Lethal effect of mononuclear cells derived from human umbilical cord blood differentiating into dendritic cells after in vitro induction of cytokines on neuroblastoma cells

BACKGROUND： Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human granulocyte-macrophage colony stimulating factor （rhG-MCSF） and recombinant human interleukin-4 （rhlL-4） can generate a great many dendritic cells and promote the lethal effect of T cells on human neuroblastoma, but it is unclear that whether the lethal effect is associated with the most proper concentration of dendritic cells. OBJEETIVE： To investigate the lethal effect of human umbilical cord blood mononuclear cells induced in vitro by cytokines differentiating into dendritic cells on human neuroblastoma, and its best concentration range. DESIGN ： Open experiment SEI-FING： Department of Pediatrics, the Medical School Hospital of Qingdao University MATERIALS ： The study was carried out in the Shandong Provincial Key Laboratory （Laboratory for the Department of Pediatrics of the Medical School Hospital of Qingdao University） during September 2005 to May 2006. Human umbilical cord blood samples were taken from the healthy newborn infants of full-term normal delivery during October to November 2005 in the Medical School Hospital of Qingdao University, and were voluntarily donated by the puerperas. Main instruments： type 3111 CO2 incubator （Forma Scientific, USA）, type 550 ELISA Reader （Bio-Rad, USA）. Main reagents： neuroblastoma cell line SK-N-SH （Shanghai Institute of Life Science, Chinese Academy of Sciences）, RPMI-1640 culture fluid and fetal bovine serum （Hyclone）, rhlL-4 （Promega, USA）, rhG-MCSF （Harbin Pharmaceutic Group Bioengineering Co.Ltd）, rat anti-human CDla monoclonal antibody and FITC-labeled rabbit anti-rat IgG （Xiehe Stem cell Gene Engineering Co.Ltd）. METHODS： ① Human umbilical cord blood mononuclear cells obtained with attachment methods differentiated into human umbilical cord blood dendritic cells, presenting typical morphology of dendritic cells after in vitro induction by rhG-MCSF and rhlL-4. ② Different concentrations of dendritic cells[ dendritic cells： neuroblastoma cells=20：1,50：1,100：1 （2×10^8 L^-1,5×10^8 L^-1,1×10^9 L^-1）], 1×10^9 L^-1 T cells and 1×10^7 L^-1 neuroblastoma cells were added in the experimental group. 1 ×10^9 L^-1 T cells and 1 ×10^7 L^-1 neuroblastoma cells were added in the control group. ③ Main surface marker CDla molecules of dendritic cells were detected with indirect immunofluorescence, and the percent rate of dendritic cells was counted with ultraviolet light and expressed as the expression rate of CD1a^＋ cells. ④Single effector cells and target cells were respectively set in the experimental group and control group to obtain the lethal effect. The lethal effect of dendritic cells on neuroblastoma cells was indirectly evaluated by detecting cellular survival with MTT assay. The lethal effect（%）= （1-A experimentat well-A effector cell /A target cell well）×100%.⑤The expenmental data were presented as Mean ±SD, and paired t test was used. MAIN OUTCOME MEASURES： ① Morphological characters of dendritic cells in the process of induction and differentiation. ②CD1a^＋ cellular expression rate. ③Lethal effect of dendntic cells on neuroblastoma cells. RESULTS： ①Morphological characters of dendritic cells in the process of induction and differentiation： On the 15^th day after human umbilical cord blood mononuclear cells were induced by rhG-MCSF and rhlL-4, typical morphology of dendritic cells could be seen under an inverted microscope. ②Expression rate of CD1a^＋ cells was （43.12±5.83）%. ③Lethal effect of dendritic cells on neuroblastoma cells： Lethal effect of dendritic cells stimulated T cells in each experimental group （ dendritic cells： neuroblastoma cells=100：1,50：1, 20：1 respectively） on neuroblastoma cells was significantly higher than that in control group[（31.00 ±4.41 ）%, （30.92±5.27）%,（33.57±5.35）%,（26.23±5.20）%, t=3.51,2.98,4.24, P〈 0.01 ）; But the lethal effect of dendntic cells on neuroblastoma was significantly lower when their ratio was 100：1 and 50：1 in comparison with 20：1 （t=2.01,2.36, P 〈 0.05）, and no significant difference in lethal effect existed between the ratio at 100：1 and 50：1 （t=0.06,P 〉 0.05）. CONCLUSION： Dendritic cells differentiated from human umbilical cord blood mononuclear cells after in vitro induction of cytokines can promote the lethal effect of T cells on neuroblastoma cells. The lethal effect is associated with the concentration of dendritic cells within some range.

How does ethanol induce apoptotic cell death of SK-N-SH neuroblastoma cells?

A body of evidence suggests that ethanol can lead to damage of neuronal cells. However, the mechanism underlying the ethanol-induced damage of neuronal cells remains unclear. The role of mitogen-activated protein kinases in ethanol-induced damage was investigated in SK-N-SH neuroblastoma cells. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide cell viability assay, DNA fragmentation detection, and flow cytometric analysis showed that ethanol induced apoptotic cell death and cell cycle arrest, characterized by increased caspase-3 activity, DNA fragmentation, nuclear disruption, and G1 arrest of cell cycle of the SK-N-SH neuroblastoma cells. In addition, western blot analysis indicated that ethanol induced a lasting increase in c-Jun N-terminal protein kinase activity and a transient increase in p38 kinase activity of the neuroblastoma cells. c-Jun N-terminal protein kinase or p38 kinase inhibitors significantly reduced the ethanol-induced cell death. Ethanol also increased p53 phosphorylation, followed by an increase in p21 tumor suppressor protein and a decrease in phospho-Rb （retinoblastoma） protein, leading to alterations in the expressions and activity of cyclin dependent protein kinases. Our results suggest that ethanol mediates apoptosis of SK-N-SH neuroblastoma cells by activating p53-related cell cycle arrest possibly through activation of the c-Jun N-terminal protein kinase-related cell death pathway.

Biological Analysis of HSV-1 Immediate-early Proteins ICP0, ICP22, and ICP27 in Neuroblastoma Cells

The three immediate-early proteins of HSV-1, ICP0, ICP22, and ICP27, have specific and pivotal functions in transcriptional activation and inhibition, multiple regulatory and control processes of viral genes. In this paper, the expression and localization of these three proteins were studied in neuroblastoma cells using biochemical assays, and their possible and potential interactive functions are discussed. The data show that the three proteins are localized in different structures, specifically in the PML-NB-associated structure, which is a specific nuclear structure composed of many protein molecules and bound tightly to the nuclear matrix in neuroblastoma cells. The results suggest that the activating and suppressive functions of ICPs are mostly dependent on their transcriptional and regulatory roles, including the PML-NB-associated structure.

Activation of Sonic Hedgehog Signaling Pathway in S-type Neuroblastoma Cell Lines

The effects of Sonic hedgehog(Shh) signaling pathway activation on S-type neuroblastoma(NB) cell lines and its role in NB tumorigenesis were investigated.Immunohistochemistry was used to detect the expression of Shh pathway components— Patched1(PTCH1) and Gli1 in 40 human primary NB samples.Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Gli1 in three kinds of S-type NB cell lines(SK-N-AS,SK-N-SH and SHEP1),respectively.Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway.S-type NB cell lines were treated with different concentrations of Shh or/and cyclopamine for different durations.Cell viability was measured by using MTT method.Apoptosis rate and cell cycle were assayed by flow cytometry.The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice.High-level expression of PTCH1 and Gli1 was detected in both NB samples and S-type NB cell lines.Cyclopamine decreased the survival rate of the three cell lines while Shh increased it,and the inhibition effects of cyclopamine could be partially reversed by shh pre-treatment.Cyclopamine induced the cell apoptosis and the cell cycle arrest in G0/G1 phase,while Shh induced the reverse effects and could partially prevent effects of cyclopamine.Cyclopamine could also inhibit the growth of NB in vivo.Our studies revealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle,suggesting a new therapeutic approach to NB.

The inhibitory effect of dihydroartemisinin on the growth of neuroblastoma cells

Objective:To evaluate the inhibitory effect of dihydroartemisinin on neuroblastoma cell line SH-SY5 Y,explore the possible mechanism of dihydroartemisinin against neuroblastoma cells.Methods:The cell viability of dihydroartemisinin treated SH-SY5 Y cells was examined by MTT assay and morphology of cells was observed by using inverted microscope.Cell cycle was examined with flowcytometry assay,then cyclin D1 and caspase-3 proteins expression was detected by ELISA and western blotting assay.Results:MTT analysis results showed that cell viability significantly decreased after exposure to 0.05,0.50,5.00 and 50.00 mmol/L dihydroartemisinin in a dose-dependent manner,and the lower density of cells was observed in treated groups.The number of cells in sub-G1 phase was increased after treatment with different doses of dihydroartemisinin compared with the control group.The expression of cyclin D1 protein was decreased,while the expression of caspase-3 protein was increased in treated group.Conclusions:Dihydroartemisinin could inhibit the proliferation through stopping the cell cycle and inducing the apoptosis in neuroblastoma SH-SY5 Y cells.

Transplantation of human placental chorionic plate-derived mesenchymal stem cells for repair of neurological damage in neonatal hypoxic-ischemic encephalopathy

Neonatal hypoxic-ischemic encephalopathy is often associated with permanent cerebral palsy,neurosensory impairments,and cognitive deficits,and there is no effective treatment for complications related to hypoxic-ischemic encephalopathy.The therapeutic potential of human placental chorionic plate-derived mesenchymal stem cells for various diseases has been explored.However,the potential use of human placental chorionic plate-derived mesenchymal stem cells for the treatment of neonatal hypoxic-ischemic encephalopathy has not yet been investigated.In this study,we injected human placental chorionic plate-derived mesenchymal stem cells into the lateral ventricle of a neonatal hypoxic-ischemic encephalopathy rat model and observed significant improvements in both cognitive and motor function.Protein chip analysis showed that interleukin-3 expression was significantly elevated in neonatal hypoxic-ischemic encephalopathy model rats.Following transplantation of human placental chorionic plate-derived mesenchymal stem cells,interleukin-3 expression was downregulated.To further investigate the role of interleukin-3 in neonatal hypoxic-ischemic encephalopathy,we established an in vitro SH-SY5Y cell model of hypoxic-ischemic injury through oxygen-glucose deprivation and silenced interleukin-3 expression using small interfering RNA.We found that the activity and proliferation of SH-SY5Y cells subjected to oxygen-glucose deprivation were further suppressed by interleukin-3 knockdown.Furthermore,interleukin-3 knockout exacerbated neuronal damage and cognitive and motor function impairment in rat models of hypoxic-ischemic encephalopathy.The findings suggest that transplantation of hpcMSCs ameliorated behavioral impairments in a rat model of hypoxic-ischemic encephalopathy,and this effect was mediated by interleukin-3-dependent neurological function.

Non-catalytic roles for TET1 protein negatively regulating neuronal differentiation through srGAP3 in neuroblastoma cells

The methylcytosine dioxygenases TET proteins （TET1, TET2, and TET3） play important regulatory roles in neural function. In this study, we investigated the role of TET proteins in neuronal differentiation using Neuro2a cells as a model. We observed that knockdown of TET1, TET2 or TET3 promoted neuronal differentiation of Neuro2a cells, and their overexpression inhibited VPA （valproic acid）-induced neuronal differentiation, suggesting all three TET proteins negatively regulate neu- ronal differentiation of Neuro2a cells. Interestingly, the inducing activity of TET protein is independent of its enzymatic activity. Our previous studies have demon- strated that srGAP3 can negatively regulate neuronal differentiation of Neuro2a cells. Furthermore, we revealed that TET1 could positively regulate srGAP3 expression independent of its catalytic activity, and srGAP3 is required for TET-mediated neuronal differentiation of Neuro2a cells. The results presented here may facilitate better understanding of the role of TET proteins in neuronal differentiation, and provide a possible therapy target for neuroblastoma.

DOWN－REGULATION OF C－MYC ONCOGENE DURING NGF－INDUCED DIFFERENTIATION OF NEUROBLASTOMA CELL LINES

There may be a close relationship between myc oncogenes and carcinogenesis of human neuroblastoma. In previous studies, we were able to induce differentiation of certain neuroblastoma cell lines with NGF. In order to study gene regulation during differentiation, N-myc and c-myc cDNA probe were hybridized with RNA extracted from different cell lines before and after NGF treatment. It was found that cell lines which expressed N-myc did not express c-myc while those with c-myc did not express N-myc except for SHEP cell line which had neither c-myc nor N-myc expression. In NGF-induced differentiated neuroblastoma cells, c-myc oncogene was down-regulated in comparison with the control samples. The time course of c-myc down-regulation was concomitant with the appearance of morphological differentiation. In situ hybridization also showed remarkable reduction of c-myc oncogene expression in NGF-induced differentiated cells as compared with the untreated control cells. These results indicate that down-regulation of c-myc oncogene may be a key event during NGF-induced differentiation and overexpression of c-myc oncogene may, at least partially, be responsible for the genesis of neuroblastoma.

Detection of ROS and translocation of ERP-57 in apoptotic induced human neuroblastoma(SH-SY5Y)cells

Several toxic compounds are known to induce apoptosis in mammalian cell lines.The human neuroblastoma cells(SHSH-SY5Y)were exposed to the phosphatase inhibiting toxin okadaic acid(OA)or hydrogen peroxide(H2O2)to induce apoptosis as well as generate reactive oxygen species(ROS).Mitoxantrone(MXT)was used as a positive control for apoptosis.The SHSH-SY5Y cells were transfected with eukaryotic expression plasmid pHyPer-dMito encoding mitochondrial-targeted fluorescent or pHyPer-dCito encoding cytoplasmic-targeted fluorescent sensor for hydrogen peroxide(HyPer).The ERp57,also called GRP58(Glucose-regulated protein 58),is a stress protein induced in conditions like glucose starvation and viral infection.Recently ERp57 was shown to translocate from the endoplasmatic reticulum to the cell surface in anthracycline-induced apoptotic cells.ERp57 co-translocation together with calreticulin has been suggested to be crucial for recognizing tumor cells to induce immunogenic cell death.ERp57 translocation after exposure to okadaic acid was studied using immunofluorescence and confocal microscopy.These studies indicated that okadaic acid has induced the translocation of ERp57 to the cellular membrane.

The cultivation and identification of tumor stem cells from neuroblastoma derived tumor spheres

Background and Objective: Since the proposal of the tumor stem cell hypothesis, considerable interest has been devoted to the isolation and purification of tumor stem cells. Tumor stem cell enrichment from primary tumor derived cell spheres has been demonstrated in specific, serum-free media. This goal of this study is to establish a method of cultivating floating tumor spheres from neuroblastoma cells and to confirm that neuroblastoma spheres are rich in tumor stem cells. Methods: Bone marrow aspirates were obtained from pediatric patients diagnosed with stage IV neuroblastoma. Primary tumor cells were isolated and cultivated in serum-free, stem cell-selective medium. Single sphere-forming cells were cultivated under serum-free conditions; their cloning efficiency and monoclonal tumor sphere formation rates were calculated. The expression of stem cell marker genes Oct-4 and Bmi-1 was detected by RT-PCR in sphere-forming cells and parental neurolastoma cells. Sphere-forming cells were injected into the armpit of nude mice with subsequent assessment for tumor growth. Sphere-forming cells were cultivated in differentiation medium containing 5 μmol/L 13-cis retinoic acid; changes in cell morphology were observed. Results: Neuroblastoma cells formed non-adherent neurospheres under serum-free, stem cell-selective conditions after a period of 4 to 6 days. A single cell dissociated from a neurosphere could reform a monoclonal sphere; cloning efficiency and monoclonal sphere formation rates were 55.3% and 26.3%, respectively. RT-PCR results revealed heightened tumor sphere expression of Oct-4 and Bmi-1 as compared with parental tumor cells. Fourteen days after injection of 104 sphere-forming cells into nude mice, a neuroblastoma xenograft formed. Treatment of sphere-forming cells with 13-cis retinoic acid induced a gradual differentiation to neuronal cell morphology. Conclusions: Neuroblastoma derived tumor spheres enrich tumor stem cells and the cultivation of primary neuroblastoma cells in serum-free, stem cell-selective medium is an effective method to dissociate and purify tumor stem cells in vitro.

Treatment of Esthesioneuroblastoma and Nonsmall Cell Lung Cancer with Phenylbutyrate

Esthesioneuroblastoma is a malignant tumor, arising in the upper nasal cavity, that could spread to the frontal lobe of the brain as well as metastasize to the lymph nodes. Due to the low incidence of this tumor, FDA-approved treatment modalities do not exist and clinical trials have not been performed. We present an interesting case of a 66-year-old female, diagnosed with Kadish stage B esthesioneuroblastoma and stage IIA nonsmall cell carcinoma of the lung, who benefited from our treatment. Both malignancies were diagnosed in 2002 at which time the patient consented to undergo left upper lobectomy for her lung cancer, but she refused the craniofacial resection and radiation therapy recommended for treatment of her esthesioneuroblastoma. From 2003 to 2004 she received treatment at the Burzynski Clinic with oral sodium phenylbutyrate (0.2 g/kg/day). She tolerated the treatment very well without significant adverse events. Gradual reduction in her tumor size was confirmed by repeat MRIs. From treatment start in March 2003 to December 2003 her tumor decreased by 40%. Subsequent MRI from March 2004 revealed increased tumor size, which, however, was still a 13% reduction from the baseline MRI. What is important to mention is that in addition to shrinkage of the esthesioneuroblastoma, the patient obtained the clinical benefit of 3.5-years longer survival than was predicted for her lung cancer—whereas the median survival for a patient with stage IIA adenocarcinoma of the left upper lobe of the lung is approximately two years, our patient survived more than five and a half years. The effect of phenylbutyrate (PB) and its metabolite phenylacetate on neuroblastoma and lung cancer is documented by numerous preclinical studies and is also evident in this case. It is proposed that the activity of these two compounds is mediated through increased expression of the p21 tumor suppressor gene. p21 is a strong inhibitor of cyclin-D and cyclin-dependent kinase 4, which contribute to undifferentiated phenotype in neuroblastoma and are instrumental in cell cycle progression from G1 to S phase. It is hoped that future research and combination of PB with other chemotherapeutic and targeted agents will provide better control of esthesioneuroblastoma and lung cancer.

多参数MRI鉴别鼻腔鼻窦嗅神经母细胞瘤与弥漫大B细胞淋巴瘤的价值

目的探讨MRI多参数在鉴别嗅神经母细胞瘤(olfactory neuroblastoma,ONB)和鼻腔鼻窦弥漫大B细胞淋巴瘤(diffusion large B cell lymphoma,DLBCL)中的价值。方法34例鼻腔鼻窦ONB和29例DLBCL患者治疗前行MRI检查。MRI评估指标包括常规征像和半定量参数。连续变量比较采用独立样本t检验或秩和检验,分类变量比较采用χ^(2)检验。二元Logistic回归用于识别最有诊断能力的MRI指标。结果ONB T2WI呈等稍高信号,累及嗅裂,易形成颅内肿块(P=0.004);DLBCL T2WI呈等略低信号(P<0.001),常累及上颌窦、眼眶(P<0.001,P=0.024)。ONB的鼻甲外移、边缘分叶征较DLBCL多见(P=0.001,P=0.004)。ONB的ADC_(ave)值更高(P<0.001),CI_(max)、WR更大(P<0.001、P=0.011),TTP小(P=0.003),TIC曲线流出型更多(P=0.022)。Logistic回归显示部位(累及嗅裂或上颌窦)、T2WI信号、ADC0.82值及以TTP52是鉴别ONB和DLBCL的独立影响因素。结论MRI的多参数联合有助于鉴别鼻腔鼻窦ONB和DLBCL。

富含半胱氨酸和甘氨酸蛋白2在神经母细胞瘤恶性进展中的功能和机制

目的:探究富含半胱氨酸和甘氨酸蛋白2(cysteine and glycine-rich protein 2,CSRP2)在神经母细胞瘤(neuroblastoma,NB)恶性进展中的功能和作用机制。方法:利用R2数据库分析NB临床样本中CSRP2基因的mRNA水平与NB患儿临床预后的相关性;在NB细胞系SK-N-BE(2)和SH-SY5Y中利用靶向小干扰RNA(small interfering RNA,siRNA)干扰CSRP2的表达或利用质粒转染过表达CSRP2;通过结晶紫染色和实时无标记动态细胞分析技术观察NB细胞的增殖情况;采用克隆形成方法观察NB细胞长时间的克隆形成能力;利用免疫荧光实验检测细胞增殖标记物Ki-67的水平;利用碘化丙啶(propidium iodide,PI)染色流式细胞术分析细胞周期比例,Annexin V/7AAD染色分析细胞凋亡比例;采用划痕实验观察细胞的迁移能力;利用Western blot或实时荧光定量PCR(quantitative real-time PCR,RT-qPCR)检测NB原发肿瘤组织和细胞系中蛋白和基因的表达水平。结果:NB临床数据库中,国际神经母细胞瘤分期(international neuroblastoma staging system,INSS)为高危险度3/4期的NB组织中CSRP2的mRNA水平显著高于低危险度的1/2期,且高表达水平组NB患儿的生存期显著低于低表达组;Western blot结果显示,CSRP2在3/4期NB组织中的蛋白水平显著高于1/2期。NB细胞中敲低CSRP2,细胞的活力减弱、增殖能力降低;NB细胞中过表达CSRP2促进细胞增殖;敲低CSRP2后,sub-G1、G0/G1和S期细胞的比例增加,Annexin V阳性细胞的比例增多;敲低CSRP2的NB细胞的划痕愈合率显著小于对照组。机制研究发现,敲低CSRP2后细胞增殖标记分子Ki-67和细胞外信号调节激酶1/2(extracellular signal-regulated kinases 1/2,ERK1/2)磷酸化水平显著低于对照组。结论:CSRP2在高危险度3/4期NB组织中高表达,表达水平与NB患儿生存期呈负相关;CSRP2通过促进ERK1/2活化,促进NB细胞的增殖和迁移,抑制细胞凋亡,表明CSRP2通过激活ERK1/2促进NB进展,为高危NB的靶向治疗提供了潜在的靶点。

PTCSC3对谷氨酸诱导SH-SY5Y神经细胞凋亡的影响

目的探讨乳头状甲状腺癌易感性候选基因3(papillary thyroid carcinoma susceptibility candidate 3,PTCSC3)对谷氨酸(glutamicacid,Glu)诱导人神经母细胞瘤细胞(SH-SY5Y)凋亡的影响及其机制。方法体外培养SH-SY5Y细胞,谷氨酸损伤SH-SY5Y细胞24 h,转染技术对PTCSC3进行抑制及过表达,采用Hoechst 33258染色法、流式细胞技术、AO/EB染色及四甲基偶氮唑盐比色(methylthiazolyl tetrazolium,MTT)法检测抑制或过表达PTCSC3后对各组细胞凋亡、生存的影响;实时荧光定量PCR(quantitative real-time PCR,qPCR)技术检测各组细胞中PTCSC3、CCAAT/增强子结合蛋白β(CAAT/enhancer binding protein beta,C/EBP-β)及Cbp/p300结合转化激活因子4(Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 4,CITED4)的基因表达情况;Western blot检测各组细胞中C/EBP-β、脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)、蛋白激酶B(protein kinase B,Akt)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)及CITED4、细胞周期蛋白D1(Cyclin D1)、原癌基因n-Myc的蛋白表达水平。结果2000μmol·L^(-1)Glu处理24 h对PTCSC3细胞有明显的损伤作用;与模型组比较,PTCSC3抑制剂可有效改善Glu诱导SH-SY5Y细胞凋亡率的增加和细胞存活率的下降;明显降低C/EBP-β基因及蛋白、Bax蛋白的表达,促进BDNF、p-Akt、Bcl-2蛋白的表达;增加CITED4、Cyclin D1和n-Myc的表达水平。反之,过表达PTCSC3则出现与上述结果相反的结果。结论PTCSC3可能通过调控C/EBP-β,进而调控下游信号通路BDNF/Akt和CITED4/Cyclin D1信号通路,改善Glu诱导SH-SY5Y神经细胞的凋亡和生存状态。

脂多糖调节SH-SY5Y细胞线粒体融合在阿尔茨海默病中的作用

目的 探讨脂多糖(LPS)调节人神经母细胞瘤细胞(SH-SY5Y细胞)线粒体融合在阿尔茨海默病(AD)中的作用。方法 选取SH-SY5Y细胞株进行常规培养,于细胞对数生长期以25、50、100μg/ml的LPS处理SH-SY5Y细胞48 h,分别设置为25μg/ml组、50μg/ml组、75μg/ml组,同时设置空白对照组。采用细胞计数试剂(CCK-8)法检测细胞存活率;光镜检测细胞内线粒体损伤情况;酶联免疫吸附测定法(ELISA)检测超氧化物歧化酶(SOD)、丙二醛(MDA)、一氧化氮(NO)和肿瘤坏死因子-α(TNF-α)变化;采用实时荧光定量PCR(Real-time PCR)法检测动力相关蛋白1 (Drp1)、线粒体融合蛋白1(Mfn1)、线粒体融合蛋白2(Mfn2)和视神经萎缩因子1(Opa1)微小核糖核酸(mRNA)表达和蛋白表达水平。分析LPS对SH-SY5Y细胞存活率和β-淀粉样蛋白(Aβ)含量, SH-SY5Y细胞线粒体形态, SH-SY5Y细胞内SOD、MDA、NO和TNF-α, SH-SY5Y细胞内mRNA表达的影响。结果 CCK-8法检测结果显示,与空白对照组相比,在48 h给药时间下, 25、50、100μg/ml组中SH-SY5Y细胞存活率明显下降, Aβ含量显著升高;随着LPS剂量的增加, SH-SY5Y细胞存活率呈剂量依赖性降低, Aβ含量呈剂量依赖性增长,差异具有统计学意义(P<0.05)。光镜结果显示,与空白对照组相比,在48 h给药时间下, 25、50、100μg/ml组中SH-SY5Y细胞内线粒体出现明显变大变圆,体积增大,线粒体出现肿胀和线粒体空泡化等改变,并伴有线粒体自噬的发生。ELISA结果显示,与空白对照组相比,在48 h给药时间下, 25、50、100μg/ml组中SH-SY5Y细胞内SOD表达分别显著降低24.16%、69.08%和123.78%, MDA表达分别显著增加37.65%、52.13%和101.85%, NO表达分别显著增加54.34%、119.08%和167.78%, TNF-α表达分别显著增加54.34%、119.08%和167.78%,随着LPS剂量的增加, SOD含量变化具有剂量依赖性降低, NO、MDA、TNF-α含量变化呈剂量依赖性增长,差异具有统计学意义(P<0.05)。Real-Time PCR结果显示,与空白对照组相比,在48 h给药时间下, 25、50、100μg/ml组中SH-SY5Y细胞内Drp1、Mnf1和Mnf2 mRNA含量随着LPS剂量的增加而显著增加, SH-SY5Y细胞内Opa1 mRNA含量随着LPS剂量的增加而显著降低,差异具有统计学意义(P<0.05)。结论 LPS可明显加速SH-SY5Y细胞线粒体融合分裂,降低细胞的存活率,在阿尔茨海默病的发病机制中具有重要作用。

双去甲氧基姜黄素对小鼠脑神经母瘤细胞的促神经分化作用及机制研究

目的研究姜黄素衍生物双去甲氧基姜黄素(BC)对小鼠脑神经母瘤细胞Neuro-2a(N2a)的促神经分化作用及机制。方法采用MTT法检测BC(1、2、4、6、8、10μmol/L)对N2a细胞存活率的影响,确定药物处理浓度范围。设对照组、视黄酸(RA)组(10μmol/L)和BC组(1、2、4μmol/L),培养48、72 h后,对分化细胞的神经突起长度进行测量并计算细胞分化率;采用Western blot法检测4μmol/L BC作用5、15、30、60、120 min后细胞中蛋白激酶B(Akt)、细胞外调节蛋白激酶1/2(ERK1/2)、p38丝裂原活化蛋白激酶(p38)蛋白的磷酸化水平。以抑制剂LY294002(LY)和PD98059(PD)干预后,进一步验证BC对Akt和ERK蛋白磷酸化水平及促神经分化的影响。结果根据MTT实验确定后续诱导细胞分化的BC浓度为1、2、4μmol/L。分化48 h后,与对照组比较,RA组和BC 1、2、4μmol/L组细胞分化率及BC 4μmol/L组细胞神经突起长度均显著升高/增加(P<0.05或P<0.01);BC继续诱导分化至72 h后,与对照组比较,RA组细胞分化率和神经突起长度、BC 4μmol/L组细胞分化率和BC 2μmol/L组细胞神经突起长度均显著升高/增加(P<0.05或P<0.01)。与0 min组比较,BC 4μmol/L作用5、15、30、60、120 min组细胞中Akt、ERK1/2、p38蛋白的磷酸化水平均有不同程度升高,部分差异有统计学意义(P<0.05或P<0.01)。加入抑制剂LY/PD后,与BC组比较,PD+BC组细胞中ERK1/2蛋白的磷酸化水平显著降低(P<0.01),LY组、LY+BC组、PD组、PD+BC组细胞分化率均显著降低(P<0.01)。结论BC可以促进N2a细胞分化,增加细胞分化率和神经突起长度,其机制可能与激活MEK/ERK和PI3K/Akt信号通路有关。

miR-145-5p对口腔鳞癌细胞增殖的调控作用及其机制

目的 探究miR-145-5p对口腔鳞状细胞癌(OSCC)细胞增殖的调控作用及其机制。方法 利用GEO数据库构建OSCC的miRNA差异表达谱;通过生物信息学技术预测miR-145-5p靶基因并对其进行GO、KEGG及PPI分析;以人OSCC细胞株(HSC-3)为实验对象,将实验分为CN组(未做处理)、NC组(转染mimic NC)和miR-145-5p组(转染miR-145-5p mimic),通过CCK-8法检测不同剂量(50、100、200 nmol/L)miR-145-5p对HSC-3细胞增殖的影响;JC-1和Annexin V-FITC/PI染色结合流式细胞术检测miR-145-5p对HSC-3细胞的凋亡诱导情况;RT-qPCR检测miR-145-5p调控神经母细胞瘤鼠肉瘤同系物(NRAS)、C-JUN氨基端激酶(C-JUN)、信号转导和转录激活因子1(STAT1)的表达情况。结果 miRNA差异表达谱显示,miR-145-5p在OSCC中表达下调(P<0.05);GO和KEGG分析显示,miR-145-5p靶基因主要调控MAPK信号通路;PPI蛋白互作显示,NRAS是miR-145-5p的关键靶基因之一;CCK-8结果显示,50、100、200 nmol/L的miR-145-5p转染HSC-3细胞后,细胞活力较CN组降低(P<0.05);JC-1染色结果显示,miR-145-5p组HSC-3细胞线粒体膜电位低于CN组(P<0.05);Annexin V-FITC/PI染色结果显示,miR-145-5p组HSC-3细胞凋亡率较CN组明显增加(P<0.05);RT-qPCR结果显示,与CN组比较,miR-145-5p组NRAS表达降低(P<0.05),而MAPK信号通路关键转录因子C-JUN和STAT1表达升高(P<0.05)。结论 miR-145-5p可通过靶向NRAS,并经过MAPK信号通路诱导细胞凋亡,从而抑制OSCC细胞增殖。

SARS-CoV-2刺突糖蛋白对神经细胞的损伤效应及机制

目的探讨严重急性呼吸综合征冠状病毒2(SARS-CoV-2)刺突糖蛋白(S蛋白)对人神经母细胞瘤细胞(SH-SY5Y)的损伤效应及其机制。方法用S蛋白0(细胞对照组),25,50,75和100 mg·L^(-1)处理SH-SY5Y 24 h,CCK-8法检测细胞活力;比色法检测乳酸脱氢酶(LDH)释放率;EdU试剂盒检测细胞增殖;荧光素酶发光法检测细胞内ATP含量;JC-1荧光探针法检测细胞线粒体膜电位(MMP);Seahorse XF检测细胞糖酵解及线粒体氧化磷酸化水平。结果与细胞对照组相比,S蛋白25,50,75和100 mg·L^(-1)组细胞活力显著降低(P<0.01),半数抑制浓度(IC_(50))为65.05 mg·L^(-1);LDH释放率显著增加(P<0.01);EdU阳性细胞比例显著降低(P<0.01);S蛋白75和100 mg·L^(-1)组细胞内ATP含量显著降低(P<0.01);S蛋白50和75 mg·L^(-1)组细胞内MMP显著降低(P<0.05,P<0.01);S蛋白50 mg·L^(-1)组基础糖酵解水平和糖酵解能力的最大值显著升高(P<0.05,P<0.01),S蛋白25和50 mg·L^(-1)组呼吸能力最大值显著升高(P<0.05,P<0.01)。SH-SY5Y细胞活力与细胞内ATP含量和MMP均呈正相关(r^(2)=0.9209,P=0.001;r^(2)=0.6170,P=0.0025);与反映细胞糖酵解水平的细胞基础糖酵解水平和糖酵解能力最大值呈负相关(r^(2)=0.5194,P=0.0285;r^(2)=0.6664,P=0.0073),与反映线粒体氧化磷酸化水平的ATP生成能力呈负相关(r^(2)=0.8204,P=0.0008)。结论S蛋白使SH-SY5Y细胞活力下降,抑制细胞增殖,其机制可能与干扰神经细胞内能量代谢密切相关。

TBM预处理方案自体造血干细胞移植治疗高危神经母细胞瘤患儿疗效分析

目的研究TBM[即塞替派(thiotepa)+白舒菲(busulfan)+美法伦(melphalan)]预处理方案自体造血干细胞移植治疗高危神经母细胞瘤患儿的临床疗效。方法选取2021年11月5日至2022年9月8日于新疆维吾尔自治区人民医院血液病科移植中心接受TBM预处理自体造血干细胞移植患儿7例为研究对象,评估造血重建情况及移植相关不良反应。分析7例高危神经母细胞瘤患儿临床疗效和疗效相关指标,以及随访结果和治疗前后的营养指标。结果患儿治疗后及末次随访不同时期的临床疗效比较,差异无统计学意义(P>0.05)。7例高危神经母细胞瘤患儿中位年龄6.2岁(2~13岁),所有患儿均移植回输前期采集冻存自体干细胞,中位计数单个核细胞(MNC)为12.17×10^(8)/kg[(8.65~28.81)×10^(8)/kg],CD34^(+)为4.15×10^(6)/kg[(2.11~13.12)×10^(6)/kg]。所有患儿均获得造血重建,中性粒细胞中位植入时间为11 d(9~18 d),血小板植入时间为16 d(13~31)d。随访期间所有患儿均存活,其中发生Ⅲ级以上黏膜炎4例,发生率为51.7%;发生Ⅲ~Ⅳ级呕吐腹泻等胃肠道不良反应5例,发生率为71.4%;发生药物性肝损伤2例,发生率为28.5%;未见神经系统、肾脏以及肝窦隙阻塞综合征等预处理相关毒性反应。患儿治疗前后的前白蛋白(PAB)、清蛋白(ALB)及血红蛋白(Hb)等营养指标比较,差异无统计学意义(P>0.05)。结论TBM方案用于高危神经母细胞瘤患儿自体造血干细胞移植预处理中的疗效较佳,且不良反应常见可控,然而仍需进一步扩大病例数临床验证。