香菇C91-3菌丝发酵液蛋白LFP91-3C小鼠体内抗肿瘤免疫机制研究

目的探讨香菇C91-3菌丝发酵液蛋白LFP91-3C的体内抗肿瘤免疫机制。方法H22瘤细胞荷瘤纯系BALB／C小鼠建立动物模型,随机分为生理盐水（NS）对照组、环磷酰胺（CTX）治疗组和LFP91-3C治疗组,观察LFP91-3C对荷瘤小鼠生存期、实体瘤块生长抑制及病理、荷瘤小鼠免疫细胞（NK细胞活性、淋巴细胞增殖率）和免疫因子（血清IL-2、IFN-γ含量）的影响。结果LFP91-3C能显著延长荷瘤小鼠的生存期,抑制实体肿瘤的生长,可在病理切片看到大量的炎细胞浸润,以及NK细胞活性、淋巴细胞增殖率、血清IL-2和IFN-γ含量也显著提高。与NS对照组和CTX治疗组比较差异有显著性。结论LFP91-3C能通过激活机体免疫系统来实现其抗肿瘤的作用。     

灵芝多糖对荷瘤小鼠肿瘤免疫系统的影响

目的:观察灵芝多糖对荷瘤小鼠免疫系统的影响.方法:纯系BALB/c小鼠30只,用U14瘤细胞荷瘤建立动物模型,随机平均分为三组:灵芝多糖治疗组、环磷酰胺治疗组、生理盐水对照组.观察灵芝多糖对荷瘤小鼠NK细胞活性、淋巴细胞转化率、TNF-α等免疫指标的影响.结果:灵芝多糖能够显著提高治疗组小鼠的NK细胞活性、淋巴细胞转化率和血清中TNF-α、IL-2的含量.结论:灵芝多糖能够显著提荷瘤小鼠免疫系统的活性.     

香菇C91-3菌丝发酵液提取蛋白抗小鼠宫颈癌U14的初步探讨

目的 探讨香菇C91-3菌丝发酵液提取蛋白对小鼠宫颈癌的作用.方法 观察香菇C91-3菌丝发酵液提取蛋白对小鼠宫颈癌U14荷瘤小鼠生存期的影响和对体外培养的小鼠宫颈癌U14细胞的抑杀作用.结果 香菇C91-3菌丝发酵液提取蛋白能明显延长小鼠宫颈癌U14荷瘤小鼠的生存期并能对体外培养的小鼠宫颈癌U14细胞有直接抑杀作用.结论 香菇C91-3菌丝发酵液提取蛋白对机体有调节、增强机体免疫系统功能的作用.     

香菇C91-3菌丝发酵蛋白对H22肿瘤细胞体内外抗肿瘤机制的初探

目的探讨香菇C91-3菌丝发酵蛋白(LFP91-3)对H22肿瘤细胞抑瘤作用及相关机制。方法应用不同剂量LFP91-3(50、100和150μg)对H22荷瘤小鼠腹水瘤模型和腋下实体瘤进行治疗,观察其生存期和体内抑瘤作用;并用MTT法(LFP91-3浓度5、10、15μg/mL)和流式细胞仪对经LFP作用的H22肿瘤细胞进行观察和检测。结果香菇C91-3菌丝发酵提取蛋白(LFP91-3)能延长H22荷瘤小鼠的生存期,对体外培养的H22有直接杀伤作用,抑瘤率出现对浓度和时间的依赖性。LFP91-3能将H22肿瘤细胞株细胞周期阻滞到S期并诱导出细胞凋亡。结论 LFP91-3在体内、外对H22肿瘤细胞有较好的抑瘤作用,主要是诱导其调亡。     

灵芝发酵液多糖提取物对荷瘤小鼠细胞免疫的动态观察

目的:观察灵芝发酵液多糖提取物对S180荷瘤小鼠部分免疫指标的动态调节作用,探讨其抗肿瘤机制.方法:S180瘤细胞荷瘤昆明小鼠80只建立动物模型,生理盐水组(NS组)与灵芝发酵液多糖组(GFG组)各40只,分别于荷瘤后第4,7,10,13,16天每组各处死8只小鼠,检测GFG对NK细胞活性、淋巴细胞转化率的影响.结果:GFG能显著提高荷瘤小鼠NK细胞活性和淋巴细胞转化率.随荷瘤时间延长,GFG组较NS组能维持较高水平(P＜0.01),但总体呈下降趋势.结论:灵芝发酵液多糖提取物能显著提高NK细胞活性和淋巴细胞转化率,并维持一定水平.     

刹毒草合剂对免疫抑制小鼠免疫功能的影响

目的:研究刹毒草合剂对免疫抑制小鼠免疫功能的影响。方法:BALB/C小鼠50只随机分为5组(n=10):空白组、模型组、刹毒草合剂低、中、高剂量组。除空白对照组外,其余各组小鼠腹腔注射环磷酰胺40 mg/kg建立免疫抑制模型。空白组和模型组灌胃生理盐水,刹毒草合剂低、中、高剂量组每天分别用刹毒草合剂10、20、40ml/kg灌胃,连续给药15 d后,检测小鼠外周血白细胞数和白介素-2(IL-2)、肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)含量;检测小鼠脾脏指数、NK细胞活性。结果:与模型组比较,刹毒草合剂能显著增加外周血白细胞数和IL-2、TNF-α、IFN-γ含量、提高脾脏指数和NK细胞活性,差异均有统计学意义(P0.05,P0.01)。结论:刹毒草合剂可明显增强环磷酰胺诱导的免疫抑制小鼠的免疫功能。     

羧甲基茯苓多糖的制备及体内抗肿瘤作用的实验研究

目的 :探讨羧甲基茯苓多糖的制备方法及体内抗肿瘤作用。方法 :选择性氧化茯苓菌发酵液得羧甲基茯苓多糖 ,检测三个剂量的羧甲基茯苓多糖 (2 mg/ ml、4mg/ ml、6mg/ ml)对 H2 2 荷瘤小鼠淋巴细胞转化率和 NK细胞杀伤活性以及血清中 TNF-α含量的影响。结果 :羧甲基茯苓多糖三个剂量组均可提高荷瘤小鼠的淋巴细胞转化率和 NK细胞杀伤活性 ,与生理盐水组相比差异有显著性 (P<0 .0 5) ,中剂量组抗肿瘤的效果显著 ,与其他两组相比差异有显著性 (P<0 .0 5) ;羧甲基茯苓多糖三个剂量组均可提高荷瘤小鼠血清中 TNF-α的含量 ,与生理盐水组相比差异有显著性 (P<0 .0 5) ,但三个剂量组之间比较差异无显著性 (P<0 .0 5)。结论 :羧甲基茯苓多糖可改善荷瘤小鼠的免疫功能 ,具有抗肿瘤作用 ,且功效与作用剂量间有一定的关系 ,在最佳剂量时活性最高     

火针层孔菌（桑黄）粗多糖对荷瘤小鼠的免疫调节研究

研究了火针层孔菌不同粗多糖对荷瘤小鼠的免疫调节作用。荷瘤小鼠随机分为四组:胞外粗多糖组、菌丝体粗多糖组、子实体粗多糖组和生理盐水阴性对照组。给药10d后测定荷瘤小鼠脾NK细胞活性和脾淋巴细胞的增殖率。结果显示火针层孔菌粗多糖组与阴性对照组相比能够提高小鼠脾NK细胞活性和脾淋巴细胞的增殖率(P<0.01),表明火针层孔菌液体发酵粗多糖和子实体粗多糖对荷瘤小鼠免疫功能均有调节作用。     

板蓝根多糖体内抗肿瘤作用与免疫功能调节实验研究

本研究主要探讨板蓝根多糖(RIP)对荷瘤小鼠的抗肿瘤作用及其对免疫功能的影响。通过建立S180小鼠移植瘤和腹水瘤模型,以环磷酰胺(CTX)为阳性对照药,观察RIP对其的影响。连续给药12 d后,测定移植瘤小鼠的瘤重,计算肿瘤生长抑制率和胸腺、脾脏指数;检测脾淋巴细胞的转化功能和NK细胞杀伤活性以及血清中的TNF-α、INF-γ、IL-2水平;并对肿瘤组织进行HE染色和细胞周期分析;对进行相同给药周期的腹水瘤小鼠继续正常饲养,记录各组小鼠自然死亡的时间。结果显示,不同剂量的RIP均可明显抑制小鼠肿瘤的生长,其100 mg/kg和50 mg/kg剂量组的抑瘤率分别为35.4%,38.5%;各剂量组移植瘤小鼠胸腺指数和脾指数与对照组比较有所提高,还能刺激脾淋巴细胞的转化及增强NK细胞的杀伤活性,升高血清中TNF-α、INF-γ和IL-2的含量,其50 mg/kg组与模型对照组相比有统计学差异(P0.01)。此外,移植瘤组织HE染色观察可见各给药组肿瘤组织坏死面积呈不同比例增大,流式细胞仪检测出给药后G_0/G_1期细胞比例增加,S期细胞比例降低,G_2/M期细胞周期未见明显变化。由此提示,RIP能够增强荷瘤小鼠的免疫功能,对荷瘤小鼠具有抗肿瘤作用,能延长荷瘤小鼠的生存时间。     

GM-CSF与IL-21基因共转染人卵巢癌细胞在裸鼠体内的抗瘤效应

目的:研究GM-CSF与IL-21基因共转染人卵巢癌细胞系SKOV3后在裸鼠体内的抗肿瘤效应.方法:将体外培养的SKOV3、SKOV3/Neo、SKOV3/GM-CSF、SKOV3/IL21及SKOV3/GM-CSF-IL21细胞分别皮下接种BALB/c裸小鼠,监测荷瘤鼠肿瘤生长情况,观察GM-CSF与IL-21单独及联合诱导的抗肿瘤效应.ELISA法检测血清IFN-γ和TNF-α含量,MTT比色法测定裸鼠脾细胞NK活性,RT-PCR检测裸鼠脾细胞中NKG2D分子的表达.结果:与SKOV3及SKOV3/Neo相比,用SKOV3/GM-CSF、SKOV3/IL21和SKOV3/GM-CSF-IL21攻击的裸鼠,体内肿瘤形成受到明显抑制,其中SKOV3/GM-CSF-IL21组更明显,与其他各组比较均有显著差异;用SKOV3/GM-CSF-IL21攻击的裸鼠外周血,白细胞总数、中性粒细胞数、单核细胞数及IFN-γ和TNF-α含量均显著升高,脾细胞NK活性及NKG2D表达增强.结论:GM-CSF与IL-21基因共转染SKOV3细胞后,可在裸鼠体内诱导明显的抗肿瘤效应,其效果显著优于单-GM-CSF或IL-21基因转染的SKOV3细胞.     

Potent induction of TNF-α during interaction of immune effectors with oral tumors as a potential mechanism for the loss of NK cell viability and function

The inhibitory role of TNF-α on survival of naïve and IL-2 treated NK cells has been demonstrated in the past. However, its effect on the function of these cells against tumor cells, in particular against oral tumors has not been established. We investigated the significance of secreted TNF-α in death and functional loss of splenocytes and NK cells in ex-vivo cultures with oral tumors. Oral tumors trigger potent secretion of TNF-α by human and murine immune effectors. Absence of TNF-α increases the cytotoxic activity and secretion of IFN-γ by IL-2 treated splenocytes and NK cells in co-cultures with MOK L2D1+/p53?/? oral tumor cells. IL-2 treated splenocytes and NK cells from TNF-α ?/? mice survive and proliferate more when compared to cells from TNF-α +/+ mice. Cell death induced by F. nucleatum, an oral bacteria, in TNF-α ?/? splenocytes are considerably lower than that induced in TNF-α +/+ splenocytes where potent release of TNF-α is reproducibly observed. Addition of exogenous rTNF-α to IL-2 treated splenocytes and NK cells decreased survival and function of splenocytes and NK cells obtained from TNF-α ?/? mice against oral tumors. These findings suggest that potent induction of TNF-α during interaction of immune effectors with oral tumors and/or oral bacteria is an important factor in decreasing the function and survival of cytotoxic immune effectors. Strategies to neutralize TNF-α may be beneficial in the treatment of oral cancers.     

Alleviation of lipopolysaccharide/d-galactosamine-induced liver injury in leukocyte cell-derived chemotaxin 2 deficient mice

Leukocyte cell-derived chemotaxin 2 (LECT2) is a secreted pleiotropic protein that is mainly produced by the liver. We have previously shown that LECT2 plays an important role in the pathogenesis of inflammatory liver diseases. Lipopolysaccharide/d-galactosamine (LPS/d-GalN)-induced acute liver injury is a known animal model of fulminant hepatic failure. Here we found that this hepatic injury was alleviated in LECT2-deficient mice. The levels of TNF-α and IFN-γ, which mediate this hepatitis, had significantly decreased in these mice, with the decrease in IFN-γ production notably greater than that in TNF-α. We therefore analyzed IFN-γ-producing cells in liver mononuclear cells. Flow cytometric analysis showed significantly reduced IFN-γ production in hepatic NK and NKT cells in LECT2-deficient mice compared with in wild-type mice. We also demonstrated a decrease in IFN-γ production in LECT2-deficient mice after systemic administration of recombinant IL-12, which is known to induce IFN-γ in NK and NKT cells. These results indicate that a decrease of IFN-γ production in NK and NKT cells was involved in the alleviation of LPS/d-GalN-induced liver injury in LECT2-deficient mice.     

C5a regulates NKT and NK cell functions in sepsis

Complement, NKT, and NK cells play critical roles in the first line defense against pathogens. Functional roles for both C5a receptors, that is, complement receptor C5a (C5aR) and C5a receptor-like 2 (C5L2), in sepsis have been demonstrated. However, the role of C5a in innate lymphocyte activation during sepsis remains elusive. In this article, we show that naive NKT and NK cells already express high levels of C5aR and minor levels of C5L2 mRNA, but no protein. Upon Escherichia coli-induced sepsis, we found C5aR surface expression on subpopulations of NKT and NK cells, suggesting rapid translation into C5aR protein on bacterial encounter. Importantly, significantly increased survival in the absence of C5aR, NKT, and NK cells, but not of C5L2, was associated with reduced IFN-γ and TNF-α serum levels. Sepsis induction in C5aR(+)/C5aR(-) mixed bone marrow chimeras identified cognate engagement of C5aR on NKT cells as an important factor for the recruitment of NKT cells. Furthermore, we found synergistic interaction between C5aR and TLRs enhancing the production of TNF-α and IFN-γ from NKT and NK cells in cocultures with dendritic cells. Our results identify C5aR activation as a novel pathway driving detrimental effects of NKT and NK cells during early experimental sepsis.     

Activation of TOLLIP by porin prevents TLR2-associated IFN-γ and TNF-α-induced apoptosis of intestinal epithelial cells

Interferon (IFN)-γ and tumor necrosis factor (TNF)-α cause chronic inflammation of the intestine leading to progression of inflammatory bowel disease (IBD), which is manifested through rapid apoptosis of the intestinal epithelial cells (iECs). Here, we show inhibition of IFN-γ and TNF-α-induced apoptosis of INT-407 cells by porin, a microbe-associated molecular pattern (MAMP) with affinity for toll-like receptor (TLR)2 and commonly present in Gram-negative bacteria. Proinflammatory cytokines induce apoptosis by activation of caspase 8 that triggers caspase 9 through Bax finally leading to activation of caspase 3, the executioner caspase. Interestingly, while IFN-γ and TNF-α promotes Bax expression, in contrast porin up-regulates anti-apoptotic Bcl-xL resulting in iEC survivability. We show elevated expression of TLR2 is a key requisite for IFN-γ and TNF-α mediated caspase 8 up-regulation that contributes to apoptosis of iECs. Down-regulation of TLR2 expression is central for checking apoptosis which is achieved by elevated level of toll-interacting protein (TOLLIP) in presence of porin. Attempts to limit IBD is in progress with anti-IFN-γ and anti-TNF-α Abs or use of IL-10. Although probiotic bacterial proteins have shown to successfully reduce IFN-γ and TNF-α mediated apoptosis, the exact mechanism of their action has remained elusive. This study identifies the underlying sequential events of transient TLR2 stimulation followed by its blocking in response to the bacterial outer membrane protein, which advocates intervention at TLR-juncture is crucial for controlling IBD.     

Cytokine release of human NK cells solely triggered with Poly I:C

Natural killer (NK) cells play a crucial role in innate immunity as effectors against tumor cells and pathogen-infected cells. Our data show for the first time that NK cells produce high levels of cytokines interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in response to stimulation with the artificial RNA analogue Poly I:C without additional cytokines or contact to other types of immune cells. An incubation period of 48 h is necessary to induce cytokine release by Poly I:C. These data suggest Poly I:C as a competent direct activator and immunomodulator of NK cell functions.     

Effects of inflammatory factors on mesenchymal stem cells and their role in the promotion of tumor angiogenesis in colon cancer

Mesenchymal stem cells (MSCs), which are modulated by cytokines present in the tumor microenvironment, play an important role in tumor progression. It is well documented that inflammation is an important part of the tumor microenvironment, so we investigated whether stimulation of MSCs by inflammatory cytokines would contribute to their ability to promote tumor growth. We first showed that MSCs could increase C26 colon cancer growth in mice. This growth-promoting effect was further accelerated when the MSCs were pre-stimulated by inflammatory factors IFN-γ and TNF-α. At the same time, we demonstrated that MSCs pre-stimulated by both inflammatory factors could promote tumor angiogenesis in vivo to a greater degree than untreated MSCs or MSCs pre-stimulated by either IFN-γ or TNF-α alone. A hen egg test-chorioallantoic membrane (HET-CAM) assay showed that treatment of MSC-conditioned medium can promote chorioallantoic membrane angiogenesis in vitro, especially treatment with conditioned medium of MSCs pretreated with IFN-γ and TNF-α together. This mechanism of promoting angiogenesis appears to take place via an increase in the expression of vascular endothelial growth factor (VEGF), which itself takes place through an increase in signaling in the hypoxia-inducible factor 1α (HIF-1α)-dependent pathway. Inhibition of HIF-1α in MSCs by siRNA was found to effectively reduce the ability of MSC to affect the growth of colon cancer in vivo in the inflammatory microenviroment. These results indicate that MSCs stimulated by inflammatory cytokines such as IFN-γ and TNF-α in the tumor microenvironment express higher levels of VEGF via the HIF-1α signaling pathway and that these MSCs then enhance tumor angiogenesis, finally leading to colon cancer growth in mice.     

香菇C_（91－3）菌发酵液小鼠体内外抗肿瘤作用的研究

香菇Ｃ_（９１－３）菌是我们多年筛选出的一株经生物发酵后,其发酵液具有明显的抗肿瘤、抗细菌作用的真菌。研究结果表明：该发酵液具有明显体外抗肿瘤作用的活性物质。对ＭＨ_（１３４）、Ｘ５５５３、Ｃａ７６１／Ｌ、ＹＡＣ－１、Ｈ_（２２）、Ｋ５６２抑瘤率为７６．７～１００％。在体内抗肿瘤实验中,采用Ｈ_（２２）、Ｓ１８０腹水瘤的研究中,小鼠存活率分别为４０％、４５％。本文还对香菇Ｃ_（９１－３）菌发酵液抗肿瘤的机理进行了探讨。