Study Of The Immunological Enhancement Function And Safety Of CpG-ODN

Objective: Via investigating the level of humoral immunity and cellular immunity and the change of histomorphology of the spleens of BAL b/c mice immunized with HBsAg singly or association with CpG-ODN, estimate the systemic and local effectiveness of CpG-ODN, investigate the immunological enhancement function and the larvate safety problem of CpG-ODN. Further more, via investigating the dynamic change of proliferative activity of lymphocytes separated from the spleens of mice which were induced with HBsAg singly or association with CpG-ODN in vitro, estimate CpG-ODN whether or not can act as an immunological adjuvant to enhance the immunostimulation of HBsAg.Methods: The plamids of CpG-ODN were transformed into E.coli DH5α, the LB plate containing 100μg/ml ampicillin was spraided with E.coli DH5αand cultivanted at 37℃for 12h. Pick several colony from the freshly plate to a 2-5ml LB medium containing 100μg/ml ampicillin and cultivate the colony at 37℃with vigorous shaking for 12h. The plamids were extracted from the bacterial culture by lye clearage method and identified with enzyme shearing and agarose electrophoresis. A large scale culture was preperated when the plamids were identified correctly. The plamids of CpG-ODN were extracted from the bacterial culture by Endo-Free Plamid Maxi Kit. And the content and purity of plamids were identified with ultraviolet spectrophotometry. BAL B/c mice were randomly divided into five groups and three times immunized with different drugs every 14 days. The first group was immunized with HBsAg and the high dose of CpG-ODN, the second group was immunized with HBsAg and the moderate dose of CpG-ODN, the third group was immunized with HBsAg and the low dose of CpG-ODN, the fourth group was immunized with HBsAg singly, the fifth group was immunized with physiologic saline. From the first week to the eighth week, the titre of HBsAb once every week and the content of IFN-γonce every two week were detected from the peripheral blood of immunized mice using ELISA as method. In the beginning of the ninth week, the lymphocytes of the spleens of mice were separated and cultivanted with ConA or HBsAg in vitro for 24 hours, and then the proliferative activity of lymphocytes was detected by XTT/PMS assay. Simultaneously, the paraffin sections of the spleens of mice were preperated, and then the change of histomorphology of spleens of mice was observered by hematoxylin-eosin staining. Lymphocytes separated from the spleen of mouse were divided into six groups in vitro: the first group was induced with ConA, the second group was induced with HBsAg, the third group was induced with HBsAg and CpG-ODN, the fourth group was induced with HBsAg and pUC18, the fifth group was induced with pUC18, the sixth group was induced with nothing, as cells control group. Another group of 1640 medium with 10% FCS but cells was added, as medium control group. The influence of HBsAg and/or CpG-ODN on the proligerative activity of lymphocytes was estimated by using XTT/PMS assay.Results: The content of plamid of CpG-ODN is 3.75mg, the ratio of OD260 and OD280 is 1.77. The titre of HBsAb of the first group to the third group was both significantly increased than that of the fourth group (p<0.05); Compare with the fourth group and the fifth group, the content of IFN-γand the proliferative activity of lymphocytes were both remarkably increased in the first group and the second group(p<0.05). Campare with the fourth group and fifth group, from the first group to the third group, the hyperplasia of spleens of mice was obvious by Hematoxylin-eosin staining: the margin of white pulp and red pulp was clear, and the periarterial lymphatic sheath obviously thickened, the quantity and volume of splenic corpuscle remarkably increased, the germinal center of splenic corpuscle was evidently observed; the hyperemia of red pulp was severe, the margin of splenic cord and splenic sinus was blear. The thickness of the periarterial lymphatic sheath and the volume of splenic corpuscle of spleens of mice of the first group and the second group significantly increased than those of the third group; nevertheless, the severity of the hyperemia of red pulp of the first group and second group was lower than that of the third group. For the first group, the histological structure was clear and intact, the abnormality of splenic corpuscle was not observed. By XTT/PMS assay, it was discovered that the most suitable final concentration of CpG-ODN is 5-10μg/mL; the best cultivate time of lymphocytes after drugs added is 72 hours.Conclusion: The results show that the cellular and humoral immune responses of the immunized mice are significantly enhanced by the association of HBsAg and CpG-ODN. The severe immunologic injury in the spleens of mice immunized with HBsAg and the high dose of CpG-ODN was not discovered. CpG-ODN can remarkably enhance the immunostimulation of HBsAg on the lymphocytes of the spleen of mouse, and will be used as an effective immunological adjuvant for HBsAg.