OBJECTIVE:Canakinumab is an interleukin (IL)-1β inhibitory antibody. Recently, a large trial of canakinumab in cardiac patients described lower lung cancer incidence in patients treated with canakinumab compared to controls. This finding is the basis for ongoing clinical trials of canakinumab in lung cancer. To address the underlying mechanism, we established lung cancer co-cultures to investigate the interactions between lung cancer cells and immunocyte macrophages as related to the expression of IL-1β and the effect of IL-1β on the NF-κB pathway on lung cancer cells.METHODS:Lung cancer cell lines H838 and H1975 and macrophages were mono-cultured separately as control groups. Lung cancer cell lines and macrophages were co-cultured respectively in a ratio of 5:1 under the conditions of 37°C in a humidified atmosphere of 5% CO2 for seven days. Cell culture supernatants were collected at predetermined time points, and cell morphology was observed and photographed by microscopy. IL-1β was detected by ELISA. H838 and H1975 cells were treated with PBS or IL-1β for 24 hours. Cells were harvested and lysed, then analyzed in a proteome profiler array.RESULTS:Cells in co-cultures initially grew well. IL-1β was almost undetectable in lung cancer cell lines and macrophage monoculture groups but was highly expressed in co-cultures after 24h and declined at the 7th day. In the H838 and H1975 co-culture group, the lung cancer cells occupied a great majority. IL-1β activates the NF-κB pathway on H838 and H1975 cells.CONCLUSION:Our data showed that in a lung cancer co-culture incorporating lung cancer cells and macrophages lead to a higher expression of IL-1β than monoculture. It is possible that such dynamic changes in IL-1β expression may also occur in vivo in response to changes in the tumor microenvironment and interactions with immune cell populations. These interactions are likely important components to be considered when studying and modeling the expression of IL-1β as a potential therapeutic target in lung cancer.

Mesenchymal stromal cells are proven to be likely induce the angiogenic response in multiple myeloma and thus represent an enticing target for antiangiogenesis therapies for multiple myeloma. Substantial evidence indicates that angiogenesis in multiple myeloma is complex and involves direct production of angiogenic cytokines by abnormal plasma cells and these B-cell neoplasia generated pathophysiology change within the microenvironment. In this study, we demonstrated that mesenchymal stromal cells cultured with U266/Lp-1 under hypoxic conditions resulted in an increased α-smooth muscle actin expression and high productive levels of both hypoxia-inducible factor-2α and integrin-linked kinase proteins. Moreover, inhibition of hypoxia-inducible factor-2α by Small interfering RNA (siRNA) in mesenchymal stromal cells decreased the protein levels of both α-smooth muscle actin and integrin-linked kinase after mesenchymal stromal cells cultured with U266 under hypoxic conditions. We further demonstrated that transfection of integrin-linked kinase-siRNA reduced the protein level of α-smooth muscle actin and attenuated angiogenesis in vitro by decreasing the attachment of Q-dot labeled cells and secretion of angiogenic factors. In conclusion, our research showed that mesenchymal stromal cells cultured with myeloma cells under hypoxia participated in the angiogenesis of multiple myeloma, which is regulated by the hypoxia-inducible factor-2α-integrin-linked kinase pathway. Thus, targeting integrin-linked kinase may represent an effective strategy to block hypoxia-inducible factor-2α-induced angiogenesis in the treatment of multiple myeloma.

IFN-γ-induced PD-L1 expression represents the existence of tumor-specific T cells, which predicts high-response rate to anti-PD-1/L1 therapy, but loss-of-function of IFN signals (e.g., JAK mutation) induces adaptive immune resistance in patients with low-response rate. Interferon regulatory factors (IRF) are frequently epigenetic silenced in carcinogenesis, while the role of methylation in anti-PD-1/L1 therapy remains unclear. We here investigated the methylation status of IFN-γ related genes IRF1/8 and IFN-α/β-related genes IRF3/7 in lung cancer tissues and found that only highly methylated IRF1 and 7 negatively correlated to cd274 (coding PD-L1) expression, similar to JAK mutation. Interestingly, decitibine (DAC) as methylation inhibitor could hypomethylate IRF1/7 to restore PD-L1 level. Meanwhile, IRF7 enhanced constitutive PD-L1 expression, which was independent of IFN-γ though directly promote transcription of PD-L1, leading to abrogating cytotoxic T lymphocytes (CTLs) generation which could be restored by anti-PD-L1 antibody, or siRNA-IRF7. The supplement of DAC to anti-PD-1 therapy in vivo improve the efficiency of anti-tumor with less methylated IRF1/7, more interferon-related genes expression (e.g., CXCL9) and IFN-γ/CD8+ T-cells infiltrations, suggesting that additional treatment of DAC could rescue the ability to response to IFN in lung cancer patients with anti-PD-1/L1 therapy resistance.

目的 探讨A549和H358细胞株经表观治疗后,对其生长、侵袭和凋亡的影响,分析可能的作用机制.方法 5-氮杂胞苷处理A549和H358细胞株24和48 h后,运用流式细胞仪检测细胞增殖,Transwell方法检测细胞侵袭能力,赫斯特染色荧光显微镜观察细胞凋亡;RT-PCR和Western blot检测处理后DNMT1、RASSFA和APC基因在肺癌、BEAS-2B细胞株中的mRNA转录和蛋白表达.结果 两细胞株中DNMT1在mRNA转录和蛋白表达水平较BEAS-2B细胞株高;治疗后,RASSFA和APC基因活性增加,DN-MT1基因活性则受抑制,A549和H358细胞株的生长和侵袭能力减弱,凋亡增加,且与处理时间呈正比关系.结论 5-氮杂胞苷可以通过抑制DNMT1活性而使肺癌细胞功能减弱甚至丧失,RASSFA和APC基因去甲基化可能参与其中.

非小细胞肺癌(NSCLC)的发生、发展是一个多基因突变的过程,同时又由多种因素触发和维持,目前限于低剂量CT筛查的低敏感性和低特异性,NSCLC确诊时大多已处于中晚期.现在已经初步认清了NSCLC在增殖、分化和凋亡过程的多种基因、危险因素和生物标志物;然而,对于这些患者,尚未建立统一有效的医疗模型去实施个性化的治疗.分析文献可知:常见的灭活肿瘤的抑制基因的表达,切除修复交叉互补基因1(ERCC1)和核苷酸还原酶M1(RRM1)等促癌基因的表达,表皮生长因子受体(EGFR)、棘皮动物微管相关类蛋白4融合间变性淋巴瘤激酶基因(EML4-ALKA)和柯尔斯顿禽类肉瘤基因(KRAS)的突变,以及微小RNA (miRNAs)、血浆或血清中循环DNA、循环肿瘤细胞(CTCs)等,均已被证明是NSCLC的潜在生物标志物,也是新治疗策略的潜在靶标.这些涉及基因分子在健康和恶性肿瘤患者中的临床及实验室研究将有助于对NSCLC新生物标志物的加入,这将有助于以后更简便快捷地诊断,采取更具体的治疗方法,并预测治疗的效果.本文将对NSCLC早期诊断及评估预后有前景的上述生物标志物做一浅析.

Hypermethylation of tumor suppressor genes (TSGs) promoters by DNA methyltransferase (DNMT) can be observed in almost all cancers which represent a hallmark of carcinogenesis, including lung cancer. DNMT inhibitors (e.g.5-Aza-CR/CdR) reactivate TSGs to exert anti-cancer activity and have been applied into the clinical. However, it is cytotoxic even at low concentrations, which might be not directly related to DNA methylation. We here investigated an alternative strategy in the lung cancer therapy and aimed to estimate and compare its efficiency and side effects of knockdown of DNMT1 in vitro and in vivo. Lung cancer tissues (n=20) showed enhanced expression of DNMT1 than corresponding non-neoplastic tissues. Similar results were found in lung cancer cell lines A549 and H538. The treatment of 5-Aza-CR or knockdown of DNMT1 in vitro could inhibit the expressions of DNMT1 but restore the TSGs expressions including the Ras association domain family 1A (RASSF1A) and the adenomatous polyposis coli (APC) via the demethylation of its promoter region, which results in the decreased proliferation, increased apoptosis and impaired ability of migration. Importantly, knockdown of DNMT1 by siRNA in vivo also effectively demethylated the RASSF1A and APC promoter, elevated their expressions and limited tumor growth, which functioned like 5-Aza-CR but with alleviated side effects, suggesting that knockdown of DNMT1 might be potential strategy for the treatment of lung cancer with better tolerability.

Both mesenchymal stromal cells (MSCs) and myeloid-derived suppressor cells (MDSCs) have immunosuppressive properties, and their presence may confer a worse prognosis upon cancer patients. However, whether MSCs can enhance the immunosuppressive effects of MDSCs in MM remains unknown. We evaluated the influence of MSCs on MDSCs growth, apoptosis, and functions. Our results show that MSCs promote proliferation and inhibit apoptosis in MDSCs. Additionally, MSCs enhance the ability of MDSCs by inhibiting T-cell proliferation and IFN-γ production. Furthermore, both the mRNA and protein levels of Arg1 and NOS2 were upregulated in MDSCs. These results suggest that MSCs may exert immunomodulatory effects on MDSCs by upregulating Arg1 and NOS2.

The peripheral blood absolute lymphocyte to monocyte ratio (LMR) has been regarded as a prognostic marker of the tumor microenvironment in various cancers. The aim of this study was a retrospective analysis of the clinical features, efficacies, survival rates and prognostic factors. Also, we explored the prognostic impact of LMR in 62 newly-diagnosed MM patients with extramedullary (EM) disease (group A). Other 83 multiple myeloma MM (full name?) patients without EM (group B) were selected as the control. Compared with group B, the incidence of EM was associated with a higher level of beta(2)-MG, lower LMR and more extensive bone disease. The common location of EM (full name?) in order was soft tissues, intracranial, lung, pleural, skin and spinal canal. The estimated overall survival (OS) in group A was significantly shorter than those in group B (36 vs. 43 months, P=0.032). Logrank univariate analysis showed that the number of osteolytic lesions >= 3 (P=0.043), beta(2)-microglobulin (beta(2)-MG) >= 5.5 mg/L (P=0.000), LMR < 2.9 (P=0.015) and hemoglobin <= 110 g/L (P=0.023) were poor prognostic factors in group A. Multivariate analysis with Cox model showed newly beta(2)-MG >= 5.5 mg/L (95% CI: 0.158-0.624) and LMR < 2.9 (95% CI: 1.312-4.774) were statistically significant. We concluded the prognosis remained poor despite intensive treatment in these newly-diagnosed MM patients with EM invasion. LMR could be an efficient prognostic factor for this cohort of disease.

Development of multiple drug resistance has been attributed to the overexpression of the ATP-binding cassette B1 (ABCB1) gene. In this study, the major purpose was to assess the expression and methylation levels of ABCB1 in human lung adenocarcinoma and to reveal the relationship between these processes and acquisition of cisplatin (DDP) resistance in the human cancer cell line A549. Methylation and expression levels of the ABCB1 gene ABCB1 in clinical human lung tissue were assessed using bisulphite sequencing, reverse transcription real-time PCR (RT2 -PCR) and Western blot methods. Cell viability, DDP resistance and apoptosis of A549 cells were evaluated using the Cell Counting Kit-8 and fluorescence-activated cell sorter analysis. Our results showed that the onset of resistance to the cisplatin analogue, DDP, was associated with hypermethylation of the ABCB1 gene. Expression of the ABCB1 gene was enhanced at both mRNA and protein levels. Treatment with 5-Aza-C contributed to the hypomethylation of the ABCB1 gene and decreased ABCB1 protein expression in A549 cells. In conclusion, this in vitro and human tissue study of lung adenocarcinoma cells demonstrated that hypermethylation of the ABCB1 gene correlated with increased gene expression and was associated with the acquisition of resistance to the cisplatin analogue, DDP in human lung adenocarcinoma cells. Taken together, our study highlighted the connection between increased ABCB1 methylation level and upregulated expression of the gene in lung cancer. Moreover, the abnormally high expression of ABCB1 in A549 cells contributed to the development of the DDP resistance.

目的 观测豫北地区黄牛心脏左心室的解剖学结构,比较与人心左心室的异同,重点分析假腱索的临床意义.方法 10％甲醛溶液固定新鲜黄牛心脏28例,解剖后观测左心室主要结构.结果 黄牛心脏左心室呈锥体形,其室壁厚度为(24.51±4.29)mm,与其他哺乳动物相似,分为左心室流入道及流出道,流入道主要结构及参数分别为:二尖瓣环周径为(145.24±20.80)mm,二尖瓣前尖、后尖宽度及高度分别为(56.68±11.65)mm、(30.48±5.96)mm和(93.48±14.86)mm、(25.59±5.33)mm,前尖、后尖层数为(6.16±0.93)层、(6.52±1.12)层；前乳头肌为1群的有19例(67.86％),2群的有9例(32.14％),后乳头肌为1群的有11例(39.29％),2群的有17例(60.71％)；左心室流出道主要结构及参数为:主动脉瓣环周径平均为(99.61±21.13)mm,左瓣高度、右瓣高度、后瓣高度分别为(32.58±6.90)mm、(33.12±3.74)mm和(35.32±5.46)mm,其宽度分别为(12.35±1.05)mm、(12.00±0.98)mm和(13.45±3.00)mm；前乳头肌腱索至外侧连合、前尖、后尖的条数依次为(1.48±0.85)条、(4.84±1.70)条、(3.35±1.36)条；后乳头肌腱索至后内侧连合、前尖、后尖的条数分别为(1.71±0.78)条、(5.23±1.41)条、(3.55±1.41)条；假腱索分布于乳头肌与室壁或室壁与室壁之间,出现率为100％,平均条数为(22.90±8.71)条.结论 黄牛心脏左心室结构在二尖瓣复合体结构与人有一定相似性,假腱索出现率为高,是研究假腱索的良好模型.

Extracellular domain gene of Human Epidermal Growth Factor Receptor 2(HER2) was amplified by RT-PCR from human breast cancer cell line SK-BR-3.The recombinant expression vector pET30-HER2(Ex) was constructed by inserting the extracellular domain gene of HER2 into the pET-30a vector,and then was transformed into competent E.coli BL21(DE3) cells.After induced by IPTG,the expression of HER2 extracellular domain protein was obtained in E.coli.Proteins from culture medium fraction,periplasmic fraction,soluble cytoplasmic fraction and inclusion body fraction were extracted and analyzed by SDS-PAGE for the localization of HER2 extracellular domain protein.Result of SDS-PAGE revealed that HER2 extracellular domain protein localized in the inclusion body of E.coli cytoplasm.Optimum temperature,IPTG concentration,E.coli cell density(OD600) and induction time were determined for the highest production of HER2 extracellular domain protein.The results showed that when OD600 reached 1.0,induced with 0.1 mmol/L IPTG at 37℃ for 4 h,where the production of recombinant protein was highest.Recombinant protein in the inclusion body of E.coli was extracted with ultrasonic disruption and was purified by Ni2+ affinity chromatography.The obtainment of HER2 extracellular domain protein with high purity and high production lays the foundation for the development of anti-HER2 antibody and the study of HER2 overexpressing tumor vaccine.

淋巴管不同于血管,其壁薄且淋巴液无色,用肉眼不易在牛心表面观察其大体形态及分布范围[1-5].早在1692年Nuck首次将水银注入淋巴管,推动了淋巴系统的研究.进入20世纪以来,Funaoka和Monterio等用不同的造影剂直接或间接地注入淋巴管内,获得了淋巴管显影的成功.近年来,汤凤彩等[6]用日本产的合成树脂(蓝色的Mercox CL-2B-5和红色的Mercox CL-2R)在胃淋巴管铸型标本中获得成功,但其铸形剂价格昂贵,来源困难,操作繁琐,并有其一定的局限性,为了寻求更好的显示方法,笔者对查阅到的资料及铸型剂配方[5-6]进行了改良,并对牛心浅表淋巴结进行了灌注观察,经腐蚀后效果比较满意,现将方法介绍如下.

Objective To observe the anatomic structure of the right ventricle in cattle,in order to accumulate data for comparative anatomy.Methods Thirty-one hearts of cattle were fixed with formaldehyde solution(1.33 mol·L-1),then the structures of right ventricles were observed with gross anotomy method.Results The right ventricle of cattle heart was a cone and the average thickness of its wall was(10.53±1.70)mm.The average circumference of tricuspid was(145.84±27.22)mm.The average heights of anterior cusp,posterior cusp and septal cusp were(35.40±7.78),(30.18±6.64),(30.37±5.75)mm respectively,the average width were(45.42±11.73),(52.15±13.98),(63.17±15.10)mm respectively,and the average strata were 4.58±0.96,4.97±0.89 and 5.90±1.30 respectively.There were many and exiguous chordae tendineae.The average numbers of chordae tendineae form anterior papillary muscles to anterior cusp,posterior cusp and lateral were 4.03±1.30,3.81±1.56 and 1.48±0.81 respectively.The average numbers of chordae tendineae form posterior papillary muscles to anterior cusp,septal cusp and posteriomedialis were 4.16±1.55,4.48±1.67 and 1.58±0.67 respectively.The average numbers of chordae tendineae from septal papillary muscles to anterior cusp,septal cusp and anteriomedialis were 3.71±1.35,5.55±1.61 and 1.52±0.10 respectively.False tendons existed between papillary muscles and the wall of right ventricle or between the walls of right ventricle commonly and the frequency of false tendons was 100%,and the average number of false tendons was 30.19±10.90.Columna papillares appeared in group,and the average numbers of anterior papillary muscles,posterior papillary muscles and septal papillary muscles were 1.00±0.00,1.77±0.76 and 1.06±0.25 respectively.The average length of moderator band was(44.33±10.98) mm and the average diameter was(4.76±3.69)mm.The main structures of the right ventricular outflow were composed of pulmonary valve ring,pulmonary valve and sinus arteriae pulmonalis.The average circumference of pulmonary valve ring was(100.18±13.11)mm,the average height of left,right and anterior pulmonary valve were(13.75±2.75),(13.59±2.58) and(14.11±2.50)mm respectively,the average width of them were(34.99±7.02),(35.41±3.92) and(39.04±6.75)mm respectively.Conclusion The right ventricle of cattle is similar to that of human heart in structure,but there were differences in size and the total number of chordae tendineae and false tendon.

Objective To explore a new method to display the superficial lymphatic vessels of the cattle cardia.Methods The superficial lymphatic vessels were displayed by new type of lymphatic casting agent perfusion method and acid corrosion technology.Results The superficial lymphatic vessels could be seen with naked eyes after perfusion,and there were eighteen strips of integrity lymphatic stereo cast structure and lymphatic nodes after acid corrosion.Conclusion The new perfusion method can show the distribution of lymphatic vessels very well,and the specimen can be preserved for a long-term.