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TAKARA RetroNectin® 重組人纖維蛋白片段，Recombinant Human Fibronectin Fragment，F(xiàn)N CH-296

產(chǎn)品英文名：TAKARA RetroNectin®(Recombinant Human Fibronectin Fragment)，F(xiàn)N CH-296

產(chǎn)品中文名：TAKARA RetroNectin®重組人纖維蛋白片段，F(xiàn)N CH-296

RetroNectin® 重組人纖維蛋白片段

RetroNectin^®是在大腸桿菌中產(chǎn)生的重組人纖維蛋白片段FN CH-296。 當(dāng)包被在培養(yǎng)瓶和培養(yǎng)板表面時(shí)，RetroNectin^®能顯著增強(qiáng)逆轉(zhuǎn)錄病毒介導(dǎo)的基因轉(zhuǎn)移到哺乳動(dòng)物細(xì)胞中。

RetroNectin^®重組人纖維蛋白片段和激發(fā)型CD3單抗可分別與T淋巴細(xì)胞上的VLA和TCR結(jié)合，兩者共同作用激活酪氨酸激酶pp125FAK，通過(guò)Ras途徑刺激T細(xì)胞的增殖和分化，使其獲得上千倍的增殖。

RetroNectin®

(Recombinant Human Fibronectin Fragment)

組份

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

儲(chǔ)存

-20℃

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

*

訂購(gòu)RetroNectin歡迎您咨詢(xún)華雅再生醫(yī)學(xué)旗艦公司：紅榮微再（上海）生物工程技術(shù)有限公司 。紅榮微再授權(quán)代理TAKARA CellArtis ，Rubicon產(chǎn)品。紅榮微再（上海）生物工程技術(shù)有限公司以“傳遞科學(xué)價(jià)值，服務(wù)科學(xué)研究”為宗旨，主營(yíng)干細(xì)胞、醫(yī)療、細(xì)胞治療、器官再生四大板塊的產(chǎn)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逆轉(zhuǎn)錄技術(shù)操作流程

RetroNectin技術(shù)可以顯著提高逆轉(zhuǎn)錄病毒的轉(zhuǎn)染效率，這一發(fā)現(xiàn)克服了向造血干細(xì)胞中導(dǎo)入基因的困難?，F(xiàn)在，RetroNectin技術(shù)已成為使用逆轉(zhuǎn)錄病毒進(jìn)行轉(zhuǎn)染操作的常規(guī)選擇，其操作流程大致如下：

1. 注射用水或滅菌蒸餾水充分溶解RetroNectin，調(diào)節(jié)濃度至1mg/ml。

2. 0.22 μm濾膜過(guò)濾RetroNectin溶液，分裝后-20 ℃保存。

3. 用緩沖液將RetroNectin溶液稀釋至20～100 μg/ml。

4. 將RetroNectin稀釋液加入24孔板，每孔0.5 ml (建議用PBS緩沖液稀釋RetroNectin后包被Non-Tissue Cluture Treated plate。

5. 室溫放置4小時(shí)或4 ℃，避光，過(guò)夜。

6. 吸出RetroNectin溶液，每孔加入PBS終止液0.5 ml。

7. 室溫放置30分鐘。

8. 加入適量PBS清洗孔板。

9. 吸出清洗液，得到RetroNectin包被的孔板 (如果暫不使用，可以在孔中加入適量PBS，用膠膜將平板蓋密封，避免RetroNectin干燥，可于4 ℃保存4周。使用前將PBS吸出) 。

10. 逆轉(zhuǎn)錄病毒保存液 (-80 ℃) 于37 ℃水浴融解。

11. 將病毒液加入包被后的24孔板，每孔300 μl。

12. 32 ℃放置4小時(shí)。

13. 吸出病毒保存液。

14. 每孔加入待轉(zhuǎn)染的細(xì)胞懸液400 μl (通常細(xì)胞濃度為1 × 105/ml) 。

RetroNectin增強(qiáng)T細(xì)胞擴(kuò)增技術(shù)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單克隆抗體包被培養(yǎng)袋。

2.  同時(shí)，來(lái)自PBMC的T細(xì)胞用GT-T551培養(yǎng)基+IL-2+血漿培養(yǎng)。

3.  第4天，T細(xì)胞轉(zhuǎn)移到培養(yǎng)袋，繼續(xù)用GT-T551培養(yǎng)基+IL-2+血漿培養(yǎng)。

4.  第7天，添加更多GT-T551培養(yǎng)基+IL-2。

5.  第10天，分裝兩個(gè)培養(yǎng)袋，添加更多GT-T551培養(yǎng)基+IL-2。

PBMC：人外周血單個(gè)核細(xì)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)

參考文獻(xiàn)

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]蔣盼,曠世佳,劉興光,肖棟濤,楊菁,張雁,汪華.冷凍聯(lián)合iCIK細(xì)胞治療口腔惡性黑色素瘤的T細(xì)胞免疫效應(yīng)分析[J].中華臨床醫(yī)師雜志(電子版),2013,7(11):4944-4949.

[3]徐本玲,袁龍,高全立,范瑞華,張成娟,宋永平.重組纖維連接蛋白誘導(dǎo)自體CIK聯(lián)合IFN-α治療晚期肝癌療效[J].鄭州大學(xué)學(xué)報(bào)(醫(yī)學(xué)版),2011,46(05):694-696.

[4]張興華. 剔除調(diào)節(jié)性T細(xì)胞增強(qiáng)RAK過(guò)繼免疫治療機(jī)制的研究[D].北京協(xié)和醫(yī)學(xué)院,2011.

[5]張興華,袁偉,趙晨,馬潔,趙平.R etronectin和CD3mAb聯(lián)合培養(yǎng)CD56~+細(xì)胞用于治療胰腺癌的研究[J].實(shí)用腫瘤雜志,2011,26(02):113-118.

[6]韓穎,于津浦,李慧,曹水,任寶柱,齊靜,安秀梅,張廼寧,任秀寶,郝希山.重組人纖維蛋白片段對(duì)CIK細(xì)胞增殖的影響及其可能機(jī)制研究[J].中國(guó)腫瘤臨床,2010,37(02):71-75.

[7]杜微麗. 纖維連接蛋白誘導(dǎo)的CIK細(xì)胞的生物學(xué)特性和殺瘤活性的實(shí)驗(yàn)研究[D].中國(guó)醫(yī)科大學(xué),2009.

[8]任瑋,王志華.重組人纖維蛋白片段誘導(dǎo)CIK的增殖及對(duì)肺癌耐順鉑細(xì)胞的殺傷作用[J].生物工程學(xué)報(bào),2008(08):1373-1380.

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RetroNectin®

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

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