ROS敏感納米粒靶向釋放SDF-1α趨化BMSCs歸巢治療電燒傷血管損傷
發(fā)布時間：2018-08-09 09:46
【摘要】：研究背景:電燒傷是現(xiàn)代工業(yè)社會特有的意外傷害,雖然發(fā)病率不高,但因其損傷是立體的,并具有夾心樣壞死和漸進性壞死的特點,造成局部組織損害嚴重,住院患者的截肢率超過30%,肢體往往遺留不同程度功能障礙,臨床危害性大。血管損傷所致的組織缺血缺氧性壞死是電燒傷組織漸進性壞死的重要原因。目前臨床上并無針對性的減輕血管損傷、促進血管修復的治療。如何促進電燒傷局部血管快速修復,對減輕組織壞死、改善預后具有重要意義。骨髓間充質(zhì)干細胞(BMSCs)具有向多種細胞分化的潛能。在燒創(chuàng)傷等病理狀態(tài)下,BMSCs能從骨髓中迅速動員進入外周循環(huán),在損傷局部歸巢并分化為內(nèi)皮祖細胞,進一步分化為血管內(nèi)皮細胞或直接分化為內(nèi)皮細胞參與血管損傷的修復。BMSCs的動員、趨化、聚集主要依賴于基質(zhì)細胞衍生因子-1α(SDF-1α)的趨化作用,而這種趨化作用依賴于局部高濃度的SDF-1α以及循環(huán)中的SDF-1α濃度梯度。因此,局部形成高濃度的SDF-1α以及循環(huán)中的SDF-1α濃度梯度是趨化并捕捉干細胞參與血管修復的必要條件。然而,如何在損傷局部形成高濃度的SDF-1α以及循環(huán)中的SDF-1α濃度梯度是一個難題。直接血管內(nèi)注射SDF-1α會迅速被血液稀釋進入全身循環(huán)而不能在損傷局部形成持久的有效濃度;直接組織內(nèi)注射SDF-1α分布不均勻,易降解,且進入循環(huán)的效率不確定。隨著藥物載體材料技術(shù)的發(fā)展,將SDF-1α通過可生物降解的納米粒系統(tǒng)給藥,不僅能有效防止其在體內(nèi)快速降解,還可以將SDF-1α靶向輸送至體內(nèi)有效部位,達到定向緩釋的目的。實現(xiàn)納米粒藥物的靶向釋放,關(guān)鍵是確定病變部位特殊的物理、化學或生物學特性�；钚匝�(ROS)作為生物體內(nèi)病理性存在的致病因子,為納米粒藥物的靶向釋放提供了靶點。在我們的前期研究中,利用對ROS敏感的硫醇縮酮聚合物PPADT為納米粒載體,將SDF-1α蛋白包載其中,研制出了SDF-1α-PPADT納米粒,該納米�？梢虿∽兘M織內(nèi)高氧自由基濃度發(fā)生裂解而釋放藥物,從而達到靶向治療目的。本研究中,我們通過制備大鼠電燒傷血管損傷模型,尾靜脈注射SDF-1α-PPADT納米粒,評價其通過靶向釋放SDF-1α、定向趨化BMSCs歸巢對電燒傷血管損傷修復的作用。研究方法:1、電燒傷血管損傷模型的制備自制電燒傷設(shè)備,220V的電壓持續(xù)電擊大鼠6s。2、電燒傷血管損傷的鑒定傷后沿近心端方向切取電擊部位含主干血管肌肉的組織,假電燒傷組同樣部位取材,HE染色和CD31免疫組化染色觀察血管損傷情況。3、損傷局部組織ROS檢測ROS熒光染色和ROS的Real Time PCR測定,觀察傷后局部ROS水平變化。4、損傷局部組織SDF-1α含量測定調(diào)節(jié)電擊時間的長短造成不同程度的組織損傷,ELISA測定局部SDF-1α水平變化。5、SDF-1α-PPADT納米粒的制備根據(jù)前期研究結(jié)果制備納米粒,先合成對ROS敏感的納米材料PPADT分子片段,然后通過復乳溶媒萃取法制備包含SDF-1α蛋白的載藥納米粒。6、ROS敏感的SDF-1α靶向釋放效應的驗證傷后輸注SDF-1α-PPADT納米粒,分別從SDF-1α的在體分布和局部SDF-1α蛋白水平變化觀察納米粒體內(nèi)靶向釋放SDF-1α的情況。7、BMSCs的定向趨化和歸巢效應驗證傷后輸注SDF-1α-PPADT納米粒,同時輸注外源性的綠色熒光蛋白GFP標記的GFP-BMSCs,免疫熒光染色觀察GFP的分布,驗證納米粒對BMSCs的定向趨化歸巢效應。8、SDF-1α-PPADT納米粒對血管修復的作用傷后輸注SDF-1α-PPADT納米粒,HE染色和CD31免疫組化染色觀察血管損傷修復情況。研究結(jié)果:1、電燒傷局部的大體觀察電燒傷后所有大鼠均存活良好,電極板處形成Ⅲ度燒傷創(chuàng)面,大鼠電擊后表現(xiàn)為雙后肢跛行。2、電燒傷可致血管損傷電燒傷大鼠血管內(nèi)皮細胞呈釘突樣凸向管腔,連續(xù)性中斷,內(nèi)膜剝脫,管腔縮窄。3、損傷局部ROS含量顯著升高電燒傷大鼠血管損傷局部ROS綠色熒光分布明顯,抗氧化酶SOD、CAT、GSH-Px的m RNA表達水平增高。4、一定范圍內(nèi)局部電燒傷程度與SDF-1α水平成反比輕度電燒傷組織局部SDF-1α產(chǎn)生增多,但是隨著電燒傷損傷程度的加重,組織局部SDF-1α產(chǎn)生下降,電擊6s時損傷局部組織SDF-1α持續(xù)處于低水平狀態(tài)。5、SDF-1α-PPADT納米粒具有良好的靶向藥物釋放效應輸注納米粒后,Cy5熒光顯示SDF-1α僅靶向分布于血管損傷局部,且損傷局部SDF-1α水平顯著提高。6、SDF-1α-PPADT納米粒定向趨化BMSCs歸巢輸注納米粒后第7天,可觀察到GFP-BMSCs陽性細胞在血管損傷局部明顯聚集。7、SDF-1α-PPADT納米粒促進血管損傷修復輸注納米粒后第10天,電燒傷大鼠血管形態(tài)基本完整,內(nèi)皮細胞排列較為整齊連續(xù),血管數(shù)較多,管腔呈圓形或橢圓形。研究結(jié)論:1、使用自制的電燒傷設(shè)備,220V持續(xù)電擊大鼠6s能造成明顯的血管損傷。2、用ROS敏感材料PPADT包裹SDF-1α制備的納米粒,可在血管損傷部位靶向釋放SDF-1α,趨化BMSCs歸巢,從而促進電燒傷血管損傷的修復。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R647
本文編號：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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R647
【參考文獻】
相關(guān)期刊論文 前1條
1 萬立華,馬智華,張佐才;血管電損傷的實驗研究[J];中國法醫(yī)學雜志;2001年01期
，本文編號：2173690
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