HIV-Tat-CLIO标记对大鼠骨髓间充质干细胞生物学行为的影响及MRI示踪效果研究
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摘要
第一部分HIV-Tat-CLIO标记对大鼠骨髓间充质干细胞生物学行为的影响
目的：
探讨HIV-Tat蛋白转导域交联氧化铁复合物(HIV transduction domaincross-linked iron oxide,HIV-Tat-CLIO)标记对体外培养的大鼠骨髓间充质干细胞(rat marrow-derived mesenchymal stem cells,rMSCs)的可行性、剂量关系以及对细胞活力、增殖、分化和迁移能力等生物学行为的影响。
材料与方法：
原代培养rMSCs至第3代，检测CD34，CD45和CD90的表达以鉴定之。不同浓度的HIV-Tat-CLIO标记rMSCs后利用普鲁士铁染色、体外MRI显像及台盼兰细胞活性测定等方法确定最佳标记条件。用CLIO 25μg／ml和HIV-Tat-CLIO 25μg／ml分别标记rMSCs各24h，通过普鲁士兰铁染色比较两组纳米铁的细胞标记效率。运用普鲁士兰染色及扫描电镜技术观察铁颗粒的细胞内存在部位。MTT实验测定该标记方法对细胞增殖能力的影响；流式细胞仪测定标记前后细胞表面标记物的表达及细胞周期的变化；通过油红0和碱性磷酸酶染色测定这种标记方法对rMSCs细胞分化能力的影响；Transwell实验测定其对干细胞迁移能力的变化。
结果：
培养的第3代rMSCs CD34(-)，CD45(-)和CD90(+)。HIV-Tat-CLIO最佳标记浓度为25μg／ml。HIV-Tat-CLIO组较CLIO组在rMSCs的标记效率上获得明显改善。细胞标记后，A570波长的光吸收值无明显变化；细胞表面CD34、CD45和CD90分子的表达无明显差异；处于S期的MSCs数量(S％)、G_1％、增殖指数PrI值(S+G_2M)％以及细胞凋亡情况均无显著改变；这种标记物对细胞分化和运动迁移能力均无显著影响。
结论：
HIV-Tat-CLIO标记大鼠MSCs后对其生物学行为无明显影响，标记后的干细胞可以正常增殖、分化及运动迁移，为磁共振活体监测干细胞移植后的动态变化提供有力的保证。
第二部分干细胞移植治疗大鼠后肢缺血中HIV-Tat-CLIO标记磁共振体内示踪的研究
目的：
探讨磁共振成像(Magnetic Resonance Imaging,MRI)无创体内示踪HIV-Tat-CLIO(HIV transduction domain cross-linked iron oxide)标记的移植大鼠骨髓间充质干细胞(rat Marrow-derived Mesenchymal Stem cells,rMSCs)的可行性和有效性，并且对该标记后的rMSCs移植治疗大鼠后肢缺血的效果进行客观评价。
材料和方法：
原代培养6周龄雄性SD大鼠的rMSCs至第3代，检测CD34，CD45和CD90的表达以鉴定。用HIV-Tat-CLIO 25μg／ml标记rMSCs 24h。另有一组细胞继续以5-溴脱氧尿苷(BrdU)3μg／ml复合标记rMSCs 24h。于大鼠右后肢缺血模型建立后的第7天进行rMSCs细胞移植。选用6周龄雄性SD大鼠28只，分为4组，每组各7只。A组为移植HIV-Tat-CLIO标记的rMSCs；B组为移植HIV-Tat-CLIO及BrdU复合标记的rMSCs；C组为移植未标记的rMSCs；D组为注射同移植体积的无菌PBS液。分别在移植术后3d、1周、2周、4周进行MRI检查示踪HIV-Tat-CLIO标记的rMSCs分布情况(C组和D组仅于术后第3d进行MRI扫描1次)。在第4周各组均采用非致冷红外热成像仪检测大鼠后肢皮肤温度，进行缺血后肢的损伤评分，并进行DSA造影，之后处死所有大鼠进行组织学检查。进行HE染色和普鲁士兰铁染色，BrdU、Ⅷ因子免疫组化染色，并计算毛细血管密度。
结果
培养的第3代rMSCs CD34(-)，CD45(-)和CD90(+)。B组和C组各有1只实验鼠因切口感染坏死，分别于术后第5天和第8天死亡，B组另有1只于移植术后第2周MRI检查时麻醉意外死亡。其余25只均存活至术后第4周。MRI检查：A组和B组在MRI上的显示接近。C组和D组在MRI上无特殊信号变化。A组和B组在术后第3d，MRI显示注射部位圆形低强度信号影；术后1周，MRI显示原低信号灶呈中心向周围扩散趋势，信号呈颗粒状，部位与随后的组织学检查结果一致；术后第2周见低密度影进一步扩散，信号强度有所弱化；术后第4周信号强度进一步弱化。低密度颗粒扩散范围局限于原部位7mm处。非致冷红外热成像仪检测大鼠后肢皮肤温度发现：A、B、C和D组的右后肢平均皮肤温度分别为32.12±0.51℃、32.39±0.61℃，31.97±0.56℃、31.16±0.67℃。A、B、C这3组间平均皮肤温度无显著性差异(P＞0.05)，A组与D组，B组与D组，C组与D组间均有显著性差异(P＜0.05)。缺血后肢的损伤评分：A、B、C和D组的缺血后肢损伤评分分别为0.68±0.17、0.65±0.24、0.67±0.11、1.79±0.22。A、B、C这3组间缺血后肢损伤评分无显著性差异(P＞0.05)，A组与D组，B组与D组，C组与D组间均有显著性差异(P＜0.05)。DSA造影：A、B、C和D组的造影显示大鼠缺血侧后肢大腿部从股骨大转子至股骨下段区域内的血管评分平均各为：2.56±0.37、2.51±0.29、2.58±0.26、1.33±0.41。A、B、C这3组间缺血后肢DSA造影血管评分无显著性差异(P＞0.05)，A组与D组，B组与D组，C组与D组间均有显著性差异(P＜0.05)。组织学检查：A、B、C和D组的缺血后肢肌肉组织中毛细血管密度分别为570.35±68.17／mm~2、563.02±61.26／mm~2、576.97±77.87／mm~2、513.01±70.68／mm~2。A、B、C这3组间毛细血管密度无显著性差异(P＞0.05)，A组与D组，B组与D组，C组与D组间均有显著性差异(P＜0.05)。BrdU染色、普鲁士铁染色、Ⅷ因子免疫荧光及MRI的结果有良好的相关性。
结论
HIV-Tat-CLIO标记的rMSCs移植入大鼠后肢缺血模型后可以运用MRI进行动态监测，根据低强度信号的范围变化可以比较清晰的显示移植后的rMSCs在宿主体内的迁移和分布。rMSCs移植能够提高大鼠后肢缺血组织的血管再生能力，促进缺血肢体的血运重建和功能恢复，而HIV-Tat-CLIO标记并不影响rMSCs的治疗作用。
Part One Effects of HIV-Tat-CLIO on the physiological manner of rat marrow-derivedmesenchymal stem cells
Objective
To evaluate the feasibility of HIV transduction domain cross-linked iron oxide (HIV-Tat-CLIO) labeling technique and its labeling efficiency, toxicity and effect on cellular function of rat marrow-derived mesenchymal stem cells (rMSCs).
Materials and Methods
On the basis of the difference of cellular adherent ability, MSCs from primary cultures of rat marrow were isolated and purified by the preplate technique. To verify the nature of cultured rMSCs, the 3rd passage cells were labeled against CD34, CD45 and CD90 and analyzed by flow cytometry. We chose the 3rd passage rMSCs to follow the later study. The cells were labeled by the HIV-Tat-CLIO with 25μg /ml for 24h or by CLIO with 25μg/ml for 24h. The labeling percentage was assessed by the Prussian blue staining to compare the effects of different tag technique. After labeling of rMSCs with HIV-Tat-CLIO complexes, Prussian blue staining and electron microscopy confirmed the presence of iron oxide nanoparticles inside the cells and cellular toxicity, functional capacity were determined by trypan blue exclusion, MTT assay, oil 0 and AKP staining and FITC-and PE-conjugated monoclonal antibodies directed against CD34, CD45 and CD90．We used transwell test to assess the ability of migration.
Results
After 3 passages, rMSCs were negative for CD34 and CD45 and positive for CD90．The labeling percentage of HIV-Tat-CLIO was better than the CLIO group. HIV-Tat-CLIO labeled cells demonstrated no short or long-term toxicity, changes in differentiation capacity of the rMSCs, or changes in phenotype when compared with unlabeled cells. There was also no change on the manifestation of cells capability for differentiation and migration.
Conclusion
The HIV-Tat-CLIO incorporation did not affect the capability for cell viability, proliferation, differentiation and migration of rMSCs which can be tracked lively by MRI. It was an efficient and effective technique for incorporating the nanoparticles within endosomes, thereby labeling cells that can be detected by MRI. Cell labeling using these methods was likely to enhance the development of cell-based strategies for the repair or replacement of tissues and other novel therapies.
Part TwoMagnetic Resonance Tracking of Transplanted Rat Marrow-derived MesenchymalStem Cells Labeled by HIV-Tat-CLIO in Rat Model of Hindlimb Ischemia
Objective
To investigate the feasibility and effect of using HIV-Tat-CLIO to label rMSCs for monitoring their temporal and spatial migration in vivo by magnetic resonance imaging (MRI) and evaluate the treatment efficiency of the implantation of HIV-Tat-CLIO labeled rMSCs in the model of rat hindlimb ischemia.
Materials and Methods
MSCs from primary cultures of rat marrow were isolated and cultured to the 3rd passage. The cells were labeled against CD34, CD45 and CD90 and analyzed by flow cytometry to verify the nature of cultured rMSCs. The cells were labeled by the HIV-Tat-CLIO with 25μg /ml for 24h and another cells were double labeled by BrdU (5-bromodeoxyuridine) with 3μg /ml for 24h, respectively. Twenty eight SD rats were randomly divided into 4 groups, containing 7 ones in each. The femoral arteries of the rats were excised to establish the model of right hindlimb ischemia and the model was check as successful according to the DSA imaging. At the 7th day after the operation, rMSCs implantation was accomplished with the technique of intramuscularly injection. We established 4 group, including A, B, C and D individually injected with HIV-Tat-CLIO labeling rMSCs, double labeled rMSCs with HIV-Tat-CLIO and BrdU, unlabeled rMSCs and same volume PBS liquids. MRI was performed at 4 different time points after the ischemia model procedure (the 3rd day, 1st week, 2nd week and the 4th week, individually) and the group C and D were only checked once on the 3rd day. That is, in vivo MR imaging was used to track their fate. At the 4th week, the skin temperatures of the ischemic hindlimb were measured with infrared thermography and the impaired score of limb function were assessed. Additionally, we carried out DSA to evaluate the hindlimb angiography and used immunohistochemistry technique to record the capillary density of the ischemic muscular tissue to observe the formation of new blood vessels. We united the staining techniques of Prussian blue and BrdU and MRI to assess the labeled effected of transplanted rMSCs.
Results
The 3rd passage rMSCs were negative for CD34 and CD45 and positive for CD90．During our experiment, there were 3 rats dead. Two rats in group B and C died of incision infection and the other in group B was due to the anethesia incident. The implanted cells were visible on MR images as a hypointense area at the injection site on the 3rd day after the rMSCs implantation. Then gradually separated into small particles and dispersed around the injection site during the follow 3 weeks. The skin temperature of the ischemic hindlimb was higher in A (32．12±0．51℃),B (32．39±0．61℃)and C (31．97±0．56℃) group than in group D (31．16±0．67℃) 4 weeks after the implantation procedure (P<0．05) . There were no group difference between group A and B, B and C or A and C (P>0．05) . Impaired score of limb function was significantly lower in group A (0．68±0．17) , B (0．65±0．24) and C (0．67±0．11) than in group D (1．79±0．22) 4 weeks after the implantation procedure (P < 0．05) . There were no obviously group difference between group A and B, B and C or A and C (P> 0．05) . The hindlimb angiography showed that the average angiographic score was 2．56±0．37 in group A, 2．51±0．29 in group B, 2．58±0．26 in group C and1．33±0．41 in group D. Group A, B and C were significantly higher than group D (P < 0．05) . There were no obviously group difference between group A and B, B and C or A and C (P>0．05) . The capillary density was 570．35±68．17/mm~2 in group A, 563．02±61．26/mm~2 in group B, 576．97±77．87/mm~2 in group C and 513．01 + 70．68/mm~2 in group D. Group A, B and C were significantly higher than group D (P < 0．05) . There were no obviously group difference between group A and B, B and C or A and C (P>0．05) .
Conclusion
The technique of MRI in vivo tracking transplanted rMSCs preoperatively labeled by HIV-Tat-CLIO was feasible and effective. The HIV-Tat-CLIO labeled rMSCs implantation could stimulate the formation of the new blood vessels and promote the foundation of the blood flow and rehabilitation of limb function in the model of rat hindlimb ischemia. Using the HIV-Tat-CLIO combination for magnetic cellular labeling should facilitate translation of this approach into clinical trials.
目的：
探讨HIV-Tat蛋白转导域交联氧化铁复合物(HIV transduction domaincross-linked iron oxide,HIV-Tat-CLIO)标记对体外培养的大鼠骨髓间充质干细胞(rat marrow-derived mesenchymal stem cells,rMSCs)的可行性、剂量关系以及对细胞活力、增殖、分化和迁移能力等生物学行为的影响。
材料与方法：
原代培养rMSCs至第3代，检测CD34，CD45和CD90的表达以鉴定之。不同浓度的HIV-Tat-CLIO标记rMSCs后利用普鲁士铁染色、体外MRI显像及台盼兰细胞活性测定等方法确定最佳标记条件。用CLIO 25μg／ml和HIV-Tat-CLIO 25μg／ml分别标记rMSCs各24h，通过普鲁士兰铁染色比较两组纳米铁的细胞标记效率。运用普鲁士兰染色及扫描电镜技术观察铁颗粒的细胞内存在部位。MTT实验测定该标记方法对细胞增殖能力的影响；流式细胞仪测定标记前后细胞表面标记物的表达及细胞周期的变化；通过油红0和碱性磷酸酶染色测定这种标记方法对rMSCs细胞分化能力的影响；Transwell实验测定其对干细胞迁移能力的变化。
结果：
培养的第3代rMSCs CD34(-)，CD45(-)和CD90(+)。HIV-Tat-CLIO最佳标记浓度为25μg／ml。HIV-Tat-CLIO组较CLIO组在rMSCs的标记效率上获得明显改善。细胞标记后，A570波长的光吸收值无明显变化；细胞表面CD34、CD45和CD90分子的表达无明显差异；处于S期的MSCs数量(S％)、G_1％、增殖指数PrI值(S+G_2M)％以及细胞凋亡情况均无显著改变；这种标记物对细胞分化和运动迁移能力均无显著影响。
结论：
HIV-Tat-CLIO标记大鼠MSCs后对其生物学行为无明显影响，标记后的干细胞可以正常增殖、分化及运动迁移，为磁共振活体监测干细胞移植后的动态变化提供有力的保证。
第二部分干细胞移植治疗大鼠后肢缺血中HIV-Tat-CLIO标记磁共振体内示踪的研究
目的：
探讨磁共振成像(Magnetic Resonance Imaging,MRI)无创体内示踪HIV-Tat-CLIO(HIV transduction domain cross-linked iron oxide)标记的移植大鼠骨髓间充质干细胞(rat Marrow-derived Mesenchymal Stem cells,rMSCs)的可行性和有效性，并且对该标记后的rMSCs移植治疗大鼠后肢缺血的效果进行客观评价。
材料和方法：
原代培养6周龄雄性SD大鼠的rMSCs至第3代，检测CD34，CD45和CD90的表达以鉴定。用HIV-Tat-CLIO 25μg／ml标记rMSCs 24h。另有一组细胞继续以5-溴脱氧尿苷(BrdU)3μg／ml复合标记rMSCs 24h。于大鼠右后肢缺血模型建立后的第7天进行rMSCs细胞移植。选用6周龄雄性SD大鼠28只，分为4组，每组各7只。A组为移植HIV-Tat-CLIO标记的rMSCs；B组为移植HIV-Tat-CLIO及BrdU复合标记的rMSCs；C组为移植未标记的rMSCs；D组为注射同移植体积的无菌PBS液。分别在移植术后3d、1周、2周、4周进行MRI检查示踪HIV-Tat-CLIO标记的rMSCs分布情况(C组和D组仅于术后第3d进行MRI扫描1次)。在第4周各组均采用非致冷红外热成像仪检测大鼠后肢皮肤温度，进行缺血后肢的损伤评分，并进行DSA造影，之后处死所有大鼠进行组织学检查。进行HE染色和普鲁士兰铁染色，BrdU、Ⅷ因子免疫组化染色，并计算毛细血管密度。
结果
培养的第3代rMSCs CD34(-)，CD45(-)和CD90(+)。B组和C组各有1只实验鼠因切口感染坏死，分别于术后第5天和第8天死亡，B组另有1只于移植术后第2周MRI检查时麻醉意外死亡。其余25只均存活至术后第4周。MRI检查：A组和B组在MRI上的显示接近。C组和D组在MRI上无特殊信号变化。A组和B组在术后第3d，MRI显示注射部位圆形低强度信号影；术后1周，MRI显示原低信号灶呈中心向周围扩散趋势，信号呈颗粒状，部位与随后的组织学检查结果一致；术后第2周见低密度影进一步扩散，信号强度有所弱化；术后第4周信号强度进一步弱化。低密度颗粒扩散范围局限于原部位7mm处。非致冷红外热成像仪检测大鼠后肢皮肤温度发现：A、B、C和D组的右后肢平均皮肤温度分别为32.12±0.51℃、32.39±0.61℃，31.97±0.56℃、31.16±0.67℃。A、B、C这3组间平均皮肤温度无显著性差异(P＞0.05)，A组与D组，B组与D组，C组与D组间均有显著性差异(P＜0.05)。缺血后肢的损伤评分：A、B、C和D组的缺血后肢损伤评分分别为0.68±0.17、0.65±0.24、0.67±0.11、1.79±0.22。A、B、C这3组间缺血后肢损伤评分无显著性差异(P＞0.05)，A组与D组，B组与D组，C组与D组间均有显著性差异(P＜0.05)。DSA造影：A、B、C和D组的造影显示大鼠缺血侧后肢大腿部从股骨大转子至股骨下段区域内的血管评分平均各为：2.56±0.37、2.51±0.29、2.58±0.26、1.33±0.41。A、B、C这3组间缺血后肢DSA造影血管评分无显著性差异(P＞0.05)，A组与D组，B组与D组，C组与D组间均有显著性差异(P＜0.05)。组织学检查：A、B、C和D组的缺血后肢肌肉组织中毛细血管密度分别为570.35±68.17／mm~2、563.02±61.26／mm~2、576.97±77.87／mm~2、513.01±70.68／mm~2。A、B、C这3组间毛细血管密度无显著性差异(P＞0.05)，A组与D组，B组与D组，C组与D组间均有显著性差异(P＜0.05)。BrdU染色、普鲁士铁染色、Ⅷ因子免疫荧光及MRI的结果有良好的相关性。
结论
HIV-Tat-CLIO标记的rMSCs移植入大鼠后肢缺血模型后可以运用MRI进行动态监测，根据低强度信号的范围变化可以比较清晰的显示移植后的rMSCs在宿主体内的迁移和分布。rMSCs移植能够提高大鼠后肢缺血组织的血管再生能力，促进缺血肢体的血运重建和功能恢复，而HIV-Tat-CLIO标记并不影响rMSCs的治疗作用。
Part One Effects of HIV-Tat-CLIO on the physiological manner of rat marrow-derivedmesenchymal stem cells
Objective
To evaluate the feasibility of HIV transduction domain cross-linked iron oxide (HIV-Tat-CLIO) labeling technique and its labeling efficiency, toxicity and effect on cellular function of rat marrow-derived mesenchymal stem cells (rMSCs).
Materials and Methods
On the basis of the difference of cellular adherent ability, MSCs from primary cultures of rat marrow were isolated and purified by the preplate technique. To verify the nature of cultured rMSCs, the 3rd passage cells were labeled against CD34, CD45 and CD90 and analyzed by flow cytometry. We chose the 3rd passage rMSCs to follow the later study. The cells were labeled by the HIV-Tat-CLIO with 25μg /ml for 24h or by CLIO with 25μg/ml for 24h. The labeling percentage was assessed by the Prussian blue staining to compare the effects of different tag technique. After labeling of rMSCs with HIV-Tat-CLIO complexes, Prussian blue staining and electron microscopy confirmed the presence of iron oxide nanoparticles inside the cells and cellular toxicity, functional capacity were determined by trypan blue exclusion, MTT assay, oil 0 and AKP staining and FITC-and PE-conjugated monoclonal antibodies directed against CD34, CD45 and CD90．We used transwell test to assess the ability of migration.
Results
After 3 passages, rMSCs were negative for CD34 and CD45 and positive for CD90．The labeling percentage of HIV-Tat-CLIO was better than the CLIO group. HIV-Tat-CLIO labeled cells demonstrated no short or long-term toxicity, changes in differentiation capacity of the rMSCs, or changes in phenotype when compared with unlabeled cells. There was also no change on the manifestation of cells capability for differentiation and migration.
Conclusion
The HIV-Tat-CLIO incorporation did not affect the capability for cell viability, proliferation, differentiation and migration of rMSCs which can be tracked lively by MRI. It was an efficient and effective technique for incorporating the nanoparticles within endosomes, thereby labeling cells that can be detected by MRI. Cell labeling using these methods was likely to enhance the development of cell-based strategies for the repair or replacement of tissues and other novel therapies.
Part TwoMagnetic Resonance Tracking of Transplanted Rat Marrow-derived MesenchymalStem Cells Labeled by HIV-Tat-CLIO in Rat Model of Hindlimb Ischemia
Objective
To investigate the feasibility and effect of using HIV-Tat-CLIO to label rMSCs for monitoring their temporal and spatial migration in vivo by magnetic resonance imaging (MRI) and evaluate the treatment efficiency of the implantation of HIV-Tat-CLIO labeled rMSCs in the model of rat hindlimb ischemia.
Materials and Methods
MSCs from primary cultures of rat marrow were isolated and cultured to the 3rd passage. The cells were labeled against CD34, CD45 and CD90 and analyzed by flow cytometry to verify the nature of cultured rMSCs. The cells were labeled by the HIV-Tat-CLIO with 25μg /ml for 24h and another cells were double labeled by BrdU (5-bromodeoxyuridine) with 3μg /ml for 24h, respectively. Twenty eight SD rats were randomly divided into 4 groups, containing 7 ones in each. The femoral arteries of the rats were excised to establish the model of right hindlimb ischemia and the model was check as successful according to the DSA imaging. At the 7th day after the operation, rMSCs implantation was accomplished with the technique of intramuscularly injection. We established 4 group, including A, B, C and D individually injected with HIV-Tat-CLIO labeling rMSCs, double labeled rMSCs with HIV-Tat-CLIO and BrdU, unlabeled rMSCs and same volume PBS liquids. MRI was performed at 4 different time points after the ischemia model procedure (the 3rd day, 1st week, 2nd week and the 4th week, individually) and the group C and D were only checked once on the 3rd day. That is, in vivo MR imaging was used to track their fate. At the 4th week, the skin temperatures of the ischemic hindlimb were measured with infrared thermography and the impaired score of limb function were assessed. Additionally, we carried out DSA to evaluate the hindlimb angiography and used immunohistochemistry technique to record the capillary density of the ischemic muscular tissue to observe the formation of new blood vessels. We united the staining techniques of Prussian blue and BrdU and MRI to assess the labeled effected of transplanted rMSCs.
Results
The 3rd passage rMSCs were negative for CD34 and CD45 and positive for CD90．During our experiment, there were 3 rats dead. Two rats in group B and C died of incision infection and the other in group B was due to the anethesia incident. The implanted cells were visible on MR images as a hypointense area at the injection site on the 3rd day after the rMSCs implantation. Then gradually separated into small particles and dispersed around the injection site during the follow 3 weeks. The skin temperature of the ischemic hindlimb was higher in A (32．12±0．51℃),B (32．39±0．61℃)and C (31．97±0．56℃) group than in group D (31．16±0．67℃) 4 weeks after the implantation procedure (P<0．05) . There were no group difference between group A and B, B and C or A and C (P>0．05) . Impaired score of limb function was significantly lower in group A (0．68±0．17) , B (0．65±0．24) and C (0．67±0．11) than in group D (1．79±0．22) 4 weeks after the implantation procedure (P < 0．05) . There were no obviously group difference between group A and B, B and C or A and C (P> 0．05) . The hindlimb angiography showed that the average angiographic score was 2．56±0．37 in group A, 2．51±0．29 in group B, 2．58±0．26 in group C and1．33±0．41 in group D. Group A, B and C were significantly higher than group D (P < 0．05) . There were no obviously group difference between group A and B, B and C or A and C (P>0．05) . The capillary density was 570．35±68．17/mm~2 in group A, 563．02±61．26/mm~2 in group B, 576．97±77．87/mm~2 in group C and 513．01 + 70．68/mm~2 in group D. Group A, B and C were significantly higher than group D (P < 0．05) . There were no obviously group difference between group A and B, B and C or A and C (P>0．05) .
Conclusion
The technique of MRI in vivo tracking transplanted rMSCs preoperatively labeled by HIV-Tat-CLIO was feasible and effective. The HIV-Tat-CLIO labeled rMSCs implantation could stimulate the formation of the new blood vessels and promote the foundation of the blood flow and rehabilitation of limb function in the model of rat hindlimb ischemia. Using the HIV-Tat-CLIO combination for magnetic cellular labeling should facilitate translation of this approach into clinical trials.
引文
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