血管内皮祖细胞移植提高裸鼠缺血皮瓣存活率

目的探讨血管内皮祖细胞(endothelialprogenitorcell,EPC)移植促进早期断蒂的缺血皮瓣的血管新生,从而提高皮瓣存活率的可能性。方法体外分离、培养人脐血中EPCs,免疫组织化学进行鉴定,然后移植于裸鼠随意皮瓣,皮瓣早期断蒂,裸鼠分两组:实验组(EPCs移植)、对照组(M199注射),术后皮瓣4d断蒂。观察皮瓣的存活率、激光多普勒血液监测仪监测血流灌注、CD34免疫组织化学检测皮瓣毛细血管密度、激光共聚焦检测EPCs在皮瓣内的密度。结果脐血中分离培养的EPCs表达CD34、KDR及CD133,实验组EPCs移植裸鼠皮瓣后,整合到缺血部位新生血管中,与对照组的皮瓣存活率分别为(60.3±2.1)%、(34.2±1.8)%(P<0.05);而且两组毛细血管密度、血流灌注差异均有统计学意义(P<0.05);实验组术后第7、11天时二组皮瓣中的EPCs密度分别为(75.2±6.5)个/mm2、(305.4±26.5)个/mm2,而对照组均为0个/mm2(P<0.05)。结论脐血中的EPCs体外培养后移植体内可促进缺血皮瓣的血管新生,提高存活率。     

血管内皮祖细胞促进游离移植脂肪颗粒组织存活的实验

目的探讨血管内皮祖细胞（endothelial progenitor cell，EPC）移植促进游离移植的脂肪颗粒组织的血管新生，提高移植脂肪颗粒组织存活率。方法体外分离、培养人脐血中EPCs，然后与来自人体的脂肪颗粒组织混合移植于裸鼠背部。结果脐血中分离培养的EPCs表达血管内皮细胞生长因子受体（KDR）及细胞表面标记CD34、CD133，EPCs与脂肪颗粒组织混合移植到裸鼠3个月后，EPCs整合到缺血部位新生血管中，与对照组的脂肪颗粒组织存活率分别为（89．3±6．8）％、（42．2±2．5）％（P〈0．05），而且实验组与对照组脂肪颗粒组织周边区毛细血管密度差异有统计学意义（P〈0．05）。术后3个月2组脂肪颗粒组织周边区的EPCs密度分别为（95．2±10．5）个／mm^2、0个／mm^2（P％O．05）。结论体外培养的脐血EPCs移植体内可促进游离移植的脂肪颗粒组织的血管新生，提高存活率。     

转染血管内皮生长因子基因的小鼠NIH3T3细胞移植促进皮瓣早期血运重建的研究

目的探讨转染血管内皮生长因子（VEGF）基因的小鼠NIH3T3细胞移植对缺血皮瓣的血管新生和皮瓣存活率的影响。方法体外PcDNA3．1（-）／VEGF165质粒转染小鼠NIH3T3细胞,免疫组化方法检测小鼠NIH3T3细胞体外表达VEGF的情况,CM-DiI标记小鼠NIH3T3细胞。将小鼠随机分为3组：A组[PcDNA3．1（-）／VEGF165质粒转染的NIH3T3细胞移植]、B组（单纯NIH3T3细胞移植）、C组（单纯DMEM培养基注射）。每只小鼠背侧皮下按组分别注射细胞悬液和培养基,注射后酶联免疫吸附（ELISA）法连续检测大鼠血浆VEGF浓度,注射后第4天掀起一个蒂在尾侧的4．0cm×1．5cm的随意皮瓣。术后第7天分别观察皮瓣的存活率、血流灌注、皮瓣毛细血管密度、NIH3T3细胞在皮瓣内的分布和存活情况。结果转染VEGF165基因的小鼠NIH3T3细胞体外和体内检测均高表达VEGF165蛋白。A组的皮瓣存活率、毛细血管密度、血流灌注比值均显著高于另外两组（P〈0．05）。结论转染VEGF基因的小鼠NIH3T3细胞皮下移植可促进缺血皮瓣的血管新生,提高存活率。     

转基因的骨髓间充质干细胞治疗大鼠缺血皮瓣

目的探讨转染血管内皮细胞生长因子(VEGF)基因的大鼠骨髓间充质干细胞(MSCs)同种异体移植促进缺血皮瓣的血管新生,从而提高皮瓣存活率的可能性。方法体外分离、培养、鉴定SD大鼠MSCs,PcDNA3.1(-)/VEGF165质粒转染MSCs,免疫荧光方法检测MSCs体外表达VEGF的情况,CM-DiI标记MSCs。SD大鼠随机分3组:A组[PcDNA3.1(-)/VEGF165质粒转染的MSCs移植]、B组(单纯MSCs移植)、C组(DMEM-F12培养基)。每只大鼠背侧皮下按组分别注射细胞悬液和培养基,注射后ELISA法连续检测大鼠血浆VEGF浓度,注射后第4天掀起1个蒂在尾侧的9 cm×2 cm的随意皮瓣。在术后第14天分别观察皮瓣的存活率、激光多普勒血液监测仪监测血流灌注、CD34免疫组织化学检测皮瓣毛细血管密度、荧光显微镜检测MSCs在皮瓣内的分布和存活状况。结果转染VEGF165基因的MSCs体外和体内检测均高表达VEGF165蛋白。A、B、C三组的皮瓣存活率分别为(83.1±2.6)%、(66.4±6.1)%、(51.5±7.5)%(P< 0.05);A、B、C三组的毛细血管密度(条/mm2)分别为:89.2±6.1、57.1±4.7、28.7±2.8(P< 0.05);血流灌注比值A组高于B、C两组,B组高于C组(P<0.05);转染VEGF165基因的MSCs移植SD大鼠皮瓣后,MSCs存活并参与血管新生。结论转染VEGF基因的大鼠MSCs体外培养后异体移植可促进缺血皮瓣的血管新生,提高存活率。     

bFGF微球对大鼠背部任意皮瓣存活的影响

目的：研究局部注射碱性成纤维细胞生长因子（bFGF）复合明胶微球对SD大鼠背部任意皮瓣存活的影响。方法：采用改良的乳化冷凝法交联制备复合bFGF明胶缓释微球,将其注射于大鼠背部任意皮瓣,24只SD大鼠随机分为bFGF微球组（A组）、bFGF治疗组（B组）和对照组（C组）,术后7天分别进行皮瓣存活率、新生血管计数的检测。结果：术后7天皮瓣的存活率分别为（65.42±2.19）%,（54.38±4.52）%,（45.43±2.71）%,微球组存活率显著高于对照组（P〈0.05）且存活质量最好;皮瓣内新生血管计数分别为28.75±2.36,21.28±3.82,18.68±5.44,具有显著性差异。结论：bFGF缓释微球可以促进缺血皮瓣的血管新生,提高皮瓣存活率。     

血管内皮祖细胞和血管内皮生长因子促进预构皮瓣血管新生作用的比较

目的 比较血管内皮祖细胞(endothelial progenitor cells,EPCs)与血管内皮生长因子(vascular endothelial growth factor,VEGF)在促进预构皮瓣血管新生作用上的差异,探讨EPCs移植提高预构皮瓣存活面积的可行性.方法 分离雄性Wistar大鼠(45只)一侧股血管柬,转位植入腹部皮下,建立预构皮瓣实验模型.将体外诱导分化的EPCs(组Ⅰ,n=15)和VEGF(组Ⅱ,n=15)分别注射于皮瓣局部,对照组仅注射PBS溶液(组Ⅲ,n=15).4周后形成以植入血管为蒂的岛状皮瓣,原位缝合;术后7 d对皮瓣存活率、血管密度计数进行检测.结果 组Ⅰ、组Ⅱ、组Ⅲ的皮瓣存活率分别为(87.26±10.13)%、(66.13±9.9)%、(55.59±13.06)%,组Ⅰ分别与组Ⅱ和组Ⅲ比较,差异均有统计学意义(P<0.001);微血管密度分别为:(38.67±9.52)个/mm~2、(25.83±6.33)个/mm~2、(26.5±5.61)个/mm~2(P<0.05).结论 EPCs促进预构皮瓣血管新生的作用优于VEGF,局部应用骨髓来源的EPCs可以有效地提高预构皮瓣存活面积.     

转染TGF-β1的骨髓间充质干细胞移植对预构皮瓣存活影响的实验研究

目的:探讨TGF-β1转染骨髓间充质干细胞(MSC)移植对预构皮瓣存活的影响及机制。方法:分离并培养大鼠MSC,采用脂质体介导技术将真核表达载体pc DNA3.1(+)/TGF-β1转染至MSC,对转染后MSC的表型及体外成管进行鉴定;选取12只SD大鼠,于其背部两侧对称建立5 cm×1 cm随意皮瓣模型,于两侧皮瓣下分别注射转染后的MSC(实验侧)与溶媒(对照侧),观察皮瓣存活情况计算皮瓣存活率,免疫组化染色及HE染色检测皮瓣血管密度。结果:流式细胞仪分析表明,转染TGF-β1后MSC仍具有MSC的特性,且具有良好的体外成管功能。注射转染TGF-β1的MSC的实验侧皮瓣存活率明显高于对照侧(82.83%vs.61.33%,P0.05),同时皮瓣内毛细血管的密度明显也明显高于对照侧(19.69个vs.8.19个,P0.05)。结论:转染TGF-β1的MSC可以促进皮瓣内毛细血管生成,提高皮瓣的存活率。     

肝细胞生长因子对大鼠背部超长任意皮瓣存活率影响的实验研究

目的：探讨肝细胞生长因子对大鼠背部超长任意皮瓣血管生成的作用及皮瓣存活率的影响。方法：建立大鼠背部超长任意皮瓣8.0cm×2.0cm动物模型,直接皮下注射50ng/ml肝细胞生长因子溶液,术后7天观察皮瓣颜色、质地、毛细血管回流、坏死范围、切割皮瓣出血情况;计算皮瓣的存活面积;免疫组化染色后在显微镜下计算毛细血管平均密度。通过自身前后对照、与生理盐水组对照,比较两者的差异。结果：肝细胞生长因子组皮瓣的存活率81.45%±2.74%,与对照组的42.82%±7.03%比较,差别有统计学意义（P〈0.05）;实验前实验组和对照组毛细血管数分别为12.70±1.35、12.31±1.22,两者差别无统计学意义（P〉0.05）;实验后实验组和对照组毛细血管数分别为39.67±4.83、21.50±1.87,两者差别有统计学意义（P〈0.05）;实验组及对照组实验后毛细血管密度高于实验前,差别有统计学意义（P〈0.05）。结论：肝细胞生长因子能促进皮瓣血管内皮细胞的生成,提高皮瓣的存活率。     

血管内皮细胞生长因子基因转染血管内皮祖细胞移植提高移植脂肪存活率的实验研究

目的探讨血管内皮细胞生长因子165(VEGF165)基因转染血管内皮祖细胞(EPCs)移植促进游离移植的脂肪组织的血管新生,提高移植脂肪组织存活率。方法体外分离、培养人脐血中EPCs,利用脂质体介导VEGF165基因体外转染EPCs,然后与来自人体的脂肪组织混合移植于裸鼠背部,裸鼠随机分为3组:VEGF165基因转染组、EPCs组及M199培养基对照组。结果脐血中分离培养的EPCs表达CD34、血管内皮细胞生长因子受体及CD133;VEGF165基因转染EPCs体外及体内检测均有VEGF165蛋白的表达。VEGF165基因转染组、EPCs组中,EPCs整合到缺血部位新生血管中,与对照组的脂肪组织存活率分别为(96.2±8.6)%、(75.3±6.8)%和(40.2±2.5)%(P<0.05),VEGF165基因转染组与EPCs组脂肪组织周边区毛细血管密度有显著差异(P<0.05),均高于对照组(P<0.05)。术后3个月时3组脂肪组织周边区的EPCs密度分别为(196±16)个/mm2、(95±11)个/mm2、0个/mm2(P<0.05)。结论脐血中的EPCs体外培养后移植体内可促进游离移植的脂肪组织的血管新生,提高存活率,而转染VEGF165基因的EPCs具有更强的促血管新生的作用。     

血管内皮生长因子基因转染血管内皮祖细胞治疗肢体缺血的实验研究

目的利用血管内皮生长因子（vascular endothelial growth factor,VEGF-165）基因转染体外诱导的血管内皮祖细胞（endothelial progenitor cells,EPCs）,并移植到下肢缺血的新西兰兔体内,观测其促进血管新生,改善肢体缺血的效果。方法（1）梯度离心法分离兔骨髓单个核细胞,然后用含有VEGF、bFGF、IGF-1的M199培养液诱导培养EPCs。并以免疫荧光、透射电镜等方法进行鉴定。（2）用携带VEGF165基因的腺病毒质粒（Adv-GFP-VEGF165）转染所培养的细胞,ELISA法检测上清液中VEGF蛋白的表达。（3）制作兔下肢缺血模型,并将其随机分为A、B、C 3组,分别移植EPCs、VEGF165基因转染后的EPCs、M199培养基,多种方法检测移植效果及局部整合情况。结果（1）自兔骨髓诱导出梭形贴壁细胞,免疫荧光及电镜检测证实为EPCs。（2）Adv-GFP-VEGF165成功转染EPCs,ELISA法检测转染VEGF165后的EPCs其上清液中VEGF蛋白浓度明显升高。（3）Brdu示踪显示移植细胞整合到缺血局部,CTA及免疫组化检查显示VEGF-165基因转染后的EPCs移植后其改善肢体缺血效果优于其他两组。结论VEGF基因转染EPCs后能改进EPCs质量,移植后促血管新生能力增强,其效果优于未转染组。     

骨髓单个核细胞与骨髓间充质干细胞促进大鼠皮瓣存活率的比较

目的比较来源于等量骨髓的单个核细胞与经扩增的间充质干细胞,促进大鼠随意皮瓣成活率的效果。方法取等质等量的大鼠骨髓,一半直接离心获得骨髓单个核细胞,一半经体外培养获得骨髓间充质干细胞,注射相等数量(n=6)的大鼠随意皮瓣。术后测量并计算皮瓣成活面积,取材,行组织学检测,计数CD31阳性血管数量。结果未经培养的骨髓单个核细胞组皮瓣的平均存活率为(71.6±8.4)%,培养的骨髓间充质干细胞组平均存活率为(66.2±3.1)%,这两组存活率均显著高于注射平衡液的对照组(55.9±3.4)%;注射细胞组之间平均存活率没有统计学差异。组织学血管密度计数显示,骨髓单个核细胞组和骨髓间充质干细胞组的微血管数量分别是(58.2±6.8)和(42.7±5.1),都显著高于PBS对照组(22.8±3.1),而骨髓单个核细胞组也显著高于骨髓间充质干细胞组。结论与不经培养的骨髓单个核细胞相比,通过体外扩增的骨髓间充质细胞,未能显著提高大鼠随意皮瓣的成活率。     

Transplanted endothelial progenitor cells augment the survival areas of rat dorsal flaps

Endothelial progenitor cells (EPCs) have been identified in peripheral blood, and have been reported to be incorporated into ischemic regions such as the ischemic hindlimb. In this study, we examined whether or not transplantation of EPCs is useful for salvaging surgical flaps in vivo. At the same time, we quantitatively compared the neovascularization ability of transplanted EPCs and that of mature endothelial cells (ECs). ECs obtained from the aorta of rats by explantation and passaged several times were used in the present study. EPCs were obtained from the blood of rat hearts. The blood samples were separated by density gradient centrifugation. Light-density mononuclear cells (MNCs) were collected and cultured on plastic plates coated with rat plasma vitronectin. Cells attached at day 7 of culture were deemed to be EPCs. Then PBS (control), ECs, or EPCs (3.0 x 10(5) suspended in 1.0 ml PBS) were injected at the middle of a flap. Seven days after surgery, the survival lengths of the flaps were evaluated. EPC-transplanted groups revealed statistically significant augmentation of survival length compared with the other two groups (p < 0.003). EPC-transplanted groups had significantly more angiographically detectable blood vessels (p < 0.003) and significantly higher capillary density (p < 0.03) than the other two groups. Confocal microscopy revealed that EPCs were incorporated into enhanced neovascularization. These results suggest that transplantation of EPCs may be useful for salvaging surgical flaps, and EPCs are superior to ECs in neovascularization ability.     

Transplantation of endothelial progenitor cells transferred by vascular endothelial growth factor gene for vascular regeneration of ischemic flaps

BACKGROUND: Neovascularization occurs through two mechanisms: angiogenesis and vasculogenesis. Therefore, there are two strategies to promote neovascularization: therapeutic angiogenesis and therapeutic vasculogenesis (endothelial progenitor cells therapy). MATERIALS AND METHODS: In this study, we examined whether or not endothelial progenitor cells combined with vascular endothelial growth factor (VEGF) gene therapy is useful for ischemia surgical flaps in vivo. At the same time, we quantitatively compared the neovascularization ability of transplanted endothelial progenitor cells (EPCs) transducted with VEGF165 gene and EPCs alone. EPCs were isolated from cord blood of healthy human volunteers, cultured in vitro for 7 days and identified by immunofluorescence. After transduced with VEGF165 gene in vitro, proliferative activity of EPCs was assessed using MTT assay. CM-DiI was used to trace EPCs in vivo 4 days after injection of 5 x 10(5) VEGF-transduced EPCs(VEGF-transduced EPCs group, n = 10), 5 x 10(5) EPCs (non-transduced EPCs group, n = 10) in 500 microL EBM-2 media, or 500 microL EBM-2 media (EBM-2 media group, n = 10) local, a cranially based flap was elevated on the back of nude mice. The percent flap survival, neovasculariztion and blood flow recovery of flaps was detected. RESULTS: EPCs expressed cell markers CD34, KDR, and CD133. A statistically significant increase in percent flap survival was observed in mice of VEGF-transduced EPCs group as compared with that of non-transduced EPCs group: 67.99 +/- 6.64% versus 59.43 +/- 4.69% (P < 0.01), and 41.24 +/- 2.44% in EBM-2 media group (P < 0.01). The capillary density and blood flow recovery of flaps in VEGF-transduced EPCs group were both improved. CM-DiI-labeled VEGF-transduced EPCs were observed in vivo and the numbers of cells increased. CONCLUSION: EPCs from human cord blood can increased neovascularization of ischemic flaps and augmented the survival areas, and VEGF-transduced EPCs have more powerful ability of promoting neovascularization in animal model of ischemic flaps.     

Autologous administration of adipose stromal cells improves skin flap survival through neovascularization: An experimental study

One of the most severe complications in aesthetic and reconstructive surgeries is the partial or total necrosis of a skin flap. In our experimental study, we demonstrated the use of adipose‐derived stem cells in the increase of skin flap survival rates. Stem cells were isolated from the fat of Wistar rats and genetically modified to permanently produce a green fluorescent protein (GFP). Two random‐pattern skin flaps (2 cm × 8 cm) were elevated on the dorsal area of the spine, and after being separated from the surgical wounds with a thin silicone sheet, they were placed back onto their original location. Then, the autologous GFP‐producing cells were injected intradermally into the dorsal area of the rats. At the seventh day, after the implantation of the stem cells, a clinical and immunohistochemical control was performed. The fluorescence microscopy revealed green vascular formations, suggesting that autologous GFP stromal cells were converted into endothelial cells through neovascularization. In the control skin flaps, where no stromal cells were used, no fluorescence was observed. The statistical analysis showed significantly lower necrosis rates in the right‐sided flaps (i.e., the flaps where adipose‐derived stromal cells were injected) compared with the left‐sided ones. Findings from our study demonstrate that adipose‐derived stem cells play an important role in the improvement of skin flap survival. Neovascularization is an effective way of achieving it.     

Bone marrow cell implantation improves flap viability after ischemia-reperfusion injury

This study attempted to clarify the effects of therapeutic neovascularization by bone marrow cells for salvaging flaps after ischemia-reperfusion injury. Bone marrow mononuclear cell layer (endothelial progenitor cell-enriched fraction) was isolated from the mouse femur and tibia. Symmetrical double flaps were elevated in mice. Each flap topically received phosphate buffered saline (PBS) or bone marrow cells in PBS. Flaps were subjected to 6-hour ischemia and subsequent reperfusion. On the seventh postoperative day, the flap survival area was measured (n = 27). The mean survival area of bone marrow cells-transplanted flaps was 66.3 +/- 18.0%, whereas control flaps showed a survival area of 49.7 +/- 22.2%. The difference was highly significant (P = 0.000209). Histologic examination revealed the average vascular density of bone marrow cells-transplanted flaps had significantly increased. The present study proved bone marrow cells acted with significant efficacy in promoting the survival of ischemia-reperfusion-mediated damaged tissue.     

Vascular endothelial growth factor (VEGF) expression and the effect of exogenous VEGF on survival of a random flap in the rat.

The induction of endogenous vascular endothelial growth factor (VEGF) production in the skin flap with ischemic injury and the effect of exogenous VEGF on survival of the ischemic skin flap were studied in rats. A dorsal flap model (3x10 cm(2)) was used in this study. In Part I, biopsies were taken from the flap at 2.5, 5.5, and 8.5 cm distances from the distal edge at 0, 6, 12, and 24 h after the flaps were sutured. Malonyldialdehyde (MDA) and VEGF(165) protein level were measured. In Part II, exogenous VEGF (1 microg/ml) was injected subdermally into the flaps in 14 rats before the flaps were replaced. Flaps that received a saline injection were used as the controls. The skin paddle survival was measured on postoperative day five. The results showed that the MDA level in the distal part of the flap significantly increased at 24 h postoperatively when compared to MDA in other parts of the flap. However, VEGF levels in the distal part of the flap significantly decreased when compared to the middle part of the flap. Subdermal injection of exogenous VEGF to the distal area of the flap could significantly improve survival of the distal flap (89% of total skin paddle) when compared to the control, which had a 64% mean percent survival. We conclude that production of endogenous VEGF protein is significantly increased in the skin flap with mild ischemia, but decreased in the flap with severe ischemia. Administration of exogenous VEGF could significantly enhance survival of ischemic flaps.     

Improvement of the skin flap survival with the bone marrow‐derived mononuclear cells transplantation in a rat model

Partial necrosis of skin flaps remains a significant problem in plastic and reconstructive surgery. In this study we attempted to evaluate the effect of bone marrow‐derived mononuclear cells (BM‐MNCs) transplantation on improvement of skin flap survival in a rat random pattern skin flap model. Thirty Wistar rats were divided into three groups with each consisting of 10 rats. BM‐MNCs and the adipose‐derived stem cells (ADSCs) were transplanted into the subcutaneous tissue in the area where the flap would be dissected. The flaps were then raised two days after cells transplantation. The animals receiving the preoperative Dulbecco's Modified Eagle Medium (DMEM) treatment were used as the controls. On the 7th postoperative day, the survival areas of flaps were measured and tissues were collected for examinations. The results showed that the mean survival areas were 46.33 ± 13.46% in the ADSCs group and 50.06 ± 13.82% in the BM‐MNCs group as the percentages of the total skin flaps, which were significantly higher than that in the control group (26.33 ± 7.14%) (P < 0.05). Histological analysis showed increased neovascularization in the flap treated with BM‐MNCs when compared with ADSCs transplantation. Survival BM‐MNCs and ADSCs were detected in the flap tissues. Higher levels of the basic fibroblast growth factor (bFGF) and vascular endothelium growth factor (VEGF) were found in the BM‐MNCs transplantation group (P < 0.05). The findings from this study demonstrated that preoperative treatment with BM‐MNCs transplantation could promote neovascularization and improve flap survival. These effects of BM‐MNCs on flap survival were comparable with ADSCs transplantation, but without necessity of in vitro cells expansion. © 2010 Wiley‐Liss, Inc. Microsurgery, 2010