临床肾脏穿刺超微病理诊断透射电镜制样的方法改进

肾脏疾病是临床常见的疾病之一,严重地威胁着人类的健康.随着肾脏穿刺组织病理活检技术的广泛开展,电镜超微病理诊断已成为肾脏疾病病理诊断中常规的检查方法,研究显示电镜观察对30%～50%的肾活检病例的诊断提供有价值的信息,电镜技术从过去的单纯应用于科研走向临床辅助检查,对肾脏疾病阐明病因、发病机制发挥了重要作用.同时,电镜对一些光镜认识不清的肾病能起到确定诊断、辅助诊断和分型的作用,对其他组织肿瘤的诊断、分型、识别组织来源、判断预后等,以及对肌肉的疾病、肝脏淤胆性疾病的诊断方面也都起到重要的作用.

Several studies have demonstrated that L-carnitine exhibits neuroprotective effects on injured sciatic nerve of rats with diabetes mellitus. It is hypothesized that L-carnitine exhibits neuro-protective effects on injured sciatic nerve of rats. Rat sciatic nerve was crush injured by a forceps and exhibited degenerative changes. After intragastric administration of 50 and 100 mg/kg L-carnitine for 30 days, axon area, myelin sheath area, axon diameter, myelin sheath diameter, and numerical density of the myelinated axons of injured sciatic nerve were similar to normal, and the function of injured sciatic nerve also improved signiifcantly. These ifndings suggest that L-carnitine exhibits neuroprotective effects on sciatic nerve crush injury in rats.

Objective. To observe the regulating effects of vascular endothelial growth factor (VEGF) and angiotensinⅡ (ANG II) on the frog's pericardium, lymphatic stomata and angiogenesis so as to reveal their effects and mechanism on the mesothelial permeability, lymphatic stoma regulation and myocardial hypertrophy. Methods. VEGF and ANGⅡ were injected into the frog's peritoneal cavity so as to examine the changes of the pericardial stromata by using transmission electron microscopy, scanning electron microscopy and computerized imaging analysis. Results. Scattered distributed pericardial stomata were found on the parietal pericardium of the frog with a few sinusoid mesothelial cells, whose blood supply was directly from the cardiac chambers flowing into the trabecular spaces of the myocardium (because there are no blood vessels in the myocardium of the frog). The average diameters of the pericardial stomata in VEGF and ANGⅡ groups were 1.50 μ m and 1.79 μ m respectively, which were much larger than those in the control group (0.72 μ m, P< 0.01); the average distribution densities of the stomata were 8.25/0.1 mm2 and 12.80/0.1 mm2 in VEGF and ANGⅡ groups, which were also much higher than those in the control group (3.57/0.1 mm2, P< 0.01); the sinusoid areas in VEGF and ANGⅡ groups were 2442.95 μ m2/0.1 mm2 and 2121.79 μ m2/0.1 mm2, which were larger than that in the control group (995.08 μ m2 /0.1 mm2 , P< 0.01); no angiogenesis was found in the frogs of the experimental groups. Conclusions. VEGF and ANGⅡ could strongly regulate the pericardial stomata by increasing their numbers and openings with larger diameters and higher distribution density. They could also increase the sinusoid areas with the result of the higher permeability of the pericardium, which clearly indicated that VEGF and ANGⅡ could speed up the material transfer of the pericardial cavity and play an important role in preventing myocardial interstitial edema. Yet there was no strong evidence to show the angiogenesis in the myocardium.

七个鸭种携带鸭乙型肝炎病毒的研究

鸭乙型肝炎病毒(DHBV)与人乙型肝炎病毒(HBV)同属嗜肝DNA病毒科(Hepadnavi-ridae).DHBV感染的鸭可作为研究HBV致病机理的动物模型.自Mason[1]与周翊钟[2]发现DHBV以来,研究者们已对DHBV进行了大量的研究[3～6].国外学者们相继报道在该地饲养多年的北京鸭中仍可检出DHBV,感染率为1～10%[1,7,8].在美国其它鸭种亦可感染DHBV,其感染率均低于北京鸭[9],而我国江苏省启东县的鸭群DHBV感染率可高达50%[9,10].

慢性病毒性肝炎的综合诊断与电镜检测的意义

慢性病毒性肝炎临床表现多样而复杂,诊断除根据临床资料外,尚须结合流行病学、病原学、免疫学以及肝活组织光镜、电镜和免疫组织化学检查等多种观察指标作综合判断.本文选择我院1979年6月～1983年4月间住院患者中上述各项检测较完整的70例,作回顾分析,着重对慢迁肝(CPH)和慢活肝(CAH)进行比较,现将结果报告如下.

输精管植入聚电解质避孕剂对人和猴子精子的生物物理学机制调控的时间依赖性影响

Aim: To determine the short and long-term morphological effects on sperm as induced by intra-vas alteration of pH and electrical charge. Methods: Desired biophysical influences were obtained by injection of reversible inhibition of sperm under guidance (RISUG) into the lumen of the vas deferens of human subjects and the monkey. RISUG is a polyelectrolyte hydrogel complex of styrene maleic anhydride (SMA) and dimethyl sulfoxide (DMSO) which generates an electrostatic charge and also lowers in a near space of pH domain. The morphology of sperm was examined by light microscopy, scanning and transmission electron microscopy. Human study enabled semen collection by masturbation as early as 3 h after injection and studies extended up to 6 months. In the monkey, on vas excision after RISUG implantation, sperm characteristics were examined in serial sections. Results: Semenology in clinical studies and histological data of the monkey showed a time-sequenced sperm plasma membrane, tail mitochondria and nuclear decondensation alterations in sperm structural components, which beared marked similarity to changes in the sperm head and tail during capacitation and entry into the ovum. Conclusion: The findings provide a means of causing such changes in the sperm that inhibit the fertilizing ability before the nucleus is affected. Therefore achieving non-obstructive vas-based contraception, without genotoxic or teratogenic effects caused by infertile sperm passing into the semen, is feasible.

PCR联合透射电镜诊断口唇初发单纯疱疹一例

患者男,66岁,因上唇水疱、糜烂伴疼痛7d就诊.取水疱及痂皮,利用单纯疱疹病毒(HSV)特异性引物做实时荧光PCR检测,发现HSV-1阳性、HSV-2阴性.取疱壁做透射电镜观察,见表皮细胞核内有空心型、颗粒型或致密型3种类型的核衣壳,细胞核外大量成熟病毒颗粒,鉴定为疱疹病毒.诊断:口唇单纯疱疹.PCR分子分型结合透射电镜是确诊初发单纯疱疹的有效方法.

银屑病患者皮肤屏障功能受损的研究

目的 探讨银屑病患者皮肤屏障功能受损的实验依据,以指导临床辅助治疗银屑病.方法 60例银屑病患者运用无创性皮肤生理功能测试仪检测皮损角质层含水量、皮脂含量及经表皮水分流失(TEWL).电镜观察皮损处细胞超微结构,同时运用免疫组化方法检测皮损处酸性神经酰胺酶的表达.结果 与正常皮肤比较,银屑病皮损角质层含水量降低(P＜0.01),TEWL值增加(P＜0.01),皮脂含量差异无统计学意义.电镜下,皮损颗粒层角质形成细胞中板层小体数量较正常对照组减少,分布紊乱,体积大小不等；免疫组化染色显示酸性神经酰胺酶表达明显减少.结论 银屑病皮肤屏障功能明显受损,因此,恢复皮肤屏障功能,加强保湿是银屑病重要的辅助治疗手段之一.

先天性肾积水输尿管狭窄段的病理及超微结构

本文对28例30只小儿肾盂输尿管狭窄段进行了光学显微镜及电子显微镜检查.提示狭窄段的主要变化是平滑肌肥厚和纤维组织增生,并见肌纤维排列和走行紊乱现象.在透视电镜下平滑肌细胞显示形态、大小、排列和密度有明显改变,肌细胞内可见线粒体,高尔基氏器、粗面内质网等,但缺少基底膜、肌丝及致密体,提示为合成型的平滑肌细胞.本文对先天性肾积水的病因、发病机制以及临床有关的几个问题加以讨论.

Microvascular endothelial cell (MVEC) is one of the target cells of TNFα (TNF effect). The dysfunction of MVEC induced by TNF plays an important role in some cardio-cerebral vascular diseases. ① Cell proliferation kinetic: Using flow cytometry, we found cell count ［(4.30±0.34)×107／L)］ in TNF group (4×105 U/L) was obviously less than that in control ［(5.23±0.50)×107／L, P＜0.01］. The cells of G1 phase were more than those of the control, while the cells of G2, S and M phase became less (P＜0.05). ② Coagulant and anticoagulant: 72 h after MVEC cultued in the media, the content of 6-keto-PGF1α (RIA) and activity of PAI decreased significantly in TNF (4×105 U/L) group (P＜0.01, vs control). The difference between TXB2 content and t-PA activity in groups was not significant (P＞0.05). ③ Adhesive molecule: The effect of low concentration TNF (＜4×105 U/L) on adhesion between cultured MVEC and leukocytes was not signficant, but when the concentration of TNF reached 8×105 U/L or more, 12 h after culture the adhesion rate between MVEC and neutrophil increased 30.8%±4.5%. If adding monoclonal antibody of ICAM-1/CD11 into media, the adhesion rate of leukocytes decreased significantly (from 31.2% to 63.4%). ④ NO: The level of nitrite in culture media (Griess reaction) was higher than that of control (P＜0.05) after pretreatment of TNF (2×106 U/L) for 6 h. Adding L-NMMA, Dexamethasone or Cycloheximide in media could block the increase of nitrite induced by TNF, while L-Arg could enhance it. The expression of iNOS mRNA of PMVEC increased significantly after treated with TNF (2×106 U/L) for 24 h (quantitative RT/PCR). Pretreatment with Dexamethasone or Cycloheximide could block the increase (P＜0.05). Meanwhile, the expression of eNOS mRNA decreased significantly compared with control, the decrease can be blocked by Cycloheximide but not by Dexamethasone. So that TNF can induce the expression of iNOS mRNA in PMVEC, but inhibited the expression of eNOS mRNA. NO production in PMVEC can be time-dependently induced by TNF. ⑤ Apoptosis: Adding high concentration TNF(＞1.2×106 U/L) in culture media for 12 h can induce apoptosis of PMVEC with electron microscopy, flow cytometry (PI/AnnexinV stain), TUNEL and DNA ladder eletrophoresis. Meanwhile, expression of apoptosis-associated gene Bcl-2 mRNA decreased and that of Fas mRNA increased (Northern blot). The expression of FADD, caspase 3 and caspase 8 enhanced too. So that signal of apoptosis induced by TNF may be transmitted following the cascade of Fas→FADD→caspase8→caspase3. In conclusion, it should be paid attention to metabolism inability and dysfunction induced by TNF which can not found easyily using light microscope. High concentration TNF can induce apoptosis of endothelial cells regulated by apoptosis-associated genes. The changes mentioned above are common pathway in pathogenesis of some diseases related to TNF.

AIM:To investigate the effects of human induced pluripotent stem cell-derived exosomes (hiPSC-exo) on cell viability, capillary-like structure formation , and senescence in endothelial cells exposed to high glucose .METHODS: Exosomes were isolated from the conditional medium of hiPSCs and confirmed by transmission electron microscopy , nanoparticle tracking analysis , and Western blot analysis using Alix and CD63 as markers.hiPSC-exo were labeled with PKH26 for tracking.Cultured HUVECs were treated with high glucose (33 mmol/L) with or without hiPSC-exo (20 mg/L) for 48 h, and cell viability, capillary tube formation, and senescence were assessed .RESULTS:hiPSC-exo showed a typical cup shape and could be taken up by human umbilical vascular endothelial cells (HUVECs) in a concentration-dependent manner.When exposed to high glucose, viability and tube formation in HUVECs was signifi-cantly reduced, whereas the proportion of senescent cells was higher compared to that in control HUVECs (P<0.01).Furthermore, hiPSC-exo restored cell viability and capillary-like structure formation , and reduced senescence in HUVECs exposed to high glucose (P<0.01).However, hiPSC-exo had minimal effects on normal HUVECs.Therefore, stem cell-derived exosomes can promote cell proliferation, enhance capillary-like structure formation , and reduce senescence in endothelial cells exposed to high glucose . CONCLUSION:Our study highlights the role of exosomes derived from hiPSC and may provide a new strategy for maintaining vascular health, preventing vascular aging , and avoiding pathological vascular remodeling that occurs in many diseases .

AIM:Early calcification of atherosclerotic plaques are colocalized with macrophage and high mobility group box 1 (HMGB1), a cytokine associated with biomineralizing process under physiological and pathological conditions .Our study aims to evaluate whether HMGB1 induces ectopic mineralization via promoting the secretion of matrix vesicles ( MVs) from macrophages .METHODS:HMGB1 was added to the medium of macrophages , the secretion of MVs in the supernatant was tested by flow cytometry analysis .The mineral deposition in calcifying medium was detected by Alizarin Red staining and von Kossa staining .Transmission electron microscopy showed the formation of hydroxyapatite crystals in MVs .Then we subcutaneous injection into mice with MVs to induce regional minera-lization.RESULTS:HMGB1 significantly promoted secretion of MVs from macrophages as raveled by flow cytometry analysis .TNAP activity, considered as a marker of MVs maturation , was higher in HMGB1-induced MVs compared to the control-MVs.HMGB1-MVs also led to mineral deposition in an in vitro MVs-collagen mineralization model .Subcutaneous injection into mice with MVs derived from HMGB1-treated cells showed a greater potential to initiate regional mineralization .Mechanistic experiments revealed that HMGB 1 activated neutral sphingomyelinase 2 ( nSMase2 ) that involved the receptor for advanced glycation end products ( RAGE ) and p38 MAPK (upstream of nSMase2).Inhibition of nSMase2 with GW4869 or p38 MAPK with SB-239063 prevented MVs secretion and min-eral deposition .CONCLUSIONS: HMGB1 induces MVs secretion from macrophages at least in part , via the RAGE/p38 MAPK/nSMase2 signaling pathway .Our findings thus reveal a novel mechanism by which HMGB 1 may participated in the early calcification of atherosclerotic plaques .