体外原代培养纯化新生鼠嗅鞘细胞的实验方法：差速贴壁＋阿糖胞苷＋胰酶法

目的:通过细胞形态学观察及生物学特性鉴定,建立一种经济实用的体外原代培养纯化新生鼠嗅鞘细胞的实验方法。方法:实验于2006-06在武汉理工大学完成。①阿糖胞苷(Sigma,批号w10562);胎牛血清(杭州四季青产品);胰蛋白酶(Amresco);多聚赖氨酸(Sigma);胶质纤维酸性蛋白,神经生长因子受体蛋白抗体(博士德)。②选取出生3d内的Wistar大鼠5只,乙醇浸泡后断头处死,取嗅球的最外两层,通过1.25g/L胰蛋白酶消化分离嗅鞘细胞,体外原代培养。③采用Nash差速贴壁 阿糖胞苷 胰酶法纯化嗅鞘细胞。培养18h后将未贴壁的细胞悬液转种于另一未涂层的器皿中,再培养36h,重复上述步骤移入0.1g/L多聚赖氨酸包被的塑料培养瓶中进行培养,纯化后的细胞在24h内陆续贴壁,常规培养2d,加入终浓度为2mg/L的阿糖胞苷,作用48h后,换上新的培养基,继续培养1d,弃去培养基,用无钙镁的Hanks液清洗2次,然后用1.25g/L的胰酶消化10min,待细胞突起回缩、胞体变圆时,立刻加入纯血清终止,其血清终浓度为20%,制备单细胞悬液。④倒置显微镜下观察其形态变化,苏木精-伊红染色,同时行神经生长因子受体蛋白和胶质纤维酸性蛋白免疫组化染色鉴定嗅鞘细胞,并计算嗅鞘细胞阳性百分率。结果:①活细胞形态观察:分离培养的嗅鞘细胞具有双极或多极突起,且突起细长,相互交织。②嗅鞘细胞的苏木精-伊红染色鉴定:细胞呈三角形或梭形,有长的突起,胞浆被染成粉红色,胞核呈蓝紫色,胞核内深染的为核仁,有1～3个核仁。③嗅鞘细胞的胶质纤维酸性蛋白免疫组化染色鉴定:细胞呈现棕黄色,胞核淡染,阳性细胞成网状连接,胞体多为三角形,有细长突起,阳性率91.5%。④嗅鞘细胞的P75免疫组化染色鉴定:阳性细胞呈绿色,多数细胞染色阳性,阳性率89%。结论:实验所采用的Nash差速贴壁 阿糖胞苷 胰酶法分离纯化培养嗅鞘细胞切实可行,为神经诱导修复材料的研究提供种子细胞。     

认知矫正治疗对慢性精神分裂症患者临床症状和社会功能的影响

目的:观察认知矫正治疗对慢性精神分裂症患者临床症状和社会功能的改善作用。方法:选择2003-01/08在北京回龙观医院住院的慢性精神分裂症患者104例。均符合CCMD-Ⅲ及DSM-Ⅳ关于精神分裂症诊断标准;年龄25～55岁;病程≥2年;病情稳定,处于迁延、残留或部分缓解状态;药物治疗状况稳定,近期无换药打算;纳入对象或家属同意入组并签署知情同意书。应用随机数字表法将患者分认知矫正治疗组和对照组,每组52例。在相近药物治疗的基础上,认知矫正治疗组以Ann Delahunty和Rodney Morice等制定的神经认知矫正手册(汉化)为治疗工具,在治疗师的指导下进行认知作业练习,内容包括认知灵活性、工作记忆、计划执行功能3大功能模块。对照组予以相同时间的工娱治疗,主要包括有治疗师指导的操作性音乐治疗和舞蹈治疗。治疗前后两组患者分别进行PANSS、住院精神患者社会功能缺陷量表和护士观察量表的评定。结果:实验共纳入慢性精神分裂症患者104例,认知矫正治疗组44例,对照组46例进入结果分析,14例脱落。①治疗前后两组患者PANSS量表总分以及阴性症状量表、复合量表、一般精神病理量表、反应缺乏量表4个分量表的评分均有下降,组内比较差异有显著性意义(t=2.12～4.59,P<0.05);减分情况在两组间差异不明显(P>0.05)。②两组患者的社会功能缺陷量表总分在治疗后均有下降,与治疗前比较,差异有显著性意义(t=3.89,2.04,P<0.05);两组间比较,差异无显著性意义(P>0.05)。认知矫正治疗组治疗后护士观察量表的总病情以及总消极、迟滞2个分量表评分下降,与治疗前比较差异有显著性意义(t=1.49,1.19,2.81,P<0.05);其中迟滞项的减分在两组间比较,差异具有显著性意义(F=4.97,P<0.05)。③社会功能量表的改善与词语流畅性的改善呈正相关(R2=0.36,P<0.05),护士观察量表中总病情与积极两项评分的改善也与言语流畅性测验的改善正相关(R2=0.37,0.34,P<0.05)。结论:认知矫正治疗能在一定程度上改善精神分裂症患者的社会功能,并与部分认知功能的改善相关,但对临床症状无明显改善作用。     

Role of ATP and related purines in inhibitory neurotransmission to the pig urinary bladder neck

Background and purpose:

As adenosine 5′-triphosphate (ATP) is one of the inhibitory mediators of the bladder outflow region, this study investigates the possible release of ATP or related purines in response to electrical field stimulation (EFS) and the purinoceptor(s) involved in nerve-mediated relaxations of the pig urinary bladder neck.

Experimental approach:

Urothelium-denuded and intact phenylephrine-precontracted strips were mounted in organ baths containing physiological saline solution at 37°C and gassed with 95% O₂ and 5% CO₂ for isometric force recordings.

Key results:

EFS, in the presence of atropine, guanethidine and N^G-nitro-L-arginine, and exogenous purines, produced frequency- and concentration-dependent relaxations respectively. Adenosine 5′-diphosphate (ADP) and adenosine were more potent than ATP in producing relaxation, while uridine 5′-triphosphate, uridine 5′-diphosphate and α,β-methylene ATP were less effective. The non-selective P2 antagonist suramin, and the P2Y₁ and P1 receptor blockers 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium and 8-(p-sulphophenyl)theophylline, respectively, inhibited the responses to EFS and ATP. The P1 agonist''s potency was: 5′-N-ethylcarboxamidoadenosine (NECA)>4-2[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene propanoic acid hydrochloride>2-chloro-N⁶-cyclopentyladenosine>-2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide = adenosine. 4-(-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol, an A_2A antagonist, reduced the relaxations to EFS, adenosine and NECA. In urothelium-intact samples, relaxations to EFS and purines were smaller than in urothelium-denuded preparations. Neuronal voltage-gated Na⁺ channels blockade failed to modify ATP relaxations. At basal tension, EFS- and ATP-induced contractions were resistant to desensitization or blockade of P2X₁ and P2X₃ receptors.

Conclusions and implications:

ATP is involved in the non-adrenergic, non-cholinergic, non-nitrergic inhibitory neurotransmission in the pig bladder neck, producing relaxation largely through muscle A_2A receptors after breakdown to adenosine, and P2Y₁ receptors after breakdown to ADP. Antagonists of these receptors may be useful for urinary incontinence treatment produced by intrinsic sphincteric deficiency.     

Direct identification of Yersinia enterocolitica in blood by polymerase chain reaction amplification

Primers based on the nucleotide sequence of the virF gene in the pYV plasmid and the chromosomal ail gene were used in polymerase chain reaction (PCR) amplifications to directly identify Yersinia enterocolitica in blood. Approximately 500 bacteria seeded into 100 microL of blood can be extracted and amplified by PCR to yield positive results. PCR analyses of seven Y. enterocolitica isolates previously implicated in blood contaminations showed that only one isolate harbored the plasmid-borne virF gene; however, all seven isolates were identified effectively by the PCR product amplified from the chromosomal gene. The PCR assay has the potential for use in the identification of Y. enterocolitica contamination in stored units of blood or in the rapid diagnosis of transfusion-related bacteremia caused by Y.     

Retroperitoneal sarcomas: A review of disease spectrum,radiological features,characterisation and management

Retroperitoneal sarcomas are a rare disease. The overall 5‐year survival rate for these lesions remains low, and surgical management offers the only option for effective treatment and potential for cure. Radiotherapy is increasingly being employed in addition to standard surgical treatment. Improvements in cross‐sectional imaging have also facilitated better characterisation of lesions, preoperative planning and long‐term follow‐up. This article reviews the current literature and documents the various types of retroperitoneal sarcomas with a particular approach to their imaging features. We also highlight the pathology, diagnostic methods and most current management of these tumours.     

Joining the dots: a plea for precise estimates of the maternal mortality ratio in sub-Saharan Africa

Monitoring of maternal mortality levels in sub-Saharan Africa (SSA) to assess the achievements of safe motherhood programmes and for MDG-5 has been made difficult because of the lack of precise estimates of the maternal mortality ratio (MMR). Projections based on the slow rate of decline of the MMR indicate that MDG-5 may not be reached before the end of this century in this region. Measurements done using demographical and health surveys, statistical modelling and censuses are imprecise and do not allow trends in individual countries to be established. SSA countries should be encouraged to measure mortality levels from their own resources, using methods that produce precise estimates such as population-based surveys. Establishment of the trends will lead to country-specific program targets. The less frequent but more precise measurements can be afforded by SSA countries, as a case study from Zimbabwe shows.     

Development of hatching blastocysts from immature human oocytes following in-vitro maturation and fertilization using a co-culture system

Recently, in-vitro maturation (IVM) of immature human oocytes recovered from non-stimulated follicles has been applied in the treatment of infertility. However, in previous reports, very few embryos cultured in conventional medium have reached the expanded blastocyst stage following in-vitro maturation and fertilization (IVM/IVF). The objective of this study was to investigate whether the developmental competence of human embryos following IVM/IVF could be enhanced by the use of a human ampullary cell co-culture system. Immature human oocytes were aspirated from small follicles at Caesarean section and then cultured in medium containing human menopausal gonadotrophin for 36 to 48 h, followed by insemination. Zygotes were randomly cultured either in conventional culture medium alone or in the co-culture system. Of 48 embryos cultured in conventional medium alone, all arrested at the 2-16- cell stage on day 3 after insemination. Of 46 embryos cultured in the co-culture system, 26 embryos (56.5%) arrested at the 2-16-cell stage. Six embryos (13%) developed to the morula stage. Fourteen embryos (30.4%) developed to expanded blastocysts and two blastocysts were hatching on day 7 after insemination. We conclude that co-culture significantly enhances the development of blastocysts in embryos resulting from IVM/IVF.