Effect of myeloid-derived suppressor cells on guanylate binding protein 1 in promoting the proliferation of glioma U87 cells and its mechanism / 肿瘤研究与临床

Objective:To investigate the effect of myeloid-derived suppressor cells (MDSC) on guanylate binding protein 1 (GBP1) in promoting the proliferation of glioma U87 cells and its mechanism.Methods:Glioma cells U87 with GBP1 overexpression (U87-GBP1) and control cells U87-Lacz transfected with empty vector were used as experimental cells. The mRNA and protein expressions of GBP1 and chemokine (C-C motif) ligand 2 (CCL2) in two groups of cells were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blot and enzyme linked immunosorbent assay (ELISA), and the proliferation of U87 cells were detected by CCK-8. CD14 + monocytes and CD3 + T lymphocytes were isolated from peripheral blood of healthy people by immunomagnetic beads. The CD14 + monocytes were treated with culture medium of U87-Lacz cells or U87-GBP1 cells, and then the cells were divided into U87-Lacz culture medium group and U87-GBP1 culture medium group. The proportion of MDSC in CD14 + monocytes was analyzed by flow cytometry. CD14 + monocytes treated by two culture medium groups were cocultured with activated CD3 + T lymphocytes, and flow cytometry was used to detect the proliferation of activated CD3 + T lymphocytes. Monocytes untreated by U87 cells culture medium or activated CD3 + T lymphocytes were used as the control group. CD14 + monocytes were treated with U87-Lacz or U87-GBP1 cell culture medium anti-human CCL2 antibody, which were U87-Lacz+anti-CCL2 culture medium group and U87-GBP1 + anti-CCL2 culture medium group, and the proportion of MDSC in CD14 + monocytes was analyzed by flow cytometry. U87-GBP1 and U87-Lacz cells were inoculated into BALB/c nude mice to cause tumors in the brain. One week later, they were divided into chemokine (C-C motif) receptor 2 (CCR2) inhibitor RS504393 treatment group (U87-Lacz + RS nude mice group and U87-GBP1+RS nude mice group) and untreated control group (U87-Lacz nude mice group and U87-GBP1 nude mice group). After 30 days, the mice were sacrificed and the brain, spleen and bone marrow were isolated. Hematoxylin-eosin (HE) staining was used to observe the transplanted tumors in the brain of nude mice, and the volume of transplanted tumor was calculated, and flow cytometry was used to detect the proportion of MDSC in the tissues. Results:The protein expression of GBP1 in U87-GBP1 cells was significantly higher than that in U87-Lacz cells, but there was no significant difference in cell proliferation level between the two groups in vitro ( P > 0.05). The proportion of MDSC in U87-GBP1 culture medium group was significantly higher than that of U87-Lacz culture medium group [(7.75±0.80)% vs. (4.50±0.08)%], and both groups were higher than that of control group [(2.55±0.31)%)] ( F = 18.27, P = 0.002). The percentage of activated CD3 + T lymphocytes in U87-GBP1 culture medium group was lower than that in U87-Lacz culture medium group [(47.38±0.08)% vs. (61.70±5.05)%, P = 0.040]. The relative expression of CCL2 mRNA in U87-GBP1 cells and the expression level of CCL2 protein in U87-GBP1 cell culture medium [30.66±0.17 and (1 005.00±12.23) ng/L] were higher than those in U87-Lacz cells [1.29±0.15 and (111.60±11.44) ng/L] (both P < 0.01), the proportions of MDSC in U87-Lacz + anti-CCL2 culture medium group and U87-GBP1 + anti-CCL2 culture medium group was lower than those in U87-Lacz culture medium group and U87-GBP1 culture medium group (all P < 0.05). The volume of transplanted brain tumor in U87-GBP1 nude mice group was larger than that in U87-Lacz nude mice group; the volume of transplanted brain tumor in U87-GBP1 + RS nude mice group and U87-Lacz + RS nude mice group increased more slowly than the corresponding nude mice group without treatment, and the differences were statistically significant (all P < 0.05); the proportions of MDSC in transplanted brain tumor, spleen and bone marrow in U87-GBP1 nude mice group were higher than those in U87-Lacz nude mice group, and the proportions of MDSC in each tissue of U87-GBP1 + RS nude mice group and U87-Lacz + RS nude mice group were lower than those in the untreated by RS504393 corresponding nude mice group, and the differences were statistically significant (all P < 0.05). Conclusion:GBP1 might increase the expression of CCL2 in glioma U87 cells and recruit MDSC to form immunosuppression in glioma microenvironment, thus promoting the proliferation of glioma U87 cells in vivo.

The role of myeloid-derived suppressor cells in glioma microenvironment / 生物医学工程学杂志

Glioma is one of the most common primary tumors in the human brain with poor prognosis. The local and systemic immunosuppressive environment created by glioma cells enables them to evade immunosurveillance. Myeloid-derived suppressor cells (MDSCs) are a critical component of the immunosuppression system. They are a heterogeneous cell population composed of early myeloid progenitor cells and precursor cells. Although the cells are diverse in phenotypes and functions, they all have strong immunosuppressive functions. MDSCs are extensively infiltrated into tumor tissues and play an important role in the glioma immunosuppressive microenvironment, which also hinders the immunotherapeutic effects of glioma. This article will review the phenotypic characteristics of MDSCs in the glioma microenvironment and their role in the progression of glioma. It is of positive significance to better understand the pathogenesis of glioma and explore effective comprehensive treatments.

Risk factors for perioperative ischemic stroke in patients with atherosclerotic cerebrovascular stenosis undergoing percutaneous transluminal angioplasty and stenting / 临床麻醉学杂志

Objective To explore the predictors of perioperative ischemic stroke following percutaneous transluminal angioplasty and stenting.Methods We retrospectively evaluated data on 416 percutaneous transluminal angioplasty and stenting (PTAS) procedures at (334 males,82 females,aged 40-85 years,falling into ASA Ⅰ-Ⅲ) a single institution.Logistic regression was used to analyze the role of clinical,angiographic and hemodynamic variables on periprocedural ischemic strokes.Results Among 328 patients underwent PTAS for the treatment of extracranial stenosis,10 patients (3.0％) had perioperative ischemic stroke.Among the 88 stenting for intracranial stenosis,6 patients (6.8 ％) had perioperative ischemic stroke.Multivariable predictors of perioperative ischemic stroke for stenting for extracranial stenosis were the presence of untreated intracranial artery stenosis (OR =9.44,95％CI 2.36-37.71,P=0.001) and intraoperative absolute minimal SBP＜90 mm Hg (OR=9.13,95％CI 1.35-61.76,P =0.023).The independent predictors of perioperative ischemic stroke following PTAS for intracranial stenosis included the patients' increasing age (OR =1.25,95 ％ CI 1.04-1.51,P=0.021),presence of calcific plaques (OR=11.02,95％CI 1.11-109.25,P=0.040) and untreated intracranial artery stenosis (OR =44.81,95％ CI 1.99-1 011.84,P =0.017).Conclusion For patients with extracranial stenosis,suffering from the presence of untreated intracranial artery stenosis and intraoperative absolute minimal SBP＜90 mm Hg are the independent risk factors for perioperative ischemic stroke.The patients' increasing age,presence of calcific plaques and untreated intracranial artery stenosis were the independent risk factors for this complication in patients with intracranial stenosis.

Study of the relationships between myeloid-derived suppressor cells (MDSC) and nonresolving inflammation (NRI) in tumor microenvironment / 中华微生物学和免疫学杂志

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immunosuppressive cells derived from bone marrow stem cells.MDSCs can not only strongly inhibit the anti-tumor immune reactions mediated by T cells,but also directly accelerate angiogenesis,tumor progression and metastasis.Nonresolving inflammation (NRI),a major driving factor in the occurrence and development of tumor,and MDSCs are present in tumor microenvironment and become hot topics in recent years.However,the relationships between MDSCs and NRI,especially in the relevant molecular regulatory networks,have not been fully elucidated.The relationships between MDSCs and NRI,the molecular regulatory networks,the key regulatory points and the strategies for targeted treatment are reviewed in this paper based on the current literatures and the work achieved by our research team.

Expression of NF-E1b in colorectal cancer tissues and its clinical significance / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To explore the expression of NF-E1b in colorectal cancer tissues and its association with various clinicopathological parameters and prognosis of the patients.</p><p><b>METHODS</b>Clinicopathological and follow-up data of 168 colorectal cancer patients undergoing radical operation at Department of Gastrointestinal Surgery, Peking University Cancer Hospital and Institute from 2005 to 2012 were retrospectively analyzed, including 96 males and 72 females, with mean age of (57.8±11.2) years. The expression of NF-E1b protein was detected in samples of 168 resected colorectal cancer tissues and 45 adjacent non-cancerous tissues by immunohistochemistry. The expression rates of NF-E1b were compared among different clinicopathological features. Moreover, the association between NF-E1b expression and prognosis was analyzed.</p><p><b>RESULTS</b>The expression of NF-E1b protein located mainly in cytoplasm. Positive rate of NF-E1b expression in adjacent non-cancerous tissues was 17.8% (8/45), which was obviously lower than 67.9%(114/168) of cancer tissues with significant difference (χ(2)=36.376, P=0.000). Clinicopathological parameters analysis suggested that the expression level of NF-E1b in cancer tissues was associated with age (χ(2)=4.862, P=0.030), TNM staging (χ(2)=10.969, P=0.002), lymph node metastasis (χ(2)=7.390, P=0.008) and distal metastasis (χ(2)=17.887, P=0.000). The median follow-up time was 23(1-77) months. The overall 5-year survival of this cohort was 33.3%. Colorectal cancer patients with high levels of NF-E1b expression showed a worse overall survival compared with those with low levels of NF-E1b expression (18.4% vs. 56.6%, P=0.000). Univariate Cox regression analysis showed that tumor location (P=0.034), tumor size (P=0.003), TNM staging (P=0.000), depth of tumor invasion (P=0.009), lymph node metastasis (P=0.000), distant metastasis (P=0.000) and NF-E1b expression level (P=0.001) were associated with the prognosis of colorectal cancer patients. Multivariate Cox regression analysis revealed that tumor diameter >4 cm (HR=2.193,95% CI:1.334 to 3.603, P=0.002), distant metastasis (HR=2.064, 95% CI:1.160 to 3.672, P=0.014) and high NF-E1b expression (HR=1.994,95% CI:1.068 to 3.724, P=0.030) were independent risk factors of predicting poor prognosis of colorectal cancer patients.</p><p><b>CONCLUSIONS</b>NF-E1b expression up-regulates in colorectal cancer tissues. High expression of NF-E1b is associated with poor prognosis of colorectal cancer patients. NF-E1b may serve as a potential target of the treatment for colorectal cancer.</p>

Tumor angiogenesis promoted by fusion of glioma stem/progenitor cells with bone marrow mesenchymal stem cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>The aim of this study was to clarify whether the fusion of bone marrow mesenchymal stem cells (MSCs) with tumor cells can promote tumor angiogensis.</p><p><b>METHODS</b>Human glioma stem/progenitor cells (GSPCs) (SU3 cells) were transfected with red fluorescent protein (RFP) gene. Bone marrow mesenchymal stem cells (MSCs) were harvested from nude mice with whole-body green fluorescent protein (GFP) gene expression. Then the two kinds of cells were co-cultured in vitro. At the same time SU3-RFP was transplanted into the brain of GFP-expressing nude mice to establish xenograft tumors. The co-cultured cells, GFP/RFP double positive (yellow) cells and blood vessels obtained from the xenograft tumors were observed under fluorescent microscope and laser scanning confocal microscope.</p><p><b>RESULTS</b>After five passages in vitro, MSCs maintained the proliferative activity and highly expressed CD105. CD105 was also expressed in the femurs of GFP-expressing nude mice, tumor cells, blood vessels of SU3 xenograft tumors, and clinical malignant gliomas. When MSCs were co-cultured with SU3-RFP, the ratio of yellow cells co-expressing RFP and GFP was significantly increased after extended time and continuous passages. According to the flow cytometry, yellow cells co-expressing RFP and GFP were 83.7% of the cultured cells. In tissue slices of the xenograft tumors, bundles of yellow vessel-like structure and cross-sectioned yellow vascular wall structures including vascular wall stroma cells were observed with RFP and GFP expression, and were identified as de novo formed vessels derived from fusion of MSCs with SU3-RFP cells.</p><p><b>CONCLUSION</b>Cell fusion occurs between tumor cells and host MSCs and it promotes tumor angiogenesis.</p>

Malignant transformation of fusion cells derived from co-culturing human glioma cell line SU3 with murine macrophages / 中华微生物学和免疫学杂志

Objective To investigate whether the malignant transformation of macrophages ( Mφ) in glioma mesenchyme was induced by the fusion of glioma cells ( SU3 ) and Mφ.Methods SU3 cells transfected with red fluorescent protein genes were co-cultured in vitro with Mφexpressing enhanced green fluorescent protein.The cell lineages with RFP+/GFP+dual-color fluorescence were established by using monoclonal selection method.A series of tests for analyzing cancer-related phenotypes, tumorigenicity and specific markers for murine macrophage were performed.Results (1) A few of dual-color fluorescent cells were observed in the co-culture.Three monoclonal cell lineages (C3, C4 and C12) were obtained success-fully.(2) Three types of cells including RFP+, EGFP+and RFP+/EGFP+cells were formed during the cul-ture of monoclonal C12 cell lineage.The percentage of EGFP+cells was increased along the extended culture time and increased passages.Then, EGFP+cells gradually became the predominant cell population.Nota-bly, the percentage of RFP+/EGFP+cells were decreased and maintained at a low level, but the RFP+cells almost disappeared.(3) EGFP+cells from monoclonal C12 cell lineage showed the malignant characteristics such as loss of contact inhibition, rapid proliferation andchromosome aneuploidy, as well as high tumorigenic rate in nude mice (5/5).They also expressed macrophage specific marker CD68 and showed a large number of telocentric chromosomes.Conclusion The results of this study suggested that the malignant transforma-tion of host macrophages as previously observed in solid tumor might be induced by cell fusion occurred be-tween human glioma cells and macrophages.Along with the previous evidences showing the isolation of the malignantly transformed macrophages ( ihCTC) from solid tumor tissue of tumor-bearing mice, the results confirmed an objective existence of malignant transformation of host macrophages in tumor microenvironment. The malignant transformation of host cells induced by fusion with tumor cells revealed not only a new under-standing for the progression of tumor and cancer heterogeneity, but also new targets for cancer therapy.

Establishment and characterization of dual-color fluorescence nude mouse models of glioma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To establish red-green dual-color fluorescence glioma model in nude mice and to explore its practical values.</p><p><b>METHODS</b>CM-DiI-stained rat glioma C6 cells (C6-CM- DiI cells) expressing red fluorescence were inoculated into the brain of athymic nude mice expressing green fluorescence protein (NC-C57BL/6J-EGFP). Then the whole-body dual-color fluorescence imaging was detected dynamically. Finally whole brains of the tumor-bearing mice were removed and 5 µm thick serial frozen slices were made. Light microscopy, fluorescence microscopy and confocal laser scanning microscopy were performed to observe the transplanted tumor tissue structure and fluorescent cells.</p><p><b>RESULTS</b>Tumor mass with red fluorescence increased gradually under continuous in-vivo fluorescence imaging monitoring. Under the fluorescence microscope, cells with red, green and yellow fluorescence were observed in the frozen sections of transplanted tumor tissue and the mutual structural relationship among them could be defined. The tumor cells migration, implantation and cell fusion between transplanted tumor cells and host cells could be observed. It could be distinguished according to the fluorescence, that blood vessels of tumor-origin displayed red fluorescence, blood vessels of host-origin displayed green fluorescence and mosaic blood vessels appeared yellow fluorescence. It was depicted that host innate astrocytes and oligodendrocytes in the microenvironment at the tumor periphery could be activated and dedifferentiated into nestin-positive cells.</p><p><b>CONCLUSIONS</b>In contrast to traditional animal model, the dual-color fluorescence imaging of nude mouse models of glioma possesses enormous advantages in investigating tumor mass in-vivo fluorescence imaging, tumor cells migration and metastasis, tumor angiogenesis and reactive activation of host innate cells in the microenvironment at tumor periphery, thus, has highly practical application value.</p>

Hydrogen-rich saline alleviation on the oxidative stress and early-phase radiation-induced brain injury in rats / 中华放射医学与防护杂志

Objective To investigate the effect of hydrogen on radiation-induced acute injury in rat brain.Methods Forty-five mature Sprague-Dawley rats were randomly divided into three groups:saline therapy group,hydrogen therapy group and healthy control group.The whole brain of SD rat was irradiated with single dose of 20 Gy by 4 MeV electrons.Rats in therapy group were injected with hydrogen-rich saline after irradiation and were sacrificed at 1,3,7,14 d post-irradiation.The changes of malonaldehyde (MDA),superoxidase dismutase (SOD) and 8-hydroxydeoxygunosine (8-OHdG) in brain homogenate and the pathological changes in brain hippocampus were observed.Results The brain water content (t=3.78,3.18,P＜0.05) and the contents of 8-OHdG (t=2.33,2.71,2.33,P＜0.05) in the therapy group was lower than the control group at 7 d and 14 d post-irradiation.The contents of SOD were significantly higher(t =2.41-2.92,P ＜ 0.05) from 1 to 7 day,while the contents of MDA were significantly lower in therapy group than those in the control group from 1 to 14 day post-irradiation (t =4.01-6.20,P ＜ 0.05).Moreover,the damage degree in the nerve cells of hippocampus was less compared to the control group.Conclusions The hydrogen-rich saline could have protection role in irradiation-induced acute brain injury in rats.

Laparoscopic Habib 4X bipolar radiofrequency device in laparoscopic liver resection / 中华肝胆外科杂志

Objective To evaluate the efficacy of laparoscopic Habib 4X (Habib 4X,Angio Dynamics US),a new bipolar radiofrequency (RF) device,in laparoscopic liver resection.Methods Thirty one patients who underwent laparoscopic liver resection using the laparoscopic Habib 4X from Sept 2009 to Apr 2012 were studied retrospectively.Results The laparoscopic Habib 4X was success fully used in 30 patients (malignant,n=18; benign,n=12).The procedures performed included left lateral sectionectomy (n=12),left hemi-hepatectomy (n=1),Ⅴ or Ⅵ segmentectomy (n=9),Ⅴ and Ⅵ bi-segmentectomy (n=2) and wedge exclusion (n=6).The time required for precoagulation and resection was 10～68 min (median 24 min).The mean intraoperative blood loss was 145±75ml (range 8-370 ml).Mild abnormal liver function which returned to normal in 3 to 5 days was detected postoperatively.The mean hospital stay was 7.8±2.6 d (range 3～12 days).There was no patient who developed postoperative bleeding,bile leakage or abdominal abscess.For cancer patients,there was no local recurrence on follow-up.Conclusion Laparoscopic Habib 4X,a device when used in laparoscopic liver resection,resulted in minimal blood loss and quick recovery.It had only mild effect on liver function and it had low morbidity.In addition,it might reduce the risk of local recurrence in malignant tumours.

Influences of carbamazepine on the neuromuscular block of rocuronium / 中国医师进修杂志

Objective To study the influence of carbamazepine on the neuromuscular block induced by rocuronium. Methods Thirty-two ASA Ⅰ- Ⅱ patients scheduled for neurosurgical operations were studied: 16 cases suffered from epilepsy were treated with carbamazepine for 24 to 180 months (carbamazepine group); the others suffered from intracranial tumors without antiepileptic treatment (noncarbamazepine group). Anesthesia was induced and maintained with target controlled with propofol 5 μ g/ml was monitored using train-of-four (TOF) stimulation. The onset time and times of muscle relaxation recovery 25%,50%,75% was recorded. Results The onset time was no difference between two groups (P＞0.05),but the times of muscle relaxation recovery 25%, 50%, 75% in carbamazepine group [(23.9±3.8 ), ( 29.2 ±4.5 ), ( 36.0 ± 5.4 ) min] were shorter than those in non-carbamazepine group [( 34.0 ± 2.8 ), (40.5 ± 4.6),( 49.9 ± 5.3 ) min] ( P ＜ 0.01 ). The recovery index in carbamazepine group [( 12.1 ± 2.9 ) min] was shorter than that in non-carbamazepine group [(15.8 ± 3.1) min](P＜0.05 ). Conclusion The duration of the rocuronium-induced neuromuscular block is significantly shortened by preceding chronic carbamazepine therapy.

Changes of function and ultrastructure of cerebral microvascular endothelial cells early after traumatic brain injury in rats / 中华创伤杂志

Objective To investigate the functional and ultrastructural changes of cerebral microvascular endothelial cells at early stage following traumatic brain injury (TBI) in rats. Methods The rat models with closed brain injury were established with the improved Marmarous method. The expressions of thrombomodulin (TM) and von Willebrand factor (vWF) were determined by immunohistochemical techniques (5 rats per group) and real-time quantitative RT-PCR (5 rats per group) respectively at 1, 4, 8, 12, 24 hours and at days 3 and 7 after injury. Results TM and vWF started expression at 4 hours, reached peak at 24 hours and recovered to normal at day 7 after TBI. The expression levels of TM and vWF at different time points in sham control group showed statistical difference compared with damage group (P < 0.05). Conclusion The activation of the cerebral microvascular endothelial cells at early stage after TBI is the main mechanism of early secondary brain injury.

cDNA array in the establishment of a gene expression profile associated with differentiation inducing the glioma cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>Establishment of a gene expression profile associated with differentiation inducing the glioma cells was made possible.</p><p><b>METHOD</b>The expression level of 18 000 genes in glioma cells was evaluated before and after induction with sodium phenyl-butyrate for 2 hours or 6 days by cDNA array technique, with the results proved by multi-dot blot.</p><p><b>RESULTS</b>Ninety-eight gene expressions in the glioma cells were changed after the induction, with some genes in transcription and translation systems down-regulated, some oncogenes down-regulated, and some differentiation or apoptosis genes up-regulated. Eighteen unknown EST fragments were changed also.</p><p><b>CONCLUSION</b>A gene expression profile associated with differentiation-inducing the glioma cells including 98 genes has been established.</p>

Construction and expression of anti-glioma single-chain Fv/human tumor necrosis factor α fusion gene / 细胞与分子免疫学杂志

Aim To construct and express the fused gene of anti-glioma single chain Fv(39ScFv)/ human tumor necrosis factor α (TNF-α ). Methods The mature peptide cDNA of TNF-α was fused to 3′ -terminus of ScFv gene. The 39ScFv-TNFα fusion gene was cloned into expression vector pET20b(＋ ) and induced to express in E.coli. Results The fusion protein expressed in E.coli account for 20％ of the total bacterial protein. It displayed a single band of Mr 44 000 by reducing SDS-PAGE and Western blot, and retained both of its anti-glioma immunoreactivity and biological activity of TNF. Conclusion The fusion gene was constructed and expressed successfully in E.coli. The fusion protein possessed bifunctional activities, it may prove useful in targeting immunotherapy against glioma.