大鼠胚胎脑源性神经干细胞的分离培养和鉴定

目的：分离培养大鼠胚胎脑源性神经千细胞，并进行增殖分化鉴定。 方法：实验于2005—06／12在中国科学技术大学生命科学学院周江宁研究室进行。①自孕14dWistar大鼠胚胎分离大脑皮质及皮质下组织，经机械吹打分离成单细胞后，利用无血清原代及传代培养方法，在添加重组人碱性成纤维细胞生长因子、重组人表皮生长因子和B27添加剂的DMEM／F12（1：1）培养基中进行悬浮培养，获得具有克隆能力的细胞群；用有限稀释法，得到来源于同一细胞的亚细胞系克隆。②从培养基中撤除生长因子和B27添加剂，换成含体积分数为0．1的胎牛血清的DMEM／F12培养基，将培养的细胞球接种于预先铺有多聚赖氨酸盖玻片的培养皿，诱导分化。③以免疫荧光细胞化学技术，用神经干细胞的特异性单克隆抗体（巢蛋白）和增殖细胞单克隆抗体（5一溴脱氧尿苷嘧啶）鉴定克隆细胞，以鉴定神经干细胞的自我更新增殖能力。④以免疫荧光细胞化学技术，用神经细胞的特异性抗体（神经元特异性烯醇化酶、胶质纤维酸性蛋白、半乳糖脑苷脂）鉴定分化细胞，以鉴定神经干细胞的多向分化潜能。 结果：①从胎鼠大脑皮质及皮质下分离的组织，经原代和传代培养均可形成细胞克隆，并具有增殖的能力，表达神经上皮干细胞蛋白抗原和增殖细胞抗原。②在血清诱导下，分化后的细胞表达神经元、星形胶质细胞和少突胶质细胞3种神经细胞的特异性抗原。 结论：运用上述方法分离培养的细胞具有自我更新和增殖能力，并具有多向分化潜能和具有神经干细胞的特异性抗原。     

肠造口病人生活质量的研究进展

介绍了肠造口病人生活质量状况的研究进展。从影响肠造口病人生活质量的相关因素及所采取的措施进行了综述。     

Sequential treatment with lamivudine and interferon-alpha monotherapies in hepatitis B e antigen-negative Chinese patients and its suppression of lamivudine-resistant mutations

OBJECTIVES: To assess the efficacy of sequential treatment with lamivudine and interferon-alpha monotherapies in Chinese patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B. METHODS: One hundred and sixty-two patients with HBeAg-negative chronic hepatitis B were included in this study. Ninety-eight were treated with lamivudine alone (100 mg per day) for 48 weeks (group B). Sixty-four were treated with lamivudine alone (100 mg per day) for 20 weeks, then combined with interferon-alpha-2b (5 million units three times per week) for 4 weeks and then treated for another 24 weeks with interferon-alpha-2b alone (5 million units three times per week) (group A). All patients were followed for an additional 24 weeks. RESULTS: After 48 weeks of treatment, the percentage of patients with normalization of alanine aminotransferase (ALT) levels or hepatitis B virus (HBV) DNA levels below 1000 copies/mL was not significantly different between the lamivudine monotherapy group (55.10% and 55.10%, respectively) and the sequential treatment group (59.36% and 56.25%, respectively). The percentage of patients with normalized ALT levels was significantly higher in group A (53%) than in group B (36%) at week 72 (P<0.05). The percentage of patients with lamivudine-resistant mutations was significantly higher with lamivudine monotherapy (22.45%) than with sequential therapy (P<0.05). CONCLUSIONS: Sequential treatment of chronic hepatitis B with lamivudine and interferon-alpha monotherapies is as effective as lamivudine-alone treatment in Chinese patients. However, sequential treatment can significantly suppress the emergence of lamivudine-resistant mutations.     

Spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ZSET1446/ST101) treatment rescues olfactory bulbectomy-induced memory impairment by activating Ca2+/calmodulin kinase II and protein kinase C in mouse hippocampus

Olfactory bulbectomy (OBX) in mice elicits impaired memory and cognitive functions. Here, we found that chronic oral administration of spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ZSET1446/ST101) (0.1-1 mg/kg/day), a novel cognitive enhancer, significantly improved memory deficits as assessed by Y-maze and novel object recognition tasks in OBX mice. Immunostaining of cholinergic neurons in the medial septum by using an anti-choline acetyltransferase antibody indicated that chronic ZSET1446 treatment did not rescue cholinergic neurons. However, chronic treatment significantly restored OBX-induced decreases both in calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) phosphorylation without improving decreased extracellular signal-regulated kinase phosphorylation in the hippocampal CA1 region. Consistent with enhanced CaMKII and PKC phosphorylation, ZSET1446 treatment improved glutamate receptor 1 (Ser-831) phosphorylation in the hippocampal CA1 region. ZSET1446 treatment also significantly rescued impaired long-term potentiation (LTP) in the hippocampal CA1 region of OBX mice. Taken together, the cognition-enhancing effect of ZSET1446 is probably mediated in part by stimulation of CaMKII and PKC activities, which in turn rescue impaired hippocampal LTP in OBX mice.     

胶原膜引导下同种骨颗粒修复兔桡骨节段性缺损

背景:胶原膜是一种可吸收引导成骨材料,同种异体骨是较为理想的骨替代材料,但单独应用均有其不足,拟将两种材料结合使用以期达到理想效果.目的:利用膜引导组织再生技术,观察胶原膜复合同种骨颗粒修复兔桡骨节段性骨缺损的成骨速度和质量,并与单纯胶原膜进行比较.设计、时间及地点:同体对照观察实验,于2004-09/2005-02在中国辐射防护研究院医用组织库中心实验室完成.材料:胶原膜规格20 mm×20 mm,由北京天新福医疗器材有限公司提供.同种异体骨颗粒按美国组织库标准制备,直径0.2～0.7 mm,由山西省医用组织库提供.方法:健康成年新西兰大白兔40只,制备双侧15 mm桡骨缺损模型,采用同体对照方法,实验侧(左侧)缺损区移植胶原膜及同种骨粒,对照侧(右侧)仅移植胶原膜.主要观察指标:光镜组织学观察骨缺损愈合情况,计算机图像分析仪计算骨小梁的成骨面积,扫描电镜观察缺损再生修复情况.结果:①光镜组织学变化:实验侧新骨增生明显,迅速,以膜性化骨及软骨内化骨的方式由骨缺损周围向中央成骨或以同种骨粒为中心由周围向骨粒内成骨,最后骨膜形成,新髓腔不断扩大、再通,骨重建顺利,缺损修复完成.对照侧成骨方式以软骨化骨为主,新骨形成较同期实验侧慢,新骨成熟度不如实验组.②成骨面积图像分析:结果显示术后2,4,8周新生骨小梁成骨面积均大于同期对照侧(P<0.05).③扫描电镜观察:术后8周实验侧有较多分泌胶原的成骨细胞和带有较多突起的骨细胞,钙盐沉积,骨质排列开始有序.对照侧软骨与类骨质互相存在,低倍下骨基质排列无序,胶原纤维编织样排列.术后12周实验侧骨质排列有序,胶原粗大排列整齐,大量骨细胞方向有序,大量钙盐沉积,可见哈佛氏管和血管.对照侧骨质排列有序,但胶原纤维较细、基质钙化程度不如实验侧.结论:两种方法均能成骨修复缺损,但胶原膜复合同种异体骨粒组成骨速度快,且成骨质量优于单纯胶原膜组.     

纳米羟基磷灰石粒子引起红细胞聚集的体外实验

背景：目前在纳米羟基磷灰石生物安全性实验中发现其引起红细胞聚集现象，是纳米羟基磷灰石血液相容性研究中亟待解决的问题。目的：探讨体外纳米羟基磷灰石引起红细胞聚集的机制。设计、时间及地点：体外细胞形态学观察实验，于2004-03／2006-06在武汉理工大学生物材料与工程研究中心实验室完成。材料：两种不同粒径羟基磷灰石粒子粉体由武汉理工大学生物医学材料与工程研究中心提供。方法：溶血实验评价材料对兔红细胞的影响，并对材料与红细胞共培养后做细胞形态学观察，Bialsche法检测纳米羟基磷灰石粒子与唾液酸的吸附量，绘制吸附等温线，纳米羟基磷灰石粒子与唾液酸共吸附作红外光谱分析。主要观察指标：纳米羟基磷灰石粒子对红细胞形态影响和超微结构观察，不同粒径的羟基磷灰石粒子对唾液酸的吸附量，纳米羟基磷灰石与唾液酸吸附后的红外光谱分析。结果：纳米羟基磷灰石粒子与兔红细胞共培养后可导致红细胞聚集，并且对细胞表面的唾液酸有强烈的吸附作用，红外光谱结果表明两者有明显的相互作用。结论：纳米粒子与红细胞体外共培养后，降低了细胞的Zeta电位和表面电荷密度，引起红细胞悬浮性下降，可能是导致聚集的发生的重要因素。     

Comparative analysis of transient receptor potential channel 5 opposite strand-induced gene expression patterns and protein-protein interactions in triple-negative breast cancer

In patients with triple-negative breast cancer (TNBC), high tumour mutation burden and aberrant oncogene expression profiles are some of the causes of poor prognosis. Therefore, it is necessary to identify aberrantly expressed oncogenes, since they have the potential to serve as therapeutic targets. Transient receptor potential channel 5 opposite strand (TRPC5OS) has been previously shown to function as a novel tumour inducer. However, the underlying mechanism of TRPC5OS function in TNBC remain to be elucidated. Therefore, in the present study TRPC5OS expression was first measured in tissue samples of patients with TNBC and a panel of breast cancer cell lines (ZR-75-1, MDA-MB-453, SK-BR-3, JIMT-1, BT474 and HCC1937) by using qRT-PCR and Western blotting. Subsequently, the possible effects of TRPC5OS on MDA-MB-231 cells proliferation were determined using Cell Counting Kit-8 and 5-Ethynyl-2′-deoxyuridine assays after Lentiviral transfection of MDA-MB-231. In addition, potential interaction partners of TRPC5OS were explored using liquid chromatography-mass spectrometry (LC-MS)/MS. Gene expression patterns following TRPC5OS overexpression were also detected in MDA-MB-231 cells by using High-throughput sequencing. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis were then used to systematically verify the potential interactions among the TRPC5OS-regulated genes. The potential relationship between TRPC5OS-interacting proteins and gene expression patterns were studied using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis. TRPC5OS expression was found to be significantly higher in TNBC tumour tissues and breast cancer cell lines compared with luminal tumour tissues and ZR-75-1. In addition, the overexpression of TRPC5OS significantly increased cell proliferation. High-throughput sequencing results revealed that 5,256 genes exhibited differential expression following TRPC5OS overexpression, including 3,269 upregulated genes and 1,987 downregulated genes. GO analysis results indicated that the functions of these differentially expressed genes were enriched in the categories of ‘cell division’ and ‘cell proliferation’ regulation. KEGG analysis showed that the TRPC5OS-regulated genes were associated with processes of ‘homologous recombination’ and ‘TNF signalling pathways’. Subsequently, 17 TRPC5OS-interacting proteins were found using LC-MS/MS and STRING analysis. The most important protein among interacting proteins was ENO1 which was associated with glycolysis and regulated proliferation of cancer. In summary, data from the present study suggest that TRPC5OS overexpression can increase TNBC cell proliferation and ENO1 may be a potential target protein mediated by TRPC5OS. Therefore, TRPC5OS may serve as a novel therapeutic target for TNBC.     

A novel fluorescent digitonin derivative for non-invasive skin cholesterol detection: potential application in atherosclerosis screening

There is a great demand for the rapid and non-invasive atherosclerosis screening method. Cholesterol content in the epidermis of the skin is an early biomarker for atherosclerosis. Risk assessment of atherosclerosis can be achieved by measuring cholesterol in the epidermis. Here, we synthesised a new fluorescent digitonin derivative (FDD) for the non-invasive detection of skin cholesterol. The results of fluorescence spectroscopy studies indicated that the probe exhibited desirable selectivity for cholesterol. The proof-of-concept preclinical study confirmed that FDD can detect different concentrations of skin cholesterol; patients diagnosed with atherosclerotic cardiovascular disease and the at-risk atherosclerosis group exhibited higher skin cholesterol content than the normal group. The area under the ROC curve for distinguishing the normal/disease group was 0.9228 (95% confidence interval, 0.8938 to 0.9518), and the area under the ROC curve for distinguishing the normal/risk group was 0.9422 (95% confidence interval, 0.9178 to 0.9665). We anticipate that this non-invasive skin cholesterol test may be used as a risk assessment tool for atherosclerosis screening in a large population for further examination and intervention in high-risk populations.

Digitonin was used to synthesise a fluorescence probe for the non-invasive detection of skin cholesterol. This non-invasive skin cholesterol method may be used as a risk assessment tool for atherosclerosis screening in a large population.     

乳腺癌外周血细胞角蛋白19 mRNA的检测及临床价值

目的应用荧光定量RT-PCR检测乳腺癌外周血中的表达情况,并评价其表达的临床意义。方法用荧光定量RT-PCR方法检测49例乳腺癌患者外周血CK19mRNA表达。结果49例乳腺癌患者外周血中CK19mRNA检测阳性28例,阳性率57.1%,统计结果显示乳腺癌患者外周血CK19mRNA的表达阳性率在患者不同年龄、病理类别、临床分期、ER、PR状态的各组差异无显著性(P>0.05),在淋巴结转移状态间有显著差异。结论CK19mRNA可作为RT-PCR法检测乳腺癌患者外周血微转移的分子标志物,有助于早期预测乳腺癌发生血道微转移,指导临床治疗。     

青年与老年结直肠癌临床病理与预后比较

为了探讨青年与老年结直肠癌的临床病理特征和预后的不同,回顾性分析1991年1月～2002年12月天津医科大学附属肿瘤医院手术治疗、经病理资料确诊且随访资料完整的青年结直肠癌（≤40岁）278例,老年结直肠癌（〉60岁）693例,应用SPSS10．0软件进行统计分析。结果示青年组结直肠癌与老年组结直肠癌相比出现症状时间较短,发病部位以直肠癌多见,病理类型较差,淋巴结转移常见,Duke分期较晚,但青年组与老年组之间总的生存期没有差别。初步研究结果提示,青年组与老年组结直肠癌患者临床病理特征有差异;但相同临床期别结直肠癌青年组预后并不比老年组差。