不同材料包被的SPIO对巨噬细胞的生物学影响及磁共振成像效应

发布时间：2017-12-31 15:15

本文关键词：不同材料包被的SPIO对巨噬细胞的生物学影响及磁共振成像效应　出处：《浙江大学》2010年硕士论文　论文类型：学位论文

【摘要】： 背景介绍与研究目的炎症反应在机体内的部分心血管疾病及神经系统疾病的发展及转归过程中发挥着重要作用,包括T细胞,B细胞及大多数巨噬细胞在内的白细胞均可迁移到病变部位并聚集。通常评价这些疾病的炎症反应需要组织学检查,但这种检查只能观察到病变部位炎症存在的静止情况,不能动态的观察炎症细胞迁移到病变部位的过程,而且具有创伤性。如果能实时并非创性观察这些疾病的炎症反应,则对疾病的治疗及预后有重要意义。近年来分子影像的迅速发展使其成为可能。通过超顺磁性氧化铁(SPIO)对巨噬细胞进行标记,从而示踪其在体内的迁移及分布,能够动态非创性的评价体内的炎症反应。当SPIO通过静脉注入体内后,随血液在人体内循环。在体内SPIO主要通过肝脏的网状内皮系统和血液中的吞噬细胞摄取清除,当SPIO被血液中的吞噬细胞摄取后,并不能再从细胞内排出,这样便对吞噬细胞进行了标记,而标记的吞噬细胞可以游走到炎症所在部位并聚集,在磁共振扫描时,会造成炎症所在部位的T_2弛豫时间缩短,从而在磁共振图像上显示为信号降低。一般来讲,SPIO主要是由内核及包被材料两部分构成,纳米颗粒内径从十几纳米到几百纳米。内核由Fe_3O_4和/或γFe_2O_3构成,包被材料的种类很多,包被材料的存在提高了SPIO的稳定性及生物相容性,而且有包被材料的存在,还可以便于对SPIO进行进一步的修饰,从而能够使SPIO具有靶向性或特异性。到目前为止,已有很多学者利用SPIO来标记被活化的或没有被活化的巨噬细胞,并对体内炎症反应进行评价。这些研究都充分说明利用SPIO标记巨噬细胞对体内炎症反应进行评价具有很大优势。虽然如此,由于SPIO包被材料和内径存在很大差异性,在这些研究中所利用的SPIO的包被材料及内径大小均不相同,所以得到的结果可比性较差。如果能定义一种最佳的SPIO用来标记巨噬细胞,不仅标记效率高,而且生物相容性高,则对进一步的研究有重要意义。但这方面的研究还很少。在本研究中,我们在体外比较了柠檬酸和葡聚糖包被的SPIO对小鼠巨噬细胞的标记效率的不同,并对标记后细胞功能的变化及磁共振成像效应进行了评价。从而尝试了解哪种SPIO更适于标记巨噬细胞,为进一步的生物应用提供试验基础。材料和方法按照共沉淀法合成柠檬酸及葡聚糖包被的SPIO。分离6周龄左右的雌性ICR小鼠腹腔巨噬细胞。在巨噬细胞与浓度为0.25mg/ml SPIO共同孵育1小时后,通过普鲁士蓝染色对两种材料包被的SPIO标记巨噬细胞的效率进行观察。浓度分别为0.1、0.25、0.5、0.75、1.0mg/ml的SPIO对巨噬细胞进行标记后,采用Calcein-AM法对不同浓度的SPIO标记后的巨噬细胞活性进行评价。巨噬细胞与浓度为0.5mg/ml的SPIO共同孵育1小时后,通过细胞划痕试验对细胞的迁移能力的变化进行观察,用imageJ进行像素分析来评价细胞迁移能力的变化。巨噬细胞与浓度为0.5mg/ml的SPIO共同孵育1小时后,通过酶联标记法测定标记细胞促炎症因子IL6的24小时分泌量。0.1、0.25、0.5、0.75、1.0mg/ml四种浓度的SPIO与巨噬细胞共同孵育1小时后,用0.5%琼脂凝胶固定于96孔板,在PHILIPS 3.0T磁共振全身扫描仪上进行扫描,对不同孵育浓度时弛豫时间的变化进行评价。SPIO孵育浓度对细胞弛豫时间及细胞活性的影响用双向方差分析,然后进行多重比较分析。SPIO对细胞迁移能力及促炎症因子的分泌量的影响通过student t检验进行。结果利用经典共沉淀法合成的SPIO为棕黑色胶状液体,柠檬酸包被的SPIO水合内径约100nm,柠檬酸包被的SPIO水合内径约150nm,纳米粒度分布较好。4℃保存SPIO稳定性好。普鲁士蓝染色结果显示未与SPIO共同孵育的巨噬细胞被染成均质红色,而与SPIO共同孵育1小时后的巨噬细胞质内出现蓝色铁颗粒,柠檬酸包被的SPIO明显比葡聚糖包被的SPIO更易进入到细胞内,即标记效率高。细胞的活性测定结果显示细胞活性随SPIO孵育浓度的增加而降低,当SPIO孵育浓度为0.25mg/ml起,两种材料包被的SPIO对细胞均存在毒性作用。另外通过比较同种孵育浓度两种包被材料的SPIO细胞活性,发现两种包被材料的SPIO在相同的浓度时对巨噬细胞的影响无统计学意义。细胞迁移能力测定结果显示两种包被材料的SPIO标记的细胞的迁移能力无明显变化,这便说明SPIO不会影响细胞的迁移能力。促炎症因子分泌量测定结果显示柠檬酸包被的SPIO标记的巨噬细胞促炎症因子的分泌量增大而葡聚糖包被的SPIO标记的细胞则无增大。在磁共振扫描的结果中显示柠檬酸包被的SPIO标记的巨噬细胞可以明显引起T_2弛豫时间的降低,而葡聚糖包被的SPIO在最高孵育浓度时才会引起T_2弛豫时间的降低。结论体外试验证实,柠檬酸包被的SPIO更适于标记巨噬细胞。柠檬酸包被的SPIO相对于葡聚糖包被的SPIO具有更高的巨噬细胞标记效率,更易引起磁共振信号的变化。在对细胞功能影响方面,柠檬酸包被的SPIO标记的巨噬细胞的细胞活性及细胞迁移能力的变化与葡聚糖包被的SPIO标记的巨噬细胞相同但促炎症因子分泌量会增大。
[Abstract]:Background and research purposes
Play a important role in the process of development and prognosis of inflammatory reaction in the body part of the cardiovascular and nervous system diseases, including T cells, B cells and white blood cells are macrophages, most migrate to the lesion and aggregation. Inflammatory reaction usually evaluate these diseases need histological examination, but still can only observe the inspection to the lesion inflammation, can dynamically observe migration of inflammatory cells to lesion, but also has real time. If not a traumatic inflammatory response observed in these diseases, treatment and prognosis of the disease has important significance. The rapid development of molecular imaging in recent years makes it possible. By superparamagnetic iron oxide (SPIO) to mark macrophages, thereby tracing in the migration and distribution in vivo, non invasive evaluation to dynamic in vivo anti inflammation When injected into the SPIO. Through the vein, with the blood circulation in the human body. In vivo SPIO mainly by the reticuloendothelial system in the liver and blood phagocytic uptake clearance, when SPIO is in the blood after ingestion of phagocytic cells, and can not be discharged from the cells, thus the phagocytic cells were labeled, and mark phagocytes can walk to the location of inflammation and aggregation in magnetic resonance scanning, can cause inflammation of the location of the T_2 relaxation time is shortened, thus reducing display signal in magnetic resonance images. Generally speaking, SPIO is mainly composed of two parts by the kernel and package material, nano particle diameter from 10 nm to a few hundred nanometers. The kernel by Fe_3O_4 and / or gamma Fe_2O_3 composition, coated materials are of many kinds, coated materials could improve the compatibility of SPIO stability and biological, and coated materials, can also be Further modification of SPIO in order to enable the SPIO targeted or specific. So far, many scholars use SPIO to mark is activated or not activated macrophages, and to evaluate the inflammatory reaction in vivo. These studies are fully illustrated by SPIO markers to evaluate in vivo macrophage inflammation has great advantages. However, due to the SPIO coating material and diameter there is a big difference, used in these studies of SPIO coated materials and size are not the same, so get worse. If you can define a best SPIO used to mark macrophages, not only the labeling efficiency high, and high biocompatibility, it has important significance for further research. But there is little research in this aspect. In this study, we compared the citric acid and dextran in vitro The labeling efficiency of coated SPIO on mouse macrophages is different, and the changes of cell function and MRI effect are evaluated after labeling. We try to understand which SPIO is more suitable for labelling macrophages, and provide experimental basis for further biological applications.
Materials and methods
According to the co precipitation synthesis of citric acid and dextran coated SPIO. isolated from female ICR mice peritoneal macrophages in about 6 weeks of age. The concentration of 0.25mg/ml in macrophages and SPIO were incubated for 1 hours, the two kinds of materials package SPIO labeled macrophages are the efficiency were observed by Prussian blue staining. The concentration of SPIO was 0.1,0.25,0.5,0.75,1.0mg/ml to mark macrophages, Calcein-AM method was used to evaluate the SPIO markers after different concentration of macrophages and macrophage activity. The concentration of 0.5mg/ml SPIO after 1 hours of incubation, the changes were observed by cell scratch test on the migration ability of cells, the change pixel was analyzed by imageJ to evaluate the ability of cell migration of macrophages. With the concentration of 0.5mg/ml SPIO after 1 hours of incubation by ELISA method for the determination of proinflammatory cytokines IL6 labeled cells The 24 hour production of.0.1,0.25,0.5,0.75,1.0mg/ml four concentrations of SPIO and macrophages were incubated for 1 hours, with 0.5% agar gel fixed in 96 hole plate scanning at PHILIPS 3.0T MR scanner, evaluate the changes of.SPIO were different incubating concentration when the relaxation time of the influence of different concentration on cell relaxation time and the cell activity analysis by two-dimensional variance, then the effect of multiple comparison of secretion of.SPIO on cell migration and proinflammatory cytokines by Student t test.
Result
SPIO co precipitation synthesis method for brown black colloidal liquid using classic citric acid coated SPIO hydration diameter about 100nm, citric acid coated SPIO hydration diameter about 150nm, nano size.4 C better save SPIO stability. Prussian blue staining results showed that SPIO were incubated with macrophages stained by homogeneous red however, SPIO were incubated with blue iron particles in macrophages within 1 hours after the matter, citric acid coated SPIO than dextran coated SPIO more easily into the cell, namely high labeling efficiency. The determination results of cells showed increased cell activity with SPIO incubation concentration decreased when SPIO incubating concentration of 0.25mg/ml, two kinds of materials coated by SPIO are toxic to cells. In addition, by comparing the same incubation concentration of two kinds of coated SPIO cell active material, found two kinds of coated materials in the same SPIO The concentration of macrophages was not statistically significant. The cell migration assay showed that two kinds of package is the migration ability of SPIO cells labeled material had no obvious change, this shows that SPIO does not affect cell migration ability. Proinflammatory cytokines increased in dextran labeled with SPIO secretion determination results show SPIO markers the citric acid coated macrophage proinflammatory cytokine secretion of the cells is increased. The SPIO markers displayed citric acid coated macrophages can significantly decrease the T_2 relaxation time in magnetic resonance imaging results, and dextran coated SPIO were incubated at the highest concentration will cause a decrease in T_2 relaxation time.
conclusion
In vitro test showed that citric acid coated SPIO is more suitable to mark macrophages. Citric acid coated SPIO with dextran coated SPIO with macrophage markers more efficient, more easy to cause the change of magnetic resonance signal. The effect on cell function change and dextran cell activity and cell migration of SPIO labeled citric acid the package was the macrophages package SPIO labeled macrophages but same proinflammatory cytokine secretion will increase.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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