions Gelatin microspheres containing VEGF could release VEGF persistently.
Part Two
Gelatin microspheres containing vascular endothelial growth factor benefits to therapy of myocardial infarction
Objective The present study was aimed to investigate whether gelatin microspheres contained VEGF could benefit to myocardial infarction.
Methods Gelatin microspheres were prepared through glutaraldehyde crosslinking of gelatin in aqueous solution dispersed in an oil phase.The gelatin microspheres were lable by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarboc yanine perchlorate(DiI).The release character of VEGF-incorporating gelatin microspheres was assessed in vitro and in vivo. Eighteen Chinese mini swines were subjected to open-chest experimental myocardial infarction and were randomized into three groups.Each group received intramyocardial injection of phosphate buffered saline(control group),vacant gelatin microspheres(MS group),and gelatin microspheres incorporating VEGF(VEGF-MS group),respectively. Twenty-four hours and three weeks later,left ventricular function was assessed by MRI. After final MRI examination,animals were humanely killed and the hearts were removed for histologic study.
Results All the gelatin microspheres were labled by Dil and showed red fluorescence. Twenty-four hours and three weeks after transplantation,there was no statistically significant difference in infarct size,left ventricular ejection fraction,end-diastolic volume,end-systolic volume,and anterior left ventricular wall thickening among three groups.At 3 weeks,severe fibrosis was observed in gelatin microspheres injection sites of the control group and the MS group.In contrast,there was less fibrosis in microspheres injection sites with more surviving myocardium in the VEGF-MS group. Histologically,the micro vascular density of VEGF-MS group was higher than that of the MS group and the control group(9.8±1.5/HPF vs 4.1±0.9 and 3.7±1.1/HPF,P<0.05).
Conclusions VEGF incorporated gelatin microspheres can induce vascularization in ischemic myocardium.And sustained VEGF release by gelatin microspheres could benefit to myocardial infarction.
Part Three
Gelatin microspheres containing vascular endothelial growth factor enhances the benefits of bone marrow mesenchymal stem cells transplantation
Objective The present study was to investigate the efficacy of transplantation of mesenchymal stem cells(MSCs) with gelatin microspheres containing vascular endothelial growth factor in ischemic myocardium.
Methods Eighteen Chinese mini swines were subjected to open-chest experimental myocardial infarction and were randomized into three groups.Each group received intramyocardial injection of phosphate buffered saline(control group),autogenetic mesenchymal stem cells(MSCs group),and MSCs with gelatin microspheres incorporating VEGF(VEGF-MSCs group) in the pefi-infarction area of left ventricular wall,respectively.Three weeks later,left ventricular function was assessed by means of magnetic resonance imaging(MRI).The contrast of the MSCs hypointense lesion was determined using the difference in signal intensity between the hypointense and normal myocardium divided by signal intensity of the normal region.
Results The mean diameter of the microspheres is(104±22.6)μm.At 24 hours,injection sites of MSCs were identified by MRI as large intramyocardial signal voids that persisted at 3 weeks.Between two groups,there were no significant difference in the contrast of the lesions and in the size of the lesions at 24 hours.At 3 weeks after injection,the size of the lesions diminished(P<0.0001) and the contrast of the lesion decreased(P<0.0001). Histology(at 3 weeks) revealed the iron inclusion from Prussian Blue staining matches DAPI fluorescent dyes on adjacent histological sections at×400 magnification at 3 weeks after MSCs injection,indicating partial ferumoxide particles is still contained within original MSCs.In the MSCs-VEGF microsphere group,the capillary density of the injection site was significantly more than that in MSCs group(42.2±13.9/HPF vs 29.5±15.4/HPF,P<0.0001).There were much more dense fluorescently labled MSCs per high power fields in injection sites of MSCs-VEGF microsphere group than that in injection sites of MSCs group(354±83/HPF vs 278±97/HPF,P<0.0001).Moreover,the apoptosis rate of MSCs of MSCs-VEGF microsphere group is more than that of MSCs group[(6.4±4.1)%vs(11.9±4.8)%,P<0.0001].
Conclusion VEGF incorporated microspheres benefits to the survival of MSCs transplanted to ischemic myocardium.Microenvironment if one of the most important factors that influence the survival of transplanted MSCs
Part Four Dynamic MRI of ferumoxide-labeled bone mesenchymal stem cells after transplantation in myocardium
Objective To investigate the potential ability of magnetic resonance imaging(MRI) in tracking magnetically labeled mesenchymal stem cells(MR-MSCs) in a swine myocardial infarction(MI) model.
Methods Adult Chinese mini pigs(n=6) were subjected to open-chest experimental MI. Their autogeneic bone marrow-derived mesenchymal stem cells(MSCs) was cultured and doubly labled with ferumoxides and DAPI.At the 14~(th) day after MI,labeled MSCs were injected intramyocardially into peri-infarct zone and normal myocardium.The contrast and the volume of the MR-MSCs hypointense lesion from the FGRE images, acquired at 24 and 3 weeks after injection,was determined using the difference in signal intensity between the hypointense and normal myocardium divided by signal intensity of the normal region.After final MRI examination,animals were humanely killed and the hearts were removed for histologic study.The heart was excised and histology corresponding to MRI slices that demonstrated MR-MSCs lesions was performed. Repeated-measures ANOVA and a paired t test were used for comparison of the contrast and the volume of the MR-MSCs hypointense lesion at different time points. Comparisons between independent groups were performed with the standard Student t test.
Results Before tranlplantation,Prussian Blue staining of ferumoxides-labeled MSCs revealed 100%of MSCs were labled by ferumoxides particles.And all the MSCs were also labeled by DAPI.At the 14~(th) day after MSCs transplantation,DE-MRI showed the infarct in all animals.The contrast of the MR-MSCs hypointense lesion from the FGRE images,acquired at 24 hours and 3 weeks after injection,was determined using the difference in signal intensity between the hypointense and normal myocardium divided by signal intensity of the normal region.Images of MR-MSCs injection sites at 24 hours after injection appeared as ovoid hypoenhancing lesions with sharp borders.At 24h after injection,the contrast[(67±5.48)%vs(61.92±7.76)%t=1.65 P=0.1158) and the size [(0.56±0.24)cm~2 vs(0.52±0.25)cm~2,t=0.39,P=0.7044)]of the lesions showed no statistical difference between in peri-infarct zone and in normal myocardium.At 3 weeks after injection,the contrast of the lesions decreased and the size of the lesions diminished both in peri-infarct zone and in normal myocardium.Moreover,the contrast of the lesions in peri-infarct zone decreased rapidly than that in normal myocardium (26.88±7.27 vs 15±4.51,F=20.08,P=0.0003).Post mortem analysis show fluorescently labeled MSCs was demonstrated on histological sections and There were much more dense fluorescently labled MSCs per high power fields in injection sites of normal myocardium than that in injection sites of peri-infarct zone(106±25/HPF vs 143±31/HPF,t=-2.47,P=0.0293).At 3 weeks,severe fibrosis observed in peri-infarct zone in control sites.In MSCs injection sites of the peri-infarct zone,the capillary density was significantly more than that in control sites(13.4±4.0/HPF vs 9.4±3.1/HPF, t=2.49,P=0.0229).When cryopreserved sections with DAPI positive nuclears were stained by hematoxylin-eosine and Prussian Blue,blue ferumoxides particles were detected around partial nuclears of MSCs.
Conclusion Magnetic resonance imaging of MR-MSCs represents a method for noninvasively tracking the quantity and location of intramyocardial delivery in myocardium.The microenvironment is one of the most important facors for transplanted MSCs to survive.
Part Five
The study of mechanism of clearing ferumoxide particles by ferumoxide-labled mesenchymal stem cells
Objective The study tried to illustrate how the division and proliferation of mesenchymal stem cells(MSCs) labeled by superparamagnetic iron oxide(SPIO) influence the SPIO labeling rate.
Methods Swine bone marrow-derived mesenchymal stem cells were labeled with SPIO by incubation with ferumoxides injectable solution(25μg Fe/mL,Feridex) in culture medium for 48 hours with 375 ng/mL poly-L-lysine(PLL;average MW=275 kDa). Prussian blue staining revealed that all of the cells were efficiently labeled with SPIO nanoparticles.Then,the MSCs was divided into two groups.One group was cultured in high glucose Dulbecco's modified Eagle medium(DMEM) plus 15%fetal calf serum (FCS) at 37℃and 5%CO_2,and the other group was maintained with low glucose DMEM plus 5%FCS.The labeling efficiency was tested through Prussian blue staining at 24 hours after every passage until the forth passage after SPIO labeling.
Results Prussian Blue staining revealed the presence of numerous blue ferumoxide particles in the cytoplasm around cell nucleus of all the MSCs after incubation with Feridex and PLL for 48 hours.The SPIO labeling rate of MSCs decreased with time in both groups.The MSCs maintained in high glucose DMEM plus 15%FCS reached 80% confluent at 72 hours after the former passage.At 24 hours after passage,the SPIO labeling rate was(98±4.39)%.MSCs maintained in high glucose DMEM experienced a passage every 72 hours.At 24 hours after each passage,the SPIO labeling rate decreased to(88.67±3.93)%,(44.5±7.74)%,and(6.17±2.64)%,respectively.However,the MSCs maintained in low glucose DMEM plus 5%FCS reached 80%confluent at 96 hours after the former passage.At 24 hours after the former passage,the SPIO labeling rate was (97±0.86)%.Then,MSCs maintained in low glucose DMEM experienced a passage every 120h or 144h.At 24 hours after each passage,the SPIO labeling rate decreased to (87±4.29)%,(46.7±7.06)%.At 24 hours after the forth passage after SPIO labeling,the SPIO labeling rate was(5.17±3.06)%,which was not statistically different from the labeling rate at the 24h after the forth passage after SPIO labeling in the MSCs maintained in high glucose DMEM[(5.17±3.06)%比(6.17±2.64)%,P=0.558].
Conclusion Division and proliferation are the main mechanism of clearing SPIO by MSCs.The faster the MSCs divided and proliferated,the faster the SPIO was cleared.
血管内皮生长因子缓释明胶微球制备及性能评价
目的:制备血管内皮生长因子明胶微球并评价其缓释性能。方法:以乳化冷凝法制备VEGF缓释明胶微球。20%的明胶溶液滴加于橄榄油中搅拌至形成乳浊液,丙酮固化,异丙醇悬浮后以不同孔径筛网筛分。离心干燥后,戊二醛交联,清洗后冷冻干燥,得淡黄色明胶微球。钴60照射灭菌。将微球浸泡于磷酸盐缓冲液中24小时,微球充分溶胀后,以显微镜自带软件测量微球粒径。取灭菌冻干明胶微球9 mg,平均分成3组,将1.5μCi~(125)Ⅰ-VEGF溶于30μl PBS中,平均滴加于3组明胶微球,4℃过夜。PBS离心洗涤2次。将微球重悬于2ml PBS中,于Γ-计数器检测总放射性强度。之后将重悬的微球置于37℃恒温摇床中,每24 h全量更换溶出液。以Γ-计数器检测各时点微球中的剩余放射性强度,绘制释放曲线。昆明小鼠42只,随机分为14组,每组3只。取明胶微球冻干粉126 mg,平均分为42份,将21μCi~(125)Ⅰ-VEGF溶于420μl PBS中,平均滴加于每份明胶微球,4℃过夜。将每份微球重悬于100μl PBS中,注射于小鼠左后肢皮下组织中。每24 h处死一组小鼠,剪取左下肢,以Γ-计数器检测组织中的剩余放射性强度。以每组放射性强度的平均值绘制~(125)Ⅰ-VEGF释放曲线。
结果:溶胀后的明胶微球平均粒径为(202.0±44.7)μm。微球体外缓释性能测试实验中,明胶微球在前4天释放VEGF速度较快,在第96小时,已由微球释放于溶出液中的VEGF的累积量占微球中初始VEGF总含量的48.73%。之后释放速度渐减慢,于第144小时微球中~(125)Ⅰ-VEGF的剩余量为初始量的46.98%。此后微球中VEGF的剩余量达到平台期。微球体内缓释性能测试实验中,微球中~(125)Ⅰ-VEGF剩余量占初始总含量的百分比呈逐渐下降的趋势。在第96小时,小鼠后肢~(125)Ⅰ-VEGF的剩余量为51.9%,于第144小时,小鼠后肢~(125)Ⅰ-VEGF的剩余量为41.89%,在第288小时,~(125)Ⅰ-VEGF的剩余量为9.98%。
结论:明胶微球包载VEGF后能够实现VEGF的缓慢释放。血管内皮生长因子缓释微球对缺血性心脏病治疗作用的实验研究
目的:探讨血管内皮生长因子(vascular endothelial growth factor,VEGF)缓释明胶微球能否诱导缺血心肌血管新生。
方法:以乳化冷凝法制备VEGF缓释明胶微球,并以1,1'-双十八烷-3,3,3',3'-四甲基-吲哚-羧花青-高氯酸盐(DiI)荧光标记。中华小型猪18头,按照随机化表将动物分为VEGF缓释微球移植组、空载微球移植组及对照组。开胸结扎冠状动脉左前降支制备小型猪心梗模型。心肌梗死模型建立后14 d磁共振检查后行二次开胸,暴露左室前壁近心尖部,于VEGF缓释微球移植组动物梗死周边区心肌注射包载VEGF的缓释微球,每只动物注射3点,每点注射量3 mg(含VEGF 3μg);于空载微球组梗死周边区心肌注射等量未包裹VEGF的缓释微球,每只动物注射3个点。对照组注射含等量VEGF的磷酸盐缓冲液。微球移植后24 h(基线)及21 d(终点),电影MRI和增强MRI采集动物心功能参数并测量心梗面积。终点指标检测结束后处死动物进行组织学检测,免疫组化法检测微球移植局部缺血心肌的纤维化程度、毛细血管密度及微球降解情况。
结果小型猪左前降支结扎后第14天,延迟增强磁共振成像显示左室前壁、心尖部及室间隔均有延迟增强灶。在微球移植后24h及与移植后21d,各组间心肌梗死面积及心功能参数均无统计学差异。组织学结果提示在微球移植于缺血心肌3周后,VEGF缓释微球移植组微球移植点处心肌组织纤维化程度低于空载微球移植组及对照组。VEGF缓释微球移植组微球注射点处心肌毛细血管密度高于空载微球移植组及对照组(9.8±1.5/HPF vs 4.1±0.9 and 3.7±1.1/HPF,P<0.05)。明胶微球移植于缺血心肌3周后,组织间隙中可见明胶微球降解碎片。
结论血管内皮生长因子缓释明胶微球能够诱导缺血心肌区新生血管形成。细胞因子缓释微球加强骨髓间充质干细胞移植对心肌梗死疗效的研究
目的本研究通过观察血管内皮生长因子(vascular endothelial growth factor,VEGF)缓释明胶微球与自体骨髓间充质干细胞(mesenchymal stem cells,MSCs)联合移植于猪心肌梗死模型梗死周边区3周后MSCs的生存情况,结合磁共振成像(magneticresonance imaging,MRI),阐明改善局部微环境对自体MSCs移植于缺血心肌后转归的影响。
方法以乳化冷凝法制备VEGF缓释明胶微球。中华小型猪18头。抽取髂骨骨髓并制备自体MSCs,以注射用顺磁性氧化铁颗粒(superparamagnetic iron oxide particles,SPIO)、4',6-二脒基-2-苯基吲哚(4',6-Diamidino-2-phenylindole dihydrochloride,DAPI)标记细胞并进行标记率检测。按照随机化表将动物分为对照组、MSCs移植组(MSCs组)及MSCs-VEGF缓释微球联合移植组(MSCs-VEGF组)。开胸结扎冠状动脉左前降支制备小型猪心肌梗死模型。模型建立后第14天,磁共振延迟增强成像(delatedenhancement MRI,DE-MRI)检测梗死面积后行二次开胸,直视经心外膜于MSCs组动物心肌梗死周边区注射MSCs 3个点(3×10~7/点),于MSCs-VEGF缓释微球组每只动物注射3个点,每点注射MSCs 3×10~7及VEGF缓释明胶微球3mg(含人重组VEGF165 3μg),于对照组动物心梗周边区相应部位注射培养基DMEM。微球移植后24 h(基线)及21 d(终点),电影MRI和增强MRI采集动物心功能参数包括射血分数、左室舒张末期容积、左室收缩末期容积、左室前壁室壁增厚率,并测量心梗面积。终点指标检测结束后处死动物进行组织学检测,免疫组化法检测微球移植局部缺血心肌的纤维化程度、毛细血管密度及干细胞存活情况。
结果DAPI对MSCs的标记率达100%。心梗模型建立后第14天,小型猪心肌梗死面积为33.6%±8.9%。细胞移植后24 h,三组间心梗面积及心功能参数均无统计学差异。细胞移植3周后,MSCs组及MSCs-VEGF组左室前壁室壁增厚率高于对照组(-5.23±3.82和-1.52±4.81比-15.53±3.42,P<0.05),MSCs-VEGF组左室收缩末期容积小于对照组(31.20±3.56 vs 37.05±6.26,P<0.05)。MSCs-VEGF组细胞注射点心肌毛细血管密度高于MSCs组及对照组(18.18±6.6比10±3.58和12.81±4.26/HPF;P<0:05)。MSCs-VEGF缓释微球组MSCs注射点存活的移植干细胞数多于MSCs组(383.07±96.33/高倍视野比285.17±73.97/高倍视野,P<0.0001),而MSCs凋亡率则低于MSCs组5.24±5.44%vs 10.24±5.04%,P<0.05]。
结论VEGF缓释明胶微球可以提高移植于缺血心肌区3周MSCs的生存率。移植干细胞所生存的微环境是影响其转归的重要因素。磁共振在体示踪氧化铁纳米颗粒标记移植干细胞的量化分析及可靠性评价
目的通过观察移植于猪心肌梗死模型梗死周边区及正常心肌区顺磁性氧化铁颗粒标记的自体骨髓间充质干细胞(mesenchymal stem cells,MSCs)所形成低信号区的信号变化情况,结合组织学检查结果,评价MRI对干细胞移植于缺血心肌后数量演变的监测价值。
方法中华小型猪6头,抽取髂骨骨髓并制备自体骨髓间质干细胞,以注射用顺磁性氧化铁颗粒(superparamagnetic iron oxide particles,SPIO)和4',6-二脒基-2-苯基吲哚(4',6-Diamidino-2-phenylindole dihydrochloride,DAPI)标记细胞并进行标记率检测。开胸结扎冠状动脉左前降支制备小型猪心肌梗死模型。模型建立后14 d,行二次开胸,直视经心外膜于每只动物心肌梗死周边区和正常心肌区各注射标记MSCs 2个点,每点注射MSCs 3×10~7。同时分别于各区注射培养基2个点作为对照点。细胞移植后24 h及21 d,快速梯度回波序列(FGRE序列)T_2~*像检测干细胞移植点低信号区的面积及信号强度。T_2~*信号减低区范围的测量由FGRE面积法实现,其信号减低的程度以正常心肌区和该区的T_2~*值之差与正常心肌区T_2~*值的百分比表示。病理组织学检查心肌细胞形态、瘢痕形成、毛细血管密度及干细胞存活情况。不同时点MSCs注射点低信号区面积及T_2~*信号强度的比较采用重复测量方差分析及配对t检验。干细胞注射点与对照点毛细血管密度的比较、梗死周边区及正常心肌区MSCs注射点信号衰减幅度的比较采用成组资料t检验。
结果DAPI及SPIO对MSCs的标记率均达100%。细胞移植后24 h,SPIO标记MSCs注射点于MRI显示为边界清晰的卵圆形T_2~*低信号区,MSCs注射后24 h,梗死周边区与正常心肌区MSCs注射点T_2~*低信号区的信号强度[(67±5.48)%vs(61.92±7.76)%t=1.65 P=0.1158)]及面积[(0.56±0.24)cm~2 vs(0.52±0.25)cm~2,t=0.39,P=0.7044)]均无统计学差异。移植3周后,T_2~*低信号区与正常心肌区的对比程度均较前下降,梗死周边区降低至(40.12±5.93)%(t=9.53,P<0.0001);正常心肌区降低至(46.92±6.25)%(t=11.03,P<0.0001)。且梗死周边区MSCs注射点低信号区T_2~*减低的程度大于正常心肌区MSCs注射点且两组间信号强度减弱的幅度具有显著性差异(26.88±7.27 vs 15±4.51,F=20.08,P=0.0003),此时梗死周边区MSCs注射点T_2~*低信号区的信号对比度低于正常心肌区MSCs注射点(t=-2.48,P=0.0234)。同时,T_2~*低信号区的面积亦均较前降小,但低信号区面积减小的程度在梗死周边区及正常心肌区无显著性差异(F=1.15,P=0.2982)。正常心肌区MSCs注射点组织中荧光标记的细胞核分布密度高于梗死边缘区MSCs注射点(106±25/HPF vs143±31/HPF,t=-2.47,P=0.0293)。梗死边缘区MSCs注射点组织中毛细血管分布密度高于该区对照点(13.4±4.0/HPF vs 9.4±3.1/HPF,t=2.49,P=0.0229)。
结论MRI可在一定时间内实现对移植干细胞的示踪并反映移植MSCs在心肌局部的数量变化趋势。移植干细胞所生存的微环境是影响其生存时间的重要因素。氧化铁颗粒标记的间充质干细胞排除铁颗粒机制的初步探讨
目的本研究从细胞学实验的角度探讨在间充质干细胞分裂增殖过程中标记于干细胞胞浆中的超顺磁性氧化铁颗粒(superparamagnetic iron oxide,SPIO)标记率的演变,以明确干细胞分裂增殖对SPIO标记率变化的影响。
方法抽取猪髂骨骨髓,密度梯度离心后获取单个核细胞层,以差时贴壁的方法纯化培养骨髓间充质干细胞(MSCs)。第一代细胞达到80%融合时,以1:3比例接种于两组六孔板中。以含有10%胎牛血清的高糖DMEM培养。在细胞达60-70%融合时,换以含铁25 ng/L及多聚赖氨酸375 pg/L的培养基为细胞换液,并培养细胞48 h。之后,细胞达到80%融合。将另一六孔板中的细胞以1:3比例传代至新的3块六孔板中,其中两块板的六个孔底部放置灭菌盖玻片。其中一组细胞换以含15%胎牛血清的高糖DMEM培养,而另一组细胞换以含5%胎牛血清的低糖DMEM培养。每隔24小时取出相应组别的细胞爬片进行固定、染色、检测。其余细胞分别以两种不同的培养基继续培养传代。将制备好的细胞爬片进行普鲁士蓝染色并估测细胞氧化铁颗粒标记率。
结果:SPIO标记干细胞48小时后,细胞爬片普鲁士蓝染色可见所有细胞中均含有蓝染颗粒,位于细胞核周围。将SPIO标记的细胞以不同条件的培养基继续培养,两组细胞SPIO标记率均逐渐减低,细胞中铁颗粒的量逐渐减少。以含15%胎牛血清的高糖DMEM继续培养的细胞组,标记后72小时细胞达到80%融合,进行第一次传代,传代后24小时,SPIO标记率较前略有降低(98±4.39)%(P=0.0409),培养72小时后,第二次传代,传代后24小时,细胞SPIO标记率降低为(88.67±3.93)%,与第一次传代后相比有统计学差异(P=0.0004)。继续培养72小时后,第三次传代,传代后24小时可见SPIO标记率进一步降低至(44.5±7.74)%,与第二次传代后24小时相比有统计学差异(P<0.0001)。第四次传代后24小时,SPIO标记率为(6.17±2.64)%。以含5%FBS的低糖DMEM继续培养的细胞组,标记后96小时细胞达到80%融合,进行第一次传代,传代后24小时,SPIO标记率较前略有降低(97±0.86)%(P=0.0172),培养120小时后,第二次传代,传代后24小时,细胞SPIO标记率降低为(87±4.29)%,与第一次传代后相比有统计学差异(P=0.0004)。继续培养144小时后,第三次传代,传代后24小时可见SPIO标记率进一步降低至(46.7±7.06)%,与第二次传代后24小时相比有统计学差异(P<0.0001)。第四次传代后24小时,SPIO标记率为第四次传代后24小时,SPIO标记率为(5.17±3.06)%。在两组细胞,均在标记以后第四次传代后24小时,SPIO的标记率下降至10%以下,在高糖DMEM组历时9天,而在低糖DMEM组历时16天。在标记后第四次传代后24小时,两组细胞SPIO标记率无统计学差异[(5.17±3.06)%比(6.17±2.64)%,P=0.558]。
结论SPIO标记的间充质干细胞排除SPIO的机制主要为细胞的分裂增殖。细胞分裂增殖越快,细胞清除SPIO的速率也越快。
Part One
The study of the sustained release performance of gelatin microspheres containing vascular endothelial growth factor
Objective The present study was aimed to investigate whether gelatin microspheres containing VEGF could release VEGF persistently.
Methods Gelatin microspheres were prepared through glutaraldehyde crosslinking of gelatin in aqueous solution dispersed in an oil phase.The gelatin microspheres were sterilized by ~(60)Co radiation.The mean diameter of these microspheres was measured after swelling in phosphate buffer saline for 24h.The in vitro release test of ~(125)Ⅰ-VEGF from ~(125)Ⅰ-VEGF-impregnated gelatin microspheres was carred out on a shaker at 37℃. The VEGF-impregnated microspheres were placed in phosphate buffer solution and the buffer was changed periodically.The amount of ~(125)Ⅰ-VEGF was measured by a gamma counter.The release character of ~(125)Ⅰ-VEGF-incorporating gelatin microspheres was also assessed in vivo.Gelatin microspheres were incorporating ~(125)Ⅰ-labeled VEGF was subcutaneously injected into the mouse hind limb.As a control,the same amount of ~(125)Ⅰ-VEGF in aqueous solution was subcutaneously.The mice were killed at intervals, and the limbs with the microspheres injection sites were cut and measured on a gamma counter to evaluate the time profile of in vivo degradation of the gelatin microspheres.
Results The diameter of the gelatin microspheres was 202.2±44.7μm.The remaining radioactivity of microspheres decreased with time in vitro and in vivo.In vitro, ~(125)Ⅰ-VEGF was released from the gelatin microspheres within 96 hours up to 48.73%. The residual radioactivity of ~(125)Ⅰ-VEGF at 144 hours remained 46.98%.From then on, the remaining radioactivity of ~(125)Ⅰ-VEGF did nearly not decrease with time.The remaining radioactivity of ~(125)Ⅰ-VEGF in vivo decreased with time.At 96 hours,144 hours,and 288 hours after gelatin microspheres transplantation,the residual radioactivity of ~(125)Ⅰ-VEGF remained 51.9%,41.89,and 9.98%,respectively.
Conclusions Gelatin microspheres containing VEGF could release VEGF persistently.
Part Two
Gelatin microspheres containing vascular endothelial growth factor benefits to therapy of myocardial infarction
Objective The present study was aimed to investigate whether gelatin microspheres contained VEGF could benefit to myocardial infarction.
Methods Gelatin microspheres were prepared through glutaraldehyde crosslinking of gelatin in aqueous solution dispersed in an oil phase.The gelatin microspheres were lable by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarboc yanine perchlorate(DiI).The release character of VEGF-incorporating gelatin microspheres was assessed in vitro and in vivo. Eighteen Chinese mini swines were subjected to open-chest experimental myocardial infarction and were randomized into three groups.Each group received intramyocardial injection of phosphate buffered saline(control group),vacant gelatin microspheres(MS group),and gelatin microspheres incorporating VEGF(VEGF-MS group),respectively. Twenty-four hours and three weeks later,left ventricular function was assessed by MRI. After final MRI examination,animals were humanely killed and the hearts were removed for histologic study.
Results All the gelatin microspheres were labled by Dil and showed red fluorescence. Twenty-four hours and three weeks after transplantation,there was no statistically significant difference in infarct size,left ventricular ejection fraction,end-diastolic volume,end-systolic volume,and anterior left ventricular wall thickening among three groups.At 3 weeks,severe fibrosis was observed in gelatin microspheres injection sites of the control group and the MS group.In contrast,there was less fibrosis in microspheres injection sites with more surviving myocardium in the VEGF-MS group. Histologically,the micro vascular density of VEGF-MS group was higher than that of the MS group and the control group(9.8±1.5/HPF vs 4.1±0.9 and 3.7±1.1/HPF,P<0.05).
Conclusions VEGF incorporated gelatin microspheres can induce vascularization in ischemic myocardium.And sustained VEGF release by gelatin microspheres could benefit to myocardial infarction.
Part Three
Gelatin microspheres containing vascular endothelial growth factor enhances the benefits of bone marrow mesenchymal stem cells transplantation
Objective The present study was to investigate the efficacy of transplantation of mesenchymal stem cells(MSCs) with gelatin microspheres containing vascular endothelial growth factor in ischemic myocardium.
Methods Eighteen Chinese mini swines were subjected to open-chest experimental myocardial infarction and were randomized into three groups.Each group received intramyocardial injection of phosphate buffered saline(control group),autogenetic mesenchymal stem cells(MSCs group),and MSCs with gelatin microspheres incorporating VEGF(VEGF-MSCs group) in the pefi-infarction area of left ventricular wall,respectively.Three weeks later,left ventricular function was assessed by means of magnetic resonance imaging(MRI).The contrast of the MSCs hypointense lesion was determined using the difference in signal intensity between the hypointense and normal myocardium divided by signal intensity of the normal region.
Results The mean diameter of the microspheres is(104±22.6)μm.At 24 hours,injection sites of MSCs were identified by MRI as large intramyocardial signal voids that persisted at 3 weeks.Between two groups,there were no significant difference in the contrast of the lesions and in the size of the lesions at 24 hours.At 3 weeks after injection,the size of the lesions diminished(P<0.0001) and the contrast of the lesion decreased(P<0.0001). Histology(at 3 weeks) revealed the iron inclusion from Prussian Blue staining matches DAPI fluorescent dyes on adjacent histological sections at×400 magnification at 3 weeks after MSCs injection,indicating partial ferumoxide particles is still contained within original MSCs.In the MSCs-VEGF microsphere group,the capillary density of the injection site was significantly more than that in MSCs group(42.2±13.9/HPF vs 29.5±15.4/HPF,P<0.0001).There were much more dense fluorescently labled MSCs per high power fields in injection sites of MSCs-VEGF microsphere group than that in injection sites of MSCs group(354±83/HPF vs 278±97/HPF,P<0.0001).Moreover,the apoptosis rate of MSCs of MSCs-VEGF microsphere group is more than that of MSCs group[(6.4±4.1)%vs(11.9±4.8)%,P<0.0001].
Conclusion VEGF incorporated microspheres benefits to the survival of MSCs transplanted to ischemic myocardium.Microenvironment if one of the most important factors that influence the survival of transplanted MSCs
Part Four Dynamic MRI of ferumoxide-labeled bone mesenchymal stem cells after transplantation in myocardium
Objective To investigate the potential ability of magnetic resonance imaging(MRI) in tracking magnetically labeled mesenchymal stem cells(MR-MSCs) in a swine myocardial infarction(MI) model.
Methods Adult Chinese mini pigs(n=6) were subjected to open-chest experimental MI. Their autogeneic bone marrow-derived mesenchymal stem cells(MSCs) was cultured and doubly labled with ferumoxides and DAPI.At the 14~(th) day after MI,labeled MSCs were injected intramyocardially into peri-infarct zone and normal myocardium.The contrast and the volume of the MR-MSCs hypointense lesion from the FGRE images, acquired at 24 and 3 weeks after injection,was determined using the difference in signal intensity between the hypointense and normal myocardium divided by signal intensity of the normal region.After final MRI examination,animals were humanely killed and the hearts were removed for histologic study.The heart was excised and histology corresponding to MRI slices that demonstrated MR-MSCs lesions was performed. Repeated-measures ANOVA and a paired t test were used for comparison of the contrast and the volume of the MR-MSCs hypointense lesion at different time points. Comparisons between independent groups were performed with the standard Student t test.
Results Before tranlplantation,Prussian Blue staining of ferumoxides-labeled MSCs revealed 100%of MSCs were labled by ferumoxides particles.And all the MSCs were also labeled by DAPI.At the 14~(th) day after MSCs transplantation,DE-MRI showed the infarct in all animals.The contrast of the MR-MSCs hypointense lesion from the FGRE images,acquired at 24 hours and 3 weeks after injection,was determined using the difference in signal intensity between the hypointense and normal myocardium divided by signal intensity of the normal region.Images of MR-MSCs injection sites at 24 hours after injection appeared as ovoid hypoenhancing lesions with sharp borders.At 24h after injection,the contrast[(67±5.48)%vs(61.92±7.76)%t=1.65 P=0.1158) and the size [(0.56±0.24)cm~2 vs(0.52±0.25)cm~2,t=0.39,P=0.7044)]of the lesions showed no statistical difference between in peri-infarct zone and in normal myocardium.At 3 weeks after injection,the contrast of the lesions decreased and the size of the lesions diminished both in peri-infarct zone and in normal myocardium.Moreover,the contrast of the lesions in peri-infarct zone decreased rapidly than that in normal myocardium (26.88±7.27 vs 15±4.51,F=20.08,P=0.0003).Post mortem analysis show fluorescently labeled MSCs was demonstrated on histological sections and There were much more dense fluorescently labled MSCs per high power fields in injection sites of normal myocardium than that in injection sites of peri-infarct zone(106±25/HPF vs 143±31/HPF,t=-2.47,P=0.0293).At 3 weeks,severe fibrosis observed in peri-infarct zone in control sites.In MSCs injection sites of the peri-infarct zone,the capillary density was significantly more than that in control sites(13.4±4.0/HPF vs 9.4±3.1/HPF, t=2.49,P=0.0229).When cryopreserved sections with DAPI positive nuclears were stained by hematoxylin-eosine and Prussian Blue,blue ferumoxides particles were detected around partial nuclears of MSCs.
Conclusion Magnetic resonance imaging of MR-MSCs represents a method for noninvasively tracking the quantity and location of intramyocardial delivery in myocardium.The microenvironment is one of the most important facors for transplanted MSCs to survive.
Part Five
The study of mechanism of clearing ferumoxide particles by ferumoxide-labled mesenchymal stem cells
Objective The study tried to illustrate how the division and proliferation of mesenchymal stem cells(MSCs) labeled by superparamagnetic iron oxide(SPIO) influence the SPIO labeling rate.
Methods Swine bone marrow-derived mesenchymal stem cells were labeled with SPIO by incubation with ferumoxides injectable solution(25μg Fe/mL,Feridex) in culture medium for 48 hours with 375 ng/mL poly-L-lysine(PLL;average MW=275 kDa). Prussian blue staining revealed that all of the cells were efficiently labeled with SPIO nanoparticles.Then,the MSCs was divided into two groups.One group was cultured in high glucose Dulbecco's modified Eagle medium(DMEM) plus 15%fetal calf serum (FCS) at 37℃and 5%CO_2,and the other group was maintained with low glucose DMEM plus 5%FCS.The labeling efficiency was tested through Prussian blue staining at 24 hours after every passage until the forth passage after SPIO labeling.
Results Prussian Blue staining revealed the presence of numerous blue ferumoxide particles in the cytoplasm around cell nucleus of all the MSCs after incubation with Feridex and PLL for 48 hours.The SPIO labeling rate of MSCs decreased with time in both groups.The MSCs maintained in high glucose DMEM plus 15%FCS reached 80% confluent at 72 hours after the former passage.At 24 hours after passage,the SPIO labeling rate was(98±4.39)%.MSCs maintained in high glucose DMEM experienced a passage every 72 hours.At 24 hours after each passage,the SPIO labeling rate decreased to(88.67±3.93)%,(44.5±7.74)%,and(6.17±2.64)%,respectively.However,the MSCs maintained in low glucose DMEM plus 5%FCS reached 80%confluent at 96 hours after the former passage.At 24 hours after the former passage,the SPIO labeling rate was (97±0.86)%.Then,MSCs maintained in low glucose DMEM experienced a passage every 120h or 144h.At 24 hours after each passage,the SPIO labeling rate decreased to (87±4.29)%,(46.7±7.06)%.At 24 hours after the forth passage after SPIO labeling,the SPIO labeling rate was(5.17±3.06)%,which was not statistically different from the labeling rate at the 24h after the forth passage after SPIO labeling in the MSCs maintained in high glucose DMEM[(5.17±3.06)%比(6.17±2.64)%,P=0.558].
Conclusion Division and proliferation are the main mechanism of clearing SPIO by MSCs.The faster the MSCs divided and proliferated,the faster the SPIO was cleared.
引文
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