清开灵注射液动物过敏模型建立方法学考察

发布时间：2018-05-26 16:35

本文选题：清开灵注射液 + 过敏模型　；参考：《南方医科大学》2010年硕士论文

【摘要】： 目的：中药含有治疗疾病的各种有效成分,是我国传统医学的瑰宝,但由于传统制剂工艺的局限,严重阻碍了中药的临床应用。近年来，，随着现代制剂技术的发展,使得中药的质量和临床疗效得到显著的提高,尤其是中药注射液的出现更是开辟了中药制剂的全新局面。中药注射液的研制不仅继承了传统方剂疗效确切和价格低廉的优点,又显著提高了中药有效成分的生物利用度和达峰速度,很好的实现了临床中急重症的治疗,并得到了临床医生的广泛认同而迅速发展。但随着品种扩增和病患使用比例不断提升,与其相关的一系列药物不良反应出现的频次也不断攀升,严重者甚至发生休克、死亡,并导致了部分品种使用“叫停”的局面,清开灵注射液便是其中一种,其不良反应发生率在中药注射剂中排第3位。如何提高中药注射液的安全性、解决中药注射液重症不良反应的问题,已经引起了国家高度重视和关注。本课题的总体思路拟从清开灵注射液中过敏原的筛查入手,通过建立动物过敏模型,从模型中采集富含抗原-抗体复合物的血清作分离提取相关抗体的物质基础,利用免疫沉淀、电泳分离、质谱检测、相关数据库分析等技术,实现清开灵注射液过敏原物质的筛查。而本论文的主要工作是通过多种处理方式建立清开灵注射液动物过敏模型,为后续实验研究提供基础。方法：1清开灵注射液过敏原初步筛查药物中大分子类物质是常见的过敏原。初步考察清开灵注射液的大分子致敏物质：使用三氯乙酸(TCA)和乙醇提取清开灵注射液中的蛋白,然后用SDS-聚丙烯酰胺凝胶(SDS-PAGE)电泳检测蛋白；将清开灵注射液冷冻干燥后根据需要调整浓度,琼脂糖凝胶电泳检测核酸；利用高效液相色谱／质谱判断清开灵注射液的分子量范围。 2清开灵注射液、清开灵注射液+弗氏佐剂建立动物过敏模型由于Balb/c小鼠承受的注射剂量有限,清开灵注射液使用在正常成年人上的常规剂量体积较大,因此先把清开灵注射液冷冻干燥后调整浓度。Balb/c小鼠随机分成清开灵注射液实验组(QKL)、清开灵注射液+弗氏佐剂实验组(QKL+FCA)、卵白蛋白(Ovalbumin OVA)阳性组和生理盐水阴性组,每组6只。QKL组皮下多点注射0.3mL清开灵注射液,QKL+FCA(完全弗氏佐剂Freund's Complete Adjuvant)组皮下多点注射0.3mL清开灵注射液与完全弗氏佐剂比例为1：1的混合溶液(两组给予的清开灵注射液含量等于正常成年人的常规剂量),OVA组皮下多点注射0.3mg/mL的OVA0.3mL,第14d后QKL+FCA组皮下注射QKL+IFCA(不完全弗氏佐剂Freund's Incomplete Adjuvant), QKL、OVA组则相同的物质和途径加强免疫,生理盐水组则按相同处理方式给予等体积的生理盐水。加强免疫后第7d、14d、21d采血,分离血清,-20℃存储备用。血清中总IgE和特异性IgE分别使用酶联免疫吸附实验(Enzyme-linked immunosorbent assay ELISA)和大鼠被动皮肤过敏试验(Passive Cutaneous Anaphylaxis Reaction PCA)进行检测,判断模型建立。 3应用Mannich反应建立过敏模型清开灵注射液中小分子化合物可能是致敏原，但是小分子物质必须和大分子的载体偶联才能诱导免疫应答。本实验采用阳离子化牛血清白蛋白(Cationized Bovine Serum Albumin cBSA)作为载体蛋白,通过Mannich反应偶联清开灵注射液中小分子致敏原制备cBSA-清开灵注射液偶联物。偶联比例称取清开灵注射液冻干粉和cBSA,使cBSA-清开灵注射液的组成之比分别为1：1、1：2、1：3、1：5,混合均匀后加入37%的甲醛0.05mL,37℃孵育过夜。观察混合、滴加甲醛、孵育后沉淀的情况,离心取上清,用葡聚糖凝胶柱分离各组分,以相同方法处理清开灵注射液和cBSA做为对照组。以0.5mL为一管收集流出物,紫外分光光度计扫描每组分离出来的组分,对照清开灵注射液和cBSA主要成分流出时间和紫外吸收峰,判断偶联的情况。偶联物建立模型成功制备的偶联物用生理盐水溶解后以体积比1：1加入佐剂明矾作为致敏原。Balb/c小鼠随机分为QKL+cBSA偶联物实验组、卵白蛋白(0Vh)阳性组和生理盐水对照组。实验组皮下多点注射0.3mL偶联物与佐剂混合溶液(含0.05mg偶联物),OVA组给予0.3mg/mL的OVAO.3mL,生理盐水组给予等体积的生理盐水。每组于第14d后相同方法加强免疫,加强免疫后30min观察小鼠行为,眼后眦静脉采血检测组胺。再分别于加强免疫后第7d、14d、21d采血,分离血清用于检测总IgE,解剖小鼠取肺部组织浸泡于10%的甲醛中制作病理切片。 4 cBSA致敏的可能性考虑偶联后残留的cBSA可能对动物造成刺激,导致IgE升高,造成实验假阳性。因此用实验剂量(0.05mg)的cBSA对Balb/c小鼠进行刺激,通过总IgE和特异性IgE评价该剂量的cBSA对小鼠的影响程度。Balb/c小鼠随机分成4组,(1) cBSA组、生理盐水组；(2) cBSA+明矾、生理盐水组,每组6只。两种致敏方法：(1) cBSA组首次腹腔注射免疫小鼠,尾静脉加强免疫；(2) cBSA+明矾组皮下注射免疫小鼠,相同途径加强免疫；两组的对照组以相同方式给予等体积的生理盐水。每组均于加强免疫后第7d、14d、21d采血,分离血清,ELISA法检测总IgE, PCA法检测特异性IgE,判断该剂量的cBSA是否能刺激小鼠。结果：1清开灵注射液的TCA和乙醇提取物经SDS-PAGE-电泳检测,发现只有Marker出现明显条带,提取的物质没有出现蛋白条带；琼脂糖凝胶电泳在紫外下观察发现清开灵注射液没有出现核酸条带；高效液相色谱／质谱判断清开灵注射液的分子量范围在1—1.5KD之间。 2清开灵注射液、清开灵注射液+佐剂建立过敏模型使用清开灵注射液和清开灵注射液+佐剂皮下免疫小鼠,小鼠自发性活动减少,实验组与生理盐水对照组总IgE检测结果经统计学比较无统计学意义(P0.05),阳性组与生理盐水对照组有统计学意义(P0.05)。特异性IgE结果与总IgE结果一致,只有阳性组出现较大的蓝色斑点,说明只有OVA使小鼠过敏,总IgE升高,产生特异性IgE。 3制备偶联物不同比例的cBSA-清开灵注射液混合的过程中发现1：5组出现沉淀,其他比例组没有出现沉淀。加入37%的甲醛偶联剂后,1：5组出现较多沉淀,1：3组出现少量絮状沉淀,1：2和1：1组没有沉淀。反应后1：5组出现大量沉淀,沉淀沉入试管底部,1：2和1：3组出现絮状沉淀,1：1组没有沉淀。紫外图谱显示分子量大的cBSA在第5-6管内全部流出,收集的流出物在277nm波长处有最大吸收峰；清开灵注射液的分子量小,第5-6管没有组分被分离出来,第23管的流出物在275nm和314nm波长有最大吸收峰；1：1组和1：5组在第5或6管的流出物没有出现吸收峰,1：2组和1：3组的第5管流出物不仅在274nm波长处有最大吸收峰,而且与单纯cBSA相比,在313nm波长处也有最大吸收峰,说明cBSA与清开灵注射液中的半抗原偶联成新的物质。1：3组的吸光度A明显低于1：2组,所以认为1：2组的偶联率在4个比例中是最高。偶联物建立模型加强免疫后偶联物组小鼠出现瘙痒、立毛、扭体等过敏体症,总IgE结果说明3个时间点的血样的总IgE升高,与生理盐水组有统计学意义(P0.01),第14d的总IgE水平与OVA组比较无统计学意义(P0.05),推测这个时间点偶联物致敏总IgE水平与OVA的致敏效果相当。组胺结果显示血清样本稀释10倍后组胺水平与生理盐水组有统计学意义(P0.05)。病理切片显示偶联物组与生理盐水组比较,肺部受到明显损伤：肺泡融合、中性粒细胞浸润、肺部充血、气管壁有轻度破损。 4 cBSA致敏通过2种方法刺激小鼠后于第7d、14d、21d各时间采集的血清测得总IgE效价与生理盐水组比较均无统计学意义(P0.05), PCA实验中大鼠背部也没有出现蓝色斑点。表明该剂量的cBSA不会对小鼠造成刺激产生IgE。结论：清开灵注射液中不含有大分子蛋白、多肽和核酸。分子量范围在1—1.5KD之间,说明清开灵注射液的各组分是小分子化合物。单纯使用清开灵注射液、清开灵注射液+佐剂无法建立动物过敏模型,因为小分子量的物质无法直接诱导免疫应答。通过应用Mannich反应制备载体蛋白与清开灵注射液的偶联物,致敏小鼠后的小鼠出现瘙痒、立毛、扭体等过敏体征,总IgE、组胺升高,肺部病理切片显示肺泡融合、充血、气管壁破损,说明偶联物成功使Balb/c小鼠。利用Mannich反应建立清开灵注射液动物过敏模型是可行的。
[Abstract]:Objective: Traditional Chinese medicine contains various effective components in the treatment of diseases, which is a gem of traditional medicine in our country. However, due to the limitations of the traditional preparation technology, the clinical application of traditional Chinese medicine is seriously hindered. In recent years, with the development of modern preparation technology, the quality and clinical efficacy of traditional Chinese medicine have been greatly improved, especially the appearance of traditional Chinese medicine injection. It has opened up a new situation of traditional Chinese medicine preparation. The development of traditional Chinese medicine injection not only inherits the advantages of the traditional prescription, but also significantly improves the bioavailability and peak speed of the effective components of traditional Chinese medicine. It has well realized the treatment of acute severe clinical treatment, and has been widely recognized by clinicians and developed rapidly. However, with the increasing proportion of the varieties and the increasing proportion of the patients, the frequency of a series of adverse drug reactions associated with them is also rising, and the serious people even have shock and death, which leads to the use of "call stop" in some varieties. Row third. How to improve the safety of traditional Chinese medicine injection and solve the problem of severe adverse reaction of traditional Chinese medicine injection has aroused great attention and concern of the country. The general idea of this subject is to start from the screening of allergen in Qingkailing injection and establish an animal allergy model to collect antigenic antibody complex from the model. The main work of this paper is to establish the allergen of Qingkailing Injection by using immunoprecipitation, electrophoretic separation, mass spectrometric detection and related database analysis. The main work of this paper is to establish the animal allergy model of Qingkailing injection through various treatments, and provide the follow-up experimental research. Basics.
Methods: 1 Qingkailing injection allergens were used to screen the large molecular substances in the drug as common allergens. The large molecular sensitizing substances of Qingkailing injection were preliminarily investigated by using three chloroacetic acid (TCA) and ethanol to extract the protein in Qingkailing injection and then SDS- polyacrylic acid gel (SDS-PAGE) electrophoresis was used to detect the protein; The molecular weight range of Qingkailing injection was determined by high performance liquid chromatography / mass spectrometry (HPLC / MS).
2 Qingkailing injection, Qingkailing injection and Freund's adjuvant set up an animal allergy model because the injection dose of Balb/c mice was limited. The conventional dose of Qingkailing injection was large in normal adults. So the concentration of Qingkailing injection after freeze drying was first randomly divided into Qingkailing Injection in.Balb/c mice. Test group (QKL), Qingkailing injection + Freund adjuvant experimental group (QKL+FCA), ovalbumin (Ovalbumin OVA) positive group and physiological saline negative group, each group of 6.QKL groups were injected subcutaneously 0.3mL Qingkailing injection, QKL+FCA (complete Freund's Freund's Complete Adjuvant) group subcutaneous injection 0.3mL Qingkailing injection and complete Freund Zuo The mixture of 1:1 (two groups of Qingkailing injection content was equal to the normal dose of normal adults), group OVA was injected subcutaneously with 0.3mg/mL OVA0.3mL, and QKL+FCA group 14d after 14d was subcutaneously injected with QKL+IFCA (incomplete Freund's adjuvant Freund's Incomplete Adjuvant), QKL, and OVA group was immune to the same substance and way. The normal saline group was given equal volume of normal saline in the same way. After immunization, 7d, 14d, 21d were collected, serum was collected, serum was separated and stored at -20 C. The serum total IgE and specific IgE were used in enzyme linked immunosorbent assay (Enzyme-linked immunosorbent assay ELISA) and rat passive skin allergy test (Passive Cutaneous). Laxis Reaction PCA) to detect and establish the model.
3 the application of Mannich reaction to the establishment of an allergy model, Qingkailing injection, may be a sensitizing agent, but small molecules must be coupled with the carrier of large molecules to induce immune response. This experiment uses cationic bovine serum albumin (Cationized Bovine Serum Albumin cBSA) as a carrier protein and Mannich reaction couple. CBSA- Qingkailing injection conjugate was prepared from small molecule allergens of Qingkailing injection.
The coupling ratio is called Qingkailing injection freeze-dried powder and cBSA, and the composition ratio of cBSA- Qingkailing injection is 1:1,1:2,1:3,1:5 respectively. After mixing and evenly adding 37% formaldehyde 0.05mL and incubating for the night at 37 C, the mixture, the drop of formaldehyde, the precipitation after incubation, the centrifuge supernatant, and the Sephadex column are used to separate the components, and the same is the same as the glucan gel column. Methods Qingkailing injection and cBSA were treated as the control group. 0.5mL was used as a collection of exudates, and the UV spectrophotometer was used to scan each group. The outflow time and ultraviolet absorption peak of the main components of Qingkailing injection and cBSA were compared with the UV absorption peaks.
The coupling substance was successfully prepared by the coupling establishment model. After dissolved in the saline solution, the coupling agent was added to the alum as the allergenic alum as the sensitized.Balb/c mice and randomly divided into the QKL+cBSA coupling experimental group, the ovalbumin (0Vh) positive group and the normal saline control group. The experimental group was subcutaneously injected with the mixed solution of the 0.3mL coupling and the adjuvant (containing 0.05mg pairs). Group OVA was given 0.3mg/mL OVAO.3mL and saline group was given equal volume of normal saline. Each group was immunized with the same method after 14d. After immunization, 30min observation of mice behavior and posterior canthus vein blood collection were used to detect histamine. Then the blood was collected after strengthening immunization, 7d, 14d, 21d, and isolated serum was used to detect total IgE and dissected the lung of mice. The tissue was soaked in formaldehyde of 10% to make pathological sections.
The possibility of 4 cBSA sensitization is that the residual cBSA may cause stimulation to the animal after coupling, resulting in the increase of IgE and the false positive of the experiment. Therefore, the Balb/c mice were stimulated with the cBSA of the experimental dose (0.05mg). The effect of cBSA on mice was evaluated by the total IgE and specific IgE, and.Balb/c mice were randomly divided into 4 groups, (1) cBSA group. Normal saline group (2) cBSA+ alum, saline group, each group of 6. Two kinds of sensitization methods: (1) group cBSA for the first time intraperitoneal injection of immune mice, the tail vein to strengthen the immune; (2) cBSA+ alum group subcutaneous injection immunized mice, the same way to strengthen the immunization; the two groups of the same way to give the same volume of saline. Each group is added. After strong immunization, blood samples were collected from 7d, 14d and 21d. The total IgE was detected by ELISA. The specific IgE was detected by PCA method, and whether the dose cBSA could stimulate mice was detected.
Results: 1 the TCA and ethanol extracts of Qingkailing injection were detected by SDS-PAGE- electrophoresis. Only the obvious strip was found in Marker, and the extracted substance did not appear in the strip. The agarose gel electrophoresis found that the Qingkailing injection did not appear in the purplish band. The molecular weight range is between 1 and 1.5KD.
2 Qingkailing injection, Qingkailing injection + adjuvant set up an allergy model using Qingkailing injection and Qingkailing injection + adjuvant subcutaneous immunization mice, the mice spontaneous activity decreased, the experimental group and the normal saline control group total IgE detection results were not statistically significant (P0.05), positive group and normal saline control group have statistics Learning significance (P0.05). The results of specific IgE were in accordance with the total IgE results. Only the positive group had larger blue spots, indicating that only OVA was allergic to the mice, the total IgE increased, and the specific IgE. was produced.
3 in the process of mixing cBSA- Qingkailing Injection in different proportions of the coupling, it was found that the 1:5 group appeared precipitation, the other proportion group did not appear precipitation. After adding 37% formaldehyde coupling agent, the 1:5 group appeared more precipitation, the 1:3 group appeared a small amount of floc precipitation, 1:2 and 1:1 group did not precipitate. After the reverse response, a large amount of precipitation, precipitation and precipitation occurred in the 1:5 group. At the bottom of the test tube, the 1:2 and 1:3 groups were flocculated, and the 1:1 group had no precipitation. The UV atlas showed that the large molecular weight of cBSA was all outflow in the 5-6 tube, and the collected exudates had the maximum absorption peak at the 277nm wave length. The molecular weight of the Qingkailing injection was small, the 5-6 tube had no components separated, and the twenty-third tube exudate was in 275nm and 314n. The M wavelength has the maximum absorption peak, and there is no absorption peak in the fifth or 6 tubes of the 1:1 and 1:5 groups. The fifth tube exudates of the 1:2 and 1:3 groups not only have the maximum absorption peak at the 274nm wave length, but also have the maximum absorption peak at the 313nm wavelength compared with the pure cBSA, indicating that the coupling of the cBSA and the antigen in the Qingkailing injection is new. The absorbance A of substance.1:3 group was significantly lower than that of 1:2 group, so the coupling rate of 1:2 group was the highest in 4 proportions.
The model of the conjugate was established to strengthen the mice with pruritus, erect hair, and torsional body, and the total IgE results showed that the total IgE of the blood samples at 3 time points increased, and was statistically significant with the saline group (P0.01). The total IgE level of 14d was not statistically significant (P0.05) compared with that of the OVA group (P0.05), and that the total Ig was sensitized to the coupling substance at this time point. The E level was similar to that of OVA. Histamine results showed that the histamine level after 10 times dilution of serum samples was statistically significant (P0.05). Pathological sections showed that the lung was significantly damaged by the coupling group and the saline group: pulmonary alveolus fusion, neutrophils infiltration, pulmonary congestion, and mild damage to the trachea wall.
4 cBSA sensitization was made by 2 methods to stimulate the mice after 7d, 14d, and 21d, and the total IgE titer was not statistically significant compared with that of the normal saline group (P0.05). There was no blue spot in the back of the rat in the PCA experiment. It showed that the dose of cBSA did not produce the IgE. in mice.
Conclusion: Qingkailing injection does not contain large molecular protein, polypeptide and nucleic acid. The molecular weight range is between 1 and 1.5KD, indicating that the components of Qingkailing injection are small molecular compounds. Pure Kailing injection and Qingkailing injection + adjuvant can not establish an animal allergy model because small molecular weight substances can not be induced directly. The Mannich reaction was used to prepare the coupling between the carrier protein and the Qingkailing injection. After sensitizing the mice, the mice appeared pruritus, erect hair, twisting body and other allergic signs. The total IgE, histamine increased, pulmonary pathological sections showed alveolar fusion, congestion, and damaged trachea wall, and the coupling substance successfully made Balb/c mice. The use of Mannich reaction to Jian Liqing The animal allergy model of Kailing injection is feasible.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R-332

【相似文献】