前列腺癌早期对雄激素剥夺治疗(ADT)治疗敏感,表现为雄激素依赖性前列腺癌(ADPC),表达腺癌标记物,后可发展为去势抵抗性前列腺癌(CRPC).前列腺癌经ADT治疗后易发生神经内分泌转化,甚至演变为前列腺神经内分泌癌(NEPC),表达神经内分泌标记物.研究表明,N-Myc为NEPC驱动基因,可促进前列腺癌的神经内分泌转化,且高表达N-Myc的患者生存率普遍较低,靶向N-Myc相关信号通路可以为治疗前列腺癌、延缓疾病进程提供新的方向和思路.

Objective To explore the clinical significance of NSE,BNP and D-dimer levels in predicting a-cute exacerbation of COPD (AECOPD). Methods 87 patients with AECOPD,87 patients with COPD and 50 healthy volunteers who met with the criteria were enrolled in this study. Their serum levels of NSE,BNP and D-dimer were determined by immunoradiometric assay,fluorescence immunoassay,and Turbidimetric inhibition immunoassay separately. Results The levels of NSE, BNP and D-dimer in the AECOPD group were significantly higher than those in the control group (P<0.05),and those in the stable COPD patients (P<0.05). Conclusion Dynamic monitoring of plasma NSE,BNP and D-dimer levels can predict the acute exacerbation of COPD.

Objective To establish the rat C6 glioma model engineered by green fluorescent protein and explore its effect.Methods C6 cells were transfected with pEGFP-C1 plasmid expressing enhanced green fluorescent protein ( EG-FP) gene.Then the transfected cells were selected by G418 to obtain the stable C6 cell clones ( pEGFP-C1-C6) .pEGFP-C1-C6 cell suspension was injected into intracal of 16 SD rats to establish rat C6 glioma model with green fluorescent protein by stereotaxic apparatus.After inoculation, cell life cycle was observed and tumor growth status was dynamically documen-ted by magnetic resonance imaging.At 7, 14, 21 day after glioma model established, glioma tumor volumes were detected and measured by MRI.Brain specimens were observed by HE staining.Confocal laser scanning microscopy was used to ob-serve the infiltration and invasion of glioma cell.Results Rat C6 glioma model with green fluorescent protein was estab-lished successfully.Tumor cells invasion can be easily detected by green fluorescent protein imaging and MRI.Tumor vol-ume was (244.60 ±36.06)mm3.It was easier to observe the micro-metastasis and invasive.Conclusions Rat C6 glioma model could be successful established with EGFP-C1-C6 cells, which was more sensitive in showing invade, metastasis and single invasive tumor cell.

Objective To explore the rat model after 6-OHDA administration by multi-spots in corpus striatum.Methods The equal amount of 6-OHDA was administrated into corpus striatum by different targets(two spots,three spots and four spots) by means of stereotaxis technique,lateral-rotation by apomorphine and TH immunohistochemistry were observed at 2,3,4 and 6 weeks after 6-OHDA induction.The successful rat model was considered to accord with the follow standardization: the number of lateral-rotation turns should be more than 7r/min(210r/min),and TH apoptosis by immunostaining was decreased to be 70% at least in iso-lateral substantia nigra and corpus striatum.Results The successful ratioes of rat models were exhibited as 43.3%(13/30) in two spots lane,70%(21/30) in three spots lane,and 66.7%(20/30) in four spots lane.The ratio of rat model was significent between two spots and three spots lanes(P=0.012),not between three spots and four spots(P=0.078).3 rats died in four spots lane,not occurred in other lanes.TH apoptosis in substantia nigra and corpus striatum were significent between two-and three spots lanes(P=0.028),not occurred between three-and four spots lanes(P=0.078).Conclusion It was an effective measure to induced rat model of PD by three spots administration of 6-OHDA in the corpus striatum.

Objective To discuss the inhibition of CDNF in 6-OHDA-induced apoptosis effect.Methods The CDNF been transfected to MSCs by Lip2000.After the screening by G418,MSCs will be stabilize expression CDNF(also calls CDNF-MSCs cells).Through the MTT analysis and Annexin Ⅴ-FITC cell apoptosis analysis detects the supernatant to 6-OHDA induction of PC12 cell apoptosis.Results Through the screening by G418,MSCs transfected cells will stably express CDNF.The supernatant of CDNF-MSCs which have a inhibition effect for it pretreatment of PC12 cells,can effectively reduce the rate of apoptosis.Conclusion CDNF which is a secreted protein will striking reduce the 6-OHDA-damaged PC12 cells.

Parkinson’s disease (PD) is a common progressive neurodegenerative disorder whose core symptoms are tremor, bradykinesia, rigidity, and postural instability. Currently available treatment of PD is mainly based on dopamine replacement strategy to provide relief of motor symptoms, but cannot halt or reverse the degenerative processes of disease. Considerable in vitro and in vivo studies have found that neurotrophic factor (NTF) has neuroprotective or even neurorestorative properties on dopaminergic (DA) system, promoting them become promising candidates for the treatment of PD. However, the precise mechanism of NTF’s effect in PD remains to be elucidated. Though the etiopathogenesis of PD has remained elusive, recently, compelling evidence has converged to suggest that failure of the ubiquitin–proteasome system (UPS) to degrade unwanted proteins may underlie nigralstrital degeneration and Lewy body (LB) formation which occurs in PD. In support of this, proteasome inhibitor has been successfully induced a PD modal both in vivo and in vitro. Many NTFs have been proved to posses definitely a therapy effect in a PD animal modal. Whether NTF can co-function with UPS that accomplishes the aim to protect and reserve dopaminergic neurons’ function from neurotoxicity injury induced by proteasome inhibitor? If this hypothesis could be confirmed, it will represent a valuable advancement in the study of PD. Moreover, investigation of the functional link between UPS and NTF should also provide useful information for understanding the pathogenesis of PD.

Objective To investigate the neuroprotective effect and mechanism of pre-heat shock treatment on 6-hydroxydopamine(6-OHDA)-induced neurotoxicity in SH-SY5Y cells.Methods Following the pre-heat shock at 42℃±0.5℃,6-OHDA-induced neurotoxicity was conducted in experimental group.In control group only 6-OHDA-induced neurotoxicity was carried out.Expressions of glucose regulated protein(GRP78 and GRP94) in SH-SY5Y cells were detected at different time points(15 min,30 min,60 min) by reverse transcription-polymerase chain reaction(RT-PCR).The morphological changes of the cells were observed under microscope and the cell viability was identified by MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.Results 6-OHDA induced the neurotoxicity of SH-SY5Y cells in a dose-and time-dependent manner.6-OHDA-induced neurotoxicity on SH-SY5Y cells was less in experiment group compared with that in control group and the statistic difference among different time points in experimental group was significant(P0.05).The mRNA levels of GRP78 and GRP94 showed a time-dependent relationship with the extension of pre-heat shock time(P0.05).Conclusion Pre-heat shock treatment can reduce 6-OHDA-induced neurotoxicity of SH-SY5Y cells and the expressions of GRP78 and GRP94 are increased during the heat shock treatment.

保守性多巴胺能神经营养因子(CDNF)是一类新型神经营养因子,可能对帕金森病(PD)具有一定的治疗作用[1-2].我们通过bac-to-bac杆状病毒表达系统,在sf9昆虫细胞中成功诱导表达出重组CDNF蛋白.

Objective To explore the differentiation-inducing effect of quercetin on rat adrenal pheochromocytoma PC12 cells.Methods Proliferation of PC12 cells was evaluated by MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide] assay after treatment with nerve growth factor(NGF) and different concentrations of quercetin(5,12.5,25,50μmol/L) which were administrated to induce differentiation of PC12 cell at the same time,and the cells treated with PBS served as the control group.The differentiation of PC12 cells was observed under light microscope and recorded three days after treatment.The morphology parameters such as cell differentiation rate(the percent of cells whose neuraxial length was equal or longer than its diameter),the number of cells with neuritis,average neuraxial number of per cell and percentage of fusiform cells were analyzed statistically.Results MTT assay showed that 50 μmol/L quercetin inhibited proliferation of PC12 cells significantly(P<0.05).Compared with the control group,the cells in NGF and different concentrations of quercetin(5,12.5,25μmol/L) groups showed significant differentiation characteristics(all P<0.05).There was no significant difference in capacity of inducing differentiation of PC12 cells between different concentrations of quercetin(5,12.5,25μmol/L).Conclusions The quercetin can induce the differentiation of PC12 cells,but has no significant dose-independence.

我们通过bac-to-bac杆状病毒表达系统,在sf9昆虫细胞中诱导表达出重组CDNF蛋白,现报道如下. 一、材料与方法 1.材料:Cellfectin Ⅱ转染剂、SFM900Ⅱ基础培养基、Grace's Insect Cell Culture Medium(Unsupplemented)培养基购自Invitrogen 公司,胎牛血清购自杭州四季青公司,质粒小量抽提试剂盒购自北京天根公司,蛋白相对分子质量Marker购自Ferment公司,抗His单克隆抗体购自北京中杉金桥生物公司,抗生素均购自Sigma公司,其他试剂均为国产分析纯.pFastBacHTb-CDNF为本室冻存,sf9昆虫细胞、DH10Bac感受态细胞购自Invitrogen公司.

帕金森病的治疗方法一直处于探索中,近20年来,神经营养因子在帕金森病的研究中受到极大的关注。大量实验研究表明,神经营养因子对中脑多巴胺能神经元具有很好的保护作用,有望成为帕金森病的治疗靶点,本文就应用神经营养因子治疗帕金森病的研究作一综述。

BACKGROUND: PEX gene can interfere with the invasion acts of malignant glioma. Bone marrow mesenchymal stem cells (MSCs) are a new type of targeted cell vector on cancer therapy. OBJECTIVE: To construct MSCs stably expressing PEX gene. METHODS: PEX eukaryotic expression vector was constructed by molecular cloning, and identified the recombinant plasmid pcDNA3.1(+)-PEX by restriction endonuclease digestion and sequencing. After transfected with MSCs, the eukaryotic expression vector expression in MSCs was verified by immunocytochemical method. The MSCs stably expressing PEX was established by G418 selection, and then was detected by using reverse transcriptase-polymerase chain reaction. RESULTS AND CONCLUSION: The MSCs stably expressing PEX gene is successfully established, in which PEX gene is highly expressed at both gene level and protein level.

Objective To study the effect of PEX gene-modified mesenchymal stem cells (MSC) on C6 glioma cells in vitro and explore its mechanism.Methods PEX eukaryotic expression vector was constructed by molecular cloning and transfected MSC,then the MSC stably expressing PEX gene (MSC-PEX) were obtained by G418 selection.Variable numbers of MSC-PEX were added to C6 glioma cells by coculture experiments.Effects of MSC-PEX on C6 glioma cell growth were observed by water soluble tetrazolium (WST-1) cell proliferation and cytotoxicity assay kit.Morphological changes of C6 glioma cell apoptosis were observed by Annexin-Ⅴ-FITC and propidium iodide (Annexin-Ⅴ-FITC/PI) staining.Apoptosis rate was detected by Annexin-Ⅴ-FITC/PI dual staining assay.Results MSC-PEX was constructed successfully.MSC-PEX could inhibit the growth of C6 glioma cell line remarkably.Significant morphological changes in C6 glioma cells were observed by Annexin-Ⅴ-FITC/PI staining.The apoptosis rates of MSC-PEX transfection group and DMEM control group were 16.7% and 1.3% respectively,indicating a significant difference (P < 0.05).Conclusions PEX gene-modified mesenchymal stem cells inhibit glioma cell proliferation and induce its apoptosis,which lay a theoretical foundation for the treatment of glioma.

Objective To construct and identify a recombinant baculovirus transfer vector of mouse conserved dopamine neurotrophic factor (mCDNF): pFastBacHTb-mCDNF. Methods Mouse total RNA was isolated by using Trizol reagent, and then, first-strand cDNAs were synthesized by reverse transcriptase. Overall length of CDNF (564 bp) was amplified by two rounds of PCR introducing appropriate restriction sites (BamH Ⅰ, Xho Ⅰ). The PCR products were cloned into pGEM-T vector and sequenced to confirm PCR fidelity. The mCDNF was sub-cloned into pFastBacHTb vector to create pFastBacHTb-mCDNF vector, then the vector was transferred into the E. coli DH5α competent cells. The clone was selected using amicillin resistance and then this vector was sequenced and identified by double digests. Results Agarose gel electrophoresis after RT-PCR showed a 564 bp band being consistent with the anticipation size. Positive clone of pGEM-T-CDNF was screened by blue/white and antibiotic resistance selection. Recombinant plasmid pGEM-T-mCDNF was identified by PCR and sequence.Recombinant plsmid pGEM-T-mCDNF and pFastBacHTb vector were cut by BamH Ⅰ and XhoⅠ restriction enzyme, and then, recombinant plasmid pFastBacHTb-mCDNF was constructed and successfully identified by double digestion of Xho Ⅰ and BamH Ⅰ restriction enzyme or single digestion of BamH Ⅰ, PCR and sequence. Conclusion We successfully constructe the recombinant baculovirus transfer vector pFastBacHTb-mCDNF, laying the foundation for further research of this neurotrophic factor.

帕金森病是一种好发于中老年人的神经变性疾病,其发病机制不明。目前,应用蛋白酶体抑制剂诱导的帕金森病动物模型,其行为学、影像学、病理学和生化特征都与帕金森病的表现极为相似,它的出现将会为帕金森病发病机制及治疗方法的探讨提供新途径。