针灸提高大鼠对疫苗的免疫效应及延缓衰老的实验研究
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摘要
目的：检测“双固一通”电针法延缓D-半乳糖诱导衰老模型大鼠的免疫衰老(实验一)；探讨“双固一通”针灸法对不同浓度百白破疫苗免疫大鼠增强其疫苗免疫效应的作用,摸索针灸提高疫苗免疫效应的最佳疫苗浓度(实验二)；检测D-半乳糖诱导衰老模型大鼠衰老相关指标的改变,鉴定免疫衰老模型构建成功(实验三)；将百白破疫苗免疫衰老模型及正常大鼠,采用“双固一通”针灸法处理,探讨针灸是否能提高大鼠对疫苗的免疫应答效应,发挥疫苗佐剂的作用及延缓免疫衰老的效应(实验四)。
方法：
实验一：雄性SPF级SD大鼠40只,随机取30只大鼠经皮下注射D-半乳糖42d制作衰老大鼠模型,剩余10只为正常对照组。造模结束后模型组再随机分为双固一通组(“关元”、“后三里”穴接电针,“百会”穴毫针针刺)、针刺对照组(“委中”、“水分”穴接电针,“印堂”穴毫针针刺)、衰老模型组,每组10只大鼠。经3周治疗后处死动物检测脾脏指数、胸腺指数、流式细胞法检测CD8+T细胞占T细胞的百分率及CD8+CD28-T占CD8+T细胞的百分率。
实验二：雌性SPF级Wistar大鼠48只,随机分组为六组：1/10疫苗免疫针灸组、1/10疫苗免疫对照组、1/20疫苗免疫针灸组、1/20疫苗免疫对照组、1/40疫苗免疫针灸组、1/40疫苗免疫对照组。每组注射相应浓度的百白破疫苗,针灸组电针刺激大鼠“后三里”、“百会”、“关元”穴,并施以艾灸处理3周,第5周取材,心脏采血分离血清,用Vero细胞法测定白喉抗毒素的效价；MTS比色法检测脾细胞特异性增殖能力；流式细胞法检测CD4+T细胞／CD8*T细胞比值；RT-PCR检测脾细胞热休克蛋白70(HSP70) mRNA的相对表达量(同GAPDH比较)。
实验三：雌性SPF级Wistar大鼠20只,随机取10只大鼠皮下注射D-半乳糖42d制作衰老大鼠模型,剩余10只为正常对照组。造模结束后,进行水迷宫实验,实验结束后处死动物,检测血清超氧化物歧化酶(SOD)的活性及甘油三脂(TG),胆固醇(CHOL)的含量,RT-PCR检测脾细胞HSP70mRNA的相对表达量。
实验四：雌性SPF级Wistar大鼠40只,随机分成4组：模型免疫针灸组、模型免疫对照组、正常免疫针灸组、正常免疫对照组。模型免疫组用D-半乳糖颈背部皮下注射6周,构建衰老大鼠模型。正常免疫组注射等量生理盐水,第7周所有大鼠注射百白破疫苗,电针刺激大鼠“后三里”、“百会”、“关元”穴,并施以艾灸处理3周,针灸治疗结束后再继续喂养2周,至第12周处死所有大鼠,心脏采血,分离血清检测SOD活性、血脂和蛋白含量,用Vero细胞法测定白喉抗毒素的效价；流式细胞法检测CD4+T细胞／CD8+T细胞比值；RT-PCR检测脾细胞HSP70mRNA的相对表达量。
结果：
实验一：衰老模型组大鼠脾脏指数、胸腺指数低于正常对照组(P<0.05); CD8+CD28-T细胞占CD8+T细胞百分率衰老模型组明显高于正常对照组(P<0.01)；CD8+T细胞占T细胞百分率衰老模型组亦明显高于正常对照组(P<0.01)。双固一通组脾脏指数、胸腺指数较衰老模型组升高(P<0.05),而CD8+CD28T细胞占CD8+T细胞百分率显著降低(P<0.01)；针刺对照组CD8+CD28-T细胞占CD8+T细胞的百分率较衰老模型组亦降低(P<0.01)；但双固一通组比针刺对照组的CD8+CD28-T细胞占CD8+T细胞比值降低得更为显著(P<0.05)。
实验二：1/20疫苗免疫针灸组的抗体效价明显高于1/20疫苗免疫对照组(P<0.01)；1/10疫苗免疫针灸组和1/10疫苗免疫对照组抗体效价均高,无明显差异；而1/40疫苗免疫针灸组和1/40疫苗免疫对照组抗体效价亦无明显差异。脾细胞增殖实验表明疫苗本身对细胞有毒性,不适宜做特异性增殖实验,流式细胞法检测1/20疫苗免疫针灸组CD4+T细胞/CD8+T细胞比值较其对照组略有增加(P>0.05)；RT-PCR结果显示1/20疫苗针灸组脾细胞HSP70mRNA的相对表达量较其对照组略有升高(P>0.05)。
实验三：衰老模型组大鼠水迷宫实验(空间探索实验)经过平台次数(P<0.05)和有效区停留时间(P<0.01)明显少于正常对照组；血清SOD活性明显低于正常对照组(P<0.01),TG和CHOL含量高于正常对照组(P<0.05)；脾细胞HSP70mRNA相对表达量低于正常对照组(P<0.05)。
实验四：模型免疫对照组较正常免疫对照组的SOD活性明显降低(P<0.01)；TG和CHOL的含量升高(P<0.05)；总蛋白(TP)和球蛋白(GLB)含量降低(P<0.05)；抗体效价明显降低(P<0.01)；CD4+T细胞／CD8+T细胞比值明显降低(P<0.01)；脾细胞HSP70mRNA的相对表达量明显降低(P<0.01)。模型免疫针灸组较模型免疫对照组的SOD活性升高(P<0.05)；TG和CHOL含量降低(P<0.05)；TP和GLB含量升高(P<0.05)；抗体效价明显升高(P<0.01)；CD4+T细胞/CD8+T细胞比值升高(P<0.05)；脾细胞HSP70mRNA的相对表达量升高(P<0.05)。正常免疫针灸组较正常免疫对照组的TG和CHOL含量降低(P<0.05)；TP和GLB含量升高(P<0.05)；抗体效价升高(P<0.05)。
结论：
实验一：电针可通过提高胸腺指数、脾脏指数及调节T细胞亚群的比例,延缓D-半乳糖诱导衰老模型大鼠的免疫衰老。
实验二：针灸能提高大鼠对百白破疫苗的免疫效应,且最佳疫苗免疫浓度为1/20倍,具有作为新型疫苗佐剂的潜能。
实验三：D-半乳糖能降低大鼠认知水平,增高血脂,降低氧化应激水平及应激蛋白HSP70mRNA表达,是一种构建衰老动物模型的较好方法。
实验四：针灸能改善衰老大鼠的衰老相关生化指标,且能提高衰老机体对百白破疫苗的体液免疫和细胞免疫应答效应,其机制可能与通过增加内源性佐剂物质HSP70mRNA表达有关。
综上所述,针灸能有效延缓免疫衰老,且能通过诱生内源性佐剂HSP70的表达,提高衰老模型大鼠和正常大鼠对百白破疫苗的体液免疫应答和细胞免疫应答效应,发挥疫苗佐剂的效用及延缓免疫衰老的作用。
Objective:
(1) To discuss the Double-reinforcing and One-unblocking acupuncture method whether could delay the immunosenescence course in aging model rats induced by D-galactose;(2) To discuss the improvement of the Double-reinforcing and One-unblocking acupuncture method on the vaccine response in aging model rats induced by different concentrations of DTaP vaccine;(3) To test the involved indexes of aging model rats induced by D-galactose, and to identify the models are performed;(4) To use the Double-reinforcing and One-unblocking acupuncture method on both aging model rats and normal rats, to discuss acupuncture whether could improve the immune effect on rats and could be used as adjuvant, moreover, to discuss acupuncture whether could delay the immunosenescence course in aging model rats.
Methods:
(1)40SD rats, male only,30rats were selected randomly to make the ageing model rats by injected subcutaneously with D-galactose for42d, and were divided into3groups:double reinforcing-one unblocking acupuncture group (GuanyuanCV4, HousanliST36and BaihuiGV20points, used electro-acupuncture therapy on GuanyuanCV4and HousanliST36only); acupuncture control group (WeizhongBL40, ShuifenCV9and YintangGV29, used electro acupuncture therapy on WeizhongBL40and ShuifenCV9only);ageing model group, then, the last10rats were in normal control group. With3-week treatment, spleen index and thymus index were observed; and the percentage of CD8+T/T cell and the percentage of CD8+CD28-T/CD8+T cell were test by flow cytometer.
(2)Wistar female rats48was randomly divided into6groups, namely:1/10vaccine immune acupuncture group;1/10vaccine immune control group;1/20vaccine immune acupuncture group;1/20vaccine immune control group;1/40vaccine immune acupuncture group;1/40vaccine immune control group. Every group were injected with DTaP vaccine, acupuncture groups were treated with electro-acupuncture therapy and moxibustion on BaiHuiGV20HousanliST36and GuanyuanCV4for3-week. At5th week, all rats were collected whole blood, serum separation, diphtheria endotoxins antibody titer were detected by Vero cell separation. The spleen lymphocyte specific proliferation index was tested by MTS colorimetric method, CD4+T/CD8+T lymphocyte ratio was tested by flow cytometer and the relative expression of splenocyte HSP70mRNA was detected by RT-PCR method(compared with GAPDH)
(3)Wistar rats20, female only,10rats were randomly selected to make aging model by injected subcutaneously with D-galactose for42d, the rest is in normal control group. After the Morris water maze, the serum SOD activity, the serum lipid, TG and the GHOL level were tested, the relative expression of splenocyte HSP70mRNA was detected by RT-PCR method.
(4)Wistar rats48, female only, all rats were randomly divided into4groups, namely:model immune acupuncture and moxibustion group; immune model control group; normal immune acupuncture and moxibustion group and normal immune control group. Normal saline was injected subcutaneously to rats in the normal immune group, and D-galactose was injected subcutaneously to rats in the immune model group for6weeks. At7th week, all rats were injected with DTaP vaccine, and treated with electro-acupuncture stimulation and moxibustion on GuanyuanCV4, HousanliST36and BaihuiGV20points for3-week expect control groups. After feed for2weeks, all rats were collected whole blood, serum separation, diphtheria endotoxins antibody titer were detected by Vero cell separation; The blood lipid, protein level and the activity of serum SOD were tested; CD4+/CD8+T cell ratio was tested by flow cytometer and the relative expression of splenocyte HSP70mRNA was detected by RT-PCR method.
Results:
(1)Compare with normal control group, in ageing model group, spleen index and thymus index were lower (P<0.05); the percentage of CD8+CD28T/CD8+T cell were higher remarkably (P<0.01); the percentage of CD8+T/T cell were higher remarkably (P<0.01). Compare with ageing model group, in double reinforcing-one unblocking acupuncture group, spleen index and thymus index increased (P<0.05); the percentage of CD8+CD28-T/CD8+T cell decreased remarkably (P<0.01). Compare with ageing model group, in acupuncture control group, the percentage of CD8+CD28-T/CD8+T cell decreased remarkably too(P<0.01). However, the percentage of CD8+CD28-/CD8+T cell in double reinforcing-one unblocking acupuncture group decreased much more significantly.
(2) Compared with1/20vaccine immune control group, the antibody titer in1/20vaccine immune acupuncture group is significantly higher (P<0.01); the antibody titer in1/10vaccine immune acupuncture group and1/10vaccine immune controls both are higher, and there was no significant difference between each other; the antibody titer of1/40vaccine immune acupuncture group and1/40vaccine immune control group have no significant difference. The splenocyte proliferation experiment found vaccine is toxic for cells and is not suitable for the specific proliferation experiment. Compared with control group, the percentage of CD4+T/CD8+T cell in1/20vaccine immune acupuncture group is increased slightly(P>0.05); the relative expression of spenocyte HSP70mRNA in1/20vaccine immune acupuncture group detected by RT-PCR increased slightly (P>0.05)
(3)Compare with normal control group, the explore space and search latency of aging model rats in water maze test shortened remarkably (P<0.01); serum SOD activity decreased significantly (P <0.01); the TG and the GHOL level increased (P<0.05); the relative expression of splenocyte HSP70mRNA decreased (P<0.05).
(4)Compare with normal immune control group, in model immune control group, SOD activity decreased remarkably (P<0.01), TG and CHOL level increased (P<0.05); TP and GLB decreased (P<0.05); antibody titer decreased significantly (P<0.01); CD4+T/CD8+T cell decreased remarkably (P<0.01); the relative expression of spenocyte HSP70mRNA decreased remarkably (P<0.01). Compare with model immune control group, in immune model acupuncture and moxibustion group, TG and CHOL decreased (P<0.05); TP and GLB increased (P<0.05); SOD activity increased (P<0.05); antibody titer increased significantly (P<0.01); CD4+T/CD8+T increased (P <0.01); the relative expression of spenocyte HSP70mRNA expression increased (P<0.01). Compare with normal immune control group, in normal immune acupuncture and moxibustion group, TG and CHOL decreased (P<0.05); TP and GLB increased (P<0.05); antibody titer increased (P<0.05).
Conclusion:
(1)Electro-acupuncture treatment can delay immunosenescence course in aging model rats induced by D-galactose, by improving of spleen index and thymus index, and regulating the proportion of T cell subgroup.
(2) Acupuncture and moxibustion treatment could be a new adjuvant, by enhancing the vaccine response in rats, and the optimum concentration is1/20.
(3) Subcutaneous injection of D-galactose can impair the learning and memory in rats, increase the blood lipid, reduce HSP70mRNA expression, thus it is a better method on the construction of aging model rats.
(4) Probably based on increase of HSP70mRNA level, acupuncture and moxibustion treatment could improve the involved indexes, and enhance the immune effect in humoral immunity and cellular immunity on aging rats.
To sum up, according to induce the HSP70expression, acupuncture and moxibustion treatment can delay immunosenescence, improve the involved indexes, and enhance the immunity effect in humoral immunity and cellular immunity on aging rats and normal rats, and as vaccine adjuvant in the process.
方法：
实验一：雄性SPF级SD大鼠40只,随机取30只大鼠经皮下注射D-半乳糖42d制作衰老大鼠模型,剩余10只为正常对照组。造模结束后模型组再随机分为双固一通组(“关元”、“后三里”穴接电针,“百会”穴毫针针刺)、针刺对照组(“委中”、“水分”穴接电针,“印堂”穴毫针针刺)、衰老模型组,每组10只大鼠。经3周治疗后处死动物检测脾脏指数、胸腺指数、流式细胞法检测CD8+T细胞占T细胞的百分率及CD8+CD28-T占CD8+T细胞的百分率。
实验二：雌性SPF级Wistar大鼠48只,随机分组为六组：1/10疫苗免疫针灸组、1/10疫苗免疫对照组、1/20疫苗免疫针灸组、1/20疫苗免疫对照组、1/40疫苗免疫针灸组、1/40疫苗免疫对照组。每组注射相应浓度的百白破疫苗,针灸组电针刺激大鼠“后三里”、“百会”、“关元”穴,并施以艾灸处理3周,第5周取材,心脏采血分离血清,用Vero细胞法测定白喉抗毒素的效价；MTS比色法检测脾细胞特异性增殖能力；流式细胞法检测CD4+T细胞／CD8*T细胞比值；RT-PCR检测脾细胞热休克蛋白70(HSP70) mRNA的相对表达量(同GAPDH比较)。
实验三：雌性SPF级Wistar大鼠20只,随机取10只大鼠皮下注射D-半乳糖42d制作衰老大鼠模型,剩余10只为正常对照组。造模结束后,进行水迷宫实验,实验结束后处死动物,检测血清超氧化物歧化酶(SOD)的活性及甘油三脂(TG),胆固醇(CHOL)的含量,RT-PCR检测脾细胞HSP70mRNA的相对表达量。
实验四：雌性SPF级Wistar大鼠40只,随机分成4组：模型免疫针灸组、模型免疫对照组、正常免疫针灸组、正常免疫对照组。模型免疫组用D-半乳糖颈背部皮下注射6周,构建衰老大鼠模型。正常免疫组注射等量生理盐水,第7周所有大鼠注射百白破疫苗,电针刺激大鼠“后三里”、“百会”、“关元”穴,并施以艾灸处理3周,针灸治疗结束后再继续喂养2周,至第12周处死所有大鼠,心脏采血,分离血清检测SOD活性、血脂和蛋白含量,用Vero细胞法测定白喉抗毒素的效价；流式细胞法检测CD4+T细胞／CD8+T细胞比值；RT-PCR检测脾细胞HSP70mRNA的相对表达量。
结果：
实验一：衰老模型组大鼠脾脏指数、胸腺指数低于正常对照组(P<0.05); CD8+CD28-T细胞占CD8+T细胞百分率衰老模型组明显高于正常对照组(P<0.01)；CD8+T细胞占T细胞百分率衰老模型组亦明显高于正常对照组(P<0.01)。双固一通组脾脏指数、胸腺指数较衰老模型组升高(P<0.05),而CD8+CD28T细胞占CD8+T细胞百分率显著降低(P<0.01)；针刺对照组CD8+CD28-T细胞占CD8+T细胞的百分率较衰老模型组亦降低(P<0.01)；但双固一通组比针刺对照组的CD8+CD28-T细胞占CD8+T细胞比值降低得更为显著(P<0.05)。
实验二：1/20疫苗免疫针灸组的抗体效价明显高于1/20疫苗免疫对照组(P<0.01)；1/10疫苗免疫针灸组和1/10疫苗免疫对照组抗体效价均高,无明显差异；而1/40疫苗免疫针灸组和1/40疫苗免疫对照组抗体效价亦无明显差异。脾细胞增殖实验表明疫苗本身对细胞有毒性,不适宜做特异性增殖实验,流式细胞法检测1/20疫苗免疫针灸组CD4+T细胞/CD8+T细胞比值较其对照组略有增加(P>0.05)；RT-PCR结果显示1/20疫苗针灸组脾细胞HSP70mRNA的相对表达量较其对照组略有升高(P>0.05)。
实验三：衰老模型组大鼠水迷宫实验(空间探索实验)经过平台次数(P<0.05)和有效区停留时间(P<0.01)明显少于正常对照组；血清SOD活性明显低于正常对照组(P<0.01),TG和CHOL含量高于正常对照组(P<0.05)；脾细胞HSP70mRNA相对表达量低于正常对照组(P<0.05)。
实验四：模型免疫对照组较正常免疫对照组的SOD活性明显降低(P<0.01)；TG和CHOL的含量升高(P<0.05)；总蛋白(TP)和球蛋白(GLB)含量降低(P<0.05)；抗体效价明显降低(P<0.01)；CD4+T细胞／CD8+T细胞比值明显降低(P<0.01)；脾细胞HSP70mRNA的相对表达量明显降低(P<0.01)。模型免疫针灸组较模型免疫对照组的SOD活性升高(P<0.05)；TG和CHOL含量降低(P<0.05)；TP和GLB含量升高(P<0.05)；抗体效价明显升高(P<0.01)；CD4+T细胞/CD8+T细胞比值升高(P<0.05)；脾细胞HSP70mRNA的相对表达量升高(P<0.05)。正常免疫针灸组较正常免疫对照组的TG和CHOL含量降低(P<0.05)；TP和GLB含量升高(P<0.05)；抗体效价升高(P<0.05)。
结论：
实验一：电针可通过提高胸腺指数、脾脏指数及调节T细胞亚群的比例,延缓D-半乳糖诱导衰老模型大鼠的免疫衰老。
实验二：针灸能提高大鼠对百白破疫苗的免疫效应,且最佳疫苗免疫浓度为1/20倍,具有作为新型疫苗佐剂的潜能。
实验三：D-半乳糖能降低大鼠认知水平,增高血脂,降低氧化应激水平及应激蛋白HSP70mRNA表达,是一种构建衰老动物模型的较好方法。
实验四：针灸能改善衰老大鼠的衰老相关生化指标,且能提高衰老机体对百白破疫苗的体液免疫和细胞免疫应答效应,其机制可能与通过增加内源性佐剂物质HSP70mRNA表达有关。
综上所述,针灸能有效延缓免疫衰老,且能通过诱生内源性佐剂HSP70的表达,提高衰老模型大鼠和正常大鼠对百白破疫苗的体液免疫应答和细胞免疫应答效应,发挥疫苗佐剂的效用及延缓免疫衰老的作用。
Objective:
(1) To discuss the Double-reinforcing and One-unblocking acupuncture method whether could delay the immunosenescence course in aging model rats induced by D-galactose;(2) To discuss the improvement of the Double-reinforcing and One-unblocking acupuncture method on the vaccine response in aging model rats induced by different concentrations of DTaP vaccine;(3) To test the involved indexes of aging model rats induced by D-galactose, and to identify the models are performed;(4) To use the Double-reinforcing and One-unblocking acupuncture method on both aging model rats and normal rats, to discuss acupuncture whether could improve the immune effect on rats and could be used as adjuvant, moreover, to discuss acupuncture whether could delay the immunosenescence course in aging model rats.
Methods:
(1)40SD rats, male only,30rats were selected randomly to make the ageing model rats by injected subcutaneously with D-galactose for42d, and were divided into3groups:double reinforcing-one unblocking acupuncture group (GuanyuanCV4, HousanliST36and BaihuiGV20points, used electro-acupuncture therapy on GuanyuanCV4and HousanliST36only); acupuncture control group (WeizhongBL40, ShuifenCV9and YintangGV29, used electro acupuncture therapy on WeizhongBL40and ShuifenCV9only);ageing model group, then, the last10rats were in normal control group. With3-week treatment, spleen index and thymus index were observed; and the percentage of CD8+T/T cell and the percentage of CD8+CD28-T/CD8+T cell were test by flow cytometer.
(2)Wistar female rats48was randomly divided into6groups, namely:1/10vaccine immune acupuncture group;1/10vaccine immune control group;1/20vaccine immune acupuncture group;1/20vaccine immune control group;1/40vaccine immune acupuncture group;1/40vaccine immune control group. Every group were injected with DTaP vaccine, acupuncture groups were treated with electro-acupuncture therapy and moxibustion on BaiHuiGV20HousanliST36and GuanyuanCV4for3-week. At5th week, all rats were collected whole blood, serum separation, diphtheria endotoxins antibody titer were detected by Vero cell separation. The spleen lymphocyte specific proliferation index was tested by MTS colorimetric method, CD4+T/CD8+T lymphocyte ratio was tested by flow cytometer and the relative expression of splenocyte HSP70mRNA was detected by RT-PCR method(compared with GAPDH)
(3)Wistar rats20, female only,10rats were randomly selected to make aging model by injected subcutaneously with D-galactose for42d, the rest is in normal control group. After the Morris water maze, the serum SOD activity, the serum lipid, TG and the GHOL level were tested, the relative expression of splenocyte HSP70mRNA was detected by RT-PCR method.
(4)Wistar rats48, female only, all rats were randomly divided into4groups, namely:model immune acupuncture and moxibustion group; immune model control group; normal immune acupuncture and moxibustion group and normal immune control group. Normal saline was injected subcutaneously to rats in the normal immune group, and D-galactose was injected subcutaneously to rats in the immune model group for6weeks. At7th week, all rats were injected with DTaP vaccine, and treated with electro-acupuncture stimulation and moxibustion on GuanyuanCV4, HousanliST36and BaihuiGV20points for3-week expect control groups. After feed for2weeks, all rats were collected whole blood, serum separation, diphtheria endotoxins antibody titer were detected by Vero cell separation; The blood lipid, protein level and the activity of serum SOD were tested; CD4+/CD8+T cell ratio was tested by flow cytometer and the relative expression of splenocyte HSP70mRNA was detected by RT-PCR method.
Results:
(1)Compare with normal control group, in ageing model group, spleen index and thymus index were lower (P<0.05); the percentage of CD8+CD28T/CD8+T cell were higher remarkably (P<0.01); the percentage of CD8+T/T cell were higher remarkably (P<0.01). Compare with ageing model group, in double reinforcing-one unblocking acupuncture group, spleen index and thymus index increased (P<0.05); the percentage of CD8+CD28-T/CD8+T cell decreased remarkably (P<0.01). Compare with ageing model group, in acupuncture control group, the percentage of CD8+CD28-T/CD8+T cell decreased remarkably too(P<0.01). However, the percentage of CD8+CD28-/CD8+T cell in double reinforcing-one unblocking acupuncture group decreased much more significantly.
(2) Compared with1/20vaccine immune control group, the antibody titer in1/20vaccine immune acupuncture group is significantly higher (P<0.01); the antibody titer in1/10vaccine immune acupuncture group and1/10vaccine immune controls both are higher, and there was no significant difference between each other; the antibody titer of1/40vaccine immune acupuncture group and1/40vaccine immune control group have no significant difference. The splenocyte proliferation experiment found vaccine is toxic for cells and is not suitable for the specific proliferation experiment. Compared with control group, the percentage of CD4+T/CD8+T cell in1/20vaccine immune acupuncture group is increased slightly(P>0.05); the relative expression of spenocyte HSP70mRNA in1/20vaccine immune acupuncture group detected by RT-PCR increased slightly (P>0.05)
(3)Compare with normal control group, the explore space and search latency of aging model rats in water maze test shortened remarkably (P<0.01); serum SOD activity decreased significantly (P <0.01); the TG and the GHOL level increased (P<0.05); the relative expression of splenocyte HSP70mRNA decreased (P<0.05).
(4)Compare with normal immune control group, in model immune control group, SOD activity decreased remarkably (P<0.01), TG and CHOL level increased (P<0.05); TP and GLB decreased (P<0.05); antibody titer decreased significantly (P<0.01); CD4+T/CD8+T cell decreased remarkably (P<0.01); the relative expression of spenocyte HSP70mRNA decreased remarkably (P<0.01). Compare with model immune control group, in immune model acupuncture and moxibustion group, TG and CHOL decreased (P<0.05); TP and GLB increased (P<0.05); SOD activity increased (P<0.05); antibody titer increased significantly (P<0.01); CD4+T/CD8+T increased (P <0.01); the relative expression of spenocyte HSP70mRNA expression increased (P<0.01). Compare with normal immune control group, in normal immune acupuncture and moxibustion group, TG and CHOL decreased (P<0.05); TP and GLB increased (P<0.05); antibody titer increased (P<0.05).
Conclusion:
(1)Electro-acupuncture treatment can delay immunosenescence course in aging model rats induced by D-galactose, by improving of spleen index and thymus index, and regulating the proportion of T cell subgroup.
(2) Acupuncture and moxibustion treatment could be a new adjuvant, by enhancing the vaccine response in rats, and the optimum concentration is1/20.
(3) Subcutaneous injection of D-galactose can impair the learning and memory in rats, increase the blood lipid, reduce HSP70mRNA expression, thus it is a better method on the construction of aging model rats.
(4) Probably based on increase of HSP70mRNA level, acupuncture and moxibustion treatment could improve the involved indexes, and enhance the immune effect in humoral immunity and cellular immunity on aging rats.
To sum up, according to induce the HSP70expression, acupuncture and moxibustion treatment can delay immunosenescence, improve the involved indexes, and enhance the immunity effect in humoral immunity and cellular immunity on aging rats and normal rats, and as vaccine adjuvant in the process.
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