本文選題：血管內(nèi)皮生長(zhǎng)因子 + 轉(zhuǎn)染　； 參考：《河北醫(yī)科大學(xué)》2015年博士論文

【摘要】：糖尿病外周血管病變(diabetic peripheral artery disease,PAD)是糖尿病(diabetic mellitus,DM)嚴(yán)重的慢性并發(fā)癥之一,與非DM患者相比,其主要病理改變?yōu)閯?dòng)脈粥樣硬化,且多累及下肢遠(yuǎn)端動(dòng)脈,病變范圍更廣、呈多節(jié)段彌漫性的狹窄或閉塞,是導(dǎo)致DM足部壞疽、截肢的主要原因,嚴(yán)重影響DM患者的生存質(zhì)量。目前傳統(tǒng)內(nèi)科藥物、介入和手術(shù)治療等對(duì)遠(yuǎn)端血管閉塞、流出道差的DM患者效果不佳,不能從根本上解決問(wèn)題,并且此類(lèi)DM患者多數(shù)高齡體弱,手術(shù)風(fēng)險(xiǎn)大,合并多種心腦血管病變,病情復(fù)雜,因此臨床治療上相當(dāng)棘手,迫切需要尋求新的技術(shù)手段,如何促進(jìn)血管新生實(shí)現(xiàn)血運(yùn)重建成為治療關(guān)鍵。近年來(lái)干細(xì)胞移植成為發(fā)展迅速的一種全新治療模式,其中骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,BMSCs)已被證實(shí)是一類(lèi)具有多向分化潛能的干細(xì)胞,可以在一定的誘導(dǎo)條件下分化為骨、軟骨、脂肪、神經(jīng)、心肌、血管內(nèi)皮細(xì)胞等,參與不同組織的修復(fù),但目前仍缺乏移植細(xì)胞在體內(nèi)存活、分化、轉(zhuǎn)歸及參與血管再生的實(shí)驗(yàn)依據(jù)可循,且骨髓MSCs采集風(fēng)險(xiǎn)較大,對(duì)患者年齡、身體條件、心理接受程度要求較高,在實(shí)際應(yīng)用中,由于高糖、氧化應(yīng)激、低氧等微環(huán)境使移植后MSCs的存活率非常低,新生血管形成速度慢等,極大地限制了干細(xì)胞移植治療的效果。相比之下,臍帶間充質(zhì)干細(xì)胞(human umbilical cord mesenchymal stem cells,h UC-MSCs)與BMSCs在生物學(xué)特性方面極為相似,由于其來(lái)源更廣泛,采集方便,擴(kuò)增力可塑性強(qiáng),無(wú)免疫原性,不存在倫理學(xué)爭(zhēng)議,具有更加有效的MSCs潛能,有可能成為BMSCs的理想替代物,成為目前誘導(dǎo)分化最佳的種子細(xì)胞。對(duì)血循環(huán)的重建而言,目前已知有多種細(xì)胞因子和生長(zhǎng)因子參與促進(jìn)血管生成作用,其中血管內(nèi)皮生長(zhǎng)因子(Vascular endothelial growth factor,VEGF)通過(guò)與其血管內(nèi)皮特異性受體結(jié)合,可顯著促進(jìn)內(nèi)皮增生及血管生成作用,被認(rèn)為是機(jī)體內(nèi)最強(qiáng)的促血管生長(zhǎng)因子,但直接應(yīng)用VEGF治療存在很多不足,如半衰期短、提純較為困難、應(yīng)用量大、成本昂貴等,限制其臨床應(yīng)用。因此,如何提高M(jìn)SCs在缺血組織存活、分化,增加局部組織生長(zhǎng)因子分泌量,促進(jìn)新生血管形成,提高M(jìn)SCs治療PAD療效是目前亟待解決的主要問(wèn)題。本研究利用基因工程構(gòu)建VEGF-EGFP基因表達(dá)載體,通過(guò)腺病毒轉(zhuǎn)染到h UC-MSCs細(xì)胞中,觀察基因轉(zhuǎn)染后對(duì)h UC-MSCs的生長(zhǎng)增殖及目的基因VEGF表達(dá)情況;并利用增強(qiáng)型綠色熒光蛋白(enhanced green fluorescent protein,EGFP)實(shí)現(xiàn)轉(zhuǎn)染后h UC-MSCs的活體示蹤定位;同時(shí)建立高脂喂養(yǎng)2型糖尿病SD大鼠下肢缺血模型,將轉(zhuǎn)染后的h UC-MSCs局部肌肉注射,觀察其向血管內(nèi)皮分化促血管新生,側(cè)支循環(huán)的建立,改善下肢缺血的實(shí)驗(yàn)療效;本研究應(yīng)用h UC-MSCs作為VEGF基因治療平臺(tái),不僅可通過(guò)提高VEGF持續(xù)分泌,達(dá)到局部穩(wěn)定的治療濃度,并通過(guò)VEGF的抗炎、抗氧化應(yīng)激、抗凋亡、促進(jìn)血管生成等作用改善缺血后微環(huán)境,為h UC-MSCs增殖、分化等過(guò)程提供最佳的生存空間;同時(shí)可放大h UC-MSCs的旁分泌作用,減少h UC-MSCs凋亡,提高h(yuǎn) UC-MSCs移植后的定位歸巢和存活率等,有效發(fā)揮VEGF與h UC-MSCs在血管再生功效方面的協(xié)同倍增作用,從而為更好的發(fā)揮h UC-MSCs移植療效提供實(shí)驗(yàn)基礎(chǔ)。本研究分為三部分:第一部分腺病毒介導(dǎo)VEGF轉(zhuǎn)染臍帶間充質(zhì)干細(xì)胞的實(shí)驗(yàn)研究目的:探討腺病毒載體介導(dǎo)VEGF轉(zhuǎn)染h UC-MSCs的可行性,以及VEGF轉(zhuǎn)染對(duì)h UC-MSCs形態(tài)及功能的影響。方法:構(gòu)建VEGF-EGFP重組基因腺病毒載體,分離和培養(yǎng)h UC-MSCs,分為VEGF-EGFP轉(zhuǎn)染組、EGFP空載組及對(duì)照組,熒光倒置顯微鏡觀察細(xì)胞轉(zhuǎn)染效果,流式細(xì)胞儀測(cè)定細(xì)胞轉(zhuǎn)染效率,確定最佳病毒感染復(fù)數(shù)(multiplicities of infection,MOI);依據(jù)最佳MOI值轉(zhuǎn)染并收集細(xì)胞,應(yīng)用蛋白印跡法(Western blot)、RT-PCR檢測(cè)轉(zhuǎn)染后目的基因VEGF的蛋白及m RNA表達(dá)情況;酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)細(xì)胞培養(yǎng)上清VEGF蛋白水平,MTT法及流式細(xì)胞術(shù)評(píng)價(jià)基因轉(zhuǎn)染對(duì)h UC-MSCs增殖和細(xì)胞周期的影響。結(jié)果:熒光顯微鏡及流式細(xì)胞儀檢測(cè)提示腺病毒介導(dǎo)的VEGF-EGFP基因能夠成功轉(zhuǎn)染h UC-MSCs,且轉(zhuǎn)染效率高,對(duì)細(xì)胞結(jié)構(gòu)形態(tài)無(wú)影響;目的基因VEGF在細(xì)胞內(nèi)能夠轉(zhuǎn)錄和表達(dá),并能分泌到細(xì)胞外,轉(zhuǎn)染后24h采用ELISA法在細(xì)胞培養(yǎng)上清中就檢測(cè)到有VEGF的表達(dá)和分泌,96h仍穩(wěn)定表達(dá);與對(duì)照組和EGFP空載組相比,通過(guò)Western blot、RT-PCR檢測(cè)VEGF-EGFP轉(zhuǎn)染組VEGF表達(dá)水平升高顯著,且表達(dá)穩(wěn)定,可提高h(yuǎn) UC-MSCs的抗凋亡及存活能力;MTT及流式細(xì)胞法檢測(cè)結(jié)果顯示VEGF基因轉(zhuǎn)染可提高h(yuǎn) UC-MSCs增殖和分化能力。結(jié)論:腺病毒介導(dǎo)VEGF基因能夠成功轉(zhuǎn)染h UC-MSCs,能持續(xù)穩(wěn)定、高效表達(dá)VEGF,改善生存微循環(huán),提高h(yuǎn) UC-MSCs的增殖及分化、存活能力,為開(kāi)展VEGF基因轉(zhuǎn)染h UC-MSCs移植治療改善糖尿病下肢血管病變的可行性提供理論依據(jù)。第二部分高脂喂養(yǎng)2型糖尿病大鼠下肢缺血模型建立目的:目前普遍采用大鼠后肢股動(dòng)脈結(jié)扎離斷的方法來(lái)制備后肢急性缺血模型,但對(duì)于如何建立及評(píng)估糖尿病高脂高血糖慢性動(dòng)脈硬化閉塞癥的缺血狀態(tài),尚無(wú)一個(gè)穩(wěn)定有效慢性缺血模型及方法。方法:將大鼠20只隨機(jī)分為2組,DM組10只予以高脂喂養(yǎng)6個(gè)月,腹腔注射(Streptozocin,STZ)(35mg/kg)誘發(fā)糖尿病模型,對(duì)照組(10只)予以普食喂養(yǎng)6個(gè)月,成模DM組及對(duì)照組大鼠在麻醉后,消毒鋪巾,沿右下肢正中的皮膚縱行切開(kāi),于腹股溝下分離出股動(dòng)脈,在腹股溝韌帶下切斷股動(dòng)脈,近端結(jié)扎,隨后向遠(yuǎn)端銳性剝離直至膝關(guān)節(jié),分離和結(jié)扎股動(dòng)脈的所有分支,造成右下肢缺血模型,術(shù)后3d、7d、14d、28d觀察大鼠患肢活動(dòng)狀況、肢體顏色、皮溫等,并于術(shù)后1d、3d、14d、28d常規(guī)麻醉后,保持溫度、光線(xiàn)相對(duì)恒定下,應(yīng)用Peri Scan PIM3激光多普勒(Laser Doppler Perfusion Imaging,LDPI)行下肢血流監(jiān)測(cè)。術(shù)后28d麻醉動(dòng)物后,切開(kāi)后腹膜,于腹主動(dòng)脈留置套管針,肝素鈉抗凝,以2 ml/s的速率注射造影劑約1.5 ml進(jìn)行CT下肢血管造影。每組動(dòng)物在血管造影后處死,分別取其健側(cè)和患側(cè)股四頭肌和腓腸肌行蘇木精-伊紅染色及CD31免疫組織化學(xué)染色、Western blot測(cè)定肌肉組織VEGF含量。結(jié)果:DM組有(8只)對(duì)照組(10只)制備成后肢缺血模型,術(shù)后第1d,兩組大鼠多普勒血流及CT血管造影均呈顯著降低提示缺血造模成功,兩組術(shù)后第7d和14d行激光多普勒提示缺血肢體血流有逐漸恢復(fù)趨勢(shì),術(shù)后第28d DM組血流恢復(fù)較對(duì)照組顯著遲緩(P0.05);CT血管造影:DM組右下肢股動(dòng)脈結(jié)扎處近端僅有少量血管代償性增加,遠(yuǎn)心端仍無(wú)明顯血流;病理組織及免疫組化染色:術(shù)后第28d DM組缺血部位肌肉組織出現(xiàn)組織結(jié)構(gòu)破壞,炎性細(xì)胞浸潤(rùn),毛細(xì)血管密度患側(cè)低于健側(cè);缺血肌肉組織VEGF較對(duì)照組的蛋白表達(dá)明顯增加(P0.05)。結(jié)論:長(zhǎng)期高脂喂養(yǎng)糖尿病模型基礎(chǔ)上,制作股動(dòng)脈結(jié)扎離斷的下肢缺血模型,更接近于糖尿病下肢慢性缺血的情況,320排CT血管造影技術(shù)可以更立體直觀評(píng)估缺血狀態(tài),為進(jìn)一步探討糖尿病下肢缺血病理機(jī)制及干細(xì)胞治療促血管新生等提供了較為理想的動(dòng)物模型及評(píng)估指標(biāo)。第三部分VEGF-EGFP轉(zhuǎn)染臍帶間充質(zhì)干細(xì)胞移植治療改善糖尿病大鼠下肢缺血的實(shí)驗(yàn)研究目的:通過(guò)腺病毒將重構(gòu)的VEGF-EGFP基因轉(zhuǎn)染到h UC-MSCs細(xì)胞中,同時(shí)建立高脂喂養(yǎng)2型糖尿病SD大鼠下肢缺血模型,將轉(zhuǎn)染后h UC-MSCs局部下肢肌肉注射,觀察血管新生,側(cè)支循環(huán)建立,下肢缺血改善的實(shí)驗(yàn)療效。方法:利用高脂飲食STZ誘導(dǎo)的2型糖尿病SD大鼠下肢股動(dòng)脈結(jié)扎建立缺血模型后,將分組觀察VEGF-EGFP-h UC-MSCs、EGFP-h UC-MSCs、h UC-MSCs及PBS局部肌肉注射到下肢缺血部位,熒光倒置顯微鏡觀察移植細(xì)胞存活及定位;在治療后2、4周,激光多普勒(瑞典Peimed,LDPI)檢測(cè)局部血流;觀察下肢活動(dòng)度和缺血情況;4周后利用腹主動(dòng)脈結(jié)扎行下肢動(dòng)脈CTA(東芝320層CT機(jī))造影檢測(cè)雙下肢血管側(cè)支循環(huán)的形成;肌肉組織HE染色和CD31免疫組化檢測(cè)新生毛細(xì)血管數(shù)量及密度;RTPCR、Western blot等檢測(cè)組織標(biāo)本中VEGF及MMP2,MMP9,TIMP1,TIMP2,ERK,AKt相關(guān)基因m RNA及蛋白的表達(dá)等。結(jié)果:移植后1、2、4周熒光顯微鏡下發(fā)現(xiàn),在下肢缺血部位有移植的EGFP標(biāo)記的h UC-MSCs存活;移植后2、4周LDPI血流圖顯示VEGF-EGFP轉(zhuǎn)染組血流灌注的恢復(fù)水平要明顯高于EGFP空載組,移植4周后CT血管造影顯示VEGF-EGFP轉(zhuǎn)染組有新生側(cè)支循環(huán),HE及免疫組化染色顯示新生毛細(xì)血管明顯高均于空載組,RT-PCR及Western blot檢測(cè)組織標(biāo)本VEGF-EGFP轉(zhuǎn)染組VEGF、ERK、AKt、MMP2和MMP9 m RNA和蛋白水平較其他兩組顯著增高,TIMP1、TIMP2的表達(dá)無(wú)明顯差異。結(jié)論:肌肉注射移植VEGF基因修飾后h UC-MSCs可在下肢缺血組織中定植存活,高效表達(dá)VEGF,促進(jìn)內(nèi)皮修復(fù),比單純h UC-MSCs更能有效地促進(jìn)血管新生,明顯改善糖尿病下肢缺血狀態(tài),為h UC-MSCs聯(lián)合基因治療糖尿病下肢血管病變提供新的理論依據(jù)。
[Abstract]:Diabetic peripheral vascular disease (diabetic peripheral artery disease, PAD) is one of the serious chronic complications of diabetes (diabetic mellitus, DM). Compared with non DM, the main pathological changes are atherosclerosis and many of the distal arteries of the lower extremity are involved in a wider range of lesions, with multiple segmental diffuse stenosis or occlusion, which leads to DM. Foot gangrene, the main cause of amputation, seriously affects the quality of life of DM patients. At present, the traditional medicine, intervention and surgical treatment are not effective for the distal vascular occlusion and the DM patients with poor outflow, and can not solve the problem fundamentally, and most of these DM patients are weak in age, the operation risk is large, and many kinds of cardiovascular and cerebrovascular diseases are merged. The clinical treatment is very difficult, so it is urgent to seek new techniques. How to promote blood vessel revascularization is the key to the treatment. In recent years, stem cell transplantation has become a rapid development of a new treatment model, in which bone marrow mesenchymal stem cells (BMSCs) has been proved to be A class of stem cells with multiple differentiation potential can differentiate into bone, cartilage, fat, nerve, myocardium, vascular endothelial cells and so on under certain induction conditions, and participate in the repair of different tissues. However, there is still a lack of experimental basis for the survival of the transplanted cells in the body, differentiation, transformation and involvement of vascular regeneration, and the risk of collecting bone marrow MSCs is more than that of the bone marrow. In practical application, the survival rate of MSCs after transplantation is very low and the rate of angiogenesis is slow, which greatly restricts the effect of stem cell transplantation. In contrast, umbilical cord mesenchymal stem cells (human umbilical). Cord mesenchymal stem cells, H UC-MSCs) and BMSCs are very similar in biological characteristics. Because of their more extensive origin, convenient collection, strong extenability, no immunogenicity, no ethical controversy, more effective MSCs potential, may become an ideal substitute for BMSCs and become the best seed to induce differentiation at present. For the reconstruction of blood circulation, a variety of cytokines and growth factors are known to be involved in promoting angiogenesis, in which Vascular endothelial growth factor (VEGF) can significantly promote endothelial proliferation and angiogenesis by combining with its vascular endothelial specific receptor. It is considered to be within the body. The strongest angiogenic growth factor, but the direct application of VEGF has many shortcomings, such as short half-life, more difficult purification, large amount of application, high cost and so on, which restrict its clinical application. Therefore, how to improve the survival and differentiation of MSCs in the ischemic tissue, increase the secretion of local tissue growth factor, promote the formation of new blood vessels and improve the treatment of MSCs for the treatment of PAD The effect is the main problem to be solved at present. This study uses gene engineering to construct VEGF-EGFP gene expression vector and transfect the adenovirus into H UC-MSCs cells, observe the growth and proliferation of H UC-MSCs and the expression of the target gene VEGF after gene transfection; and use the enhanced green color fluorescent protein (enhanced green fluorescent protein,) EGFP) in vivo tracer localization of H UC-MSCs after transfection; at the same time, a high fat feeding type 2 diabetic SD rat lower limb ischemia model was established, and the transfected h UC-MSCs was injected locally to observe its angiogenesis to vascular endothelium, the establishment of collateral circulation, and the improvement of the experimental efficacy of lower extremity blood deficiency. This study used h UC-MSCs as a VEGF base. Because of the treatment platform, it can not only improve the concentration of local stable treatment by increasing the continuous secretion of VEGF, but also improve the microenvironment after ischemia by the anti-inflammatory, antioxidant stress, anti apoptosis, and angiogenesis of VEGF, and provide the best living space for H UC-MSCs proliferation, differentiation and other processes. At the same time, the paracrine effect of H UC-MSCs can be amplified and reduced. Less h UC-MSCs apoptosis, improve the localization and survival rate after H UC-MSCs transplantation, effectively play a synergistic multiplier effect of VEGF and H UC-MSCs in vascular regeneration, thus providing an experimental basis for the better efficacy of H UC-MSCs transplantation. This study is divided into three parts: the first part of adenovirus mediated VEGF transfection of umbilical cord mesenchymal stem cells Objective: To investigate the feasibility of adenovirus vector mediated VEGF transfection of H UC-MSCs and the effect of VEGF transfection on the morphology and function of H UC-MSCs. Methods: construct VEGF-EGFP recombinant adenovirus vector, isolate and culture h UC-MSCs, divide into VEGF-EGFP transfection group, EGFP empty load group and control group, and observe cell transformation by fluorescence inverted microscope. The transfection efficiency was determined by flow cytometry, the optimal number of multiplicities of infection (MOI) was determined, and the cells were transfected and collected according to the optimum MOI value. The expression of the egg Rhizoma Bletillae m RNA expression of the target gene VEGF after transfection was detected by the Western blot (Western blot), and the enzyme linked immunosorbent assay (ELISA) was finely detected. Cell culture supernatant VEGF protein level, MTT method and flow cytometry to evaluate the effect of gene transfection on the proliferation and cell cycle of H UC-MSCs. Results: fluorescence microscopy and flow cytometry showed that adenovirus mediated VEGF-EGFP gene could transfect h UC-MSCs successfully, and the transfection efficiency was high, and the cell structure morphology was not affected; the target gene VEGF was found. The cells can be transcribed and expressed, and can be secreted out of the cell. After transfection, the expression and secretion of VEGF are detected by ELISA method in cell culture supernatant, and the expression of 96h is still stable. Compared with the control group and the EGFP empty load group, the expression level of VEGF in the VEGF-EGFP transfer group is increased by Western blot and RT-PCR, and the expression is stable, and the expression is stable and can be extracted. The expression of 24h is stable and can be extracted. The anti apoptosis and survival ability of Gao H UC-MSCs, and the results of MTT and flow cytometry showed that VEGF gene transfection could improve the proliferation and differentiation of H UC-MSCs. Conclusion: adenovirus mediated VEGF gene can successfully transfect h UC-MSCs, can continue to be stable, efficiently express VEGF, improve the survival microcirculation, improve the proliferation and differentiation of H UC-MSCs, and the viability, To provide a theoretical basis for the feasibility of VEGF gene transfection of H UC-MSCs transplantation in the treatment of diabetic lower extremity vascular lesions. Second the purpose of establishing the lower limb ischemia model of type 2 diabetic rats is to establish an acute ischemia model of the hind limbs by the method of ligature of the femoral artery in the hind limbs of the rat. And to assess the ischemic state of chronic arteriosclerosis obliterans with hyperglycemia and hyperglycemia, there was no stable and effective chronic ischemia model and method. Methods: 20 rats were randomly divided into 2 groups, 10 in group DM were fed with high fat for 6 months, and the diabetes model was induced by intraperitoneal injection (Streptozocin, STZ) (35mg/ kg), and the control group (10) was fed by common food (6). After anaesthesia, rats in the model DM group and the control group sterilize the tissue and cut the skin along the right lower extremities, separate the femoral artery under the groin, cut the femoral artery under the groin, and ligate the femoral artery near the inguinal ligament. Then the distal end is stripped to the knee, and all branches of the femoral artery are separated and ligated to cause ischemic model of the right lower limb. After the operation, 3D, 7d, 14d, 28d were used to observe the condition of limb movement, body color, skin temperature and so on. After the routine anesthesia of 1D, 3D, 14d, 28d, the temperature was maintained. The light ray was relatively constant, and the Peri Scan PIM3 laser Doppler was used to monitor the blood flow of the lower limbs. After the operation, the peritoneum was opened and the peritoneum was opened and the abdominal initiative was taken after the operation. Vein indwelling trocar, heparin sodium anticoagulant, CT lower extremity angiography was performed by injection of contrast agent about 1.5 mL at 2 ml/s rate. Each group of animals died after angiography. The healthy side and the affected lateral femoral four head and the gastrocnemius muscle were stained with hematoxylin eosin staining and CD31 immunohistochemical staining, and Western blot was used to determine the VEGF content in muscle tissue. Results: DM Group (8) control group (10 rats) was prepared to form a hind limb ischemia model. After operation 1D, the Doppler blood flow and CT angiography in the two groups were significantly reduced to suggest that the ischemia model was successful. The two groups of 7D and 14d after the operation showed that the blood flow of the ischemic limb was gradually restored, and the blood flow recovery of group 28d DM after operation was significantly slower than that of the control group (P0 .05); CT angiography: in group DM, only a small amount of blood vessels were compensated for the proximal femoral artery ligation at the right lower extremities, and there was no obvious blood flow in the distal end of the heart. Pathological tissue and immunohistochemical staining: tissue structure destruction, inflammatory cell infiltration, capillary density side lower than the healthy side, and VEGF of ischemic muscle tissue in group 28d DM after operation. The expression of protein in the control group was significantly increased (P0.05). Conclusion: on the basis of long-term high fat feeding diabetes model, the ischemia model of lower extremity of the femoral artery ligation is closer to the condition of chronic ischemia in the lower limbs of diabetes. The 320 row CT angiography can be more stereoscopic to assess the ischemic state more stereoscopic, to further explore the deficiency of the lower extremity of diabetes. Blood pathological mechanism and stem cell therapy provide an ideal animal model and evaluation index. Third VEGF-EGFP transfection of umbilical cord mesenchyme stem cell transplantation to improve lower limb ischemia in diabetic rats: transfection of recombinant VEGF-EGFP gene into H UC-MSCs cells by adenovirus. To establish the lower limb ischemia model of SD rats with type 2 diabetes mellitus by high fat feeding, the local muscle injection of H UC-MSCs after transfection was injected to observe the effects of angiogenesis, collateral circulation and lower limb ischemia improvement. Methods: after the ischemia model was established by ligation of the femoral artery in the lower extremities of type 2 diabetic SD rats induced by high fat diet STZ, the group was divided into groups to observe VEGF-EG. FP-h UC-MSCs, EGFP-h UC-MSCs, H UC-MSCs and PBS were injected into the ischemic parts of the lower extremities. The survival and location of the transplanted cells were observed by fluorescence inverted microscope. The local blood flow was detected by laser Doppler (Sweden Peimed, LDPI) after the treatment. The lower extremity activity and the blood deficiency were observed. 4 weeks later, the lower extremity artery was ligated by abdominal aorta ligature. The formation of collateral circulation of both lower extremity vessels was detected by Toshiba 320 layer CT. The number and density of newborn capillaries were detected by HE staining and CD31 immunohistochemistry in muscle tissue; VEGF and MMP2, MMP9, TIMP1, TIMP2, ERK, AKt related genes and protein expression in tissue specimens, such as RTPCR, Western blot, etc. The EGFP labeled h UC-MSCs transplanted in the lower limb of the lower extremities was found to survive, and the LDPI blood flow map of 2,4 weeks after transplantation showed that the recovery level of the blood flow perfusion in the VEGF-EGFP transfected group was significantly higher than that of the EGFP no-load group. After 4 weeks, the CT angiography showed that there was a new collateral circulation in the VEGF-EGFP transfection group, and HE and immunohistochemical staining showed the newborn capillary. RT-PCR and Western blot detected VEGF-EGFP transfection group VEGF, ERK, AKt, MMP2 and MMP9 m significantly higher than the other two groups. VEGF, which promotes endothelial repair, is more effective than h UC-MSCs in promoting angiogenesis, obviously improving the state of lower limb ischemia in diabetes, and providing a new theoretical basis for the treatment of diabetic lower extremity vascular lesions by H UC-MSCs combined gene.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R587.2

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