Effect of Ionizing Radiation on the Expression of p16, CyclinDI and CDK4 in Mouse Thymocytes and Splenocytes

Objective To investigate the effect of ionizing radiation on the expression of p16, CyclinDl, and CDK4 in mouse thymocytes and splenocytes. Methods Fluorescent staining and flow cytometry analysis were employed for the measurement of protein expression. Results In time course experiments, it was found that the expression of p16 protein was significantly increased at 8, 24, and 48 h for thymocytes (P<0.05, P<0.01, and P<0.05, respectively) and at 24 h for splenocytes (P<0.05) after whole body irradiation (WBI) with 2.0 Gy X-rays. However, the expression of CDK4 protein was significantly decreased from 8 h to 24 h for thymocytes (P<0.05,P<0.01) and from 8 h to 72 h for splenocytes (P<0.05-P<0.01). In dose effect experiments, it was found that the expression of p16 protein in thymocytes and splenocytes was significantly increased at 24 h after WBI with 1.0, 2.0, and 4.0 Gy (P<0.05-P<0.01), whereas the expression of CDK4 protein was significantly decreased with 2.0Gy for thymocytes (P<0.05) and 0.5-6.0 Gy for splenocytes (P<0.05-P<0.01). Results also showed that the expression of CyclinDl protein decreased markedly in both thymocytes and splenocytes after exposure. Conclusion The results indicate that the expression of p 16 protein in thymocytes and splenocytes can be induced by ionizing radiation, and the p16-CyclinD1/CDK4 pathway may play an important role for G1 arrest of thymocytes induced by X-rays.

Immunomodulating property of Ocimum sanctum by regulating the IL-2 production and its mRNA expression using rat's splenocytes

Objective:To investigate the regulatory effect of aqueous extract of leaves of Ocimum sanctum on IL-2 cytokine production in vivo and in vitro,and the effect of leaves extract on general blood picture including T& B lymphocytes.Methods:For in vivo studies albino Wistar rats were treated with aqueous crude leaves extract of Ocimum sanctum for 20 consecutive days.Spleen cells were harvested and assayed for IL-2 production by using sandwich enzyme-linked immunosorbent assay(ELISA) and mRNA expression methods.For in vitro study aqueous Ocimum sanctum leaves extract= in different concentrations(25-500μ/mL) was added into culture plates containing ConA stimulated splenocytes.To study the overall effect on blood picture,density gradient purified lymphocytes analysis and conventional methodology for total and differential leukocyte count and hemoglobin level were also done.Results:It indicated that the rats treated with Ocimum sanctum leaves extract had significantly enhanced(P【0.001) ability of spleen cells to secrete IL-2.Investigation in vitro also showed regulation of IL-2 production.Blood study exhibited leucocytosis and augmentation of T& B lymphocytes by 25%approximately.4-5%increase in Hemoglobin value was also noticed.Conclusion:Aqueous Ocimum sanctum leaves extract may have stimulatory effect on T & B lymphocytes particularly on Th 1 subset of lymphocytes as shown by enhancement in IL-2 production.

Immunopotentiating Activity of Dendrobium Species in Mouse Splenocytes

This study aimed to explore a pharmacological activity marker for quality assurance of Dendrobium species. The immunopotentiating activity in aqueous extracts prepared from four Dendrobium species, including D. officinalis, was assessed by an in vitro assay of concanavalin A (Con A)-stimulated proliferation of mouse splenocytes. Four samples of commercially available Dendrobii Caulis were also analyzed for comparison. The results indicated that the aqueous extract of D. officinalis produced immunopotentiating action, as evidenced by the increase in Con A-stimulated proliferation of mouse splenocytes, with the extent of stimulation being more prominent than those of other tested Dendrobium species and Dendrobii Caulis samples. In conclusion, an in vitro immunopotentiation assay may be used for assessing the pharmacological activity of Dendrobium species. The finding that D. officinalis produced a more potent immunopotentiating action is consistent with its 'yin-nourishing' action in Chinese medicine, which is more effective than other Dendrobium species in clinical use.

A murine model of mixed bone marrow transplant:importance of the splenocytes to balance HVG and GVH reactions

Objective: A murine model of mixing syngeneic and haploidentical major histocompatibility complex (MHC) matched bone marrow cells transplant was used to evaluate the effect of splenocytes in graft-versus-host disease (GVHD) and host-versus-graft reaction (HVGR). Methods: BALB/C recipient mice were lethally conditioned with 8.5 Gy and injected with different grafts which consisted of syngeneic bone marrow cells plus splenocytes (SPLCs) and haploidentical MHC matched bone marrow cells (BMCs)plus different doses of splenocytes. Recipient mice were detected for the percentage of haploidentical MHC matched mouse origin cells in the peripheral blood cells and checked daily for the appearance of GVHD symptoms. Histopathological examination of multiple organs from moribund mice was used to evaluate the grades of GVHD. Results: Recipient mice infused with 10 × 106 haploidentical MHC matched SPLCs and 5×106 syngeneic splenocytes showed a higher level and more stable chimerism with GVHD Ⅱ degree histopathological alterations. Histopathological results of GVHD in other group's hosts were not obvious, and the levels of chimerism were unstable. All of the mice survived over 150 d. Conclusion:The proportion and dose of syngeneic and haploidentical MHC splenocytes are of importance for inducing stable engraftment on the basis of nonlethal GVHD and to balance GVHD and HVGR.

Remarkably Decreased CD11b Positive Splenocytes in Aquaporin 3-Null Mice

Aquaporins（AQPs） are molecular water channels that play important physiological roles in fluid trans-porting organs. The expression and function of AQPs in the immune system are largely unknown. CD 11 （a-d）/CD18 integrins are adhesion molecules expressed on leukocytes, which play a critical role in leukocyte adhesion, migration and host defense. In the present study, we discovered the expression of aquaporin-3（AQP3） on spleen CD1 lb positive cells, and the content of CDllb positive splenocytes in aquaporin 3-null mice is significantly decreased. Further analysis suggested remarkably decreased monocyte/macrophage subpopulation and significantly decreased granulocyte subpopulation. It is the first report suggesting an important role of AQP in the development and maturation of imrnunocytes.

PROLIFERATION OF ANTI－CD_3 McAb AND IL－2 INDUCED SPLENOCYTES AND ANTITUMOR EFFECT OF THEIR CULTURE SUPERNATANTS

The proliferation of splenocytes from health adults was induced by anti-CD3 McAb and IL-2.The proliferative potential of the splenocytes and antitumor activity of their culture supernatants of splenocytes were studied.The results showed that anti-CD3 McAb not only enhanced the proliferation of the splenocytes directly,but also enhanceil that of induced by IL-2.Their enhancing effect was more significant when the incubation time in vitro was prolonged.The culture supernatants of anti-CD3 and IL-2 induced splenocytes also had the antitumor activity and enhancing capability to the antitumor activity of LAK tells.The results suggested that LAK cells could secret lymphokine,and this effect would be synergically promoted when anti-CD3 and IL-2 were simultaneously used.

RESPONSES OF HUMAN FETAL SPLENOCYTES AND THYMOCYTES TO INTERLEUKIN-2: LAK ACTIVITY AND PROLIFERATION

Using cytotoxicity and thymidine uptake assays, we investigated the effects of human recombinant in-terleukin-2 (rIL-2) on the induction of lympholine-activated killer (LAK) activity and cellular proliferation in splenocytes and thymocytes from human fetuses (18-22 weeks). We observed that fetal splenocytes and thymocytes incubated with low doses of rIL-2 (10-100 U ml) developed broad antitumor activity (LAK activity) although the kinetics and magnitudes of the responses were different. It indicated the LAK precursors are present in fetal spleen and thymus. Further, rIL-2 induced a strong proliferative response in splenocytes, but not in thymocytes. On the basis of the findings, we conclude that the responses of fetal splenocytes and thymocytes to IL-2 are different.

Differential Effects of Ursolic Acid and Oleanolic Acid on Mitochondrial ATP Generation in H9c2 Cardiomyocytes and Lipopolysaccharide-Induced Cell Proliferation in Mouse Splenocytes: Yang versus Yin

In the present study, two cell-based systems for assessing Yang and Yin activities were for the first time used to investigate the effect of ursolic acid (UA) and oleanolic acid (OA). The results indicated that while UA was only active in the Yang assay, OA produced activity in the Yin assay. The Yang/Yin activity of UA/OA may be attributed to their distinct molecular structures, which confer their differential ability to interact with mitochondrial membrane or cellular membrane lipids, with resultant membrane fluidization and potentiation of biological responses.

Pharmacological Investigation on the Qi-Invigorating Action of Schisandrin B: Effects on Mitochondrial ATP Generation in Multiple Tissues and Innate/Adaptive Immunity in Mice

Schisandrae Fructus, containing schisandrin B (Sch B) as its main active component, is recognized in traditional Chinese medicine (TCM) for its Qi-invigorating properties in the five visceral organs. Our laboratory has shown that the Qi-invigorating action of Chinese tonifying herbs is linked to increased mitochondrial ATP generation and an enhancement in mitochondrial glutathione redox status. To explore whether Sch B can exert Qi-invigorating actions across various tissues, we investigated the effects of Sch B treatment on mitochondrial ATP generation and glutathione redox status in multiple mouse tissues ex vivo. In line with TCM theory, which posits that Zheng Qi generation relies on the Qi function of the visceral organs, we also examined Sch B’s impact on natural killer cell activity and antigen-induced splenocyte proliferation, both serving as indirect measures of Zheng Qi. Our findings revealed that Sch B treatment consistently enhanced mitochondrial ATP generation and improved mitochondrial glutathione redox status in mouse tissues. This boost in mitochondrial function was associated with stimulated innate and adaptive immune responses, marked by increased natural killer cell activity and antigen-induced T/B cell proliferation, potentially through the increased generation of Zheng Qi.

低HMGB1含量的肿瘤细胞裂解物增强树突状细胞的抗肺癌作用

目的探讨低含量高迁移率族蛋白B1(HMGB1)的肿瘤细胞裂解物(TCL)对树突状细胞抗肺癌作用的影响。方法反复冻融的方法制备Lewis肺癌细胞TCL。Western blot检测TCL中HMGB1蛋白的表达情况。用30 nmol/L甘草酸(GA)抑制Lewis肺癌细胞中HMGB1的表达,进而制备低含量HMGB1的TCL(LH-TCL)。用未处理的Lewis肺癌细胞制备正常含量HMGB1的TCL(NH-TCL)。分为不同剂量NH-TCL负载DC组、LH-TCL负载DC组及PBS对照组,用流式细胞术检测体外诱导的小鼠树突状细胞(DC)表面CD11b、CD11c、CD86表达或细胞凋亡,用酶联免疫吸附试验检测DC细胞IL-12分泌情况。分为NH-TCL刺激DC活化的脾细胞组、LH-TCL刺激DC活化的脾细胞组及PBS对照组,用流式细胞术检测小鼠脾细胞表面CD69的表达情况,酶联免疫吸附试验检测小鼠脾细胞TNF-β的分泌。分为NH-TCL刺激DC活化的脾细胞组、LH-TCL刺激DC活化的脾细胞组及PBS对照组,并设5∶1、10∶1、20∶1三个效靶比进行体外杀伤实验,采用CCK8法检测小鼠脾细胞杀伤肿瘤细胞的活性。分为未活化DC组、NH-TCL活化的DC组、LH-TCL活化的DC组及PBS对照组,通过体内抑瘤实验,评估DC过继免疫细胞疗法抑制小鼠皮下移植瘤生长的效果。结果GA作用后的Lewis肺癌细胞制备的TCL中,HMGB1的含量显著降低(P<0.05)。与NH-TCL相比,LH-TCL具有更好的减少DC凋亡的能力(P<0.001),并促进DC表面CD86的表达和IL-12的分泌,而负载LH-TCL的DC也能更好地激活脾淋巴细胞并诱导更强的抗肿瘤免疫作用(P<0.01)。在体外,负载LH-TCL的DC激活脾淋巴细胞对Lewis肺癌细胞的杀伤活性显著提高(P<0.01);在实验动物体内,免疫负载LH-TCL的DC可明显抑制肿瘤组织生长(P<0.05)。结论用LH-TCL负载DC,可以增强DC诱导的抗肿瘤免疫作用,提高DC过继免疫细胞治疗的疗效,为该免疫治疗方法的改进提供了一个新的途径。

高强度超声介入治疗对宫颈癌小鼠模型肿瘤增殖、免疫功能及脾细胞Th1/Th2亚群的影响

目的 探讨高强度超声介入治疗对宫颈癌小鼠模型肿瘤增殖、免疫功能及脾细胞Th1/Th2亚群的影响。方法 42只宫颈癌小鼠按随机数字表法分为3组-模型组、超声1组与超声2组,每组14只。超声1组与超声2组均给予进行局部高强度超声治疗,超声1组的单点单次治疗时间为10 s,超声2组的单点单次治疗时间为20 s,共治疗15次;模型组不给予超声治疗。所有小鼠均治疗观察4周,记录3组小鼠的体重、肿瘤体积、肿瘤增殖、免疫功能及脾细胞Th1/Th2亚群变化情况。结果 超声1组与超声2组治疗第2周与第4周的小鼠体重均高于模型组,肿瘤体积均小于模型组,超声2组较超声1组变化更显著,差异均有统计学意义(P<0.05)。超声1组与超声2组治疗第4周与第8周的小鼠肿瘤细胞增殖率均低于模型组,超声2组低于超声1组,差异均有统计学意义(P<0.05)。超声1组与超声2组治疗第4周与第8周的CD4^(+)T细胞比例和CD8^(+)T细胞比例高于模型组,超声2组高于超声1组,差异均有统计学意义(P<0.05)。超声1组与超声2组治疗第4周与第8周的Bcl-2、MMP-9蛋白相对表达水平低于模型组,超声2组低于超声1组,差异均有统计学意义(P<0.05)。结论 高强度超声介入在宫颈癌小鼠的应用能抑制Bcl-2、MMP-9蛋白的表达,改善脾细胞Th1/Th2亚群功能,提高小鼠的免疫功能,抑制肿瘤增殖,从而发挥很好的抗癌作用。

Differential Effects of Yin- and Yang-Chinese Tonifying Herbs on Innate and Adaptive Immunity

The present study investigated the effect of treatment with methanolic extracts of Yin- and Yang-Chinese tonifying herbs on concanavalin A (Con A)/lipopolysaccharide (LPS)-stimulated splenocyte proliferation (adaptive immunity) and natural killer (NK) cell activity (innate immunity) in an ex vivo mouse model. The results indicated that while treatment with most Yin herbal extracts potentiated the Con A/LPS-stimulated splenocyte proliferation, only Yang (but not Yin) herbal extracts stimulated NK cell activity. The differential effects of Yin- and Yang-Chinese tonifying herbs on innate and adaptive immunity are consistent with the Chinese medicine theory which depicts the Yin and Yang functional components of Zheng Qi (vital energy), with the Yang component being responsible for the first line of defense against invading microorganisms (i.e., innate immunity) and the Yin oner serving as a follow-up defensive response (adaptive immunity).

复方幺川调免消瘰颗粒的制备与鉴别及促小鼠脾淋巴细胞体外增殖的研究

目的筛选复方幺川调免消瘰颗粒的制备工艺,获得薄层色谱鉴别方法,以及对小鼠脾淋巴细胞体外增殖的影响。方法采用水提工艺获得复方幺川调免消瘰提取物冻干粉,通过正交实验法优化颗粒剂的处方;利用薄层色谱鉴别方法鉴别复方颗粒;用CCK-8法检测复方幺川调免消瘰颗粒对小鼠脾淋巴细胞体外增殖的影响。结果正交实验结果显示,辅料可溶性淀粉与提取粉末用量比为1∶1,使用70%的乙醇作为润湿剂,乙醇占冻干粉的重比为50%;获得复方中甘草与皂角刺的薄层色谱鉴别方法;复方幺川调免消瘰颗粒对LPS诱导下的小鼠脾淋巴细胞增殖具有促进作用。结论筛选工艺优化得到复方幺川调免消瘰颗粒,薄层色谱法鉴别出特征斑点,专属性良好,该复方对小鼠脾淋巴细胞体外增殖具有促进作用。

钩吻素子对小鼠脾细胞增殖反应及体液免疫反应的抑制作用

钩吻素子(Kou) 体外对混合淋巴细胞培养反应、刀豆蛋白A(ConA 2μg/ml) 或细菌脂多糖(LPS10μg/ ml) 诱发的小鼠脾细胞增殖反应均有不同程度的抑制作用,最低抑制浓度分别为10 、5 、40μg/ml。Kou(10 、20 、40 mg/kg ×7d)ip 可降低小鼠血清溶血素的活性,高浓度时Kou

黄芪中一种新葡聚糖的分离纯化与化学结构研究

目的 从中药黄芪中提取免疫活性多糖并研究其化学结构。方法 用乙醇分级沉淀、脱蛋白、CTAB选择性沉淀、离子交换及凝胶过滤柱层析的方法从黄芪水提物提取均一多糖 ;运用凝胶过滤法确定多糖的均一性和分子量 ;运用糖组成分析、甲基化分析、Sm ith降解、IR及 1 3 CNMR等方法研究多糖的结构。结果 得到均一多糖A2 Nb。其平均分子量为 36 0 0 0 0 ,由 D-葡萄糖组成 ,主要连接方式为 α- D- (1→ 4) - Glc,在每 2 5个葡萄糖中有一个 6 -O位上的分枝。结论 从黄芪中首次分离到具有促脾细胞增殖作用的高分子量葡聚糖 A2 Nb。

锌对大鼠胸腺、脾脏淋巴细胞增殖功能影响的研究

从哺乳期开始建立大鼠缺锌（ＺＤ）和高锌（ＺＥ）模型，应用流式细胞术（ＦＡＣＳ）研究微量元素锌对胸腺、脾脏淋巴细胞增殖功能的影响。结果表明：（１）ＺＤ组和ＺＥ组胸腺、脾脏静止期（Ｇ_０、Ｇ_１期）细胞明显增高，而增殖期（Ｓ＋Ｇ_２＋Ｍ期）细胞及增殖指数明显降低，与自由喂养组（ＡＬ）及配对组（ＰＦ）相比差别非常显著。（２）ＺＤ组及ＺＥ组胸腺、脾脏细胞内ＲＮＡ及蛋白质含量降低；细胞内ｃＡＭＰ含量及ｃＡＭＰ／ｃＧＭＰ比值明显增高，脾脏细胞内ｃＧＭＰ含量明显降低，与ＡＬ组及ＰＦ组比较差别非常显著。结果提示：不适量锌可通过影响胸腺、脾脏淋巴细胞内环核苷酸水平，影响正常的细胞合成，使细胞周期正常进行受阻，胸腺、脾脏淋巴细胞增殖受抑，机体免疫功能降低。

白芍总甙对白细胞介素-2产生的影响

白芍总甙(0.5～62.5mg/L)对ConA(3mg/L)诱导的大鼠脾细胞产生白细胞介素-2具有双向调节作用。同时用活化小鼠脾细胞检测了ConA诱导大鼠脾细胞产生白细胞介素-2的纯度,表明,主要以白细胞介素-2为主。

枸杞子活性多糖的研究

目的 对 4个均一枸杞多糖 :LBP 1a 1,LBP 1a 2 ,LBP 3a 1和LBP 3a 2进行结构研究和药理评价。方法采用凝胶柱色谱、酸水解、过碘酸氧化和波谱学等方法测定多糖样品的分子量、单糖组成及连接方式 ,并以小鼠脾淋巴细胞增殖反应为免疫活性指标对多糖样品进行活性评价。结果 4个多糖样品的分子量分别为 11 5× 10 4 ,9 4×10 4 ,10 3× 10 4 ,8 2× 10 4 ,LBP 1a 1,LBP 1a 2为 1,6连接的葡聚糖 ;LBP 3a 1,LBP 3a 2具有 1,4连接的多聚半乳糖醛酸主链 ,及微量的半乳糖和阿拉伯糖分支。 4个多糖均具有免疫促进作用。结论 4种多糖首次从枸杞子中分离得到 ,具有 1。

黄芪总提物的免疫调节作用

目的:研究黄芪总提物(TAE)的免疫调节作用。方法:以DNFB诱导小鼠迟发超敏(DTH)反应作为模型,用耳肿胀度作为主要的检测指标。以3H-TdR掺入法研究TAE体外对ConA或LPS诱导的小鼠脾淋巴细胞增殖反应;以活化脾细胞法检测IL-2。结果:TAE3个剂量对Cy降低或升高的小鼠DTH反应均能恢复或接近正常水平;TAE对亚适浓度ConA或LPS诱导小鼠脾淋巴细胞的增殖反应与产生IL-2均有明显的促进作用,量效曲线均呈典型的钟罩型。结论:TAE具有机能和浓度依赖性的双向免疫调节作用。

当归多糖硫酸酯的合成及其对脾细胞增殖的作用

目的 合成不同取代度的当归多糖硫酸酯 ;探讨氯磺酸对当归多糖硫酸酯化过程的影响和当归多糖及其硫酸酯对小鼠脾细胞增殖反应的作用 .方法 采用氯磺酸 -吡啶法进行硫酸化修饰 ;Ba SO4比浊法测定修饰物的硫含量 ;MTT比色法研究当归多糖及其硫酸酯对小鼠脾细胞增殖反应的影响 .结果 得到 8种不同取代度的当归多糖硫酸酯 ,硫含量为 93.7～ 171.4mg· g- 1 ,产率为 0 .737～ 0 .884g· g- 1 .当归多糖及其硫酸酯在剂量为 5 0 0 mg· L- 1 和 15 0 0 mg· L- 1时 ,给药组光密度明显高于对照组 (P<0 .0 1) ,作用与剂量有良好的正相关性 .结论 提高氯磺酸的比例 ,可以提高产物的硫含量 ,但收率降低 .当归多糖及其硫酸酯对活化的