摘要
研究背景和目的
创伤愈合是一个修复组织完整性的多相过程。包含了多种细胞,细胞外基质,细胞因子和生长因子之间的机能协调及相互作用。异常的创伤愈合过程,如：烧伤,挫伤和外科手术导致的不规则的皮肤创伤愈合,会导致瘢痕疙瘩的发生。瘢痕疙瘩不仅影响患者的躯体功能,常导致焦虑等心理问题。临床治疗治疗后复发率高,对于整形外科医生,是一个很大的挑战。
目前,关于瘢痕疙瘩的研究集中在细胞因子,生长因子和基因治疗等方面。转化生长因子-β1 (Transforming growth factor-betal, TGF-β1)是一种多功能的生长因子,与创面愈合和瘢痕形成密切相关。核因子κB (Nuclear factor kappa B,NF-κB),结缔组织生长因子(connective tissue growth factor, CTGF)和TGF-βreceptorⅡ(TβRⅡ)是TGF-β1信号的重要的细胞内介质。TGF-β1通过这些通路调节成纤维细胞的增殖。阻断TGF-β1的信号通路是一种有效防治瘢痕疙瘩的方法。
噬菌体肽库(Peptide library)技术是噬菌体展示技术的一个非常重要的分支。其原理是通过把大量的随机肽段与丝状噬菌体的外壳蛋白(PⅧ或PⅢ)融合表达、组装,从而展示于噬菌体颗粒表面以组成每个噬菌体都带有一个不同肽段的重组噬菌体库,然后用目的蛋白来筛选与之相互作用的噬菌体肽。通过分析所筛选到的噬菌体肽的结构和序列,为蛋白质分子之间(如抗原与抗体、受体和配体、酶与底物)的相互作用机理提供理论依据与实验支持。噬菌体随机肽库日益受到人们的重视,噬菌体随机肽库技术也日趋成熟,并根据应用目的不同而构建了各种不同的肽库,其已成为探索受体与配体之间相互作用结合位点、寻求高亲合力生物活性的配体分子、探测未知蛋白空间结构表位的工具,在蛋白分子相互识别的研究、新型疫苗的研制以及药物的开发等领域具有广泛的应用前景。已从噬菌体随机肽库中筛选获得多种生长因子的特异性的配体和抗原表位,如CTGF, EGF, FGF和VEGF等。
本研究的目的是从噬菌体随机12肽库中筛选获得TGF-β1噬菌体模拟肽。噬菌体上表达的特异性的模拟肽可能包含类似于TGF-β1的外源性多肽,可能是TGF-β1的受体阻断剂,与TGF-β1竞争性结合其受体,从而抑制瘢痕疙瘩成纤维细胞的增殖。应用噬菌体随机肽库筛选获得TGF-β1的肽抑制剂,有助于抑制TGF-β1的作用,可能为防治瘢痕疙瘩提供新的机遇。
材料和方法
第一部分应用噬菌体随机十二肽库筛选TGF-β1相关模拟肽的实验研究
以人TGF-β1单克隆抗体为靶,进行4轮生物淘选,富集包含特异性模拟肽的噬菌体。对洗脱物和扩增物进行滴度测定。宿主菌划线接种、培养进行噬菌体扩增。接种宿主菌单菌落于培养基中,计数蓝斑,计算噬菌体滴度。随机挑选第4轮生物淘选的噬菌体蓝斑,扩增后计算噬菌体产出/投入比,评估噬菌体的回收率。应用酶联免疫吸附(ELISA)法检测单克隆噬菌体的结合力,将噬菌体DNA沉淀、离心,进行琼脂糖凝胶电泳检测DNA提取质量。噬菌体DNA和-96gⅢ测序引物一起送北京华大基因有限公司,进行染料示踪的双脱氧法自动测序。应用核酸Blast系统检测模拟肽DNA的相似性。
第二部分TGF-β1噬菌体模拟肽抑制瘢痕疙瘩成纤维细胞增殖的实验研究
以人TGF-β1单克隆抗体包被孔板,加肽库,共筛选4轮,富集包含特异性模拟肽的噬菌体。由北京华大基因有限公司进行染料示踪的双脱氧法自动测序。应用核酸相似性比较数据库(Blast系统)检测模拟肽DNA的相似性。共获得4个噬菌体模拟肽,进行下一步的离体试验。
从经过病理学鉴定的瘢痕疙瘩培养获得瘢痕疙瘩成纤维细胞。应用组织块培养法获得瘢痕疙瘩成纤维细胞。
噻唑蓝(MTT)法检测活细胞数量,检测TGF-β1对细胞增殖的影响。共设13组：包括阴性对照组、噬菌体M13对照组、TGF-β1对照组各1组和噬菌体模拟肽组10组。
应用Annexin V-FITC/PI凋亡检测试剂盒和流式细胞技术检测细胞的凋亡。共设5组：阴性对照组和4组噬菌体模拟肽组。Annexin V-FITC阳性和PI阴性表示细胞处于凋亡早期阶段,二者均为阳性表示细胞处于凋亡晚期阶段,二者均阴性为未凋亡细胞。
模拟肽的细胞亲和力检测：共设13组：包括阴性对照组、噬菌体M13对照组(1012 pfu/ml)、TGF-β1对照组(5 mg/L)各1组和噬菌体模拟肽组10组(1012pfu/ml)。按照免疫荧光检测试剂盒的操作指导说明进行操作。以人单克隆TGF-β1抗体(红色)和M13外壳蛋白(绿色)为一抗。
采用实时定量PCR法对瘢痕疙瘩成纤维细胞核因子κB(NF-κB)和结缔组织生长因子(CTGF)的表达进行检测。共设13组：包括阴性对照组、噬菌体M13对照组、TGF-β1对照组各1组和噬菌体模拟肽组10组。应用RNAiso试剂提取表皮细胞的总RNA。应用PrimeScriptTM RT试剂盒反转录获得cDNA,应用SYBR Premix Ex TaqTM II进行PCR反应。反应条件为：30 s,95℃；95℃5 s,58℃31 s(40个循环)。引物序列为：(1)β-肌动蛋白：上游：5'TGACGTGGACATCCGCAAAG 3',下游：5'CTGGAAGGTGGACAGCGAGG3'(204 bp)；(2)NF-κB：上游：5'CTGTAACTGCTGGACCCAAGG 3',下游：5'CTTTTTCCCGATCTCCCAGCT 3' (209 bp);(3)CTGF：上游：5'CCTCTTCTGTGACTTCGGCTC 3',下游：5'GAACGTCCATGCTGCACAG 3'(188 bp)。
应用SPSS16.0软件进行统计学分析。结果数据以x±s表示,应用单因素方差分析法(one-way ANOVA)(?)(?)Dunnet's检测方法分析各组间的差异,以P<0.05为差异有统计学意义。
结果
1,应用噬菌体随机十二肽库筛选TGF-β1相关模拟肽的实验研究
经过4轮生物淘选,获得的噬菌体总量及其产率均逐渐升高,包含有特异性模拟肽的噬菌体获得了富集, ELISA检测获得TGF-β1的标准品-吸光度值的曲线。结果显示筛选获得的噬菌体具有较好的结合活性,测序获得10个与促进成纤维细胞增殖或抑制细胞增殖有关的碱基序列。经过Blast系统检测,其中1个模拟肽(No.1)与TGF-β1相似,3个模拟肽(No.2,5,8)与TGF-β2相似,5个模拟肽(No.3,6-8,10)与TβRⅢ受体相似,3个模拟肽(No.4,5,8)与TGF-β诱导因子相似,1个模拟肽(No.9)与NF-κB相似,6个模拟肽(No.4-9)与细胞分裂原素活化蛋白激酶(mitogen-activated protein kinase, MAPK)相似。
2,转化生长因子-β1噬菌体模拟肽抑制瘢痕疙瘩成纤维细胞增殖的实验研究
在570 nm处检测吸光度A值,MTT结果显示阴性对照组的A值为0.22(保留小数点后两位数字M13对照组也是0.22),TGF-β1对照组不同浓度(0.05、0.5、5μg/L)的A值分别为0.29、0.31、0.35,单因素方差分析和Dunnet's方法分析结果显示TGF-β1对照组与阴性对照组间差异有统计学意义。TGF-β1对照组能够显著地促进瘢痕疙瘩成纤维细胞增殖(P<0.01)。统计分析结果显示在阴性对照组和噬菌体M13对照组之间差异无统计学意义(P>0.05),在阴性对照组和第5、6组噬菌体模拟肽组之间,以及阴性对照组和低浓度(1010 pfu/m1)的第7、10组噬菌体模拟肽组之间差异无统计学意义(P>0.05),但是阴性对照组和其他噬菌体模拟肽组之间差异有统计学意义(P<0.05),结果提示4种噬菌体模拟肽(第7-10组)能够抑制瘢痕疙瘩成纤维细胞增殖。
对MTT结果中能够抑制瘢痕疙瘩成纤维细胞增殖的4种噬菌体模拟肽(第7-10组),进一步评估其促使瘢痕疙瘩成纤维细胞凋亡的作用。相对于阴性对照组,这4种噬菌体模拟肽(第7-10组)能够轻度促使瘢痕疙瘩凋亡,能够诱导瘢痕疙瘩成纤维细胞凋亡的晚期阶段。
免疫荧光检测结果显示TGF-β1对照组和10种噬菌体模拟肽组能够与瘢痕疙瘩成纤维细胞相结合,但是噬菌体M13对照组不能与瘢痕疙瘩成纤维细胞相结合。
实时荧光定量PCR检测TGF-β1对照组和各噬菌体模拟肽组(1～10组)NF-κB及CTGF的表达水平,与阴性对照组(表达量设为1)相比,TGF-β1对照组和第1-4噬菌体模拟肽组NF-KB和CTGF的表达升高,第5-6噬菌体模拟肽组变化不明显,而第7-10噬菌体模拟肽组表达减弱。其中NF-κB的表达量分别是阴性对照组的0.28、0.26、0.46、0.30倍,CTGF的表达量分别是阴性对照组的0.26、0.60、0.34、0.17倍。
结论
1,从噬菌体随机十二肽库中可淘选到与TGF-β1相关的噬菌体模拟肽。
2,4种噬菌体模拟肽能够与瘢痕疙瘩成纤维细胞相结合,并轻度促使瘢痕疙瘩成纤维细胞发生轻度的晚期凋亡。
3,噬菌体模拟肽可能是通过调节NF-κB及CTGF的表达,调节瘢痕疙瘩成纤维细胞的增殖。
Background and Objective
Wound healing and tissue repair is the integrity of a multiphase process. Contains a variety of cells, extracellular matrix, cytokines and growth factors co-ordination between the functions and interactions. Abnormal wound healing may lead to keloid. Abnormal wound healing, such as:burns, contusions, and surgery lead to irregular skin wound healing, may lead to keloid. Keloids affect the patient's physical function and appearance, and lead to the psychological problems. It is a challenge for the plastic surgeon to face with because of its high recurrence rate after treatment.
Currently, research is focused on keloids in cytokines, growth factors and gene therapy. Transforming growth factor-betal (Transforming growth factor-betal, TGF-β1) is a multifunctional growth factor, and wound healing and scar formation are closely related. Nuclear factor kappa B (NF-κB), connective tissue growth factor (CTGF) and TGF-beta receptor II (T(3RII) is an important intracellular medium in TGF-β1 signaling pathway. TGF-β1 via these pathways regulating fibroblast proliferation. Blocking of TGF-β1 signaling pathway is an effective method of prevention and treatment of keloids.
Phage random peptide library is is a very important branch of phage display technology. The principle is to abtain recombinant phage through a large number of random peptides with the filamentous phage coat protein (PⅧor PⅢ) fusion expression, assembly, and thus displayed on the phage particles.The target protein is used to screen phage peptide interacted with. Through the course of analyzing the screened phage peptide structure and sequence, it provides a theoretical basis and experimental support for the protein molecules (such as antigens and antibodies, receptor and ligand, enzyme and substrate) interaction mechanism. Phage random peptide library increasing people's attention, phage random peptide library technology has built a variety of different peptide libraries depending on the application and different purposes. It has become a effective mathods to explore the interaction between the receptor and ligand binding sites, for the biological activity of high affinity ligand to detect spatial structure of the unknown protein epitope of the tool.It has a wide range of applications in the study of protein molecules recognize each other, the new vaccine and drug development and other areas. From random peptide phage library was screened to obtain a variety of growth factors specific ligands and epitopes, such as CTGF, EGF, FGF and VEGF, etc.
The purpose of this study is from a random 12-peptide phage library was screened to obtain TGF-β1 peptide phage simulation. Phage-specific expression similar to the analog peptide may contain exogenous TGF-β1 peptide, may be of TGF-β1 receptor antagonist, and TGF-β1 competitive binding its receptor, thereby inhibiting keloid fibroblast proliferation. Random peptide phage library screening to obtain peptide inhibitor of TGF-β1, to help curb the role of TGF-β1, may provide new opportunities for prevention and treatment of keloids.
Materials and methods
Part one TGF-betal related model peptides isolated from a phage display 12-mer peptide library
Human TGF-β1 monoclonal antibody for the target, the four bio-panning enriched phage peptide contains the specific simulation. Eluate and the amplicon of the titer determination. Crossed the host bacteria inoculated, cultured for phage amplification. Single colonies were inoculated in the host cell culture medium, count the blue spot, calculate the phage titer. Randomly selected the first four biological panning phage blue plaques, amplified phage were calculated output/ input ratio to assess the recovery of phage. Enzyme-linked immunosorbent assay (ELISA) to detect the binding of monoclonal phage, the phage DNA precipitation, centrifugation, agarose gel electrophoresis detection of DNA extract quality. Phage DNA and-96g III sequencing primers together to send Beijing Genomics Institute Co., Ltd., the dye tracer dideoxy automated sequencing. Application of peptide nucleic acid analog Blast system detects DNA similarity.
Part two The study of tgf-β1 phage model peptides on inhibiting keloid fibroblasts proliferation
Human TGF-β1 monoclonal antibody-coated plates, plus peptide library, were selected four, including enrichment of specific phage peptide analog. Limited by the Beijing Genomics Institute of the dideoxy dye tracer automated sequencing. Application of nucleic acid similarity comparison database (Blast System) test the similarity of DNA analog peptides. Total of four phage peptide analog, the next step in vitro test.
Identified through the pathology of keloid from training to obtain keloid fibroblasts. Application of tissue culture method to obtain keloid fibroblasts. Thiazolyl blue (MTT) assay the number of living cells to detect TGF-β1 on cell proliferation. A total of 13 groups:including the negative control group, the phage M13 group, TGF-β1 group and the control group and 1 analog peptide phage group 10 group.
Application Annexin V-FITC/PI apoptosis detection kit and flow cytometry apoptosis. A total of 5 groups:negative control group and 4 analog peptide phage group. Annexin V-FITC positive and PI-negative cells in that early stage of apoptosis, both of which are positive that the cells in the late stages of apoptosis, both of which are not negative as apoptotic cells.
Cell affinity peptide analog detection:A total of 13 groups:including the negative control group, control group M13 phage (1012 pfu/ml), TGF-β1 control group (5 mg/L) and 1 analog peptide group and phage group 10 group (1012 pfu/ ml). Immunofluorescence detection kit according to the operating instructions carefully. Human TGF-β1 monoclonal antibody (red) and M13 coat protein (green) as the primary antibody.
Real-time quantitative PCR of keloid fibroblast nuclear factorκB (NF-κB) and connective tissue growth factor (CTGF) expression were detected. A total of 13 groups:including the negative control group, the phage M13 group, TGF-β1 group and the control group and 1 analog peptide phage group 10 group. Application RNAiso reagent extracted epidermal cells of total RNA. Application PrimeScriptTM RT reverse transcription kit obtained cDNA, SYBR Premix Ex TaqTM II application for PCR reaction.
Reaction conditions:30 s,95℃; 95℃5 s,58℃31s (40 cycles). Primer sequence:
(1)β-actin:upstream:5 TGACGTGGACATCCGCAAAG 3', downstream:5 'CTGGAAGGTGGACAGCGAGG 3' (204 bp);
(2) NF-κB:upstream:5'CTGTAACTGCTGGACCCAAGG 3', downstream:5 'CTTTTTCCCGATCTCCCAGCT 3'(209 bp);
(3) CTGF:upstream:5 'CCTCTTCTGTGACTTCGGCTC 3', downstream:5 'GAACGTCCATGCTGCACAG 3'(188 bp).
Application of SPSS 16.0 software for statistical analysis. Result data to said single factor analysis of variance (one-way ANOVA) and Dunnet's test to analyze differences between the groups, with P<0.05 was considered statistically significant.
Results
1, TGF-betal related model peptides isolated from a phage display 12-mer peptide library
After 4 rounds of bio-panning, phage obtained and the production rate gradually increased the total, containing specific to obtain analog peptide phage enrichment, ELISA detection of TGF-β1 received standard-absorbance curve. The results obtained phage display screening has good binding activity, sequencing to obtain 10 to promote fibroblast proliferation and inhibition of cell proliferation or the base sequence. Blast through the detection system, in which an analog peptide (No.1) and TGF-β1 is similar to three analog peptides (No.2,5,8) and TGF-beta2 is similar to five analog peptides (No.3,68,10) and TGF-betaⅡreceptor similar to the three analog peptides (No.4,5,8) and TGF-β-inducible factor similar to an analog peptide (No.9) and NF-κB similar to 6 analog peptide (No.4-9) and cell division of the original activated protein kinase (mitogen-activated protein kinase, MAPK) is similar.
2, The study of tgf-β1 phage model peptides on inhibiting keloid fibroblasts proliferation
Detection at 570 nm absorbance value of A, MTT results showed that the negative control group A of 0.22 (two digits after the decimal point to retain control group M13 is 0.22), TGF-β1 group with different concentrations (0.05,0.5,5μg/L) a-values were 0.29,0.31,0.35, single-factor analysis of variance and Dunnet's method of analysis showed that TGF-β1 and the negative control group was statistically significant difference between the groups. TGF-β1 group can significantly contribute to keloid fibroblasts (P<0.01). Statistical analysis showed that the M13 phage negative control group and the control group, no significant difference between (P> 0.05), in the negative control group and the analog peptide phage group 5 and 6 between the groups, as well as the negative control group and the low concentration of (1010 pfu/ml) of section 7,10 analog peptide phage group no significant difference between groups (P> 0.05), but the negative control group and the other phage peptide analog difference between groups was statistically significant (P<0.05), simulation results suggest that four kinds of phage peptides (7 to 10 groups) can inhibit the proliferation of keloid fibroblasts.
The results of MTT to inhibit keloid fibroblast proliferation of the four phage peptide analog (7 to 10 group), further assessment of keloid fibroblasts to promote apoptosis. Relative to the negative control group, these four phage peptide analog (7 to 10 groups) to mild keloid promote apoptosis, can induce apoptosis in keloid fibroblasts in the late stage.
Immunofluorescence showed that TGF-β1 control group and 10 analog peptide phage group with a combination of keloid fibroblasts, but not the control group phage M13 and keloid fibroblasts combined.
Real-time PCR detection of TGF-β1 control group and the analog peptide phage group (1 to 10 groups) NF-κB and the expression of CTGF levels and negative control (expression set to 1) compared to, TGF-β1 group and 1 to 4 analog peptide phage group of NF-κB and increased expression of CTGF,5 to 6 analog peptide phage group did not change significantly, and the first 7 to 10 analog peptide phage group expression decreased. One expression of NF-κB is a negative control group were 0.28,0.26,0.46,0.30 times, CTGF expression is the negative control group were 0.26,0.60,0.34,0.17 times.
Conclusions
1, twelve from phage random peptide library can be panned to the TGF-β1 associated with the analog peptide phage.
2, Four kinds of phage peptide analog with a combination of keloid fibroblasts and keloid fibroblasts promote mild mild late apoptosis.
3, phage peptides may be simulated by regulating NF-κB and the expression of CTGF to regulate the proliferation of keloid fibroblasts.
引文
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