基于非基因工程技术的细胞表面修饰策略研究进展

细胞表面成分与结构在维持细胞内代谢环境稳定、控制细胞内外物质交换和促进细胞间通讯方面发挥着至关重要的作用,因此,调节或改变细胞表面成分对研究细胞命运具有重要意义。基因工程是目前调节细胞表面成分最常用的技术,但是由于这种方法具有转染效率低、存在突变风险等问题,其应用范围仍然有限。相比之下,近些年发展起来的多种非基因工程细胞表面修饰技术,具有简便、易操作、适用范围广等优势,为调控细胞生命活动、赋予细胞新的性质和功能提供了更多新的选择,因而其在基础生物学研究和新药研发中具有广泛应用前景。对目前非基因工程技术的原理、特点以及在细胞治疗领域的应用等进行了综述和讨论,期望有助于深入了解这一新技术及其在生物医学领域中的应用。     

修饰性肽核酸的细胞转运

肽核酸是人工合成的寡核苷酸类似物,以N-(2-氨乙基)甘氨酸结构单元替代DNA分子中的戊糖-磷酸结构。与天然核酸相比,肽核酸可以更高效地与DNA或RNA特异性杂交,在分子生物学和基因药物领域具有良好的应用前景。但是,肽核酸骨架呈电中性,难以高效穿过细胞膜,这成为工程应用的最大障碍。为了改善肽核酸的细胞转运性能,对肽核酸进行化学修饰是近年来的研究热点。结合近十年来文献报道和本实验室的工作,对肽核酸的骨架修饰和配合物结合修饰两类增强细胞转运的修饰方法进行综述,并对修饰性肽核酸细胞转运研究中存在的问题以及未来的研究趋势及其应用提出了见解。     

遗传修饰方法在克服Gal表位介导的异种移植排斥反应中的应用

本文介绍了Gal表位和异种天然抗体在异种移 植排斥中的作用,综述了目前针对Gal表位遗传修饰在异种移植中的研究进展,并分析了这一策略存在的问题及其可能的解决途径。     

氧化修饰在调控细胞凋亡信号转导中的作用

氧化修饰是细胞内的活性氧诱导生物大分子发生氧化反应引起的结构及构象改变,发挥调控信号转导和对应激作出反应的功能。氧化修饰发生在凋亡信号转导中的多个生物大分子,包括凋亡相关蛋白质的氧化,如caspase-9、线粒体通透性转变孔及电压依赖的阴离子通道(voltagedependent anion channel,VDAC),同时也包括膜磷脂的氧化修饰,如磷脂酰丝氨酸及线粒体特异的心磷脂。氧化修饰作用也涉及凋亡诱导因子、促凋亡的凋亡信号调控激酶1(apoptosis signalregulatin gkinasel,ASK1)信号转导途径及抗凋亡的转录因子NF—kB的激活和活性。所以氧化修饰可能是调控凋亡信号转导机制中除磷酸化、泛素化外的另一个新的分子机制。     

诱导性调节性T细胞抑制移植排斥反应的作用研究

目的:通过体外诱导的方法将幼稚CD4+T细胞(na觙ve CD4+T cell)转化为调节性T细胞(RegulatoryT cells,Tregs),并验证其在小鼠异体皮片移植模型上对移植排斥反应的抑制作用。方法:分选na觙ve CD4+T细胞并在体外诱导其转化为Tregs,流式检测细胞确定其转化率。将诱导性Treg(induced Treg,iTreg)与效应T细胞(Effective T cells,Teffs)以不同比例共同培养检测其对T细胞增殖的抑制能力。建立C57bl/6到Balb/c小鼠的异体皮片移植模型,植皮术后将iTreg经由股静脉输注入受体(Balb/c小鼠)体内,观察皮片存活情况,绘制皮片存活曲线。同时于皮片移植术后11天对皮片进行病理切片,观察移植排斥反应状况。结果:体外诱导na觙ve CD4+T细胞转化为iTreg的比例约44%,在iTreg:Teff比例大于1:4时,iTreg具有明显地抑制Teff增殖的作用,且这种抑制作用具有剂量依赖性。植皮小鼠输注iTreg后皮片存活时间较对照组延长约2.4天,病理切片显示排斥反应减轻,但皮片在14天左右时仍被排斥。结论:体外诱导的iTreg能够在体外抑制Teff增殖,且能有效抑制小鼠异体皮片移植后排斥反应。     

耐受性树突状细胞的研究进展

耐受性树突状细胞(tolerogenic dendritic cell,tolDC)在器官移植和自体免疫性疾病中对免疫耐受调节至关重要.与免疫抑制剂相比,tolDC用于治疗移植后排斥反应的副作用较低.此外,tolDC可通过多种途径培养生成,且不同的培养方式具有独特功效.目前,tolDC作为一种诱导器官移植耐受的辅助治疗...     

氧化修饰使HDL促动脉平滑肌细胞胆固醇流出减少

为了研究氧化修饰对高密度脂蛋白（ＨＤＬ）转运细胞胆固醇地＾３Ｈ－胆固醇负荷的培养人动脉平滑肌细胞（ＳＭＣ）分别与天然ＨＤＬ及Ｃｕ＾２＋ａｋｇ ＨＯＣｌ氧化修饰的ＨＤＬ在３７℃温育不同时间后,分别测定细胞＾３Ｈ－胆固醇清除率。结果发现,温育２４ｈ后,经Ｃｕ＾２＋或ＨＯＬｌ氧修饰后的ＨＤＬ其细胞胆因醇清除率分别较天然ＨＤＬ下降了３０．０％和４３．１％（ｐ〈０．０１）。结果还发现,Ｃｕ＾２＋或ＨＯＣｌ氧     

氧化修饰LDL诱导U937细胞凋亡及其机制探讨

用氧化修饰低密度脂蛋白（ox-LDL）诱导人髓系白血病细胞株U937细胞凋亡,并研究其作用机制.用脱氧核苷酸转移酶介导的dUTP切口末端标记技术(TUNEL法)、流式细胞仪和DNA断裂分析检测细胞凋亡;用免疫组化检测c-fos、c-jun和c-myc蛋白表达,RT-PCR显示c-fos、c-jun和c-myc mRNA表达水平.结果表明ox-LDL可致U937细胞凋亡,其作用具有浓度效应;ox-LDL可以上调c-fos、c-jun和c-myc基因表达,使c-fos、c-jun和c-myc蛋白合成增多,最终诱导U937细胞凋亡.     

微生物谷氨酰胺转氨酶催化细胞色素c赖氨酸残基的定点修饰

研究微生物谷氨酰胺转氨酶(mTG)催化细胞色素c(Cytc)的PEG定点修饰的可行性,并优化修饰条件,研究PEG修饰对Cytc性质的影响。将单甲氧基聚乙二醇氨(mPEG-NH_2)与N-苄氧羰基-谷氨酰胺-甘氨酸(CBZ-QG)共价结合制备含谷氨酰胺残基的甲氧基聚乙二醇衍生物(N-苄氧羰基-谷氨酰胺-甘氨酰-单甲氧基聚乙二醇,CBZ-QG-mPEG);mTG分别催化mPEG-NH_2、CBZQG-mPEG(mTG)修饰Cytc,研究酶法定点修饰Cytc残基的可行性;改变酶的用量、温度、反应时间和p H等反应条件优化谷胺酰胺转氨酶催化修饰Cytc的条件。研究结果表明:(1)mPEG-NH_2不能作为mTG的底物修饰Cytc,甲氧基聚乙二醇氨(mPEG-NH_2)分子上引入谷氨酰胺残基后,在mTG的催化作用下了实现Cytc的PEG修饰,而且基于mTG的底物特异性实现了PEG定点修饰Cytc的赖氨酸(Lys)残基;(2)37℃温度下,p H 8.0的溶液中,1mg/ml的mTG催化修饰反应2h是最佳修饰反应条件;(3)化学法PEG修饰Cytc产物复杂,是多种多点修饰产物的混合物,酶法催化PEG修饰Cytc只产生单一产物;(4)与天然Cytc相比,修饰后Cytc的活力、稳定性都有所提高。提出的谷胺酰胺转胺酶催化修饰法解决了蛋白质Lys残基难以定点修饰的难题,拓展了mTG在蛋白质修饰方面的应用。     

细胞色素c在氨基酸及二肽修饰金电极上的电化学反应

系统地研究了细胞色素ｃ在多种氨基酸和多肽修饰电极上的电化学反应。并对影响加速细胞色素ｃ电化学反应的因素进行了讨论。     

Surface modification of polymers: chemical, biological and surface analytical challenges

Surface modification methods can optimise the biocompatibility or the specificity of biointeraction of a biosensor or medical device. With only the surface modified, the manufacture and implantation protocol remain unchanged. This review article summarises some of the chemical, surface analytical and biological challenges associated with surface modification of biosensors and biomedical devices.     

Effect of chemical modification on struture and activity of glucoamylase fromAspergillus candidus andRhizopus species

The histidine, tyrosine, tryPtoPhan and carboxyl grouPs in the enzyme glucoamylase fromAsPergillus Candidus andRhizoPus sPecies were modified using grouP sPecific reagents. Treatment of the enzyme with diethylPyrocarbonate resulted in the modification of 0.3 and 1 histidine residues with only a slight loss in activity (10% and 35%) of glucoamylase fromAsPergillus candidus andRhizoPus sPecies resPectively. Modification of tyrosine either by N-acetylimidazole or [I¹²⁵]-leads to a Partial loss of activity. Under denaturing conditions, maltose did not helP in Protecting the enzyme against tyrosine modification or inactivation. Treatment with 2-Hydroxy-5-nitro benzyl bromide in the Presence of urea, Photooxidation at PH 9.0, N-bromosuccinamide at PH 4.8 resulted in a comPlete loss of activity. However, the results of exPeriments in the Presence of maltose and at PH 4.8 Photooxidation and N-bromosuccinamide treatment suggested the Presence of two tryPtoPhan residues at the active site. There was a comPlete loss of enzyme activity when 10 and 28 carboxyl grouPs fromAsPergillus candidus andRhizoPus, resPectively were modified. Modification in the Presence of substrate maltose, showed at least two carboxyl grouPs were Present at the active site of enzyme and that only one active center seems to be involved in breaking ally 3 tyPes of α-glucosidic linkages namely α-1, 4, α-1, 6 and α-l, 3.     

Acid glycosaminoglycans and their chemical modification

The modern state of chemical modification of hyaluronic acid, chondroitin sulfates, and heparin is considered, and the possible application of modified glycosaminoglycans as potential drugs is discussed.     

Effects of chemical modification of lysine residues on the sweetness of lysozyme

Lysozyme is a sweet-tasting protein with a sweetness threshold value of around 7 microM. To clarify the effect of basicity at the side chain of lysine residues on the threshold values of sweetness, charge-specific chemical modifications such as guanidination, acetylation and phosphopyridoxylation of lysine residues were performed. Sensory analysis showed that the sweetness threshold value of lysozyme was not changed by guanidination, whereas it was increased markedly by acetylation and phosphopyridoxylation. To confirm the importance of the basicity in the lysine residues in detail, purification of acetylated (Ac-) and phosphopyridoxylated (PLP-) lysozymes using SP-ion exchange column chromatography was performed. The threshold values were not changed by modification with fewer than two residues (approximately 7 microM), whereas the threshold values significantly increased to 15 and 34 microM when tetra-Ac and tri-PLP, respectively. Furthermore, sweetness was not detected at 30 microM (hexa-, penta-Ac and tetra-PLP). It should be noted that removal of the negative charges of the phosphate groups in the tri-PLP lysozyme by acid phosphatase resulted in the recovery of sweetness (6.4 microM), indicating that basicity at the position of the lysine residues is responsible for lysozyme sweetness and that strict charge complementarities might be required for interaction to its putative receptor.     

重组L-门冬酰胺酶工程菌的表达和PEG的化学修饰

目的提高重组L-门冬酰胺酶(rL-ASP)工程菌的表达量,分离纯化rL-ASP并对之进行PEG化学修饰。方法将带有编码rL-ASP的基因的质粒(pKA)导入不同的宿主菌中,挑出高表达菌株,同时优化发酵培养基,分离纯化获得的高纯度rL-ASP再用PEG进行化学修饰,SDS-PAGE检测修饰效果。结果在pH7.0的条件下,宿主菌为JMl09的工程菌pKA/JMl09酶活力最高,三角瓶振摇培养的酶活力可达216×103IU/L;发酵罐发酵培养,酶活力达312×103IU/L。纯化后的rL-ASP比活力为220IU/mg,rL-ASP经过PEG化学修饰生成rL-ASP-PEG,分子量发生改变。结论改变目标蛋白表达的宿主菌和优化发酵工艺,提高了rL-ASP的表达量,纯化的rL-ASP经过PEG化学修饰后分子量增大。     

Elevated temperature and chemical modification selectively abolishes levan forming activity of levansucrase of Zymomonas mobilis

A levansucrase (SacB) of Zymomonas mobilis was purified to electrophoretic homogeneity from a recombinant Escherichia coli. The 55 kDa enzyme hydrolysed -fructosides but not -glucosides and catalysed levan formation from sucrose as well as raffinose. The optimum temperature for polymerase activity (30°C ) was lower than that for hydrolase activity (50°C ). In contrast to other levansucrases, polymerase activity of levansucrase was inhibited by para- chloromercuribenzoate (1 mM) but with little or no effect on hydrolase activity. Selective modulation of polymerase activity by this inhibitor will be useful in revealing the mechanism of levansucrase catalysis.     

Two forms of trehalase in rabbit enterocyte: Purification and chemical modification

Trehalase found to be associated with the brush border membrane vesicles and the Ca₂₊ aggregated basolateral membrane vesicles were purified to homogeneity. They were found to differ in their molecular weight, subunit structure, heal stability, N-terminal residues, amino acid composition and also the active site residues. Chemical modification showed the presence of a histidine and tyrosine at the active site of brush border membrane vesicle trehalase and two histidines at the active site of basolateral membrane vesicle.     

A preliminary study of modification of gait in real-time using surface electromyography

Modification of abnormal gait was attempted in real-time using a surface electromyography-based protocol to teach recruitment of the anterior tibialis at the correct time in the gait cycle. Two children diagnosed with cerebral palsy were able to learn volitional control of the anterior tibialis as demonstrated by improved clearance of the toe on the swing phase of the gait and newly learned ability to recruit and relax the anterior tibialis. The children were able to walk with the new gait pattern and reproduce the old one at will. Implications for future research in this area are discussed.     

不同剂量的He—Ne激光对Raji细胞膜表面SH的影响

本文以离体培养的Ｒａｊｉ细胞为材料,采用ＥＬＬＭＡＮ方法检测了不同剂量的Ｈｅ－Ｎｅ激光对Ｒａｊｉ细胞膜表面ＳＨ含量的影响。发现０．５Ｊ／ｃｍ＾２Ｈｅ－Ｎｅ激光能量可明显增加膜表面ＳＨ含量（Ｐ〈０．０５）。大于或小于此剂量的Ｈｅ－Ｎｅ激光对膜表面ＳＨ含量的影响均不明显（Ｐ〉０．０５）。提示０．５Ｊ／ｃｍ＾２的激光能量对膜有刺激作用。