ROS敏感纳米粒靶向释放SDF-1α趋化BMSCs归巢治疗电烧伤血管损伤
发布时间：2018-08-09 09:46
【摘要】：研究背景:电烧伤是现代工业社会特有的意外伤害,虽然发病率不高,但因其损伤是立体的,并具有夹心样坏死和渐进性坏死的特点,造成局部组织损害严重,住院患者的截肢率超过30%,肢体往往遗留不同程度功能障碍,临床危害性大。血管损伤所致的组织缺血缺氧性坏死是电烧伤组织渐进性坏死的重要原因。目前临床上并无针对性的减轻血管损伤、促进血管修复的治疗。如何促进电烧伤局部血管快速修复,对减轻组织坏死、改善预后具有重要意义。骨髓间充质干细胞(BMSCs)具有向多种细胞分化的潜能。在烧创伤等病理状态下,BMSCs能从骨髓中迅速动员进入外周循环,在损伤局部归巢并分化为内皮祖细胞,进一步分化为血管内皮细胞或直接分化为内皮细胞参与血管损伤的修复。BMSCs的动员、趋化、聚集主要依赖于基质细胞衍生因子-1α(SDF-1α)的趋化作用,而这种趋化作用依赖于局部高浓度的SDF-1α以及循环中的SDF-1α浓度梯度。因此,局部形成高浓度的SDF-1α以及循环中的SDF-1α浓度梯度是趋化并捕捉干细胞参与血管修复的必要条件。然而,如何在损伤局部形成高浓度的SDF-1α以及循环中的SDF-1α浓度梯度是一个难题。直接血管内注射SDF-1α会迅速被血液稀释进入全身循环而不能在损伤局部形成持久的有效浓度;直接组织内注射SDF-1α分布不均匀,易降解,且进入循环的效率不确定。随着药物载体材料技术的发展,将SDF-1α通过可生物降解的纳米粒系统给药,不仅能有效防止其在体内快速降解,还可以将SDF-1α靶向输送至体内有效部位,达到定向缓释的目的。实现纳米粒药物的靶向释放,关键是确定病变部位特殊的物理、化学或生物学特性。活性氧(ROS)作为生物体内病理性存在的致病因子,为纳米粒药物的靶向释放提供了靶点。在我们的前期研究中,利用对ROS敏感的硫醇缩酮聚合物PPADT为纳米粒载体,将SDF-1α蛋白包载其中,研制出了SDF-1α-PPADT纳米粒,该纳米粒可因病变组织内高氧自由基浓度发生裂解而释放药物,从而达到靶向治疗目的。本研究中,我们通过制备大鼠电烧伤血管损伤模型,尾静脉注射SDF-1α-PPADT纳米粒,评价其通过靶向释放SDF-1α、定向趋化BMSCs归巢对电烧伤血管损伤修复的作用。研究方法:1、电烧伤血管损伤模型的制备自制电烧伤设备,220V的电压持续电击大鼠6s。2、电烧伤血管损伤的鉴定伤后沿近心端方向切取电击部位含主干血管肌肉的组织,假电烧伤组同样部位取材,HE染色和CD31免疫组化染色观察血管损伤情况。3、损伤局部组织ROS检测ROS荧光染色和ROS的Real Time PCR测定,观察伤后局部ROS水平变化。4、损伤局部组织SDF-1α含量测定调节电击时间的长短造成不同程度的组织损伤,ELISA测定局部SDF-1α水平变化。5、SDF-1α-PPADT纳米粒的制备根据前期研究结果制备纳米粒,先合成对ROS敏感的纳米材料PPADT分子片段,然后通过复乳溶媒萃取法制备包含SDF-1α蛋白的载药纳米粒。6、ROS敏感的SDF-1α靶向释放效应的验证伤后输注SDF-1α-PPADT纳米粒,分别从SDF-1α的在体分布和局部SDF-1α蛋白水平变化观察纳米粒体内靶向释放SDF-1α的情况。7、BMSCs的定向趋化和归巢效应验证伤后输注SDF-1α-PPADT纳米粒,同时输注外源性的绿色荧光蛋白GFP标记的GFP-BMSCs,免疫荧光染色观察GFP的分布,验证纳米粒对BMSCs的定向趋化归巢效应。8、SDF-1α-PPADT纳米粒对血管修复的作用伤后输注SDF-1α-PPADT纳米粒,HE染色和CD31免疫组化染色观察血管损伤修复情况。研究结果:1、电烧伤局部的大体观察电烧伤后所有大鼠均存活良好,电极板处形成Ⅲ度烧伤创面,大鼠电击后表现为双后肢跛行。2、电烧伤可致血管损伤电烧伤大鼠血管内皮细胞呈钉突样凸向管腔,连续性中断,内膜剥脱,管腔缩窄。3、损伤局部ROS含量显著升高电烧伤大鼠血管损伤局部ROS绿色荧光分布明显,抗氧化酶SOD、CAT、GSH-Px的m RNA表达水平增高。4、一定范围内局部电烧伤程度与SDF-1α水平成反比轻度电烧伤组织局部SDF-1α产生增多,但是随着电烧伤损伤程度的加重,组织局部SDF-1α产生下降,电击6s时损伤局部组织SDF-1α持续处于低水平状态。5、SDF-1α-PPADT纳米粒具有良好的靶向药物释放效应输注纳米粒后,Cy5荧光显示SDF-1α仅靶向分布于血管损伤局部,且损伤局部SDF-1α水平显著提高。6、SDF-1α-PPADT纳米粒定向趋化BMSCs归巢输注纳米粒后第7天,可观察到GFP-BMSCs阳性细胞在血管损伤局部明显聚集。7、SDF-1α-PPADT纳米粒促进血管损伤修复输注纳米粒后第10天,电烧伤大鼠血管形态基本完整,内皮细胞排列较为整齐连续,血管数较多,管腔呈圆形或椭圆形。研究结论:1、使用自制的电烧伤设备,220V持续电击大鼠6s能造成明显的血管损伤。2、用ROS敏感材料PPADT包裹SDF-1α制备的纳米粒,可在血管损伤部位靶向释放SDF-1α,趋化BMSCs归巢,从而促进电烧伤血管损伤的修复。
[Abstract]:Background: electrical burn is a peculiar accidental injury in modern industrial society. Although the incidence is not high, the injury is stereoscopic, with the characteristics of sandwich like necrosis and progressive necrosis, which causes serious local tissue damage, the amputation rate of the hospitalized patients exceeds 30%, the limbs are left to different degrees of dysfunction, and the clinical harm is great. Tissue ischemic necrosis caused by tube injury is an important cause of progressive necrosis of electrical burn tissue. At present, there is no targeted reduction of vascular injury and promoting the treatment of vascular repair. How to promote rapid repair of local blood vessels in electrical burns is of great significance to reduce tissue necrosis and improve the prognosis. B MSCs) has the potential to differentiate into a variety of cells. In the pathological state of trauma and other pathological conditions, BMSCs can quickly mobilize from the bone marrow into the peripheral circulation, differentiate into the endothelial progenitor cells and differentiate into vascular endothelial cells or directly differentiated into endothelial cells to participate in the mobilization, chemotaxis and aggregation of.BMSCs. The chemotaxis depends on the chemotaxis of the stromal cell derived factor -1 alpha (SDF-1 alpha), which depends on the local high concentration of SDF-1 alpha and the concentration gradient of the SDF-1 alpha in the circulation. Therefore, the local formation of high concentration of SDF-1 A and the concentration gradient of SDF-1 alpha in the circulation are necessary for chemotactic and capture stem cells to participate in vascular repair. However, how to form high concentration of SDF-1 A and the concentration gradient of SDF-1 alpha in the circulation is a difficult problem. The direct intravascular injection of SDF-1 alpha will quickly be diluted into the circulation of the blood and can not form a lasting effective concentration in the local injury. Direct intravascular injection of SDF-1 alpha is inhomogeneous, easy to degrade, and enters the cycle. The efficiency is uncertain. With the development of drug carrier material technology, SDF-1 alpha can not only effectively prevent its rapid degradation in the body, but also can be transported to the effective part of the body to achieve the goal of directed release. The key is to determine the target release of the nanoparticles, and the key is to determine the lesion. The specific physical, chemical, or biological properties of the site. Active oxygen (ROS), as a pathogenetic pathogenic factor in the organism, provides target for the targeting release of nanoscale drugs. In our previous study, the ROS sensitive thiol ketal polymer PPADT was used as the nanoparticle carrier, and the SDF-1 alpha protein was loaded into SDF-1, and SDF-1 was developed. Alpha -PPADT nanoparticles, which can release drugs due to the fragmentation of the hyperoxic radical concentration in the diseased tissue, can be targeted for target therapy. In this study, we prepared SDF-1 alpha -PPADT nanoparticles in the tail vein by preparing the rat electrical burn vascular injury model, and evaluated its targeted release of SDF-1 alpha by targeting the chemotaxis of BMSCs homing to the electricity. The function of repair of vascular injury in burn. 1, the electrical burn equipment was prepared by the model of vascular injury of electric burn. The voltage of 220V was continuously struck by electric shock in rats 6s.2. After the injury of the vascular injury of the electric burn, the tissues of the main artery and muscle were cut along the proximal end of the heart. The same parts of the sham burn group were taken from the same site, HE staining and CD31 exemption. The damage of blood vessels was observed by immunohistochemical staining.3, the local tissue ROS was detected by ROS fluorescence staining and the Real Time PCR of ROS was measured. The changes of local ROS level after injury were observed, and the length of the local tissue SDF-1 a was determined to regulate the length of the electric shock time. Nanoparticles were prepared according to the previous study. First, the ROS sensitive nanomaterials PPADT fragment was synthesized. Then the drug loaded nanoparticles containing SDF-1 alpha protein.6 were prepared by the reemulsion solvent extraction method. The ROS sensitive SDF-1 alpha targeting release effect was validated after the injection of SDF-1 alpha -PPADT nanoparticles, respectively, from the SDF-1 alpha in body. The distribution and local SDF-1 alpha protein level changes to observe the target release of SDF-1 alpha in the nanoparticles in vivo.7. The directional chemotaxis and homing effect of BMSCs are verified after the injection of SDF-1 alpha -PPADT nanoparticles, and the exogenous green fluorescent protein GFP marked GFP-BMSCs is injected, and the distribution of GFP is observed by immunofluorescence staining, and the determination of nanoparticles to BMSCs is verified. The effect of chemotactic homing and homing effect.8, SDF-1 alpha -PPADT nanoparticles on vascular repair after injecting SDF-1 a -PPADT nanoparticles, HE staining and CD31 immunohistochemical staining to observe the repair of vascular injury. The results of the study were as follows: 1, all rats with electric burn were well observed after electrical burn, and all rats were formed at the electrode plate at the third degree burn wound, and rats were formed at the electrode plate. After electric shock, the claudication of double hind limbs was.2, electrical burn could cause vascular injury in electric burn rats, the vascular endothelial cells were nailed to the lumen, the continuous interruption, the exfoliation of the endometrium, the narrowing of the lumen.3, the local ROS content of the injured rats increased significantly in the local ROS green fluorescein distribution in the vascular injury of the electric burn rats, and the RNA expression of the antioxidant SOD, CAT, GSH-Px m. The level of local electrical burn in a certain range and the level of SDF-1 a in a certain range is inversely proportional to the increase of local SDF-1 alpha in the mild electrical burn tissue, but with the aggravation of the damage degree of the electrical burn, the local SDF-1 alpha in the tissue decreases and the SDF-1 a in the local tissue of the injured 6S is at a low level of.5, and the SDF-1 a -PPADT nanoparticles are good at 6S. After the target drug release effect was injected into the nanoparticles, Cy5 fluorescence showed that SDF-1 a only was localized in the local vascular damage, and the level of local SDF-1 alpha was significantly increased by.6. SDF-1 alpha -PPADT nanoparticles targeted BMSCs homing to the nanoparticles after seventh days, and GFP-BMSCs positive cells obviously aggregated.7 in the local vascular damage and SDF-1 alpha -PPA. DT nanoparticles promoted vascular injury to repair the delivery of the nanoparticles tenth days later. The vascular morphology of the electric burn rats was basically complete, the endothelial cells were arranged neatly and continuously, the number of blood vessels was more, the cavity was round or oval. The conclusion: 1, using the self-made electrical burn equipment, the 220V sustained electric shock rat 6S could cause obvious vascular injury.2, sensitive to ROS sensitivity. PPADT coated SDF-1a nanoparticles can release SDF-1a targeting at the site of vascular injury and chemotaxis BMSCs homing, thus promoting the repair of electrical burn vascular injury.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R647
本文编号：2173690
[Abstract]:Background: electrical burn is a peculiar accidental injury in modern industrial society. Although the incidence is not high, the injury is stereoscopic, with the characteristics of sandwich like necrosis and progressive necrosis, which causes serious local tissue damage, the amputation rate of the hospitalized patients exceeds 30%, the limbs are left to different degrees of dysfunction, and the clinical harm is great. Tissue ischemic necrosis caused by tube injury is an important cause of progressive necrosis of electrical burn tissue. At present, there is no targeted reduction of vascular injury and promoting the treatment of vascular repair. How to promote rapid repair of local blood vessels in electrical burns is of great significance to reduce tissue necrosis and improve the prognosis. B MSCs) has the potential to differentiate into a variety of cells. In the pathological state of trauma and other pathological conditions, BMSCs can quickly mobilize from the bone marrow into the peripheral circulation, differentiate into the endothelial progenitor cells and differentiate into vascular endothelial cells or directly differentiated into endothelial cells to participate in the mobilization, chemotaxis and aggregation of.BMSCs. The chemotaxis depends on the chemotaxis of the stromal cell derived factor -1 alpha (SDF-1 alpha), which depends on the local high concentration of SDF-1 alpha and the concentration gradient of the SDF-1 alpha in the circulation. Therefore, the local formation of high concentration of SDF-1 A and the concentration gradient of SDF-1 alpha in the circulation are necessary for chemotactic and capture stem cells to participate in vascular repair. However, how to form high concentration of SDF-1 A and the concentration gradient of SDF-1 alpha in the circulation is a difficult problem. The direct intravascular injection of SDF-1 alpha will quickly be diluted into the circulation of the blood and can not form a lasting effective concentration in the local injury. Direct intravascular injection of SDF-1 alpha is inhomogeneous, easy to degrade, and enters the cycle. The efficiency is uncertain. With the development of drug carrier material technology, SDF-1 alpha can not only effectively prevent its rapid degradation in the body, but also can be transported to the effective part of the body to achieve the goal of directed release. The key is to determine the target release of the nanoparticles, and the key is to determine the lesion. The specific physical, chemical, or biological properties of the site. Active oxygen (ROS), as a pathogenetic pathogenic factor in the organism, provides target for the targeting release of nanoscale drugs. In our previous study, the ROS sensitive thiol ketal polymer PPADT was used as the nanoparticle carrier, and the SDF-1 alpha protein was loaded into SDF-1, and SDF-1 was developed. Alpha -PPADT nanoparticles, which can release drugs due to the fragmentation of the hyperoxic radical concentration in the diseased tissue, can be targeted for target therapy. In this study, we prepared SDF-1 alpha -PPADT nanoparticles in the tail vein by preparing the rat electrical burn vascular injury model, and evaluated its targeted release of SDF-1 alpha by targeting the chemotaxis of BMSCs homing to the electricity. The function of repair of vascular injury in burn. 1, the electrical burn equipment was prepared by the model of vascular injury of electric burn. The voltage of 220V was continuously struck by electric shock in rats 6s.2. After the injury of the vascular injury of the electric burn, the tissues of the main artery and muscle were cut along the proximal end of the heart. The same parts of the sham burn group were taken from the same site, HE staining and CD31 exemption. The damage of blood vessels was observed by immunohistochemical staining.3, the local tissue ROS was detected by ROS fluorescence staining and the Real Time PCR of ROS was measured. The changes of local ROS level after injury were observed, and the length of the local tissue SDF-1 a was determined to regulate the length of the electric shock time. Nanoparticles were prepared according to the previous study. First, the ROS sensitive nanomaterials PPADT fragment was synthesized. Then the drug loaded nanoparticles containing SDF-1 alpha protein.6 were prepared by the reemulsion solvent extraction method. The ROS sensitive SDF-1 alpha targeting release effect was validated after the injection of SDF-1 alpha -PPADT nanoparticles, respectively, from the SDF-1 alpha in body. The distribution and local SDF-1 alpha protein level changes to observe the target release of SDF-1 alpha in the nanoparticles in vivo.7. The directional chemotaxis and homing effect of BMSCs are verified after the injection of SDF-1 alpha -PPADT nanoparticles, and the exogenous green fluorescent protein GFP marked GFP-BMSCs is injected, and the distribution of GFP is observed by immunofluorescence staining, and the determination of nanoparticles to BMSCs is verified. The effect of chemotactic homing and homing effect.8, SDF-1 alpha -PPADT nanoparticles on vascular repair after injecting SDF-1 a -PPADT nanoparticles, HE staining and CD31 immunohistochemical staining to observe the repair of vascular injury. The results of the study were as follows: 1, all rats with electric burn were well observed after electrical burn, and all rats were formed at the electrode plate at the third degree burn wound, and rats were formed at the electrode plate. After electric shock, the claudication of double hind limbs was.2, electrical burn could cause vascular injury in electric burn rats, the vascular endothelial cells were nailed to the lumen, the continuous interruption, the exfoliation of the endometrium, the narrowing of the lumen.3, the local ROS content of the injured rats increased significantly in the local ROS green fluorescein distribution in the vascular injury of the electric burn rats, and the RNA expression of the antioxidant SOD, CAT, GSH-Px m. The level of local electrical burn in a certain range and the level of SDF-1 a in a certain range is inversely proportional to the increase of local SDF-1 alpha in the mild electrical burn tissue, but with the aggravation of the damage degree of the electrical burn, the local SDF-1 alpha in the tissue decreases and the SDF-1 a in the local tissue of the injured 6S is at a low level of.5, and the SDF-1 a -PPADT nanoparticles are good at 6S. After the target drug release effect was injected into the nanoparticles, Cy5 fluorescence showed that SDF-1 a only was localized in the local vascular damage, and the level of local SDF-1 alpha was significantly increased by.6. SDF-1 alpha -PPADT nanoparticles targeted BMSCs homing to the nanoparticles after seventh days, and GFP-BMSCs positive cells obviously aggregated.7 in the local vascular damage and SDF-1 alpha -PPA. DT nanoparticles promoted vascular injury to repair the delivery of the nanoparticles tenth days later. The vascular morphology of the electric burn rats was basically complete, the endothelial cells were arranged neatly and continuously, the number of blood vessels was more, the cavity was round or oval. The conclusion: 1, using the self-made electrical burn equipment, the 220V sustained electric shock rat 6S could cause obvious vascular injury.2, sensitive to ROS sensitivity. PPADT coated SDF-1a nanoparticles can release SDF-1a targeting at the site of vascular injury and chemotaxis BMSCs homing, thus promoting the repair of electrical burn vascular injury.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R647
【参考文献】
相关期刊论文 前1条
1 万立华,马智华,张佐才;血管电损伤的实验研究[J];中国法医学杂志;2001年01期
，本文编号：2173690
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