NT-3基因转染对骨髓间充质干细胞增殖及向神经元分化的影响

目的研究神经营养素-3(neurotrophin-3,NT-3)对骨髓间充质干细胞(mesenchymal stem cells,MSCs)增殖和向神经元分化的影响,探讨转染NT-3基因的MSCs体内移植治疗脊髓损伤的可行性。方法体外培养稳定表达NT-3蛋白的MSCs,通过MTT法检测NT-3对MSCs增殖的影响;应用免疫细胞化学和免疫印迹方法检测转染NT-3的MSCs表达神经元标志物NeuN蛋白的情况。结果转染NT-3基因的MSCs生长曲线上移,细胞增殖率显著升高,与对照组相比,两者有显著差异(P<0.05),表明转染NT-3后细胞生长加快。免疫荧光染色结果显示转染NT-3的MSCs的NeuN阳性细胞数明显增多,与对照组比较有显著差异(P<0.05);Western印迹检测结果显示转染NT-3的MSCs内NeuN蛋白表达明显增多,与对照组比较有显著差异(P<0.05),表明NT-3促进MSCs向神经元方向分化。结论 NT-3基因转染可促进MSCs的增殖,并诱导其向神经元方向分化。     

不同他汀类药物标准剂量对稳定性斑块的影响

目的探讨不同他汀类药物标准剂量对稳定性斑块的影响。方法入选经冠状动脉造影和血管内超声检查确定为稳定性斑块的135例患者,分为3组：辛伐他汀20mg组47例,阿托伐他汀20mg组45例,瑞舒伐他汀10mg组43例。随访3-6个月,观察给予不同他汀类药物标准剂量的3组血清低密度脂蛋白-胆固醇（10wdensitvlipoprotein-cholesterol,LDL—C）、高密度脂蛋白-胆固醇（highdensitylipoprotein—cholesterol,HDL．C）、高敏C反应蛋白（high—sensitivityC．reactionprotein,hs—CRP）浓度变化及斑块坏死核所占百分比、斑块体积的动态变化。结果3组血清LDL—C、HDL—C、hs．CRP浓度及斑块坏死核所占百分比、斑块体积的基线资料比较,差异无统计学意义（P〉0．05）。随访3-6个月后,他汀类药物标准剂量治疗的3组血清LDL．C浓度与基线相比均有明显下降,均达标（P〈0．01）,且瑞舒伐他汀最优,阿托伐他汀其次,辛伐他汀最后（P均〈0．01）。辛伐他汀组血清HDL-C浓度与基线比较。差异无统计学意义（P〉0．05）,但阿托伐他汀组、瑞舒伐他汀组明显高于基线,差异有统计学意义（P=0．001．P=0．048）,且两组间比较,差异无统计学意义（P=0．852）。辛伐他汀组血清hs—CRP浓度与基线比较,差异无统计学意义（P〉0．05）,但阿托伐他汀组、瑞舒伐他汀组明显低于基线,差异有统计学意义（P=0．015,P=0．025）．且两组间比较,差异无统计学意义（P=0．411）。通过血管内超声虚拟组织成像技术（intravascularuhrasound—virtualhistology,IVUS—vH）检测,斑块坏死核所占百分比在辛伐他汀组明显高于基线,差异有统计学意义（16．33％-＋15．38％眦7．87％-＋1．04％,P〈0．01）;阿托伐汀组治疗后与基线相比,斑块坏死核所占百分比明显减少,差异有统计学意义（6．64％±3．25％帆7．91％±1．27％,P=0．007）;瑞舒伐他汀组斑块坏死核所占百分比与基线相比,差异无统计学意义（P=0．133）。斑块体积在辛伐他汀组治疗前、后比较,差异无统计学意义（P=O．286）;但阿托伐他汀组、瑞舒伐他汀组斑块体积明显低于基线,差异有统计学意义[（28．65±10．77）mm。vs．（33．31±10．75）mm3,P=0．041;（30．69_＋8．12）mm3ys．（36．337-＋12．15）mm。,P=0．013],且治疗后两组体积比较,差异有统计学意义（P=0．322）。结论不同种类他汀类药物标准剂量对稳定性斑块疗效不同：对于LDL—C,辛伐他汀、阿托伐他汀、瑞舒伐他汀标准剂量治疗后均可达标;阿托伐他汀、瑞舒伐他汀能升高HDL—C,降低hs—CRP;辛伐他汀无遏制斑块向不稳定进展的作用,阿托伐他汀有促进斑块稳定,逆转斑块的作用,瑞舒伐他汀可阻止斑块进展及逆转斑块的作用。     

谷胱甘肽过氧化物酶1高表达对β淀粉样蛋白介导PC12细胞损伤的保护作用

目的探讨β淀粉样蛋白(Aβ)所致阿尔茨海默病发病的分子机制,以及谷胱甘肽过氧化物酶1(GPX1)高表达时对其所致细胞损伤的保护作用。方法将PC12细胞转染合人GPX1基因的plncx质粒及空载体plncx质粒,对PCl2、GPXl-PCl2、plncx-PCl2 3组细胞,分别给予Aβ_(25-35)和亚硒酸钠+Aβ_(25-35)2种干预方式,MTT法检测细胞的存活情况,免疫细胞化学法观察磷酸化环磷酸腺苷反应元件结合蛋白(pCREB)的表达情况。结果 PCl2组与plncx-PCl2组细胞存活率比较,差异无统计学意义(P>0.05);GPXl-PC12组较plncx-PCl2组细胞存活率明显增高(P<0.01);Aβ_(25-35)干预后,plncx-PC12组和GPX1-PC12组pCREB蛋白阳性率明显下降,plncx-PC12组较GPX1-PCl2组下降更明显(P<0.05)。亚硒酸钠+Aβ_(25-35)干预后,plncx-PC12组和GPX1-PC12组pCREB蛋白阳性率明显上升,GPX1-PC12组较plncx-PC12组增高更明显(P<0.01)。结论 GPX1基因高表达对Aβ_(25-35)所介导的PCl2细胞损伤有明显保护作用;Aβ_(25-35)可下调pCREB在PCl2细胞中表达,而GPXl与pCREB共同参与保护PCl2细胞,减少Aβ_(25-35)所引起的细胞损伤。     

Biological Effects of Lentivirus-Mediated shRNA Targeting Collagen Type I on the Mesangial Cells of Rats

Aim: To investigate the effects of lentivirus-mediated shRNA targeting collagen type I on the mesangial cells of rats and the feasibility of lentivirus-mediated shRNA delivery through renal parenchyma injection. Methods: Anti-collagen type I shRNA lentiviral vector was constructed, and rat mesangial cells were transfected with transfection enhancer (control group), blank lentiviral vectors (pSC-GFP group), and pSC-GFP/Col I lentiviral vectors (pSC-GFP/Col I group). Transfection efficiency and cell cycle were determined by flow cytometry. RT-PCR and Western blot were performed to detect the mRNA and protein expressions of Col I. Cell proliferation was evaluated by 3-(4,5)-dimethylthiahiazo-3, 5-di-phenytetrazolium-romide (MTT) assay and direct counting, and apoptosis was detected using AnnexinV/PE staining. The feasibility of renal parenchyma injection of lentiviral vectors was assessed. Results: The transfection efficiency was 75.42%. The expressions of collagen type I in pSC-GFP/Col I group was markedly decreased when compared with the other two groups. PSC-GFP/Col I group was higher than pSC-GFP group in the inhibition efficiency of mesangial cell after transfection. Results revealed that pSC-GFP/Col I transfection induced apoptosis to a certain extent. The proportion of cells in G2/M phase in pSC-GFP/Col I group and pSC-GFP group was higher than that in control group after of transfection. Moreover, cells arrested in S phase were markedly increased. Our results also revealed renal injection of lentivirus-mediated shRNA was feasible. Conclusion: Lentivirus-mediated shRNA targeting collagen type I could stably and efficiently transfect rat mesangial cells and significantly suppressed collagen type I expressions with acceptable safety. Renal injection of Col I lentivirus-mediated shRNA was also feasible.     

The conjunctive trace

Memories serve to establish some permanence to our inner lives despite the fleeting nature of subjective experience. Most neurobiological theories of memory assume that this mental permanence reflects an underlying cellular permanence. Namely, it is assumed that the cellular changes which first occur to store a memory are perpetuated for as long as the memory is stored. But is that really the case? In an opinion piece in this issue of Hippocampus, Aryeh Routtenberg raises the provocative idea that the subjective sense of memory persistence is not in fact a result of persistence at the cellular level, rather, that “supple synapses” and multiple “evanescent networks” that are forever changing are responsible for our memories. On one level, his proposal could be construed as a radical challenge to some of our most fundamental theories of the neurobiology of memory, including Donald Hebb's proposal that memories are stored by networks that strengthen their connections to increase the likelihood of the same activity patterns being recreated at a later date. However, it could also be seen as a moderating call, a call for a greater acknowledgement of the dynamic, stochastic, and distributed nature of neural networks. In this response to Routtenberg's article, we attempt to provide a clarification of the dividing line between these two interpretations of his argument, and in doing so, we provide some overview of the empirical evidence that bears on this subject. We argue that the data that exists to date favors the more moderate interpretation: that memory storage involves a process in which activity patterns are made more likely to reoccur, but that an under‐appreciated reality is that mnemonic traces may continue to change and evolve over time. © 2013 Wiley Periodicals, Inc.     

Synthesis of stable-isotope-labeled N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-(3-dimethylaminopropyl)-N′-ethylurea

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) is a carbodiimide coupling reagent commonly used for the preparation of amides from carboxylic acids and amines. Because of initial concerns regarding the genotoxicity of EDC and its use in GMP syntheses at Bristol Myers Squibb, the quantitation of residual EDC and its by-product N-(3-dimethylaminopropyl)-N′-ethylurea (EDU) by liquid chromatography–mass spectrometry (LCMS) impurity analysis was required. These analyses required the use of stable-isotope-labeled EDC and EDU to serve as internal standards. To meet this need, stable-isotope-labeled EDC 9 and EDU 10 were prepared from [1,2-¹³C₂] ethylene glycol and [¹³C,¹⁵N] potassium cyanide in overall yields of 6% and 8%, respectively.     

Yttrium-90 Transarterial Radioembolization for Chemotherapy-Refractory Intrahepatic Cholangiocarcinoma: A Prospective,Observational Study

PurposeTo evaluate the safety and efficacy of yttrium-90 transarterial radioembolization (TARE) for the treatment of unresectable, chemotherapy-refractory intrahepatic cholangiocarcinoma (ICC).MethodsA prospective, observational study was carried out in 10 centers between 2013 and 2017. TARE plus standard care was delivered to patients with unresectable, chemotherapy-refractory or chemotherapy-intolerant ICC. Primary outcome was overall survival. Secondary outcomes included safety, progression-free survival (PFS), and liver-specific progression-free survival (LPFS).ResultsSixty-one patients were treated with TARE. Patients were 53% male; median age was 64 years; 91% had performance status 0/1; 92% had received prior chemotherapy; and 59% had no extrahepatic disease. Median follow-up was 13.9 months (95% confidence interval [CI], 9.6–18.1). Overall survival was 8.7 months (95% CI, 5.3–12.1), and 37% of patients survived to 12 months. PFS was 2.8 months (95% CI, 2.6–3.1), and LPFS was 3.1 months (95% CI, 1.3–4.8). One severe complication (abdominal pain) occurred at the time of the TARE procedure. Thirty patients experienced a total of 49 adverse events, of which 8% were grade ≥3; most common were grade 1–2 fatigue and abdominal pain. A total of 77 abnormal laboratory value events were recorded, of which 4% were grade ≥3.ConclusionsPatients with advanced ICC have limited therapeutic options and a poor prognosis. This prospective study examined the survival of patients with unresectable, chemotherapy-refractory primary ICC treated with TARE in real-world practice. The results demonstrate that this treatment merits further investigation in this patient cohort in a larger study, including collection of patient-reported outcomes.     

siRNA沉默c－myc对肾上腺皮质癌SW－13细胞增殖的影响

目的：应用siRNA干扰肾上腺皮质癌SW－13细胞c－myc基因的表达,探讨siRNA沉默c－myc对肾上腺皮质癌细胞增殖的影响。方法将siRNA－c－myc转染入SW－13细胞中,采用荧光定量PCR和Wes-tern blot检测转染后c－myc的表达,MTT分析SW－13细胞的增殖,荧光定量PCR检测转染后FHIT在SW－13细胞中的表达。结果 c－myc mRNA在转染48 h后明显下降,c－myc蛋白在转染72 h后明显下降。 SW－13细胞增殖能力在转染24 h后开始下降,48 h和72 h后受到明显抑制。沉默c－myc基因后,FHIT在SW－13细胞的表达有所升高。结论 siRNA沉默c－myc使肾上腺皮质癌细胞增殖受到抑制。 siRNA沉默c－myc基因为肾上腺皮质癌的靶向治疗提供新的理论依据。     

补肺活血胶囊治疗慢性阻塞肺疾病稳定期临床观察

目的观察补肺活血胶囊治疗稳定期慢性阻塞肺疾病(COPD)患者临床疗效。方法将80例COPD患者随机分为两组,对照组给予西药沙美特罗替卡松粉吸入治疗,治疗组在对照组治疗的基础上加以补肺活血胶囊治疗,治疗2个月后,采用圣·乔治呼吸问卷(SGRQ)评分评定治疗前后生活质量改善情况,并比较两组治疗前后肺功能、动脉血气变化、中医证候总积分治疗疗效。结果治疗组治疗总有效率显著高于对照组(P0.05);两组患者治疗后的SGRQ评分、肺功能和动脉血气指标较治疗前均有显著改善(P0.05),且治疗组较对照组改善明显(P0.05);两组治疗后的中医症状总积分较治疗前显著下降,但治疗组下降更为明显(P0.05)。结论在西药常规治疗基础上,加以补肺活血胶囊治疗稳定期COPD较单独使用西药治疗更为有效,可以显著提高患者生活质量和改善肺功能。     

基于临床参考及基因因素建立华法林稳定维持剂量预测模型

目的 探讨华法林应用的临床参考因素及基因因素与华法林稳定维持剂量的相关性,并尝试构建适用于非瓣膜病心房纤颤（non-valvular-disease atrial fibrillation,NVAF）患者华法林稳定维持剂量的预测模型。方法 按照纳入标准共纳入126例患者,应用测序反应通用试剂盒及荧光检测仪检测细胞色素P450 2C9和维生素K环氧化物还原酶基因多态性,同时记录华法林应用的临床参考因素：年龄、体质量、房颤栓塞风险评分系统（CHA₂DS₂-VASc）评分、房颤出血风险评分系统（HAS-BLED）评分、谷丙转氨酶（glutamic-pyruvic transaminase,ALT）、肾小球滤过率（glomerular filtration rate,GFR）、左心室射血分数（left ventricular ejection fraction,LVEF）、二尖瓣环水平左室侧壁组织多普勒S波;采用相关性分析探讨临床参考因素及基因多态性与华法林稳定维持剂量的相关性,并通过多元线性回归建立了华法林稳定维持剂量预测模型。结果 体质量、ALT水平、二尖瓣环水平左室侧壁组织多普勒S波与华法林稳定维持剂量成正相关,年龄、CHA₂DS₂-VASc评分、HAS-BLED评分则成负相关,而LVEF、GFR未显示明显的相关性。建立的预测模型对已有样本验证准确率达55.6%。结论 该模型可用于预测NVAF患者华法林稳定维持剂量。