TAKARA RetroNectin® 重组人纤维蛋白片段，Recombinant Human Fibronectin Fragment，FN CH-296

产品英文名：TAKARA RetroNectin®(Recombinant Human Fibronectin Fragment)，FN CH-296

产品中文名：TAKARA RetroNectin®重组人纤维蛋白片段，FN CH-296

RetroNectin® 重组人纤维蛋白片段

RetroNectin^®是在大肠杆菌中产生的重组人纤维蛋白片段FN CH-296。 当包被在培养瓶和培养板表面时，RetroNectin^®能显著增强逆转录病毒介导的基因转移到哺乳动物细胞中。

RetroNectin^®重组人纤维蛋白片段和激发型CD3单抗可分别与T淋巴细胞上的VLA和TCR结合，两者共同作用激活酪氨酸激酶pp125FAK，通过Ras途径刺激T细胞的增殖和分化，使其获得上千倍的增殖。

RetroNectin®

(Recombinant Human Fibronectin Fragment)

组份

RetroNectin (Recombinant Human Fibronectin Fragment)  0.5 mg (Cat. #T100A)

RetroNectin (Recombinant Human Fibronectin Fragment)  2.5 mg (Cat. #T100B)

RetroNectin is provided as a sterile 1 mg/ml solution.

[Form]　Sterile solution containing 12.5 mM sodium citrate (pH 6.2) and 1.25% sucrose

储存

-20℃

Caution:  Freezing and thawing can be repeated up to 10 times

*

订购RetroNectin欢迎您咨询华雅再生医学旗舰公司：红荣微再（上海）生物工程技术有限公司 。红荣微再授权代理TAKARA CellArtis ，Rubicon产品。红荣微再（上海）生物工程技术有限公司以“传递科学价值，服务科学研究”为宗旨，主营干细胞、医疗、细胞治疗、器官再生四大板块的产品。

RetroNectin®

(Recombinant Human Fibronectin Fragment，rFN-CH-296)

RetroNectin原理

RetroNectin reagent is a recombinant human fibronectin fragment (rFN-CH-296) composed of three functional domains: the cell-binding domain (C-domain), heparin-binding domain (H-domain),and CS-1 sequence. The fragment enhances retroviral-mediated gene transduction by aiding the co-localization of target cells and virions. Specifically, virus particles bind RetroNectin reagent via interaction with the H-domain, and target cells bind mainly through the interaction of cell surface integrin receptor VLA-5 and VLA-4 with the fibronectin C-domain and CS-1 site, respectively.By facilitating close proximity, RetroNectin reagent can enhance retroviral-mediated gene transfer to target cells expressing integrin receptors VLA-4 and/or VLA-5.*1

There are two RetroNectin-mediated infection protocols: the supernatant (SN) infection method and the RetroNectin-bound virus (RBV) infection method.5),*2 With the SN infection method, cells are mixed with virus supernatant and loaded on a RetroNectin-coated plate. In the RBV method, the retrovirus is first bound to the RetroNectin-coated plate, and cells are added after removing the retrovirus supernatant. Removal of the supernatant reduces inhibitory molecules (e.g., molecules secreted from the producer cells such as proteoglycans and/or viral envelope proteins) that can reduce the efficiency of viral-mediated gene transduction.

*1  RetroNectin can also enhance lentiviral-mediated gene transfer.

*2  Both methods can be used for efficient gene transduction. Although the RBV infection method is widely applicable, some modification might be required depending on the target cells,

vectors, and/or target genes.

RetroNectin®

(Recombinant Human Fibronectin Fragment，rFN-CH-296)

RetroNectin逆转录技术操作流程

RetroNectin技术可以显著提高逆转录病毒的转染效率，这一发现克服了向造血干细胞中导入基因的困难。现在，RetroNectin技术已成为使用逆转录病毒进行转染操作的常规选择，其操作流程大致如下：

1. 注射用水或灭菌蒸馏水充分溶解RetroNectin，调节浓度至1mg/ml。

2. 0.22 μm滤膜过滤RetroNectin溶液，分装后-20 ℃保存。

3. 用缓冲液将RetroNectin溶液稀释至20～100 μg/ml。

4. 将RetroNectin稀释液加入24孔板，每孔0.5 ml (建议用PBS缓冲液稀释RetroNectin后包被Non-Tissue Cluture Treated plate。

5. 室温放置4小时或4 ℃，避光，过夜。

6. 吸出RetroNectin溶液，每孔加入PBS终止液0.5 ml。

7. 室温放置30分钟。

8. 加入适量PBS清洗孔板。

9. 吸出清洗液，得到RetroNectin包被的孔板 (如果暂不使用，可以在孔中加入适量PBS，用胶膜将平板盖密封，避免RetroNectin干燥，可于4 ℃保存4周。使用前将PBS吸出) 。

10. 逆转录病毒保存液 (-80 ℃) 于37 ℃水浴融解。

11. 将病毒液加入包被后的24孔板，每孔300 μl。

12. 32 ℃放置4小时。

13. 吸出病毒保存液。

14. 每孔加入待转染的细胞悬液400 μl (通常细胞浓度为1 × 105/ml) 。

RetroNectin增强T细胞扩增技术流程

T lymphocytes (T cells) play a specialized, critical role in the antigen-specific immune response. When activated, these cells can recognize and directly kill virally infected or malignant cells. Although T cells can be isolated efficiently from blood and grown ex vivo for cancer therapy (e.g., in adoptive cell therapy with chimeric antigen receptors), they are difficult to manipulate genetically and to efficiently expand to therapeutically relevant numbers.

In vitro T-cell expansion can be boosted by the addition of growth factor interleukin 2 (IL-2) in combination with anti-CD3 monoclonal antibody (OKT3 clone). CD3 pathway activation renders T cells susceptible to IL-2 receptor stimulation, triggering proliferation. Introducing anti-CD3 antibodies disrupts antigen receptors on the surface of T cells that are associated with the CD3 complex, inducing antigen receptor activation (Tsoukas et al. 1985). In conjunction with IL-2 and anti-CD3 antibody, RetroNectin reagent has been shown to further increase the expansion of naïve CD8+ T cells from peripheral blood mononuclear cells (PBMCs), and to improve the expansion and post-transplant persistence of genetically modified T cells (Yu et al. 2008). RetroNectin improves CD8+ T-cell expansion through the stimulation of the integrins VLA-4 and VLA-5, enhancing effector multifunctionality and in vivo memory formation (Hosoi et al. 2014).

RetroNectin Recombinant Human Fibronectin Fragment and RetroNectin GMP grade reagents have widespread use as enhancers of retroviral or lentiviral gene transfer into hard-to-transduce cell types. Here, we present data on an additional application, the improved expansion of T cells from PBMCs and increased production of naïve T cells, using a combination of our products: RetroNectin reagent, GT-T551 T-cell medium, CultiLife culture bags, and anti-CD3 antibody.

T-cell expansion protocol using RetroNectin reagent, GT-T551 T-cell medium, CultiLife bags, and anti-CD3 mAb.

1.  RetroNectin和CD3单克隆抗体包被培养袋。

2.  同时，来自PBMC的T细胞用GT-T551培养基+IL-2+血浆培养。

3.  第4天，T细胞转移到培养袋，继续用GT-T551培养基+IL-2+血浆培养。

4.  第7天，添加更多GT-T551培养基+IL-2。

5.  第10天，分装两个培养袋，添加更多GT-T551培养基+IL-2。

PBMC：人外周血单个核细胞

Fibronectin：is a high-molecular weight (~440kDa) glycoprotein of the extracellular matrix that binds to membrane-spanning receptor proteins called integrins. Similar to integrins, fibronectin binds extracellular matrix components such as collagen, fibrin, and heparan sulfate proteoglycans (e.g. syndecans).

Fibronectin exists as a protein dimer, consisting of two nearly identical monomers linked by a pair of disulfide bonds.The fibronectin protein is produced from a single gene, but alternative splicing of its pre-mRNA leads to the creation of several isoforms.

Fibronectin plays a major role in cell adhesion, growth, migration, and differentiation, and it is important for processes such as wound healing and embryonic development.Altered fibronectin expression, degradation, and organization has been associated with a number of pathologies, including cancer and fibrosis.

RetroNectin®

(Recombinant Human Fibronectin Fragment)

参考文献

1)  Kimizuka F,et al.Production and characterization of functional domains of human

fibronectin expressed inEscherichia coli. J Biochem. (1991) 110: 284-291.

2)  Hanenberg H, et al. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells.Nat Med. (1996) 2: 876-882.

3)  Hanenberg H, et al. Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells.Hum Gene Ther. (1997) 8: 2193-2206.

4)  Pollok KE,et al.High-efficiency gene transfer into normal and adenosine deaminase-deficient T lymphocytes is mediated by transduction on recombinant fibronectin fragments.J Virol. (1998) 72: 4882-4892.

5)  Chono H, et al. Removal of inhibitory substance with recombinant fibronectin-CH-296 plates enhances the retroviral transduction efficiency of CD34+CD38- bone marrow cells.J Biochem. (2001) 130: 331-334.

6）  Barbara Savoldo,et al.CD28 costimulation improves expansion and persistence of chimeric antigen receptor–modified T cells in lymphoma patients.J Clin Invest.( 2011) May 2; 121(5): 1822–1826.

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[2]蒋盼,旷世佳,刘兴光,肖栋涛,杨菁,张雁,汪华.冷冻联合iCIK细胞治疗口腔恶性黑色素瘤的T细胞免疫效应分析[J].中华临床医师杂志(电子版),2013,7(11):4944-4949.

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[4]张兴华. 剔除调节性T细胞增强RAK过继免疫治疗机制的研究[D].北京协和医学院,2011.

[5]张兴华,袁伟,赵晨,马洁,赵平.R etronectin和CD3mAb联合培养CD56~+细胞用于治疗胰腺癌的研究[J].实用肿瘤杂志,2011,26(02):113-118.

[6]韩颖,于津浦,李慧,曹水,任宝柱,齐静,安秀梅,张廼宁,任秀宝,郝希山.重组人纤维蛋白片段对CIK细胞增殖的影响及其可能机制研究[J].中国肿瘤临床,2010,37(02):71-75.

[7]杜微丽. 纤维连接蛋白诱导的CIK细胞的生物学特性和杀瘤活性的实验研究[D].中国医科大学,2009.

[8]任玮,王志华.重组人纤维蛋白片段诱导CIK的增殖及对肺癌耐顺铂细胞的杀伤作用[J].生物工程学报,2008(08):1373-1380.

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RetroNectin®

(Recombinant Human Fibronectin Fragment，rFN-CH-296))

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