Toll样受体-9的研究进展

Toll样受体-9(Toll-like receptor 9,TLR9)是哺乳动物TLRs家族中一员,作为细胞表面的天然模式识别受体,主要参与免疫刺激序列(CpG序列)激活免疫细胞的信号传导,从而在天然抗感染免疫及联系天然免疫和获得性免疫中发挥重要作用。通过对TLR9-CpG作用通路的研究,将促进天然免疫机制研究的进一步深入,有利于解决诸如:CpG佐剂、DNA疫苗、CpG抗感染、抑制肿瘤、预防过敏反应等实际应用过程中存在的问题。     

鼻咽癌组织中巨噬细胞移动抑制因子和MMP-2、MMP-9表达的关系

目的　研究巨噬细胞移动抑制因子 (macrophagemigrationinhibitoryfactor,MIF)和MMP 2、MMP 9在鼻咽癌组织中的表达水平及相互关系 ,探讨鼻咽癌细胞早期侵袭转移的机制。方法　收集 4 5例确诊的鼻咽原发癌活检组织标本 ,采用免疫组化LSAB法检测鼻咽癌组织中MIF和MMP 2、MMP 9的表达 ,并分析患者的临床参数的关系。结果　在 4 5例鼻咽原发癌组织中 ,MIF、MMP 2和MMP 9的阳性表达率分别为 77 8% (35 / 4 5 )、6 4 4 % (2 9/ 4 5 )和 71 1% (32 / 4 5 )。其中 ,癌细胞MIF和MMP 9的表达水平均显示与淋巴结转移有关 ,伴有淋巴结转移的癌组织中二者表达水平均高于无淋巴结转移的癌组织 (P值均 <0 0 5 )。MIF阳性组的癌细胞MMP 9的表达 (5 0 2 %± 33 5 % )明显高于MIF阴性病例 (11 7%± 2 2 7% ) ,两者差异有显著性 (P <0 0 1) ,且MIF的表达与MMP 9的表达亦呈正相关 (rs=0 .4 92 ,P <0 0 1) ,但癌细胞MMP 2的表达与MIF、MMP 9的表达以及是否有淋巴结转移则均未显示相关性。以Schmincke型生长方式分布的癌细胞MIF表达水平 (6 7 4 %±35 2 % )也高于以Regaud型方式分布的癌细胞 (32 9%± 2 9 7% ) ,差异有显著性 (P <0 0 1)。结论　鼻咽癌组织中癌细胞的MIF和MMP 9同步过表达 ,可能在鼻咽癌细胞的转移     

低氧抑制猪肺动脉平滑肌细胞MMP-2和MMP-9的表达

目的探讨低氧对猪肺动脉平滑肌细胞（PASMC）分泌基质金属蛋白酶（MMPs）的影响。方法采用酶谱法测定PASMC培养基中MMP-2和MMP-9的酶活性，免疫印迹法检测培养基中MMP-2和MMP-9的蛋白表达，免疫组化法测定细胞原位MMP-2和MMP-9的蛋白表达，RT-PCR法检测mRNA的表达。结果低氧后PASMC分泌的MMP-2酶活性、细胞内外蛋白表达量、mRNA表达量均下降；MMP-9酶活性、细胞外蛋白表达量下降，而细胞内蛋白表达无变化。结论低氧可抑制PASMC分泌MMP-2和MMP-9的酶活性，其机制可能是低氧影响PASMC中MMP-2基因的转录、影响MMP-9蛋白表达后的分泌与活化，导致MMP-2和MMP-9酶活性的改变。     

人脐血血清对巨噬细胞Toll样受体4表达及功能的影响

目的研究人脐血血清(cord blood serum,CBS)对巨噬细胞RAW264.7 Toll样受体4(Toll like receptor4,TLR4)表达及功能的影响。方法取足月孕妇脐带血和外周血血清,与RAW264.7细胞共孵育,电镜下观察形态学变化,MTT法检测血清对RAW264.7细胞活性的影响;RT-PCR和流式细胞术检测RAW264.7细胞TLR4基因和蛋白表达水平;免疫荧光技术检测RAW264.7细胞I_κB_α水平,RR-PCR检测RAW264.7细胞COX-2的表达,以反映TLR4信号途径活化程度。结果人脐血血清可下调RAW264.7细胞TLR4的mRNA和蛋白表达水平;人脐血血清预孵育可抑制由LPS诱导的RAW264.7细胞NF-_κB激活和COX-2的表达。结论人脐血血清能抑制巨噬细胞TLR4的表达及其下游信号传递,这为阐明脐血血清对脐血细胞免疫功能调节的机制提供了新的实验线索。     

Toll样受体2和4在髋关节滑膜巨噬细胞中的表达

目的　探讨TLR2和TLR4在髋关节滑膜巨噬细胞中的表达。 方法　收集2007年至　2010年因髋关节疾病在我院行髋关节手术患者的髋关节滑膜标本共47例,其中股骨颈骨折24例（A组）,股骨头坏死18例（B组）,人工髋关节置换术后假体无菌性松动5例（C组）。采用免疫组化SP法检测TLR2和TLR4在3组的髋关节滑膜巨噬细胞中的表达,在高倍镜（×400）视野下对阳性的巨噬细胞的进行观察及计数,并作统计分析。 结果　各组用histoscore计算阳性巨噬细胞百分数得分,每高倍视野下A组中TLR2（0.27±0.33）,TLR4 （0.69±0.18）;B组中TLR2 （0.31±0.19）,TLR4 （0.71±0.31）;C组中　TLR2 （1.78±0.18）,TLR4 （2.00±0.39）。TLR2和TLR4在C组中表达较A组、B组高（P＜0.001）,而且3组中TLR4均较TLR2表达要高（P＜0.001）。 结论　TLR2及TLR4在人工关节无菌性松动患者的假体周围组织巨噬细胞中表达明显增多,可能参与了巨噬细胞介导的假体无菌性松动过程。     

小檗碱对2型糖尿病大鼠巨噬细胞MMP-9和TIMP-1表达的影响

目的： 探讨小檗碱对2型糖尿病大鼠肺泡巨噬细胞（AM）和腹腔巨噬细胞（PM）基质金属蛋白酶-9（MMP-9）、基质金属蛋白酶组织抑制因子-1（TIMP-1）表达的影响。 方法：SD大鼠随机分成4组，正常对照组、高脂组、糖尿病组、小檗碱治疗组。治疗12周后，测定各组大鼠血清葡萄糖、胰岛素和血脂含量，并检测肺泡和腹腔巨噬细胞氧化修饰低密度脂蛋白（ox-LDL）含量、MMP-9和TIMP-1的基因表达水平。结果：糖尿病组大鼠血糖、血胰岛素、血总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白（LDL）浓度明显高于对照组，AM和PM内ox-LDL浓度、MMP-9 mRNA表达明显高于对照组；而TIMP-1 mRNA的表达低于对照组。小檗碱治疗组大鼠血糖、血胰岛素、LDL、TC、TG浓度低于糖尿病组，AM、PM内MMP-9 mRNA的表达低于糖尿病组，而TIMP-1 mRNA的表达高于糖尿病组。 结论：小檗碱具有降低血糖、调节血脂、防治2型糖尿病动脉粥样硬化的作用，其机制可能与其减少ox-LDL生成，直接下调巨噬细胞中MMP-9的表达有关。     

肿瘤相关巨噬细胞在肿瘤脉管生成中的研究进展

肿瘤相关巨噬细胞(TAM)在肿瘤微环境中占有重要地位,对细胞外基质重塑、肿瘤细胞生长和转移、肿瘤脉管的生成和免疫抑制具有促进作用。脉管生成是肿瘤进展的重要过程,TAM在肿瘤组织中的表达显著高于癌旁组织,并且与肿瘤组织内病理性血管、淋巴管的生成密切相关。TAM促进肿瘤血管生成和淋巴管生成的方式具有一定相似性,能够通过分泌血管内皮生长因子(VEGF)、表达内弹力膜内皮细胞激酶2(TIE2),缺氧环境刺激、分泌多种细胞因子和蛋白、分化为内皮细胞等促进肿瘤脉管的生成,是今后抗肿瘤血管和淋巴管生成的重要研究方向。     

中性粒细胞分泌MMP-2和MMP-9发挥促肿瘤作用

目的肿瘤相关中性粒细胞(tumor-associated neutrophils,TANs)分为抗肿瘤的N1型和促肿瘤的N2型,N2型TANs的作用机制尚不清楚。本研究探讨以胃癌细胞培养上清模拟胃癌微环境或用干扰素β抗体抑制细胞干扰素β,能否诱导正常人中性粒细胞分泌基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)和基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2),影响肿瘤的发生发展。方法收集51例正常人外周血,用中性粒细胞分离液分离中性粒细胞,分别用胃癌MGC-803细胞培养上清干预正常人中性粒细胞;用抗干扰素β(interferon beta,IFN-β)抗体干预正常人中性粒细胞。用Real-time PCR法检测2种干预前后中性粒细胞MMP-9和MMP-2 m RNA的变化,以ELISA法检测2种干预前后中性粒细胞分泌MMP-9和MMP-2的情况,明胶酶谱法检测2种干预前后中性粒细胞分泌的MMP-9和MMP-2的酶活性。结果中性粒细胞在MGC-803上清诱导下,MMP-9和MMP-2 m RNA水平、酶含量和酶活性均增加(P<0.01);中性粒细胞经抗IFN-β抗体干预后,MMP-9和MMP-2 m RNA水平、酶含量和酶活性亦显著高于对照组(P<0.01)。结论胃癌微环境或抑制干扰素β均可使中性粒细胞MMP-9和MMP-2分泌量和酶活性增加,促进肿瘤发展,发挥N2型TANs的作用。     

Toll样受体在卵巢癌中作用的研究进展

卵巢恶性肿瘤是病死率最高的妇科疾病,严重威胁女性的健康.固有性免疫系统发挥重要免疫防御作用,作用的关键是对病原体的识别,这一识别主要是通过Toll样受体(TLRs)完成的.TLRs是近年免疫学研究的焦点,其不仅通过对病原微生物的病原相关分子模式的识别激活固有性免疫应答,还引起细胞因子的释放,上调共刺激分子的表达,为适应性免疫的启动提供必要的活化信号.因此,TLRs在很多疾病的发生和进展中起重要作用.阐明TLRs和固有免疫在卵巢恶性肿瘤中的作用可能会为研究者提供一个更好地了解这种疾病的分子机制.此外,利用TLRs的激动剂或拮抗剂有希望成为对抗卵巢恶性肿瘤的新的免疫治疗方法.     

脂多糖刺激对牛单核细胞由来的巨噬细胞Toll样受体表达的影响

目的:研究牛末梢血中单核细胞由来的巨噬细胞在受到LPS刺激后细胞表面Toll样受体表达的变化.方法:试验采取3头日本黑牛外周血,进行分离得到外周血单核细胞,并用Repcell进行巨噬细胞的分离培养,7天后用脂多糖(lipopolysaccharide,LPS)刺激细胞24小时后,通过RT-PCR测定巨噬细胞表面Toll样受体mRNA的表达以及细胞因子mRNA的表达.结果:在LPS刺激24小时后,IL-6、 TNF-α和IL-1βmRNA的表达量显著升高(P＜0.05),与此同时IL-8和IL-12p40 mRNA的表达量相对提高;TLR1和TLR10的转录物显著下降(P＜0.05),然而TLR2、4和6保持稳定.结论:巨噬细胞在受到病原刺激后通过提高IL-6、 TNF-α和IL-1β mRNA的表达量,从而提高了天然免疫的作用.该研究为探讨巨噬细胞在无然免疫中的作用奠定了基础.     

Fibronectin activates matrix metalloproteinase-9 secretion via the MEK1-MAPK and the PI3K-Akt pathways in ovarian cancer cells

Cell adhesion to the extracellular matrix appears to trigger a cascade of intracellular signalings. We have previously shown that treatment of ovarian cancer cells, NOM1, with fibronectin (FN) stimulated matrix metalloproteinase (MMP)-9 secretion and thereby activated the invasiveness of cells via the FAK/Ras signaling pathway. By use of chemical inhibitors, we investigated the downstream effectors critical for FN-dependent secretion of MMP-9. Treatment of cells with MEK1 inhibitors, U0126 and PD98059, dramatically suppressed the secretion of MMP-9 activated by FN. Similarly, PI-3 kinase inhibitors, Wortmannin and LY294002, strongly suppressed the FN-dependent secretion of MMP-9 together with the inhibition of Akt activation. In contrast, a specific PKC inhibitor (GF109203X) showed no inhibitory effect on the FN-dependent MMP-9 secretion. Moreover, we found that both the MEK1 inhibitor and the PI3-K inhibitor, but not the PKC inhibitor, strongly suppressed the invasiveness of NOM1 cells. Taken together, our results suggest that activation of dual signaling pathways, MEK1-MAPK and PI3K-Akt, is required for the FN-dependent activation of MMP-9 secretion. Our results suggest the importance of these signaling molecules as a chemotherapeutic target for cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Increased matrix metalloproteinase-9 activity in human ovarian cancer cells cultured with conditioned medium from human peritoneal tissue

Ovarian cancer cells disseminate by attachment to the peritoneal mesothelial cell surface of the abdominal cavity. We therefore investigated the influence of conditioned medium (CM) from human peritoneal tissues and mesothelial cells on the secretion of matrix metalloproteinases (MMPs) by ovarian cancer cells. The molecular weights of MMPs stimulating factors derived from human peritoneal tissues and mesothelial cells were estimated using microconcentrators with various cut-off membranes. Human peritoneal tissues were obtained from 12 surgical patients, and mesothelial cells were isolated from three peritoneal specimens. Exposure to CM from peritoneal tissue caused a concentration-dependent increase of the MMP-2 and MMP-9 bands in CM from NOM1 ovarian cancer cells, as shown by zymography. There was a significant difference in the increase of MMP-2 and MMP-9 (2.46-fold and 7.14-fold, respectively, at 0.4mg/ml protein; P < 0.005). CM from mesothelial cells also significantly increased the secretion of MMP-9 by NOM1 cells. The molecular size of possible MMP-9-stimulating factors secreted by peritoneal tissues and mesothelial cells was above M 100000. Further, CM of peritoneal tissues and mesothelial cells also induced the invasiveness of NOM1 cells. These findings suggest that mesothelial cells may secrete some factors which predominantly induce the MMP-9 production and increase invading cell numbers.     

Participation of macrophages in glomerular sclerosis through the expression and activation of matrix metalloproteinases

In order to investigate the role of macrophages in glomeruli in the progression of glomerular sclerosis, methyl-cellulose (MC) was administered intraperitoneally to Wistar rats, in addition to intravenous injection of anti-thy1-1 antibody. In this group of rats (Thy-1 + MC group), many macrophages infiltrated in the lytic mesangium accompanied by rupture of capillary loops at an early stage and stayed with abundant deposition of mesangial matrices until day 35, whereas the proliferative lesions following mesangiolysis almost vanished in the rats treated with anti-thy1-1 antibody alone (Thy-1 group). In immunostaining, matrix metalloproteinase (MMP)-9 was expressed along regenerating capillaries of the Thy-1 group and in extracapillary lesions of the Thy-1 + MC group after day 7. In gelatin zymography, the gelatinolytic band for MMP-9 was expressed much more strongly in the Thy-1 + MC group than in the Thy-1 group at day 3, but it was expressed a little more strongly in the Thy-1 group than in the Thy-1 + MC group at day 7. The bands for an active form of MMP-2 were more strongly expressed in the Thy-1 + MC group than in the Thy-1 group throughout the experimental period. These results suggest that persistent accumulation of macrophages in mesangium induces glomerular sclerosis through expression and activation of MMP.     

Expression of matrix metalloproteinases and their inhibitors in human brain tumors

Sixty human brain tumors, classified according to the New World Health Organization (WHO) classification including, grade I schwannomas, meningiomas and pilocytic astrocytomas, grade II astrocytomas, grade III anaplastic astrocytomas, grade IV glioblastomas, grade III anaplastic oligodendrogliomas and grade IV glioblastomas and lung and melanoma metastases were analyzed for the expression of three matrix metalloproteinases (MMPs), two tissue inhibitors of MMPs (TIMPs) and for MMP activity. Some correlation was found between MMP expression and the degree of malignancy. Western blotting analysis revealed a more uniform pattern of distribution of MMP-2 (gelatinase A) than of MMP-9 (gelatinase B) and MMP-12 (metalloelastase) among tumors. MMP-9 levels were found to be significantly higher in grade III anaplastic astrocytomas and anaplastic oligodendrogliomas than those in grade I schwannomas and meningiomas. Anaplastic astrocytomas and Grade IV glioblastomas expressed significantly higher levels MMP-12 than grade I meningiomas. All sixty tumors showed a similar pattern of activity in zymography, proMMP-9 being the major species detected. Interestingly, TIMP-1 and TIMP-2 expression levels were especially low in tumors of grade II and grade III but significantly higher in tumors of grade I, particularly in schwannomas. Taken together, these data suggest that: 1) a balance between MMPs and TIMPs has an important role to play in human brain tumors; 2) TIMP expression may be valuable markers for tumor malignancy. This revised version was published online in July 2006 with corrections to the Cover Date.     

肿瘤相关巨噬细胞microRNA表达谱及生物信息学分析

 目的 研究肿瘤相关巨噬细胞（TAM）microRNA的表达谱。 方法 建立小鼠乳腺癌细胞系4T1移植瘤模型,从移植瘤组织中分离TAM,用基因芯片检测microRNA表达谱,实时荧光定量PCR（Real-time PCR）验证芯片结果并进行生物信息学分析,以小鼠腹腔巨噬细胞（PEC）为阴性对照。 结果 与阴性对照细胞相比,TAM中有59个microRNAs表达量出现显著变化,其中23个microRNAs表达上调,有36个microRNAs表达下调;实时荧光定量PCR对miR-146a、miR-222、miR-31和miR-877的表达进行了验证,其结果与基因芯片检测结果一致;这些microRNAs参与了多个信号通路的调控。 结论 microRNA表达谱及生物信息学分析表明microRNA在TAM分化过程的调控中有重要作用。     

New insights into the roles of matrix metalloproteinases in colorectal cancer development and progression

This review outlines new concepts that are emerging for the functions of matrix metalloproteinases in colorectal cancer development and progression. The two main concepts that will be discussed are the role of matrix metalloproteinases in the early stages of colorectal tumour development and the functional mechanisms by which matrix metalloproteinases contribute to colorectal tumour invasion and metastasis. The matrix metalloproteinases are a group of enzymes, which have been best characterized for their ability to degrade extracellular matrix proteins and thus they have been extensively studied in tumour invasion. It is now becoming recognized that the matrix metalloproteinases have key roles in a variety of biological processes that are distinct from their well-defined role in matrix degradation. This group of enzymes has been shown to interact with a broad range of non-matrix proteins including growth factors and their receptors, mediators of apoptosis, and cell adhesion molecules. The elucidation of novel biological roles for the matrix metalloproteinases also challenges the current predominant concept of matrix metalloproteinases as enzymes only involved in matrix degradation. Recent studies have shown that several matrix metalloproteinases, especially matrilysin (MMP-7), interact with the specific molecular genetic and signalling pathways involved in colorectal cancer development. In particular, matrilysin is activated at an early stage of colorectal tumourigenesis by the beta-catenin signalling pathway. Furthermore, studies are now elucidating specific mechanisms by which individual matrix metalloproteinases, especially membrane-type matrix metalloproteinases, interact with specific cell adhesion molecules and cytoskeletal proteins and thus contribute dynamically to colorectal tumour invasion.     

Toll-like receptors 3, 7, 8, and 9 in gastric cancer

Toll-like receptors (TLRs) have been shown to have anti-tumor, pro-tumor, or even dual effects in cancer, and are thus potential prognostic biomarkers and immunotherapeutic targets. The present study aimed to evaluate associations between endosomal TLRs, namely TLR3, TLR7, TLR8, and TLR9, expression and clinicopathological variables and survival in gastric cancer. A total of 564 gastric adenocarcinoma patients were included in this retrospective cohort study. Samples and clinicopathological data were retrieved and organized into tissue microarray blocks. Protein expressions were detected by immunohistochemical staining. The patients were divided into low expression and high expression groups by median values of expression. Cox regression provided hazard ratios (HR) with 95% confidence intervals (CI), adjusted for confounders. Patients with high nuclear TLR3 expression had significantly poorer 5-year survival than the low nuclear TLR3 expression group in the univariable analysis (crude HR 1.31, 95% CI 1.07–1.60). With radically resected patients, poor prognosis was also seen in the multivariable analysis (adjusted HR 1.38, 95% CI 1.08–1.77). Cytoplasmic TLR3, TLR7, TLR8, and TLR9 were not associated with 5-year survival. In conclusion, high nuclear TLR3 expression seems to have prognostic impact in gastric cancer, while TLR7, TLR8, and TLR9 do not.     

Matrix metalloproteinases 7 and 9 and their types 1 and 4 tissue inhibitors in tumors and plasma of patients with colorectal cancer

Enzyme immunoassays showed significantly elevated content of matrix metalloproteinase 7 and type 1 tissue inhibitor of metalloproteinases in tumors compared to adjacent histologically unchanged mucosa of patients with colorectal cancer; the levels of metalloproteinase 9 and type 4 tissue inhibitor of metalloproteinases were virtually the same in the tumors and mucosa. Plasma concentrations of the studied proteins did not correlate with their levels in the tumor, did not surpass the normal, and did not decease after removal of the primary tumor in the majority of patients. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 4, pp. 438–441, April, 2007