淫羊藿苷体内外免疫调节作用的实验研究
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摘要
目的:研究淫羊藿苷(icariin,ICA)体内外对免疫细胞及免疫系统的影响,探讨淫羊藿苷的免疫调节活性及其作用机制,为淫羊藿苷应用于临床治疗肿瘤等疾病提供实验依据。
方法:
1在无菌条件下采集健康足月妊娠产妇脐血,用密度梯度离心法获得脐血单个核细胞,用GM-CSF、IL-4诱导获得未成熟的树突状细胞(dendritic cells, DCs)。
2采用光镜及透射电镜(TEM)观察未处理组、TNF-α刺激组、ICA刺激组DCs的形态学变化。
3采用流式细胞技术(flow cytometry,FCM)检测未处理组、TNF-α刺激组、ICA刺激组DCs表面CD1a、CD83、CD80、CD86的表达情况;检测经不同浓度ICA(12.5、25、50、100μg/ml)作用12小时后人子宫内膜癌耐药细胞株B-MD-C1(ADR+/+)表面CD54、CD18的表达情况以及细胞周期分布的情况。
4采用ELISA方法检测未处理组、TNF-α刺激组、ICA刺激组DCs培养上清中IL-12和IFN-γ水平。
5采用四甲基偶氮唑蓝法(MTT)检测未处理组、TNF-α刺激组、ICA刺激组DCs刺激T细胞增殖的能力;检测不同浓度ICA(12.5、25、50、100μg/ml),处理不同时间(24、48和72h)对B-MD-C1(ADR+/+)细胞增殖的抑制作用。
6采用乳酸脱氢酶(LDH)释放法检测B-MD-C1(ADR+/+)细胞经不同浓度ICA(12.5、25、50、100μg/ml)作用12小时后,CIK细胞对其杀伤活性的变化。
7采用逆转录-聚合酶链反应(revers transcription PCR,RT-PCR)半定量检测不同浓度ICA(0、12.5、25、50μg/ml)作用12小时后,B-MD-C1(ADR+/+)细胞CD54、CD18 mRNA表达的变化。
8将60只7-8周龄C57BL/6j小鼠随机分为6组,建立小鼠免疫抑制模型,其中实验组、阳性对照组和模型对照组均于腹腔注射环磷酰胺(Cy,300mg/kg);第二天,实验组通过灌胃方式给予不同剂量ICA(150、80、40mg/kg·day),阳性对照组小鼠经腹腔注射参芪扶正注射液,(1ml/day)。模型对照组小鼠给予等量生理盐水(NS),连续给药10d,正常对照组小鼠不做任何处理,正常饲养。每天观察小鼠生存状态,于末次给药12小时后引颈处死小鼠,分离胸腺、脾、腹腔巨噬细胞、骨髓、外周血,备用。
9采用HE染色光镜下观察小鼠胸腺组织结构变化。
10采用MTT法检测小鼠脾淋巴细胞的增殖反应。
11采用ELISA方法检测小鼠腹腔巨噬细胞产生TNF-α、IL-12的水平。
12采用乳酸脱氢酶(LDH)释放法检测小鼠腹腔巨噬细胞杀伤能力的变化。
13采用瑞氏-姬姆萨染色法观察小鼠骨髓和外周血涂片细胞形态变化。
14采用全自动血液分析仪检测小鼠外周血红细胞、白细胞和血小板水平的变化。
15光镜下计数小鼠单根股骨髓细胞(BMC)数量变化。
结果:
1人脐血单个核细胞(CBMC)经GM-CSF、IL-4诱导5天后呈现典型未成熟树突状细胞形态,细胞体积较小,表面毛刺状突起较少,细胞成簇分布,呈集落样生长。
2 CBMC在体外诱导培养10天时,经ICA刺激5d产生具有典型成熟特征的DCs,细胞体积较大,形态不规则,表面有大量细而长的树突状突起,胞浆线粒体、粗面内质网丰富,核异染色质边集于核膜下,溶酶体和空泡少见。
3表型分析发现,ICA刺激组DCs具有成熟的特征性表面分子CD1a、CD83、CD80和CD86的表达,表达水平与未处理组DCs相比均显著增加(P<0.05),CD80表达水平最高达(90.44±0.81)%;随着ICA作用浓度的增加,B-MD-C1(ADR+/+)细胞表面CD54、CD18表达水平逐渐升高,各浓度处理组之间细胞的CD54、CD18表达水平具有显著性差异(P<0.01),各浓度处理组与对照组细胞之间CD54、CD18的表达水平具有显著性差异(P<0.01)。流式细胞分析结果显示:ICA能使B-MD-C1(ADR+/+)细胞生长周期被阻滞在S期。
4 ICA刺激组DCs培养上清中IL-12和IFN-γ的含量均明显高于对照组DCs(P<0.01),但与TNF-α刺激组DCs无显著性差异(P>0.05)。
5经ICA刺激获得的DCs可明显刺激T细胞增殖,且随DCs与T细胞比例增加而增强;在12.5~100μg/ml浓度范围内ICA能显著抑制B-MD-C1(ADR+/+)体外增殖反应(P<0.05),抑制率与ICA呈明显的浓度和时间依赖性,其中100μg/ml ICA作用72h的抑制率最高,达到78.74%。
6与非ICA处理组相比,12.5~100μg/ml浓度的ICA对B-MD-C1(ADR+/+)细胞对CIK具有明显的杀伤增敏作用。在不同效靶比5∶1、10∶1、20∶1时,CIK对B-MD-C1(ADR+/+)细胞的杀伤率随着ICA浓度及效靶比的增加而增加,并与对照组相比具有显著性差异(P<0.01);
7随着ICA作用浓度的增加,B-MD-C1(ADR+/+)细胞CD54、CD18 mRNA表达水平逐渐升高,各浓度处理组与对照组细胞之间CD54、CD18 mRNA的表达水平具有显著性差异(P<0.05);
8成功建立了环磷酰胺诱导的小鼠免疫抑制模型,模型组小鼠活动能力差,进食饮水状况不佳,在第6天时有一只死亡;正常对照组、阳性对照组、实验组小鼠的活动、进食、饮水均正常活跃,全部存活。
9模型对照组小鼠胸腺组织结构发生明显改变,胸腺周围出现脂肪细胞变性和坏死,低浓度(40mg/kg·day)ICA处理组小鼠胸腺组织仍可见部分脂肪组织填充,但未见明显坏死组织。参芪扶正注射液和中、高剂量ICA处理组小鼠胸腺组织形态正常,未见明显改变。
10 ConA可明显促进高(150mg/kg·day)、中剂量(80mg/kg·day)ICA和参芪扶正注射液处理组小鼠脾脏T细胞增殖反应,与模型对照组相比,有显著性差异(P<0.05);LPS可促进高剂量ICA组小鼠B细胞的增殖反应,与模型对照组相比有显著性差异(P<0.05),中、低剂量ICA处理后,对小鼠脾B细胞增殖反应无显著影响(P>0.05)。
11与正常组小鼠相比,模型组小鼠腹腔巨噬细胞产生TNF-α、IL-12的水平明显降低(P<0.01),经ICA处理10天后,实验组小鼠腹腔巨噬细胞产生TNF-α、IL-12的水平与阳性对照组及正常对照组相比无显著性差异(P>0.05)。
12与正常组小鼠相比,模型对照组小鼠腹腔巨噬细胞的杀伤活性显著降低(P<0.01),不同浓度ICA均能提高巨噬细胞的杀伤活性,与模型对照组相比具有显著性差异(P<0.05);高浓度ICA处理组和阳性对照组小鼠巨噬细胞的杀伤活性与正常对照组相比,无明显差异(P>0.05)。
13小鼠骨髓和外周血涂片可见,正常组小鼠骨髓涂片中有核细胞增生活跃,模型对照组小鼠骨髓呈现造血抑制状态。经ICA处理后,实验组小鼠骨髓涂片和外周血涂片中均可见骨髓抑制状态有所改善。
14外周血常规分析结果显示:模型组小鼠WBC、RBC和PLT较对照组明显降低(P<0.01),而高、中、低浓度ICA均可明显提高实验组小鼠外周血WBC、RBC和PLT数量。
15于小鼠腹腔注射Cy10天后,盐水对照组小鼠单根股骨骨髓细胞数量为7.03×106/femur,与对照组小鼠(10.36×106/femur)相比明显降低,差异有显著性(P<0.01),经高、中浓度ICA和参芪扶正注射液治疗后,小鼠单根股骨髓细胞数量与对照组相比,无显著性差异(P>0.05)。
结论:
1 ICA可在体外诱导CBMC来源的DCs成熟,上调DCs表面分子表达,促进DCs细胞因子的分泌,增强其刺激T细胞增殖的能力。
2 ICA可以上调B-MD-C1(ADR+/+)细胞CD54、CD18蛋白和基因的表达,增强该细胞免疫原性,有利于免疫活性细胞的识别杀伤。
3在一定浓度范围内ICA能抑制B-MD-C1(ADR+/+)细胞的生长,能使B-MD-C1(ADR+/+)细胞的生长周期被阻滞在S期,具有明显的量效和时效关系。
4 ICA对免疫抑制小鼠的免疫器官具有很好的保护作用。
5 ICA可显著刺激小鼠脾T、B细胞的增殖,增强腹腔巨噬细胞的杀伤能力,显著增加免疫抑制小鼠腹腔巨噬细胞产生TNF-α和IL-12等细胞因子的含量,可明显增强免疫抑制小鼠细胞免疫和体液免疫功能。
6 ICA可显著改善免疫抑制小鼠的骨髓造血状态,提高骨髓细胞和外周血细胞数量,可明显恢复和改善造血功能。
7 ICA显著增强免疫功能,提示其可作为抗肿瘤药物,并具有良好的安全性,有潜在的临床应用价值。
Objective: In order to study the effect of ICA on immune system and immunocyte in vitro and in vivo. Meanwhile, we approached immunoregulation mechanism of ICA initially. For the final purpose, this study provides some experimental bases to clinical application of ICA to cure certain diseases, such as tumor.
Methods:
1 The cord blood monocytes, which were collected from health and uterogestation parturient were isolated by density gradient centrifugation using lymphocyte isolating solution under axenic condition, and dendritic cells (DCs) were induced by GM-CSF and IL-4.
2 The morphological characteristics of DCs from untreated group, TNF-αgroup and ICA group were observed under inverted microscope and transmission election microscope.
3 Expressions of CD1a, CD83, CD80 and CD86 on DCs of untreated group, TNF-αgroup and ICA group, and the cell cycles and expressions of CD54, CD18 on B-MD-C1 (ADR+/+) cells treated by ICA (0, 12.5, 25, 50, 100μg/ml) for 12hours were detected by flow cytometry.
4 The level of IL-12 and IFN-γin the culture supernatant of DCs from untreated group, TNF-αgroup and ICA group were detected by ELISA.
5 The proliferation of T cells stimulated by DCs, and the inhibition effect of ICA on cell growth of B-MD-C1 (ADR+/+) in treated or untreated groups (12.5, 25, 50, 100μg/ml ICA and control) for different treatment times (24h, 48h and 72h) were determined by MTT method.
6 The cytotoxicity of CIK cells against B-MD-C1 (ADR+/+) cell, which were treated by ICA (0, 12.5, 25, 50, 100μg/ml) for 12hours were investigated by LDH release method.
7 Expression of CD54, CD18 mRNA in B-MD-C1 (ADR+/+) cells after treated with ICA (0, 1.25, 25, 50μg/ml) for 12h were detected by semi-quantitative RT-PCR.
8 The sixty C57BL/6j mice of 7-8-week-old were divided into six groups randomly, and constructed immunosuppression models on them besides control group, the others were divided into experimental group, positive group and model group, and then the three groups were injected with cyclophosphamide (Cy) in abdominal cavity one time (300mg/kg). In the second day, the mice of experimental group were given ICA (150, 80, 40 mg/kg·day) by intragastric administration, the positive group were given Shenqi Fuzheng Injection (1ml/day); the model group were given normal saline (NS). All the mice were treated for 10 days. The mice of control group were raised normally. To observe the survival condition of the mice everyday, after the lasted given drugs for 12 hours, all the mice were killed, and the thymus, spleen, peritoneal macrophage, bone marrow and peripheral blood were removed for the succedent experiments.
9 Morphology of thymus of the mice were observed under optical microscope after HE staining.
10 The proliferation of lymphocyte of the mice were determined by MTT method.
11 The level of TNF-αand IL-12 that were produced by peritoneal macrophage of mice were detected by ELISA.
12 The cytotoxicity of the peritoneal macrophage of the mice were investigated by LDH release method.
13 Morphological images of bone marrow and peripheral blood smears of the mice were detected by Wright-Giemsa’s staining.
14 Population of WBC, RBC and PLT in the peripheral blood were detected with automated blood cell counter (ABCC).
15 The number of bone marrow cells in the single femur was counted under microscope.
Results:
1 After 5 days cultured with GM-CSF and IL-4, the human CBMC developed into immature DCs with typical morphological characteristics, the cells were small and a little dendrites on the cellular surface, and they were grown in clustering.
2 After 10 days cultured with cytokines and ICA, the CBMC that were stimulated by ICA for 5 days developed into mature DCs with typical morphological characteristics, the cells enlarged and were irregular in shape, many thin and long dendrites on the cellular surface, abundance of cytoplasm with more mitochondrion and rough endoplasmic reticulum, the caryon heterochromatin were under the karyolemma, and fewer lysosome and vacuolus.
3 The result of phenotype analysis showed that the expressions of CD1a, CD83, CD80 and CD86 on mature DCs surface were up-regulated significantly by ICA, compared to the control group untreated with stimulating factor(P<0.05), in which most significant change occurred at CD80, reached (90.44±0.81)%. The expression of CD54 and CD18 on B-MD-C1 (ADR+/+) cells surface were also up-regulate by ICA in concentration-dependent manner. The expression of CD54 and CD18 on B-MD-C1 (ADR+/+) cells surface between every treated group, the difference was significant(P<0.01), the cells after treatment with different doses of ICA, compared to control group, the difference was significant(P<0.01). The results of FCM showed that ICA could arrest cell cycle of B-MD-C1 (ADR+/+) cells at S phase.
4 ICA enhanced obviously the IL-12 and IFN-γproduction by DCs(P<0.01), but there was no significance difference between ICA group and TNF-αgroup(P>0.05).
5 ICA-treated DCs stimulated markedly the proliferation of T cell (P<0.05), and the ability displayed rising tendency with the DC/T ratio. ICA (12.5~100μg/ml) could inhibited the proliferation of B-MD-C1 (ADR+/+) cells in vitro (P<0.05), the growth inhibition was in a time-and dose-dependent manner. The greatest inhibition rate was 78.74% observed in the treatment group of B-MD-C1 (ADR+/+) cells with the concentration of 100μg/ml ICA for 72 hours.
6 ICA could enhance the susceptibility of B-MD-C1 (ADR+/+) cells to CIK in concentration-dependent manner and E/T-dependent manner, compared to control group, the difference was significant (P<0.05).
7 The results analized by semi-quantitative RT-PCR showed that CD54, CD18 mRNA were up regulated after B-MD-C1 (ADR+/+) cells were treated with different concentrations of ICA for 12h, there was statistically significant differences compared to the control group (P<0.05).
8 The immunosuppression models of the mice that were induced by cyclophosphamide were successfully established. The locomotor activity of the mice of the model group were feebly, the foodintake and hydroposia were not so good, one of them was died in the 6th day, however, the control group, positive group and experimental group were normal, none of them was died.
9 Great changes have taken place in thymus of the mice of model group, and there were apomorphosis and necrosis of adipocyte around the thymus; there were partial fill of adipose tissue in the thymus of ICA low dose group, and no necrosis obviously, there were no changes in thymus of high dose, middle dose and Shenqi Fuzheng Injection groups.
10 ConA enhanced obviously the proliferation of spleen T cell of high dose of ICA, middle dose and Shenqi Fuzheng Injection groups, compared to model group, the difference was significant (P<0.05); and LPS could enhanced the proliferation of spleen T cell of high dose of ICA, compared to model group, the difference was significant (P<0.05), however, it has no effects on the proliferation of spleen B cell of middle dose of ICA and low dose(P>0.05).
11 Compared with control group, the level of TNF-αand IL-12 that were produced by peritoneal macrophage of the mice of model group was decreased markedly, after treated with ICA for 10 days, there was no significance difference in the experimental group, positive group and control group(P>0.05).
12 The cytotoxicity of peritoneal macrophage of the mice of model group was decreased markedly(P<0.01), ICA of different concentration could all enhanced the cytotoxicity of peritoneal macrophage, compared to model group, the difference was significant(P<0.05); high dose and positive group, compared with control group, there was no significance difference(P>0.05).
13 In the bone marrow and peripheral blood smear, we can see the karyocyte hyperplasia in the control group, but hematopoietic depression in the model group; after treated with ICA, the hematopoietic depression was improved.
14 Result of automated blood cell counter (ABCC) revealed that the WBC, RBC and PLT of model group were all decreased markedly (P<0.01), after treated with ICA for 10 days, all of them were improved.
15 After treated with Cy for 10 days, the number of bone marrow cells in single femur of NS group was 7.03×106/femur, compared with control group (10.36×106/femur) was decreased markedly(P<0.01), after treated with middle dose, high dose of ICA and Shenqi Fuzheng Injection, the number of bone marrow cells in the single femur, compared with control group, there was no significance difference(P>0.05).
Conclusion:
1 ICA induced the differentiation and maturation of DCs derived from CBMC in vitro, up-regulated the surface markers, cytokines production and increased the ability to stimulate the proliferation of T cell.
2 ICA could up-regulated the expression of LFA-1/ICAM-1 on the B-MD-C1 (ADR+/+) cells, enhance the immunogenicity of them, and make them easy to recognized by immunologically competent cell.
3 ICA protected immune organs of immunosuppression mice.
4 ICA could inhibit the proliferation of B-MD-C1 (ADR+/+) cells in vitro and in a dose-and time-dependent manner, and arrest cell cycle of B-MD-C1 (ADR+/+) cells at S phase.
5. ICA enhanced T and B cell proliferation of mice spleen significantly, and the killing ability of peritoneal macrophage of the mice as well, stimulated the production of TNF-αand IL-12, enhanced the function of cellular immunity and humoral immunity of immunosuppression mice.
6 ICA improved the hematopoiesis of bone marrow of the mice, raised the number of bone marrow and peripheral blood cells, and indicated that it was good at improving the hematopoiesis of bone marrow.
7 ICA possessed immunologic enhancement significantly, and great potent in clinical application.
方法:
1在无菌条件下采集健康足月妊娠产妇脐血,用密度梯度离心法获得脐血单个核细胞,用GM-CSF、IL-4诱导获得未成熟的树突状细胞(dendritic cells, DCs)。
2采用光镜及透射电镜(TEM)观察未处理组、TNF-α刺激组、ICA刺激组DCs的形态学变化。
3采用流式细胞技术(flow cytometry,FCM)检测未处理组、TNF-α刺激组、ICA刺激组DCs表面CD1a、CD83、CD80、CD86的表达情况;检测经不同浓度ICA(12.5、25、50、100μg/ml)作用12小时后人子宫内膜癌耐药细胞株B-MD-C1(ADR+/+)表面CD54、CD18的表达情况以及细胞周期分布的情况。
4采用ELISA方法检测未处理组、TNF-α刺激组、ICA刺激组DCs培养上清中IL-12和IFN-γ水平。
5采用四甲基偶氮唑蓝法(MTT)检测未处理组、TNF-α刺激组、ICA刺激组DCs刺激T细胞增殖的能力;检测不同浓度ICA(12.5、25、50、100μg/ml),处理不同时间(24、48和72h)对B-MD-C1(ADR+/+)细胞增殖的抑制作用。
6采用乳酸脱氢酶(LDH)释放法检测B-MD-C1(ADR+/+)细胞经不同浓度ICA(12.5、25、50、100μg/ml)作用12小时后,CIK细胞对其杀伤活性的变化。
7采用逆转录-聚合酶链反应(revers transcription PCR,RT-PCR)半定量检测不同浓度ICA(0、12.5、25、50μg/ml)作用12小时后,B-MD-C1(ADR+/+)细胞CD54、CD18 mRNA表达的变化。
8将60只7-8周龄C57BL/6j小鼠随机分为6组,建立小鼠免疫抑制模型,其中实验组、阳性对照组和模型对照组均于腹腔注射环磷酰胺(Cy,300mg/kg);第二天,实验组通过灌胃方式给予不同剂量ICA(150、80、40mg/kg·day),阳性对照组小鼠经腹腔注射参芪扶正注射液,(1ml/day)。模型对照组小鼠给予等量生理盐水(NS),连续给药10d,正常对照组小鼠不做任何处理,正常饲养。每天观察小鼠生存状态,于末次给药12小时后引颈处死小鼠,分离胸腺、脾、腹腔巨噬细胞、骨髓、外周血,备用。
9采用HE染色光镜下观察小鼠胸腺组织结构变化。
10采用MTT法检测小鼠脾淋巴细胞的增殖反应。
11采用ELISA方法检测小鼠腹腔巨噬细胞产生TNF-α、IL-12的水平。
12采用乳酸脱氢酶(LDH)释放法检测小鼠腹腔巨噬细胞杀伤能力的变化。
13采用瑞氏-姬姆萨染色法观察小鼠骨髓和外周血涂片细胞形态变化。
14采用全自动血液分析仪检测小鼠外周血红细胞、白细胞和血小板水平的变化。
15光镜下计数小鼠单根股骨髓细胞(BMC)数量变化。
结果:
1人脐血单个核细胞(CBMC)经GM-CSF、IL-4诱导5天后呈现典型未成熟树突状细胞形态,细胞体积较小,表面毛刺状突起较少,细胞成簇分布,呈集落样生长。
2 CBMC在体外诱导培养10天时,经ICA刺激5d产生具有典型成熟特征的DCs,细胞体积较大,形态不规则,表面有大量细而长的树突状突起,胞浆线粒体、粗面内质网丰富,核异染色质边集于核膜下,溶酶体和空泡少见。
3表型分析发现,ICA刺激组DCs具有成熟的特征性表面分子CD1a、CD83、CD80和CD86的表达,表达水平与未处理组DCs相比均显著增加(P<0.05),CD80表达水平最高达(90.44±0.81)%;随着ICA作用浓度的增加,B-MD-C1(ADR+/+)细胞表面CD54、CD18表达水平逐渐升高,各浓度处理组之间细胞的CD54、CD18表达水平具有显著性差异(P<0.01),各浓度处理组与对照组细胞之间CD54、CD18的表达水平具有显著性差异(P<0.01)。流式细胞分析结果显示:ICA能使B-MD-C1(ADR+/+)细胞生长周期被阻滞在S期。
4 ICA刺激组DCs培养上清中IL-12和IFN-γ的含量均明显高于对照组DCs(P<0.01),但与TNF-α刺激组DCs无显著性差异(P>0.05)。
5经ICA刺激获得的DCs可明显刺激T细胞增殖,且随DCs与T细胞比例增加而增强;在12.5~100μg/ml浓度范围内ICA能显著抑制B-MD-C1(ADR+/+)体外增殖反应(P<0.05),抑制率与ICA呈明显的浓度和时间依赖性,其中100μg/ml ICA作用72h的抑制率最高,达到78.74%。
6与非ICA处理组相比,12.5~100μg/ml浓度的ICA对B-MD-C1(ADR+/+)细胞对CIK具有明显的杀伤增敏作用。在不同效靶比5∶1、10∶1、20∶1时,CIK对B-MD-C1(ADR+/+)细胞的杀伤率随着ICA浓度及效靶比的增加而增加,并与对照组相比具有显著性差异(P<0.01);
7随着ICA作用浓度的增加,B-MD-C1(ADR+/+)细胞CD54、CD18 mRNA表达水平逐渐升高,各浓度处理组与对照组细胞之间CD54、CD18 mRNA的表达水平具有显著性差异(P<0.05);
8成功建立了环磷酰胺诱导的小鼠免疫抑制模型,模型组小鼠活动能力差,进食饮水状况不佳,在第6天时有一只死亡;正常对照组、阳性对照组、实验组小鼠的活动、进食、饮水均正常活跃,全部存活。
9模型对照组小鼠胸腺组织结构发生明显改变,胸腺周围出现脂肪细胞变性和坏死,低浓度(40mg/kg·day)ICA处理组小鼠胸腺组织仍可见部分脂肪组织填充,但未见明显坏死组织。参芪扶正注射液和中、高剂量ICA处理组小鼠胸腺组织形态正常,未见明显改变。
10 ConA可明显促进高(150mg/kg·day)、中剂量(80mg/kg·day)ICA和参芪扶正注射液处理组小鼠脾脏T细胞增殖反应,与模型对照组相比,有显著性差异(P<0.05);LPS可促进高剂量ICA组小鼠B细胞的增殖反应,与模型对照组相比有显著性差异(P<0.05),中、低剂量ICA处理后,对小鼠脾B细胞增殖反应无显著影响(P>0.05)。
11与正常组小鼠相比,模型组小鼠腹腔巨噬细胞产生TNF-α、IL-12的水平明显降低(P<0.01),经ICA处理10天后,实验组小鼠腹腔巨噬细胞产生TNF-α、IL-12的水平与阳性对照组及正常对照组相比无显著性差异(P>0.05)。
12与正常组小鼠相比,模型对照组小鼠腹腔巨噬细胞的杀伤活性显著降低(P<0.01),不同浓度ICA均能提高巨噬细胞的杀伤活性,与模型对照组相比具有显著性差异(P<0.05);高浓度ICA处理组和阳性对照组小鼠巨噬细胞的杀伤活性与正常对照组相比,无明显差异(P>0.05)。
13小鼠骨髓和外周血涂片可见,正常组小鼠骨髓涂片中有核细胞增生活跃,模型对照组小鼠骨髓呈现造血抑制状态。经ICA处理后,实验组小鼠骨髓涂片和外周血涂片中均可见骨髓抑制状态有所改善。
14外周血常规分析结果显示:模型组小鼠WBC、RBC和PLT较对照组明显降低(P<0.01),而高、中、低浓度ICA均可明显提高实验组小鼠外周血WBC、RBC和PLT数量。
15于小鼠腹腔注射Cy10天后,盐水对照组小鼠单根股骨骨髓细胞数量为7.03×106/femur,与对照组小鼠(10.36×106/femur)相比明显降低,差异有显著性(P<0.01),经高、中浓度ICA和参芪扶正注射液治疗后,小鼠单根股骨髓细胞数量与对照组相比,无显著性差异(P>0.05)。
结论:
1 ICA可在体外诱导CBMC来源的DCs成熟,上调DCs表面分子表达,促进DCs细胞因子的分泌,增强其刺激T细胞增殖的能力。
2 ICA可以上调B-MD-C1(ADR+/+)细胞CD54、CD18蛋白和基因的表达,增强该细胞免疫原性,有利于免疫活性细胞的识别杀伤。
3在一定浓度范围内ICA能抑制B-MD-C1(ADR+/+)细胞的生长,能使B-MD-C1(ADR+/+)细胞的生长周期被阻滞在S期,具有明显的量效和时效关系。
4 ICA对免疫抑制小鼠的免疫器官具有很好的保护作用。
5 ICA可显著刺激小鼠脾T、B细胞的增殖,增强腹腔巨噬细胞的杀伤能力,显著增加免疫抑制小鼠腹腔巨噬细胞产生TNF-α和IL-12等细胞因子的含量,可明显增强免疫抑制小鼠细胞免疫和体液免疫功能。
6 ICA可显著改善免疫抑制小鼠的骨髓造血状态,提高骨髓细胞和外周血细胞数量,可明显恢复和改善造血功能。
7 ICA显著增强免疫功能,提示其可作为抗肿瘤药物,并具有良好的安全性,有潜在的临床应用价值。
Objective: In order to study the effect of ICA on immune system and immunocyte in vitro and in vivo. Meanwhile, we approached immunoregulation mechanism of ICA initially. For the final purpose, this study provides some experimental bases to clinical application of ICA to cure certain diseases, such as tumor.
Methods:
1 The cord blood monocytes, which were collected from health and uterogestation parturient were isolated by density gradient centrifugation using lymphocyte isolating solution under axenic condition, and dendritic cells (DCs) were induced by GM-CSF and IL-4.
2 The morphological characteristics of DCs from untreated group, TNF-αgroup and ICA group were observed under inverted microscope and transmission election microscope.
3 Expressions of CD1a, CD83, CD80 and CD86 on DCs of untreated group, TNF-αgroup and ICA group, and the cell cycles and expressions of CD54, CD18 on B-MD-C1 (ADR+/+) cells treated by ICA (0, 12.5, 25, 50, 100μg/ml) for 12hours were detected by flow cytometry.
4 The level of IL-12 and IFN-γin the culture supernatant of DCs from untreated group, TNF-αgroup and ICA group were detected by ELISA.
5 The proliferation of T cells stimulated by DCs, and the inhibition effect of ICA on cell growth of B-MD-C1 (ADR+/+) in treated or untreated groups (12.5, 25, 50, 100μg/ml ICA and control) for different treatment times (24h, 48h and 72h) were determined by MTT method.
6 The cytotoxicity of CIK cells against B-MD-C1 (ADR+/+) cell, which were treated by ICA (0, 12.5, 25, 50, 100μg/ml) for 12hours were investigated by LDH release method.
7 Expression of CD54, CD18 mRNA in B-MD-C1 (ADR+/+) cells after treated with ICA (0, 1.25, 25, 50μg/ml) for 12h were detected by semi-quantitative RT-PCR.
8 The sixty C57BL/6j mice of 7-8-week-old were divided into six groups randomly, and constructed immunosuppression models on them besides control group, the others were divided into experimental group, positive group and model group, and then the three groups were injected with cyclophosphamide (Cy) in abdominal cavity one time (300mg/kg). In the second day, the mice of experimental group were given ICA (150, 80, 40 mg/kg·day) by intragastric administration, the positive group were given Shenqi Fuzheng Injection (1ml/day); the model group were given normal saline (NS). All the mice were treated for 10 days. The mice of control group were raised normally. To observe the survival condition of the mice everyday, after the lasted given drugs for 12 hours, all the mice were killed, and the thymus, spleen, peritoneal macrophage, bone marrow and peripheral blood were removed for the succedent experiments.
9 Morphology of thymus of the mice were observed under optical microscope after HE staining.
10 The proliferation of lymphocyte of the mice were determined by MTT method.
11 The level of TNF-αand IL-12 that were produced by peritoneal macrophage of mice were detected by ELISA.
12 The cytotoxicity of the peritoneal macrophage of the mice were investigated by LDH release method.
13 Morphological images of bone marrow and peripheral blood smears of the mice were detected by Wright-Giemsa’s staining.
14 Population of WBC, RBC and PLT in the peripheral blood were detected with automated blood cell counter (ABCC).
15 The number of bone marrow cells in the single femur was counted under microscope.
Results:
1 After 5 days cultured with GM-CSF and IL-4, the human CBMC developed into immature DCs with typical morphological characteristics, the cells were small and a little dendrites on the cellular surface, and they were grown in clustering.
2 After 10 days cultured with cytokines and ICA, the CBMC that were stimulated by ICA for 5 days developed into mature DCs with typical morphological characteristics, the cells enlarged and were irregular in shape, many thin and long dendrites on the cellular surface, abundance of cytoplasm with more mitochondrion and rough endoplasmic reticulum, the caryon heterochromatin were under the karyolemma, and fewer lysosome and vacuolus.
3 The result of phenotype analysis showed that the expressions of CD1a, CD83, CD80 and CD86 on mature DCs surface were up-regulated significantly by ICA, compared to the control group untreated with stimulating factor(P<0.05), in which most significant change occurred at CD80, reached (90.44±0.81)%. The expression of CD54 and CD18 on B-MD-C1 (ADR+/+) cells surface were also up-regulate by ICA in concentration-dependent manner. The expression of CD54 and CD18 on B-MD-C1 (ADR+/+) cells surface between every treated group, the difference was significant(P<0.01), the cells after treatment with different doses of ICA, compared to control group, the difference was significant(P<0.01). The results of FCM showed that ICA could arrest cell cycle of B-MD-C1 (ADR+/+) cells at S phase.
4 ICA enhanced obviously the IL-12 and IFN-γproduction by DCs(P<0.01), but there was no significance difference between ICA group and TNF-αgroup(P>0.05).
5 ICA-treated DCs stimulated markedly the proliferation of T cell (P<0.05), and the ability displayed rising tendency with the DC/T ratio. ICA (12.5~100μg/ml) could inhibited the proliferation of B-MD-C1 (ADR+/+) cells in vitro (P<0.05), the growth inhibition was in a time-and dose-dependent manner. The greatest inhibition rate was 78.74% observed in the treatment group of B-MD-C1 (ADR+/+) cells with the concentration of 100μg/ml ICA for 72 hours.
6 ICA could enhance the susceptibility of B-MD-C1 (ADR+/+) cells to CIK in concentration-dependent manner and E/T-dependent manner, compared to control group, the difference was significant (P<0.05).
7 The results analized by semi-quantitative RT-PCR showed that CD54, CD18 mRNA were up regulated after B-MD-C1 (ADR+/+) cells were treated with different concentrations of ICA for 12h, there was statistically significant differences compared to the control group (P<0.05).
8 The immunosuppression models of the mice that were induced by cyclophosphamide were successfully established. The locomotor activity of the mice of the model group were feebly, the foodintake and hydroposia were not so good, one of them was died in the 6th day, however, the control group, positive group and experimental group were normal, none of them was died.
9 Great changes have taken place in thymus of the mice of model group, and there were apomorphosis and necrosis of adipocyte around the thymus; there were partial fill of adipose tissue in the thymus of ICA low dose group, and no necrosis obviously, there were no changes in thymus of high dose, middle dose and Shenqi Fuzheng Injection groups.
10 ConA enhanced obviously the proliferation of spleen T cell of high dose of ICA, middle dose and Shenqi Fuzheng Injection groups, compared to model group, the difference was significant (P<0.05); and LPS could enhanced the proliferation of spleen T cell of high dose of ICA, compared to model group, the difference was significant (P<0.05), however, it has no effects on the proliferation of spleen B cell of middle dose of ICA and low dose(P>0.05).
11 Compared with control group, the level of TNF-αand IL-12 that were produced by peritoneal macrophage of the mice of model group was decreased markedly, after treated with ICA for 10 days, there was no significance difference in the experimental group, positive group and control group(P>0.05).
12 The cytotoxicity of peritoneal macrophage of the mice of model group was decreased markedly(P<0.01), ICA of different concentration could all enhanced the cytotoxicity of peritoneal macrophage, compared to model group, the difference was significant(P<0.05); high dose and positive group, compared with control group, there was no significance difference(P>0.05).
13 In the bone marrow and peripheral blood smear, we can see the karyocyte hyperplasia in the control group, but hematopoietic depression in the model group; after treated with ICA, the hematopoietic depression was improved.
14 Result of automated blood cell counter (ABCC) revealed that the WBC, RBC and PLT of model group were all decreased markedly (P<0.01), after treated with ICA for 10 days, all of them were improved.
15 After treated with Cy for 10 days, the number of bone marrow cells in single femur of NS group was 7.03×106/femur, compared with control group (10.36×106/femur) was decreased markedly(P<0.01), after treated with middle dose, high dose of ICA and Shenqi Fuzheng Injection, the number of bone marrow cells in the single femur, compared with control group, there was no significance difference(P>0.05).
Conclusion:
1 ICA induced the differentiation and maturation of DCs derived from CBMC in vitro, up-regulated the surface markers, cytokines production and increased the ability to stimulate the proliferation of T cell.
2 ICA could up-regulated the expression of LFA-1/ICAM-1 on the B-MD-C1 (ADR+/+) cells, enhance the immunogenicity of them, and make them easy to recognized by immunologically competent cell.
3 ICA protected immune organs of immunosuppression mice.
4 ICA could inhibit the proliferation of B-MD-C1 (ADR+/+) cells in vitro and in a dose-and time-dependent manner, and arrest cell cycle of B-MD-C1 (ADR+/+) cells at S phase.
5. ICA enhanced T and B cell proliferation of mice spleen significantly, and the killing ability of peritoneal macrophage of the mice as well, stimulated the production of TNF-αand IL-12, enhanced the function of cellular immunity and humoral immunity of immunosuppression mice.
6 ICA improved the hematopoiesis of bone marrow of the mice, raised the number of bone marrow and peripheral blood cells, and indicated that it was good at improving the hematopoiesis of bone marrow.
7 ICA possessed immunologic enhancement significantly, and great potent in clinical application.
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2.宁鹤丽,林洪生.关于肿瘤化疗辅助中药新药临床疗效指标的研究与分析[J].中国新药杂志,2008, 17(11):821-824
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