VEGF转染脐带间充质干细胞促血管新生改善糖尿病下肢缺血的实验研究

发布时间：2018-05-12 14:01

本文选题：血管内皮生长因子 + 转染　；参考：《河北医科大学》2015年博士论文

【摘要】：糖尿病外周血管病变(diabetic peripheral artery disease,PAD)是糖尿病(diabetic mellitus,DM)严重的慢性并发症之一,与非DM患者相比,其主要病理改变为动脉粥样硬化,且多累及下肢远端动脉,病变范围更广、呈多节段弥漫性的狭窄或闭塞,是导致DM足部坏疽、截肢的主要原因,严重影响DM患者的生存质量。目前传统内科药物、介入和手术治疗等对远端血管闭塞、流出道差的DM患者效果不佳,不能从根本上解决问题,并且此类DM患者多数高龄体弱,手术风险大,合并多种心脑血管病变,病情复杂,因此临床治疗上相当棘手,迫切需要寻求新的技术手段,如何促进血管新生实现血运重建成为治疗关键。近年来干细胞移植成为发展迅速的一种全新治疗模式,其中骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)已被证实是一类具有多向分化潜能的干细胞,可以在一定的诱导条件下分化为骨、软骨、脂肪、神经、心肌、血管内皮细胞等,参与不同组织的修复,但目前仍缺乏移植细胞在体内存活、分化、转归及参与血管再生的实验依据可循,且骨髓MSCs采集风险较大,对患者年龄、身体条件、心理接受程度要求较高,在实际应用中,由于高糖、氧化应激、低氧等微环境使移植后MSCs的存活率非常低,新生血管形成速度慢等,极大地限制了干细胞移植治疗的效果。相比之下,脐带间充质干细胞(human umbilical cord mesenchymal stem cells,h UC-MSCs)与BMSCs在生物学特性方面极为相似,由于其来源更广泛,采集方便,扩增力可塑性强,无免疫原性,不存在伦理学争议,具有更加有效的MSCs潜能,有可能成为BMSCs的理想替代物,成为目前诱导分化最佳的种子细胞。对血循环的重建而言,目前已知有多种细胞因子和生长因子参与促进血管生成作用,其中血管内皮生长因子(Vascular endothelial growth factor,VEGF)通过与其血管内皮特异性受体结合,可显著促进内皮增生及血管生成作用,被认为是机体内最强的促血管生长因子,但直接应用VEGF治疗存在很多不足,如半衰期短、提纯较为困难、应用量大、成本昂贵等,限制其临床应用。因此,如何提高MSCs在缺血组织存活、分化,增加局部组织生长因子分泌量,促进新生血管形成,提高MSCs治疗PAD疗效是目前亟待解决的主要问题。本研究利用基因工程构建VEGF-EGFP基因表达载体,通过腺病毒转染到h UC-MSCs细胞中,观察基因转染后对h UC-MSCs的生长增殖及目的基因VEGF表达情况;并利用增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)实现转染后h UC-MSCs的活体示踪定位;同时建立高脂喂养2型糖尿病SD大鼠下肢缺血模型,将转染后的h UC-MSCs局部肌肉注射,观察其向血管内皮分化促血管新生,侧支循环的建立,改善下肢缺血的实验疗效;本研究应用h UC-MSCs作为VEGF基因治疗平台,不仅可通过提高VEGF持续分泌,达到局部稳定的治疗浓度,并通过VEGF的抗炎、抗氧化应激、抗凋亡、促进血管生成等作用改善缺血后微环境,为h UC-MSCs增殖、分化等过程提供最佳的生存空间;同时可放大h UC-MSCs的旁分泌作用,减少h UC-MSCs凋亡,提高h UC-MSCs移植后的定位归巢和存活率等,有效发挥VEGF与h UC-MSCs在血管再生功效方面的协同倍增作用,从而为更好的发挥h UC-MSCs移植疗效提供实验基础。本研究分为三部分:第一部分腺病毒介导VEGF转染脐带间充质干细胞的实验研究目的:探讨腺病毒载体介导VEGF转染h UC-MSCs的可行性,以及VEGF转染对h UC-MSCs形态及功能的影响。方法:构建VEGF-EGFP重组基因腺病毒载体,分离和培养h UC-MSCs,分为VEGF-EGFP转染组、EGFP空载组及对照组,荧光倒置显微镜观察细胞转染效果,流式细胞仪测定细胞转染效率,确定最佳病毒感染复数(multiplicities of infection,MOI);依据最佳MOI值转染并收集细胞,应用蛋白印迹法(Western blot)、RT-PCR检测转染后目的基因VEGF的蛋白及m RNA表达情况;酶联免疫吸附试验(ELISA)检测细胞培养上清VEGF蛋白水平,MTT法及流式细胞术评价基因转染对h UC-MSCs增殖和细胞周期的影响。结果:荧光显微镜及流式细胞仪检测提示腺病毒介导的VEGF-EGFP基因能够成功转染h UC-MSCs,且转染效率高,对细胞结构形态无影响;目的基因VEGF在细胞内能够转录和表达,并能分泌到细胞外,转染后24h采用ELISA法在细胞培养上清中就检测到有VEGF的表达和分泌,96h仍稳定表达;与对照组和EGFP空载组相比,通过Western blot、RT-PCR检测VEGF-EGFP转染组VEGF表达水平升高显著,且表达稳定,可提高h UC-MSCs的抗凋亡及存活能力;MTT及流式细胞法检测结果显示VEGF基因转染可提高h UC-MSCs增殖和分化能力。结论:腺病毒介导VEGF基因能够成功转染h UC-MSCs,能持续稳定、高效表达VEGF,改善生存微循环,提高h UC-MSCs的增殖及分化、存活能力,为开展VEGF基因转染h UC-MSCs移植治疗改善糖尿病下肢血管病变的可行性提供理论依据。第二部分高脂喂养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动脉结扎离断的方法来制备后肢急性缺血模型,但对于如何建立及评估糖尿病高脂高血糖慢性动脉硬化闭塞症的缺血状态,尚无一个稳定有效慢性缺血模型及方法。方法:将大鼠20只随机分为2组,DM组10只予以高脂喂养6个月,腹腔注射(Streptozocin,STZ)(35mg/kg)诱发糖尿病模型,对照组(10只)予以普食喂养6个月,成模DM组及对照组大鼠在麻醉后,消毒铺巾,沿右下肢正中的皮肤纵行切开,于腹股沟下分离出股动脉,在腹股沟韧带下切断股动脉,近端结扎,随后向远端锐性剥离直至膝关节,分离和结扎股动脉的所有分支,造成右下肢缺血模型,术后3d、7d、14d、28d观察大鼠患肢活动状况、肢体颜色、皮温等,并于术后1d、3d、14d、28d常规麻醉后,保持温度、光线相对恒定下,应用Peri Scan PIM3激光多普勒(Laser Doppler Perfusion Imaging,LDPI)行下肢血流监测。术后28d麻醉动物后,切开后腹膜,于腹主动脉留置套管针,肝素钠抗凝,以2 ml/s的速率注射造影剂约1.5 ml进行CT下肢血管造影。每组动物在血管造影后处死,分别取其健侧和患侧股四头肌和腓肠肌行苏木精-伊红染色及CD31免疫组织化学染色、Western blot测定肌肉组织VEGF含量。结果:DM组有(8只)对照组(10只)制备成后肢缺血模型,术后第1d,两组大鼠多普勒血流及CT血管造影均呈显著降低提示缺血造模成功,两组术后第7d和14d行激光多普勒提示缺血肢体血流有逐渐恢复趋势,术后第28d DM组血流恢复较对照组显著迟缓(P0.05);CT血管造影:DM组右下肢股动脉结扎处近端仅有少量血管代偿性增加,远心端仍无明显血流;病理组织及免疫组化染色:术后第28d DM组缺血部位肌肉组织出现组织结构破坏,炎性细胞浸润,毛细血管密度患侧低于健侧;缺血肌肉组织VEGF较对照组的蛋白表达明显增加(P0.05)。结论:长期高脂喂养糖尿病模型基础上,制作股动脉结扎离断的下肢缺血模型,更接近于糖尿病下肢慢性缺血的情况,320排CT血管造影技术可以更立体直观评估缺血状态,为进一步探讨糖尿病下肢缺血病理机制及干细胞治疗促血管新生等提供了较为理想的动物模型及评估指标。第三部分VEGF-EGFP转染脐带间充质干细胞移植治疗改善糖尿病大鼠下肢缺血的实验研究目的:通过腺病毒将重构的VEGF-EGFP基因转染到h UC-MSCs细胞中,同时建立高脂喂养2型糖尿病SD大鼠下肢缺血模型,将转染后h UC-MSCs局部下肢肌肉注射,观察血管新生,侧支循环建立,下肢缺血改善的实验疗效。方法:利用高脂饮食STZ诱导的2型糖尿病SD大鼠下肢股动脉结扎建立缺血模型后,将分组观察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,荧光倒置显微镜观察移植细胞存活及定位;在治疗后2、4周,激光多普勒(瑞典Peimed,LDPI)检测局部血流;观察下肢活动度和缺血情况;4周后利用腹主动脉结扎行下肢动脉CTA(东芝320层CT机)造影检测双下肢血管侧支循环的形成;肌肉组织HE染色和CD31免疫组化检测新生毛细血管数量及密度;RTPCR、Western blot等检测组织标本中VEGF及MMP2,MMP9,TIMP1,TIMP2,ERK,AKt相关基因m RNA及蛋白的表达等。结果:移植后1、2、4周荧光显微镜下发现,在下肢缺血部位有移植的EGFP标记的h UC-MSCs存活;移植后2、4周LDPI血流图显示VEGF-EGFP转染组血流灌注的恢复水平要明显高于EGFP空载组,移植4周后CT血管造影显示VEGF-EGFP转染组有新生侧支循环,HE及免疫组化染色显示新生毛细血管明显高均于空载组,RT-PCR及Western blot检测组织标本VEGF-EGFP转染组VEGF、ERK、AKt、MMP2和MMP9 m RNA和蛋白水平较其他两组显著增高,TIMP1、TIMP2的表达无明显差异。结论:肌肉注射移植VEGF基因修饰后h UC-MSCs可在下肢缺血组织中定植存活,高效表达VEGF,促进内皮修复,比单纯h UC-MSCs更能有效地促进血管新生,明显改善糖尿病下肢缺血状态,为h UC-MSCs联合基因治疗糖尿病下肢血管病变提供新的理论依据。
[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.

【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.2

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