三聚β肽聚乙二醇化后抗肿瘤转移生物活性研究

目的:比较聚乙二醇(PEG)修饰对三聚β肽(β3)抗肿瘤转移活性的影响。方法:采用黏附试验观察β3及其PEG修饰物(β3-PEG)对肝癌细胞株与纤连蛋白(FN)黏附能力的影响;采用人工基底膜胶观察β3和β3-PEG对肿瘤细胞侵袭重组基底膜能力的影响。结果:β3和β3-PEG对肿瘤细胞SMMC-7721和HCCLM6与FN黏附能力均有显著的抑制作用(P<0.05),且呈剂量及时间正相关;PEG修饰可增强β3对2种肿瘤细胞的黏附抑制作用(P<0.05)。β3和β3-PEG对2种肿瘤细胞迁移和侵袭均有显著的抑制作用(P<0.05)。结论:PEG修饰可增强β3抗黏附作用,但对细胞迁移和侵袭的抑制作用无明显影响。     

聚乙二醇化多聚β肽抗肝癌转移的体内外实验研究

目的：观察多聚β肽及其聚乙二醇（PEG）修饰物对肝癌细胞株侵袭黏附能力和对裸鼠移植人肝癌早期切除术后转移复发的影响。方法：以MTT法测量多聚β肽和其PEG修饰物对肝癌细胞与纤连蛋白（FN）黏附的影响。以细胞侵袭实验观察多聚β肽及其PEG修饰物对肝癌细胞侵袭能力的影响。以LCI-D20裸鼠人肝癌转移模型为材料，观察多聚β肽及其PEG修饰物对早期肝癌切除术后转移复发的影响。结果：β2、β2-PEG、β3和β3-PEG对SMMC-7721细胞与FN黏附抑制率分别为31．3％、39．2％、45．7％和57．1％，侵袭抑制率分别为33．6％、35．9％、38．3％和41．2％；对HCCLM6细胞与FN的黏附抑制率分别为48．7％、60．9％、63．4％和79．3％，侵袭抑制率分别为36．8％、46．6％、45．6％和50．8％。多聚β肽及其PEG修饰物组切缘复发肿瘤重量和肺转移灶个数显著低于对照组（P％0．05）。结论：多聚β肽及其PEG修饰物能抑制肿瘤细胞的黏附和侵袭能力，亦能防治裸鼠移植人肝癌早期切除术后的转移和复发。     

新型定点修饰的聚乙二醇化重组人生长激素修饰位点研究

目的:建立新型定点修饰的聚乙二醇化重组人生长激素(PEG-rhGH)修饰位点的研究方法,为该类新型修饰技术产物的质量评价提供依据。方法:采用MALDI-TOF质谱分析修饰产物中聚乙二醇(PEG)的修饰个数及修饰肽段的质量数;采用LC-Q-TOF质谱分析修饰位点,分析色谱柱为UPLC色谱柱(Protein ACQUITY BEH C₄ Column, 150 mm×2.1 mm, 1.7μm, 300?),以0.1%甲酸水溶液-0.1%甲酸乙腈溶液为流动相,进行梯度洗脱,柱温为40℃。质谱数据采集条件为MS~E模式,一级质谱能量4 eV,二级碎裂电压30～55 eV。结果:修饰蛋白中PEG的平均相对分子质量为30 000,并且每个PEG-rhGH分子中仅存在1个PEG修饰;原型蛋白重组人生长激素中第134位精氨酸被替换为赖氨酸,且该赖氨酸的ε-氨基与连接子HOOC-O-CH₂-CH₂-N₃中的羧基端共价结合,连接子另一端的叠氮基团与活化后的PEG偶联后生成修饰产物。结论:联合运用多种质谱技术,通过比对...     

聚乙二醇化脑啡肽的镇痛药效和体内分布

目的探讨聚乙二醇(PEG)修饰改善甲硫氨酸脑啡肽(MEK)体内半衰期短和对中枢神经系统(CNS)释放的作用。方法热板致痛小鼠侧脑室注射3.1μmol.kg-1MEK及其mPEG2000和mPEG5000修饰物,醋酸致痛小鼠尾静脉注射31μmol.kg-1同上受试物,比较镇痛活性;小鼠尾静脉注射0.2 ml.(10 g)-1容积5.55～7.40 GBq.L-1浓度的125I标记MEK及其mPEG2000修饰物,比较体内分布。结果 MEK及mPEG修饰物3组间F检验P<0.05或0.01(热板模型15 min除外);mPEG5000修饰物作用强于mPEG2000修饰物及未修饰MEK,镇痛活性可持续至120 min(P<0.01)。mPEG2000修饰物10 min时肝脏分布低于MEK、240 min时血液分布高于MEK(P<0.01),血液半衰期(T12)是原型肽的3倍、清除率(CL)降低2.2倍。结论适当分子量PEG修饰可降低MEK肝脏清除、增强镇痛活性、延长半衰期和镇痛时间,对改善脑啡肽成药性有积极意义;未直接证实mPEG修饰改善MEK的CNS释放。     

聚乙二醇修饰聚酰胺-胺的合成、表征及其人角膜上皮细胞毒性

目的:研究不同相对分子质量聚乙二醇(PEG)修饰不同代数树枝状大分子聚酰胺-胺(PAM-AM)得到的产物,测定其对人体角膜上皮细胞(HCECs)的毒性。方法:采用硝基苯氯甲酸酯(p-NPC)将单甲氧基聚乙二醇(mPEG,相对分子质量为750,2 000,5 000)活化成PEG碳酸酯,对第4,5代PAMAM大分子进行修饰;目标产物用FT-IR、1H-NMR进行结构表征;采用WST-8法考察PEG修饰对PAMAM的人角膜上皮细胞(HCECs)毒性的影响。结果:PAMAM经较低浓度PEG修饰后,对HCECs的毒性减弱不明显,经较高浓度PEG修饰后,对HCECs减毒明显减弱,不同相对分子质量的PEG修饰对PAMAM的减毒作用无明显差异。结论:经PEG修饰后,可以降低PAMAM对HCECs的毒性,作为新型眼部给药载体具有良好的前景。     

紫杉醇对肺腺癌细胞侵袭能力抑制作用的研究

目的 :探讨纤连蛋白 (fibronectin ,FN )对非小细胞肺癌的粘附和迁移能力的影响及紫杉醇的干预效果。方法 :以肺腺癌细胞系A5 49为研究对象 ,先观察FN对癌细胞增殖活性的影响及不同浓度紫杉醇的抑制作用。其次 ,在FN包被的培养板和FN滤膜的Boyden浸润小室等体外侵袭模型中 ,检测紫杉醇预处理细胞与FN粘附及迁移能力的变化。设立牛血清清蛋白作为对照组。结果 :FN明显提高肺癌细胞活性 ,紫杉醇 6nmol·L- 112nmol·L- 1和 2 4nmol·L- 1均能有效抑制这种活性的升高。此外 ,肺癌细胞在FN培养板粘附数及小室中穿膜数远高于对照组 ,而各浓度紫杉醇预处理细胞的粘附及迁移能力明显下降。结论 :FN能促进肺腺癌细胞的增殖活性、粘附和迁移能力。这些作用可被紫杉醇 6～ 2 4nmol·L- 1有效阻断。     

聚乙二醇修饰水蛭素的分离纯化与活性分析

用活化的聚乙二醇(PEG)对水蛭素进行化学修饰，质谱分析表明，修饰产物是修饰度不同的分子质量相差5000u的水蛭素的混合物。用离子交换层析和凝胶过滤层析对反应产物进行分离纯化，离子交换层析难以达到完全分离；而采用凝胶过滤层析方法可较好地分离修饰度不同的各组份。生物学活性分析表明，不同的PEG修饰度对水蛭素的活性具有显著影响，连接1～2个PEG的水蛭素保持了原有活性，而连接3个PEG的水蛭素活性显著下降。     

聚乙二醇修饰重组人干扰素α-2b修饰产物的初步分析研究

目的 对聚乙二醇(PEG)修饰重组人干扰素α-2b(rhIFNa-2b)的修饰产物进行了初步分析研究。方法以单甲氧基聚乙二醇甲酸琥珀酰亚胺酯对rhIFNα-2b进行修饰,修饰产物以Superdex 75 Highload制备型凝胶色谱柱进行分离、纯化,SDS-PAGE鉴定各组分,采用Mariner电喷雾飞行时间质谱对单修饰IFNα-2b(Inono-PEG-IFNα-2b)进行分析。结果 分别获得了纯化的mono-PEG-IFNα-2b和多修饰IFNα-2b(poly-PEG-IFNα-2b)。ESI-TOF质谱也证明mono-PEG-IFNα-2b分子量比rhIFNα-2b多5 000左右;紫外光谱分析表明,PEG修饰产物的结构没有发生改变。结论获得了纯化的PEG修饰产物。     

聚乙二醇化海葵神经毒素的修饰反应条件及药效研究

目的优化聚乙二醇(PEG)修饰海葵神经毒素rhk2a(rhk2a)的反应条件,并考察修饰后产物PEG化海葵神经毒素rhk2a(mPEG-rhk2a)的药效性质。方法本研究选用分子量为5 000的PEG丙醛(mPEG-ALD)对rhk2a的N-末端氨基进行修饰;通过单因素考察和正交试验筛选出最佳的修饰反应条件。同时,采用HPLC检测修饰后产物中mPEG-rhk2a的纯度,并通过建立急性心力衰竭豚鼠模型,考察mPEG-rhk2a对豚鼠的强心作用。结果优选出的mPEG-ALD对rhk2a的N-末端氨基的修饰反应条件是:当pH为5.0、rhk2a与mPEG反应摩尔比1∶20、还原剂氰基硼氢化钠为30μL、反应时间为12 h时,所得修饰产物mPEG-rhk2a的得率较高。采用SP-650S强阳离子交换层析法对修饰反应后产物进行分离,纯化后产物中mPEG-rhk2a占83.287%。对于心衰豚鼠,给mPEG-rhk2a溶液后,左心室收缩压、左心室压力最大下降速率明显增加,20 min后呈持续的平稳状态。其强心作用虽然不如rhk2a强,但波动起伏较小,而且强心效果明显高于乙酰毛花苷注射液。结论优选出mPEG修饰rhk2a反应的适宜条件,所得的mPEG-rhk2a强心作用较为理想。     

聚乙二醇修饰天花粉蛋白的初步研究

目的:研究聚乙二醇(PEG)修饰天花粉蛋白(TCS)的条件、纯化方法、生物活性及致过敏反应程度。方法:用SDS-PAGE检测反应条件对产物成分的影响,利用离子交换和分子筛凝胶层析对修饰产物进行分离纯化,通过SDS-PAGE和HPLC检测聚乙二醇化天花粉蛋白(PEG-TCS)的纯度,小鼠引产实验测定PEG-TCS体内生物学活性,用TCS和PEG-TCS作为抗原对豚鼠进行致敏和攻击。结果:在PEG修饰TCS的反应中,PEG与TCS的投料(质量)比和pH值是影响修饰效率的主要因素,修饰反应一般在24～48h内达到平衡;纯化的PEG-TCS经SDS-PAGE检测纯度大于95%,HPLC显示单一峰;小鼠引产实验表明,经过PEG修饰后的TCS,引产活性加强;豚鼠过敏反应实验表明,PEG-TCS具有极低的全身性过敏反应。结论:PEG修饰TCS的最佳反应条件为:TCS与PEG投料比为1:6,pH6．0,反应时间为48h,催化剂氰基硼氢化钠浓度为40mmol·L~(-1);PEG-TCS纯度大于95%; PEG-TCS体内活性能较好的保留,且全身性过敏反应降低。     

Antiadhesive sites present in the fibronectin type III-like repeats of human plasma fibronectin

We have found that fibronectin (FN) has a functional cryptic site opposing cell adhesion to extracellular matrix (ECM): a synthetic FN peptide derived from the 14th FN type III-like (FN-III) repeat, termed peptide FNIII14, inhibits cell adhesion to the FN without binding to beta1 integrins. This antiadhesive activity of peptide FNIII14 depends on its C-terminal amino acid sequence YTIYVIAL. A 50-kDa membrane protein (p50) has been detected as a specific binding protein of peptide FNIII14. Here we showed that antiadhesive activity of peptide FNIII14 was depedent upon the presence of p50 on cell surfaces. Furthermore, we found that there exists a sequence, analogous to the YTIYVIAL, in the 10th FN-III repeat of the FN molecule and that a FN peptide containing this analogous sequence, termed peptide FNIII10, inhibited cell adhesion to the FN. Peptide FNIII10 appeared to share p50 with peptide FNIII14 in expressing the antiadhesive activity. As a physiological consequence of decreased adhesion, peptides FNIII10 and FNIII14 accelerated the anoikis-like apoptosis of normal fibroblasts by down-regulating Bcl-2 expression through blocking the FAK/PI3K/Akt signaling pathway. Thus, the YTIYVIAL-related sequences of the FN molecule may be involved in cell regulation by modulating negatively cell adhesion to the ECM, in which p50 probably serves as a membrane receptor.     

Synthesis of symmetrically and asymmetrically branched pegylating reagents

Abstract: A solution‐phase procedure using an orthogonal protection scheme was developed for the synthesis of a novel family of multi‐pegylating reagents. The procedure was exemplified by the synthesis of bis‐ and tris‐pegylating reagents prepared by stepwise insertion of the poly(ethylene glycol) units thereby enabling the preparation of both symmetrical and asymmetrical pegylating reagents. Asymmetrical pegylation and tris‐pegylation of peptides and proteins introduces new variables for use in the optimization of pegylated peptides and proteins. These reagents are ideally suited for conjugation to peptides and proteins as they possess a required functional group and will be useful intermediates for the synthesis of a new generation of pegylated products. Tris‐pegylation can also provide more effective protection from proteolysis by shielding the protein surface from approaching macromolecules. To illustrate this potential, conditions were developed for the successful coupling of the tris‐pegylating reagent to a model pentapeptide.     

Carboxyalkylated Histidine Is a pH-Dependent Product of Pegylation with SC-PEG

PURPOSE: Pegylation of therapeutic protein usually results in a mixture of monopegylated proteins with differing sites of modification. With rh-interferon-alpha2A pegylation, we have found that this heterogeneity includes two classes of pegylation site chemistry, the relative proportions of which can be adjusted by reaction pH. METHODS: The effect of pegylation reaction pH on the relative proportion of three peaks produced was investigated. Products were purified and characterized by peptide mapping, chemical stability to neutral hydroxylamine, and biologic activity. RESULTS: Reactions at basic pH levels produced a mixture of products pegylated at lysine residues as has been observed elsewhere. However, the dominant product of reactions at mildly acidic levels of pH showed distinct chemistry and higher cytopathic effect activity. The primary site of modification at this pH was His34. We developed a quantitative assay using sensitivity to neutral hydroxylamine to measure the proportion of urethane bonds involving carboxyalkylated histidines. This assay showed that histidine was pegylated preferentially at low pH levels with another protein, rh-Interleukin-10. CONCLUSIONS: Reaction pH can be used to select the preferred pegylation site chemistry.     

Carboxyl-directed Pegylation of Brain-derived Neurotrophic Factor Markedly Reduces Systemic Clearance with Minimal Loss of Biologic Activity

Purpose. Brain-derived neurotrophic factor (BDNF) was modified by carboxyl-directed protein pegylation in order to both retain biologic activity of the neurotrophin and reduce the rate of systemic clearance of this cationic protein in vivo. Since the modification of surface lysine residues of neurotrophins results in loss of biologic activity, the present studies examine the feasibility of placing polyethyleneglycol (PEG) polymers on carboxyl residues of surface glutamate or aspartate residues of BDNF. Methods. PEG molecules with terminal hydrazide (Hz) moieties of molecular weight 2,000 (PEG²⁰⁰⁰-Hz) or 5,000 (PEG⁵⁰⁰⁰-Hz) Daltons were coupled to BDNF carboxyls using carbodiimide. Results. The systemic clearances of the BDNF-PEG²⁰⁰⁰ and BDNF-PEG⁵⁰⁰⁰ were reduced 67% and 91%, respectively, compared to unconjugated BDNF. The brain volume of distribution (V_D) of BDNF-PEG⁵⁰⁰⁰ was not significantly different from the cerebral plasma volume. Cell survival studies and TrkB auto-phosphorylation assays showed that the biologic activity of BDNF was not changed following pegylation with PEG²⁰⁰⁰, and was minimally impaired following pegylation with PEG⁵⁰⁰⁰. Conclusions. These experiments describe the first carboxyl-directed pegylation of a neuropeptide, and show this formulation substantially reduces the systemic distribution and elimination of the neurotrophic factor. The biologic activity of the neurotrophin is retained with carboxyl-directed pegylation.     

Optimization of phage heptapeptide library-screening process for developing inhibitors of the isocitrate lyase homologue from Mycobacterium tuberculosis

The aims of this study were to screen peptide inhibitors specific for isocitrate lyase (ICL) using a phage peptide library and computer molecular docking and to explore the relevant mechanisms. Using ICL as a target, the phage peptide library was screened to obtain peptides with specific binding affinity. Based on the three-dimensional crystal structure of ICL(pdb:1F8I), the obtained polypeptides were docked to the 1F8I using the computer-simulated molecular docking technique. The successfully docked polypeptides were synthesized using the Fmoc solid-phase synthesis method, and the ICL inhibition rate of these peptides was measured. Finally, the possible mechanism underlying the inhibition was explored by Binding Site Analysis. A total of 29 heptapeptides were obtained through screening the phage peptide library. We found that 12 out of the 29 peptides were successfully docked to the 1F8I, and all 12 peptides could obviously inhibit the ICL activity, of which three heptapeptides showed an inhibiting (extent of inhibition over 50 %), IC50 value of 126 μM. Structural analysis revealed that the ICL tetramer has a large cavity in the center, and the polypeptides bind to ICL at amino acid residue 119’s GLN of the ICL monomer. We successfully obtained peptide inhibitors specific for ICL, and analyzed the mechanism underlying the interaction between the peptides and ICL. Our study provides scientific evidence for the development of antituberculosis peptide drugs targeting ICL.     

Pegylated peptides II

General procedures are presented for the site-specific pegylation of peptides at the NH₂-terminus, side-chain positions (Lys or Asp/Glu) or COOH-terminus using solid-phase Fmoc/tBu methodologies. A model tridecapeptide fragment of interleukin-2, IL-2(44-56)-NH₂, was chosen for this study since it possesses several trifunctional amino acids which serve as potential sites for pegylation. The pegylation reagents were designed to contain either Nle or Orn, which served as diagnostic amino acids for confirming the presence of 1 PEG unit per mole of peptide. NH₂-Terminal pegylation was carried out by coupling PEG-CH₂CO-Nle-OH to the free NH₂-terminus of the peptide-resin. Side-chain pegylation of Lys or Asp was achieved by one of two pathways. Direct side-chain pegylation was accomplished by coupling with Fmoc-Lys(PEG-CH₂CO-Nle)-OH or Fmoc-Asp(Nle-NH-CH₂CH₂-PEG)-OH, followed by solid-phase assemblage of the pegylated peptide-resin and TFA cleavage. Alternatively, allylic protective groups were introduced via Fmoc-Lys(Alloc)-OH or Fmoc-Asp(O-Allyl)-OH, and selectively removed by palladium-catalyzed deprotection after assemblage of the peptide-resin. Solid-phase pegylation of the side-chain of Lys or Asp was then carried out in the final stage with PEG-CH₂CO-Nle-OH or H-Nle-NH-(CH₂)₂-PEG, respectively. COOH-Terminal pegylation was achieved through the initial attachment of Fmoc-Orn(PEG-CH₂CO)-OH to the solid support, followed by solid-phase peptide synthesis using the Fmoc/tBu strategy. The pegylated peptides were purified by dialysis and preparative HPLC and were fully characterized by analytical HPLC, amino acid analysis, ¹H-NMR spectroscopy and laser desorption mass spectrometry.     

化学合成肽修饰钛种植体表面的抗菌效果研究

摘要：目的　探讨化学合成肽修饰钛种植体表面的抗菌效果。方法　利用源于人类β防御素3（hBD3）的一个片段（hBD3-1）与肽TBP-1化学结合,形成合成肽TBP-1-hBD3-1。Peptide Calculator软件分析合成肽的基本性质;标准微量肉汤稀释法检测TBP-1-hBD3-1对牙龈卟啉单胞菌（P. gingivalis）的最小抑菌浓度（MIC）、最小杀菌浓度（MBC）,微量滴定法检测生物膜对照组和生物膜实验组光密度（OD600）值差异;共聚焦激光扫描显微镜（CLSM）检测TBP-1-hBD3-1修饰在钛种植体表面的黏附、活性情况。结果　TBP-1-hBD3-1具有较好的溶解性及亲水性,并有疏水性基团,带有一定的正电荷数（4+）。TBP-1-hBD3-1对P. gingivalis的MIC值和MBC值分别为400 mg/L和1 000 mg/L。生物膜实验组较生物膜对照组OD600值降低（P＜0.05）。CLSM结果表明,被AMC标记成蓝色的TBP-1-hBD3-1能够牢固地黏附于钛样本表面。结论　合成肽TBP-1-hBD3-1能够修饰在钛样本表面并发挥抗菌和抑制细菌生物膜形成的作用。     

Modulation of the Cellular Junction Protein E-Cadherin in Bovine Brain Microvessel Endothelial Cells by Cadherin Peptides

Cadherins are calcium-dependent glycoproteins involved in homophilic cell-cell adhesion of tight intercellular junctions. The ability of cadherin peptides to inhibit cadherin-mediated cell-cell adhesion of bovine brain microvessel endothelial cells (BBMECs) was investigated. This was accomplished by using two cadherin function assays, the inhibition of calcium-dependent reaggrega-tion and the dissociation of BBMECs. Peptides that exhibit inhibitory and dissociating properties are presumably bound to cadherins on the surface of BBMECs, inhibiting cadherin-cadherin interactions. We have found six peptides from the EC-1 domain of E-and N-cadherins that inhibit cell-cell adhesion of BBMECs. A very significant inhibitory activity was displayed by a 24-mer peptide (3) derived from the human-E-cadherin sequence. One hexapeptide (7) derived from the E-cadherin sequence can effectively inhibit aggregation of BBMECs. These results will improve our ability to design peptides that can modulate cell-cell adhesion in the intercellular tight junctions.