本文選題：肝細胞生長因子 + 血管內(nèi)皮祖細胞��； 參考：《天津醫(yī)科大學》2012年碩士論文

【摘要】：目的 體外條件下從大鼠骨髓中分離出單個核細胞,使用特定的培養(yǎng)基使其向內(nèi)皮祖細胞(endothelial progenitor cells,EPCs)分化生長,以Ad-GFP缺陷性腺病毒為載體將HGF基因轉染原代培養(yǎng)的大鼠血管內(nèi)皮祖細胞,之后注入缺血后肢裸鼠體內(nèi),觀察其體內(nèi)血管生成能力。 方法 1.取4-6周齡Wistar大鼠(120-150g)雙下肢股骨及脛骨骨髓,利用密度梯度離心法分離單個核細胞,并使用10%胎牛血清(FBS)DMEM誘導培養(yǎng),使其分化為血管內(nèi)皮祖細胞。 2.以缺陷性腺病毒(Ad-GFP)為載體介導HGF基因轉染血管內(nèi)皮祖細胞并計算轉染效率 3.建立裸鼠缺血后肢模型后,將裸鼠分為3組,分別為空白對照組、EPCs組、HGF病毒轉染EPCs組(Ad-HGF-EPCs),損傷后即刻及24h后分別將生理鹽水、內(nèi)皮祖細胞、攜帶HGF基因的EPCs以2×108cells細胞數(shù)由尾靜脈注入裸鼠尾靜脈。控制各鼠都輸入同樣生理鹽水。觀察期缺血肢體表現(xiàn),ELISA檢測血清HGF表達水平,激光多普勒(LDPI)檢測血流灌注,CD31免疫組化檢測毛細血管密度。 結果 1.成功分離和培養(yǎng)出大鼠骨髓血管內(nèi)皮祖細胞,EPCs攝取Dil-acLDL、結合FITC-UEA-1雙熒光細胞陽性率為(88.0±5.0)%。 2.成功將HGF基因轉染大鼠骨髓血管內(nèi)皮祖細胞,熒光顯微鏡下可見綠色熒光蛋白的表達,轉染72h后,轉染效率高達80%以上。 3.實驗中成功建立缺血后肢模型。術后2天,三組裸鼠缺血后肢功能評分迅速升至高峰,證實建模成功。HGF基因轉染EPCs移植后,肢體自截率及壞死率明顯降低,缺血后肢功能評分明顯較EPCs組低,證實移植后有利于肢體存活和功能恢復。而且ELISA證實Ad-HGF-EPCs移植后體內(nèi)HGF蛋白持續(xù)高表達,術后2天后即達峰值,此后逐漸降低,但仍高于未轉染EPCs移植及空白對照,并至少持續(xù)21天。激光多普勒及CD31免疫組化檢測發(fā)現(xiàn)HGF基因轉染EPCs移植后,缺血后肢血流灌注量和血管密度顯著增加,作用遠強于EPCs單獨移植。 結論 腺病毒介導的HGF基因轉染血管內(nèi)皮祖細胞移植缺血后肢裸鼠后,可在體內(nèi)持續(xù)過表達HGF蛋白,促進缺血后肢的血管生成,增加血管密度和血流量,改善后肢循環(huán),有利于肢體存活和功能恢復。
[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 鹵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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

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本文編號：1940094