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Study of the Immunological Enhancement Function and Safety of CpG-ODN

Author: GuoXiaoLei
Tutor: MengMinJie
School: Guangdong College of Pharmacy
Course: Pathogen Biology
Keywords: CpG-ODN HBsAg Humoral immunity Cellular immunity Histomorphological XTT / PMS
CLC: R392
Type: Master's thesis
Year: 2008
Downloads: 106
Quote: 0
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Abstract


Purpose by the hepatitis B surface antigen and CpG-ODN alone or combined immunodeficiency BAL b / c mice, the morphological changes observed humoral immune and cellular immune response to the level of their spleen, evaluation of CpG-ODN in mice systemic and immunological effects of CpG-ODN immune spleen local produce enhancements and possible security problems; further in vitro observation by the hepatitis B surface antigen with CpG-ODN alone or the Commonwealth of extracellular stimuli mouse spleen lymphocyte proliferation activity dynamic change the evaluation of CpG-ODN immune to hepatitis B surface antigen enhancements, and then explore the CpG-ODN as an immune adjuvant of hepatitis B surface antigen. CpG-ODN plasmid transformed into E. coli DH5α coated ampicillin Tablet cultured at 37 ℃, picked colonies at 37 ° C a small amount of liquid culture, plasmids were extracted by alkaline lysis method, identification of enzyme electrophoresis; colonies to expand the identification of successful training a large number of spent endotoxin plasmid extraction kit plasmid extraction, of UV spectrophotometry identification plasmid content and purity. BAL b / c mice were randomly divided into five groups: HBsAg and high-dose CpG-ODN combined immune group, the middle dose group of HBsAg with CpG-ODN HBsAg and low-dose CpG-ODN combined immunodeficiency group HBsAg alone group and the saline control groups, respectively, at 0,2,4 weeks immunization BAL B / c mice. Tail blood collection in the first 1-8 weeks, HBsAb and levels of IFN-γ ELISA assay peripheral blood of mice; spleen at 9 weeks, isolated from mouse spleen lymphocytes, respectively of ConA and HBsAg stimulated in vitro for 24h XTT / PMS assay mouse spleen lymphocyte proliferation; mouse spleen tissue paraffin sections were prepared at the same time, HE staining was observed in mice spleen morphological changes. Through the establishment of ConA group, HBsAg, HBsAg combined with CpG-ODN group, HBsAg and pUC18 combination group, CpG-ODN group the pUC18 group, cell blank control and medium blank control group, XTT / PMS method evaluation HBsAg and / or CpG ODN on the proliferation of mouse spleen lymphocytes. Results CpG-ODN plasmid content to 3.75mg of OD260/OD280 ratio of 1.77. HBsAg and high doses of CpG-ODN United immunohistochemistry The HBsAg and medium dose CpG-ODN joint immunohistochemistry HBsAg and low-dose CpG-ODN United immunohistochemistry HBsAb were significantly higher than HBsAg alone group (p lt; 0.05); HBsAg and high doses of CpG-ODN combined immunodeficiency group, IFN-γ levels and spleen lymphocyte proliferation of HBsAg and the dose of CpG-ODN combined immunodeficiency group were significantly higher than the the HBsAg group and the saline control group (p lt; 0.05 ). HE staining, compared with HBsAg alone group and the saline control group, HBsAg and high-dose CpG-ODN joint immunohistochemistry, the HBsAg and medium doses of CpG-ODN combined immunodeficiency group HBsAg and low-dose CpG-ODN combined immunodeficiency group mouse spleen white pulp, red pulp boundaries clear, white pulp periarterial lymphatic sheath thickening, an increase in the number of splenic volume increased spleen bodies showed obvious germinal center; red pulp congestion significantly, splenic cord spleen sinusoids boundaries blurred. Contrast, HBsAg and CpG-ODN dose group in the spleens of mice periarterial lymphatic sheath thicker, the splenic larger, HBsAg and low-dose CpG-ODN joint immunized mice spleen red pulp hyperemia more obvious. HBsAg and high doses of CpG-ODN joint immunohistochemistry of mouse spleen organizational structure is still clear and complete, not observed in the morphology of the splenic abnormalities. XTT / PMS detected, CpG-ODN optimum final concentration 5-10μg/mL; optimal stimulating culture time 72h. Conclusion CpG-ODN can significantly improve the mice generated humoral immunity against hepatitis B surface antigen and cellular immune response level; CpG-ODN impact of the high dose did not produce serious damage to the immune-induced mouse spleen; CpG-ODN can significantly enhance the HBsAg stimulation of mouse spleen lymphocytes can be used as the immunoadjuvant of the hepatitis B surface antigen.

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