發(fā)布時間：2018-05-26 16:35

  本文選題：清開靈注射液 + 過敏模型��； 參考：《南方醫(yī)科大學》2010年碩士論文

【摘要】： 目的：中藥含有治療疾病的各種有效成分,是我國傳統(tǒng)醫(yī)學的瑰寶,但由于傳統(tǒng)制劑工藝的局限,嚴重阻礙了中藥的臨床應用。近年來，，隨著現(xiàn)代制劑技術(shù)的發(fā)展,使得中藥的質(zhì)量和臨床療效得到顯著的提高,尤其是中藥注射液的出現(xiàn)更是開辟了中藥制劑的全新局面。中藥注射液的研制不僅繼承了傳統(tǒng)方劑療效確切和價格低廉的優(yōu)點,又顯著提高了中藥有效成分的生物利用度和達峰速度,很好的實現(xiàn)了臨床中急重癥的治療,并得到了臨床醫(yī)生的廣泛認同而迅速發(fā)展。但隨著品種擴增和病患使用比例不斷提升,與其相關(guān)的一系列藥物不良反應出現(xiàn)的頻次也不斷攀升,嚴重者甚至發(fā)生休克、死亡,并導致了部分品種使用“叫停”的局面,清開靈注射液便是其中一種,其不良反應發(fā)生率在中藥注射劑中排第3位。如何提高中藥注射液的安全性、解決中藥注射液重癥不良反應的問題,已經(jīng)引起了國家高度重視和關(guān)注。本課題的總體思路擬從清開靈注射液中過敏原的篩查入手,通過建立動物過敏模型,從模型中采集富含抗原-抗體復合物的血清作分離提取相關(guān)抗體的物質(zhì)基礎(chǔ),利用免疫沉淀、電泳分離、質(zhì)譜檢測、相關(guān)數(shù)據(jù)庫分析等技術(shù),實現(xiàn)清開靈注射液過敏原物質(zhì)的篩查。而本論文的主要工作是通過多種處理方式建立清開靈注射液動物過敏模型,為后續(xù)實驗研究提供基礎(chǔ)。 方法：1清開靈注射液過敏原初步篩查藥物中大分子類物質(zhì)是常見的過敏原。初步考察清開靈注射液的大分子致敏物質(zhì)：使用三氯乙酸(TCA)和乙醇提取清開靈注射液中的蛋白,然后用SDS-聚丙烯酰胺凝膠(SDS-PAGE)電泳檢測蛋白；將清開靈注射液冷凍干燥后根據(jù)需要調(diào)整濃度,瓊脂糖凝膠電泳檢測核酸；利用高效液相色譜／質(zhì)譜判斷清開靈注射液的分子量范圍。 2清開靈注射液、清開靈注射液+弗氏佐劑建立動物過敏模型由于Balb/c小鼠承受的注射劑量有限,清開靈注射液使用在正常成年人上的常規(guī)劑量體積較大,因此先把清開靈注射液冷凍干燥后調(diào)整濃度。Balb/c小鼠隨機分成清開靈注射液實驗組(QKL)、清開靈注射液+弗氏佐劑實驗組(QKL+FCA)、卵白蛋白(Ovalbumin OVA)陽性組和生理鹽水陰性組,每組6只。QKL組皮下多點注射0.3mL清開靈注射液,QKL+FCA(完全弗氏佐劑Freund's Complete Adjuvant)組皮下多點注射0.3mL清開靈注射液與完全弗氏佐劑比例為1：1的混合溶液(兩組給予的清開靈注射液含量等于正常成年人的常規(guī)劑量),OVA組皮下多點注射0.3mg/mL的OVA0.3mL,第14d后QKL+FCA組皮下注射QKL+IFCA(不完全弗氏佐劑Freund's Incomplete Adjuvant), QKL、OVA組則相同的物質(zhì)和途徑加強免疫,生理鹽水組則按相同處理方式給予等體積的生理鹽水。加強免疫后第7d、14d、21d采血,分離血清,-20℃存儲備用。血清中總IgE和特異性IgE分別使用酶聯(lián)免疫吸附實驗(Enzyme-linked immunosorbent assay ELISA)和大鼠被動皮膚過敏試驗(Passive Cutaneous Anaphylaxis Reaction PCA)進行檢測,判斷模型建立。 3應用Mannich反應建立過敏模型清開靈注射液中小分子化合物可能是致敏原，但是小分子物質(zhì)必須和大分子的載體偶聯(lián)才能誘導免疫應答。本實驗采用陽離子化牛血清白蛋白(Cationized Bovine Serum Albumin cBSA)作為載體蛋白,通過Mannich反應偶聯(lián)清開靈注射液中小分子致敏原制備cBSA-清開靈注射液偶聯(lián)物。 偶聯(lián)比例稱取清開靈注射液凍干粉和cBSA,使cBSA-清開靈注射液的組成之比分別為1：1、1：2、1：3、1：5,混合均勻后加入37%的甲醛0.05mL,37℃孵育過夜。觀察混合、滴加甲醛、孵育后沉淀的情況,離心取上清,用葡聚糖凝膠柱分離各組分,以相同方法處理清開靈注射液和cBSA做為對照組。以0.5mL為一管收集流出物,紫外分光光度計掃描每組分離出來的組分,對照清開靈注射液和cBSA主要成分流出時間和紫外吸收峰,判斷偶聯(lián)的情況。 偶聯(lián)物建立模型成功制備的偶聯(lián)物用生理鹽水溶解后以體積比1：1加入佐劑明礬作為致敏原。Balb/c小鼠隨機分為QKL+cBSA偶聯(lián)物實驗組、卵白蛋白(0Vh)陽性組和生理鹽水對照組。實驗組皮下多點注射0.3mL偶聯(lián)物與佐劑混合溶液(含0.05mg偶聯(lián)物),OVA組給予0.3mg/mL的OVAO.3mL,生理鹽水組給予等體積的生理鹽水。每組于第14d后相同方法加強免疫,加強免疫后30min觀察小鼠行為,眼后眥靜脈采血檢測組胺。再分別于加強免疫后第7d、14d、21d采血,分離血清用于檢測總IgE,解剖小鼠取肺部組織浸泡于10%的甲醛中制作病理切片。 4 cBSA致敏的可能性考慮偶聯(lián)后殘留的cBSA可能對動物造成刺激,導致IgE升高,造成實驗假陽性。因此用實驗劑量(0.05mg)的cBSA對Balb/c小鼠進行刺激,通過總IgE和特異性IgE評價該劑量的cBSA對小鼠的影響程度。Balb/c小鼠隨機分成4組,(1) cBSA組、生理鹽水組；(2) cBSA+明礬、生理鹽水組,每組6只。兩種致敏方法：(1) cBSA組首次腹腔注射免疫小鼠,尾靜脈加強免疫；(2) cBSA+明礬組皮下注射免疫小鼠,相同途徑加強免疫；兩組的對照組以相同方式給予等體積的生理鹽水。每組均于加強免疫后第7d、14d、21d采血,分離血清,ELISA法檢測總IgE, PCA法檢測特異性IgE,判斷該劑量的cBSA是否能刺激小鼠。 結(jié)果：1清開靈注射液的TCA和乙醇提取物經(jīng)SDS-PAGE-電泳檢測,發(fā)現(xiàn)只有Marker出現(xiàn)明顯條帶,提取的物質(zhì)沒有出現(xiàn)蛋白條帶；瓊脂糖凝膠電泳在紫外下觀察發(fā)現(xiàn)清開靈注射液沒有出現(xiàn)核酸條帶；高效液相色譜／質(zhì)譜判斷清開靈注射液的分子量范圍在1—1.5KD之間。 2清開靈注射液、清開靈注射液+佐劑建立過敏模型使用清開靈注射液和清開靈注射液+佐劑皮下免疫小鼠,小鼠自發(fā)性活動減少,實驗組與生理鹽水對照組總IgE檢測結(jié)果經(jīng)統(tǒng)計學比較無統(tǒng)計學意義(P0.05),陽性組與生理鹽水對照組有統(tǒng)計學意義(P0.05)。特異性IgE結(jié)果與總IgE結(jié)果一致,只有陽性組出現(xiàn)較大的藍色斑點,說明只有OVA使小鼠過敏,總IgE升高,產(chǎn)生特異性IgE。 3制備偶聯(lián)物不同比例的cBSA-清開靈注射液混合的過程中發(fā)現(xiàn)1：5組出現(xiàn)沉淀,其他比例組沒有出現(xiàn)沉淀。加入37%的甲醛偶聯(lián)劑后,1：5組出現(xiàn)較多沉淀,1：3組出現(xiàn)少量絮狀沉淀,1：2和1：1組沒有沉淀。反應后1：5組出現(xiàn)大量沉淀,沉淀沉入試管底部,1：2和1：3組出現(xiàn)絮狀沉淀,1：1組沒有沉淀。紫外圖譜顯示分子量大的cBSA在第5-6管內(nèi)全部流出,收集的流出物在277nm波長處有最大吸收峰；清開靈注射液的分子量小,第5-6管沒有組分被分離出來,第23管的流出物在275nm和314nm波長有最大吸收峰；1：1組和1：5組在第5或6管的流出物沒有出現(xiàn)吸收峰,1：2組和1：3組的第5管流出物不僅在274nm波長處有最大吸收峰,而且與單純cBSA相比,在313nm波長處也有最大吸收峰,說明cBSA與清開靈注射液中的半抗原偶聯(lián)成新的物質(zhì)。1：3組的吸光度A明顯低于1：2組,所以認為1：2組的偶聯(lián)率在4個比例中是最高。 偶聯(lián)物建立模型加強免疫后偶聯(lián)物組小鼠出現(xiàn)瘙癢、立毛、扭體等過敏體癥,總IgE結(jié)果說明3個時間點的血樣的總IgE升高,與生理鹽水組有統(tǒng)計學意義(P0.01),第14d的總IgE水平與OVA組比較無統(tǒng)計學意義(P0.05),推測這個時間點偶聯(lián)物致敏總IgE水平與OVA的致敏效果相當。組胺結(jié)果顯示血清樣本稀釋10倍后組胺水平與生理鹽水組有統(tǒng)計學意義(P0.05)。病理切片顯示偶聯(lián)物組與生理鹽水組比較,肺部受到明顯損傷：肺泡融合、中性粒細胞浸潤、肺部充血、氣管壁有輕度破損。 4 cBSA致敏通過2種方法刺激小鼠后于第7d、14d、21d各時間采集的血清測得總IgE效價與生理鹽水組比較均無統(tǒng)計學意義(P0.05), PCA實驗中大鼠背部也沒有出現(xiàn)藍色斑點。表明該劑量的cBSA不會對小鼠造成刺激產(chǎn)生IgE。 結(jié)論：清開靈注射液中不含有大分子蛋白、多肽和核酸。分子量范圍在1—1.5KD之間,說明清開靈注射液的各組分是小分子化合物。單純使用清開靈注射液、清開靈注射液+佐劑無法建立動物過敏模型,因為小分子量的物質(zhì)無法直接誘導免疫應答。通過應用Mannich反應制備載體蛋白與清開靈注射液的偶聯(lián)物,致敏小鼠后的小鼠出現(xiàn)瘙癢、立毛、扭體等過敏體征,總IgE、組胺升高,肺部病理切片顯示肺泡融合、充血、氣管壁破損,說明偶聯(lián)物成功使Balb/c小鼠。利用Mannich反應建立清開靈注射液動物過敏模型是可行的。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R-332

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