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In vivo Fluorescein Isothiocyanate-dextran (FD4) Permeability Assay
体内异硫氰酸荧光素右旋糖酐(FD4)渗透性分析   

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Abstract

Using pluripotent stem cells, it is now becoming possible to develop tissue models of organ systems within the body. These organs will allow for the study of organ function, physiology, embryology, and even pathologic processes. Recently, our group developed a model of human small intestine developed from human pluripotent stem cells which when transplanted in vivo, produce a mature, cystic intestinal structure that has digestive functions similar to that of native small intestine (Watson et al., 2014). Intestinal permeability is a primordial function of both the epithelium and associated tight junctions to control nutrient intake and prevent the passage of pathogens. One way to study gastrointestinal paracellular permeability is by determining the ability of fluorophores-conjugated macromolecules (i.e., fluorescein isothiocyanate-dextran (FITC-dextran; or FD4) to cross from the lumen and into circulation (Dong et al., 2014). We were able to test the intestinal permeability by injecting FITC-dextran directly into the lumen of the bioengineered intestine and determining the fluorescence within the blood of the murine host at various time points after injection.

Materials and Reagents

  1. EDTA blood collection tubes (BD Biosciences, catalog number: 365873 )
  2. 1 ml syringes
  3. 30 gauge needles
  4. Microhematocrit Capillary Tubes (Thermo Fisher Scientific, catalog number: 22-362-566 )
  5. 1 ml Eppendorf tubes (USA Scientific, catalog number: 1615-5510 )
  6. 96-well microplates (Greiner Bio-One GmbH, catalog number: 655101 )
  7. Immune deficient NOD-SCID IL-2Rγnull (NSG) mice, 8-16 weeks of age (bred in house), transplanted with a bioengineered intestine (maturation of transplant then allowed for 6-8 weeks)
  8. Fluorescein isothiocyanate-conjugated dextran (FITC-dextran 3-5 Kda) (Sigma-Aldrich, catalog number: FD4 )
  9. Sterile water
  10. Phosphate-buffered saline (PBS)
  11. Isoflurane for inhaled anesthesia (Henry Schein, catalog number: 050033 )
  12. Buprenorphine (Midwest Veterinary Supply, catalog number: 790.06700.3 )
  13. Isopropyl alcohol
  14. Povidone-Iodine
  15. Small surgical kit (scissors, ringed forceps, needle driver/holder)
  16. 3-0 Vicryl suture (Ethicon US, catalog number: VR416 )
  17. GLUture tissue adhesive 1.5 ml 1/Tb (Skin closure glue) (Henry Schein, catalog number: 034418 )

Equipment

  1. Spectrophotofluorometer (BioTek Instruments, model: Synergy H1 )
  2. Dissecting Stereomicroscope (Leica Microsystems)
  3. Microcentrifuge
  4. Hair clipper/trimmer

Procedure

  1. Dissolve FITC-Dextran in sterile water at a final concentration of 20 mg/ml.
  2. Trim the hair over the abdomen of a transplanted NSG mouse then clean the skin with isopropyl alcohol and povidone-iodine to create a sterile field.
  3. Induce anesthesia via nose cone with 2% inhaled Isofluorane.
  4. Once the mouse is anesthetized, cut the skin of the abdomen using scissors for several centimeters over the region of the transplanted tissue.
  5. Bring and secure the desired transplanted intestinal tissue up into the incision field using a dissecting microscope.
  6. Draw 100 μl of the FITC-Dextran solution into a 1 ml syringe equipped with a 30 gauge injection needle.
  7. Inject the lumen of the transplant with 100 μl of FITC-Dextran solution.
  8. Seal the injection site with skin glue to prevent leakage of the solution into the peritoneal cavity.
  9. Close the skin of the mouse using 3-0 vicryl suture in a running fashion.
  10. Give the mice a subcutaneous injection of buprenorphine (0.05 mg/kg) for analgesia.
  11. Remove the mouse from anesthesia and place the mouse into a clean, warm area for recovery.
  12. At 30 min after injection, return the injected mouse to the anesthesia machine for inhaled isoflurane anesthesia.
  13. Collect blood in EDTA blood collection tubes via retro-orbital bleeding using heparinized capillary tubes. Mix by inverting the tube 3-4 times.
  14. Return the mouse to the recovery area until the second timepoint of 4 h from the initial injection.
  15. Repeat step 13 at 4 h after injection.
  16. Sacrifice the mice using a CO2 exposure and confirm the euthanasia by cervical dislocation and collect tissues for histology at this time if desired. All animal euthanasia must be performed in accordance with the IACUC guidelines and an approved animal protocol within the institution.
  17. Centrifuge the EDTA tubes at 300 rpm for 10 min at 4 °C using the microcentrifuge.
  18. Remove the serum supernatant from the EDTA tubes.
  19. Dilute each sample in an equivalent amount of chilled PBS.
  20. Prepare a standard curve using serial dilution of FITC-Dextran in PBS (0, 125, 250, 375, 500, 750, 1,000 µg/ml).
  21. Add 100 μl of diluted serum to a 96-well, flat-bottom plate in duplicates. Remember to include a negative control with serum from a mouse that was not injected with FITC-Dextran solution.
  22. Determine the concentration of FITC-Dextran in the serum using a spectrophotometer (Excitation: 485 nm; Emission: 528 nm).
  23. Plot the data to determine the concentration of the sample using the standard curve (Figure1).

Representative data


Figure 1. a. Standard curve obtained from serial dilution of FITC-Dextran in PBS. b. FITC-Dextran injected into engraftments in vivo increased significantly in murine serum from initial timepoint of 30 min compared with timepoint of 4 h within each mouse (n=7). Values represented in the graph represent Mean ± s.e.m.; *p <0.05.

Notes

  1. This protocol can be adapted for other experiments involving other types of transplanted tissue or even adapted for native intestinal tissues in animal models. Any hollow viscus can be injected in a similar fashion to that outlined within this protocol. Our protocol is an alternative to oral gavage of FITC-Dextran as outlined previously in Bio-protocol (Gupta et al., 2014).
  2. The skin glue used to seal our injection site on our tissue provides an adequate seal to prevent “leaking” of FITC-dextran into the peritoneal cavity of the mice. Care is taken to prevent leakage of the solution which might affect the overall results.
  3. Alternatives to suture closure of the abdomen include closure with clips/staples.

Acknowledgments

This project was supported in part by US National Institutes of Health (NIH) grants NIH-R01DK083325 (M.A.H), NIH P30 DK078392 (Digestive Health Center, Cincinnati Children’s Hospital Medical Center), and NIH UL1RR026314 (Clinical and Translational Science Awards (CTSA), University of Cincinnati).

References

  1. Dong, C. X., Zhao, W., Solomon, C., Rowland, K. J., Ackerley, C., Robine, S., Holzenberger, M., Gonska, T. and Brubaker, P. L. (2014). The intestinal epithelial insulin-like growth factor-1 receptor links glucagon-like peptide-2 action to gut barrier function. Endocrinology 155(2): 370-379.
  2. Gupta, J. and Nebreda, A. R. (2014). Analysis of intestinal permeability in mice. Bio-protocol 4 (22): e1289.
  3. Watson, C. L., Mahe, M. M., Munera, J., Howell, J. C., Sundaram, N., Poling, H. M., Schweitzer, J. I., Vallance, J. E., Mayhew, C. N., Sun, Y., Grabowski, G., Finkbeiner, S. R., Spence, J. R., Shroyer, N. F., Wells, J. M. and Helmrath, M. A. (2014). An in vivo model of human small intestine using pluripotent stem cells. Nat Med 20(11): 1310-1314.

简介

使用多能干细胞,现在可以开发机体内的器官系统的组织模型。这些器官将允许对器官功能,生理学,胚胎学,甚至病理过程的研究。最近,我们的小组开发了从人多能干细胞发育的人类小肠的模型,当其在体内移植时,产生具有类似于天然小肠的消化功能的成熟的囊性肠结构(Watson et al。,2014)。肠通透性是上皮和相关紧密连接的原始功能,以控制营养物摄取并防止病原体通过。研究胃肠道细胞旁通透性的一种方法是通过测定荧光团结合的大分子(即异硫氰酸荧光素 - 葡聚糖(FITC-葡聚糖或FD4)从管腔穿入并进入循环的能力(Dong 我们能够通过将FITC-葡聚糖直接注射到生物工程化肠的腔中并测定注射后不同时间点的鼠宿主血液中的荧光来测试肠通透性。

材料和试剂

  1. EDTA血液收集管(BD Biosciences,目录号:365873)
  2. 1 ml注射器
  3. 30号针
  4. 微血细胞毛细管毛细管(Thermo Fisher Scientific,目录号:22-362-566)
  5. 1ml Eppendorf管(USA Scientific,目录号:1615-5510)
  6. 96孔微孔板(Greiner Bio-One GmbH,目录号:655101)
  7. 使用生物工程化肠(移植成熟,然后允许6-8周)移植的8-16周龄的免疫缺陷型NOD-SCID IL-2Rγ裸细胞(NSG)小鼠,
  8. 荧光素异硫氰酸酯 - 缀合的葡聚糖(FITC-葡聚糖3-5Kda)(Sigma-Aldrich,目录号:FD4)
  9. 无菌水
  10. 磷酸盐缓冲盐水(PBS)
  11. 用于吸入麻醉的异氟烷(Henry Schein,目录号:050033)
  12. 丁丙诺啡(Midwest Veterinary Supply,目录号:790.06700.3)
  13. 异丙醇
  14. 吡啶 - 碘
  15. 小外科套件(剪刀,环镊子,针驱动器/支架)
  16. 3-0 Vicryl缝合线(Ethicon US,目录号:VR416)
  17. GLUture组织粘合剂1.5ml 1/Tb(皮肤闭合胶)(Henry Schein,目录号:034418)

设备

  1. 分光光度计(BioTek Instruments,型号:Synergy H1)
  2. 解剖立体显微镜(Leica Microsystems)
  3. 微量离心机
  4. 理发剪/修剪器

程序

  1. 将FITC-葡聚糖溶解在无菌水中,最终浓度为20mg/ml
  2. 修剪头发在移植的NSG小鼠的腹部,然后用异丙醇和聚乙烯碘清洗皮肤,以创建一个无菌区。
  3. 用2%吸入异氟烷通过鼻锥诱导麻醉
  4. 一旦小鼠麻醉,用剪刀在移植组织的区域上切割腹部几厘米的皮肤。
  5. 使用解剖显微镜将所需的移植肠组织带入并固定到切口区域
  6. 将100μl的FITC-葡聚糖溶液倒入装有30号注射针的1ml注射器中
  7. 用100μlFITC-Dextran溶液注射移植管腔。
  8. 用皮肤胶密封注射部位,以防止溶液渗入腹膜腔。
  9. 使用3-0 vicryl缝线以运行方式关闭鼠标的皮肤。
  10. 给小鼠皮下注射丁丙诺啡(0.05mg/kg)用于止痛。
  11. 从麻醉中取出鼠标,将鼠标放在干净,温暖的地方恢复。
  12. 在注射后30分钟,将注射的小鼠返回麻醉机用于吸入异氟烷麻醉。
  13. 通过使用肝素化毛细管的眼眶后出血收集EDTA血液收集管中的血液。通过翻转管3-4次混合
  14. 将鼠标返回到恢复区域,直到从最初注射起4小时的第二个时间点。
  15. 在注射后4小时重复步骤13
  16. 牺牲小鼠使用CO 2 2暴露,并确认安乐死的颈椎脱臼,收集组织的组织学在这个时候,如果需要。所有动物安乐死必须根据IACUC指南和机构内批准的动物方案进行
  17. 使用微量离心机在4℃下以300rpm离心EDTA管10分钟
  18. 从EDTA管中取出血清上清液。
  19. 在等量的冷PBS中稀释每个样品
  20. 使用在PBS中的FITC-葡聚糖(0,125,250,375,500,750,1000μg/ml)的系列稀释制备标准曲线。
  21. 加入100微升稀释的血清到96孔,平底板一式两份。记住包括来自未注射FITC-葡聚糖溶液的小鼠的血清的阴性对照
  22. 使用分光光度计(激发:485nm;发射:528nm)测定血清中FITC-葡聚糖的浓度。
  23. 绘制数据,使用标准曲线确定样品的浓度(图1)。

代表数据


图1。 a。从在PBS中的FITC-葡聚糖的系列稀释获得的标准曲线。 b。与每只小鼠(n = 7)内的4小时的时间点相比,注射入体内植入物的FITC-葡聚糖在小鼠血清中从30分钟的初始时间点显着增加。图中表示的值表示平均值±标准偏差。 * p <0.05

笔记

  1. 该方案可以适用于涉及其他类型的移植组织或甚至适合于动物模型中的天然肠组织的其他实验。任何中空的粘液可以以与本方案中概述的类似的方式注射。我们的方案是口服灌胃FITC-Dextran的替代方案,如先前在Bio-protocol中所述(Gupta等人,2014)。
  2. 用于在我们的组织上密封我们的注射部位的皮肤胶提供了足够的密封,以防止FITC-葡聚糖"渗漏"到小鼠的腹膜腔中。注意防止溶液泄漏,这可能影响整体结果。
  3. 对腹部缝合闭合的替代方案包括用夹子/U形钉闭合。

致谢

该项目部分由美国国立卫生研究院(NIH)授予NIH-R01DK083325(MAH),NIH P30 DK078392(Digestive Health Center,Cincinnati Children's Hospital Medical Center)和NIH UL1RR026314(临床和转化科学奖(CTSA) ,University of Cincinnati)。

参考文献

  1. 1.Dong,C.X.,Zhao,W.,Solomon,C.,Rowland,K.J.,Ackerley,C.,Robine,S.,Holzenberger,M.,Gonska,T.and Brubaker, 肠上皮胰岛素样生长因子-1受体将胰高血糖素样肽-2作用连接到肠屏障功能。 Endocrinology 155(2):370-379。
  2. Gupta,J。和Nebreda,A.R。(2014)。小鼠肠道通透性分析。 生物协议 4(22):e1289
  3. Watson,CL,Mahe,MM,Munera,J.,Howell,JC,Sundaram,N.,Poling,HM,Schweitzer,JI,Vallance,JE,Mayhew,CN,Sun,Y.,Grabowski,G.,Finkbeiner, SR,Spence,JR,Shroyer,NF,Wells,JM和Helmrath,MA(2014)。 使用多能干细胞的人体小肠的体内模型。/a> Nat Med 20(11):1310-1314。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Watson, C. L., Mahe, M. M. and Helmrath, M. A. (2015). In vivo Fluorescein Isothiocyanate-dextran (FD4) Permeability Assay. Bio-protocol 5(20): e1618. DOI: 10.21769/BioProtoc.1618.
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