技术领域technical field
本发明属于生物技术领域，具体涉及一种调节FGFR1活性的多肽R1-P1，及其在制备缓解或治疗骨性关节炎疾病药物中的应用。The invention belongs to the field of biotechnology, and in particular relates to a polypeptide R1-P1 regulating FGFR1 activity and its application in the preparation of medicines for alleviating or treating osteoarthritis.
背景技术Background technique
骨性关节炎(osteoarthritis，OA)是关节软骨退化、变性和继发的软骨增生，骨化为主要病理变化，以关节疼痛、活动功能障碍为主要临床表现的一种疾病，是严重影响国民健康和生活质量的重大公共卫生问题。目前关节软骨损伤退变尚无有效的治疗药物，临床上主要应用透明质酸钠和氨基葡萄糖来营养润滑关节从而缓解症状，然而无法从根本上抑制软骨退变的进程。关节炎发展到后期，常采取关节镜下关节清扫术和关节置换手术。然而无论是药物治疗还是手术治疗，均存在不同程度的缺陷，无法满足患者的医疗需求。因此，迫切需要发展新的关节损伤退变的治疗药物。Osteoarthritis (OA) is a disease characterized by the degeneration, degeneration and secondary cartilage hyperplasia of articular cartilage, ossification as the main pathological change, and joint pain and activity dysfunction as the main clinical manifestations. It is a disease that seriously affects national health. major public health issues and quality of life. At present, there is no effective treatment for articular cartilage degeneration. Sodium hyaluronate and glucosamine are mainly used clinically to nourish and lubricate joints to relieve symptoms, but they cannot fundamentally inhibit the process of cartilage degeneration. Arthritis develops to the late stage, often take arthroscopic joint dissection and joint replacement surgery. However, whether it is drug therapy or surgical treatment, there are varying degrees of defects, which cannot meet the medical needs of patients. Therefore, there is an urgent need to develop new drugs for the treatment of joint damage and degeneration.
目前药物靶点的筛选主要集中在细胞膜受体和代谢相关的酶类，如IL-1受体、前列腺素E2合酶(PGES)、TNF-α、Bmp7等。FGFR(成纤维细胞生长因子受体)是一类具有自身磷酸化活性的跨膜酪氨酸激酶受体，与其相应配体FGFs(成纤维细胞生长因子)结合调节细胞增殖、分化、迁移和存活等，在组织器官发育及损伤修复过程中发挥重要作用。目前已发现4种FGFR，即FGFR1、FGFR2、FGFR3和FGFR4。研究小组前期利用tamoxifen可诱导关节软骨特异性敲除FGFR1小鼠模型，通过分析3种关节炎模型(衰老模型、抗原诱导的关节炎模型(Antigen-induced Arthritis，AIA)、创伤诱导的关节不稳定模型(Destabilization ofthe Medial Meniscus，DMM))的关节病理及量化评分，结果显示关节软骨细胞敲除FGFR1能够延缓衰老、AIA模型导致的关节软骨基质蛋白多糖PGs的丢失并减轻DMM模型导致的关节软骨结构破坏。FGFR3表达升高、MMP-13表达下降可能是FGFR1敲除保护关节软骨的潜在机制。上述研究结果提示靶向抑制FGFR-1可能是缓解或治疗关节软骨损伤和退变的新策略。因此，以FGFR1为干预靶点，筛选FGFR1特异结合多肽，研究其在缓解或治疗骨性关节炎疾病中的功能，是一种可行的思路。At present, the screening of drug targets mainly focuses on cell membrane receptors and metabolism-related enzymes, such as IL-1 receptor, prostaglandin E2 synthase (PGES), TNF-α, Bmp7, etc. FGFR (fibroblast growth factor receptor) is a class of transmembrane tyrosine kinase receptors with autophosphorylation activity, which binds to its corresponding ligand FGFs (fibroblast growth factor) to regulate cell proliferation, differentiation, migration and survival etc., play an important role in the process of tissue organ development and injury repair. Four kinds of FGFRs have been found so far, namely FGFR1, FGFR2, FGFR3 and FGFR4. The research team used tamoxifen to induce articular cartilage-specific knockout FGFR1 mouse model in the early stage, and analyzed three arthritis models (aging model, antigen-induced arthritis model (Antigen-induced Arthritis, AIA), trauma-induced joint instability Model (Destabilization of the Medial Meniscus, DMM)) joint pathology and quantitative score, the results show that knocking out FGFR1 in articular chondrocytes can delay aging, the loss of articular cartilage matrix proteoglycan PGs caused by the AIA model and alleviate the articular cartilage structure caused by the DMM model destroy. Increased expression of FGFR3 and decreased expression of MMP-13 may be the underlying mechanism of FGFR1 knockout to protect articular cartilage. The above findings suggest that targeted inhibition of FGFR-1 may be a new strategy to alleviate or treat articular cartilage damage and degeneration. Therefore, it is a feasible idea to use FGFR1 as an intervention target to screen FGFR1-specific binding polypeptides and study their functions in alleviating or treating osteoarthritis diseases.
发明内容Contents of the invention
有鉴于此，本发明的目的在于提供一种调节FGFR1活性的多肽，其具有分子量小、制备简单、质量可控、免疫原性低等优点；本发明还提供了其在制备缓解或治疗骨性关节炎疾病药物中的应用。In view of this, the purpose of the present invention is to provide a polypeptide that regulates the activity of FGFR1, which has the advantages of small molecular weight, simple preparation, controllable quality, and low immunogenicity; Use in drugs for arthritic diseases.
本发明采取的技术方案如下：The technical scheme that the present invention takes is as follows:
1、调节FGFR1活性的多肽R1-P1，氨基酸序列为Gly-Pro-Pro-Asp-Trp-His-Trp-Lys-Ala-Met-Thr-His。1. The polypeptide R1-P1 regulating the activity of FGFR1, the amino acid sequence is Gly-Pro-Pro-Asp-Trp-His-Trp-Lys-Ala-Met-Thr-His.
2、上述调节FGFR1活性的多肽R1-P1在制备缓解或治疗骨性关节炎药物中的应用。2. The application of the above-mentioned polypeptide R1-P1 regulating FGFR1 activity in the preparation of medicaments for alleviating or treating osteoarthritis.
本发明的有益效果在于：本发明提供的R1-P1多肽能够与FGFR1特异结合，并且该多肽具有分子量小、制备简单、质量可控、免疫原性低等优点；另外，本发明通过建立小鼠膝关节炎模型并注射R1-P1肽发现，R1-P1肽可以延缓DMM模型小鼠的关节炎进程，提示其在制备缓解或治疗骨性关节炎药物中具有良好的应用价值。The beneficial effect of the present invention is that: the R1-P1 polypeptide provided by the present invention can specifically bind to FGFR1, and the polypeptide has the advantages of small molecular weight, simple preparation, controllable quality, and low immunogenicity; Knee arthritis model and injection of R1-P1 peptide found that R1-P1 peptide can delay the arthritis process in DMM model mice, suggesting that it has good application value in the preparation of drugs for relieving or treating osteoarthritis.
附图说明Description of drawings
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图：In order to make the purpose, technical scheme and beneficial effect of the present invention clearer, the present invention provides the following drawings:
图1为ELISA法验证R1-P1肽与FGFR1的结合情况。Fig. 1 is the verification of the combination of R1-P1 peptide and FGFR1 by ELISA method.
图2为荧光素酶报告基因法检测10μM R1-P1肽对FGFR1下游信号p-ERK水平的影响，**表示与空白对照组比，P<0.01；***表示与空白对照组比，P<0.001。Figure 2 shows the effect of 10 μM R1-P1 peptide on the level of p-ERK, the downstream signal of FGFR1, detected by the luciferase reporter gene method. ** indicates the comparison with the blank control group, P<0.01; *** indicates the comparison with the blank control group, P <0.001.
图3为Western blot法检10μM R1-P1肽对FGFR1主要下游信号通路MAPK中p-ERK的影响；其中，1泳道为空白对照组，2泳道为PD166866抑制剂组，3泳道为R1-P1肽组。Figure 3 shows the effect of 10 μM R1-P1 peptide on p-ERK in MAPK, the main downstream signaling pathway of FGFR1, detected by Western blot; lane 1 is the blank control group, lane 2 is the PD166866 inhibitor group, and lane 3 is the R1-P1 peptide Group.
图4为DMM模型小鼠膝关节组织切片藏红固绿染色结果。Figure 4 shows the results of saffron-fast green staining of knee joint tissue sections of DMM model mice.
图5为DMM模型小鼠膝关节组织切片关节软骨基质丢失评分结果。Fig. 5 is the result of score of loss of articular cartilage matrix in knee joint tissue slices of DMM model mice.
图6为DMM模型小鼠膝关节组织切片关节软骨退变评分结果。Fig. 6 is the result of scoring the articular cartilage degeneration of the knee joint tissue section of the DMM model mouse.
具体实施方式Detailed ways
下面对本发明的优选实施例进行详细的描述。实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。Preferred embodiments of the present invention are described in detail below. For the experimental methods that do not specify specific conditions in the examples, usually follow the conventional conditions or the conditions suggested by the manufacturer.
优选实施例中使用的FGFR1、鼠抗FGFR1抗体均购自美国Sigma公司，噬菌体12肽库筛选试剂盒(包括ER2738菌种)购自美国New England Lab公司，pcDNA3.1-Myc空载体由本课题组保存，Myc-FGFR1质粒由美国华盛顿大学David Ornitz教授惠赠。The FGFR1 used in the preferred embodiment and the mouse anti-FGFR1 antibody were all purchased from Sigma Company of the United States, the phage 12 peptide library screening kit (including ER2738 strain) was purchased from New England Lab Company of the United States, and the pcDNA3.1-Myc empty vector was provided by our research group Preserved, the Myc-FGFR1 plasmid was kindly donated by Professor David Ornitz, Washington University, USA.
实施例1、从噬菌体12肽库中淘选能与FGFR1特异性结合的多肽Example 1. Panning a polypeptide that can specifically bind to FGFR1 from a phage 12 peptide library
1、噬菌体12肽库的淘选1. Panning of phage 12 peptide library
用浓度为20μg/mL的FGFR1溶液(以浓度为0.1mol/L、pH为8.6的碳酸氢钠溶液为溶剂)150μL包被酶标板，4℃包被过夜，弃去包被液，满孔加入浓度为5mg/mL的BSA溶液(以浓度为0.1mol/L、pH为8.6的碳酸氢钠溶液为溶剂)，37℃封闭1h，弃去封闭液，用TBST(即含有Tween-20的TBS缓冲液，第一轮淘选时Tween-20的浓度为1mL/L，第二轮和第三轮淘选时Tween-20的浓度提高至5mL/L)洗涤6次，投入噬菌体(第一轮淘选时投入噬菌体12肽库原液与TBST的混合液，第二轮和第三轮淘选时投入前一轮淘选出的特异性结合的噬菌体)，室温下温和震荡孵育1h，弃去非特异性结合的噬菌体，用TBST洗涤10次，加入浓度为0.2mol/L、pH为2.2的甘氨酸-盐酸缓冲液，室温下温和震荡8min洗脱特异性结合的噬菌体，吸取洗脱液，加入浓度为1mol/L、pH为9.1的Tris-HCl缓冲液150μL中和，测定滴度，备用；Coat the microtiter plate with 150 μL of FGFR1 solution with a concentration of 20 μg/mL (sodium bicarbonate solution with a concentration of 0.1 mol/L and a pH of 8.6 as a solvent), coat at 4°C overnight, discard the coating solution, and fill the wells Add BSA solution with a concentration of 5 mg/mL (sodium bicarbonate solution with a concentration of 0.1 mol/L and a pH of 8.6 as a solvent), block at 37°C for 1 h, discard the blocking solution, and use TBST (that is, TBS containing Tween-20 buffer, the concentration of Tween-20 during the first round of panning was 1mL/L, and the concentration of Tween-20 was increased to 5mL/L during the second round and the third round of panning), washed 6 times, and dropped into phage (first round The mixture of phage 12 peptide library stock solution and TBST was put into the panning, and the specific binding phage obtained from the previous round of panning were put into the second and third rounds of panning), incubated for 1 hour with gentle shaking at room temperature, and discarded nonspecific phages. The heterosexually bound phages were washed 10 times with TBST, and the glycine-hydrochloric acid buffer solution with a concentration of 0.2 mol/L and a pH of 2.2 was added to elute the specifically bound phages by gentle shaking at room temperature for 8 min. Neutralize with 150 μL of 1mol/L Tris-HCl buffer solution with a pH of 9.1, measure the titer, and set aside;
将ER2738菌接种至LB培养基20mL中，37℃剧烈震荡培养至对数中期(OD600值约0.5)，加入前述用Tris-HCl缓冲液中和后的噬菌体洗脱液100μL，37℃震荡培养4.5h，4℃、转速10000rpm离心10min，吸取含有噬菌体的上清液，加入相当于上清液1/6体积的PEG/NaCl溶液(由浓度为200g/L的PEG8000和浓度为2.5mol/L的NaCl组成，下同)，4℃静置过夜沉淀噬菌体，4℃、转速10000rpm离心15min，弃上清液，用TBS缓冲液重悬噬菌体沉淀，再离心取上清液，加入相当于上清液1/6体积的PEG/NaCl溶液，冰浴沉淀噬菌体90min，离心弃上清液，用含有浓度为0.2g/L的叠氮钠的TBS缓冲液1mL重悬噬菌体沉淀，再离心取上清液，加入相当于上清液1/6体积的PEG/NaCl溶液，冰浴沉淀噬菌体90min，离心弃上清液，用含有浓度为0.2g/L的叠氮钠的TBS缓冲液200μL重悬噬菌体沉淀，再离心取上清液，测定滴度，作为下一轮淘选的投入噬菌体；Inoculate ER2738 bacteria into 20 mL of LB medium, culture with vigorous shaking at 37°C until the mid-logarithmic phase (OD600 value is about 0.5), add 100 μL of the phage eluate neutralized with Tris-HCl buffer, and culture with shaking at 37°C for 4.5 h, centrifuge at 4°C at 10000rpm for 10min, absorb the supernatant containing phage, add PEG/NaCl solution equivalent to 1/6 volume of the supernatant (200g/L of PEG8000 and 2.5mol/L of PEG NaCl composition, the same below), let stand overnight at 4°C to precipitate the phage, centrifuge at 10,000rpm at 4°C for 15min, discard the supernatant, resuspend the phage pellet with TBS buffer, centrifuge again to get the supernatant, and add the equivalent supernatant 1/6 volume of PEG/NaCl solution, precipitate phage in ice bath for 90min, centrifuge to discard the supernatant, resuspend the phage pellet with 1mL of TBS buffer containing 0.2g/L sodium azide, and then centrifuge to get the supernatant , add PEG/NaCl solution equivalent to 1/6 volume of the supernatant, precipitate the phage in an ice bath for 90 minutes, centrifuge and discard the supernatant, and resuspend the phage precipitation with 200 μL of TBS buffer containing sodium azide at a concentration of 0.2 g/L , then centrifuge to take the supernatant, measure the titer, and use it as the input phage for the next round of panning;
重复上述步骤共进行三轮淘选，富集能与FGFR1特异性结合的噬菌体；第三轮淘选出的噬菌体不再用ER2738菌进行扩增。The above steps were repeated for a total of three rounds of panning to enrich the phages that can specifically bind to FGFR1; the phages selected in the third round of panning were no longer amplified with ER2738 bacteria.
各轮淘选的噬菌体回收率见表1。The recovery rates of phage in each round of panning are shown in Table 1.
表1.各轮淘选过程中噬菌体的回收率Table 1. The recovery rate of phage during each round of panning
由表1可知，经过三轮淘选后，与FGFR1特异性结合的噬菌体的回收率逐渐增大，表明与FGFR1具有较高亲和性的噬菌体被有效富集。It can be seen from Table 1 that after three rounds of panning, the recovery rate of phages specifically binding to FGFR1 gradually increased, indicating that phages with higher affinity to FGFR1 were effectively enriched.
2、噬菌体的DNA提取和测序2. DNA extraction and sequencing of phage
将ER2738菌接种至LB培养基中，37℃剧烈震荡培养至对数中期，分取1mL置培养管中，加入从第三轮淘选出的噬菌体的滴度测定平板上随机挑取的噬菌体单克隆，37℃剧烈震荡培养4.5h，离心，取含有噬菌体的上清液500μL，加入PEG/NaCl溶液200μL，室温沉淀噬菌体10min，离心弃上清液，加入碘化物缓冲液100μL重悬噬菌体沉淀，再加入乙醇250μL，室温孵育10min使噬菌体DNA沉淀而大多数噬菌体蛋白保留在溶液中，离心弃上清液，DNA沉淀用浓度为700mL/L的乙醇洗涤，真空干燥后重悬于TE缓冲液30μL中。取该溶液5μL，委托上海嘉根生物科技有限公司进行测序。Inoculate ER2738 bacteria into LB medium, culture with vigorous shaking at 37°C until mid-logarithmic phase, divide 1mL into a culture tube, and add a single phage randomly picked from the phage titer assay plate selected from the third round of panning. Cloning, cultured with vigorous shaking at 37°C for 4.5 hours, centrifuged, took 500 μL of supernatant containing phage, added 200 μL of PEG/NaCl solution, precipitated phage at room temperature for 10 min, discarded supernatant by centrifugation, added 100 μL of iodide buffer to resuspend phage pellet, Then add 250 μL of ethanol and incubate at room temperature for 10 minutes to precipitate the phage DNA while most of the phage proteins remain in the solution, centrifuge to discard the supernatant, wash the DNA precipitate with ethanol with a concentration of 700 mL/L, dry it in vacuum and resuspend in 30 μL of TE buffer middle. Take 5 μL of this solution and entrust Shanghai Jiagen Biotechnology Co., Ltd. to perform sequencing.
本实施例按上述方法从第三轮淘选出的噬菌体中随机挑取23个噬菌体单克隆进行DNA提取和测序，筛选得到3种与FGFR1具有较高亲和力的12肽，挑选其中重复率最高的多肽R1-P1(氨基酸序列如SEQ ID No.1所示)进行研究，结果发现R1-P1肽能够特异性结合FGFR1。In this example, 23 single phage clones were randomly selected from the phages selected in the third round of panning according to the above method for DNA extraction and sequencing, and three 12-peptides with high affinity to FGFR1 were screened, and the one with the highest repetition rate was selected. Peptide R1-P1 (amino acid sequence shown in SEQ ID No.1) was studied, and it was found that the R1-P1 peptide can specifically bind to FGFR1.
实施例2、人工合成能与FGFR1特异性结合的多肽Example 2. Artificial synthesis of polypeptides that can specifically bind to FGFR1
根据R1-P1肽的氨基酸序列，委托上海强耀生物科技有限公司采用标准Fmoc方案固相合成R1-P1肽。经测定，合成多肽纯度高于95％，温度-70℃保存。According to the amino acid sequence of the R1-P1 peptide, Shanghai Qiangyao Biotechnology Co., Ltd. was entrusted to use the standard Fmoc protocol to synthesize the R1-P1 peptide in solid phase. It has been determined that the purity of the synthetic polypeptide is higher than 95%, and it is stored at -70°C.
实施例3、ELISA法验证合成多肽与FGFR1的结合Example 3, ELISA method to verify the combination of synthetic polypeptide and FGFR1
在96孔酶标板中，每孔加入浓度为1mmol/μL的R1-P1肽溶液20μL和包被缓冲液(取碳酸钠0.3g、碳酸氢钠0.58g和叠氮钠0.04g，加水溶解，调节pH至9.6，再加水稀释至200mL，即得)80μL，同时设置空白对照组(不加入R1-P1肽)，4℃包被过夜，弃去溶液，满孔加入浓度为50g/L的小牛血清溶液(以pH为7.4的PBS为溶剂)，37℃封闭60min，弃去溶液，用PBST(含有浓度为0.5mL/L的Tween-20的PBS，pH为7.4)洗涤3次，加入浓度为100μg/mL的FGFR1溶液30μL和pH为7.4的PBS 70μL，37℃孵育1h，用PBST洗涤3次，加入1:500稀释的鼠抗FGFR1抗体(以质量百分浓度为1％的BSA溶液为稀释溶剂)100μL，37℃孵育1h，弃去溶液，用PBST洗涤5次，加入HRP标记的兔抗鼠IgG 100μL，37℃孵育30min，弃去溶液，用PBST洗涤5次，加入四甲基联苯二胺-过氧化氢尿素溶液100μL，37℃避光显色10min，加入浓度为2mol/L的硫酸溶液终止反应，在波长450nm处测定OD450值，结果以3个复孔的均值表示。结果如图1所示，R1-P1肽与FGFR1有较高的结合率。In a 96-well ELISA plate, add 20 μL of R1-P1 peptide solution with a concentration of 1 mmol/μL and coating buffer (take 0.3 g of sodium carbonate, 0.58 g of sodium bicarbonate and 0.04 g of sodium azide, dissolve in water, Adjust the pH to 9.6, then add water to dilute to 200mL to obtain) 80μL, set up a blank control group (without adding R1-P1 peptide), coat overnight at 4°C, discard the solution, and add a concentration of 50g/L small Bovine serum solution (PBS with pH 7.4 as solvent), blocked at 37°C for 60 min, discarded the solution, washed 3 times with PBST (PBS containing Tween-20 at a concentration of 0.5 mL/L, pH 7.4), and added a concentration of 30 μL of 100 μg/mL FGFR1 solution and 70 μL of PBS with a pH of 7.4 were incubated at 37°C for 1 h, washed 3 times with PBST, and a 1:500 dilution of mouse anti-FGFR1 antibody was added (1% BSA solution with a mass percent concentration of Dilute solvent) 100 μL, incubate at 37°C for 1 h, discard the solution, wash 5 times with PBST, add 100 μL of HRP-labeled rabbit anti-mouse IgG, incubate at 37°C for 30 min, discard the solution, wash 5 times with PBST, add tetramethyl-linked 100 μL of phenylenediamine-hydrogen peroxide urea solution was developed at 37°C in the dark for 10 minutes, and a sulfuric acid solution with a concentration of 2 mol/L was added to terminate the reaction. The OD450 value was measured at a wavelength of 450 nm, and the results were expressed as the average of three replicate wells. The results are shown in Figure 1, the R1-P1 peptide has a higher binding rate to FGFR1.
实施例4、合成多肽对FGFR1活性的调节作用检测Example 4, detection of the regulation effect of synthetic polypeptide on FGFR1 activity
1、荧光素酶报告基因法检测合成多肽对FGFR1主要下游信号通路MAPK的激活1. The luciferase reporter gene method was used to detect the activation of synthetic peptides on the main downstream signaling pathway MAPK of FGFR1
转染前24h，将293T细胞接种至12孔培养板，待长至80％融合后，用高效真核转染试剂VigoFect同时转染0.1ng Myc-FGFR1质粒(以pcDNA3.1-Myc质粒作对照)和ERK通路报告基因质粒(50ng PFR-luc，50ng P-EIK-1和5ng内参PRL-TK)，3h后换液，24h后加入10μMR1-P1肽处理，同时设置空白对照组(不加入R1-P1肽)和PD166866对照组(以FGF抑制剂PD166866替代R1-P1肽)，处理24h后弃去培养基，用预冷的1×PBS漂洗细胞2次，每孔加入300μL 1×Universal Lysis Buffer(ULB)，置-80℃低温冰箱冰冻裂解1h，再室温旋涡振荡30min至细胞完全裂解，测定荧光素酶活性，记录萤火虫荧光素酶发光单位(Rlus1)和海肾荧光素酶发光单位(Rlus2)，以Rlus1/Rlus2的相对比值作为报告基因的转录相对活性值，结果以3个复孔的均值±标准差表示。结果如图2所示，10μM R1-P1肽能够显著抑制FGFR1下游p-ERK的活性，提示R1-P1肽可能是通过调节p-ERK的活性来调节细胞的生理过程。24 hours before transfection, 293T cells were inoculated into a 12-well culture plate, and after growing to 80% confluence, 0.1 ng of Myc-FGFR1 plasmid was simultaneously transfected with the high-efficiency eukaryotic transfection reagent VigoFect (the pcDNA3.1-Myc plasmid was used as a control ) and ERK pathway reporter gene plasmids (50ng PFR-luc, 50ng P-EIK-1 and 5ng internal reference PRL-TK), the medium was changed after 3h, and 10μMR1-P1 peptide was added after 24h for treatment, and a blank control group (without adding R1 -P1 peptide) and PD166866 control group (replacing R1-P1 peptide with FGF inhibitor PD166866), discard the medium after 24 hours of treatment, rinse the cells twice with pre-cooled 1×PBS, add 300 μL 1×Universal Lysis Buffer to each well (ULB), freeze and lyse in a -80°C low-temperature refrigerator for 1 hour, then vortex at room temperature for 30 minutes until the cells are completely lysed, measure luciferase activity, and record firefly luciferase luminescence units (Rlus1) and Renilla luciferase luminescence units (Rlus2 ), the relative ratio of Rlus1/Rlus2 was used as the relative transcriptional activity value of the reporter gene, and the results were expressed as the mean ± standard deviation of 3 duplicate wells. The results are shown in Figure 2, 10 μM R1-P1 peptide can significantly inhibit the activity of p-ERK downstream of FGFR1, suggesting that R1-P1 peptide may regulate the physiological process of cells by regulating the activity of p-ERK.
2、Western blot法检测合成多肽R1-P1对FGFR1主要下游信号通路MAPK中p-ERK的激活2. The activation of p-ERK in MAPK, the main downstream signaling pathway of FGFR1, by the synthetic peptide R1-P1 detected by Western blot
转染前24h，将293T细胞接种至35mm培养皿，待长至80％融合后，用高效真核转染试剂VigoFect同时转染0.1ng Myc-FGFR1质粒(以pcDNA3.1-Myc质粒作对照)，3h后换液，24h后加入10μM R1-P1肽处理，同时设置空白对照组(不加入R1-P1肽)和PD166866对照组(以FGF抑制剂PD166866替代R1-P1肽)，处理24h，收集细胞，用细胞裂解液吹打裂解细胞，于冰上收集细胞匀浆液，超声破碎仪破碎细胞(参数设定：放大倍数35％，总时间20s，每次5s，间隔3s)。蛋白样品加入等体积的2×SDS-PAGE loading buffer上样缓冲液，煮沸5min，室温冷却后进行SDS-PAGE(采用浓度为50g/L的浓缩胶、浓度为90g/L的分离胶，电泳参数为80V 20min、120V 80min)，电泳结束后，将蛋白电转印(恒流3000mA 60min)至PVDF膜上，用8％脱脂奶粉TBST(含20mM Tris-HCl，137mM NaCl，0.1％Tween-20)于37℃摇床封闭1h，再加入1:1000稀释的鼠抗FGFR1一抗，4℃过夜，弃去溶液，用TBST洗涤5次，加入HRP标记的兔抗鼠二抗，37℃孵育1h，弃去溶液，用TBST洗涤5次，ECL化学发光液(美国Pierce公司,A、B液1:1混合)曝光、显影。结果如图3所示，与对照组相比，PD166866抑制剂组完全抑制ERK的磷酸化水平，R1-P1肽组ERK的磷酸化水平显著性降低，表明R1-P1肽能够显著抑制FGFR1下游p-ERK的活性。24 hours before transfection, 293T cells were inoculated into 35mm culture dishes, and after growing to 80% confluence, 0.1ng Myc-FGFR1 plasmid was simultaneously transfected with high-efficiency eukaryotic transfection reagent VigoFect (the pcDNA3.1-Myc plasmid was used as a control) , change the medium after 3h, add 10μM R1-P1 peptide to treat after 24h, set up blank control group (without adding R1-P1 peptide) and PD166866 control group (use FGF inhibitor PD166866 to replace R1-P1 peptide), treat for 24h, collect Cells were blown and lysed with a cell lysate, the cell homogenate was collected on ice, and the cells were disrupted by a sonicator (parameter settings: magnification 35%, total time 20s, each time 5s, interval 3s). Add an equal volume of 2×SDS-PAGE loading buffer to the protein sample, boil for 5 minutes, and then perform SDS-PAGE after cooling at room temperature (use a stacking gel with a concentration of 50g/L, a separation gel with a concentration of 90g/L, electrophoresis parameters 80V 20min, 120V 80min), after electrophoresis, protein electrotransfer (constant current 3000mA 60min) to PVDF membrane, with 8% skimmed milk powder TBST (containing 20mM Tris-HCl, 137mM NaCl, 0.1% Tween-20) Shake at 37°C for 1 hour, then add 1:1000 diluted mouse anti-FGFR1 primary antibody, overnight at 4°C, discard the solution, wash 5 times with TBST, add HRP-labeled rabbit anti-mouse secondary antibody, incubate at 37°C for 1 hour, discard Remove the solution, wash 5 times with TBST, expose and develop with ECL chemiluminescence solution (Pierce Company, USA, A and B solutions 1:1 mixed). The results are shown in Figure 3. Compared with the control group, the PD166866 inhibitor group completely inhibited the phosphorylation level of ERK, and the phosphorylation level of ERK in the R1-P1 peptide group was significantly reduced, indicating that the R1-P1 peptide can significantly inhibit the downstream expression of FGFR1. - Activity of ERK.
实施例5、合成多肽对小鼠膝关节炎模型的影响Embodiment 5, the influence of synthetic polypeptide on mouse knee arthritis model
1、内侧半月板不稳定(Destabilization of the medial meniscus，DMM)模型建立1. Destabilization of the medial meniscus (DMM) model establishment
采用显微外科手术实施DMM手术构建小鼠膝关节关节炎模型。手术分为DMM手术组和假手术Sham组，各组分别设置对照组。手术主要步骤如下：DMM手术组为将小鼠麻醉固定、消毒后，打开小鼠右侧膝关节，钝性分离软组织，用显微器械切断内侧半月板平台韧带，带线缝合针缝合关节囊，关闭关节腔，最后缝合皮肤；假手术Sham组只是打开小鼠右侧膝关节，钝性分离软组织，不切断内侧半月板平台韧带，缝合皮肤。术中注意避免损伤关节软骨，术后不固定手术肢体，笼内自由活动，腹腔注射青霉素预防感染。A mouse model of knee arthritis was established by microsurgery with DMM. The operation was divided into DMM operation group and sham operation Sham group, each group was set up as a control group. The main steps of the operation are as follows: in the DMM operation group, after the mice were anesthetized, fixed, and disinfected, the right knee joint of the mice was opened, the soft tissue was bluntly separated, the medial meniscus platform ligament was cut off with microscopic instruments, and the joint capsule was sutured with suture needles. The joint cavity was closed, and the skin was finally sutured; in the sham group, the right knee joint of the mice was only opened, the soft tissue was bluntly separated, and the medial meniscal plateau ligament was not cut, and the skin was sutured. During the operation, care should be taken to avoid damage to the articular cartilage. After the operation, the operated limbs were not fixed, and they could move freely in the cage. Penicillin was injected intraperitoneally to prevent infection.
2、关节腔内注射R1-P1肽2. Intra-articular injection of R1-P1 peptide
DMM模型手术后1周DMM手术组和假手术Sham组分别向关节腔内注射R1-P1肽，其各自的对照组则注射生理盐水，一周一次，连续8周，最后同时取材。One week after the operation of the DMM model, the DMM operation group and the sham operation Sham group were injected with R1-P1 peptide into the joint cavity, and their respective control groups were injected with normal saline, once a week for 8 consecutive weeks, and finally samples were collected at the same time.
注射具体方法如下：用生理盐水将R1-P1肽稀释成1μg/μL(1mM母液)，将小鼠麻醉固定、消毒后，用显微刀片切开膝关节处皮肤，暴露关节腔，按照10μg/只的量取10μLR1-P1肽稀释液注射在关节腔中，同时按照相同的方法向对照组小鼠关节腔内注射10μL生理盐水，每周一次，连续8周。The specific injection method is as follows: Dilute the R1-P1 peptide to 1 μg/μL (1mM mother solution) with normal saline, anesthetize, fix and disinfect the mice, cut the skin at the knee joint with a microblade to expose the joint cavity, 10 μL of R1-P1 peptide dilution was injected into the joint cavity of each mouse, and 10 μL of normal saline was injected into the joint cavity of the control group mice in the same way, once a week for 8 consecutive weeks.
3、关节软骨损伤退变病理评分3. Pathological score of articular cartilage damage and degeneration
小鼠关节取材后，4％PFA固定、甲酸(快速脱钙液)脱钙、石蜡包埋切片。小鼠关节的切片有两种方式，即冠状切和矢状切，此处采用矢状切的方式。矢状切片时从内侧半月板开始切片，间隔相应的位置捞片，保证每个个体之间尽量在同样的位置捞片，4个标本/片，约25张片子(关节面出现软组织，表示已经到达切片的终点位置)。每个标本间隔选择同样位置的片子10张，进行番红O-固绿染色(番红O能使蛋白聚糖染成红色，固绿能使矿化区域染成绿色)，通过IPP软件拍照，采用关节炎评分标准进行关节软骨退变评分与基质丢失评分(选取整个关节中退变最严重的片子进行评分，并且至少两人进行双盲评分，此两种评分方法均是分值越高代表关节炎损伤退变越严重)，具体评分标准见表2和表3；与空白对照组相比，*P<0.05，**P<0.001。After the mouse joints were collected, they were fixed with 4% PFA, decalcified with formic acid (quick decalcification solution), and embedded in paraffin. There are two ways to slice mouse joints, coronal section and sagittal section, and sagittal section is used here. When slicing sagittally, slice from the medial meniscus, take slices at corresponding positions, and ensure that slices are taken at the same position as far as possible between each individual, 4 specimens/slice, about 25 slices (soft tissue on the articular surface indicates that it has been reaches the end position of the slice). Select 10 slices at the same position for each specimen, and perform Safranin O-Fast Green staining (Safranin O can stain proteoglycans red, and Fast Green can stain mineralized areas green), and take pictures with IPP software. Score articular cartilage degeneration and matrix loss using the arthritis scoring standard (choose the slice with the most severe degeneration in the entire joint for scoring, and at least two people perform double-blind scoring, and the higher the score of the two scoring methods, the higher the score of the joint The more serious the degeneration of inflammatory damage), the specific scoring standards are shown in Table 2 and Table 3; compared with the blank control group, *P<0.05, **P<0.001.
表2关节软骨退变评分Table 2 Articular cartilage degeneration score
表3基质丢失评分Table 3 Matrix loss score
藏红固绿染色和评分统计结果如图4、5、6所示，相比注射生理盐水的对照组，DMM手术后注射R1-P1肽的小鼠关节软骨退变和基质丢失程度显著性降低，表明R1-P1肽可以延缓DMM模型小鼠的关节炎进程。Saffron fast green staining and score statistical results are shown in Figures 4, 5, and 6. Compared with the control group injected with normal saline, the degree of articular cartilage degeneration and matrix loss in mice injected with R1-P1 peptide after DMM surgery was significantly reduced , indicating that the R1-P1 peptide can delay the progression of arthritis in DMM model mice.
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that it can be described in terms of form and Various changes may be made in the details without departing from the scope of the invention defined by the claims.
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