摘要
目的:初步研制一种用于紧急预防和辅助治疗甲型副伤寒的新型生物制剂,并探索其制备工艺。方法:(1)体内免疫法制备甲型副伤寒特异性转移因子(PA-STF):制备甲型副伤寒沙门菌(SPA)免疫原,采取皮下多点注射免疫山羊,免疫成功后分别取脾脏和淋巴结制备PA-STF。(2)体外免疫法制备PA-STF:采用SPA体外免疫脾细胞技术,探索PHA刺激的合适剂量、免疫原最佳浓度及最佳作用时间、免疫羊脾细胞最适培养时间,按优化条件培养羊脾细胞,制备PA-STF。(3)PA-STF的理化性质检定:采用紫外分光光度计进行多波长扫描,以苔黑酚法和改良Lowry法分别检测PA-STF的核糖及多肽含量,并进行无菌试验、热原质试验和安全试验等。(4) PA-STF的免疫学活性检定:取昆明小鼠,随机分成实验1组、实验2组、对照1组、对照2组和NS组,分别以体内法制备的PA-STF、体外法制备的PA-STF、体内法制备的非特异性转移因子(nTF)、体外法制备的nTF、生理盐水灌胃,采用淋巴细胞增殖试验、白细胞粘附抑制试验(LAIT)、吞噬细胞吞噬功能试验、吞噬细胞杀菌功能试验和免疫保护性试验等检定PA-STF的免疫学活性。免疫保护性试验中,SPA攻击后第4天仍然存活的小鼠,取5只观察攻击后2周时的一般情况,剩余小鼠在攻击后第7天处死,剪取3cm的回肠用4%甲醛固定后,HE染色,镜检,观察肠粘膜的病理学改变。结果:(1)以淋巴结制备的PA-STF的多肽浓度为1.83±0.17mg/ml,核糖浓度为0.572±0.024mg/ml,以脾脏制备的PA-STF的多肽浓度为1.59±0.22 mg/ml,核糖浓度为0.493±0.015mg/ml,两者相比,多肽之间及核糖之间浓度没有显著差异(P＞0.05)。(2) 100μg/ml的PHA与脾细胞培养6h后,加入0.2ng/ml的SPA免疫原,继续培养至96h条件下制备的PA-STF的多肽和核糖的含量,明显高于其他剂量、时间等条件下制备的PA-STF(P＜0.05)。(3)两种方法制备的PA-STF的氨基酸、多肽、核糖的含量无显著性差异(P＞0.05),无菌试验均为阴性,热原质试验和安全试验均合格。(4)两种方法制备的PA-STF在多肽浓度为0.60mg/ml时,对淋巴细胞的刺激指数(SI)明显高于其它浓度时的SI(P＜0.05);两实验组的LAIR均高于两对照组,差异有统计学意义(P＜0.05);两实验组之间比较,LAIR无显著性差异(P＞0.05);吞噬细胞的吞噬功能和杀菌功能试验中,实验组与对照组之间以及两实验组之间比较,吞噬率和杀菌率均无显著性差异(P＞0.05),各组吞噬率与杀菌率的相关系数均大于0.8;免疫保护性试验中,两实验组分别与两对照组比较,实验组小鼠存活率均明显高于对照组(P＜0.05),两实验组之间差异无统计学意义(P＞0.05)。实验组存活小鼠2周时一般情况明显好于其它组,且病理学结果显示小鼠回肠粘膜的炎性程度较其它组轻。结论:(1)体内免疫法成功制备PA-STF,脾脏和淋巴结均可作为制备原料;体外免疫法成功制备PA-STF,以100μg/ml的PHA与脾细胞共同培养6h,然后加入浓度为0.2ng/ml的PA免疫原,第96h终止培养为最佳制备条件。(2)两种方法制备的PA-STF口服制剂的理化性质均符合《中国药典》(2005版)的要求标准。(3)两种方法制备的PA-STF口服制剂均可以增强小鼠的非特异性免疫应答,能够转移针对SPA的特异性免疫效应。
Objective: To prepare a new kind of biological agent initially, which will be used to urgent prevention and adjuvant therapy for paratyphi a ; and to explore the technique of preparation. Methods: (1) preparation of paratyphi a specific factor(PA-STF) in vivo. Prepared the SPA immunogen, immunized goats subcutaneouly, took their spleens and lymph node after successful immunization and prepared PA-STF.(2) Preparation of PA-STF in vitro. With the technique of immunizing spleen cells in vitro,explored the best dosage of PHA, more proper PA immunogen concentration and acting time, and the more adequate cultivation time. And then, cultured spleen cells under the best condition.(3)Standardization of physicochemical property of PA-STF.Firstly, scaned the PA-STF with multi-wavelength using ultraviolet spectrophotometer, determined the content of polypeptide and ribose with orcinol assay and modified Lowry assay respectively,and then sterility test, pyrogen test and safty test were carried out.(4) Standardization of the immunological actvity of PA-STF.Took 420 KM mice, divided them into 5 groups,that is,experiment 1 group, experiment 2 group,control 1 group, control 2 group and NS group,and drenched them respectively with PA-STF prepared in vivo,PA-STF prepared in vitro,nTF prepared in vivo, nTF prepared in vitro and normal saline, and then detected the immunological activity of PA-STF and nTF through lymphocyte proliferation test, leukocyte adhesion inhibition test(LAIT), phagocytosis test and sterilization test and immunological protection test.After the immunological protection test,took 5 mice from the survived ones which lived for 4 days,and observed the general status at the 2nd week, the other survived mice would be killed at the 7th day,and cut out their ileums with the length of 3 centimeters,fixed them with 4% formaldehyde solution, and stained with HE,observed the pathological change of mucous membrane under microscope. Results: (1)The concentration of polypeptide and ribose of PA-STF prepared with lymph nodes was 1.83±0.17mg/ml and 0.572±0.024mg/ml respectively,the concentration of polypeptide and ribose of PA-STF prepared with spleen was 1.59±0.22 mg/ml and 0.493±0.015mg/ml respectively, there was no difference between the two materials (P>0.05). (2)The best preparation condition of in vitro was: PHA of 100μg/ml , 0.2ng/ml SPA immunogen and add it delayed 6h than PHA,and stop culture at 96h,under these conditions,the content of polypeptide and ribose of PA-STF was the highest.(3)There was no difference between the content of animo acid,polypeptide and ribose of PA-STF prepared with two methods. The results of sterility tests were all negtive,and those of pyrogen tests and safty tests were all valid.(4)Under the polypeptide concentration of 0.60mg/ml, the SIs of PA-STF prepared with two methods were obviously higher than those of other polypeptide concentration (P<0.05).In the LAIT,the two experiment groups compared with two control groups respectively,there was obviouse difference in the leukocyte adhesion inhibition rate(LAIR) (P<0.05),but there was no difference between the two experiment groups(P>0.05). The results of phagocytosis tests and sterilization tests were that there was no obviouse difference between experiment groups and control groups,however,there was good correlation between phagocytosis rate and sterilization rate(r>0.8).In the immune protection test,compared with two control groups,the living rate of mice in two experiment groups was higher (P<0.05),between the two experiment gropus,there was no difference(P>0.05).The general status of mice in the two experiment groups was better than that of the mice in other groups,and the inflammation in ileums of mice in the experiment groups was also lighter than that in other groups.Conclusions: (1)Prepared PA-STF successfully in vivo, it can be made of spleen or lymph node;prepared PA-STF succesfully in vitro,too. 100μg/ml PHA, 0.2ng/ml SPA- antigen and add it delayed 6h than PHA,and stop cultrue at 96h were the best conditions of PA-STF preparation.(2)The physico-chemical property of PA-STF prepared with two methods all accords with the creterial of Chinese Pharmacopoeia(2005 edition).(3)Both PA-STF prepared with two methods could enhance the non-specific immune response of mouse,and transfer the specific immune effect aim at SPA between them.
引文
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