载脂蛋白E亚型通过细胞外信号调节激酶途径影响轴突生长锥的生长

目的观察载脂蛋白E(apolipoprotein E,ApoE)各个亚型对神经元轴突生长锥的影响并探索其机制。方法体外培养小鼠皮质神经块,加入重组人类ApoE到神经块培养基中,免疫荧光和Western blot检测重组人类ApoE能否进入轴突及其生长锥;鬼丙环肽染色生长锥观察重组人类ApoE2、3、4对生长锥的影响;加入细胞外信号调节激酶(extracel-lular signal-regulated kinase,ERK)信号通路抑制剂,观察ERK信号通路是否参与ApoE亚型对生长锥的影响。结果免疫荧光和Western blot显示加入重组人类ApoE后的神经块轴突及其生长锥中ApoE为阳性;加入重组人类ApoE2、3、4组的荧光强度分别为(50.7±19.4)、(58.5±15.4)、(23.4±13.5),其中加入重组人类ApoE4的轴突生长锥荧光强度低于加入重组人类ApoE2、3的生长锥荧光强度(P<0.05);同时加入重组人类ApoE4和ERK信号通路抑制剂的实验组生长锥荧光强度为(32.8±13.2),而仅加入重组人类ApoE4的实验组生长锥荧光强度为(21.9±6.9),实验组生长锥荧光强度高于对照组(P<0.05)。结论重组人类ApoE能进入轴突及其生长锥,ApoE4能负性影响生长锥生长,阻断ERK信号途径能抑制ApoE4对生长锥的负面影响。     

改良贴壁组织块法与改良Ⅰ型胶原酶消法对成骨细胞增殖效果的比较研究

目的 比较改良贴壁组织块法与改良Ⅰ型胶原酶消法对SD大鼠颞下颌关节髁突软骨下骨成骨细胞的增殖效果.方法 取10只出生1 -3dSD乳鼠颞下颌关节髁突软骨下骨,将骨片平分成2份,分别采用改良贴壁组织块法和改良Ⅰ型胶原酶消法培养颞下颌关节髁突成骨细胞,并标记为Ⅰ组和Ⅱ组,通过细胞形态学观察、HE染色、ALP染色鉴定成骨细胞.取第4代成骨细胞进行细胞计数、MTT生长曲线及流式细胞仪周期分析,比较2种原代培养方法对成骨细胞增殖能力的异同.结果 （1）Ⅰ组和Ⅱ组培养的细胞经鉴定均为成骨细胞; （2） Ⅱ组成骨细胞总量约为Ⅰ组细胞总量的1.5倍; （3） Ⅱ组成骨细胞MTT生长曲线较Ⅰ组细胞提前2d进入对数生长期及达到生长高峰; （4）2组细胞在达到第4代时,细胞周期含量差异无统计学意义（P＞0.05）.结论 改良贴壁组织块法和改良Ⅰ型胶原酶消化法均适用于颞下颌关节软骨下骨成骨细胞的培养,但改良Ⅰ型胶原酶消化法可以更快速有效地获得大量成骨细胞.     

甘草不定根培养的研究

[目的]考察甘草不同外植体与外源性激素诱导不定根能力,为甘草不定根培养做基础研究。[方法]切取乌拉尔甘草无菌实生苗的根、叶、带芽茎段接种到含有不同外源性激素的不定根诱导培养基中,考察不同外植体诱导生根的能力,并在此基础上进行不定根增殖尝试。[结果]不同外植体以及不同类型和浓度的外源性生长素对不定根诱导能力不同。根和叶诱导不定根效果好于带芽茎段;萘乙酸(NAA)较吲哚丁酸(IBA)更有利于不定根的诱导,6-苄氨基嘌呤(BA)则抑制不定根的诱导;不同类型的外植体诱导生根的生长素(NAA)适宜浓度存在差异,其中根段在0.2~1 mg/L,真叶在3~5 mg/L,带芽茎段在1~3 mg/L诱导效果较好。[结论]甘草不同外植体在含有外源性生长素的培养基中均可以诱导生根,但细胞分裂素强烈的抑制生根;根段和真叶作为外植体是甘草不定根诱导较为理想的材料。     

Release of Atrial Natriuretic Factor from Intact and Hypophysectomized Rat Hypothalamic Expiants

Experiments examined release of atrial natriuretic factor (ANF), measured by radioimmunoassay, from acutely prepared explants of rat hypothalamus maintained in vitro by intra-arterial perfusion of artificial cerebrospinal fluid. Perfusates collected from intact preparations contained 6.1 ± 0.6 pg (mean ± SEM) of ANF per 2-min sample. Following a 3-min infusion of noradrenaline (60 μM), ANF release increased significantly (P<0.05) to 11.4±1.4 pg/sample. Media collected from hypophysectomized preparations showed the same basal ANF release (6.8 ± 0.9 pg/sample) as intact preparations, but demonstrated no significant increase after noradrenaline infusions. Levels of spontaneous ANF release were not appreciably affected by the absence of the paraventricular nuclei and/or the anteroventral third ventricle area.
Extracted material from the perfusate by reverse-phase high performance liquid chromatography revealed two main peaks of immunoreactive ANF: a small molecular weight form that coeluted with synthetic ANF (99–126) and with similar biological activity in a radioreceptor assay, and a larger molecular weight form with the same elution profile as the ANF (1–126) prohormone.
These observations indicate that the ANF released from perfused rat hypothalamic explants contains distinct contributions from the hypothalamus (sites undetermined) and the neurointermediate lobe.     

Localization of human breast tumors grafted in nude mice with a monoclonal antibody directed against a defined cell surface antigen of human mammary epithelial cells

Summary A mouse monoclonal antibody (BLMRL-HMFG-Mc5) prepared against a defined cell surface antigen of human mammary epithelial cells, non-penetrating glycoprotein (NPGP), was used in imaging and distribution studies in athymic nude mice grafted with human breast tumors. Forin vivo tissue distribution studies,¹²⁵I-labeled monoclonal antibody was injected into nude mice carrying simulated metastases of human tumors (breast and colon carcinomas). After 22–24 hr the amount of radioactivity per gram of tissue was 3–4 times higher in the breast tumor than in liver, brain, lung, muscle, or spleen. In contrast, colon carcinoma tissue, grafted and treated likewise, did not show higher accumulation of radioactivity relative to other tissues. At 4 days, the incorporation in breast tumors remained almost as high, while the circulating radioactive tracer and the incorporation in tissues other than breast had fallen significantly.In tumor imaging studies, breast tumor masses as small as 4 mm in diameter were clearly localized on a whole body scan using¹³¹I-labeled BLMRL-HMFG-Mc5 antibodies with a High-Purity germanium gamma camera. Normalization of¹³¹I-distribution to that of^99mTc-pertechnetate increased the specificity of this imaging methodology. The quantitative density of¹³¹I-label was 2–3 fold higher over the breast tumor than over comparable areas of the mouse. No positive localization images were obtained for similar implants of colon and lung carcinomas or melanomas after injections of¹³¹I-labeled BLMRL-HMFG-Mc5. Localization of human breast tumors in this model can be achieved with¹³¹I-labeled anti-breast epithelial monoclonal antibodies.Abbreviations NPGP non-penetrating glycoprotein - CEA carcinoembryonic antigen     

Reversal by retinoids of keratinization induced by benzo[alpha]pyrene in normal hamster tracheal explants: comparison with the assay involving organ culture of tracheas from vitamin A-deficient hamsters

In a defined culture system for hamster tracheal explants, the activity of 12 different retinoids was evaluated for reversal of keratinization induced by exposure to the carcinogen, benzo[a]pyrene (BP-HTOC assay). The effects of retinoids in this system were compared to those in a defined culture system for tracheal explants from vitamin A-deficient hamsters (standard-HTOC assay). In both assays, all-trans-retinoic acid (RA) and 13-cis-RA were the most active retinoids. For RA and 13-cis-RA, the values of ED₅₀ determined in the BP-HTOC bioassay were 4 × 10⁻¹² and 1 × 10⁻¹¹ M, respectively, whereas the corresponding values in the standard HTOC assay were 2 × 10⁻¹¹ and 3.3 × 10⁻¹⁰ M. For all 12 retinoids, the ED₅₀ values from the BP-HTOC were lower than those from the standard-HTOC assay, and there was also a statistically significant correlation between the rank-ordering of ED₅₀ values from the 2 assays. Among 3 N-(retinoyl)amino acids examined in both assays, N-(retinoyl)leucine was the most active, N-(retinoyl)phenylalanine the least active, and N-(retinoyl)alanine intermediate. Among a novel series of bifunctional retinoic acid analogues, the dicarboxyl derivative was the most active. On the basis of these results, the BP-HTOC assay appears to be one of the most sensitive assays for retinoids yet developed. This assay is an appropriate model for evaluating the chemopreventive potential of new retinoids in vitro.     

Analysis of the metabolic footprint and tissue metabolome of placental villous explants cultured at different oxygen tensions reveals novel redox biomarkers

Pre-eclampsia (PE) is a multi-system disorder of pregnancy hypothesised to arise from circulating factors derived from an unhealthy placenta. Some changes in placental phenotype seen in PE can be reproduced by culture in altered oxygen (O(2)) tension. Currently, these circulating factors are unidentified, partly due to the complexity of maternal plasma. Investigation of factors released from placental tissue provides a potential method to identify bioactive compounds. Experimental strategies to study compounds present in a biological system have expanded greatly in recent years. Metabolomics can detect and identify endogenous and secreted metabolites. We aimed to determine whether metabolites could be identified in placental cultures with acceptable experimental variability and to determine whether altered O(2) tension affects the composition of the placental metabolome. In this study we used gas-chromatography-mass spectroscopy to determine the presence of metabolites in conditioned culture medium (CCM) and tissue lysates of placental villous explants cultured in 1, 6 and 20% atmospheric O(2) for 96h. This experimental strategy had an intra-assay variation of 6.1-11.6%. Intra and inter-placental variability were 15.7-35.8% and 44.8-46.2% respectively. Metabolic differences were identified between samples cultured in 1, 6 and 20% O(2) in both CCM and tissue lysate. Differentially expressed metabolites included: 2-deoxyribose, threitol or erythritol and hexadecanoic acid. We conclude that metabolomic strategies offer a novel approach to investigate placental function. When conducted under carefully controlled conditions, with appropriate statistical analysis, metabolic differences can be identified in placental explants in response to altered O(2) tension. Metabolomics could be used to identify changes in conditions associated with placental pathology.     

A robust ex vivo model for evaluation of induction of apoptosis by rhTRAIL in combination with proteasome inhibitor MG132 in human premalignant cervical explants

Development of medical therapies for high-grade cervical intraepithelial neoplasia (CIN II/III) is hampered by the lack of CIN II/III cell lines. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis upon binding to its receptors DR4 or DR5. Proteasome inhibition by MG132 sensitized cervical cancer cell lines to recombinant human (rh)TRAIL. In our study, we aimed to develop an ex vivo model for CIN II/III and to investigate the apoptosis-inducing effect of rhTRAIL and/or MG132 in cervical explants from CIN II/III patients. A short-term ex vivo culture system was optimized for cervical biopsies, in which explants from normal cervix and CIN II/III lesions were exposed to either rhTRAIL (1 microg/ml), MG132 (5 microM) or the combination and compared to untreated explants for apoptosis induction. Normal cervix (n = 90) and CIN II/III (n = 24) explants could be reproducibly put in culture and kept viable for up to 7 days using a transwell membrane system. CIN II/III explants (n = 5) were highly sensitive to rhTRAIL plus MG132 (mean % apoptosis: 91 +/- 5) compared to normal cervix (n = 10) treated with rhTRAIL plus MG132 (mean % apoptosis: 24 +/- 10, p < 0.0001), while monotherapy with either rhTRAIL, MG132 or medium resulted in a mean % apoptosis <10 in both CIN II/III and normal cervix. Our ex vivo model system allows preclinical evaluation of (topical) medical therapies for CIN II/III. A strong synergistic apoptosis-inducing effect of the combination of rhTRAIL and MG132, especially in CIN II/III lesions indicates that rhTRAIL combined with proteasome inhibitors deserves exploration as medical treatment for CIN II/III.     

A procedure for measuring the contributions of intracellular and extracellular tyrosine pools to the rate of myocardial protein synthesis

To determine the relative contribution of intracellular and extracellular amino acid sources as precursors for protein synthesis, rat left atria were incubated in l-[³H]tyrosine at medium tyrosine concentrations ranging from 0.05 to 2.5 mm. Under conditions where protein synthesis was shown to be constant, tyrosine incorporation rates calculated from either the intracellular or extracellular tyrosine concentration. This behavior is consistent with a model which postulates that amino acids inside and outside the cell simultaneously supply a common precursor pool. A method is presented for determining the contributions of the intracellular and extracellular pools to the total protein synthesis rate. Analysis of tyrosine incorporation data according to this procedure yields a total incorporation rate of 1.14 ± 0.18 (s.e.m.) nmol tyrosine/mg protein/h, of which 61 ± 15% is attributed to a mixed intracellular source and 39 ± 7% to amino acids taken directly from the extracellular medium. During a 3-h incubation of the isolated atria protein synthesis and degradation rates were equal, and the oxygen consumption and ATP and creatine phosphate levels were similar to those of the beating, perfused heart.     

Culturing spinal cord explants in a collagen gel

A new method for culturing spinal cord slices or explants is presented which entails the use of a commercially available purified collagen, Vitrogen. Vitrogen provides a stable three-dimensional matrix for culturing spinal cord explants which is superior to the conventional method of applying explants to moist dishes coated with rat tail collagen. The use of Vitrogen facilitated the culturing of spinal cord explants which remain viable for over 2 1/2 weeks in culture, in addition to enhancing neuritic growth.