广谱炎症相关趋化因子抑制剂先导物的筛选
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摘要
炎症反应,包括急性炎症和慢性炎症的一个重要特征是白细胞在局部炎症区域的过度聚集。几乎任何改变细胞稳定因素的刺激,都能诱导趋化因子的分泌,从而募集特定的白细胞到达炎症区域,过度积聚的白细胞也会促进产生过量的细胞因子,加剧细胞毒性反应,引发炎症。在炎症前期,病毒感染刺激因子激活体内炎症效应细胞,产生大量趋化因子并引起炎性细胞的大量积聚,趋化因子高水平的持续不适当的表达,也会使募集的白细胞大量增加形成破坏性的损伤,导致自身免疫病的发生。同时,白细胞介导的损伤又使趋化因子高水平表达或使其他类型的趋化因子表达增加,产生进一步的组织损伤,这样使白细胞对机体的保护作用被破坏而引起疾病。因而,针对趋化因子及其受体的抑制剂、拮抗剂有可能成为炎症反应治疗的新的靶点。
本课题拟运用噬菌体展示技术从噬菌体肽库中筛选与炎症前期产生的相关趋化因子结合并可特异性阻断趋化因子进一步致炎作用的噬菌体克隆,近年来,随着噬菌体展示技术应用范围的不断扩大和研究的不断深入,它在炎症反应的研究中发挥的作用也越来越大。以炎症反应产生的趋化因子为靶标,筛选得到与趋化因子具有高亲和力的短肽,可以抑制炎症感染前期产生的趋化因子,从而阻断机体的炎症反应,并且能克服常规大分子药物的分子量大、组织渗透性弱、不稳定、有免疫原性及体内运输能力差的等缺点,为炎症反应药物的开发研究提供了新的方法。筛选得到的阳性克隆经DNA序列测定、多肽的分析比较研究,确定其活性,再研究其对炎症反应的抑制作用原理,为新型抗炎因子作用靶点的发掘奠定基础。
筛选出的拮抗剂先导物对感染产生的细胞因子有抑制作用,从而阻断机体的炎症反应,而且一系列的药效和毒理学实验都表明,广谱趋化因子功能抑制剂不仅具有强大抗炎效果,而且相较于甾体类抗炎药物有明显的降低毒副反应的作用,因此具有极其优秀的开发应用前景。
目的：
通过筛选出炎症前期趋化因子的抑制剂先导物,对感染产生的炎症相关趋化因子产生拮抗作用,从而阻断机体的炎症反应,为炎症感染的防治探索新途径。
研究方法：
1.常规方法抽提和培养外周血单核细胞(PBMC),以的脂多糖(LPS)刺激PBMC建立体外急性炎症模型,浓缩收集并分析上清液中趋化因子的产生情况。
2.利用噬菌体随机十二肽库进行亲和筛选,获得噬菌体阳性克隆。
3.将获得的阳性噬菌体克隆进行DNA测序,应用多种生物信息学软件将多肽序列进行比较分析,推导与炎性相关趋化因子广谱结合的的小分子十二肽。
4.通过趋化抑制试验选择抑制率高的克隆进行合成并研究其活性；
5.对合成的短肽进行MTT检测和体外活性检测,研究其对炎性趋化因子的抑制情况；
6.利用钙流试验检测短肽是否与趋化因子受体有关；
7.根据广谱趋化因子抑制剂的分子作用机制,采用RT-PCR试验检测其mRNA水平的趋化因子表达情况,以研究短肽降低趋化因子表达的作用机制。
实验结果：
1.经过4轮噬菌体亲和筛选,噬菌体克隆回收率从5.51×10-8增加到5.06×10-6,第四轮的回收率约是第一轮91.83倍,说明目标噬菌体克隆得到了有效富集。
2.效价测定实验发现有16个克隆效价较高,琼脂糖凝胶试验发现其中10个克隆的DNA处于相同的位置,条带比较清晰,对其进行测序。
3.测序结果发现有5个克隆有相同的插入序列高概率短肽,命名为P1肽,另外5个同源性较差,利用PROTPARAM生物信息学软件分析各条短肽的理化性质等。
4.趋化抑制实验发现,P1肽可显著抑制急性炎症模型中各类炎性趋化因子的表达,其他短肽的抑制率并不理想,可以认为它可能是炎性趋化因子抑制剂的基本骨架或者先导物。
5.根据短肽序列进行生物合成,利用MTT试验检测其对PBMC的活性影响,结果说明P1肽对PBMC细胞的活性并无影响。
6.通过LUMINEX芯片技术检测P1肽对急性炎症模型的趋化因子表达的影响情况,试验结果表明P1肽可以明显降低各类炎症相关趋化因子的表达。
7.钙流试验结果表明P1肽可以有效抑制炎症趋化因子与受体的结合概率,而且P1肽本身也不会引起细胞内钙离子的变化,说明P1肽的趋化抑制作用与趋化因子受体并无明显关系。
8.RT-PCR结果说明在P1肽的作用下,各类趋化因子的mRNA的表达均有明显的降低,但是TTP的表达并无影响,说明趋化因子mRNA的降低表达并不是通过TTP来实现的,可能涉及到其他功能基因的表达,这均有待于进一步的实验研究证明。
结论：
1成功筛选得到可有效抑制趋化因子表达的十二短肽；
2活性实验研究发现该短肽对细胞不会产生毒副作用；
3作用机制研究发现P1肽可以抑制各类炎症相关趋化因子mRNA的表达进而抑制趋化因子的量,但是其具体的抑制机制有待于进一步研究证明。
Inflammation, including acute inflammation and chronic inflammation,an important feature of them is that the leukocyte are over accumulation in the local inflammatory area. Almost any stimulation which can hange cell stabilizing factor could induce the secretion of chemokines, raising the specific white blood cells to the inflammatory area, the over accumulative white blood cells can induce produce excessive cytokines,intensify cell-cytotoxicity reaction, then induce inflammation. Early in inflammation, viral could infect, stimulate and activate inflammation effector cells, resulting in a large number of chemokines and causing substantial accumulation of inflammatory cells. However, the continued high level of chemokine expression is not only inappropriate, but also it will raise a significant increase in white blood cells and the formation of destructive damage, resulting in occurrence of autoimmune disease.Meanwhile,the white blood cell mediated injury also caused high expression of chemokines and the other types cytokines,which resulting in further tissue damage, destoryed the white blood cells of the body's protective effect and caused diseases. Thus, antagonists of the chemokines and chemokine receptor of inflammatory response may become a new target for therapy way.
This subject proposed to use phage display technology from phage peptide library to screen the phage clones which could bind early inflammatory chemokine production to block inflammation.In recent years, with the scope of application of phage display technology expanding and deepening in the study, it plays an increasing role in the inflammatory reaction, inflammation chemokines are as targets,screened the short peptide with high affinity which can inhibit the infection of early inflammtory chemokines production,thereby blocking the body's inflammtory response,and overcome the disadvantages of the conventional macromolecular drugs,such as,the molecular weight, the weak of the tissue permeability, unstable, vivo immunogenicity and poor transport capacity,it can provide a new way for the development of inflammatory drugs.Then,by DNA sequencing analysising, study the inflammatory response inhibition theory, the targets of it is to find a new anti-inflammatory factors, and a series of efficacy and toxicology experiments have shown that broad-spectrum inhibitor of chemokine function is not only a powerful anti-inflammatory effects, and compared with steroidal anti-inflammatory drugs significantly,it reduced the role of toxic side effects,so it has a very good development prospects.
Object:
By screening the early inflammatory chemokine antagonist lead compound,which is to inhibit the production of cytokines and to block the body's inflammatory response for exploring a new way for infection prevention and control of inflammation,.
Methods:
1. Conventional method to obtain and develop in peripheral blood mononuclear cells (PBMC), using lipopolysaccharide to stimulate PBMC to set up vitro inflammation model, then collected supernatant and analyzed the production of chemokines.
2. Using phage random peptide library to obtain positive phage clones which are screened by four rounds of affinity and initial validation.
3. Sequenced positive phage clones, used a variety of bioinformatics software to analysis peptide sequence and found the broad spectrum of inflammatory chemokine binding peptide.
4. Selected the high inhibition rate of clones and researched by chemokine inhibition test;
5. On the synthesis of short peptides of MTT testing in vitro activity and detection of its inhibition of inflammatory effects of chemokines;
6. Use of calcium flow test study the relationship the peptide with the chemokine receptor;
7. According to broad-spectrum chemokine inhibitors molecular mechanism, using RT-PCR test to detect the level of chemokine mRNA expression, to study peptide mechanism of action of reducing the expression of chemokines.
Results:
1. Using phage display screening method by 4 rounds of panning, phage clones recovery rate from 5.51×10"8 to 5.06×10-6, the fourth round phage clones recovery is about the first round of 91.83 times,it showed that the target phage clones were effectively enriched.
2. Titer determination experiments found that 16 clones had a higher titer, agarose gel study found that 10 clones of them were in the same position, relatively clear bands, so sequenced these peptides.
3. The sequencing results showed that 5 clones have the same insertion sequence, named P1 peptide, another five were poor homologous,then use of PROTPARAM bioinformatics software to analyze various physical and chemical nature of peptides.
4. Chemotactic inhibition was found that P1 peptides could significantly inhibit the expression of various types of inflammatory chemokine on acute inflammation model,, other short peptides inhibition is not ideal, it may be that the P1 is the basic framework or lead compounds of the inflammatory chemokine inhibitors.
5. The MTT test results indicated that the P1 peptide did not affect the activity of PBMC cells.
6. LUMINEX chip test results showed that the P1 peptide could significantly reduce the various types of inflammatory chemokine expression.
7. Calcium flow test results show that the P1 peptide can inhibit the inflammatory chemokine receptor binding probability, and the P1 peptide itself does not cause changes in intracellular calcium, indicating the P1 has no significant relationship with the chemokine receptors.
8.RT-PCR results showed that all kinds of mRNA of chemokines expression were significantly reduced under the action of the P1 peptide, but it did not affect by the expression of TTP, indicating the reduced mRNA expression is not achieved by the TTP, may involve the expression of other genes, which are to be further experimental studies.
Conclusion:
1 screened a short peptide successfully by 4 rounds of panning;
2 activity experimental studies found that the peptide does not produce toxic side effects on the cells;
3 Mechanism of P1 peptide was found that it could inhibit the amount of chemokines by inhibiting various inflammatory chemokine mRNA expressions but the specific inhibition mechanism needs further study to prove.
本课题拟运用噬菌体展示技术从噬菌体肽库中筛选与炎症前期产生的相关趋化因子结合并可特异性阻断趋化因子进一步致炎作用的噬菌体克隆,近年来,随着噬菌体展示技术应用范围的不断扩大和研究的不断深入,它在炎症反应的研究中发挥的作用也越来越大。以炎症反应产生的趋化因子为靶标,筛选得到与趋化因子具有高亲和力的短肽,可以抑制炎症感染前期产生的趋化因子,从而阻断机体的炎症反应,并且能克服常规大分子药物的分子量大、组织渗透性弱、不稳定、有免疫原性及体内运输能力差的等缺点,为炎症反应药物的开发研究提供了新的方法。筛选得到的阳性克隆经DNA序列测定、多肽的分析比较研究,确定其活性,再研究其对炎症反应的抑制作用原理,为新型抗炎因子作用靶点的发掘奠定基础。
筛选出的拮抗剂先导物对感染产生的细胞因子有抑制作用,从而阻断机体的炎症反应,而且一系列的药效和毒理学实验都表明,广谱趋化因子功能抑制剂不仅具有强大抗炎效果,而且相较于甾体类抗炎药物有明显的降低毒副反应的作用,因此具有极其优秀的开发应用前景。
目的：
通过筛选出炎症前期趋化因子的抑制剂先导物,对感染产生的炎症相关趋化因子产生拮抗作用,从而阻断机体的炎症反应,为炎症感染的防治探索新途径。
研究方法：
1.常规方法抽提和培养外周血单核细胞(PBMC),以的脂多糖(LPS)刺激PBMC建立体外急性炎症模型,浓缩收集并分析上清液中趋化因子的产生情况。
2.利用噬菌体随机十二肽库进行亲和筛选,获得噬菌体阳性克隆。
3.将获得的阳性噬菌体克隆进行DNA测序,应用多种生物信息学软件将多肽序列进行比较分析,推导与炎性相关趋化因子广谱结合的的小分子十二肽。
4.通过趋化抑制试验选择抑制率高的克隆进行合成并研究其活性；
5.对合成的短肽进行MTT检测和体外活性检测,研究其对炎性趋化因子的抑制情况；
6.利用钙流试验检测短肽是否与趋化因子受体有关；
7.根据广谱趋化因子抑制剂的分子作用机制,采用RT-PCR试验检测其mRNA水平的趋化因子表达情况,以研究短肽降低趋化因子表达的作用机制。
实验结果：
1.经过4轮噬菌体亲和筛选,噬菌体克隆回收率从5.51×10-8增加到5.06×10-6,第四轮的回收率约是第一轮91.83倍,说明目标噬菌体克隆得到了有效富集。
2.效价测定实验发现有16个克隆效价较高,琼脂糖凝胶试验发现其中10个克隆的DNA处于相同的位置,条带比较清晰,对其进行测序。
3.测序结果发现有5个克隆有相同的插入序列高概率短肽,命名为P1肽,另外5个同源性较差,利用PROTPARAM生物信息学软件分析各条短肽的理化性质等。
4.趋化抑制实验发现,P1肽可显著抑制急性炎症模型中各类炎性趋化因子的表达,其他短肽的抑制率并不理想,可以认为它可能是炎性趋化因子抑制剂的基本骨架或者先导物。
5.根据短肽序列进行生物合成,利用MTT试验检测其对PBMC的活性影响,结果说明P1肽对PBMC细胞的活性并无影响。
6.通过LUMINEX芯片技术检测P1肽对急性炎症模型的趋化因子表达的影响情况,试验结果表明P1肽可以明显降低各类炎症相关趋化因子的表达。
7.钙流试验结果表明P1肽可以有效抑制炎症趋化因子与受体的结合概率,而且P1肽本身也不会引起细胞内钙离子的变化,说明P1肽的趋化抑制作用与趋化因子受体并无明显关系。
8.RT-PCR结果说明在P1肽的作用下,各类趋化因子的mRNA的表达均有明显的降低,但是TTP的表达并无影响,说明趋化因子mRNA的降低表达并不是通过TTP来实现的,可能涉及到其他功能基因的表达,这均有待于进一步的实验研究证明。
结论：
1成功筛选得到可有效抑制趋化因子表达的十二短肽；
2活性实验研究发现该短肽对细胞不会产生毒副作用；
3作用机制研究发现P1肽可以抑制各类炎症相关趋化因子mRNA的表达进而抑制趋化因子的量,但是其具体的抑制机制有待于进一步研究证明。
Inflammation, including acute inflammation and chronic inflammation,an important feature of them is that the leukocyte are over accumulation in the local inflammatory area. Almost any stimulation which can hange cell stabilizing factor could induce the secretion of chemokines, raising the specific white blood cells to the inflammatory area, the over accumulative white blood cells can induce produce excessive cytokines,intensify cell-cytotoxicity reaction, then induce inflammation. Early in inflammation, viral could infect, stimulate and activate inflammation effector cells, resulting in a large number of chemokines and causing substantial accumulation of inflammatory cells. However, the continued high level of chemokine expression is not only inappropriate, but also it will raise a significant increase in white blood cells and the formation of destructive damage, resulting in occurrence of autoimmune disease.Meanwhile,the white blood cell mediated injury also caused high expression of chemokines and the other types cytokines,which resulting in further tissue damage, destoryed the white blood cells of the body's protective effect and caused diseases. Thus, antagonists of the chemokines and chemokine receptor of inflammatory response may become a new target for therapy way.
This subject proposed to use phage display technology from phage peptide library to screen the phage clones which could bind early inflammatory chemokine production to block inflammation.In recent years, with the scope of application of phage display technology expanding and deepening in the study, it plays an increasing role in the inflammatory reaction, inflammation chemokines are as targets,screened the short peptide with high affinity which can inhibit the infection of early inflammtory chemokines production,thereby blocking the body's inflammtory response,and overcome the disadvantages of the conventional macromolecular drugs,such as,the molecular weight, the weak of the tissue permeability, unstable, vivo immunogenicity and poor transport capacity,it can provide a new way for the development of inflammatory drugs.Then,by DNA sequencing analysising, study the inflammatory response inhibition theory, the targets of it is to find a new anti-inflammatory factors, and a series of efficacy and toxicology experiments have shown that broad-spectrum inhibitor of chemokine function is not only a powerful anti-inflammatory effects, and compared with steroidal anti-inflammatory drugs significantly,it reduced the role of toxic side effects,so it has a very good development prospects.
Object:
By screening the early inflammatory chemokine antagonist lead compound,which is to inhibit the production of cytokines and to block the body's inflammatory response for exploring a new way for infection prevention and control of inflammation,.
Methods:
1. Conventional method to obtain and develop in peripheral blood mononuclear cells (PBMC), using lipopolysaccharide to stimulate PBMC to set up vitro inflammation model, then collected supernatant and analyzed the production of chemokines.
2. Using phage random peptide library to obtain positive phage clones which are screened by four rounds of affinity and initial validation.
3. Sequenced positive phage clones, used a variety of bioinformatics software to analysis peptide sequence and found the broad spectrum of inflammatory chemokine binding peptide.
4. Selected the high inhibition rate of clones and researched by chemokine inhibition test;
5. On the synthesis of short peptides of MTT testing in vitro activity and detection of its inhibition of inflammatory effects of chemokines;
6. Use of calcium flow test study the relationship the peptide with the chemokine receptor;
7. According to broad-spectrum chemokine inhibitors molecular mechanism, using RT-PCR test to detect the level of chemokine mRNA expression, to study peptide mechanism of action of reducing the expression of chemokines.
Results:
1. Using phage display screening method by 4 rounds of panning, phage clones recovery rate from 5.51×10"8 to 5.06×10-6, the fourth round phage clones recovery is about the first round of 91.83 times,it showed that the target phage clones were effectively enriched.
2. Titer determination experiments found that 16 clones had a higher titer, agarose gel study found that 10 clones of them were in the same position, relatively clear bands, so sequenced these peptides.
3. The sequencing results showed that 5 clones have the same insertion sequence, named P1 peptide, another five were poor homologous,then use of PROTPARAM bioinformatics software to analyze various physical and chemical nature of peptides.
4. Chemotactic inhibition was found that P1 peptides could significantly inhibit the expression of various types of inflammatory chemokine on acute inflammation model,, other short peptides inhibition is not ideal, it may be that the P1 is the basic framework or lead compounds of the inflammatory chemokine inhibitors.
5. The MTT test results indicated that the P1 peptide did not affect the activity of PBMC cells.
6. LUMINEX chip test results showed that the P1 peptide could significantly reduce the various types of inflammatory chemokine expression.
7. Calcium flow test results show that the P1 peptide can inhibit the inflammatory chemokine receptor binding probability, and the P1 peptide itself does not cause changes in intracellular calcium, indicating the P1 has no significant relationship with the chemokine receptors.
8.RT-PCR results showed that all kinds of mRNA of chemokines expression were significantly reduced under the action of the P1 peptide, but it did not affect by the expression of TTP, indicating the reduced mRNA expression is not achieved by the TTP, may involve the expression of other genes, which are to be further experimental studies.
Conclusion:
1 screened a short peptide successfully by 4 rounds of panning;
2 activity experimental studies found that the peptide does not produce toxic side effects on the cells;
3 Mechanism of P1 peptide was found that it could inhibit the amount of chemokines by inhibiting various inflammatory chemokine mRNA expressions but the specific inhibition mechanism needs further study to prove.
引文
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[19]Studen tsov YY. Enhanced eczyme-linked inmunosoebent assay for detection of antibodies to virus-like partieles of human papillemavirus[J].j ClinMICROIOL,2002,40(5):1755-1760.
[20]WaterboerT,PSehr,M Pawlita. Suppression of non-specific binding in serological Luminexassays[J] J Immunol Methods,2006,309(1-2):200-204.
[21]Martins T R.Heterophile antibody interference in a multipleased fluore scentm icrosphere immunosaasy for auantitation of cytokines in human serum[J].Clin Dian Lab Immunol,2004,11(2):325-329.
[22]Carson RT, Vignali DA. Simultaneous quantitation of 15 cytokines using a multiplexed flow cytometric assay. J Immunol Methods,1999,30.227(1-2):41-52..
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[52]Nefertiti C.duPont, Kehui Wang, Pathik D et al. Wadhwa Validation and comparison of luminex multiplex cytokine analysis kits with ELISA:Determinations of a panel of nine cytokines in clinical sample culture supernatants. Journal of Reproductive Immunology,2005,66:175-191.
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[29]Smothers JF et al. Phage display:affinity selection from biological libraries. Science,2002,298(5593):621-622
[30]李珣,任珍珍,钟国华.噬菌体展示技术在蛋白质研究中的应用[J]生命的化学.2009.29(4)：588-594
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[32]DemangeC, LafayeP, MazieC.Reproducing the immunere sponse against the Plasmodium vivax merozoite surface protein I with mimotopes selected from a Phage displayed peptide library[J]Moleculogy Immunology,1996,33(11/12):909-916.
[33]冯烨.噬菌体展示技术及其在感染性疾病中的应用[J]中国生物制品学杂 志.2009.22(8)：834-836
[34]孙美艳,张磊,李艳.噬菌体展示技术的研究进展[J].吉林医药学院学报2009.30(2)：97-99
[35]Jens V. Stein and Cesar Nombela-Arrieta.Chemokine control of lymphocyte trafficking:a general overview.Immunology,2005,116:1-12.
[36]Kruys V, Marinx O, Shaw G, Deschamps J, et al. Translational blockade imposed by cytokine-derived UA-rich sequences. Science 1989;245:852-5.
[37]Kontoyiannis D, Pasparakis M, Pizarro TT, Cominelli F, Kollias G. Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies. Immunity 1999;10:387-98.
[38]Taylor GA, Carballo E, Lee DM, et al. A pathogenetic role for TNF in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. Immunity 1996;4:445-54.
[39]Hitoshi Sawaoka, Dan A. Dixon, John A. Oates,et al. Tristetraprolin Binds to the 3'-Untran-slated Region of Cyclooxygenase-2 mRNA.[J] Biology Chemistry 2003;278;13928-13935.
[40]Masae Maeda, Hirofumi Sawa, Minoru Tobiume et al. Tristetraprolin inhibits HIV-1 production by binding to genomic RNA. Microbes and Infection.2006;8; 2647-2656
[41]Loetscher P, Moser B, Baggiolini M. [J].Adv.immunol,2000,74:127-80.
[42]Sallusto F,Mackay C.R, Lanzavecchina A. [J].Annu.Rev.Immunol,2000,18:593-620.
[43]Margareta Krook, Klaus Mosbach, Olof Ramstrom. Novel peptides binding to the Fc-portion of immunoglobulins obtained from a combinatorial phage display peptide library.Journal of Immunological Methods,1998,221:151-157.
[44]Smothers JF et al. Phage display:affinity selection from biological libraries.Science.2002.298(5593):621-622.
[45]Margareta Krook, Klaus Mosbach, Olof Ramstro"m. Novel peptides binding to the Fc-portion of immunoglobulins obtained from a combinatorial phage display peptide library. Journal of Immunological Methods,1998,221:151-157
[46]Benharl.Biotechnological applications of phage and cell display[J].BiotechAdv,2001,19(1):1,33.
[47]MANOUTCHARIAN K,GEVORKIAN QCANO AETAL.Phage displayed biomolecules as preventive and therapeutic agents [J].Currpharm Biotechnology,2001,2(3):217-223.
[48]AINAOH,SROKATC.Therapeutica cancer targeting peptides[J].Biopolymers,2002,66(3):184-199.
[49]陈莉琴.LPS受体作用机制研究进展.中国水电医学[J].2006,3：186-190.
[50]Laffey JG, Boylan JF, Cheng DC.The systemic inflammatory response to cardiac surgery:implications for the anesthesiologist. Anesthesiology,2002,97:215-252.
[51]Johanna Dernfalk, Karin Persson Waller, Anders Johannisson.ThexMAPTMechnique can be used for detection of the inflammatory cytokines IL-1β, IL-6 and TNF-a in bovine samples.Veterinary Immunology and Immunopathology,2007,118:40-49.
[52]Nefertiti C.duPont, Kehui Wang, Pathik D et al. Wadhwa Validation and comparison of luminex multiplex cytokine analysis kits with ELISA:Determinations of a panel of nine cytokines in clinical sample culture supernatants. Journal of Reproductive Immunology,2005,66:175-191.
[53]Subhash C. Datta, Mark R. Opp.Lipopolysaccharide-induced increases in cytokines in discrete mouse brainregions are detectable using Luminex Xmap.technology Journal of Neuroscience Methods,2008,175:119-124.
[54]周先岭,徐文锐,陈贵海.利用噬菌体随机肽库筛选表达Aβ靶向肽的噬菌体.中国医药生物技术,2008,3(5)：366-369.
[55]Fan J, Heller NM, Gorospe M, et al. The role of post-transcriptional regulation in chemokine gene expression in inflammation and allergy [J]. Eur RespirJ,2005,26 (5):933-947.
[56]Hamilton TA, Novotny M, Datta S, et al. Chemokine and chemoattractant receptor expression:post-transcriptional regulation [J]. J Leukoc Biol,2007,82 (2): 213-219.
[57]Taylor GA, Carballo E, Lee DM, et al. A pathogenetic role for TNF in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. Immunity 1996;4:445-54.
[58]Fan J, Heller NM, Gorospe M, et al. The role of post-transcriptional regulation in chemokine gene expression in inflammation and allergy [J]. Eur Respir J,2005,26 (5):933-947.
[59]F. T. Ishmaell, X. Fangl, N. Hellerl,et al. Role of the RNA-binding Protein Tristetraprolin (TTP) in Glucocorticoid (GC)-mediated Gene Regulation. Environmental Health Sciences,2006,6,321-334.
[60]Ulla Jalonen, Aleksi Lahti, Riku Korhonen,et al. Inhibition of tristetraprolin expression by dexamethasone in activated macrophages. Biochemical Pharmacology.2005;69;733-740.
[61]Horuk R. OPINION Chemokine receptor antagonists:overcoming developmental hurdles [J]. Nature Reviews Drug Discovery,2009,8 (1):23-33.
[62]Schaljo B, Kratochvill F, Gratz N, et al. Tristetraprolin is required for full anti-inflammatory response of murine macrophages to IL-10 [J]. J Immunol,2009,183 (2):1197-1206.
[63]Comerford I, Nibbs RJ, et al. Post-translational control of chemokines:a role for decoy receptors [J]? Immunol Lett,2005,96 (2):163-174.
[64]Proost P, De Meester I, Schols D, et al. Amino-terminal truncation of chemokines by CD26/dipeptidyl-peptidase Ⅳ. Conversion of RANTES into a potent inhibitor of monocyte chemotaxis and HIV-1-infection [J]. J Biol Chem,1998,273 (13):7222-7227.
[65]Jamieson T, Cook DN, Nibbs RJ, et al. The chemokine receptor D6 limits the inflammatory response in vivo [J]. Nat Immunol,2005,6 (4):403-411.
[66]Graham GJ, McKimmie CS. Chemokine scavenging by D6:a movable feast [J]? Trends Immunol,2006,27 (8):381-386.