微囊化猪视网膜色素上皮细胞移植治疗帕金森病的实验研究(英文)

目的研究微囊化后的猪视网膜色素上皮细胞(retinal pigment epithelial,RPE)对帕金森病大鼠模型的移植疗效。方法原代培养RPE 并传代,高效液相色谱法测定培养液上清中多巴胺(dopamine, DA)和高香草酸(homovanillic acid, HVA)的含量,ELISA法检测脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)和胶质细胞源性神经营养因子(glial-derived neurotrophic factor, GDNF)的含量。用高压静电成囊装置制备海藻酸钠-多聚赖氨酸-海藻酸钠微囊化细胞。6-羟基多巴胺(6-hydroxydopamine, 6-OHDA)毁损内侧前脑束 (medial fore-brain bundle,MFB)建立 SD 大鼠帕金森病模型。立体定向移植 RPE+ 微囊,检验旋转实验、免疫组化和脑内生化的变化。结果 RPE 培养上清液中DA、HVA、BDNF、GDNF 的含量稳定,微囊化后细胞长期存活,活性没有明显变化。6-OHDA毁损MFB建立大鼠帕金森病模型的成模率为83%。移植微囊化的RPE后有效率为33%。结论猪 RPE 体外培养生长旺盛,持续分泌 DA、BDNF 和 GNDF,微囊化不影响其分泌功能。RPE 移植对帕金森病大鼠有一定的治疗作用。     

Age, lens transmittance, and the possible effects of light on melatonin suppression

Recently it has been suggested that a previously undetected, rhodopsin-based, visual pigment, located in some retinal ganglion cells and having a peak sensitivity around 460 nm, may be responsible for light-induced melatonin suppression and, perhaps, maintenance of the circadian rhythm. Using data from the literature, it is shown that, as absorption in the crystalline lens for shorter visible wavelengths increases substantially with age, while the pupil diameter tends to decrease, the effective retinal exposure received under the same ambient lighting conditions by the pigment is almost 10 times lower in an old, as compared with a young, eye. Interestingly, replacement of the old crystalline lens by an intraocular implant restores the exposure to youthful levels. The possible effects of these changes with age on circadian rhythms are discussed.     

灯盏细辛黄酮类成分对压力诱导的视网膜神经节细胞凋亡的影响

目的考察灯盏细辛中黄酮类成分(总黄酮、灯盏花素)对体外高压诱导的视网膜神经节细胞(retinal gan-glion cells,RGCs)凋亡的影响,探讨其视神经保护作用的物质基础。方法采用胰酶消化法将24只出生2~3d的SD(Sprague-Dawley)乳鼠视网膜制成细胞悬液,接种于经多聚鸟氨酸(HA)和层粘连蛋白(LN)包被的盖玻片中。培养72h后,将覆有细胞的血盖片转入加压装置中,加入黄酮类成分,继续培养48h,采用Fas蛋白免疫组化染色法及原位末端脱氧核苷酸转移酶标记(Tunel-POD)法进行检测,每天观察细胞形态,同时对部分细胞行NSE染色检查。结果细胞生长良好,NSE染色表明,85%以上的细胞为RGCs。给药组的Fas蛋白阳性表达指数和凋亡指数均明显低于模型组(P<0.05~0.01)。结论黄酮类成分均能对抗压力诱导的RGCs凋亡,为灯盏细辛视神经保护的有效组分。     

枸杞多糖对氧化损伤大鼠晶状体上皮细胞凋亡的调控

目的 :研究枸杞多糖 (lyciumbarbarumpolysaccha rides,LBP)对实验性晶状体氧化损伤所致晶状体上皮细胞(lensepithelialcell,LEC)凋亡的影响。方法 :复制大鼠晶状体LEC凋亡模型 ,将 2 4只透明晶状体随机分为 4组 ,空白组、H2 O2 组、白内停组、枸杞多糖组。枸杞多糖组加入终浓度为1g/L的枸杞多糖共同孵育 2 4h ,采用TUNEL法检测LEC凋亡率 ,用透射电子显微镜观察LEC超微结构改变。结果H2 O2 组LEC凋亡率 (92 .0± 2 .5 5 )显著高于空白组 (3.5± 1.84 ) ,(t=6 2 .97,P <0 .0 1) ;枸杞多糖组LEC凋亡率 (16 .6±8.11)与白内停组比较 ,差异有非常显著性 (t=15 .5 0 ,P <0 .0 1)。透射电镜观察 ,H2 O2 组胞浆明显浓缩 ,核内染色质凝聚或成块 ,枸杞多糖组多数LEC形态改变轻微 ,表现为胞浆基质密度轻度升高 ,部分线粒体水肿 ,细胞间间隙增宽。结论 :枸杞多糖可明显抑制LEC凋亡 ,从祖国医药中寻求防治白内障的有效药物具有广阔前景。     

Immunobead and Density Gradient Purification of Paget Cells: In vitro Studies of Proliferation

Previous studies using primary monolayer cultures of epithelial cells from the involved epidermis of patients with mammary and extramammary Paget's disease investigated whether Paget cells proliferate as other malignant cells do. Although epithelial monolayers from the involved skin were maintained for approximately 45 days, no permanent cell lines were established. The proportion of carcinoembryonic antigen (CEA)-positive cells did not increase in the long-term cultures. Herein, we report studies of whether there is a real reduction of Paget cell numbers or if this is merely a decrease in the expression of CEA by the cells. Furthermore, we investigated whether Paget cells survive longer when cultured free from any potential inhibitory keratinocytes or other epidermal cells. Skin samples were obtained from one patient with mammary Paget's disease and three with extramammary Paget's disease; epidermal cells were cultured in vitro. An enrichment of Paget cells was carried out from the cultured epidermal cells by combining an anti-epithelial membrane antigen monoclonal antibody, binding to immunobeads, and density gradient centrifugation in Nycodenz. The separated cells were re-cultured in Keratinocyte-SFM serum-free media. The proportion of CEA-positive cells did not increase in the cultures, and the purified cells did not show any increase in survival times compared to the non-purified cultured cells. These results suggest that the decrease of CEA-positive cells noted during culture results from a decline in expression of CEA in the Paget cells. Paget cells in the involved epidermis do not proliferate significantly and thus differ from many other malignant cells.     

Treatment of pigment epithelial detachments due to age-related macular degeneration with intra-ocular C3F8 injection*

Purpose: To flatten pigment epithelial detachments (PED) cue to age-related macular degeneration in an attempt to visualize the underlying choroidal neovascularization by fluorescein angiography (FA) and reveal a treatment target. Methods: Nine patients with PED received intravitreal gas injections via the pars plana and postured face down. Fluorescein angiograms were obtained before and after gas injection. In two patients, retinal scotopic sensitivity was also measured. Results: Eight patients demonstrated change in the shape and size of the PED following gas injection. Four patients showed a better delineation of underlying structures on FA. Three patients had focal laser treatment to the newly visible choroidal neovascular complex, but this was successful in only one patient with flattening of the PED. Conclusion: Pigment epithelial detachments can be modified by intravitreal gas injection in some patients, but this treatment did not have a major impact on overall outcome or management.     

Distribution and spatial geometry of dopamine interplexiform cells in the retina. II. External arborizations in the adult rat and monkey

The morphology and distribution of dopaminergic interplexiform cells in adult rat and monkey retinas were analyzed to determine any correlation with the function of dopamine in the outer retinal layers. The retinas were processed as whole mounts for tyrosine hydroxylase immunohistochemistry. There was a network formed by the sclerally directed processes of interplexiform cells in the inner nuclear, outer plexiform, and outer nuclear layers running throughout the retina. Their density was higher in the superior retina than in the inferior retina of the rat and was especially high in the superior temporal quadrant. The external network in this quadrant was significantly less dense in the monkey than in the rat, as are the interplexiform cells. The somata of interplexiform and other dopaminergic cells were about the same size in both rats and monkeys. Computer-assisted reconstruction of external arborizations of individual cells showed that external processes lay very close to horizontal and photoreceptor cells and also to blood capillaries. Because they were long, thin, and highly varicose; branched at right angles; and often arose from an axon hillock, the external processes were identified as axons. Therefore, we define the dopaminergic interplexiform cells as multiaxonal neurons, with at least one outwardly directed axon that reaches the outer plexiform layer. The function of the network of external processes from the interplexiform dopaminergic cells is discussed in terms of modulating the release of dopamine to external layers.     

Effect of long-term sucralfate ingestion on antral and fundic epithelial proliferation in the rat

Sucralfate accelerates the healing of chronic gastric ulcers, but its mechanism is not well understood. We studied the effect of long-term administration of sucralfate on gastric epithelial proliferation in the rat by means of tritiated thymidine autoradiography. Rats were treated perorally with 500 mg/kg sucralfate once a day. After 28 days, rats were injected with tritiated thymidine 1µCi/g body weight and sacrificed 1 hr later. Autoradiographs from antral and fundic mucosae were prepared and a number of proliferative measurements were made. Long-term sucralfate administration produced an increase in tritiated thymidine labeling of epithelial cells and expansion of the proliferative zone in antral mucosa. These results indicate that long-term sucralfate ingestion stimulates gastric antral epithelial proliferation in the rat. In light of the fact that chronic gastric ulcers are usually located in the antral region in humans, this enhanced epithelial proliferation may contribute to the beneficial effect of sucralfate in accelerating the healing of gastric ulcers.     

Pattern-reversal electroretinograms and visual evoked potentials in branch retinal vein occlusion

We recorded pattern electroretinograms and visual evoked potentials in a group of selected patients with unilateral uncomplicated branch retinal vein occlusion. To document the effects of preexisting risk factors, patients were divided into three groups: diabetes mellitus, hypertension with hyperlipidemia and no systemic disease. The transient and steady-state pattern electroretinogram and visual evoked potential amplitudes were significantly reduced and visual evoked potential peak times were delayed relative to the fellow eyes and agematched normal subjects. There was a second amplitude reduction relative to the other patient groups in both the affected and fellow eyes of the diabetes mellitus group, which was indicative of an additive effect of diabetes mellitus.Abbreviations BRVO branch retinal vein occlusion     

Bronchial hyperresponsiveness, epithelial damage, and airway eosinophilia after single and repeated allergen exposure in a rat model of anhydride-induced asthma

Bronchial hyperresponsiveness (BHR) and damage of the epithelium, as well as eosinophilia in the airway wall, induced by trimellitic anhydride (TMA) in sensitized brown Norway rats were studied. Rats were challenged once or seven times with aerosol of TMA conjugated to rat serum albumin (TMA-RSA) 3 weeks after intradermal TMA sensitization. Airway responsiveness (-log PC₃₀₀ of acetylcholine i.v.) was measured 24 h after allergen challenge. Epithelial lesion and eosinophil infiltration in the airway walls were quantified under light microscopy, and TMA-specific IgE and IgG in serum were evaluated with ELISA. High levels of TMA-specific IgE and IgG were found in all rats in the sensitized groups compared to nonsensitized groups ( P < 0.001). Repeated allergen challenges of 0.03% TMA-RSA for 7 consecutive days enhanced the level of TMA-specific IgG, compared to single challenge ( P < 0.05). Single allergen challenge of 0.3% TMA-RSA had a nonsignificant tendency to produce BHR in sensitized rats compared to nonsensitized rats ( P =0.06). However, repeated allergen challenges (0.003% and 0.03% TMA-RSA for 7 consecutive days) produced significant BHR in sensitized rats ( P < 0.05). Furthermore, repeated low-dose (0.003%) TMA-RSA challenge produced more BHR than a 10 times higher single dose (0.03%) ( P < 0.05). Slight damage of the airway epithelium was seen in sensitized and repeat-challenged groups. However, bronchial eosinophilia was found in the sensitized and single-challenged groups, but not in nonsensitized nonchallenged, and sensitized repeat-challenged groups ( P < 0.005). We conclude that the brown Norway rat can be sensitized with TMA, and that repeated low-dose allergen challenges produce slight epithelial damage and BHR which is independent of ongoing eosinophilia in the airway wall.