【摘要】：磁性細(xì)胞分選(Magnetic-activated cell separation, MACS)技術(shù)已經(jīng)成為一項(xiàng)快速發(fā)展的生物技術(shù),它是基于抗原抗體特異性反應(yīng)原理,并結(jié)合磁性材料特有的超順磁性來完成細(xì)胞分選的。德國(guó)美天旎(?)(Miltenyi);納米級(jí)葡聚糖磁性粒子和美國(guó)戴諾(?)(Dynal)微米級(jí)聚苯乙烯高分子磁性微球在磁性細(xì)胞分選領(lǐng)域的應(yīng)用已經(jīng)得到了國(guó)際認(rèn)可,而我國(guó)此項(xiàng)技術(shù)非常落后,只能依賴進(jìn)口產(chǎn)品。本課題旨在以具有自主知識(shí)產(chǎn)權(quán)的GoldMag(?)金磁微粒為載體,結(jié)合“鏈霉親和素(Streptavidin, SA)-生物素(Biotin)"的高度放大效應(yīng),基于抗原抗體特異性結(jié)合的原理建立免疫磁性細(xì)胞分選技術(shù)。 1.以金磁微粒為載體,制備鏈親和素磁性復(fù)合微粒(SA-GoldMag)。分別通過物理方法以及化學(xué)方法將鏈霉親和素偶聯(lián)于金磁微粒表面。在化學(xué)方法中首先以聚丙烯酸(polyacrylic acid, PAA)對(duì)金磁微粒表面進(jìn)行修飾并在1－(3－二甲氨基丙基)－3－乙基碳二亞胺鹽酸鹽(1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide, EDC)的作用下進(jìn)行SA偶聯(lián)形成SA-PAA-GoldMag;分別對(duì)物理以及化學(xué)方法偶聯(lián)所得的SA-GoldMag進(jìn)行篩選及評(píng)價(jià)。結(jié)果顯示：30nm金磁微粒經(jīng)物理方法偶聯(lián)所得SA-GoldMag以及50nm金磁微粒經(jīng)化學(xué)方法偶聯(lián)所得SA-PAA-GoldMag均可使用于下游的細(xì)胞分選,二者對(duì)SA的偶聯(lián)量分別為22.14μg/mg和13.54μg/mg,且SA活性高、穩(wěn)定性好。 2.以制備得到的SA-GoldMag為載體建立磁性細(xì)胞分選體系,對(duì)外周血淋巴細(xì)胞的表面CD4+的細(xì)胞進(jìn)行分選；并對(duì)細(xì)胞分選過程中所用的緩沖液、抗體用量、孵育時(shí)間以及磁珠用量進(jìn)行優(yōu)化；對(duì)免疫磁性細(xì)胞分選方法分選所得的CD4+細(xì)胞進(jìn)行評(píng)價(jià)。結(jié)果顯示：在含5%BSA、2m M EDTA的PBS緩沖液作用下,細(xì)胞與磁珠始終呈現(xiàn)良好的分散性；在0.5μg抗體、10min(4℃)抗體-細(xì)胞孵育時(shí)間、15min(4℃)磁珠-細(xì)胞孵育時(shí)間以及65μg50nm SA-PAA-GoldMag為最適用量的條件下分選CD4+細(xì)胞得率可達(dá)90.4%,純度為91.72%；分選前后細(xì)胞存活率分別為(97.3+0.9)%和(96.4+1.3)%,細(xì)胞狀態(tài)沒有受到明顯影響,且WST結(jié)果顯示磁珠對(duì)細(xì)胞沒有明顯的毒性作用,不影響細(xì)胞的正常培養(yǎng)。
[Abstract]:Magnetic cell sorting (Magnetic-activated cell separation, MACS) has become a rapidly developing biological technique, which is based on the antigen-antibody specific reaction principle and combined with the magnetic material specific superparamagnetism to complete cell sorting. The application of) (Miltenyi); nanometer-grade dextran magnetic particles and?) (Dynal) micron polystyrene magnetic microspheres in magnetic cell separation has been internationally recognized in Germany, but this technology is very backward in China. We can only rely on imported products. The purpose of this paper is to use GoldMag (?) Combining the high amplification effect of "streptavidin (Streptavidin, SA)-biotin (Biotin)", an immunomagnetic cell sorting technique was established based on the principle of specific binding of antigen and antibody. 1. Chain affinity magnetic composite particles (SA-GoldMag) were prepared with gold magnetic particles as the carrier. Streptavidin was coupled to the surface of gold magnetic particles by physical and chemical methods respectively. In the chemical method, the surface of gold particles was first modified with polyacrylic acid (polyacrylic acid, PAA) and coupled with 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide-3-ethylcarbodiimide (EDC) in the presence of 1- (3- dimethylaminopropyl) -3-ethylcarbodiimide (EDC) to form SA-PAA-GoldMag; pairs. The SA-GoldMag obtained by physical and chemical methods were screened and evaluated. The results showed that SA-GoldMag obtained by physical coupling of 30nm gold magnetic particles and SA-PAA-GoldMag obtained by chemical coupling of 50nm gold magnetic particles could be used in downstream cell sorting. The coupling amounts of SA to SA were 22.14 渭 g/mg and 13.54 渭 g / mg, respectively, and the SA activity was high. Good stability. 2. The magnetic cell sorting system was established by using the prepared SA-GoldMag as the carrier, the CD4 cells on the surface of peripheral blood lymphocytes were sorted, and the buffer solution, antibody dosage, incubation time and the amount of magnetic beads used in the cell sorting process were optimized. The CD4 cells obtained from immunomagnetic cell sorting were evaluated. The results showed that under the action of PBS buffer containing 5 BSA-2m M EDTA, the cells and magnetic beads always showed good dispersion. Under the conditions of 0.5 渭 g antibody, 10min (4 鈩，

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