南京丰年虫卵黄颗粒在离体培养下重建细胞的电子显微镜观察

本文主要叙述南京丰年虫中雌中间性以卵黄颗粒为基础在离体培养下重建核和细胞的电镜观察。观察到的核结构和细胞结构的形式是多种多样的,这可能与中雌的发育情况,不同方式的重建过程、不同培养时间以及不同发展程度有关。同时也观察到卵黄颗粒结构的变化,核重建和细胞重建的某些过渡形态等。这些结果与中雌以卵黄颗粒为基础在离体培养下重建细胞的显微缩时电影和相差定位观察是可以相互对应的,而且与中雌生殖囊内长成卵母细胞以卵黄颗粒为基础重建细胞从原位切片进行电子显微镜观察的结果也是相类似的。     

离体培养的人胎儿胃成纤维细胞的转化

用紫外线和乙基亚硝基脲先后处理离体培养的正常人胎儿胃细胞,建立了能长久传代的GTS 8502细胞系。该细胞具有典型的转化细胞的特征:核外形不规则、核质比大,核仁增多;细胞表面微绒毛较多;生长指数和分裂指数高;能形成明显的转化灶;在软琼脂培养基中能长成克隆;细胞膜蛋白性质改变;染色体异倍化,异常染色体频繁出现,异种接种肿块诱发率高。电子显微镜观察和组织化学分析等多种结果表明GTS 8502源于成纤维细胞。正常人细胞的体外转化的成功率很低。两种致癌物的协同作用,可提高转化率,从而可能使人细胞的转化成为检测环境致癌物的有效手段。哺乳类细胞的体外转化,多以啮齿类细胞为材料。也有不少实验室从事正常人细胞的体外转化试验,但成功报道仍属罕见。本实验室用两种致癌物先后处理离体培养的人胎儿胃细胞,选出转化细胞后继续培养,迄今已传至80余代,细胞形态及其他生物学特性稳定。该细胞系波命名为GTS 8502。     

荧光碳点与CdTe量子点对毕赤酵母的毒性比较

以葡萄糖为碳源合成荧光碳点;以巯基琥珀酸为稳定剂,合成CdTe量子点。通过紫外吸收光谱和荧光发射光谱对二者的荧光性能进行表征。又以毕赤酵母作为指示生物,考察了延滞期的毕赤酵母分别与荧光碳点和量子点共培养后的生长曲线,并利用血球计数板法对培养至25h的毕赤酵母细胞进行计数。研究结果表明:荧光碳点对毕赤酵母的生长抑制作用不明显,即使在高浓度(14.4 mmol/L)下,也基本不影响毕赤酵母的生长;而CdTe量子点必须控制在很低的浓度(5.1μmol/L),才表现出低的细胞毒性。     

白血病患者骨髓单个核细胞裂解气相色谱分析

]本文用裂解气相色谱PGC对10例正常人、19例各种类型白血病人及12例其它血液病人骨髓单个核细胞冻样品进行了分析,结果表明正常人骨髓单个核细胞PGC图几乎完全一致;白血病人与正常人骨髓单个核细胞PGC图之间差别非常明显;同种类型的白血病骨髓单个核细胞PGC图有共同特征;不同类型白血病存在差异;其它血液病人骨髓单个核细胞PGC图和正常人及白血病人亦有区别。结果显示出PGC在白血病诊断方面的一定价值。     

聚二甲基硅氧烷-纸复合微流控芯片上的肝癌细胞三维培养

研发了一种聚二甲基硅氧烷-纸复合型微流控芯片用于肝癌细胞三维培养.芯片使用明胶处理硝酸纤维素薄膜作为细胞培养基底,以水凝胶网格作为三维培养支撑.结合微通道主动灌流与水凝胶中的被动扩散,模拟体内的流体运输形式实现细胞与外界物质交换.实验结果显示,芯片上的液滴生成以及细胞定位种植简便可靠.连续监测显示肝癌HcpG2细胞在水凝胶微球中增殖形成类似组织的三维结构.细胞增殖动力学分析以及生化检测结果显示了芯片三维培养与二维培养的差别.这种芯片三维细胞培养方法操作简便可靠,仿真度高,适合于肿瘤细胞研究.     

电离辐射致真核细胞损伤与死亡的动态研究——乳酸脱氢酶(LDH)反应直观指示胞核损伤与异型化的动力学过程

本文用光学镜细胞化学方法与电子显微镜技术,用100R或300R~(60)Co γ射线对大白鼠进行一次全身照射,照射后以不同时间对淋巴细胞核形态及LDH反应的观察。其结果,细胞核破损率和LDH从胞核中以不同形式泄逸的反应率,在时相曲线上表现出平行关系的规律性。细胞化学彩色照片直观地揭示了LDH在淋巴细胞核中的存在。酶反应在胞核内的行为和向外泄逸的现象反映了淋巴细胞核受电离辐射后,内含酶发生动态反应。而细胞核异型化的亚显微结构变化,验证了动态发展过程。我们以动力学概念对辐射致胞核损伤与死亡的原发过程提出解说。     

亲水性修饰的量子点的结构对荧光性能的影响研究

亲水性量子点的荧光性能是其作为生物检测探针的一个重要质量指标. 不同结构的量子点在亲水性修饰过程中, 其抵抗荧光淬灭的能力差异较大. 设计与制备具有不同结构和成分的核、核壳量子点, 再通过双亲性高分子对其亲水性改性, 利用荧光光谱监测亲水性修饰过程中的荧光性能变化来度量所合成量子点的光化学稳定性. 实验结果表明,在表面亲水性修饰过程中, 未包覆壳层的裸核量子点其抵抗荧光淬灭的能力最弱; 包覆壳层的核壳量子点, 其抵抗荧光淬灭的能力增强, 且壳层越多, 抵抗能力越强. 壳层的结构和成分直接影响核壳量子点抵抗荧光淬灭的能力, 具有合理晶格匹配的核壳量子点, 其抵抗荧光淬灭的能力较强. 另外, 通过优化设计与制备的核壳量子点经表面亲水性修饰后, 再偶联叶酸, 构建出特异性生物荧光探针, 对乳腺癌细胞进行靶向性标记后, 利用流式细胞仪进行细胞检测分析. 实验结果表明, 通过优化制备的核壳量子点, 亲水性修饰后仍具有很好的荧光性能, 偶联叶酸后具有较好的细胞靶向性.     

亚微米级聚苯乙烯/聚硅氧烷核壳粒子的制备及其应用

以乳液聚合制备的聚苯乙烯乳液为种子,加入甲基三甲氧基硅烷(MTMS)水解溶液进行缩聚反应,合成亚微米级聚苯乙烯/聚硅氧烷核壳粒子,并以此作为光散射剂添加至聚甲基丙烯酸甲酯(PMMA)树脂中,制备了光散射材料;考察了亚微米级核壳粒子添加在PMMA树脂中的分散性。结果表明:经过双螺杆剪切作用的挤出加工后,可以实现核壳粒子在PMMA树脂中的良好分散。核壳粒子可以大幅度提高PMMA的雾度,当聚苯乙烯/聚硅氧烷核壳粒子(NS82)的含量为1%时,制得的PMMA样片(厚度为2 mm)的雾度为89%,透光率为69%,有效光散射系数为61%。     

非细胞体系重建核生物学活性研究——重建核植入未受精卵能够诱导卵裂并进行细胞周期性变化

将HeLa细胞的中期染色体与成熟非洲爪蟾卵的提取物混合并加入ATP再生系统,经过一定时间的温育,可以自发进行非细胞体系(体外)细胞核构建。将体外重建核从核重建体系中分离出来,再移植到非洲爪蟾卵细胞中,能够诱导卵细胞进行卵裂。体外重建核本身可以随卵细胞进行细胞周期性变化。在卵细胞中有大量星体组装。     

非细胞体系重建核染色质组装、结构和DNA复制

用lambda DNA和蛙卵提取物进行非细胞体系核重建实验,结果显示,lambda DNA参与构成了重建核的染色质;染色质组装是逐步进行的;组装的染色质含有典型的核小体串珠状一级结构、300的螺线管和染色质的更高级结构;重建核能够复制DNA。     

Determination of polybrominated diphenyl ethers in freshwater fishes from a river polluted by e-wastes

Analytical method using mass spectrometric techniques was applied for the determination of polybrominated diphenyl ethers (PBDEs) in freshwater fishes. Fish samples collected from Nanyang River contaminated by the recycling electron-wastes (e-wastes) materials were prepared by using Soxhlet extraction and multiple-step column chromatographic clean-up. PBDEs were determined by gas chromatography (GC) coupled with ion trap mass spectrometry (for mono- to hepta-BDEs) and quadrupole mass spectrometry (for BDE-209). The method performance was evaluated with the recovery of ¹³C-labeled internal standards and with the analysis of certified reference biota. The obtained recoveries ranged from 75 to 125% with a relative standard deviation of lower than 10% for 16 PBDE congeners. The total PBDE (ΣPBDE) concentrations in fishes showed the following trend: grass carp < mud carp < crucian carp < silver carp < carp. ΣPBDE concentrations in the abdomen, back and tail muscles of carp ranged from 766, 458 and 530 ng/g w.w., and 53, 52, 45 ng/g w.w. in grass carp, respectively. The ΣPBDE concentrations in abdomen muscles were no significantly higher than in back and tail muscles in carp, crucian carp, grass carp and mud carp. PBDE congener concentrations in muscles correlated well with their lipid content. BDE-47 and BDE-28 were the most abundant congeners followed by BDE-17, BDE-15, BDE-66, BDE-154 and BDE-153 in fishes collected from Guiyu.     

Fibrin glue increases the cell survival and the transduced gene product secretion of the ceiling culture-derived adipocytes transplanted in mice

The development of clinically applicable scaffolds is important for the application of cell transplantation in various human diseases. The aims of this study are to evaluate fibrin glue in a novel protein replacement therapy using proliferative adipocytes and to develop a mouse model system to monitor the delivery of the transgene product into the blood and the fate of the transduced cells after transplantation. Proliferative adipocytes from mouse adipose tissue were transduced by a retroviral vector harboring the human lecithin-cholesterol acyltransferase (lcat) gene, and were subcutaneously transplanted into mice combined with fibrin glue. The lcat gene transduction efficiency and the subsequent secretion of the product in mouse adipocytes were enhanced using a protamine concentration of 500 μg/ml. Adipogenesis induction did not significantly affect the lcat gene-transduced cell survival after transplantation. Immunohistochemistry showed the ectopic enzyme production to persist for 28 days in the subcutaneously transplanted gene- transduced adipocytes. The increased viability of transplanted cells with fibrin glue was accompanied with the decrease in apoptotic cell death. The immunodetectable serum LCAT levels in mice implanted with the fibrin glue were comparable with those observed in mice implanted with Matrigel, indicating that the transplanted lcat gene-transduced adipocytes survived and functioned in the transplanted spaces with fibrin glue as well as with Matrigel for 28 days. Thus, this in vivo system using fibrin is expected to serve as a good model to further improve the transplanted cell/scaffold conditions for the stable and durable cell-based replacement of defective proteins in patients with LCAT deficiency.     

STUDY ON THE MODE OF PRIMARY CONTROL IN THE EGG OF GYNOGENETIC CRUCIAN CARP FOR INHIBITING THE DEVELOPMENT OF TWO TYPES OF SPERM NUCLEI

By comparing the different developmental characteristics of two types of sperm nucleiwhich were from gynogenetic fish (crucian carp) and amphimictic fishes (red carp, redgoldfish and sex--reversal red carp) respectively in the eggs of gynogenetic crucian carp, itwas preliminarily revealed that there existed selective inhibiting actions of the primarycontrol in the eggs of crucian carp for inhibiting the development of the two types of spermnuclei. To homologous sperms, the primary control showed weak effect, thus leading tothe decondensation of homologous sperm nuclei at different degrees in the eggs of cruciancarp. But to heterologous sperms, the primary control showed strong effects, resulting in thetotal inhibition of the development of heterologous sperm nuclei. Moreover, our experimentalresults also showed that the different developmental behavior of the two types of spermnuclei might have a great relationship to the changes or the sex ratio in the population ofgynogenetic crucian carp. The infiltr     

Analysis of Phospholipids in Crucian Carp(Carassius auratus) Muscle by Offline HPLC-MALDI-TOF MS

The interest in the analysis of phospholipids(PLs), especially phosphatidylcholine(PC), has been increasing due to the importance of them in biochemistry as well as in industry. A method was reported based on the offline combination of MALDI-TOF MS and normal-phase HPLC for analyzing PLs extracted from crucian carp. Total PLs of crucian carp were extracted and then separated by HPLC before the collected subfractions were analyzed by MALDI-TOF MS. The mass spectra obtained show peaks of H+, Na+ and K+ adduct...     

Cation binding to parvalbumin studied by 113Cd and 23Na NMR. Peak assignment of rabbit (pI 5.5) parvalbumin

Cation binding to three apoparvalbumins was studied by means of 113Cd NMR. The 3 parvalbumins that were investigated were carp pI 4.25, rabbit pI 5.5 and pike pI 5.0. The results showed that Cd2+ ions bind to the EF and CD sites of carp apoparvalbumin pI 4.25 with about the same affinity. For rabbit (pI 5.5) apoparvalbumin, Cd2+ binds preferentially to the EF site, while for pike (pI 5.0) apoparvalbumin, it was the CD site that exhibited somewhat higher affinity for Cd2+. The effect of Mn2+ on the 113Cd signals of rabbit parvalbumin was used to assign the 113Cd NMR signals to the EF and CD sites. The Mn2+ paramagnetic effect on rabbit and pike parvalbumins differed from that obtained for carp parvalbumin. This is in agreement with the assumption that the beta-lineage parvalbumins possess a third external site of higher affinity than the alpha-lineage parvalbumins. Furthermore, 23Na NMR was used to study Na+-Mg2+ competition in the native carp (pI 4.25) parvalbumin. The results showed that Na+ and Mg2+ compete for the same site, the third external site.     

在线凝胶渗透色谱-二维气相色谱/质谱法测定鲫鱼样品中的14种农药残留

建立了在线凝胶渗透色谱-二维气相色谱/质谱(GPC-MDGC/MS)测定鲫鱼样品中14种农药残留的方法。样品用环己烷/乙酸乙酯(1：1,v/v)提取两次,合并提取液。提取液冷冻、过膜后,经在线凝胶渗透色谱净化后直接进行MDGC/MS分析,通过中心切割的方式将农药组分选择性切割进入二维色谱柱进行进一步分离分析,采用内标法进行定量。实验结果表明：14种农药在0.01~0.9 mg/L范围内具有较好的线性关系,相关系数均大于0.99。14种农药在3个添加水平(0.01、0.05、0.1 mg/kg)的加标回收率为83.0%~112.9%,相对标准偏差为3.2%~12.0%。该方法前处理简单,实现了在线GPC和MDGC的有效结合,准确度好、精密度高,具有很好的推广性。     

Contralaterally transplanted human embryonic stem cell-derived neural precursor cells (ENStem-A) migrate and improve brain functions in stroke-damaged rats

The transplantation of neural precursor cells (NPCs) is known to be a promising approach to ameliorating behavioral deficits after stroke in a rodent model of middle cerebral artery occlusion (MCAo). Previous studies have shown that transplanted NPCs migrate toward the infarct region, survive and differentiate into mature neurons to some extent. However, the spatiotemporal dynamics of NPC migration following transplantation into stroke animals have yet to be elucidated. In this study, we investigated the fates of human embryonic stem cell (hESC)-derived NPCs (ENStem-A) for 8 weeks following transplantation into the side contralateral to the infarct region using 7.0T animal magnetic resonance imaging (MRI). T2- and T2*-weighted MRI analyses indicated that the migrating cells were clearly detectable at the infarct boundary zone by 1 week, and the intensity of the MRI signals robustly increased within 4 weeks after transplantation. Afterwards, the signals were slightly increased or unchanged. At 8 weeks, we performed Prussian blue staining and immunohistochemical staining using human-specific markers, and found that high percentages of transplanted cells migrated to the infarct boundary. Most of these cells were CXCR4-positive. We also observed that the migrating cells expressed markers for various stages of neural differentiation, including Nestin, Tuj1, NeuN, TH, DARPP-32 and SV38, indicating that the transplanted cells may partially contribute to the reconstruction of the damaged neural tissues after stroke. Interestingly, we found that the extent of gliosis (glial fibrillary acidic protein-positive cells) and apoptosis (TUNEL-positive cells) were significantly decreased in the cell-transplanted group, suggesting that hESC-NPCs have a positive role in reducing glia scar formation and cell death after stroke. No tumors formed in our study. We also performed various behavioral tests, including rotarod, stepping and modified neurological severity score tests, and found that the transplanted animals exhibited significant improvements in sensorimotor functions during the 8 weeks after transplantation. Taken together, these results strongly suggest that hESC-NPCs have the capacity to migrate to the infarct region, form neural tissues efficiently and contribute to behavioral recovery in a rodent model of ischemic stroke.     

Phenotypic Changes in Cyprinus carpiovar var. Jian Introduced by Sperm-Mediated Transgenesis of Rearranged Homologous DNA Fragments

Common carp, specifically the Jian variety (Cyprinus carpiovar var. Jian), is an important Chinese and global aquatic stock for commercial foodstuff. Homologous recombination of carp gene sequences has been widely used in population genetics to broadly screen for beneficial phenotypical variations, thus optimizing artificially engineered carp stocks with Jian variety and native stock varieties. Random rearrangement of homologous DNA fragments from parent specimens of C. carpiovar var. Jian were attained by digestion of genomic DNA with MspI followed by religation and redigestion with EcoR I to specifically rearrange homologous DNA fragments of myostatin and microsatellite genes. Based on known characteristics of myostatin gene function, growth pattern changes in resultant carp mutant varieties was expected. DNA fragments were introduced into metaphase-II oocytes, resulting in one to several dozen insertions of homologous fragments into the host genome by sperm-mediated transgenesis. Introduction of rearranged homologous DNA fragments often resulted in phenotypic changes in C. carpiovar var. Jian, including significant phenotypic changes linked to growth rate at 4 months.     

DARK ADAPTATION OF HORIZONTAL CELLS IN THE TELEOST FISH RETINA

The dark adaptation behaviors of rod-driven and cone-driven horizontal cells were exam-ined by analyzing their light responses recorded intracellularly in the intact, immobilizedcarp, and compared with that of the electroretinographic b--wave recorded simultaneously.Like the b--wave, the light responsiveness of rod horizontal cells increased gradually withtime in the dark and attained a steady level at 60 min. On the other hand, cone horizontalcells initially increased in light responsiveness in the first 10 min, but thereafter decreasedsteadily so that the response amplitudes of these cells to bright light flashes were only 3--5 mV.The results suggest that cone horizontal cells are strongly suppressed in prolonged darkness.