醛糖还原酶和晚期糖基化终末产物受体对糖尿病视网膜病变神经元凋亡的影响

目的　探究醛糖还原酶和晚期糖基化终末产物受体对糖尿病视网膜病变神经元凋亡的影响。方法　Wistar大鼠36只，随机分为对照组、模型组、转染组，后两组建立糖尿病大鼠模型。模型建立成功后，构建含有晚期糖基化终末产物受体siRNA的质粒并利用慢病毒转染入转染组大鼠体内。造模后4周、8周、12周，记录各组大鼠体质量及空腹血糖。造模后9周，禁食6 h，测定口服葡萄糖耐量。造模后12周，处死全部大鼠后，TUNEL法检测各组大鼠视网膜神经元凋亡情况，荧光分光光度计测定醛糖还原酶活性，Western blotting法测定晚期糖基化终末产物受体的表达，RT-PCR检测视网膜中Bcl-2和Bax mRNA相对表达量。结果　造模后4周、8周、12周，转染组和模型组的大鼠体质量均低于对照组（均为P<0.05）；造模后12周，转染组大鼠体质量高于模型组（P<0.05）。造模后4周、8周、12周，各组内大鼠空腹血糖水平均无明显变化（均为P>0.05），转染组和模型组大鼠的空腹血糖水平均高于对照组（均为P<0.05）。模型组和转染组大鼠在口服葡萄糖后30 min时，血糖水平均高于对照组（均为P<0.05）；在120 min时分别下降至最低，但仍高于对照组（均为P<0.05）。模型组和转染组的视网膜神经元凋亡指数、醛糖还原酶活性、晚期糖基化终末产物受体和Bax mRNA相对表达量均高于对照组（均为P<0.05），且转染组均高于模型组（均为P<0.05）。模型组和转染组的Bcl-2 mRNA相对表达量均低于对照组（均为P<0.05），转染组低于模型组（P<0.05）。结论　晚期糖基化终末产物结合受体后产生大量的氧自由基损伤，可能是导致糖尿病视网膜神经元凋亡，进而导致糖尿病视网膜病变发生的机制之一。     

PBL教学法在中医急诊学教学中的运用分析

目的分析PBL教学法在中医急诊学教学中的运用效果。方法2018年3月-2018年6月选取本校2016级针推专升本学生68名进行调查,采用随机数字表法将其分为2组,各34名,观察组采取PBL教学法,对照组采取传统讲授法,对比2组学生的理论、实践考核成绩,并通过调查问卷评价学生对教学方法的认可程度。结果实施教学后,观察组学生理论、实践考核成绩较对照组学生明显提高(P<0.05)。相比于对照组,观察组学生在培养学习兴趣、提升学习技巧、增强合作能力、创新思维方式的层面上对教学方法的认可程度普遍较高(P<0.05)。结论采取PBL法实施中医急诊学教学有助于提升临床教学效果和质量。     

芎芷石膏汤治疗原发性三叉神经痛

原发性三叉神经痛是三叉神经分布区内短暂的发作性剧痛,多发于中老年人,40岁以上患者占70%~80%,发作时表现为面颊上下颌及舌部明显的剧烈电击样、针刺样、刀割样疼痛,持续时间短,突发突止,间歇期完全正常。中医将其归属于"面痛"范畴,多从风热立论,笔者跟随导师临证过程中,发现《医宗金鉴》的芎芷石膏汤治疗风热上犯型原发性三叉神经痛效果显著。     

Internalization of titanium dioxide nanoparticles by glial cells is given at short times and is mainly mediated by actin reorganization-dependent endocytosis

Many nanoparticles (NPs) have toxic effects on multiple cell lines. This toxicity is assumed to be related to their accumulation within cells. However, the process of internalization of NPs has not yet been fully characterized. In this study, the cellular uptake, accumulation, and localization of titanium dioxide nanoparticles (TiO₂ NPs) in rat (C6) and human (U373) glial cells were analyzed using time-lapse microscopy (TLM) and transmission electron microscopy (TEM). Cytochalasin D (Cyt-D) was used to evaluate whether the internalization process depends of actin reorganization. To determine whether the NP uptake is mediated by phagocytosis or macropinocytosis, nitroblue tetrazolium (NBT) reduction was measured and the 5-(N-ethyl-N-isopropyl)-amiloride was used. Expression of proteins involved with endocytosis and exocytosis such as caveolin-1 (Cav-1) and cysteine string proteins (CSPs) was also determined using flow cytometry.TiO₂ NPs were taken up by both cell types, were bound to cellular membranes and were internalized at very short times after exposure (C6, 30 min; U373, 2 h). During the uptake process, the formation of pseudopodia and intracellular vesicles was observed, indicating that this process was mediated by endocytosis. No specific localization of TiO₂ NPs into particular organelles was found: in contrast, they were primarily localized into large vesicles in the cytoplasm. Internalization of TiO₂ NPs was strongly inhibited by Cyt-D in both cells and by amiloride in U373 cells; besides, the observed endocytosis was not associated with NBT reduction in either cell type, indicating that macropinocytosis is the main process of internalization in U373 cells. In addition, increases in the expression of Cav-1 protein and CSPs were observed.In conclusion, glial cells are able to internalize TiO₂ NPs by a constitutive endocytic mechanism which may be associated with their strong cytotoxic effect in these cells; therefore, TiO₂ NPs internalization and their accumulation in brain cells could be dangerous to human health.     

Immunogenicity and immunoprotection of porcine circovirus type 2 (PCV2) Cap protein displayed by Lactococcus lactis

The capsid (Cap) protein, an important immunoprotective protein of porcine circovirus type 2 (PCV2), was expressed on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. Cap protein was fused to the peptidoglycan binding domain (known as the protein anchor domain, PA) of the lactococcal AcmA cell-wall hydrolase. The Cap protein fusion was non-covalently rebound to the surface of non-genetically modified, non-living high-binder L. lactis cells (designated Gram-positive enhancer matrix (GEM) particles). Expression of the recombinant GEM-displaying capsid protein (GEM-PA-Cap) was verified by Western blotting and immunofluorescence and transmission electron microscopy assays. To evaluate the immunogenicity of the recombinant Cap protein (rCap), 20 PCV2-seronegative piglets were immunized with the GEM-PA-Cap subunit vaccine, GEM alone, or phosphate-buffered saline (PBS, challenge control and empty control). Each group consisted of five piglets. The results showed that the level of PCV2-specific antibodies in piglets immunized with the GEM-PA-Cap subunit vaccine was significantly higher than that of the piglets immunized with GEM alone or the control group at all the time points post-vaccination (P < 0.01). After challenge with the PCV2 wild-type strain, piglets that received the GEM-PA-Cap subunit vaccine showed significantly higher average daily weight gain (DWG) and shorter fever duration than the other two groups (P < 0.001). Furthermore, a significant reduction in the gross lung lesion scores and lymph node lesion scores was noted in the GEM-PA-Cap-immunized group compared with the scores of the GEM or PBS-treated group (P < 0.01). The results suggest that recombinant rCap displayed by L. lactis GEM particles provided the piglets with significant immunoprotection from PCV2-associated disease. Thus, the novel GEM-PA-Cap subunit vaccine has potential to be considered an effective and safe candidate vaccine against PCV2 infection in piglets.     

Dominance of zoonotic genotype D of Enterocytozoon bieneusi in bamboo rats (Rhizomys sinensis)

Enterocytozoon bieneusi is an emerging zoonotic intestinal pathogen that infects humans and various animal species. Here, we aimed to determine the infection rate and genetic characteristics of E. bieneusi from bamboo rats from different regions of China using nested polymerase chain reaction-based amplification of the internal transcribed spacer region of the rRNA gene. A total of 435 bamboo rats fecal samples were collected from individual tank from Guangdong, Hunan, Jiangxi, Chongqing, and Guangxi, southeastern China. E. bieneusi was detected on 22 tanks (5.1%, 22/435), with a higher infection rate being observed among samples from Guangdong Province (10.9%, 5/46) compared with those from Hunan (9.3%, 10/107), Jiangxi (6.7%, 6/90), Chongqing (2.0%, 1/50), and Guangxi (0%, 0/142) (P < .01). Six genotypes were identified, including four known genotypes (D, EbpA, J, and PigEBITS7) and two novel genotypes (named BR1 and BR2). Of these, zoonotic genotype D was the most prevalent in the present study (n = 17). Phylogenetic analysis revealed that genotypes D, EbpA, and PigEBITS7 were clustered into Group 1, while genotypes J, BR1, and BR2 were clustered into Group 2. To our knowledge, this is the first report of E. bieneusi in bamboo rats. The identification of zoonotic genotype D as the predominant genotype in bamboo rats suggests that these animals represent a potential zoonotic risk for the transfer of the pathogen in China.     

Potential impacts of host specificity on zoonotic or interspecies transmission of Enterocytozoon bieneusi

Microsporidia are composed of a highly diverse group of single-celled, obligate intracellular fungi that colonize an extremely wide range of other eukaryotes, among which Enterocytozoon bieneusi is the most common species responsible for human microsporidiasis. Genotyping of E. bieneusi based on sequence analysis of the ribosomal internal transcribed spacer (ITS) has recognized ~500 genotypes in humans and a great variety of other mammals and birds. Those genotypes vary in genetic or hereditary characteristics and form 11 genetic groups in phylogenetic analysis of the ITS nucleotide sequences. Some of genotypes in Group 1 (e.g., D, EbpC, and type IV) and Group 2 (e.g., BEB4, BEB6, I, and J) have broad host and geographic ranges, constituting a major risk for zoonotic or cross-species transmission. By contrast, host specificity seems common in Group 3 to Group 11 whose members appear well adapted to specific hosts and thus would have minimal or unknown effects on public health. Multilocus sequence typing using the ITS, three microsatellites MS1, MS3, and MS7, and one minisatellite MS4, and population genetic analysis of Group 1 isolates reveal the occurrence of clonality, potential host adaptation, and population differentiation of E. bieneusi in various hosts. Nonetheless, it is still highly desirable to explore novel genetic markers with enough polymorphisms, to type complex or unstructured E. bieneusi populations of various host species and geographic origins, notably those belonging to Group 2 to Group 11. Additional population genetic and comparative genomic data are needed to elucidate the actual extent of host specificity in E. bieneusi and its potential impacts on zoonotic or interspecies transmission of microsporidiasis.