花背蟾蜍视网膜感光细胞分化的研究

经电镜及间接荧光抗体对花背蟾蜍视网膜感光细胞分化的研究,结果表明:视网膜感光细胞在视网膜中央首先分化,同一切片中位于中央的细胞分化程度最高,离它越远、分化越低。以中央细胞为准,胚胎发育到21期,感光细胞开始分化,22期外节出现,外节盘膜是直接以外凸折叠形成的。同时在视网膜杆锥层出现视蛋白特异性荧光,说明光敏蛋白的合成,感光细胞轴突与双极细胞建立突触联系。至此(22期末),感光细胞行使功能的结构基本形成。到25期,外节盘数达300多层,突触结构发育成熟。     

S波段高功率微波对眼组织结构和功能影响的实验研究

目的:研究5mW/cm2的S波段高功率微波(high-power microwave,HPM)对动物眼的组织结构和功能的影响,为我国高功率微波安全防护标准的制定提供参考。方法:S波段HPM模拟源以远场平面波(平均功率5mW/cm2)分三种不同的峰值功率(G1、G2、G3组)进行单次照射,并在照后7个时间点,通过眼底镜、裂隙灯观察、视网膜电图测定、组织病理学等方法研究HPM对动物的角膜、晶状体、视网膜等眼重要部位的结构与功能的影响。结果:HPM照射后角膜表面平滑无异常,未见混浊、粗糙等病理现象发生;晶状体在观察期内无肉眼可见的晶状体混浊,实质和晶状体后囊和玻璃体无异常;照射后即刻动物眼底动静脉和毛细血管稍有扩张充血,并于1天~3天后恢复正常表现;病理染色显示G1和G2组照后1天视网膜内外节排列稍有紊乱,照后3天~7天内恢复;G3组稍重,可见外核层细胞排列松散,于照后7天~14天后亦恢复正常;视网膜电图显示与照前相比,三组在照后1天的视网膜电图都有所降低,但3天后恢复正常,直至照后28天无明显改变。结论:5mW/cm2的S波段HPM单次照射对角膜、晶状体和玻璃体等部位不能造成损伤,视网膜在照后有轻微病理变化,但很快可以恢复。大鼠眼视功能在照后可出现暂时但可逆性的下降。此剂量范围的HPM照射对眼组织结构和功能无显著影响。     

模拟强日光短时间照射导致兔视网膜细胞凋亡的实验研究

目的:探讨模拟强日光短时间照射导致兔视网膜细胞凋亡的病理学改变的特征及其发生机制。方法:10只健康新西兰白兔按是否给予30000lx模拟日光照射30min随机均分为正常组和光照组;采用常规HE染色与TUNEL技术对光损伤后视网膜细胞的病理学改变进行观察。结果:光照组2d时视网膜细胞发生退行性变性,TUNEL标记结果显示光损伤后视网膜内、外核层出现大量阳性着色细胞。结论:模拟强日光短时间照射可导致视网膜细胞发生退行性变性,凋亡是视网膜内、外核层细胞退行性变性的重要发生机制。     

激光照射对角膜蛋白质二级结构影响的光谱分析

为了研究激光照射后角膜蛋白质化学组成的改变,探讨激光角膜损伤的发生机制,将日本大耳白兔随机分为正常对照组和激光损伤组,选取角膜上皮层和基质层作为研究对象,通过傅里叶变换红外光谱技术(Fou-rier transform infrared spectroscopy,FT-IR)对角膜组织酰胺I带中蛋白质二级结构各吸收峰进行定量分析,观察激光照射后蛋白质分子结构的改变情况。结果显示,激光照射后出现蛋白质二级结构吸收峰的位移和积分百分比的改变。激光照射可使角膜上皮层和基质层蛋白质构象发生改变,从而导致蛋白质结构稳定性下降和蛋白质生物功能的破坏。     

Nd:YAG激光致视网膜感光细胞损伤及相关基因的表达

目的：观察兔眼视网膜激光损伤后不同时间点感光细胞的改变以及相关基因的表达变化。方法：北京青紫蓝灰兔随机分为正常对照组和激光损伤组,在调Q倍频Nd：YAG激光损伤后不同时间采用超微结构观察、凝胶电泳、TUNEL染色、原位杂交(ISH)以及Northern blot等方法检测了感光细胞损伤特点并分析相关基因的表达改变。结果：电镜、核酸电泳和TUNEL结果表明视网膜感光细胞照射后出现明显的凋亡特征,在伤后不同时间,其分布和强度都有改变。而杂交结果表明c—fos和bax在照后表达增强,尤其以照后第1天为最,而bcl—2和p53在损伤后无明显改变。结论：大量感光细胞的凋亡是临床上低剂量倍频Nd：YAG激光致视网膜损伤、视功能下降的主要机制。c—fos和bax基因的表达可能介导了视网膜细胞凋亡的过程。     

脑红蛋白在家兔视网膜中的分布

目的探讨脑红蛋白(NGB)在家兔视网膜的分布特征。方法选择健康成年家兔5只,利用免疫组织化学染色SP法,观察NGB在家兔视网膜中的分布情况。结果 NGB在家兔视网膜的视神经纤维层、节细胞层、内网状层、外网状层、光感受器内节段和色素上皮层中有强阳性表达,在视网膜的内核层有弱阳性表达,在视网膜的外核层和光感受器外节段中未见有阳性表达,内界膜、外界膜和视神经中亦发现有NGB阳性表达。家兔视网膜NGB阳性表达的细胞类型主要有节细胞、双极细胞和光感受器细胞,其中节细胞的阳性表达定位于细胞质,胞核中未见表达。结论除外核层和光感受器外节段外,NGB在家兔视网膜其它各层中均有表达,提示NGB在维持视网膜氧代谢平衡中发挥着重要作用。     

420～750 nm超连续谱光源和532 nm激光致兔视网膜损伤修复

超连续谱(SC)光源是一种宽光谱、高亮度、眼睛危害大的新光源,有关它致视网膜损伤研究却未见报道。为了观察SC光源致视网膜损伤特点和修复过程,试验将波长420～750 nm的SC光源与532 nm激光进行比较,采用两者平行光入射,在0. 1 s照射时间,3 mm角膜光斑直径条件下,用略高于视网膜损伤阈值的功率照射青紫蓝灰兔视网膜;通过检眼镜和HE染色观察视网膜照后4 h,1、3、7、14 d损伤修复过程。SC光源和532 nm激光致视网膜的损伤在检眼镜下为灰色小圆斑。照后4 h出现视网膜外核层固缩深染,感光细胞外节与视网膜色素上皮层(RPE)粘连;随后RPE层色素增生、损伤的外核层细胞丢失,损伤进行性加重; 3 d后色素细胞开始向外核层和内核层迁移,胶质细胞填充损伤区域。由此可见,波长420～750 nm的SC光源主要损伤视网膜外核层和RPE层,后期色素颗粒和胶质细胞填充损伤区域。其视网膜损伤特点和修复过程与532 nm激光类似。     

视网膜神经网络的信息传递

可见光作用于脊椎动物视网膜时,感光细胞外段的视色素吸收光量子,经过一系列瞬时光化学反应,迅速将光能转换成电信息,并向视网膜内核层细胞(水平、双极和无足细胞)及视神经节细胞传递,后者以峰电位形式将信息传向中枢。最近十几年来,对视觉系统外周部分的信息转换和传递过程的研究十分活跃,尤其是Tomita用微电极记录视网膜细胞内反应技术的发展,以及Kaneko用荧光黄染色单个     

崇安髭蟾视网膜的组织结构

在光镜和电镜下观察了崇安髭蟾视网膜的组织结构，着重探讨感光细胞和神经节细胞的形态，计数及分布。结果表明，其视网膜组织结构符合脊椎动物的基本模式。视我膜总厚度为１４３μｍ。感光细胞总数约２５０只，几乎全是视杆，似视锥的光感受器不超过３％。神经节细胞总数２１万，从节细胞等密度图上看出在视盘背侧沿鼻颞独有一个高密区，即视条。比较神经节细胞的密度分布及光 镜和 感光细胞形态结构及其分布，认为发髭蟾视网膜的     

视神经是真正的神经吗?

神经由神经纤维组成,位于神经系统的周围部,神经纤维的外面包以神经内膜,许多条神经纤维集合成神经束,神经束外面包以神经束膜,许多神经束集合成神经,神经外面包以神经外膜. 视神经由视网膜内节细胞轴突构成.视网膜分为三层细胞.第一层为视杆细胞和视锥细胞,二层为双极细胞,第三层为节细胞.从其结构组成和功能上看,视杆细胞和视锥细胞只是光感受器细胞,双极细胞和节细胞才是真正的神经元.节细胞的轴突组成视神经. 从胚胎学角度看神经系统的发生:视神经来自脑泡,在胚胎发育第四周,前脑泡的腹     

Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media.

In this study, the quantitative fluctuation of cytoplasmic lipid droplets (LD) and cryotolerance were investigated in bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes developed in different culture systems using serum-free or serum-containing media. The serum-free cultures were grown using IVMD101 medium in conjunction with bovine cumulus/granulosa cell (BCGC) cocultures or IVD101 medium without BCGC cocultures, and the serum-containing cultures were grown in the presence of BCGC cocultures using HPM199 medium supplemented with 5% calf serum (HPM199 + CS). Large numbers of sudanophilic LD were present in the cytoplasm of bovine embryos from 2-cell to hatched blastocyst stages, and the number and size differed between the embryos cultured in serum-free and serum-supplemented media. In the embryos cultured in HPM199 + CS, large (2-6 microm in diameter) sudanophilic LD increased significantly from the morula to the blastocyst stages. Throughout the embryonic development, the embryos developed in serum-free cultures with and without BCGC cocultures had numerous sudanophilic LD, but most of these droplets were small (<2 microm in diameter) and large LD were less numerous than those embryos cultured in HPM199 + CS. Giant LD (>6 microm in diameter) were frequently observed in morulae and blastocysts (including early blastocysts) developed in HPM199 + CS. Electron microscopic observations demonstrated that large LD were abundant in the cytoplasm of trophoblast and embryonic (inner cell mass) cells of blastocysts cultured in HPM199 + CS. These large LD were identified as osmophilic LD, an indication that these lipid inclusions contained a significant proportion of unsaturated lipids. Many elongated mitochondria were found in embryos developed in IVMD101 and IVD101 at the morula and early blastocyst stages, whereas many of the mitochondria in the morulae developed in HPM199 + CS were of an immature form such as spherical or ovoid shape. The survival and hatching rates of embryos (morulae, early blastocysts, and blastocysts) produced in serum-free media (both IVMD101 and IVD101) after post-thaw culture were superior to those of embryos produced in serum-containing medium. These results showed that bovine embryos cultured in serum-containing medium abnormally accumulated cytoplasmic lipids into their cytoplasm and the excess accumulation of cytoplasmic LD in embryos may affect the cryotolerance of embryos.     

The NADPH- and iron-dependent lipid peroxidation in human placental microsomes

In pregnant females, placenta is the most important source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides is often linked to preeclampsia. In our study, we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) occurred. In the presence of Fe²⁺ ion, HPM produced small amounts of thiobarbituric acid-reactive substances (TBARS) – a final product of lipid peroxidation. NADPH caused a strong increase of iron stimulated TBARS formation. TBARS formation was inhibited by superoxide dismutase, butylated hydroxytoluene and α-tocopherol but not by mannitol or catalase. TBARS and superoxide radical production was inhibited in similar manner by cytochrome P450 inhibitors. The results obtained led us to the following conclusions: (1) microsomal lipid peroxidation next to mitochondrial lipid peroxidation may by an important source of lipid hydroperoxides in blood during pregnancy and (2) superoxide radical released by microsomal cytochrome P450 is an important factor in NADPH- and iron-dependent lipid peroxidation in HPM.     

Growth performance and histological intestinal alterations in piglets fed dietary raw and heated pigeon pea seed meal

Histological intestinal villus alterations were studied in piglets fed raw (PM) or heated (HPM) pigeon pea seed meal. The trypsin inhibition rate was 99.15% in PM and 54.31% HPM. The PM and HPM were added into the basal diet (crude protein; 176.3 g/kg, gross energy; 4.15 kcal/g, control) at 20% and 40% levels, respectively. The diets were formulated in order to adjust protein to 180 g/kg and gross energy to about 4.20 kcal/g. The feed intake was not different among groups. The daily body weight gain and feed efficiency tended to decrease with the increasing PM level, and they decreased significantly in the 40% PM group compared with the control group (P < 0.05). However, HPM groups showed a growth performance similar to the control. The villus height, cell area and cell mitosis tended to decrease with the increasing PM level, and they decreased significantly in the 40% PM group compared with the control group (P < 0.05). In HPM group, these villus height, cell area and cell mitosis were significantly higher than those of the 40% PM group (P < 0.05), and did not show a significant difference compared with the control. Compared with the duodenal villus surface of the control group, the PM groups had a smooth surface due to flat cells and the HPM group showed a rough surface due to protuberated cells. The current histological alterations of intestinal villi demonstrate that the villi might be atrophied in the piglets fed raw PM due to anti-nutritional factors, resulting in the decreased growth performance, and that heating PM might abolish such a harmful effect of the anti-nutritional factors on the villus function, resulting in a similar growth performance to the control. Raw PM could be incorporated under a level of 40%, but heated PM increases the incorporation rate up to the 40% level.     

Biochemical aspects of the visual process XXXII. Movement of sodium ions through bilayers composed of retinal and rod outer segment lipids

The leakage of Na⁺ from sonicated liposomes, composed of rod outer segment lipids, retinal lipids and a 4 : 1 phosphatidylcholine/phosphatidylserine mixture, has been studied. Both retinal and rod outer segment lipid liposomes lose Na⁺ faster than Ca²⁺ which indicates that the observed leakage occurs from closed liposomal structures.Liposomes from rod outer segment lipids are extremely leaky, losing sodium about 10 times as fast as retinal lipid liposomes and twice as fast as the phosphatidylcholine/phosphatidylserine liposomes.This high permeability of rod outer segment lipid liposomes, as compared to retinal lipid liposomes, is probably due to both the higher degree of unsaturation of the fatty acid chains and their lower cholesterol content. In the rod outer segment lipid extract 48% of the fatty acid chains consists of docosahexaenoic acid (C_22:6) against only 24% in retinal lipid extract. Rod outer segment lipids contain 4.0% cholesterol against 12.3% in retinal lipids.The sodium leakage from rod outer segment lipid liposomes is little affected by the presence of 5 mM calcium in the external dialysis medium, but with the two other types of liposomes significant decreases in permeability of about 20% are observed.The results are discussed in connection with the role of cations in visual excitation.     

Effects of cold shock and phospholipase A2 on intact boar spermatozoa and sperm head plasma membranes

Head plasma membranes (HPM) isolated from cryopreserved boar spermatozoa show an excessive fluidization, which might be involved in the loss of fertility. The current study assessed the ability of cold shock (5 degrees C) and phospholipase A2 (PA2) to duplicate these effects on membrane structure and to affect 45Ca2+ uptake and gross morphological characteristics of whole, fresh boar-sperm. The HPM from cold-shocked sperm showed a significantly greater rate of fluidization over time than did HPM from control sperm. Addition of PA2 (bee or snake venom, 0.1 or 10.0 ng/ml) to HPM from control sperm caused fluidization similar to cold shocking, but to a lesser degree (P less than 0.05). Cold-shocked intact sperm exhibited severe acrosomal disruption, loss of motility, and increased 45Ca2+ uptake relative to control sperm. Addition of PA2 (bee or snake venom, 0.1, 1.0., 10.0, and 1,000 ng/ml) to control sperm had no effect on gross morphology or motility while maintaining or increasing sperm extrusion of 45Ca2+. Therefore, although PA2 can, to some extent, duplicate the effects of cold shock on HPM molecular organization, its lipid hydrolytic action is insufficient to cause all the gross disruptions of severe thermal shock. Both PA2 and cold shock disrupted HPM structure, but only cold shock increased 45Ca2+ uptake, suggesting that cold shock may be increasing 45Ca2+ uptake in areas other than the head. Cold shock disrupts sperm on three levels; membrane molecular organization, intracellular Ca2+ regulation, and gross morphology/motility.     

穿膜肽-LacI HPM载体的构建及其DNA结合活性测定

目的：借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体（LacI HPM）高亲和力结合DNA的特性,构建新型基因转导载体。方法：PCR扩增LacI、Lacl基因前头肽序列、前头肽序列突变体、TAT序列的编码基因,构建前头肽序列突变体和TAT的原核表达载体,可溶性表达TAT-LacI HPM融合蛋白并纯化,在缇冲液中氧化获得TAT-LacI HPM二聚体并浓缩,PCR检测二聚体融合蛋白与质粒的体外结合能力。结果：获得了pET-28a（-4-）-LacI HPM及pET-28a（＋）-TAT-LacI HPM表达质粒,表达纯化并获得二聚化融合蛋白,体外结合实验确定TAT-Lac／HPM二聚体融合蛋白与检测质粒DNA具有特异的高亲和力结合活性。结论：构建了穿膜呔TAT-LacI HPM,为进一步研究其作为新型DNA转运载体的可行性奠定了基础。     

Metabolism of phosphatidylethanolamine in the frog retina

The synthesis and the turnover of phosphatidylethanolamine in frog retinal rod outer segments and microsomes were studied by monitoring the incorporation of five radioactive precursors: 32PO4, 33PO4 [3H]glycerol, [3H]serine, and [3H]ethanolamine. 1. Labeled serine was actively incorporated into phosphatidylethanolamine. The kinetics of the labeling patterns in both microsomes and rod outer segments was consistent with formation via decarboxylation of phosphatidylserine. 2. Ethanolamine was found to be an ineffective precursor of phosphatidylethanolamine, suggesting that the major pathway for phosphatidylethanolamine synthesis in the retina is via the decarboxylation reaction. 3. An active methylation of phosphatidylethanolamine to phosphatidylcholine was observed in both retinal microsomes and rod outer segments. 4. The kinetics of labeling of phosphatidylethanolamine in the rod outer segments was different for the various isotopic precursors, and was found to depend on the relative turnover times of the precursor pools. Glycerol was the only precursor that gave a true pulse of radioactivity. 5. The specific activity of phosphatidylethanolamine derived from labeled glycerol declined exponentially, demonstrating that the labeled lipid was diffusely distributed throughout the rod outer segments. The half-life of phosphatidylethanolamine in the rod outer segments was determined to be 18 days. Comparison of this value to the turnover time of rod outer segment integral proteins revealed that rod outer segment lipid is renewed at a faster rate than protein.     

Incorporating lipids into boar sperm decreases chilling sensitivity but not capacitation potential

Fresh boar sperm were incubated with small unilamellar liposomes composed of either the total lipids extracted from head plasma membranes (HPM) of fresh boar sperm or selected lipids (SL) of five defined phospholipids with specific acyl chains. To optimize fusion, liposomes with 2 mol% octadecyl rhodamine fluorophore in Beltsville Thawing Solution +/- 1 mM CaCl(2) were incubated at 35 degrees C with 1;ts 10(7) or 10(8) spermatozoa/ml and monitored over 60 min, using flow cytometry and fluorescence microscopy. The HPM fused to both sperm concentrations faster than SL but was equivalent by 30 min (10(8) sperm/ml) or 60 min (10(7) sperm/ml; 57.5 +/- 3% and 67.1 +/- 8% sperm fused to HPM and SL, respectively) +/- Ca(2+). Neither HPM nor SL affected onset of capacitation or spontaneous or ionophore-induced acrosome reactions at 0 or 3 h (chlortetracycline and fluorescein isothiocyanate-Pisum sativum agglutinin; n = 3). During cooling and after cryopreservation (n = 4 ejaculates), SL but not HPM significantly improved sperm motility and viability (Sybr14/propidium iodide staining) +/- 20% egg yolk, but egg yolk alone was more effective than SL alone. Liposomes of complex composition can fuse to boar sperm without harming in vitro capacitation or acrosome reaction and reduce sperm chilling sensitivity.     

穿膜肽介导的新型基因转运载体的功能研究

目的：借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体（LacI HPM）高亲和力结合DNA的特性,建立-种安全高效、无基因插入片段大小限制的基因转导系统。方法：在TAT-LacI HPM片段C端和N端分别添加GST标签,构建pET-28a（＋）-TAT-LacI HPM-GST和pGEX-GST-TAT-LacI HPM重组表达载体,可溶性表达TAT-LacI HPM-GST及GST-TAT-LacI HPM融合蛋白并纯化,获得TAT-LacI HPM二聚体,免疫荧光检测TAT-LacI HPM融合蛋白穿过HeLa细胞膜的情况,观察EGFP的表达,用免疫印迹检测TAT-LacI HPM融合蛋白介导质粒DNA进入细胞的能力。结果：表达、纯化并获得二聚体融合蛋白,体内实验表明其具有跨膜能力,能介导带有LacI结合序列的DNA质粒进入细胞,并在转染细胞里检测到了目的蛋白。结论：初步证实TAT-LacI HPM融合蛋白作为-种新型通用性非病毒DNA转运载体的可行性,为评价这种新型DNA疫苗载体在提高免疫效果方面的可行性奠定了前期实验基础。