摘要
为研究太子参参须多糖(RPFRP)对模式抗原卵清白蛋白(OVA)免疫小鼠的佐剂作用,本研究将60只雌性ICR小鼠随机分成空白对照组(CK组),单独免疫OVA组(OVA组),OVA+RPFRP低、中、高(50 mg/kg、100 mg/kg、200 mg/kg)剂量组(D、Z、G组)。在实验的第1 d~5 d和第16 d~20 d,除CK组和OVA组小鼠灌胃(ig)双蒸水之外,其他组小鼠分别灌胃对应剂量的RPFRP。第6 d和第21 d,除CK组小鼠经皮下注射0.2 mL PBS外,其余各组小鼠均经皮下注射OVA溶液0.2 mL(抗原含量≥99.9%),共免疫2次。第36 d迫杀各组小鼠,分别采用MTT法检测小鼠脾淋巴细胞增殖指数(SI)及NK细胞活性,结果显示:与OVA组相比,OVA+各剂量RPFRP均能够显著提高T细胞的SI值(P<0.05),其中低、中剂量的RPFRP能够显著提高淋巴细胞的SI值。OVA+各剂量RPFRP组小鼠NK细胞的杀伤活性与CK和OVA组差异均不显著(P>0.05);采用ELISA法检测各组小鼠血清中细胞因子、特异性IgG抗体及其亚类含量,结果显示,与OVA组相比,OVA+各剂量RPFRP组的细胞因子含量及血清中IgG1抗体水平均有所提高但均差异不显著,G、Z组IgG2a抗体水平显著升高(P<0.05),G组IgG和IgG2b抗体水平均显著升高(P<0.05);采用RT-qPCR法检测各组小鼠脾淋巴细胞中的细胞因子以及转录因子的转录水平,结果显示,与OVA组相比,OVA+各剂量RPFRP组IL-4、IL-10、IFN-γ、转录因子T-bet、GATA mRNA转录水平和T-bet/GATA比值均无显著提高,仅G、Z组IL-2 mRNA的转录水平显著升高(P<0.05);采用流式细胞术检测各组小鼠脾细胞中CD3、CD4^(+)、CD8^(+)T淋巴细胞亚群数量及CD4^(+)/CD8^(+)T细胞的比值,结果显示,与OVA组相比,G、Z组小鼠脾细胞中的CD3、CD8^(+)T细胞及G组CD4^(+)T细胞数量均显著升高(P<0.05),而G、Z组的CD4^(+)/CD8^(+)T细胞比值明显降低(P<0.05),接近CK组的该比值。上述结果表明,RPFRP能在一定程度上提高小鼠脾淋巴细胞的增殖水平、NK细胞杀伤活性,调节IgG及其亚类抗体的分泌水平,调节相关细胞因子、转录因子的转录水平等,从而能在一定程度上提高小鼠对OVA的免疫应答能力,表现出一定的佐剂作用。本研究可丰富RPFRP的免疫学内容,为进一步寻找高效、无毒、安全的中药免疫佐剂提供一定数据参考。
To study the adjuvant effect of radix pseudostellariae fibrous roots polysaccharide(RPFRP)on mice immunized with ovalbumin(OVA).Sixty ICR female mice were randomized into control group(CK group),OVA alone group(OVA group),OVA together with low,medium and high doses of RPFRP groups(OVA+RPFRP groups;50,100,200mg/kg;D,Z,G group).On the 1st-5th and 16th-20th days,the mice in the OVA+RPFRP groups were given the corresponding doses of RPFRP,while the mice in the CK and OVA groups were given the same does of distilled water.On the 6th and 21st days,all the mice were injected subcutaneously with 0.2mL of OVA solution(antigen purity≥99.9%)except for the CK group,which were given 0.2mL of PBS instead.Totally,twice immunicaitons were performed.On the 36th day,all the mice were killed.And the proliferation index of splenic lymphocytes,and NK cell activity were detected by MTT method.The results showed that the T cells stimulation index(SI)of mice(P<0.05)was significantly increased in OVA+RPFRP groups compared with that in the OVA alone group,and the lymphocytes stimulation index of mice was also significantly increased in the Z and D groups.Compared with the OVA and CK groups,NK cell killing activities of mice were no significant variation in OVA+RPFRP groups(P>0.05).The amounts of cytokines,specific antibodies IgG and its subclasses in serum were detected by ELISA method.Compared with the OVA group,the cytokines and antibody IgG1 of mice were not significantly increased in OVA+RPFRP groups.The antibody IgG2a of mice was significantly increased in G and Z groups(P<0.05),the antibodies IgG and IgG2b of mice were exceedingly promoted in the G group(P<0.05).The transcription levels of cytokines and transcription factors mRNAs in splenic lymphocytes were detected by RT-q PCR,the results showed that compared with the OVA group,there were no significant difference for the transcription levels of IL-4,IL-10and IFN-γmRNA,and transcription factors T-bet,GATA m RNA(P>0.05)in the OVA+RPFRP groups,as well as for the ratio of T-bet/GATA.The transcription level of IL-2 mRNA(P<0.05)was remarkably increased in the G and Z groups.The amounts of CD3,CD4^(+),CD8^(+)T cells and T lymphocyte subsets in splenic cells,and the ratio of CD4^(+)/CD8^(+)were detected by flow cytometry,the results showed that compared with the OVA group,both the amounts of CD3and CD8^(+)T cells(P<0.05)in the G and Z groups and the amounts of CD4^(+)T cells(P<0.05)in the G group were significantly increased,while,the ratios of CD4^(+)/CD8^(+)T cells(P<0.05)were obviously decreased in the G and Z groups similar to that in the CK group.Above results illustrated that RPFRP could improve the proliferation level of splenic lymphocytes and NK cells killing activity to a certain extent,regulating the secretion levels of OVA specific antibody IgG and its subclasses,regulating the transcription level of related cytokines and transcription factors.Thus,to a certain extent,the immune response of mice to OVA was improved by RPFRP showing the effect of adjuvant.The study can enrich the immunological knowledge of RPFRP,and provide some reference for further reserach to find an efficient,non-toxic and safe immune adjuvant in Traditional Chinese medicine.
作者
曾丽
甘思言
杜蓥蓥
乔石
张炎达
黄一帆
王全溪
马玉芳
ZENG Li;GAN Si-yan;DU Yin-yin;QIAO Shi;ZHANG Yan-da;HUANG Yi-fan;WANG Quan-xi;MA Yu-fang(University Key Laboratory for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province,Fuzhou 350002,China;Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health(Fujian Agriculture and Forestry University),Fuzhou 350002,China;Fujian Bedi Pharmaceutical Co.,Ltd.,Ningde 355399,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第6期648-654,681,共8页
Chinese Journal of Preventive Veterinary Medicine
基金
福州市科技创新平台项目(2021P036)
福建省重大专项专题项目(2021NZ02908)。
关键词
太子参参须多糖
免疫模式抗原
小鼠
佐剂
免疫功能
radix pseudostellariae fibrous roots polysaccharide
immune model antigen
mice
adjuvant
immune function
