紧凑型超支化聚乙二醇接枝的膜抗原受保护的功能性红血细胞
Summary
细胞膜的变形例的红血细胞（红细胞），超支化聚甘油（HPG）。修改红细胞进行了表征双水相分割，渗透脆性和补体介导的溶解。使用流式细胞仪和微的打字系统（MTS）的血液分型卡的表面蛋白抗原的伪装进行了评估。
Abstract
红细胞（RBC）输血是用于治疗急性和慢性的医疗问题，如重型地中海贫血和镰状细胞贫血1-3一些至关重要的。由于众多的RBC表面上的（〜308已知抗原4），在慢性输血治疗的患者发展由于丢失如同输血红细胞4，5的轻微上的抗原的同种抗体的抗原的存在下。接枝亲水性聚合物，如聚乙二醇（PEG）和超支化聚甘油（HPG）的形成，RBC膜排除层上防止表面抗原的抗体的相互作用，而不会影响通过小分子如氧，葡萄糖，和离子3。目前，没有一种方法是可用于普遍的红血供体细胞的产生部分是因为大量抗原（基于蛋白质和碳水化合物）的存在下对RBC表面和d提出严峻的挑战才有发展这种方法将大大提高输血的安全性，并显着提高红细胞的可用性和使用。在这份报告中，提出的实验，是用来开发抗原保护功能红细胞由HPG及其表征膜接枝。 HPGs高度生物相容性紧凑的聚合物6，7，并预期位于小区内的的糖萼围绕的脂质膜8，9和掩模RBC表面抗原10，11。
Protocol
A.超支化的聚甘油改造（SS-HPG） 地点冻干HPG 60 kDa的（0.5克，0.0083毫摩尔）在一个圆底烧瓶中，并真空下干燥过夜，在90℃下冷藏烧瓶至室温，并溶解在无水吡啶（3ml）中的干燥的HPG。 若要官能约8个羟基基团与羧基基团上HPG，添加催化量的二甲基氨基吡啶（5毫克/毫升的吡啶溶液一滴）的HPG溶液。到该混合物中，加入琥珀酸酐，（0.0067克，0.0664毫摩尔）溶解在0.5毫升吡啶在10?…
Representative Results
迷彩恒河猴D抗原和CD47 RBC表面蛋白的流式细胞仪定量，使用荧光标记的单克隆抗体，和具有代表性的结果在图1中给出。在HPG-接枝红细胞的情况下（灰色），该信号的强度降低（峰转移至左）相比，控制红细胞（红色和绿色），表示表示掩蔽的表面蛋白的细胞表面结合的抗体减少。 <img src="/files/ftp_upload/50075/50075fig1.jpg" alt="图1" fo:content-…
Discussion
万能供血者红细胞有很大的潜力，在提高血液的输血治疗的有效性和安全性。红细胞也被认为是有前途的药物运输车辆，由于其长循环和固有的生物相容性，14日，15。本文提出的实验评估丘脑-垂体-性腺修改红细胞在体外的特点。 体外性能和丘脑-垂体-性腺修改红细胞在体内循环，本集团已在最近8，11。 Dextran500K/PEG8K双水相体系中，补充盐和钠磷酸盐，红细胞…
Disclosures
The authors have nothing to disclose.
Acknowledgements
资助这项研究是由加拿大血液服务（CBS）和加拿大研究所（CIHR）健康科学的研究合作基金。作者感谢的LMB高分子枢纽的UBC血液研究中心的研究设施的使用。基础设施的支持，加拿大创新基金会（CFI）和迈克尔史密斯健康研究基金会（MSFHR）。河Chapanian是一个收件人（CIHR / CBS）在输血科学博士后奖学金和收件人的MSFHR研究实习生博士后奖学金。 JN Kizhakkedathu是一个收件人的MSFHR生涯研究学者奖。
Materials
| Glycidol | Sigma Aldrich | (ON, Canada) | |
| Trimethylolpropane | Fluka | (ON, Canada) | |
| Potassium methylate | Sigma Aldrich | (ON, Canada) | |
| Anhydrous pyridine | Sigma Aldrich | (ON, Canada) | |
| 4-Dimethylaminopyridine | Sigma Aldrich | (ON, Canada) | |
| Succinic anhydride | Sigma Aldrich | (ON, Canada) | |
| Acetone | Fisher Scientific | (ON, Canada) | |
| Anhydrous dimethyl formamide | Sigma Aldrich | (ON, Canada) | |
| N-Hydroxysuccinimide | Sigma Aldrich | (ON, Canada) | |
| N,N’-Diisopropylcarbodiimide | Sigma Aldrich | (ON, Canada) | |
| MTS cards | Micro Typing System (MTS) cards (FL, USA) | ||
| Dextran 500 kDa | Pharmacia Fine Chemicals | (Sweden) | |
| PEG 8 kDa | Sigma Aldrich | (ON, Canada) | |
| FITC monoclonal anti-Rhesus D (RhD) | Quotient Biodiagnostics | (PA, USA) | |
| PE monoclonal anti-CD47 | BD Biosciences | (NJ, USA) | |
| Drabkin’s reagent | Sigma Aldrich | (ON, Canada) | |
| Table. Chemicals and reagents used for the grafting of HPG polymers to RBC membrane and their analysis. |
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Cite This Article
Chapanian, R., Constantinescu, I., Brooks, D. E., Scott, M. D., Kizhakkedathu, J. Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols. J. Vis. Exp. (71), e50075, doi:10.3791/50075 (2013).