人脐血内皮祖细胞体内促进血管重建的实验研究

目的：通过观察人脐血内皮祖细胞（EPC）在裸鼠体内缺氧状态下分化成内皮细胞的现象，初步研究其促进血管重建的作用。方法：根据EPC表面标记CD133，采用免疫磁珠法分离培养EPC。设计EPC裸鼠成瘤实验，观察瘤体内血管增生情况；将荧光标记的EPC注射于裸鼠腹部皮瓣中，观察EPC参与皮瓣血管重建情况；将EPC接种于聚羟基乙酸（PGA）中，移植于裸鼠皮下，观察EPC参与血管重建。结果：原代EPC贴壁后呈梭形，从第7天开始数量明显增加，呈克隆状生长。透射电镜观察到成熟内皮细胞最具特征性的细胞器——weibel-Palade小体。裸鼠成瘤实验：抗人vWF免疫荧光显示大量EPC参与肿瘤血管生成；裸鼠皮瓣实验：荧光标记EPC参与裸鼠皮瓣血管生成；PGA移植实验：抗人vWF免疫酶组织化学染色显示EPC参与血管生成。结论：EPC参与血管重建，具有促进血管新生，加速缺血组织血管化的作用。     

含内皮祖细胞的组织工程皮肤体外构建及裸鼠移植研究

目的构建含内皮祖细胞（EPC）和成纤维细胞的组织工程复合皮，初步研究EPC在组织工程皮肤移植中促血管发生的作用。方法采用免疫磁珠法从人脐血中分离培养CD133＋血管内皮祖细胞，以聚羟基乙酸（PGA）为真皮基质，接种EPC和成纤维细胞，其上覆盖表皮细胞膜片，构建组织工程复合皮，覆盖裸鼠背部全层皮肤缺损创面。结果EPC和成纤维细胞能均匀贴附于PGA支架纤维材料上，并逐渐伸展为梭形或多极状。裸鼠移植实验表职，实验组创面愈合早于对照组，可见EPC参与大量新生小血管形成，真皮支架5周完全降解。结论EPC参与血管重建，具有促进血管新生，加速缺血组织血管化的作用。应用PGA＋纤维蛋白凝胶，制备含EPC和成纤维细胞的活性真皮替代物，具有良好的生物相容性、降解特性。     

人脐血来源内皮祖细胞的纯化鉴定及定向分化的研究

目的 检测CD133^＋血管内皮祖细胞（EPC）的细胞表面标志物，鉴定其生物学特性。方法 采用免疫磁珠法分离人脐血EPC，用EGM-2MV培养基[含表皮生长因子（EGF）、血管内皮生长因子（VEGF）、成纤维细胞生长因子（FGF）2等具有诱导分化作用的细胞因子]进行体外培养。应用流式细胞术、免疫组织化学等方法检测CD133^＋细胞的比例、原代EPC的生长曲线及生长特性。透射电镜查找Weibel-Palade小体。同时设计EPC裸鼠成瘤实验，观察瘤体生长及血管增生情况，以鉴定EPC的生物学特性。结果 CD133^＋细胞在免疫磁珠分离前后的比例各为0．91％及85．52％。EPC原代培养时细胞形态正常、密度均匀，前3d为相对抑制期，此后快速增殖，10d后达80％～90％融合。EPC生长曲线显示，接种后5d内细胞数量无明显变化，从第7天开始明显增加，第17天达高峰。光学显微镜下可见，原代EPC贴壁后呈梭形，接种后7d数量明显增加，呈克隆状生长。透射电镜观察到，细胞膜伸出许多伪足丝，基底膜呈多层；细胞质内可见一种短棒状小体，含平行管状的内部结构，即Weibel-Palade小体，确定其为EPC。裸鼠成瘤实验可见EPC组肿瘤瘕体及血管数量大于或多于对照组；抗人血管性假血友病因子（vWF）免疫荧光染色显示，大量EPC参与肿瘤血管生成。结论本实验分离培养的CD133^＋细胞具有分化为成熟血管内皮细胞的能力，即为EPC。裸鼠成瘤实验初步证明EPC参与血管重建，具有促进血管新生、加速缺血组织血管化的作用。     

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。     

自体内皮祖细胞移植对急性心肌梗死近、中期心肌收缩力的影响

目的比较自体内皮祖细胞（EPCs）移植急性心肌梗死区域后近、中期心功能恢复的程度。方法将SD大鼠120只随机分为实验组和对照组（每组60只），抽取自体外周血，体外分离出内皮祖细胞。开胸结扎左冠状动脉前降支，实验组用自体内皮祖细胞经局部注射移植入急性心肌梗死区域；对照组以IMDM（Iscove＇s modified Dulbecco＇s Medium）作对照。术后3、6周、6、8和12个月取标本，体外药物刺激离体肌条检测心肌收缩情况。免疫组织化学法、计算机图象分析检测组织血管内皮生长因子（VEGF），碱性成纤维生长因子（bFGF），血管内皮第八因子（Ⅷ因子）的表达。结果实验组梗死区3、6周、6个月bFGF、VEGF灰度值明显高于对照组（P〈0．01），微血管计数较对照组明显增多（P〈0．01），心肌收缩力明显较对照组增强（P〈0．01），且随时间推移恢复程度增加（P〈0．05）。但从8个月开始，实验组以上指标不再明显增加。结论自体内皮祖细胞移植可能通过血管发生、血管新生等方式增加心肌细胞的收缩力，但中期疗效并不能随着时间推移而持续增加，在较长时间内保持稳定。     

硫酸锌对皮瓣缺血再灌注损伤保护作用的研究

目的 观察外源性锌对皮瓣缺血再灌注（ischemia—reperfusion，IR）损伤的保护作用，并探讨其机制。方法 48只大鼠随机分为非-IR组、IR组和补锌-IR组。在大鼠以腹壁浅血管为蒂的岛状皮瓣缺血再灌注模型上，分别测定皮瓣组织中丙二醛（MDA）含量和髓过氧化物酶（MPO）活性。观察免疫组化切片中金属硫蛋白（metallothionein，MT）的表达，并对切片进行图像分析。应用透射电镜观察皮瓣缺血再灌注损伤后超微结构的改变，观察皮瓣成活率。结果 补锌-IR组在再灌注1h和24h，皮瓣组织中MDA含量分别较IR组降低11．3％、33．2％（P〈0．05），MPO活性分别较IR组降低14．2％、22．7％（P〈0．05），MT含量分别较IR组高41．5％、44％（P〈0．01）。在补锌-IR组掀起皮瓣即刻的标本中有一定量的MT表达。MT表达在皮瓣组织多种细胞的细胞浆中。补锌-IR组皮瓣的超微结构改变较IR组减轻，皮瓣成活率较IR组升高27．2％（P〈0．05）。结论 外源性锌能诱导皮瓣内MT产生，MT通过细胞保护作用对皮瓣缺血再灌注损伤产生一定的保护作用。     

促血管生成素家族在大肠腺癌中的表达及与微血管密度和临床病理特征的关系

目的 探讨促血管生成素-1（Ang-1）、促血管生成素-2（Ang-2）、促血管生成素受体（Tie-2）及血管内皮生长因子（VEGF）在大肠腺癌及癌旁正常组织中的表达，及与微血管密度（MVD）和临床病理特征的关系。方法 采用免疫组织化学方法检测Ang-1、Ang-2、Tie-2及VEGF在45例大肠腺癌及10例癌旁正常组织中的表达。结果 大肠腺癌组织中的Ang-2蛋白及VEGF蛋白明显高于癌旁正常组织（P〈0．01），腺癌的分化程度越低，Ang-2及VEGF蛋白的表达率越高（P〈0．05），Ang-2与VEGF蛋白的表达存在明显正相关性（r=0．997，P〈0．01）；大肠腺癌组织中的Ang-1蛋白明显低于癌旁正常组织（P〈0．01），腺癌的分化程度越高，Ang-1蛋白的表达率越高（P〈0．05）；Tie-2蛋白在大肠腺癌和癌旁正常组织中的表达差异无统计学意义（P〉0．05）。大肠腺癌的分化程度越低，MVD越高（P〈0．05），腺癌组织中Ang-1蛋白阳性表达组MVD明显低于阴性表达组（P〈0．01），Ang-2蛋白阳性表达组MVD明显高于阴性表达组（P〈0．01）。≥5cm及有淋巴结转移的大肠腺癌组织中，Ang-2蛋白的表达明显增加（x^2=8．889，P〈0．01；x^2=10．020，P〈0．01）。结论 在大肠腺癌组织中，相对Ang-1占优势的Ang-2的过度表达，可能在肿瘤的血管生成和进展过程中起着重要作用。     

非小细胞肺癌淋巴结转移和预后与CD133和CD147表达的关系

目的观察肿瘤干细胞标志物CD133和CD147在非小细胞肺癌（NSCLC）组织中的表达及其临床意义。方法用免疫组织化学方法检测77例NSCLC组织中CD133与CD147的表达，分析其与患者肿瘤大小、组织学类型、组织分化程度、淋巴结转移和预后的关系。结果77例NSCLC组织中CD133与CD147的阳性表达率分别为51．9％（40／77）和67．5％（52／77）。CD133和CD147的表达与淋巴结转移呈正相关（r=0．246，P〈0．05；r=0．295，P〈0．05）；CD133和CD147的表达均与患者术后生存时间呈负相关（P〈0．05）。CD133和CD147的表达与肿瘤大小、组织学类型和组织分化程度均无明显相关（P〉0．05）。CD133与CD147的表达之间呈正相关（r=0．315，P〈0．05）。结论肿瘤干细胞标志物CD133和CD147的表达与NSCLC的淋巴转移和患者预后密切相关，它们的高表达提示预后不良。     

重组人VEGF基因在游离组织移植中作用的实验研究

目的：研究重细人血管内皮生长因子（VEGF）基因治疗皮瓣、皮片及游离颗粒脂肪移植存活的影响。方法：将带有PCD-VEGF165的大肠杆菌接种到LB培养基中，通过碱分裂法制备PCD-VEGF165质粒，再用反蒸发法将质粒包埋于脂质体中。将108只雄性SD大鼠均分为三组，每组36只，每组再按注射的成分不同分三小组，分别为PCD-VEGF165目的基因组、PCD空白质粒组和生理盐水组，各组分别注入目的基因、空白质粒和生理盐水后在不同时间点计算组织成活率，同时取标本做成石蜡块行常规染色及免疫组化染色，检测VEGF165的表达及血管生长情况。结果：目的基因组的皮瓣及皮片成活面积较空白质粒和生理盐水组明显大（P〈0．01），游离脂肪重量减少的程度亦显著小于空白质粒组和生理盐水组（P〈0．05），三种移植组织中目的基因组VEGF的表达及微血管密度均显著高于其他两对照组（P〈0．01），目的基因组的血管直径明显小于对照组（P〈0．05），空白质粒组及生理盐水组差异无显著性意义。结论：注射转VEGF基因的质粒后可在组织中表达丰富的VEGF，诱导新血管的形成，增加血流灌注，并减少移植组织的吸收，最终促进移植皮瓣、皮片及脂肪的存活。     

骨髓干细胞移植促进缺血肢体血管新生的研究

我们通过建立大鼠肢体缺血模型。采用骨髓内皮祖细胞(EPC)移植于缺血肢体，观察缺血组织血管新生及血管内皮生长因子(VEGF)的表达情况，探讨自体骨髓干细胞移植在治疗肢体缺血性疾病中的价值。     

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。     

Enhanced survival of ischemic skin flap by combined treatment with bone marrow-derived stem cells and low-level light irradiation

The aim of this study is to examine the enhanced survival effect of ischemic skin flap by combined treatment with bone marrow-derived stem cells (BMSCs) and low-level light irradiation (LLLI). The neovasculogenic effect of BMSCs induced by LLLI was detected using a wound healing and tube formation assay. ICR mice were divided into four groups: control group, LLLI group, BMSCs group, and combine-treated group. The percentage of skin flap necrosis area was calculated on the seventh post-operative day. Specimens were harvested for histologic analyses. LLLI promoted BMSC migration and tube formation. The flap survival rate of combined treated group was significantly higher than that of the control group. Histologic results demonstrated a significant increase in neovascularization in the combined treatment group. This study demonstrates that combination treatment of BMSCs and LLLI could enhance the survival of ischemic skin flap in a mouse model.     

天然水蛭素对大鼠缺血皮瓣血管生成作用的Micro-CT观察

目的通过Micro-CT及三维重建技术观察天然水蛭素对大鼠缺血皮瓣血管生成的影响。方法选取32只成年SD大鼠,在其背部制备一8.0cm×1.8cm大小的缺血皮瓣模型,然后随机分为水蛭素组和对照组(n=16)。水蛭素组术后即刻和术后3 d内每天皮下注射天然水蛭素0.3 mL(含天然水蛭素6 ATU),对照组皮下注射等量生理盐水。术后6 d,大体观察皮瓣成活情况并测定皮瓣成活率,取材行HE染色观察皮瓣组织学变化,行Micro-CT三维重建观察并测量皮瓣血管容积、长度及数量。结果术后两组大鼠均存活至实验完成,无感染发生。术后6 d,两组皮瓣远端均发生不同程度坏死,水蛭素组皮瓣成活率为72.11%±8.97%,显著高于对照组的58.94%±4.02%,差异有统计学意义(t=3.280,P=0.008)。组织学观察示水蛭素组较对照组组织结构层次更清楚,有较多微血管生成,炎症反应及炎症细胞浸润更轻。Micro-CT三维重建示水蛭素组皮瓣血管更多、更加密集;血管容积、长度及数量均显著大于对照组(P<0.05)。结论天然水蛭素可减轻组织炎症反应、促进缺血皮瓣血管的新生与再通,从而提高皮瓣成活率。     

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     

Controlled delivery of bFGF to recipient bed enhances the vascularization and viability of an ischemic skin flap

Therapeutic angiogenesis is a promising approach to treat ischemic skin flaps. We delivered basic fibroblast growth factor (bFGF) to the recipient bed of a rat dorsal skin flap by a drug delivery system with acidic gelatin hydrogel microspheres (AGHMs), and assessed augmentation of neovascularization and flap viability. An axial skin flap was elevated on the back of male Sprague–Dawley rats, and bFGF solution or bFGF-impregnated AGHMs were injected into the recipient bed. The dose of bFGF in the bFGF solution was set to 15 (Sol-15 group), 50 (Sol-50 group), or 150 μg (Sol-150 group). Correspondingly, 2 mg AGHMs were impregnated with 15 (AGHM-15 group), 50 (AGHM-50 group), or 150 μg (AGHM-150 group) bFGF. Other groups of animals received phosphate-buffered saline (Sol-Cont group) or phosphate-buffered saline-impregnated AGHMs (AGHM-Cont group) as controls. Seven days later, analyses of the area of necrosis, microangiographic findings, and histological findings in the flap were carried out. The area of necrosis in the AGHM-150 group was significantly smaller than that in the other groups. Microangiographic and histological analyses showed that neovascularization of the ischemic skin flap significantly increased in the AGHM-150 group as compared with the Sol-150 group and the AGHM-Cont group. These findings suggest that continuous delivery of bFGF to the recipient bed by bFGF-impregnated AGHMs enhances the viability of an ischemic skin flap.     

Diabetic wound healing in a MMP9‐/‐ mouse model

Reduced mobilization of endothelial progenitor cells (EPCs) from the bone marrow (BM) and impaired EPC recruitment into the wound represent a fundamental deficiency in the chronic ulcers. However, mechanistic understanding of the role of BM‐derived EPCs in cutaneous wound neovascularization and healing remains incomplete, which impedes development of EPC‐based wound healing therapies. The objective of this study was to determine the role of EPCs in wound neovascularization and healing both under normal conditions and using single deficiency (EPC) or double‐deficiency (EPC + diabetes) models of wound healing. MMP9 knockout (MMP9 KO) mouse model was utilized, where impaired EPC mobilization can be rescued by stem cell factor (SCF). The hypotheses were: (1) MMP9 KO mice exhibit impaired wound neovascularization and healing, which are further exacerbated with diabetes; (2) these impairments can be rescued by SCF administration. Full‐thickness excisional wounds with silicone splints to minimize contraction were created on MMP9 KO mice with/without streptozotocin‐induced diabetes in the presence or absence of tail‐vein injected SCF. Wound morphology, vascularization, inflammation, and EPC mobilization and recruitment were quantified at day 7 postwounding. Results demonstrate no difference in wound closure and granulation tissue area between any groups. MMP9 deficiency significantly impairs wound neovascularization, increases inflammation, decreases collagen deposition, and decreases peripheral blood EPC (pb‐EPC) counts when compared with wild‐type (WT). Diabetes further increases inflammation, but does not cause further impairment in vascularization, as compared with MMP9 KO group. SCF improves neovascularization and increases EPCs to WT levels (both nondiabetic and diabetic MMP9 KO groups), while exacerbating inflammation in all groups. SCF rescues EPC‐deficiency and impaired wound neovascularization in both diabetic and nondiabetic MMP9 KO mice. Overall, the results demonstrate that BM‐derived EPCs play a significant role during wound neovascularization and that the SCF‐based therapy with controlled inflammation could be a viable approach to enhance healing in chronic diabetic wounds.     

骨髓血管内皮祖细胞移植提高缺血皮瓣存活率的研究

目的：探讨大鼠骨髓来源的血管内皮祖细胞（endothelial progenitor cells,EPCs）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：在Wistar大鼠背部形成2cm×8cm蒂部位于双侧髂棘连线上的随意型超比例皮瓣,建立缺血皮瓣模型。采用密度梯度离心法从Wistar大鼠骨髓中分离出骨髓单个核细胞,并在专有的诱导培养基（EG-M2MV）中培养,贴壁筛选法分离,诱导分化为EPCs;对细胞的形态、表型、功能加以鉴定。将EPCs注射移植于iWstar大鼠背部的缺血皮瓣,大体观察皮瓣的存活率,vWF免疫酶染色法检测皮瓣毛细血管密度。以缺血皮瓣单纯注射磷酸盐缓冲液（PBS）的Wistar大鼠作为对照组。结果：骨髓中分离培养的EPCs呈现典型的＂铺路石＂样外观,表达CD34、Flk-1及CDl 33表型,能够摄取DIL-ac-LDL和特异性结合FITC-UEA-1。注射EPCs组的缺血皮瓣存活率以及毛细血管密度显著高于对照组（P〈0.05）。结论：骨髓中的EPCs在体外培养后注射移植于皮瓣内,可以促进缺血性皮瓣的血管新生,提高缺血性皮瓣的存活率。