Notch信号对激光诱导的小鼠CNV生成过程中巨噬细胞极化表型及功能的调控

背景 巨噬细胞在脉络膜新生血管（CNV）中的作用尚存在争议,与其在不同微环境中的功能异质性有关,Notch信号通路参与眼内新生血管生长的调控,但其对CNV中巨噬细胞功能的调控作用尚未证实. 目的 探讨巨噬细胞在CNV生成中极化表型的变化与Notch信号通路对巨噬细胞极化表型的调控.方法 在体实验选择58只成年雄性C57BL小鼠,以随机数字表法按照造模后取材时间的不同随机分为光凝后3d组和7d组.每组选23只小鼠用视网膜光凝法诱导小鼠CNV模型,分别于光凝后3d和7d制备脉络膜铺片,采用免疫荧光染色法观察CNV面积和巨噬细胞阳性染色面积,血管内皮生长因子（VEGF）及肿瘤坏死因子α（TNF-α）的分泌情况;光凝后3d和7d对眼杯冰冻切片行免疫荧光染色,观察CNV中巨噬细胞的极化表型及其分泌VEGF、TNF-α的情况,评估巨噬细胞上Notch信号胞内段（NICD）相应分子标志物的表达情况.每组设定6只小鼠的左眼为正常对照眼,其右眼以激光光凝法建立CNV模型,采用实时荧光定量PCR（qRT-PCR）法检测眼杯组织中VEGF、TNF-α和巨噬细胞极化相关因子的表达情况.体外分离和培养C57BL小鼠的骨髓单核前体细胞,用粒细胞-巨噬细胞集落刺激因子（GM-CSF）诱导其分化为巨噬细胞,加入Notch信号通路抑制剂γ-分泌酶抑制剂（GSI）和极化诱导因子,24 h后收集细胞及培养液上清,通过qRT-PCR及ELISA法检测M1型、M2型巨噬细胞分子表面标志物的表达水平. 结果 与光凝后第3天比较,光凝后第7天CNV面积明显增大,但巨噬细胞浸润面积缩小,差异均有统计学意义（t=8.138、5.272,均P=0.000）.光凝后第3天,巨噬细胞在CNV周边和中央均有分布,第7天时局限于CNV中央部.光凝后第3天,色素上皮-脉络膜-巩膜复合体中精氨酸合酶1（Arg1）、甘露糖受体（MR）及白细胞介素-6（IL-6）mRNA的相对表达量明显高于第7天,而诱导型一氧化氮合酶（iNOS） mRNA的相对表达量无明显改变.免疫荧光结果显示,光凝后第3天F4/80＋ Arg1＋细胞（M2型巨噬细胞）数量明显多于F4/80＋ iNOS＋细胞（M1型巨噬细胞）,光凝后第7天F4/80＋ Arg1＋细胞数量明显减少,差异均有统计学意义（t=7.348,P=0.000;t5.562,P＜0.001）,但仍多于F4/80＋ iNOS＋细胞,二者间差异有统计学意义（t=4.568,P＜0.01）.在光凝后3d时巨噬细胞分泌的VEGF和TNF-α明显多于光凝后7d,差异均有统计学意义（t=5.143、4.519,均P＜0.01）.巨噬细胞中Notch信号下游分子在光凝后3d时未见表达,7d时表达量明显增加,差异均有统计学意义（均P＜0.05）.体外实验表明,阻断Notch信号后,M1型巨噬细胞分子标志物表达量明显下降,而M2型巨噬细胞分子标志物的表达量明显增加,差异均有统计学意义（均P＜0.05）.结论 巨噬细胞的极化表型在CNV模型的不同阶段发生变化,而巨噬细胞中Notch信号的激活状态调控巨噬细胞的极化表型改变和功能.

Adult endothelial progenitor cells from human peripheral blood maintain monocyte/macrophage function throughout in vitro culture

Mononuclear cells （MNCs） isolated from peripheral blood by density gradient centrifugation were plated on human fibronectin-coated culture plates and cultured in EGM-2 medium. Attached spindle-shaped cells, reported as endothelial progenitor cells （EPCs） by some investigators, had elongated from adherent round cells, but had not proliferated from a small number of cells as supposed previously. The growth curve of the primary EPCs showed that the cells had little proliferative capacity. Flow cytometry analysis showed that the cells could express some of the endothelial lineage markers, while they could also express CD 14, which is considered a marker of monocyte/macrophage lineages throughout culture. In endothelial function assays, the cells demonstrated a lower level of expression of eNOS than mature endothelial cells in the reverse transcription-polymerase chain reaction and did not show an ability to develop tube-like structures in angiogenesis assay in vitro. In this study, we identified the monocytoid function of EPCs by the combined Dillabeled acetylated low-density lipoprotein （Dil-Ac-LDL） and Indian ink uptake tests. All the cells were double positive for Dil- Ac-LDL and Indian ink uptake at days 4, 14 and 28 of culture, which means the EPCs maintained monocytoid function throughout the culture. Therefore, although adult EPCs from peripheral MNCs have some endothelial lineage properties, they maintain typical monocytic function and have little proliferative capacity.

Difference in gene expression of macrophage between normal spleen and portal hypertensive spleen idendified by cDNA microarray

AIM: To identify the difference in gene expression of microphage (Mφ) between normal spleen and portal hypertensive spleen using cDNA microarrays and find new gene functions associated with hypersplenism in portal hypertension. METHODS: The Biostar-H140s chip containing 14 112 spots of cDNAs were used to investigate the difference of the expression. The total RNA extracted from macrophages isolated from both normal spleen and portal hypertensive spleen was reversely transcribed to cDNA with the incorporation of fluorescent (cy3 and cy5) labeled dCTP to prepare the hybridization probes. After hybridization, the gene chip was scanned for the fluorescent intensity. The differentially expressed genes were screened. That was repeated three times, and only the genes which had differential expression in all three chips were considered to be associated with hypersplenism in portal hypertension. RESULTS: Eight hundred and ninety-six, 1330 and 898 genes were identified to be differentially expressed in three chips, respectively. One hundred and twenty-one genes (0.86%) were identified to be differentially expressed in all three chips, including 21 up-regulated genes and 73 down-regulated genes. The differentially expressed genes were related to ionic channel and transport protein, cyclin, cytoskeleton, cell receptor, cell signal conduct, metabolism, immune, and so on. These genes might be related to the hypersplenism in portal hypertension.CONCLUSION: The investigations based on cDNA microarray can screen differentially expressed genes of macrophages between normal spleen and portal hypertensive spleen, thus may provide a new idea in studying the pathogenesis of hypersplenism in portal hypertension.

Effects of hyperbaric air exposure on the functions of mouse peritoneal macrophages

Objective: To study the effects of hyperbaric air exposure on the functions of peritoneal macrophages of mice. Methods: Forty-eight mice were equally randomized to 6 groups: (1) normal air group (NA); (2) hyperbaric air group 1 (HA1); (3) hyperbaric air group 2 (HA2); (4) hyperbaric air group 3 (HA3); (5) hyperbaric oxygen group (HO);(6) hyperbaric nitrogen group (HN). Every group was exposed to corresponding pressure for 60 min, twice a day for 3 d. Peritoneal macrophages were obtained at the corresponding time to observe the changes of phagocytosis, acid phos-phatase, antigen presentation function and the produce of NO and TNF-α. Results: Compared with those in NA group, the activity of phagocytosis, acid phosphatase, antigen presentation function and the produce of NO and TNF-a were markedly inhibited in hyperbaric oxygen group and hyperbaric air group 1 ( P < 0.05, P < 0.01) and they changed little in HN group. These changes could disappear in 3 - 5 d. Conclusion: The functions of mice peritoneal macrophages were obviously inhibited in simulated air diving environment and hyperoxia may play an important role in it.

Determination of phagocytic functions of macrophages, vascular density and NPY-nerve within autotransplanted splenic tissue in adult rats

Objective: To estimate directly phagocytic functions of the macrophages because of the importance in innate immunity, determine blood vessel density and re-innervation density which are basis of function. Methods: Eighty adult Wistar rats were randonjy divided into experimental and control groups.The formers underwent splenotomy and a half splenie slice was transplanted into greater omentum. The latter only moved. After 6 months, examination was made as follows: ① After injection of 0.4% carbon particles by vein, spleme tissues were taken out at different times for estimating phagocytosis by light microscope. ② When splenic tissues had been intubated into left ventricle under total anesthesia, animals were perfused by formalin and India ink mixture suspension. Splenic tissues were taken out for making sections for measurement of area density of blood vessels.③ Immunohistochemical procedure was used for detecting neuropeptide Y(NPY). Results: Phagocytie functions had no difference between two groups, hut the area density of blood vessels and NPY-positive fibers re-dued (P<0.01) in experimental group. Conclusion:Autotransplanted splenic tissues show good innate immunity though regeneration of blood vessels and nerves do not reach normal level.

MicroRNAs in tumor immunity:functional regulation in tumor-associated macrophages

Tumor-associated macrophages(TAMs)are the most abundant immune cells in the tumor microenvironment(TME)and are critical for cancer initiation and progression.MicroRNAs(miRNAs)could notably influence the phenotype of TAMs through various targets and signal pathways during cancer progression due to their posttranscriptional regulation.In this review,we discuss mainly the regulatory function of miRNAs on macrophage differentiation,functional polarization,and cellular crosstalk.Firstly,during the generation process,miRNAs take part in the differentiation from myeloid cells to mature macrophages,and this maturation process directly influences their recruitment into the TME,attracted by tumor cells.Secondly,macrophages in the TME can be either tumor-promoting or tumor-suppressing,depending on their functional polarization.Large numbers of miRNAs can influence the polarization of macrophages,which is crucial for tumor progression,including tumor cell invasion,intravasation,extravasation,and premetastatic site formation.Thirdly,crosstalk between tumor cells and macrophages is essential for TME formation and tumor progression,and miRNAs can be the mediator of communication in different forms,especially when encapsulated in microvesicles or exosomes.We also assess the potential value of certain macrophage-related miRNAs(MRMs)as diagnostic and prognostic markers,and discuss the possible development of MRM-based therapies.

Kv1.3 channel blockade enhances the phagocytic function of RAW264.7 macrophages

This study aimed to comprehend the largely unknown role of voltage-gated potassium channel 1.3 （Kvl.3） in the phagocytic function of macrophages. We found that blocking of the Kv 1.3 channel with 100 pmol L 1 Stichodactyla helianthus neurotoxin （ShK） enhanced the phagocytic capacities of both resting and lipopolysaccharide （LPS）-stimulated RAW264.7 macrophages in the chicken erythrocyte system. In the fluorescein isothiocyanate （FITC）-labeled Escherichia coli k-12 system, ShK increased the phagocytic capacities of resting RAW264.7 cells, but not of the LPS-stimulated cells, as LPS alone stimulated almost satu- rated phagocytosis of the macrophages. ShK increased the nitric oxide （NO） production in LPS-activated cells, but not in rest- ing RAW264.7 cells. There was no effect of ShK alone on the cytokine secretions in resting RAW264.7 cells, but it suppressed IL-113 secretion in LPS-stimulated RAW264.7 cells. At a concentration of 100 pmol L 1, ShK did not affect the viability of the tested cells. Kv 1.3 was expressed in RAW264.7 cells; this expression was downregulated by LPS, but significantly upregulat- ed by disrupting caveolin-dependent endocytosis with filipin III. In addition, cytochalasin D, an inhibitor of actin polymeriza- tion, did not affect the Kvl.3 expression. Thus, blocking of the Kvl.3 channel enhances the phagocytic capacity and NO pro- duction of this cell line. Our results suggest that Kv 1.3 channel serves as a negative regulator of phagocytosis in macrophages and can therefore be a potential target in the treatment of macrophage dysfunction.