【作者】 党时鹏；

【导师】 李肖蓉； 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2010， 硕士

【摘要】 目的:探讨聚乙烯亚胺修饰白蛋白微泡转染真核细胞的方法,从而提高白蛋白微泡转基因效率。方法:(1)分别将25%人体白蛋白、葡萄糖和甘露醇等按一定的质量比置于10 ml瓶中,混合后用超声振动仪声振20 S(电压100 V、电流0.5 A)制得白蛋白微泡(Albumin Microbubbles,AMB);(2)将微泡包裹气体由空气换为全氟丙烷气体,并在微泡组分中添加聚乙二醇,以提高微泡稳定性;(3)在微泡组分中添加聚乙烯亚胺,获得聚乙烯亚胺修饰包裹全氟丙烷气体微泡(PAMB),通过转染CHO细胞,36 h后消化细胞流式细胞仪检测GFP阳性细胞比例,最大转基因效率时质量比即为最佳白蛋白与聚乙烯亚胺质量比;(4)使用纳米激光粒度仪和Zeta电位分析仪检测微泡表面电位;(5)将质粒DNA分别与AMB和PAMB按一定的比例混合后,加入SYBR Green,SYBR Green与DNA结合后可被激发出绿色荧光,在荧光显微镜下观察,结合质粒DNA的微泡可发出绿色荧光;如果微泡未与质粒DNA结合则只能看到绿色荧光而无微泡形态;(6)转染CHO, 293T, M231, 293, SK-Hep-1, SKBR-3, MCF-7, COS 8种细胞,使用流式细胞仪检测GFP阳性细胞比例以比较PEI、白蛋白+PEI、Lipofectamine 2000和PAMB的转基因效率;(7)使用CCK-8试剂盒检测PEI, Lipofectamine 2000和PAMB细胞毒性及转染后细胞增值能力,细胞活性= PEI组, Lipofectamine 2000组和PAMB组吸光值/未处理组吸光值,细胞增殖指数=加入转染试剂之前和加入转染试剂作用6 h之后0 h, 24 h, 48 h, 72 h吸光值/加入转染试剂之前吸光值;(8)使用COS细胞比较AMB,SonoVue微泡和PAMB在超声介导下转基因效率,超声强度为0.5 W/cm2、1.0 W/cm2和2.0 W/cm2,时间分别为30 S和60 S;(9)探讨超声在微泡转基因中的作用,制备CHO细胞爬片,分别按如下分组加入试剂:①只加入DNA荧光染料NA-Green(不能主动进入细胞);②AMB+DNA+NA-Green;③AMB+DNA+NA-Green+超声(1.0 W/cm2, 30 S) ;④PAMB+ NA-Green ;⑤PAMB+DNA+NA-Green ;⑥PAMB+DNA+ NA-Green+超声(1.0 W/cm2, 30 S),37℃、5% CO2培养6 h后吸取培养液,使用PBS冲洗三遍,每孔加入1ml 1%多聚甲醛固定15 min,用PBS冲洗两遍,然后使用共聚焦荧光显微镜观察拍照。结果:(1)将微泡包裹气体由空气换为全氟丙烷气体,并在微泡组分中添加聚乙二醇后,微泡稳定性大大提高,半衰期由原来的半小时至数天延长到6个月;(2)通过转染CHO细胞,得到最佳白蛋白与聚乙烯亚胺的质量比为125:1,此时PAMB转染CHO细胞最大效率可达55%左右;(3)纳米激光粒度仪检测显示: AMB平均直径1400 nm左右,而PAMB平均直径在600 nm左右,Zeta电位分析仪检测结果示: AMB的表面电位为(-59.28±3.48)mV,而PAMB的表面电位为(-44.56±0.75)mV;(4)结合质粒DNA的微泡(PAMB)在荧光显微镜下可发出绿色荧光, AMB可见大量绿色荧光而未见微泡形态,未添加DNA的PAMB未见荧光;(5)通过8种细胞比较PEI、白蛋白+PEI、Lipofectamine 2000和PAMB的转基因效率,发现PAMB组明显高于单纯PEI组和白蛋白+PEI组(P<0.05),而在293T、COS、SKBR-3和SK-Hep-1细胞PAMB的转基因效率高于Lipofectamine 2000,在CHO、293、M231和MCF-7细胞转基因效率低于Lipofectamine 2000(P>0.05),白蛋白+PEI的转基因效率低于单纯PEI的转基因效率(P<0.05);(6)PEI,PAMB和Lipofectamine 2000均具有细胞毒性,PAMB的细胞毒性低于PEI和Lipofectamine 2000(P<0.05),且使用PAMB转染后细胞增殖能力明显高于使用PEI和Lipofectamine 2000转染的细胞(P<0.05);(7)通过实施不同强度和时间的超声,聚乙烯亚胺修饰白蛋白微泡微泡转基因效率在各种强度和时间下均明显高于白蛋白微泡和SonoVue微泡(P<0.05),不同超声强度和时间下聚乙烯亚胺修饰白蛋白微泡微泡转基因效率在COS和CHO两种细胞均没有显著差异,且与未超声组相比略有下降,但无统计学意义(P>0.05);(8)超声介导AMB和SonoVue微泡破裂转基因时,质粒DNA依靠空化效应在细胞膜上形成的微孔进入细胞,而PAMB可携带质粒DNA主动进入细胞。结论:本研究制作的聚乙烯亚胺修饰包裹全氟丙烷气体微泡可将其携带的外源基因转入真核细胞,且体外实验证实其具有低细胞毒性和较高的转基因效率,是一种有效的体外基因转染方法,为PAMB作为体内转基因载体提供实验依据。更多还原

【Abstract】 Objective:To improve the transgene efficiency of albumin microbubbles,we investigated a method of transgene with polyethylenimine coated albumin microbubbles.Method:(1) Albumin microbubbles (AMB) were prepared by sonicating the mixture of human albumin, manose and glucose; (2) Air filled in albumin microbubbles was exchanged with perfluoropropane and polyethylene glycol (PEG) was added as one gradient of microbubbles to improve the stability of albumin microbubbles; (3) Polyethylenimine (PEI) coated albumin microbubbles (PAMB) was prepared by sonicating the mixture of human albumin, PEI, polyethylene glycol (PEG), glucose, manose and perfluoropropane. Through transfecting CHO cell line with plasmid pIRSE2-EGFP, the optimal weight ratio of albumin and PEI was insured by measureing the percentage of GFP with flow cytometry; (4) Mean particle diameter and surface potential of AMB and PAMB were determined by photon cross correlation spectroscopy with a NANOPHOX particle size analysis system and ZetaProbe 7020; (5) To identify PAMB combined with pDNA, PAMB and pDNA with addition of SYBR green were mixed in serum-free Opti-MEM?Ⅰmedium in an Eppendorf tube. When SYBR green was combined with plasmid DNA, it could be excited by blue light and emitted green fluorescence. Thereafter, if PAMB combined with pDNA, it could emit green fluorescence. Green microbubbles could be inspected with a fluorescence microscrope; (6) Through transfecting CHO, 293T, M231, 293, SK-Hep-1, SKBR-3, MCF-7 and COS cell lines with plasmid pIRSE2-EGFP, we compared the transgene efficiency of PEI, albumin+PEI, Lipofectamine 2000 and PAMB; (7) The cytotoxicity and cell proliferation ablity of PEI, Lipofectamine 2000 and PAMB were measured by cell counting kit-8. Cell proliferation index was calculated by the OD value prior to Transfection, and 0 h, 24 h, 48 h, 72 h posttransfection / OD value prior to transfection; (8) COS cell line was transfected by AMB, SonoVue and PAMB mediated by ultrasound with different ultrasound (U) intensity and time; (9) The effect of ultrasound on microbubbles transgene was investigated. After CHO cell sections were prepared, reagents were added as follows in each group:①o nly NA-Green (a kind of DNA fluorescence dye which can not enter cells actively);②AMB+DNA+NA-Green;③AMB+DNA+NA-Green+U(1.0 W/cm2, 30 S);④P AMB+ NA-Green;⑤PAMB+DNA+NA-Green;⑥P AMB+DNA+ NA-Green+U (1.0 W/cm2, 30 S). After incubation at 37℃, 5% CO2 for 6 h; the medium was removed. The sections were rinsed for three times and immobilized with 1% paraformaldehyde, then were inspected with confocal microscoup and photos were taken.Results:(1) The half time of albumin microbubbles was extended from half an hour or several days to 6 months after air that filled in albumin microbubbles was exchanged with perfluoropropane and polyethylene glycol (PEG) was added as one gradient of microbubbles; (2) The optimal weight ratio of albumin and PEI was 125:1, which was obtained through transfecting CHO cell line with plasmid pIRSE2-EGFP, and the highest transfection efficiency of PAMB was about 55% on CHO cell line; (3) The particle size analysis showed the average particle diameter of AMB and PAMB was about 1400nm and 600nm, and the surface potentials of PAMB and AMB were -44.56±0.75 mV and -59.28±3.48 mV, respectively; (4) AMB could not combine with pDNA and only emitted plenty of fluorescence without outlook of microbubbles, PAMB combined with pDNA emitted fluorescence; (5) The transgene efficiency in PAMB group was higher than that in PEI group and albumin + PEI group in each cell line (P<0.01) and there was no statistical difference between PAMB group and Lipofectamine 2000 group; (6) Cytotoxicity of PAMB was lower than PEI and Lipofectamine 2000(P < 0.01), the proliferation index in no treatment group and PAMB was higher than that in PEI and Lipofectamine 2000 group (P < 0.01), there was no statistical difference PAMB group and no treatment group (P>0.05); (7) COS cells were transfected with AMB, SonoVue and PAMB mediated by ultrasound with different ultrasound intensity and time, the results showed that the transfection efficiency of PAMB was higher than the efficiency of AMB and SonoVue (P < 0.01), there was no statistical difference between AMB and SonoVue, and there were no statistical differences between different ultrasound intensity and time, and compared with PAMB alone; (8) Destruction of AMB and SonoVue mediated by ultrasound could increase the permeability of membrane, plasmid DNA enter cells through these micropores, while PAMB could bring plasmid DNA into cells actively.Conlusion:In summary, PAMB is very useful and low toxicity gene delivery method with high transfection efficiency in vitro. Further studies are necessary to examine the detailed mechanism of the effect of ultrasound on PAMB and transfection efficiency in vivo.更多还原