Department of Vascular Surgery, Second Affiliated Hospital of Suzhou University, Suzhou, Jiangsu 215004, China.
Chin Med J (Engl). 2010 Feb 20;123(4):471-7.
The organization and recanalization of thrombi is a dynamic and complex process. The aim of this research was to study the cotherapeutic effect of stem cell transplantation and gene transfection on chronic venous thrombosis.
We constructed a recombinant adenoviral vector carrying the vascular endothelial growth factor 165 (VEGF165) gene by using the pAdEasy system, which was subsequently identified and amplified. Simultaneously, endothelial progenitor cells (EPCs) were isolated from rat bone marrow using Ficoll, cultured in EBM-2MV medium, and identified. Then, the cells were transfected with the recombinant Ad-VEGF165. The EPCs were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (Dil) before transplantation. A rat model of chronic vein thrombosis was developed by partial ligation of the inferior vena cava. The rats were randomly divided into 4 groups (n = 25, each): A, Ad-VEGF165/EPC-transplantation group received 1 ml (10(6)) of Ad-VEGF165/EPCs; B, EPC-transplantation group received 1 ml (10(6)) of EPCs; C, Ad/EPC-transplantation group received 1 ml (10(6)) of Ad/EPCs; D, control group received 1 ml of the transplantation medium. The thrombi and adjacent caval walls were harvested 28 days after transplantation; real-time quantitative polymerase chain reaction was used to detect the expression level of vascular endothelial growth factor (VEGF) mRNA; and western blotting was used to measure changes in VEGF protein expression. Hematoxylin-eosin staining and immunohistochemical staining were performed to detect recanalization. Neovascularization was detected by immunohistochemical staining using the antibody for von Willebrand factor (vWF), which is a component of endothelial cells. The capillary density was quantitatively determined by counting the capillaries under a high-power microscope.
The Ad-VEGF165 was constructed, and bone-marrow-derived EPCs were cultivated and successfully identified. We determined the optimum transfection ratio that promoted the growth of EPCs. After transfection, the EPCs secreted the VEGF protein. After transplantation, the in vivo survival of EPCs and their differentiation into endothelial cells were determined by detecting the fluorescence associated with the Dil stain. VEGF mRNA was expressed in groups A, B, C and D after transplantation, and the VEGF mRNA level in group A was significantly higher than those in groups B, C and D (P < 0.05); the VEGF mRNA levels in groups B and C were significantly higher than those in group D (P < 0.05), and there was no statistical significance between the VEGF mRNA levels in groups B and C. The recanalization capillary density in group A was significantly higher than those in groups B, C (P < 0.05) and D (P < 0.01); the recanalization capillary densities in groups B and C were significantly higher than that in group D (P < 0.05). Moreover, there was no statistical significant difference between the values for groups B and C.
The EPCs were successfully transfected by Ad-VEGF165. A suitable transfection ratio can improve the efficiency of EPCs and the possibility of promotion of angiogenesis after transplantation. Transfected EPCs caused accelerated organization and recanalization of vein thrombi.
血栓的组织和再通是一个动态而复杂的过程。本研究旨在研究干细胞移植和基因转染对慢性静脉血栓形成的协同治疗作用。
我们使用 pAdEasy 系统构建了携带血管内皮生长因子 165（VEGF165）基因的重组腺病毒载体，然后对其进行鉴定和扩增。同时，我们使用 Ficoll 从大鼠骨髓中分离内皮祖细胞（EPCs），在 EBM-2MV 培养基中培养，并进行鉴定。然后，将重组 Ad-VEGF165 转染 EPCs。在移植前，用 1,1'-二辛基-3,3,3',3'-四甲基吲哚羰花青（Dil）标记 EPCs。通过下腔静脉部分结扎建立大鼠慢性静脉血栓模型。将大鼠随机分为 4 组（每组 n = 25）：A 组：接受 1ml（10(6))的 Ad-VEGF165/EPCs；B 组：接受 1ml（10(6))的 EPCs；C 组：接受 1ml（10(6))的 Ad/EPCs；D 组：接受 1ml 的移植培养基。移植后 28 天采集血栓和相邻腔静脉壁；实时定量聚合酶链反应检测血管内皮生长因子（VEGF）mRNA 的表达水平；western blot 检测 VEGF 蛋白表达的变化。苏木精-伊红染色和免疫组织化学染色检测再通情况。使用 von Willebrand 因子（vWF）抗体（内皮细胞的组成部分）进行免疫组织化学染色检测新生血管。在高倍显微镜下通过计数毛细血管定量测定毛细血管密度。
构建了 Ad-VEGF165，培养并成功鉴定了骨髓来源的 EPCs。我们确定了促进 EPCs 生长的最佳转染比例。转染后，EPCs 分泌 VEGF 蛋白。移植后，通过检测与 Dil 染色相关的荧光来确定 EPCs 的体内存活及其向内皮细胞的分化。移植后 A、B、C 和 D 组均表达 VEGF mRNA，A 组 VEGF mRNA 水平明显高于 B、C 和 D 组（P < 0.05）；B 和 C 组 VEGF mRNA 水平明显高于 D 组（P < 0.05），B 和 C 组之间 VEGF mRNA 水平无统计学差异。A 组再通毛细血管密度明显高于 B、C 组（P < 0.05）和 D 组（P < 0.01）；B 和 C 组再通毛细血管密度明显高于 D 组（P < 0.05）。此外，B 和 C 组之间的数值无统计学差异。
EPCs 被 Ad-VEGF165 成功转染。合适的转染比例可以提高 EPCs 的效率，并增加移植后促进血管生成的可能性。转染后的 EPCs 加速了静脉血栓的组织和再通。
