IL-6、PCT、CRP及WBC与老年腹部手术患者术后肺部感染的相关性

目的研究血白细胞介素-6(IL-6)、降钙素原(PCT)、C反应蛋白(CRP)及白细胞计数(WBC)与老年腹部手术后早期肺部感染的相关性。方法收集2023年1月至2023年12月濮阳市中医医院收治的45例老年腹部手术后早期肺部感染患者作为感染组,另按1∶2比例抽取同期行腹部手术但术后早期未出现肺部感染的90例作为未感染组,所有病例术后次日清晨均采集血样完成IL-6、PCT、CRP、WBC检测,比较感染组与未感染组上述指标差异,分析早期肺部感染患者病原菌分布,比较不同感染严重程度患者上述指标的差异,分析以上各指标与肺部感染严重程度的关系,并应用受试者工作曲线(ROC)分析以上参数诊断老年腹部手术后早期肺部感染的效能。结果腹部手术后早期肺部感染病原菌分布主要以革兰阴性菌(77.78%)为主,其中铜绿假单胞菌所占比例最高(53.33%,其次为大肠埃希菌(15.56%);革兰阳性菌(17.78%)中金黄色葡萄球菌所占比例最高(11.11%);真菌占4.44%,均为白假丝酵母菌;感染组术后次日清晨血IL-6、PCT、CRP、WBC水平均高于未感染组,差异均有统计学意义(P<0.05);不同严重程度肺部感染患者术后次日各实验室指标比较差异有统计学意义,随感染严重程度的上升,IL-6、PCT、CRP、WBC水平上升,差异均有统计学意义(P<0.05);IL-6、PCT、CRP、WBC均与临床肺部感染评分(CPIS)呈正相关,差异均有统计学意义(r=0.716、0.765、0.725、0.735,P<0.05);IL-6、PCT、CRP、WBC联合检测诊断腹部手术早期肺部感染的曲线下面积(AUC)值高于单项检测,差异均有统计学意义(P<0.05)。结论IL-6、PCT、WBC及CRP对老年腹部外科术后早期肺部感染皆有一定的诊断价值,且联合检测诊断效能更高,可将其作为腹部外科术后早期肺部感染筛查的重要指标。

Bone marrow stromal cell versus neural stem cell transplantation in a C6 glioma rat model

BACKGROUND： Embryonic neural stem cells （NSCs） have provided positive effects for the treatment of glioma. However, the source for embryonic NSCs remains limited and high amplification conditions are required. Bone marrow stromal cells （BMSCs） have been proposed for the treatment of glioma. OBJECTIVE： To investigate biological changes in NSCs and BMSCs following transplantation into rat models of glioma. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Embryonic Stem Cell Research Laboratory of Yunyang Medical College from February 2006 to August 2008. MATERIALS： The rat C6 glioma cell line was purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; mouse anti-bromodeoxyuridine （BrdU） monoclonal antibody and Cy3-1abeled goat anti-mouse IgG antibody was purchased from Upstate, USA. METHODS： A total of 95 Sprag6ue Dawley rats were randomly assigned to three groups： NSC （n = 35）, transplanted with 〉 6 × 10^6 NSCs via left medial hind limb; BMSC （n = 35）, transplanted with 〉 1 × 10^6 BMSCs via left medial hind limb; model group （n = 25）, injected with the same volume of 0.1 mmol/L phosphate buffered saline. MAIN OUTCOME MEASURES： Gliomal growth and size were assessed by nuclear magnetic resonance, and glioma morphological features were observed following hematoxylin-eosin staining and BrdU immunohistochemistry 3 and 4 weeks following transplantation. RESULTS： The average survival of rats in the BMSC, NSC, and model groups was 4.03, 4.28, and 3.88 weeks. At 3 weeks, there was no significant difference in the average glioma diameter between the BMSC and model groups （P 〉 0.05）. However, gliomal diameter was significantly decreased in the NSC group compared with the model group （P 〈 0.05）. At 4 weeks, there was no statistical difference between the groups （P 〉 0.05）. BrdU immunohistochemistry revealed that BMSCs and NSCs appeared to migrate to the gliomas. CONCLUSION： NSCs inhibited glioma cell growth and prolonged rat survival. BMSCs did not significantly suppress glioma cell growth.

Effects of antigliomatin from the scorpion venom of Buthus martensii Karsch on chloride channels on C6 glioma cells

Using whole-cell patch-clamp recordings, the effects of antigliomatin were observed on chloride channels on C6 glioma cells cultured in vitro. Antigliomatin was extracted from the venom of the scorpion Buthus martensii Karsch. Chloride channels are closed under normal osmotic pressure. When osmotic pressure was reduced to 120, 110 and 100 mV, the cell volume enlarged, chloride channels opened, and the chloride channel current increased. Three minutes after antigliomatin treatment, the chloride channel current decreased in a dose-dependent manner. These results show that antigliomatin extracted from the venom of the scorpion Buthus martensii Karsch diminishes chloride channel currents on C6 glioma cells.

Heat shock induction of a 65 kDa ATP-binding proteinase in rat C6 glioma cells

The 45, 55, 65 and 100 kDa ATP-binding proteinases (ATP-BPases) of the heat-shocked (44 ℃ for 30 min, recovery for 12h) rat C6 glioma cells were purified by DEAE-ionexchange and ATP-affinity chromatography. Their molecular masses, isoelectric points (pI), pH-optima and other properties were analyzed by native proteinase gels.It was shown that the 65 kDa ATP-BPase is specifically induced by heat shock and not detectable in control cells.Its N-terminal 1-9 amino acid sequence was determined by Edman degradation, but no homologies to other proteins in the protein data bases were found. 30 and 31 kDa proteinases can be cleaved from the 45, 55 and 65 kDa proteinases to which they are linked. A possible relationship of the heat-induced 65 kDa ATP-BPase with the ATP-dependent proteinases (ATP-DPases) in prokaryotes and eukaryotes is discussed.

Lipid-albumin nanoassemblies co-loaded with borneol and paclitaxel for intracellular drug delivery to C6 glioma cells with P-gp inhibition and its tumor targeting

Successful chemotherapy with paclitaxel(PTX)is impeded by multidrug resistance(MDR)in tumor cells.In this study,lipid-albumin nanoassemblies co-loaded with borneol and paclitaxel(BOR/PTX LANs)were prepared to circumvent MDR in C6 glioma cells.The physiochemical properties including particle size,encapsulation efficiency and morphology were evaluated in vitro.Quantitative and qualitative investigations of cellular uptake were carried out in C6 glioma cells.The cytotoxicity of the BOR/PTX LANs was determined by MTT assay.After that,the tumor targeting was also evaluated in C6 glioma bearing mice by in vivo imaging analysis.BOR/PTX LANs have a higher entrapment efficiency(90.4±1.2%),small particle size(107.5±3.2 nm),narrow distribution(P.I.=0.171±0.02).The cellular uptake of PTX was significantly increased by BOR/PTX LANs compared with paclitaxel loaded lipidalbumin nanoassemblies(PTX LANs)in quantitative research.The result was further confirmed by confocal laser scanning microscopy qualitatively.The cellular uptake was energy-,timeand concentration-dependent,and clathrin-and endosome/lysosome-associated pathways were involved.The BOR/PTX LANs displayed a higher cytotoxicity agaist C6 glioma cells in comparion with PTX LANs and Taxol.Moreover,the encapsulation of BOR in LANs obviously increased the accumulation of the drug in tumor tissues,demonstrating the tumor targeted ability of BOR/PTX LANs.These results indicated that BOR/PTX LANs could overcome MDR by combination of drug delivery systems and P-gp inhibition,and shown the potential for treatment of gliomas.

白细胞介素-6、降钙素原、超敏C反应蛋白和白细胞计数在儿童细菌感染性疾病早期诊断中的价值

目的探讨白细胞介素-6(IL-6)、降钙素原(PCT)、超敏C反应蛋白(hs-CRP)和白细胞计数(WBC)在儿童细菌感染性疾病早期诊断中的价值。方法选取2021年2月至2023年2月苏州市吴江区儿童医院收治的54例细菌感染性疾病患儿作为观察组,选择同期64例非细菌感染性疾病患儿作为对照组,比较两组血清IL-6、PCT、hs-CRP、WBC水平及阳性率,采用受试者工作特征曲线分析联合检测的诊断价值。结果观察组血清IL-6、PCT、hs-CRP、WBC水平均高于对照组(P<0.05)。观察组血清IL-6、PCT、hs-CRP、WBC阳性率均高于对照组(P<0.05)。血清IL-6、PCT、hs-CRP、WBC联合检测的曲线下面积大于单一检测(P<0.05)。结论IL-6、PCT、hs-CRP和WBC联合检测在儿童细菌感染性疾病早期诊断中的价值较高,值得临床推广应用。

Temporal dynamic changes of connexin 43 expression in C6 cells following lipopolysaccharide stimulation

Connexin 43, a gap junction protein, is expressed mainly in glia in the central nervous system. Neuroinflammation plays an important role in central nervous system injury. Changes to glial connexin 43 levels and neuroinflammation may trigger brain injury and neurodegenerative diseases To illustrate the relationship between connexin 43 and neuroinflammation, this study investigated how connexin 43 expression levels change in lipopolysaccharide-stimulated rat C6 glioma cells. C6 cells were treated with 0.05, 0.25, 0.5, 1,2.5 and 5 IJg/mL lipopolysaccharide for 24 hours. The nitrite estimation-detected nitric oxide release level was elevated substantially after lipopolysaccharide stimulation. To test the transcriptional level changes of inducible nitric oxide synthase, tumor necrosis factor-a and connexin 43 mRNA, C6 cells were treated with 5 pg/mL lipopolysaccharide for 3 48 hours. Reverse transcription-PCR showed that the expression of inducible nitric oxide synthase and tumor necrosis factor-a mRNA increased over time, but connexin 43 mRNA levels increased in lipopolysaccharide-stimulated C6 cells at 3 and 6 hours, and then decreased from 12 to 48 hours. Connexin 43 protein expression was detected by immunofluorescence staining, and the protein levels matched the mRNA expression levels. These results suggest that connexin 43 expression is biphasic in lipopo^ysacchadde-induced neuroinflammation in C6 cells, which may be correlated with the connexin 43 compensatory mechanism.

Up-regulation interleukin-6 and interleukin-8 by activated protein C in lipopolysaccharide-treated human umbilical vein endothelial cells

Objective: To investigate the effect of activated protein C (APC) on inflammatory responses in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS). Methods: The second passage of collagenase digested HUVEC was divided into the following groups: serum free medium control group (SFM control), phosphate buffer solution control group (PBS control), LPS group with final concentration of 1 μg/ml (LPS group), APC group with final concentration of 7 μg/ml, Pre-APC group (APC pretreatment for 30 min prior to LPS challenge), and Post-APC group (APC administration 30 min after LPS challenge). Supernatant was harvested at 0, 4, 8, 12 and 24 h after LPS challenge. Interleukin-6 (IL-6) and Interleukin-8 (IL-8) levels were analyzed with ELISA. Cells were harvested at 24 h after LPS challenge, and total RNA was extracted. Mes-senger RNA levels for IL-6 and IL-8 were semi-quantitatively determined by RT-PCR. Results: Compared with control group, IL-6 and IL-8 levels steadily increased 4 to 24 h after LPS stimulation. APC treatment could increase LPS-induced IL-6 and IL-8 production. The mRNA levels of IL-6 and IL-8 exhibited a similar change. Conclusion: APC can further increase the level of IL-6 and IL-8 induced by LPS. The effect of these elevated cytokines is still under investigation.

Establishment of Embryonic Stem Cell Lines from C57BL/6J Mice and Generation of Chimeras

Four embryonic stem (ES) cell lines, designated CE1, CE2, CE3 and CE4, were isolated from C57BL/6J blastocysts. The ratio of normal diploid composition of these cell lines is above 70%. To examine the differentiation potential of the ES cells, the CE2 cells were injected subcutaneously into syngenic mice, and many kinds of differentiated cells were observed on the sections of the teratoma derived from this ES cell line. On the other hand, to test the chimeric ability of the ES cells, the CE2 cells were microinjected into the blastocysts of ICR mice, and a chimera was obtained among living pups. These results show that CE2 ES cells are pluripotent stem cells, which can differentiate into many kinds of cell types, and can be used as a cell system for further research.

Inhibitory effects of Pseudomonas aeruginosa mannose-sensitive hemagglutinin on rat C6 glioma cell proliferation

BACKGROUND： Pseudomonas aeruginosa mannose-sensitive hemagglutinin （PA-MSHA） parenteral injection is used as a broad-spectrum immunomodulator. It remains unclear whether PA-MSHA exhibits inhibitory effects on tumor cell growth. OBJECTIVE： To investigate inhibitory mechanisms of PA-MSHA-induced proliferation in rat C6 glioma cells in vitro. DESIGN, TIME AND SETTING： Comparative observation and in vitro experiments were performed at the Key Laboratory of Natural Medicine, Kunming Medical College, China from July 2008 to April 2009. MATERIALS： Rat C6 glioma cell line （Shanghai Institute of Cell Biology, Chinese Academy of Sciences, China） and PA-MSHA parenteral injection （Beijing Wanteer Bio-Pharmaceutical, China） were used in the present study. METHODS： Rat C6 glioma cells in logarithmic growth phase were harvested in vitro. Adherent monolayer cells were respectively treated with PA-MSHA at final colony-forming units （cfu） of 1 ×10^8 cfu/mL, 2 × 10^8 cfu/mL, 4 × 10^8 cfu/mL, 6 × 10^8 cfu/mL, and 8 ×10^8 cfu/mL following 24 hours of conventional culture. MAIN OUTCOME MEASURES： MTT colorimetric assay was utilized to determine the inhibitory rate of C6 glioma cells following treatment with various concentrations of PA-MSHA at different times. Cell apoptosis was detected by fluorescent microscopy following Hoechst 33258 staining. Flow cytometry was used to measure PA-MSHA effects on C6 cell cycle. RESULTS： Inhibitory rate of C6 glioma cells increased with prolonged time and increased dose. Hoechst 33258 staining revealed obvious morphological changes in apoptotic C6 glioma cells. Flow cytometry revealed hypodiploid peaks, Le., apoptotic peak, and the apoptotic rate in cells during S-phase significantly increased with increased concentrations in the experimental groups. CONCLUSION： With in vitro experiments, PA-MSHA preparations inhibited C6 glioma cell proliferation in a time- and dose-dependent manner. These mechanisms are likely associated with cell apoptosis induction and inhibition of the S phase.

Effects of selective cyclooxygenase-2 inhibitor on C6 glioma cell proliferation and apoptosis

BACKGROUND： Studies have shown that cyclooxygenase-2 is associated with proliferation and apoptosis of glioma cells. OBJECTIVE： To investigate the effects of selective cyclooxygenase-2 inhibitor celecoxib on proliferation and apoptosis of C6 glioma cells in vitro. DESIGN, TIME AND SETTING： A cellular, molecular, controlled study was performed at the Central Laboratory and Room of Electron Microscope, Medical School, Xi＇an Jiaotong University, China from March 2007 to March 2008. MATERIALS： C6 glioma cells during in vitro log phase were assigned to control and experimental groups. Celecoxib （Pfizer, USA）, dimethyl sulfoxide （Sigma, USA）, and MTT （Sigma, USA） were used for this study. METHODS： The control group was subdivided into blank control and dimethyl sulfoxide control groups. C6 glioma cells in the blank control and dimethyl sulfoxide control groups were incubated in Dulbecco＇s modified Eagle＇s medium supplemented with 10% calf serum and 0.3% dimethyl sulfoxide respectively. C6 glioma cells in the experimental group were separately treated with 60, 80 and 100 μmol/L celecoxib. MAIN OUTCOME MEASURES： Activity of C6 glioma cells was examined by MTT assay. C6 glioma cell cycle and apoptosis were determined by annexin V-fluorescein isothiocyanate/propidium iodide double-staining, followed by flow cytometry. Morphology and ultrastructure of C6 glioma cells were observed with an inverted microscope and a transmission electron microscope, respectively. RESULTS： Compared with the blank control group, cell density was reduced, adherence ability weakened, and irregular nuclei were visible, with the presence of chromatin condensation, margination, and some apoptotic bodies in the experimental group. Activity of C6 glioma cells was significantly decreased （P 〈 0.05）, cell number was reduced during S phase, cell number was significantly increased during G2/M phase （P 〈 0.01 ）, and the apoptotic rate was significantly increased （P 〈 0.05） in the experimental group. These results were displayed in a dose- and time-dependent fashion. The outcomes were obvious in the 100 IJmol/L celecoxib group following 72 hours of treatment. CONCLUSION： Celecoxib blocked proliferation and induced apoptosis of C6 glioma cells in a dose- and time-dependent fashion.

血清CC16、IL-37、IL-6、CRP对肺炎合并呼吸衰竭患者预后的预测价值

目的探讨血清Clara细胞分泌蛋白16(CC16)、白细胞介素(IL)-37、IL-6、C反应蛋白(CRP)对肺炎合并呼吸衰竭患者预后的预测价值。方法选取洪湖市人民医院于2020年7月至2022年7月收治的117例肺炎合并呼吸衰竭患者作为研究组,根据患者恢复情况分为预后良好组和预后不良组。另选取同期洪湖市人民医院收治的113例未合并呼吸衰竭的肺炎患者作为对照组。采用酶联免疫吸附试验检测所有患者血清CC16、IL-37、IL-6、CRP水平;采用受试者工作特征(ROC)曲线分析血清CC16、IL-37、IL-6、CRP水平对肺炎合并呼吸衰竭患者预后的预测价值。结果与对照组相比,研究组血清CC16水平较低(P<0.05),血清IL-37、IL-6和CRP水平较高(P<0.05)。与预后良好组相比,预后不良组血清CC16水平较低(P<0.05),血清IL-37、IL-6和CRP水平较高(P<0.05)。预后不良组患者白细胞计数(WBC)、中性粒细胞计数(NEU)和急性生理与慢性健康状况评价Ⅱ(APACHEⅡ)评分均高于预后良好组(P<0.05)。ROC曲线分析结果显示,血清CC16、IL-37、IL-6、CRP 4项联合预测肺炎合并呼吸衰竭患者预后不良的AUC为0.934(95%CI:0.873~0.972),大于CC16、IL-37、IL-6、CRP单项预测的AUC[0.816(95%CI:0.734~0.882)、0.750(95%CI:0.662~0.825)、0.842(95%CI:0.763~0.902)、0.827(95%CI:0.746~0.890)],差异均有统计学意义(Z=2.598、2.771、1.976、1.981,P<0.05)。结论肺炎合并呼吸衰竭患者血清CC16水平较低,IL-37、IL-6和CRP水平较高,4项指标联合检测对肺炎合并呼吸衰竭患者预后不良具有较高的预测价值。

Induction of apoptosis and inhibition of proliferation of C6 glioma cells in vitro by tamoxifen

Objective To investigate the anti-tumor effect and mechanism of tamoxifen on rat C6 glioma cells. Methods C6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 3% fetal calf serum (FCS), and treated with tamoxifen of different concentrations, i.e. group A (1.25μmol/L), group B (2.50 μmol/L), group C (5.00 μmol/L), group D (10.00 μmol/L), group E (20.00 μmol/L) and control group (0.00 μmol/L). Morphological changes, MTT assay and 5-bromo-2’-deoxyuriding labeling ratio were assessed. Apoptosis was observed by flow cytometry. Results C6 cells treated with different doses of tamoxifen for 24, 48, and 72 hours became irregular in shape, while cells treated with vehicle grew normally. MTT assay showed that tamoxifen did not suppress C6 cell growth until 72 hours after treatment. Seventy-two hours after treatment, there were significant differences in cell viable rate between group A versus groups C, D and E; so did group B versus group D as well as group E (P<0.05). BrdU incorporation assay indicated significant difference of BrdU labbled index (BrdU LI) among groups A, C, E and control group 48 hours after treatment (P<0.05). And the BrdU LI decreased with the increased concentration of tamoxifen. Flow cytometry (FCM) showed significant difference between treated group and control group at 24, 48, and 72 hours after treatment (P<0.05). Conclusion Tamoxifen significantly suppresses the growth of C6 glioma cells in a time-and dose-dependent manner. The mechanism of tamoxifen suppressing C6 glioma cells may be inhibiting proliferation and inducing apoptosis. Therefore, tamoxifen can be a candidate as a chemotherapy agent for glioma.

Preparation, Release-control and Cell Apoptosis of C_6 Glioma Cells in PEG-PLGA-Rg3 Nanoparticles

With biodegradable material poly（ethylene glycol）-poly（lactide-co-glycolide） （PEG-PLGA） as substrate, the size distribution of Rg3-NPs was approved by the scanning electron microscopy. MTT assay was used to detect the effects of Rg3-NPs on the growth rate of C6 cells at various concentrations and flow cytometry（FCM） was applied to assay the cell cycle and cell apoptosis of C6 glioma cells. Western blot analysis was used to measure the protein level of PCNA. The results show that Rg3-NPs are slick and uniformity, the average diameter of the nanoparticles is about 75-90 nm, entrapment efficiency is （89.7±1.7）%. MTT assay shows the growth of C6 Glioma Cells can be significantly inhibited by Rg3-NPs in a dose-dependence manner. FCM and Western blot analysis show Rg3 can be released from the conjugated nanoparticles to function in the cell nuclei so as to lead to the changes in the growth cycle of the cells, which results in the arrest of G0-G1 cell cycle and induces the apoptosis of C6 cells. Therefore, Rg3-NPs may be used for the auxiliary therapy of brain glioma.

RNA interference affects tumorigenicity and expression of insulin-like growth factor-1,insulin-like growth factor-1 receptor,and basic fibroblast growth factor-2 in rat C6 glioma cells

BACKGROUND： Human gliomas are more likely to express basic fibroblast growth factor-2 （FGF-2） insulin-like growth factor-1（IGF-1）, and IGF-1 receptor （IGF-1R） than normal brain tissue. These factors activate signal transduction systems of Ras/MAPK and PI3K/Akl, which promote glioma growth. OBJECTIVE： To utilize RNA interference （RNAi） technique to down-regulate FGF-2, IGF-1, and IGF-1R gene expression, and to investigate the effects of these genes on rat C6 glioma cells, as well as the feasibility of RNAi for treating glioma. DESIGN, TIME AND SETTING： This neurooncological, randomized, controlled, in vivo and in vitro experiment, which used RNAi methodology, was performed at the Laboratory of Molecular Biology, Institute of Biochemistry, Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS： Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences. Small interfering RNA （siRNA） was synthesized by Shanghai GenePharma. Anti-IGF-1, anti-IGF-1R, anti-FGF-2, anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology, USA. Four to six week-old BALB/c nude mice were purchased from the Laboratory Animal Center, Chinese Academy of Sciences. METHODS： C6 glioma cells were transfected with siRNA, which was chemically synthesized in vitro to correspond to endogenous FGF-2, IGF-1, and IGF-1R genes. The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR, and protein expression was determined by Western blot analysis. C6 glioma cell proliferation was observed using a growth curve C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry. C6 glioma cell growth regression was observed by transwell migration assay. In addition, nude mouse subcutaneous tumor models were used in this study. For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA, two blank control groups, with six mice each, were set up： A （2.5 μg siRNA was injected one week after C6 cells were inoculated, Le., when tumor volume reached 8 mm × 8 mm） and B （siRNA was injected at the same time with C6 cells were inoculated. To study the effects of FGF-2 siRNA, the groups consisted of a blank control group, negative control group, 2.6 μg siRNA group, 4 μg siRNA group, and 5.3 μg siRNA group, with six mice each. MAIN OUTCOME MEASURES： mRNA and protein inhibition ratio of FGF-2, IGF-1, and IGF-1 R; C6 glioma cell proliferation, apoptosis, and cycle growth arrest; C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS： All siRNA constructs proved to be effective. After 48 hours, transfection of 200 nmol/L siRNA resulted in a FGF-2 or IGF-1R gene inhibition ratio 〉 80% and an IGF-1 gene inhibition ratio of approximately 70%. Protein expression levels for FGF-2, IGF-1, and IGF-1R decreased in a dose-dependent manner following siRNA transfection, with an inhibition rate 〉 85%, 60%, and 50%, respectively. C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA. The apoptosis rate of C6 glioma cells induced by FGF-2, IGF-1, and IGF-1R siRNA was 39.96%, 15.07% and 22.47%, respectively （P 〈 0.01）. Transfection of 200 nmol/L IGF or IGF-1R siRNA for 48 hours suppressed C6 glioma cell migration. At 30 days after intratumoral injection of 2.6, 4, and 5.3 tJg FGF-2 siRNA, tumor growth regression rate of FGF-2 siRNA was 56%, 67%, and 86%, respectively. The tumor growth regression rate was 71.88% and 45.71%, respectively, when IGF-1 or IGF-1R siRNA was intratumorally injected 1 week after C6 glioma cell transplantation. When IGF-1 or IGF-1 R siRNA was intratumorally injected during C6 glioma cell transplantation, the tumor growth regression rate was 78.13% and 74.29%, respectively. CONCLUSION： siRNA transfection downregulated gene expression of FGF-2, IGF-1, and IGF-1R In addition, siRNA treatment markedly suppressed glioma cell proliferation, growth, and migration, and concomitantly reduced subcutaneous tumorigenicity.

Stable EGFP Gene Expression in C6 Glioma Cell Line after Transduction with HIV-1-based Lentiviral Vector

Objective： To establish a stable C6/EGFP glioma cell transfected with the human immunodeficiency virus line for studies on glioma. Methods： The C6 glioma cell line was type I （HIV-1） based lentivirus vector containing two enhancer-promoters CMV and EF1α. Enhanced green fluorescent protein （EGFP）-positive C6 cells were sorted out by fluorescence-activated cell sort. Expression of EGFP was observed by fluorescent microscopy. EGFP gene in C6 genome was assessed by Polymerase chain reaction （PCR） and DNA sequencing. Original and transfected cells were compared biologically and cytomorphologically. Results： Lentivirus vector transfection produced up to 40% EGFP-positive cells. After fluorescence-activated cell sort selection, a pure cell line C6/EGFP was established. PCR and DNA sequencing revealed integration of EGFP gene in C6 cell genome. Analysis of cell characteristics revealed no difference between transfected and original cells. Conclusion： A C6/EGFP cell line expressing EGFP as a marker is established, in which the EGFP gene is integrated into the genome. This cell line can be served as a promising tool for further basic research and gene therapy studies.

Changes of the intestinal endocrine cells in the C57BL/6 mouse after implantation of murine lung carcinoma (3LL): An immunohistochemical quantitative study

AIM: To study the distributions and frequencies of intestinal endocrine cells in the C57BL/6 mouse with immunohistochemical method using seven types of specific antisera against chromogranin A (CGA), serotonin,somatostatin, glucagons, gastrin, cholecystokinin (CCK)-8 and human pancreatic polypeptide (hPP) after abdominal subcutaneous implantation of murine lung carcinoma (3LL).METHODS: The experimental animals were divided into two groups, one is non-implanted Sham and the other is 3LL-implanted group. Samples were collected from six regions of intestinal tract at 28th d after implantation of 3LL cells (1×105 cell/mouse).RESULTS: In this study, five types of immunoreactive (IR) cells were identified except for gastrin and hPP. The regional distributions of the intestinal endocrine cells in the 3LL-implanted group were similar to those of the non-implanted Sham. However, significant decreases of IR cells were detected in 3LL-implanted group compared to those of non-implanted Sham. CGA- and serotonin-IR cells significantly decreased in 3LL-implanted groups compared to that of non-implanted Sham. Somatostatin-IR cells in the jejunum and ileum and CCK-8-IR cells in the jejunum of 3LL-implanted groups significantly decreased compared to that of non-implanted Sham. In addition,glucagon-IR cells were restricted to the ileum and colon of non-implanted Sham.CONCLUSION: Implantation of tumor cell mass (3LL)induced severe quantifiable changes of intestinal endocrine cell density and the abnormality in density of intestinal endocrine cells may contribute to the development of gastrointestinal symptoms such as anorexia and indigestion, frequently encountered in patients with cancer.

Intramedullary Spinal Cord Glioma Following Microinjection of Glioblastoma Cell Line C6 in Rats

Background: This paper describes the establishment of a rat intramedullary spinal cord tumor (IMSCT) model and histopathological characterization of the tumor model. Methods: Fourteen male Wistar rats were randomized into two groups. The rats in group 1 (control group, n = 7) received a 5 μl intramedullary injection of serum physiologic (SF). Those in group 2 (experimental group, n= 7) received a 5 μl intramedullary implantation of media containing 5 × 105 C6 glioma cells. The animals were sacrificed for histopathological examination at 21 days. Results: The control group showed normal functional and histopathological findings. The group 2 rats implanted with C6 glioblastoma cells developed hind-limb paraplegia. Pathological sections confirmed intramedullary C6 glioblastoma invading the spinal cord. Conclusions: A rat C6 IMSCT model was successfully established. This model may be useful in increasing understanding of intramedullary spinal cord gliomas in humans.

Establishment of Germ-line Competent C57BL/6J Embryonic Stem Cell Lines

Objective To establish C57BL/6J embryonic stem （ES） cell lines with potential germ- line contribution Methods ES cells were isolated from blastocyst inner cell mass of C5 7BL/6J mice, and cultured for 15 passages, and then injected into blastococels of ICR mice blastocysts to establish chimeric mice. Results Three ES cell lines （mC57ES1,mC57ES3, mC57ES7） derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker “stage-specific embryonic antigen-I” and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC57ES1 cells consistently differentiated into derivatives of all three embryonic germ layers. When mC57ES1 cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained. One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.

Effect of Different Isorhamnetin of Different Density Cultured Rat C6 Glioma Cells in Vitro

Objective To explore the influence of different concentrations of isorhamnetin on C6 glioma cell morphology.Methods Set the blank control group,blank solvent control group and reagent group of four concentration,the growth of cells were observed under microscope;MTT assay was used to test the effect of isorhamnetin on cultured C6 glioma cells,as well as calculate the cell inhibition rate and survival rate;flow cytometry was used to check the detection peak and detection rate of Isorhamnetin group and negative control apoptosis group,and analyzed the relationship between different concentrations of isorhamnetin and C6 glioma cell apoptosis rate;total protein was extracted from cells,and used Western blotting to detected total AKT protein and Ser473 AKT protein loci in cells;used SD rats to construct brain glioma model,feed isorhamnetin plain to them for five days,and then used HPLC to detect plasma,liver,brain tissue content.Results Under the observation of inverted microscope and image analysis,after using Isorhamnetin,tumor cells appear apoptosis and necrosis change.Display with different Isorhamnetin MTT colorimetric method shows that the higher the concentration of added Isorhamnetin,the worse the growth rate of C6 glioma cells in vitro,and the higher the Inhibitory rate,the lower survival rate.The flow cytometric detection shows the C6 glioma cells which is added 40 ug/ul Isorhamnetin have the highest rate of apoptosis.After adding 80μg/μl concentration of the isorhamnetin,C6 glioma cells have the lowest survival rate.Western blot test shows the AKT protein and Ser473 total site AKT protein density is in reverse proportion to the increase of the concentration of the isorhamnetin.High performance liquid chromatographic method has determined that there are isorhamnetin in both the rat plasma and brain tissue,which shows that the plasma and tissue all have different isorhamnetin distribution,and isorhamnetin mainly exist in the brain tissue.Conclusion Low concentration of isorhamnetin can induce apoptosis of C6 glioma cells,and high concentration of isorhamnetin can lead to apoptosis and necrosis of C6 glioma cells in vitro,which has obvious inhibitory effect on the growth of glioma cells,and the mechanism is closely related to PI3K/AKT pathway,and in SD rat brain glioma model,the high performance liquid chromatography was used to detect the content of plasma and brain tissue,which indicated the isorhamnetin has target in brain tissue,which provided experimental evidence for the development and utilization of isorhamnetin in mice.