TAT介导的磁性纳米脂质体对脊髓损伤区域靶向分布的实验研究
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摘要
[目的]
探讨一种FITC标记TAT介导的磁性纳米脂质体的制备和表征,在体外与雪旺细胞共培养的透膜能力；同时观察其在大鼠脊髓损伤后在脊髓组织局部聚集的情况,研究其对损伤大鼠脊髓分布的靶向性,为将来该脂质体介导药物治疗脊髓损伤确定提供实验依据。
[材料与方法]
本实验采用反相蒸发法制备兼具跨膜、长循环功能及包覆疏水性超顺磁性Fe304颗粒的TAT磁性纳米脂质体,透射电镜,粒径仪和Zeta电位仪测定脂质体粒径和基本表征。
通过结扎Wistar成年大鼠单侧隐神经激活雪旺细胞,采用双酶消化组织块法结合机械分离法分离培养雪旺细胞；应用低浓度胶原酶、胰酶快速消化法和差速贴壁法纯化雪旺细胞。用S-100抗体、DAPI染色来鉴定雪旺细胞的纯度。将雪旺细胞分别与外接TAT磁性纳米脂质体及未接TAT磁性纳米脂质体共孵育,对照组加入未接TAT的磁性纳米脂质体(59μg/ml) 0.5ml:实验组加入外接TAT的磁性纳米脂质体(59μg/ml) 0.5ml,5%CO2,37℃共孵育1小时,在荧光显微镜下观察雪旺细胞对脂质体的摄取情况。
本实验采用雌性Wistar大鼠16只,用IMPACTOR MODEL-Ⅱ打击器建立T10脊髓损伤(spinal cord injury, SCI))模型(10g×25mm)。造模成功后随机分为2组,分别于24小时通过尾静脉注射FITC标记的磁性纳米脂质体(1mg/kg),对照组注射未接TAT的磁性纳米脂质体,实验组加入外接TAT的磁性纳米脂质体注射后1小时处死大鼠,取出脊髓组织作5μm快速冰冻切片,在荧光显微镜下观察脂质体在脊髓组织中的聚集情况,同时取大鼠肝脏、脾脏、肾脏作冰冻切片并在荧光显微镜下观察脂质体被其它器官摄取情况。
成年雌性Wistar大鼠16只,随机分为两组,对照组未作脊髓损伤处理,仅采取相同剂量进行麻醉,实验组建立脊髓损伤模型,两组动物均在尾静脉途径注射外接TAT的磁性纳米脂质体(1mg/kg),实验组时间点选取为伤后24小时,对照组为麻醉后24小时加制作模型所需时间。注射后1小时处死大鼠,取出脊髓组织作5μm快速冰冻切片,在荧光显微镜下观察脂质体在脊髓组织中的聚集情况。
[结果]
1.磁性脂质体呈球形,分散性较好,粒径较小,Zeta电位较高,外接TAT组与未接TAT组粒径及Zeta电位无明显差别。
2.激活态雪旺细胞经分离、培养、纯化后,细胞生长旺盛。传至第4代时,低倍镜下几乎不见成纤维样细胞。雪旺细胞纯度达到95%以上。
3.细胞摄取实验外接TAT的脂质体更多的聚集在雪旺细胞内部,并大多聚集在细胞核周围,而未接TAT的脂质体较少进入雪旺细胞,比较二者的镜下FITC荧光的平均光密度(average optical density, AOD),二者具有统计学差异。
4.在荧光显微镜蓝色荧光激发下,FITC使磁性纳米脂质体呈现绿色荧光。在大鼠损伤脊髓组织中,外接TAT的脂质体更多的聚集在损伤脊髓周围,神经元细胞的胞浆中存在大量的荧光颗粒,聚集成片状显现出对核的趋向性,而未接TAT的脂质体则较少分布于受损脊髓。计算二者AOD值,结果具有统计学意义。
5.在荧光显微镜蓝色荧光激发下,未接TAT组的肝、脾、肾脏中均可见明亮的绿色荧光,外接TAT组绿色荧光相对较弱。
6.在荧光显微镜蓝色荧光激发下,正常大鼠脊髓组织中绿色荧光较少,而脊髓损伤大鼠的脊髓中聚集了大量的绿色荧光,计算二者AOD值,结果具有统计学意义。
[结论]
本研究证明了TAT介导的脂质体可大量进入受损脊髓,进入神经元细胞,并且具有靶向聚集受损脊髓组织的特性,为其成为装载治疗脊髓损伤药物的载体提供了可行性依据。
[Objective] Explore the FITC-TAT-mediated magnetic liposomes and character-ization biocompatibility of FITC labeled TAT-PEG magnetic cationic liposomes with Schwann cells in vitro, and analyze its ability to through the membrane by co-culture of Schwann cells in vitro, observe the local gathering in the rat spinal cord tissue after spinal cord injury, research The targeting abilityin the injured spinal, to provide experimental evidence with theliposomes mediating drugs for the treatment of spinal cord injury in the future.
[Method] In the study, long cycle and superparamagnetic TAT-magnetic nano liposomes were prepared by reverse phase evaporation. Transmission electron--microscopy, particlesize and Zeta potential analyzer measured particle size and basic characterization of liposomes. By unilateral ligating saphenous nerve of Wistar rats to activate Schwann cells, and then isolation, culture and purification of Schwann cells were performed in vitro. At last the purity of Schwann cells were identified by S-100 antibody and DAPI staining.
The Schwann cells and two types of liposomes (TAT-type, noTAT-type) were incubated together. The Schwann cells were divided into two groups:the control group (co-culture with TAT-liposome for 1 hour) (59μg/ml) 0.5ml, the experimental group (co-culture with noTAT-liposome for 1 hours) (59μg/ml) 0.5ml. Observing the uptake of liposomes by Schwann cells under fluorescence microscope was undertaken.
Sixteen of female Wistar rats were subjected to IMPACTOR MODEL-Ⅱto establish a T10 spinal cord injured model. The animals were randomly divided into two groups:24 hours after injury, respectively, by tail vein injection of FITC-magnetic nano-liposomes (1mg/kg), the control group received nano-liposomes without TAT, experimental group to join the TAT-magnetic nano-liposomes.By injecting FITC labeled new liposome (1mg/kg) into tail vein, rats were killed 1 hour later. Harvested the spinal cord was subjected to quick 5μm frozen sections.The sections of spinal cord, liver, spleen and kidney were also observed under fluorescence microscope.
Sixteen of female Wistar rats were randomly divided into two groups:control group without treatment for spinal cord injury, only to take the same dose of anesthesia, the experimental group to establish spinal cord injury model.Two groups of animals were injected in the tail vein of the TAT-magnetic nanoliposomes (1mg/kg), the time point selected as the experimental group was 24 hours after injury, control group was 24 hours after anesthesia adding the time required for production models. rats were killed 1 hour later. Harvested the spinal cord was subjected to quick 5μm frozen sections.
[Results]
1. Magnetic liposomes were spherical lipid lanes, better dispersion, smaller particle size, higher external Zeta potential. There were no significant difference between TAT-nanoliposomes group and noTAT group in the particle size and Zeta potential.
2. Activated Schwann cells were isolated, cultured. The purity was more than 95%.
3. Cellular uptake experiment show that:TAT-liposomes gathered inside Schwann cells,and most gathered around the nucleus.The noTAT-liposomes can not be observed in Schwann cells.By comparing the FITC fluorescence microscope in two groups,the average optical density(average optical density, AOD) showed a significant difference.
4. In the blue fluorescence under fluorescence microscope, FITC made the magnetic nano-liposomes showeing green fluorescence. In the rat spinal cord tissue,TAT-liposomes gathered more around the injured spinal cord side, neuronal cells present in the cytoplasm of a large number of fluorescent particles, gathered into a sheet showing the trend of the nuclear.noTAT-liposomes were then distributed less in damage side of the spinal cord. Both AOD values calculated, the result was statistically significant.
5. By excitation with blue in fluorescence microscope, in the no TAT group,in the liver, spleen, kidney, bright green fluorescence are visible.The TAT group is relatively weaker with green fluorescence.
6. By the blue fluorescence excitation, Spinal cord tissue had less green fluorescence in normal rats.Spinal cord gathered a large number of green fluorescencein injured spinal cord rats.Calculating values of the two AOD, the results have statistical significance.
[Conclusion] This study proved that TAT-mediated nanoliposomes can be poured into the damaged spinal cord, get into neurons, and have the targeting characteristics of gethering to damaged spinal cord,provided a feasible basis for becoming a load carrier of drugs for curing spinal cord injury.
探讨一种FITC标记TAT介导的磁性纳米脂质体的制备和表征,在体外与雪旺细胞共培养的透膜能力；同时观察其在大鼠脊髓损伤后在脊髓组织局部聚集的情况,研究其对损伤大鼠脊髓分布的靶向性,为将来该脂质体介导药物治疗脊髓损伤确定提供实验依据。
[材料与方法]
本实验采用反相蒸发法制备兼具跨膜、长循环功能及包覆疏水性超顺磁性Fe304颗粒的TAT磁性纳米脂质体,透射电镜,粒径仪和Zeta电位仪测定脂质体粒径和基本表征。
通过结扎Wistar成年大鼠单侧隐神经激活雪旺细胞,采用双酶消化组织块法结合机械分离法分离培养雪旺细胞；应用低浓度胶原酶、胰酶快速消化法和差速贴壁法纯化雪旺细胞。用S-100抗体、DAPI染色来鉴定雪旺细胞的纯度。将雪旺细胞分别与外接TAT磁性纳米脂质体及未接TAT磁性纳米脂质体共孵育,对照组加入未接TAT的磁性纳米脂质体(59μg/ml) 0.5ml:实验组加入外接TAT的磁性纳米脂质体(59μg/ml) 0.5ml,5%CO2,37℃共孵育1小时,在荧光显微镜下观察雪旺细胞对脂质体的摄取情况。
本实验采用雌性Wistar大鼠16只,用IMPACTOR MODEL-Ⅱ打击器建立T10脊髓损伤(spinal cord injury, SCI))模型(10g×25mm)。造模成功后随机分为2组,分别于24小时通过尾静脉注射FITC标记的磁性纳米脂质体(1mg/kg),对照组注射未接TAT的磁性纳米脂质体,实验组加入外接TAT的磁性纳米脂质体注射后1小时处死大鼠,取出脊髓组织作5μm快速冰冻切片,在荧光显微镜下观察脂质体在脊髓组织中的聚集情况,同时取大鼠肝脏、脾脏、肾脏作冰冻切片并在荧光显微镜下观察脂质体被其它器官摄取情况。
成年雌性Wistar大鼠16只,随机分为两组,对照组未作脊髓损伤处理,仅采取相同剂量进行麻醉,实验组建立脊髓损伤模型,两组动物均在尾静脉途径注射外接TAT的磁性纳米脂质体(1mg/kg),实验组时间点选取为伤后24小时,对照组为麻醉后24小时加制作模型所需时间。注射后1小时处死大鼠,取出脊髓组织作5μm快速冰冻切片,在荧光显微镜下观察脂质体在脊髓组织中的聚集情况。
[结果]
1.磁性脂质体呈球形,分散性较好,粒径较小,Zeta电位较高,外接TAT组与未接TAT组粒径及Zeta电位无明显差别。
2.激活态雪旺细胞经分离、培养、纯化后,细胞生长旺盛。传至第4代时,低倍镜下几乎不见成纤维样细胞。雪旺细胞纯度达到95%以上。
3.细胞摄取实验外接TAT的脂质体更多的聚集在雪旺细胞内部,并大多聚集在细胞核周围,而未接TAT的脂质体较少进入雪旺细胞,比较二者的镜下FITC荧光的平均光密度(average optical density, AOD),二者具有统计学差异。
4.在荧光显微镜蓝色荧光激发下,FITC使磁性纳米脂质体呈现绿色荧光。在大鼠损伤脊髓组织中,外接TAT的脂质体更多的聚集在损伤脊髓周围,神经元细胞的胞浆中存在大量的荧光颗粒,聚集成片状显现出对核的趋向性,而未接TAT的脂质体则较少分布于受损脊髓。计算二者AOD值,结果具有统计学意义。
5.在荧光显微镜蓝色荧光激发下,未接TAT组的肝、脾、肾脏中均可见明亮的绿色荧光,外接TAT组绿色荧光相对较弱。
6.在荧光显微镜蓝色荧光激发下,正常大鼠脊髓组织中绿色荧光较少,而脊髓损伤大鼠的脊髓中聚集了大量的绿色荧光,计算二者AOD值,结果具有统计学意义。
[结论]
本研究证明了TAT介导的脂质体可大量进入受损脊髓,进入神经元细胞,并且具有靶向聚集受损脊髓组织的特性,为其成为装载治疗脊髓损伤药物的载体提供了可行性依据。
[Objective] Explore the FITC-TAT-mediated magnetic liposomes and character-ization biocompatibility of FITC labeled TAT-PEG magnetic cationic liposomes with Schwann cells in vitro, and analyze its ability to through the membrane by co-culture of Schwann cells in vitro, observe the local gathering in the rat spinal cord tissue after spinal cord injury, research The targeting abilityin the injured spinal, to provide experimental evidence with theliposomes mediating drugs for the treatment of spinal cord injury in the future.
[Method] In the study, long cycle and superparamagnetic TAT-magnetic nano liposomes were prepared by reverse phase evaporation. Transmission electron--microscopy, particlesize and Zeta potential analyzer measured particle size and basic characterization of liposomes. By unilateral ligating saphenous nerve of Wistar rats to activate Schwann cells, and then isolation, culture and purification of Schwann cells were performed in vitro. At last the purity of Schwann cells were identified by S-100 antibody and DAPI staining.
The Schwann cells and two types of liposomes (TAT-type, noTAT-type) were incubated together. The Schwann cells were divided into two groups:the control group (co-culture with TAT-liposome for 1 hour) (59μg/ml) 0.5ml, the experimental group (co-culture with noTAT-liposome for 1 hours) (59μg/ml) 0.5ml. Observing the uptake of liposomes by Schwann cells under fluorescence microscope was undertaken.
Sixteen of female Wistar rats were subjected to IMPACTOR MODEL-Ⅱto establish a T10 spinal cord injured model. The animals were randomly divided into two groups:24 hours after injury, respectively, by tail vein injection of FITC-magnetic nano-liposomes (1mg/kg), the control group received nano-liposomes without TAT, experimental group to join the TAT-magnetic nano-liposomes.By injecting FITC labeled new liposome (1mg/kg) into tail vein, rats were killed 1 hour later. Harvested the spinal cord was subjected to quick 5μm frozen sections.The sections of spinal cord, liver, spleen and kidney were also observed under fluorescence microscope.
Sixteen of female Wistar rats were randomly divided into two groups:control group without treatment for spinal cord injury, only to take the same dose of anesthesia, the experimental group to establish spinal cord injury model.Two groups of animals were injected in the tail vein of the TAT-magnetic nanoliposomes (1mg/kg), the time point selected as the experimental group was 24 hours after injury, control group was 24 hours after anesthesia adding the time required for production models. rats were killed 1 hour later. Harvested the spinal cord was subjected to quick 5μm frozen sections.
[Results]
1. Magnetic liposomes were spherical lipid lanes, better dispersion, smaller particle size, higher external Zeta potential. There were no significant difference between TAT-nanoliposomes group and noTAT group in the particle size and Zeta potential.
2. Activated Schwann cells were isolated, cultured. The purity was more than 95%.
3. Cellular uptake experiment show that:TAT-liposomes gathered inside Schwann cells,and most gathered around the nucleus.The noTAT-liposomes can not be observed in Schwann cells.By comparing the FITC fluorescence microscope in two groups,the average optical density(average optical density, AOD) showed a significant difference.
4. In the blue fluorescence under fluorescence microscope, FITC made the magnetic nano-liposomes showeing green fluorescence. In the rat spinal cord tissue,TAT-liposomes gathered more around the injured spinal cord side, neuronal cells present in the cytoplasm of a large number of fluorescent particles, gathered into a sheet showing the trend of the nuclear.noTAT-liposomes were then distributed less in damage side of the spinal cord. Both AOD values calculated, the result was statistically significant.
5. By excitation with blue in fluorescence microscope, in the no TAT group,in the liver, spleen, kidney, bright green fluorescence are visible.The TAT group is relatively weaker with green fluorescence.
6. By the blue fluorescence excitation, Spinal cord tissue had less green fluorescence in normal rats.Spinal cord gathered a large number of green fluorescencein injured spinal cord rats.Calculating values of the two AOD, the results have statistical significance.
[Conclusion] This study proved that TAT-mediated nanoliposomes can be poured into the damaged spinal cord, get into neurons, and have the targeting characteristics of gethering to damaged spinal cord,provided a feasible basis for becoming a load carrier of drugs for curing spinal cord injury.
引文
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