【作者】 曹发昊；

【导师】 欧阳五庆；

【作者基本信息】 西北农林科技大学 ， 细胞生物学， 2012， 博士

【摘要】 目的：本研究通过纳米技术将人参皂甙（GS）和盐酸左旋咪唑（LH）组合成复方人参皂甙（GS-LH-NE），对其处方组成、质量性能、生物安全性以及免疫增强作用进行研究，旨在提高GS和LH稳定性的同时，开发一种复方免疫增强剂，减少LH用量，从而降低其毒副作用和药物残留。方法：（1）样品用甲醇超声处理后，过树脂将GS和LH分离，建立GS-LH-NE药物含量检测方法。（2）利用伪三元相图考察不同纳米乳体系的形成情况，根据纳米乳区大小、纳米乳稳定性以及对药物的溶解能力，筛选出GS-LH-NE药用空白纳米乳配方；然后通过脾淋巴细胞增殖和单核巨噬细胞碳廓清试验，研究不同含药量纳米乳对免疫抑制小鼠的免疫增强作用，从中筛选出免疫增强作用较好的含药纳米乳作为GS-LH-NE处方，并对其进行质量评价。（3）通过家兔皮肤和肌肉刺激试验、小鼠急性毒性试验，考察GS-LH-NE生物安全性。（4）将免疫抑制小鼠分为：正常对照组、模型对照组、空白纳米乳组、LH-NE组、GS-NE组、GS-LH水溶液组、GS-LH-NE高、中、低剂量组，对其细胞免疫、体液免疫和非特异性免疫的相关指标进行检测，研究GS-LH-NE中GS和LH合用的免疫增强效果。（5）将卵清白蛋白（OVA）免疫小鼠分为：正常对照组、OVA对照组、空白纳米乳组、LH-NE组、GS-NE组、GS-LH水溶液组、GS-LH-NE高、中、低剂量组，对其血清抗体及其亚类水平、脾淋巴细胞增殖活性、脾细胞因子产生、NK细胞活性进行检测，研究GS-LH-NE灌胃对抗原免疫的增强作用。（6）从脾淋巴细胞增殖、脾淋巴细胞因子产生以及腹腔巨噬细胞能量代谢、吞噬活性及其效应分子产生等方面，研究GS-LH-NE体外对小鼠主要免疫细胞的作用。结果：（1）比色-分光光度法测GS含量：平均回收率及其RSD分别为98.86%和0.38％，重复性试验RSD为0.46%，日内和日间精密度RSD分别为0.73%和2.38%；高效液相色谱法测LH含量：平均回收率及其RSD分别为99.02%和0.40％，重复性试验峰面积和保留时间的RSD分别为0.34%和0.77%，日内和日间精密度RSD分别为1.01%和2.03%。（2）GS-LH-NE处方为Solutol HS-15/甘油/PEG400/肉豆蔻酸异丙酯/蒸馏水（质量比为25.33:10.14:2.53:6:56），GS和LH含量分别为30.15mg/mL和30.04mg/mL。它是黄色透明O/W纳米乳，液滴呈球形，平均粒径为23.08nm，粒度分散指数为0.237，浊点为75.4℃，pH为5.67；稳定性好，有效期为18个月。（3）GS-LH-NE一次性或多次用药对完整皮肤和破损皮肤均无刺激；GS-LH-NE对股四头肌刺激反应级最高与最低之差小于2，刺激反应总分为2；GS-LH-NE对小鼠灌胃半数致死量为402.85mg/kg。（4）GS-LH-NE对免疫抑制小鼠免疫增强作用的研究GS-LH-NE中剂量组脾脏指数和胸腺指数与模型对照组比较均显著升高；较LH-NE组分别显著增加56.71%和52.76%；较GS-NE组分别显著增加52.98%和56.70%；较GS-LH水溶液组分别显著增加21.23%和20.63%；与高、低剂量比较均显著升高。GS-LH-NE中剂量组迟发型超敏反应耳重差和ConA诱导脾淋巴细胞增殖的刺激指数与模型对照组比较均显著升高；较LH-NE组分别显著增加43.02%和44.27%；较GS-NE组分别显著增加46.88%和47.66%；较GS-LH水溶液组分别显著增加18.50%和18.13%；与高、低剂量组比较均显著升高。GS-LH-NE中剂量组LPS诱导脾淋巴细胞增殖的刺激指数、血清溶血素水平和抗体形成细胞水平与模型对照组比较均显著升高；较LH-NE组分别显著增加41.27%、64.39%和41.88%；较GS-NE组分别显著增加35.88%、51.81%和39.17%；较GS-LH水溶液组分别显著增加16.34%、24.01%和22.75%；与高、低剂量组比较均显著升高。GS-LH-NE中剂量组单核巨噬细胞吞噬指数、腹腔巨噬细胞吞噬率和吞噬指数、血清溶菌酶水平和脾NK细胞杀伤率与模型对照组比较均显著升高；较LH-NE组分别显著增加33.97%、43.32%、38.46%、39.12%和36.49%；较GS-NE组分别显著增加37.80%、47.22%、44.00%、44.85%和40.69%；较GS-LH水溶液组分别显著增加14.84%、20.87%、18.03%、16.79%和14.96%；与高、低剂量组比较均显著升高。（5）GS-LH-NE对OVA免疫小鼠免疫增强作用的研究GS-LH-NE中剂量组IgG、IgG1和IL-4水平与OVA对照组和低剂量组比较均显著升高；与GS-NE组、GS-LH水溶液组、高剂量组比较均有所升高，但差异均不显著；较LH-NE组分别显著增加31.09%、45.63%和67.62%。GS-LH-NE中剂量组IgG2a和IFN-γ水平与OVA对照组比较均显著升高；较LH-NE组分别显著增加32.75%和40.94%；较GS-NE组分别显著增加34.46%和49.59%；较GS-LH水溶液组分别显著增加11.27%和11.90%；与高、低剂量组比较均显著升高。GS-LH-NE中剂量组ConA、OVA诱导脾淋巴细胞增殖的刺激指数和脾NK细胞活性与OVA对照组均显著升高；较LH-NE组分别显著增加35.96%、39.07%和31.44%；较GS-NE组分别显著增加39.64%、35.48%和36.16%；较GS-LH水溶液组分别显著增加11.91%、15.38%和16.40%；与高、低剂量组比较均显著升高。GS-LH-NE中剂量组LPS诱导脾淋巴细胞增殖的刺激指数与OVA对照组和低剂量组比较显著升高；较LH-NE组显著增加30.41%；与GS-NE组、GS-LH水溶液组和高剂量组比较有所升高，但差异不显著。（6）GS-LH-NE体外对脾淋巴细胞的作用：GS-LH-NE在3.13～12.50μg/mL既能单独又能协同ConA或LPS显著促进脾淋巴细胞增殖，促进脾淋巴细胞产生IL-2和IFN-γ，并且GS-LH-NE在3.13和6.25μg/mL时对脾淋巴细胞免疫功能的增强作用显著强于GS-LH水溶液。GS-LH-NE体外对腹腔巨噬细胞的作用：GS-LH-NE在1.57～6.25μg/mL能显著提高巨噬细胞的能量代谢水平和吞噬活性，促进巨噬细胞产生NO和IL-1β，并且GS-LH-NE在1.57μg/mL时对巨噬细胞免疫功能的增强作用显著强于GS-LH水溶液。结论：（1）GS和LH含量检测方法的回收率、重复性和精密度均能满足要求，为GS-LH-NE的制备及其质量评价提供了可靠的质检方法。（2）GS-LH-NE处方为Solutol HS-15/甘油/PEG400/肉豆蔻酸异丙酯/蒸馏水（质量比为25.33:10.14:2.53:6:56），GS和LH含量分别为30.15mg/mL和30.04mg/mL，是黄色透明的水包油纳米乳，平均粒径为23.08nm，有效期为18个月。（3）GS-LH-NE对皮肤和肌肉无刺激，对小鼠灌胃半数致死量为402.85mg/kg。（4）GS-LH-NE中GS和LH合用能协同增强免疫抑制小鼠的细胞免疫、体液免疫和非特异性免疫功能，并且其免疫增强作用显著强于GS-LH水溶液；其免疫增强作用和其剂量呈“钟罩”型量效关系。（5）GS-LH-NE灌胃能诱导OVA免疫小鼠产生Th1/Th2混合型免疫反应，增强其细胞免疫、体液免疫以及NK细胞活性；其免疫增强作用和剂量呈“钟罩”型量效关系。GS-LH-NE对细胞免疫和NK细胞活性的增强作用显著强于LH-NE、GS-NE和GS-LH水溶液；对体液免疫的增强作用显著强于LH-NE，较GS-NE和GS-LH水溶液有所增强，但无统计学意义。（6）GS-LH-NE在一定浓度范围内能增强淋巴细胞和巨噬细胞的免疫功能，并且其作用显著强于GS-LH水溶液。GS-LH-NE是一种稳定、安全、有效的复方免疫增强剂，提高了药物的免疫增强效果，有助于减少LH用量，从而降低其毒副作用和药物残留，可用于增强免疫低下机体的免疫功能和抗原的免疫效果。更多还原

【Abstract】 Objective: To improve the stability of ginsenoside (GS) and levamisole hydrochloride(LH), and develop an immunostimulants which can be used to be used to reduce the dosageof LH, thereby reducing its toxic side effects and drug residues, GS and LH were combinedinto compound ginsenoside and levamisole hydrochloride nanoemulsion (GS-LH-NE)through nanotechnology. And its prescription composition, quality performance, biologicalsafety and immunological enhancement effect were systematically studied.Methods:(1) Samples were treated with methanol under ultrasonic emulsion breakingand passed through the resin column to separate GS and LH to establish the analyticalmethod for determining the drug content of GS-LH-NE, which would be used in thepreparation and quality evaluation of GS-LH-NE.(2) Pseudo-ternary phase diagram was usedto study the formation of blank nanoemulsion systems with different components. Accordingto the nanoemulsion region size, the stability of blank nanoemulsion and the drug solubilityin it, the optimum medicinal blank nanoemulsion was screened out to preparate GS-LH-NE.The enhancement effect of nanoemulsions containing different content of drugs on theimmunologic function of immunosuppressive mice were studied through the tests of spleniclymphocytes proliferation and monocyte-macrophage carbon clearance, and the drugnanoemulsion with better immunologic enhancement effect was screened out for ascertain theprescription composition of GS-LH-NE. The quality performance of GS-LH-NE wassystematically studied.(3) The biological safety of GS-LH-NE was evaluated through rabbitskin irritation test, muscle irritation test and intragastric acute toxicity test in mice.(4)Immunosuppressive mice were divided into: normal control group, model control group,blank nanoemulsion group, LH-NE group, GS-NE group, GS-LH solution group, GS-LH-NEhigh dose group, GS-LH-NE middle dose group, GS-LH-NE low dose group. The immuneparameters correlated with cellular, humoral and nonspecific immunity were investigated tostudy the effect of GS and LH combination in GS-LH-NE on the immune function of lowimmune function animals.(5) Mice immunized with ovalbumin were divided into: normalcontrol group, ovalbumin control group, blank nanoemulsion group, LH-NE group, GS-NE group, GS-LH solution group, GS-LH-NE high dose group, GS-LH-NE middle dose group,GS-LH-NE low dose group. The immune parameters of mice, such as the level of serumantigen specific antibody and its subclasses, the proliferation activity of splenic lymphocyte,the cytokine production of splenocytes and the killing activity of NK cells on YAC-1, wereinvestigated to study the effect of oral administration with GS-LH-NE on the immunefunction of antigen or vaccine immunized animals.(6) The effect of GS-LH-NE on micesplenic lymphocytes and peritoneal macrophages were studied in vitro through spleniclymphocytes proliferation and its cytokine production, the energy metabolism level andphagocytic activity of peritoneal macrophages and its effector molecules production.Results:(1) Colorimetric-spectrophotometry method was used to determine the contentof GS, which showed that the average recovery rate was98.86%, RSD of the averagerecovery rate was0.38%, RSD of the repeatability test was0.46%, RSD of the within-dayprecision was0.73%, and RSD of the day-to-day precision was2.38%. High performanceliquid chromatograph method was used to determine the content of LH, which showed thatthe average recovery rate was99.02%, RSD of the average recovery rate was0.40%, RSD ofthe peak area in the repeatability test was0.34%, RSD of the retention time in therepeatability test was0.77%, RSD of the within-day precision was1.01%, and RSD of theday-to-day precision was2.03%.(2) The optimal prescription composition of GS-LH-NE wasSolutol HS-15/glycerol/PEG400/IPM/distilled water (with the mass ratio of25.33:10.14:2.53:6:56), in which the content of GS and LH were30.15mg/mL and30.04mg/mL, respectively. GS-LH-NE was a yellow transparent O/W nanoemulsion, which hadspherical droplets with the average particle size of23.08nm and the particle dispersion indexof0.237. And its cloud point and pH value were75.4℃and5.67, respectively. It had goodstability and was valid for18months.(3) Both single and multiple dosing of GS-LH-NE didnot show obvious irritation on the unbroken and broken rabbit skins. The difference betweenthe maximum and minimum irritation response of GS-LH-NE on the rabbit muscle was lessthan2, and the total irritation score was2, which showed that GS-LH-NE could be used forintramuscular injection. The gavage LD50of GS-LH-NE in mice was402.85mg/kg (4) Theimmunological enhancement effect of GS-LH-NE on the immunosuppressive mice thespleen index and thymus index in GS-LH-NE middle dose group were significantly higherthan model control group; were significantly increased by56.71%and52.76%than LH-NEgroup, respectively; were significantly increased by52.98%and56.70%than GS-NE group,respectively; were significantly increased by21.23%and20.63%than GS-LH solution group,respectively; were significantly higher than GS-LH-NE high dose group and low dose group,respectively. The ear weight difference of delayed-type hypersensitivity and the stimulation index of splenic lymphocytes proliferation induced by ConA in GS-LH-NE middle dosegroup were significantly higher than model control group; were significantly increased by43.02%than44.27%than LH-NE group, respectively; were significantly increased by46.88%and47.66%than GS-NE group, respectively; were significantly increased by18.50%and18.13%than GS-LH solution group, respectively; were significantly higher thanGS-LH-NE high dose group and low dose group, respectively. The stimulation index ofsplenic lymphocytes proliferation induced by LPS, the level of serum hemolysin and the levelof splenic antibody forming cell in GS-LH-NE middle dose group were significantly higherthan model control group; were significantly increased by41.27%,64.39%and41.88%thanLH-NE group, respectively; were significantly increased by35.88%,51.81%and39.17%than GS-NE group, respectively; were significantly increased by16.34%,24.01%and22.75%than GS-LH solution group, respectively; were significantly higher than GS-LH-NEhigh dose group and low dose group, respectively. The phagocytic index of monocytemacrophage, the phagocytic rate and phagocytic index of peritoneal macrophage, the level ofserum lysozyme and the activity of splenic NK cells in GS-LH-NE middle dose group weresignificantly higher than model control group; were significantly increased by33.97%,43.32%,38.46%,39.12%and36.49%than LH-NE group, respectively; were significantlyincreased by37.80%,47.22%,44.00%,44.85%and40.69%than GS-NE group, respectively;were significantly increased by14.84%,20.87%,18.03%,16.79%and14.96%than GS-LHsolution group, respectively; were significantly higher than GS-LH-NE high dose group andlow dose group, respectively.(5) The immunological enhancement effect of GS-LH-NE onthe ovalbumin-immunized mice the level of IgG, IgG1and IL-4in GS-LH-NE middledose group were significantly higher than ovalbumin control group and GS-LH-NE low dosegroup, respectively; were significantly increased by31.09%,45.63%and67.62%thanLH-NE group, respectively; were above GS-NE group (P＞0.05), GS-LH solution group (P＞0.05) and GS-LH-NE high dose group (P＞0.05). The level of IgG2a and IFN-γ inGS-LH-NE middle dose group were significantly higher than ovalbumin control group; weresignificantly increased by32.75%and40.94%than LH-NE group, respectively; weresignificantly increased by34.46%and49.59%than GS-NE group, respectively; weresignificantly increased by11.27%and11.90%than GS-LH solution group, respectively; weresignificantly higher than GS-LH-NE high dose group and low dose group, respectively. Thestimulation index of splenic lymphocytes proliferation induced by ConA and ovalbumin andthe activity of splenic NK cells in GS-LH-NE middle dose group were significantly higherthan ovalbumin control group; were significantly increased by35.96%,39.07%and31.44%than LH-NE group, respectively; were significantly increased by39.64%,35.48%and 36.16%than GS-NE group, respectively; were significantly increased by11.91%,15.38%and16.40%than GS-LH solution group, respectively; were significantly higher thanGS-LH-NE high dose group and low dose group, respectively. The stimulation index ofsplenic lymphocytes proliferation induced by LPS in GS-LH-NE middle dose group wassignificantly higher than ovalbumin control group and GS-LH-NE low dose group,respectively; was significantly increased by30.41%than LH-NE group; was above GS-NEgroup (P＞0.05), GS-LH solution group (P＞0.05) and GS-LH-NE high dose group (P＞0.05).(6) The effect of GS-LH-NE on the splenic lymphocytes of mice3.13～12.50μg/mL GS-LH-NE could directly promote the proliferation of splenic lymphocytes (P＜0.05or P＜0.01);3.13～12.50μg/mL GS-LH-NE could promote the proliferation of spleniclymphocytes induced by ConA or LPS (P＜0.05or P＜0.01);3.13～12.50μg/mLGS-LH-NE could promote the splenic lymphocytes induced by ConA to produce the cytokineIL-2and IFN-γ (P＜0.05or P＜0.01); And the immunologic enhancement effect of3.13and6.25μg/mL GS-LH-NE on the splenic lymphocytes was significantly stronger than GS-LHaqueous solution. The effect of GS-LH-NE on the peritoneal macrophages of mice1.57～6.25μg/mL GS-LH-NE could improved the energy metabolism level and thephagocytic activity of macrophages（P＜0.05or P＜0.01），promote the macrophages toproduce NO and IL-1β (P＜0.05or P＜0.01); And the immunologic enhancement effect of1.57μg/mL GS-LH-NE on the macrophages was significantly stronger than GS-LH aqueoussolution.Conclusion:(1) The established analytical methods for determining the content of GSand LH possess high recovery rate, good repetitiveness and good precision，which can beused for the preparation and quality evaluation of GS-LH-NE.(2) The prescriptioncomposition of GS-LH-NE was Solutol HS-15/glycerol/PEG400/IPM/distilled water (withthe mass ratio of25.33:10.14:2.53:6:56), in which the content of GS and LH were30.15mg/mL and30.04mg/mL, respectively. GS-LH-NE was yellow transparent oil in waternanoemulsion, which has the spherical droplets with a average particle size of23.08nm andthe pH value of5.67. It had good stability and was valid for18months.(3) GS-LH-NE hadno obvious irritation on skin and muscle. And the gavage LD50of GS-LH-NE in mice was402.85mg/kg.(4) The combination of GS and LH in GS-LH-NE can work together toenhance the cellular immunity, humoral immunity and nonspecific immunity ofimmunosuppressive mice, which is significantly stronger than the effect of GS-LH aqueoussolution, and shows a “Bell” dose-effect relationship.(5) Oral administration of GS-LH-NEcan promote the ovalbumin-immunized mice to produce a mixed Th1/Th2type immuneresponse, enhance its cellular immunity, humoral immunity and the activity of NK cells, which shows a “Bell” dose-effect relationship. The immunologic enhancement effect ofGS-LH-NE on the cellular immunity and the activity of NK cells of ovalbumin-immunizedmice are significantly stronger than LH-NE, GS-NE and GS-LH solution. Its humoralimmune enhancement is significantly stronger than LH-NE, but compared to GS-NE andGS-LH aqueous solution, its humoral immune enhancement has been enhanced, but there isno significant difference.(6) GS-LH-NE with a certain range can enhance the immunefunction of lymphocytes and macrophages, which were significantly stronger than GS-LHaqueous solution.GS-LH-NE is a stable, safe and effective immunostimulants and can improve theimmunologic enhancement effect of drugs, which indicates that it can reduce the dosage andthe residue of LH. GS-LH-NE can be used to improve the immune function ofimmunosuppressive animals and enhance the immune effect of antigen.更多还原