不同的抗原修复条件对免疫组织化学染色结果的影响

抗原是指能刺激机体产生抗体,并能与抗体相结合的物质。物质所具有的这种特性称为抗原性,它主要由分布在抗原表面的抗原决定簇决定。但是部分组织经甲醛固定、石蜡包埋后,部分甚至全部抗原决定簇被封闭,影响免疫组织化学的标记效果,误导诊断。因此,不少的研究者纷纷采用不同的方法去暴露被封闭的抗原决定簇。笔者比较了30种常用的抗体在9种不同的抗原修复条件中的表达情况,     

结节性淋巴细胞为主型霍奇金淋巴瘤

1.病例简介:患者男,70岁,偶然发现右腋下无痛性肿物3年,逐渐增大。于1997年10月24日入院。体检:右腋下肿物10cm×10cm×4cm,无触痛。质软,部分坚实,可活动。全身其余部位浅表淋巴结未扪及。肝脾肋下未及。实验室及影像学检查均未见异常。临床诊断:脂肪瘤。行肿物手术切除。2.病理检查:肉眼观察:灰红灰黄组织13·0cm×12·0cm×4·5cm,多结节状,切面灰红色,均质,质软。光镜观察:淋巴结结构破坏,低倍镜下可见多个大小不一深染模糊结节,边界不清,无生发中心(图1)。结节与结节之间较拥挤,有的结节较大,周围有少许纤维组织围绕,大多数结节间不见…     

Survival of donor epithelial cells after limbal stem cell transplantation

PURPOSE: To determine the long-term fate of donor epithelial cells after limbal allograft transplantation. METHODS: Corneal buttons and peripheral blood leukocytes were obtained at the time of penetrating keratoplasty from three patients who had undergone a successful limbal allograft transplantation. Microdissection was used to remove the corneal epithelial cells from the button. The presence of donor and recipient epithelial cells in each sample was determined by using PCR for DNA microsatellites. Phenotypic analysis of the epithelium was performed by immunohistochemistry. RESULTS: Various patterns of DNA microsatellites were observed. Nonrecipient cells (presumed to be donor) were consistently detected in all three corneal buttons. In two of the three cases, recipient cells were also detected, whereas in the third case, exclusively donor epithelial cells were found at 3.5 years after limbal allograft transplantation. Mild T-lymphocytes and macrophages were observed in one of the corneal buttons. CONCLUSIONS: This study provides evidence for the persistence of donor epithelial cells up to 3.5 years after limbal allograft transplantation and supports the use of systemic immunosuppressive therapy.     

Cannabinoids produce neuroprotection by reducing intracellular calcium release from ryanodine-sensitive stores

Exogenously administered cannabinoids are neuroprotective in several different cellular and animal models. In the current study, two cannabinoid CB1 receptor ligands (WIN 55,212-2, CP 55,940) markedly reduced hippocampal cell death, in a time-dependent manner, in cultured neurons subjected to high levels of NMDA (15 μM). WIN 55,212-2 was also shown to inhibit the NMDA-induced increase in intracellular calcium concentration ([Ca²⁺]_i) indicated by FURA-2 fluorescence imaging in the same cultured neurons. Changes in [Ca²⁺]_i occurred with similar concentrations (25–100 nM) and in the same time-dependent manner (pre-exposure 1–15 min) as CB1 receptor mediated neuroprotective actions. Both effects were blocked by the CB1 receptor antagonist SR141716A. An underlying mechanism was indicated by the fact that (1) the NMDA-induced increase in [Ca²⁺]_i was inhibited by ryanodine, implicating a ryanodine receptor (RyR) coupled intracellular calcium channel, and (2) the cannabinoid influence involved a reduction in cAMP cAMP-dependent protein kinase (PKA) dependent phosphorylation of the same RyR levels that regulate channel. Moreover the time course of CB1 receptor mediated inhibition of PKA phosphorylation was directly related to effective pre-exposure intervals for cannabinoid neuroprotection. Control studies ruled out the involvement of inositol-trisphosphate (IP3) pathways, enhanced calcium reuptake and voltage sensitive calcium channels in the neuroprotective process. The results suggest that cannabinoids prevent cell death by initiating a time and dose dependent inhibition of adenylyl cyclase, that outlasts direct action at the CB1 receptor and is capable of reducing [Ca²⁺]_i via a cAMP/PKA-dependent process during the neurotoxic event.     

非穿透小梁手术的术后早期并发症及成本-效果分析

目的评价非穿透小梁手术(NPTS)的术后早期并发症及成本效果。方法回顾性分析147例(215只眼)原发性开角型青光眼患者行滤过性手术后的临床资料,包括早期(住院期间)视力、眼压及并发症发生情况。其中NPTS组104只眼,改良小梁切除术(MT)组111只眼。根据所需治疗的例数(NNT)和手术效果,对预防不良事件的发生进行成本效果分析。结果(1)早期视力波动:术后视力下降两行以上者NPTS组25只眼(24.0%)、MT组26只眼(23.4%),两组间的视力变化差异无统计学意义(P>0.05)。(2)早期低眼压:术后第1天眼压≤5mmHg(1mmHg=0.133kPa)者NPTS组39只眼(38.2%),MT组10只眼(9.2%),两组间差异有统计学意义(P<0.01);出院时眼压≤5mmHg者分别为18只眼(27.5%)和19只眼(17.3%),两组间差异有统计学意义(P<0.05)。(3)前房出血:NPTS组20只眼(19.2%),MT组10只眼(9.0%);各组均有2只眼需行前房冲洗;两组间前房出血发生率的差异有统计学意义(P<0.05)。(4)早期浅前房:NPTS组发生Ⅱ°浅前房2只眼,Ⅰ°浅前房5只眼;MT组Ⅱ°浅前房5只眼,Ⅰ°浅前房5只眼;各组均未出现Ⅲ°浅前房者,两组间不同程度浅前房的发生率差异无统计学意义(P>0.05)。(5)其他并发症:NPTS组术中小梁穿透1只眼,遂改行小梁切除术;术后瞳孔散大6只眼;急性眼压升高1只眼;内滤口虹膜前粘连1只眼;低眼压性黄斑水肿1只眼。(6)成本效果分析:NPTS组对不良事件的绝对风险降低率(ARR)=3.0%,防止1例不良事件发生需治疗的病例数(NNT)=33.2例,较MT组多花费成本11.6万元;对于严重不良事件,NPTS组的绝对风险降低率=1.8%,NNT=55.5例,较MT组多花费成本19.4万元。结论NPTS可能在减少抗青光眼术后严重不良事件的发生方面具有一定作用,但与MT相比,其在减少并发症方面的成本较高;因此,基层医院眼科医师应重点掌握MT操作技术。     

Association of interleukin-12 p40 gene 3＇-untranslated region polymorphism and outcome of HCV infection

AIM: To investigate the effect of interleukin-12 p40 gene (IL12E 3‘-untranslated region polymorphism on the outcome of HCV infection.METHODS: A total of 133 patients who had been infected with HCV for 12-25 (18.2&#177;3.8) years, were enrolled in this study. Liver biochemical tests were performed with an automated analyzer and HCV RNA was detected by fluorogenic quantitative polymerase chain reaction. B-mode ultrasound was used for liver examination. Polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) was used for the detection of IL12B (1188A/C) polymorphism.RESULTS: Self-limited infection was associated with AC genotype (OR = 3.48; P = 0.001) and persistent infection was associated with AA genotype (OR = 0.34; P = 0.014)at site 1188 of IL12B. In patients with persistent HCV infection, no significant differences were found regarding the age, gender, duration of infection and biochemical characteristics (P&gt;0.05). According to B-mode ultrasound imaging and clinical diagnosis, patients with persistent infection were divided into groups based on the severity of infection. No significant differences were found in the frequency of IL-12 genotype (1188A/C) between different groups (P&gt;0.05).CONCLUSION: The polymorphism of II12B (1188A/C)appears to have some influence on the outcome of HCV infection.     

人类新基因CTRP4的原核表达及多克隆抗体的制备

目的:构建新基因CTRP4的原核表达载体,在大肠杆菌中表达并纯化rhCTRP4-his蛋白,制备和鉴定小鼠抗人CTRP4多克隆抗体,为进一步研究人类新基因CTRP4的生物学功能奠定基础。方法:从pcDNA3.1-myc/his(-)B-hCTRP4重组载体中通过PCR的方法扩增CTRP4基因ORF中不含信号肽的目的基因片段,将得到的片段双酶切定向插入pET-32a中,构建原核表达载体pET-32a-hCTRP4。构建好的质粒在E.coli BL21(DE3)中进行诱导、表达,通过镍亲和层析柱纯化融合蛋白,SDS-PAGE电泳分析蛋白纯度。将pcDNA3.1-myc/his(-)B-hCTRP4重组质粒和纯化好的融合蛋白依次免疫BALB/c小鼠,制备多克隆抗体。并对抗体进行纯化、效价测定以及特异性鉴定。结果:DNA测序证实构建的pET-32a-hCTRP4重组表达载体含有hCTRP4编码序列,其序列比对分析与GenBank中公布序列一致,质粒在E.coli中表达相对分子质量(Mr)为35 000的目的蛋白,SDS-PAGE电泳分析纯度为95%以上。ELISA法检测抗体效价为1∶20 000,Westernblot、免疫荧光细胞化学和免疫组化分析显示抗体能特异性识别重组hCTRP4以及天然状态hCTRP4蛋白。结论:成功构建了hCTRP4蛋白的原核表达载体,并获得较高纯度的融合蛋白,制备了高效价、高特异性的多克隆抗体。     

The roles of extracellular signal-regulated kinase 1/2 pathway in regulating osteogenic differentiation of murine preosteoblasts MC3T3-E1 cells on roughened titanium surfaces

Surface roughness of titanium-based implants may enhance osteogenic differentiation of cells in vitro and bone-to-implant contact in vivo. Nevertheless, how surface roughness regulates the signaling pathway of osteoblasts is little understood. The study intended to investigate specifically the roles of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in regulating osteogenic differentiation of MC3T3-E1 murine preosteoblast cells on Ti surfaces. Substrates applied were two groups of titanium disks: (1) sand-blasted and acid-etched rough surfaces (SLA) and (2) smooth pretreated Ti surfaces (PT). Surface morphology of the two groups was examined by scanning electron microscope, and cell morphology cultured on Ti disks was observed by confocal microscope. The levels of alkaline phosphatase (ALP) activity and calcium deposition were measured and compared between the two groups. Real-time polymerase chain reaction was applied to detect the expression levels of osteogenic genes including runt related protein 2 (Runx2), osterix (OSX), osteocalcin (OCN) and osteoprotegerin (OPN) of the cells cultured on the two groups of substrates and on SLA surfaces treated with ERK1/2 inhibitor, PD98095. ERK1/2 activities in MC3T3-T1 cells were measured by Western-blotting on the two surfaces with or without PD98095. Cells cultured on rougher SLA surfaces displayed a more differentiated morphology. ALP activities at 7 days and 14 days and the calcium deposition at 28 days were significantly higher on SLA surfaces. The expression levels of Runx2, OSX, OPN and OCN were upregulated by the effect of surface roughness and PD98095 further upregulated the expression levels of these osteogenic genes on SLA surfaces. ERK1/2 phosphorylation was continuously inhibited by surface roughness at 2 days, 4 days and 6 days. In contrast, no marked alterations in ERK1/2 phosphorylation on PT surfaces were observed. PT surfaces treated with PD98095 (50 μM) and SLA surfaces without PD98095 both demonstrated reduced ERK1/2 phosphorylation of the cells, and the inhibitive effect of SLA surfaces was milder than that of PD98095. In conclusion, ERK1/2 pathway may be a negative regulator of cell differentiation in a dosage-dependent manner, and the enhancing effect of surface roughness on osteoblastic differentiation may be mediated through inhibiting ERK1/2 pathway.