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(P>0.05),阳性组与生理盐水对照组有统计学意义(P<0.05)。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(P<0.01),第14d的总IgE水平与OVA组比较无统计学意义(P>0.05),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(P<0.05)。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(P>0.05), 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。
Objects:
With the development of extraction, separation and purification of traditional Chinese medicine by new technology, preferable Chinese materia medica preparation which has good therapeutic action now can be required. Kinds of pharmaceutical dosage form of traditional Chinese medicine have been out-of-date, and is not meet medical needs. In order to change the present situation, many conventional dosage forms are improved into injectable dosage forms, such as Qingkailing injection, Shuanghuanglian injection, Yuxingcao injection and so on. These new injections which were base on ancient prescription are not only cheap, but also can be used for mergencies and acute disease. But the incidence of side effects happened during injection process is high, which include allergy, cardinal symptoms, even drug-induced allergic shock and death. Since the prevalence of adverse event and intercurrent illnesses continue to rise,FDA had decided to stop using some traditional Chinese medicine, such as Qingkailing injection and Yuxingcao injection. In view of the above-mentioned facts, we have to find a way to solve the problems existing in traditional Chinese medicine injection to improve the security and extend clinical application of Chinese medical injection. Multiple data-sources, methods, analyses or theories should be combined to overcome the bias that comes from single informants, single-methods or single theory studies. The global property of Chinese medicine should be also paid more attention. In this study we designed to investigate the antigen in the murine allergic with Qingkailing injection.
Methods:
1 Big weight molecule is the most common antigen in the medication, for instance:protein. First of all, we inspect the probability of sensitization of big weight molecule. SDS-PAGE gel electrophoresis detects the proteins which precipitate by trichloroacetic acid (TCA) and ethanol. Meanwhile, nucleic acid was be detected by agarose gel electrophoresis. Apply the HPLC/MS to estimate the molecular weight range of Qingkailing injection.
2 6-week-old female BALB/c mice were randomly divided into 4 groups: Qingkailing injection(QKL), Qingkailing injection with Freund's adjuvant (QKL+FCA), Ovalbumin(OVA)group and physiological saline group,6 mice in each group. Qingkailing injection were dissolved in sterilized saline and emulsified with an equal volume of Freund's adjuvant, The emulsion solutions were injected 0.3mL of subcutaneously into BALB/C mice in QKL+FCA group; BALB/c mice of QKL and OVA group received subcutaneous injection of 0.3mL Qingkailing injection and OVA (0.1mg) respectively. Booster injections were carried out on the 14th day after the primary doses. QKL+FCA group followed subcutaneous injection with the same quantity of immunogen emulsified with incomplete Freund's adjuvant at 14 days intervals. QKL and OVA group were received the some doses by subcutaneous injection with Qingkailing injection and OVA. Saline group were administered the same volume of physiological saline. Mice were bled from the retro-orbital plexus just at 7,14,21days after Booster injections. After collecting, sera were allowed to repose for 30 min at 37℃and thus slightly centrifuged in a bench centrifuge during 10 min at 4℃and the clean material stored at-20℃until use.
The samples were taken to obtain plasma for analyzing the IgE by enzyme-linked immuno sorbent assay(ELISA) and Passive Cutaneous Anaphylaxis Reaction (PCA).
3 Hapten is a class of small molecules and is immunogenicity obtained only by coupling with carrier protein. The Mannich reaction is a familiar an amino-alkylated reaction in which compounds containing active hydrogen, such as esters, phenols, ketones etc., can be condensed with formaldehyde and an amido in weak acidity. The carrier protein is bovine serum albumin that has been modified by substituting anionic carboxyl groups with cationic aminoethyl-amide groups. When used as an immunogen, this cationized BSA (cBSA) stimulates a much higher antibody response than native BSA
The preparation of QKL-cBSA base on the Mannich reaction. Briefly, a quantity of QKL (2,4,6,10 mg) and 2mg of cBSA were dissolved in physiological saline and conjugation buffer (0.1 mol/1 MES pH 4.8) respectively. After dropwise addition of QKL solution into the protein solution, the mixture was stirred gently, then 50μl of formaldehyde were added into the mixture and immediately stirred gently for 24 h at 37℃. Apply the entire hapten-carrier reaction mixture directly onto the top disc of the desalting column while collecting the flow-through in a test tube. When the sample has completely entered the column and the flow has stopped, transfer the column to a new tube and add 0.5 ml of the purification buffer.Finally the sample was detected by ultraviolet spectrophotometer.
4 BALB/c mice were randomly divided into 3 groups:QKL-cBSA group, OVA group and physiological saline group,6 mice in each group. Groups of QKL-cBSA obtained QKL-cBSA conjugate was emulsified with an equal volume of Alum adjuvant. OVA group received subcutaneous injection of 0.3mL OVA (contain O.lmg OVA).Saline group were administered the same volume of physiological saline. Booster injections were carried out according to the primary doses on the 14th day after the first injection.Mice were bled from the retro-orbital plexus just at 30min and 7,14,21,days after Booster injections. After collecting, sera were allowed to repose for 30 min at 37℃and thus slightly centrifuged in a bench centrifuge during 10 min at 4℃and the clean material stored at-20℃until use. The samples were taken to obtain plasma for analyzing the IgE and histamine by ELISA:
In consideration of the cBSA as carrier protein which molecular weight is 68KD, if cBSA mix with carrier-hapten, it also can induce hypersusceptibility in mice. So we use cBSA as antigen to stimulate mice in dose of experiment by two ways: six-week-old female BALB/c mice intraperitoneal injection and boosted by vein of trail with the 0.05mg cBSA on the 14th day after the first injection. Aother group was administrated by subcutaneous injection with 0.05mg cBSA was emulsified with an equal volume of Alum adjuvant. Booster injections were carried out according to the primary doses on the 14th day after the first injection,too. Mice were bled from the retro-orbital plexus just at 7,14,21, days after Booster injections.The samples were taken to obtain plasma for analyzing the IgE by ELISA and PCA.
Results:
1 Protein content in medicine used to induce antibodies synthesis was estimated according the method of Greg. Protein profile of QKL was evaluated by polyacrylamide gel electrophoresis. According to the results, The poorest protein fraction extracted by TCA and ethanol has been not exhibits in previous works. nucleic acid was analyzed by gelose gel eelctrophoresis. As observed under the UV, There is lack of nucleic acid in QKL.The range of molecular weight is 1-1.5KD.
2 None of the fractions induced antibodies level increases when mice received QKL and QKL+FCA by subcutaneous route and thus, did not develop allergy. Nonetheless, anti-sera of mice sensitized with OVA by subcutaneous route displayed considerable immunological response while QKL and QKL+FCA did not. IgE level augmented consistently against OVA tested Possible allergenic effects induced by QKL、QKL+FCA and OVA were investigated by PCA in rats and mice to estimate IgE synthesis, respectively. The QKL、QKL+FCA were not able to induce synthesis of IgE by subcutaneous route.However, synthesis of IgE was estimated to occur blue points in animals that reCeived OVA by subcutaneous stimulation. As a rule, the OVA induce immunological response and animals receiving OVA via subcutaneous were sensitive to produce IgE.
3 Two important parameters in preparing conjugates are the hapten utilized ratio and the molar ratio between hapten and carrier protein. The former is known to estimate the efficiency of hapten utilization, which assists in the choice of a suitable initial molar ratio; the latter play an important role, with regard to the degree of immune reactivity, solubility of the final conjugate, and the sensitivity of the immunoassay based on such antibodies elicited by the corresponding conjugate. In the current study,2,4,6,10 mg of Qingkailing were mixed with 2mg of cBSA in conjugation buffer to prepare the conjugates. In summary, QKL coupled with cationized protein cBSA has been demonstrated by UV-Vis. The maximum absorbing wavelength in cBSA UV-Vis spectra, which are characteristic of protein, were slightly blue-shifted from 280 to 277 nm compared with BSA. This might be explained in terms of increased cBSA polarity due to induction of excessive cationic groups, amino-ethylaminegroups, into BSA. The spectra of QKL-cBSA conjugates showed a broad characteristic absorption band around 313 nm, attributed to QKL, and another band around 277 nm, indicating a combination band of 277 nm and 313 nm due to QKL and cBSA, respectively, but it was interesting that the absorbency of the ratio of 1:2 is higher than others When the initial molar ratio of QKL to cBSA was increased, there was a marked precipitate for the conjugate molar ratio to increase, This implies that the best molar ratio is 1:2
4 Both the total IgE and specific IgE levels of mice which stimulated with QKL, QKL+FCA, and cBSA shown that sensitizer do not make the mice allergy. But the total IgE and histamine of cBSA-QKL conjugate group which had statistically significant effect vs physiological saline group (P<0.05). According to the results, the conjugates of cBSA-QKL can provoke allergy by subcutaneous route.
Conclusion:
QKL is composed of salts and many other low molecular substances some of such already described. The very low concentration of such molecules in QKL could certainly explain absence of detectable immunological response. Using a method based on Mannich-type principles. The coupling effects were investigated with different initial molar ratios of QKL to cBSA. The conjugate molar ratio was 1:2 The cationized proteins and their conjugates were identified by UV-Vis spectra, which showed the characteristic bands of cBSA and QKL, respectively. After BALB/c mice were immunized with QKL-cBSA, a quicker immunological response and a similar sensitivity of antisera against QKL were observed, compared with immunization by QKL and QKL+_adjuvant. This suggests that the Mannich-type reaction might be an alternative method of preparation for QKL-cBSA. As a rule, the conjugate induce immunological response and animals receiving conjugate samples via subcutaneous were more sensitive to produce IgE and histamine
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