FITC标记葡聚糖
发布时间：2020-05-21 点击数：714
FITC标记葡聚糖
CAS Number: 60842-46-8
化学名称：
·Dextran(3’,6’-dihyroxy-3-Oxospiro (isobenzofuran-1-(3H), 9'-[9Hxanthen]-5 (or 6)-ylcarbamothiate.
·Fluoresceinisothiocyanate-dextran
· Fluoresceinyl thiocarbamoyl-dextran
化学名称：
·Dextran(3’,6’-dihyroxy-3-Oxospiro (isobenzofuran-1-(3H), 9'-[9Hxanthen]-5 (or 6)-ylcarbamothiate.
·Fluoresceinisothiocyanate-dextran
· Fluoresceinyl thiocarbamoyl-dextran
FITC标记葡聚糖是由荧光素异硫氰酸酯(5-异构体)与相应的葡聚糖偶联合成的。控制每个批次的分子量，取代度，干燥失重和游离FITC。TdB生产分子量4kDa到20kDa的FITC标记葡聚糖。FITC标记葡聚糖是黄色至橙色粉末，易溶于水或盐溶液，呈黄色溶液。该产品也可以溶于DMSO, 甲酰胺和其它极性有机溶剂，但在低级脂肪醇，丙酮，氯仿和DMF中基本不溶。
结构
葡聚糖是来源于肠系膜白串珠菌B-512F的分支多糖，本质上是一种α-(1-6)链接的线性葡萄糖链。分支比例大约是5%。荧光素是通过稳定的硫代氨甲酰键链接的，标记过程不会引起葡聚糖的解聚。FITC标记葡聚糖，每个葡萄糖单元对应0.002到0.008 mol的FITC。 这些低取代度使葡聚糖电荷小，这也是渗透性研究基本的要求。
图1 FITC标记葡聚糖分子结构示意图
光谱数据
FITC标记葡聚糖的荧光激发波长为493nm，检测波长为518nm（图2）。由于荧光素的电荷依赖于溶媒的pH和离子强度，荧光强度也随着这些参数而变化。在pH>8时观测到强度max。在生物媒介中测量，会影响荧光强度，增强或衰减。
图2 FITC标记葡聚糖70在pH9.0磷酸盐缓冲生理盐水中的荧光图谱
图3 FITC标记葡聚糖在pH4-9的荧光（发射波长520nm）
储存和稳定性
FITC标记葡聚糖在密封容器室温下可以保存6年以上。
已经在多种介质和不同温度条件下调研过FITC标记葡聚糖的稳定性。研究表明，FITC标记葡聚糖无论在体内或体外都很稳定。仅当pH>9和温升时，存在荧光标记水解的风险。37℃下对兔血浆、肌肉匀浆、肝匀浆和尿液的研究证实，FITC标记葡聚糖至少可稳定3天。分子量没有变化，荧光素亦未见释放。FITC标记葡聚糖在6%三氯乙酸溶液中在室温下可稳定3天。pH10-10.75时氢氧根离子对水解反应存在特异性催化作用(1)。
通过HPLC检测，硫代氨甲酰键的水解引起4-或5-氨基荧光素的增加。 一项未发表的研究考察了跨度5个月的时间里在压力容器中，温度范围8到50°C下的 FITC标记葡聚糖 70溶液。仅在50°C下观测到游离氨基荧光素略微增加了1%。 仅高压只释放了2.7%的游离氨基荧光素。另一项未发表的研究表明，FITC标记葡聚糖溶液在pH 4温度高至35°C下一个月内稳定。即使80°C和pH 4时, 硫代氨甲酰键可稳定30分钟。不过，葡聚糖可能会降解。pH 9时，1个月时间内, 相当客观的荧光标记多糖(24 %)减少了。多项研究表明在实验持续时间内FITC标记葡聚糖在体内的稳定性(2)。
毒性
小鼠实验表明, FITC标记葡聚糖静脉或腹腔注射量高达 6g/kg体重时，耐受性仍良好。 毒性模式与母体葡聚糖的一致。葡聚糖作为血浆扩容剂在临床上已经用了50多年。人体在注射了临床用的葡聚糖溶液后，观察到葡聚糖诱导的类过敏反应 (DIARs)的发生(3,4)。 FITC标记葡聚糖亦可能显示类似的行为模式，但相关实验动物出现问题很少见诸报道。
合成
来源于天然的葡聚糖B512F经荧光素标记制备了对应的荧光标记葡聚糖。荧光素是通过稳定的硫代氨甲酰键链接的，标记过程不会引起葡聚糖的解聚。FITC标记葡聚糖，每个葡萄糖单元对应0.002到0.008 mol的FITC。 这些低取代度使葡聚糖电荷很小，这也是渗透性研究基本的要求。分子量超过5000 Daltons的葡聚糖分子在溶液中呈有弹性的延长的卷状。下表1显示了不同分子量的分子空间尺寸。
|
Dextran MW
|
斯托克斯半径 (Å)
|
回转半径 (Å)
|
|
2 x 106
|
270
|
380
|
|
1 x 106
|
199
|
275
|
|
500 000
|
147
|
200
|
|
200 000
|
130
|
130
|
|
100 000
|
69
|
95
|
|
70 000
|
58
|
80
|
|
40 000
|
44.5
|
62
|
|
10 000
|
23.6
|
-
|
Table 1. 葡聚糖的分子尺寸
葡聚糖和FITC标记葡聚具有牛顿流体特征，例如粘度与剪切速率无关（图4）。在pH4-10范围的研究显示，粘度与pH无关。 在pH8-9和6.5-9.5下，经电泳，FITC标记葡聚糖的等电点未发生迁移(1)。
Fig. 4. 不同浓度下葡聚糖的粘度
产品列表
|
产品编号
|
品名
|
分子量(kDa)
|
包装
|
|
FD4-100mg
|
FITC标记葡聚糖4
|
4
|
100 mg
|
|
FD4-1g
|
1 g
|
||
|
FD4-5g
|
5 g
|
||
|
FD10-100mg
|
FITC标记葡聚糖10
|
10
|
100 mg
|
|
FD10-1g
|
1 g
|
||
|
FD10-5g
|
5 g
|
||
|
FD20-100mg
|
FITC标记葡聚糖20
|
20
|
100 mg
|
|
FD20-1g
|
1 g
|
||
|
FD20-5g
|
5 g
|
||
|
FD40-100mg
|
FITC标记葡聚糖40
|
40
|
100 mg
|
|
FD40-1g
|
1 g
|
||
|
FD40-5g
|
5 g
|
||
|
FD70-100mg
|
FITC标记葡聚糖70
|
70
|
100 mg
|
|
FD70-1g
|
1 g
|
||
|
FD70-5g
|
5 g
|
||
|
FD110-100mg
|
FITC标记葡聚糖110
|
110
|
100 mg
|
|
FD110-1g
|
1 g
|
||
|
FD110-5g
|
5 g
|
||
|
FD150-100mg
|
FITC标记葡聚糖150
|
150
|
100 mg
|
|
FD150-1g
|
1 g
|
||
|
FD150-5g
|
5 g
|
||
|
FD500-100mg
|
FITC标记葡聚糖500
|
500
|
100 mg
|
|
FD500-1g
|
1 g
|
||
|
FD500-5g
|
5 g
|
||
|
FD2000-100mg
|
FITC标记葡聚糖2000
|
2000
|
100 mg
|
|
FD2000-1g
|
1 g
|
||
|
FD2000-5g
|
5 g
|
应用
FITC标记葡聚糖主要用于渗透性和微循环研究。也可用作分子量标志，用于给药研究及其它
FITC标记葡聚糖主要用于细胞和组织中渗透性和传递研究。测定荧光可提供健康组织和病态组织渗透性的定量数据。可以通过活体荧光显微镜实时进行。该技术灵敏度较高，组织液体中浓度低至1μg/ml亦可检测出。 FITC标记葡聚糖在细胞中亦可作为一种pH探针使用(6,7)。 极化实验也表明，荧光素结合葡聚糖仍保留了较高的旋转自由度，激发态下的荧光寿命和未结合的相近(6)。
1. 常规过程
在不同的实验条件下，仓鼠颊囊的微血管是研究血浆渗漏的有效模型，例如缺血/再灌注后，或局部应用一系列炎症介质、寄生虫和细菌。利用该技术，可以实时研究血管渗透性变化，并与白细胞粘附、活化等微血管事件相关联。使用合适的滤光片（490/520nm）对颊囊进行活体荧光显微镜检查，并用数码相机拍摄图像。注射5%FITC标记葡聚糖150生理盐水溶液，（约100毫克/千克体重）(8-10)。
通过荧光结合立体显微镜对渗透性的研究，Thorball（2）报道了荧光光学显微镜。本文还包括FITC标记葡聚糖参与的组织固定技术和显微镜设置（滤镜、照明）的细节。用再生钛耳室（兔子）进行FITC标记葡聚糖参与的血液/淋巴微循环系统研究。植入后4-8周可见淋巴生长（11）。
图5. 注射FITC标记葡聚糖 150后拍摄的颊囊图像。第二张图显示的是服用组胺后微血管的渗漏
(图片版权E. Svensjö)
2.肠道组织的渗透性研究
使用FITC标记葡聚糖 150 (12)研究处于炎症期的肠上层单皮的渗透性。使用FITC标记葡聚糖4（13）研究蛋白酶抑制剂对胃肠道粘膜糜烂和上皮功能障碍的作用。Thorball做了大量使用FITC标记葡聚糖在胃肠道进行组织固定的研究(2)。FITC标记葡聚糖 (4000-70000)用于皮肤热损伤在体外使用改进型尤斯灌流室后引起的渗透性变化(14)。请参看参考资料(15,16)。
3. 大脑和神经系统的渗透性研究
有关神经系统中FITC标记葡聚糖的研究，需要示踪剂固定技术，使神经元、胶质细胞、坐骨神经内脊膜、神经节等特征具有良好的光学分辨率。采用干燥解剖组织样品冷冻、80℃甲醛蒸汽固定，石蜡或塑料包埋的方法证明效果良好（17）。在安装和检测之前，这些切片要浸入二甲苯中。
给仓鼠和小鼠静脉注射了FITC标记葡聚糖（Mw 3000到150000），研究在体内的分布。正常的大脑皮层神经周围扩散屏障，均不被上述分子量范围内FITC标记葡聚糖渗透（19）。仓鼠的注射量为0.5ml生理盐水，含浓度为5mg /10g体重的FITC记葡聚糖（18）。调查了FITC标记葡聚糖对海马体渗透性的影响。聚焦超声用于血脑屏障检查，分析FITC标谱聚糖的渗透情况（19）。
采用静脉注射FITC标记葡聚糖150（20）检测缺血再灌注损伤后，与时间相关的脑血管渗透性。通过FITC标记葡聚糖监测蛋白相关渗透性变化，进行急性肝功能衰竭后脑水肿的研究（21）。
4. 肿瘤组织的渗透性研究
Gerlowski（22）介绍了透射光荧光电视显微镜用于肿瘤组织中微血管渗透性的实时研究。在兔耳腔模型中使用了FITC记葡聚糖150。作者每隔90分钟检测浓度从0.6 mg到30 mg/100 ml的FITC标记葡聚糖的猝灭作用。只有接近浓度峰值时才会猝灭。T. Li和同事（23）使用FITC标记葡聚糖500研究聚焦超声对肉瘤细胞膜渗透性的影响。FITC标记葡聚糖还应用于卵巢癌细胞的内吞作用研究（24）。
5. 眼窝内的渗透性研究
E. Mannerma和同事（25）使用FITC标记葡聚糖40等探针研究药物在视网膜色素上皮中的渗透过程。FITC标记葡聚糖用于评估TNFF-alpha引起的屏障功能障碍（26）。S. Lightman和同事（27）详细研究了炎症模型视网膜血管的渗漏。反复多剂量的FITC标记葡聚糖，耐受性良好。研究了FITC标记葡聚糖和荧光素离开玻璃体的途径（28）。C.B.Toris和同事（29）使用FITC标记葡聚糖检查了常规眼疾的葡萄膜巩膜外流。描述了血-视网膜外屏障的体外模型（30）。
6.肾组织渗透性研究
70%剂量的FITC标记葡聚糖4在一小时内快速分泌至尿液(兔子、老鼠)(未发表数据)。Lencer和同事(31)研究报道了FITC标记葡聚糖10作为探针探测肾脏内肾小球的功能和定位。对切除组织的冷冻切片进行了荧光显微镜检查。
多种应用
FITC标记葡聚糖曾用于体外鼻粘膜的渗透性研究 (32)。该项研究还涉及带电荷的 FITC标记葡聚糖衍生物。 使用载有溶解微针的FITC标记葡聚糖20用于药物在皮肤的直接扩散研究(33)。
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3. K.G.Ljungström, H.Rench, K.Strandberg et al. Adverse reactions to dextran in Sweden 1970-1979 Acta Chir Scan, 1983; 149:253-262
4. H.Hedin, W.Richter and J.Ring. Dextran-induced anaphylactoid reactions in man: role of dextran reactive antibodies Int. Arch Allergy Appl Immun. 1976; 52:145-159.
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