HGF基因转染血管内皮祖细胞促进血管生成的动物实验研究

发布时间：2018-05-27 02:39

本文选题：肝细胞生长因子 + 血管内皮祖细胞　；参考：《天津医科大学》2012年硕士论文

【摘要】：目的体外条件下从大鼠骨髓中分离出单个核细胞,使用特定的培养基使其向内皮祖细胞(endothelial progenitor cells,EPCs)分化生长,以Ad-GFP缺陷性腺病毒为载体将HGF基因转染原代培养的大鼠血管内皮祖细胞,之后注入缺血后肢裸鼠体内,观察其体内血管生成能力。方法 1.取4-6周龄Wistar大鼠(120-150g)双下肢股骨及胫骨骨髓,利用密度梯度离心法分离单个核细胞,并使用10%胎牛血清(FBS)DMEM诱导培养,使其分化为血管内皮祖细胞。 2.以缺陷性腺病毒(Ad-GFP)为载体介导HGF基因转染血管内皮祖细胞并计算转染效率 3.建立裸鼠缺血后肢模型后,将裸鼠分为3组,分别为空白对照组、EPCs组、HGF病毒转染EPCs组(Ad-HGF-EPCs),损伤后即刻及24h后分别将生理盐水、内皮祖细胞、携带HGF基因的EPCs以2×108cells细胞数由尾静脉注入裸鼠尾静脉。控制各鼠都输入同样生理盐水。观察期缺血肢体表现,ELISA检测血清HGF表达水平,激光多普勒(LDPI)检测血流灌注,CD31免疫组化检测毛细血管密度。结果 1.成功分离和培养出大鼠骨髓血管内皮祖细胞,EPCs摄取Dil-acLDL、结合FITC-UEA-1双荧光细胞阳性率为(88.0±5.0)%。 2.成功将HGF基因转染大鼠骨髓血管内皮祖细胞,荧光显微镜下可见绿色荧光蛋白的表达,转染72h后,转染效率高达80%以上。 3.实验中成功建立缺血后肢模型。术后2天,三组裸鼠缺血后肢功能评分迅速升至高峰,证实建模成功。HGF基因转染EPCs移植后,肢体自截率及坏死率明显降低,缺血后肢功能评分明显较EPCs组低,证实移植后有利于肢体存活和功能恢复。而且ELISA证实Ad-HGF-EPCs移植后体内HGF蛋白持续高表达,术后2天后即达峰值,此后逐渐降低,但仍高于未转染EPCs移植及空白对照,并至少持续21天。激光多普勒及CD31免疫组化检测发现HGF基因转染EPCs移植后,缺血后肢血流灌注量和血管密度显著增加,作用远强于EPCs单独移植。结论腺病毒介导的HGF基因转染血管内皮祖细胞移植缺血后肢裸鼠后,可在体内持续过表达HGF蛋白,促进缺血后肢的血管生成,增加血管密度和血流量,改善后肢循环,有利于肢体存活和功能恢复。
[Abstract]:Purpose Mononuclear cells were isolated from rat bone marrow in vitro and differentiated into endothelial progenitor cells (EPCs). HGF gene was transfected into primary cultured rat vascular endothelial progenitor cells (VECs) using Ad-GFP deficient adenovirus as vector. Then the ischemic hind limb nude mice were injected to observe the angiogenesis ability in vivo. Method 1. Bone marrow of femur and tibia of 4-6 week-old Wistar rats were isolated by density gradient centrifugation. The mononuclear cells were cultured with 10% fetal bovine serum (FBS) DMEM to differentiate them into vascular endothelial progenitor cells (VECs). 2. Transfection of HGF Gene into Vascular Endothelial progenitor cells mediated by Ad-GFPV and its transfection efficiency 3. After establishing the model of hind limb ischemia in nude mice, the nude mice were divided into three groups: the blank control group was transfected with HGF virus into EPCs group, and the normal saline and endothelial progenitor cells were obtained immediately after injury and 24 hours after injury. EPCs carrying HGF gene was injected into the caudal vein of nude mice with 2 脳 108cells cells from the caudal vein. Control the rats to be given the same saline. The expression of HGF in serum was detected by Elisa and the capillary density was detected by immunohistochemistry. Result 1. EPCs from rat bone marrow vascular endothelial progenitor cells (EPCs) were successfully isolated and cultured. The positive rate of double fluorescent cells combined with FITC-UEA-1 was 88.0 卤5.0%. 2. The HGF gene was successfully transfected into rat bone marrow vascular endothelial progenitor cells. The expression of green fluorescent protein was observed under fluorescence microscope. After 72 hours of transfection, the transfection efficiency was more than 80%. 3. The model of hindlimb ischemia was established successfully. On the 2nd day after operation, the functional scores of ischemic hind limbs in the three groups increased rapidly to the peak. It was confirmed that the self-cutting rate and necrosis rate of limbs were significantly decreased after EPCs transplantation, and the functional score of ischemic hind limbs was significantly lower than that of EPCs group. It was proved that the graft was beneficial to limb survival and functional recovery. ELISA confirmed that the expression of HGF protein continued to be high after Ad-HGF-EPCs transplantation, peaked 2 days after transplantation, then decreased gradually, but still higher than that of untransfected EPCs transplantation and blank control, and lasted for at least 21 days. The results of laser Doppler and CD31 immunohistochemistry showed that the blood flow perfusion and blood vessel density of ischemic hind limb increased significantly after EPCs transplantation with HGF gene transfection, and the effect was much stronger than that of EPCs transplantation alone. Conclusion Adenovirus mediated HGF gene transfection of vascular endothelial progenitor cells into ischemic hind limb nude mice can continuously express HGF protein in vivo, promote angiogenesis of ischemic hind limb, increase vascular density and blood flow, and improve hind limb circulation. Good for limb survival and functional recovery.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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