消痰化瘀利窍方对慢性间歇性低氧小鼠认知障碍的改善作用*

目的: 探讨消痰化瘀利窍方对慢性间歇性低氧小鼠认知障碍的改善作用。方法: 48只雄性C57小鼠随机分为4组(n=12),常氧对照组(Normoxia),慢性间歇性低氧组(CIH)、慢性间歇性低氧中药干预组(Formula+CIH)、中药对照组(Formula)。Normoxia和Formula组暴露于常氧环境,CIH与Formula+CIH组暴露于间歇性低氧环境(低氧舱中前1.5 min充入氮气使舱内氧浓度降至9%,后1.5 min充入氧气使氧浓度恢复至21%,3 min/循环,每天上舱8 h,共35 d)。其中,Formula +CIH与Formula组于每日灌胃中药水煎液灌胃(26.8 g/kg),同时CIH组与Normoxia组灌胃给予同体积生理盐水。实验在26～35 d连续应用水迷宫观测各组小鼠的学习和记忆能力,35 d造模结束后,首先进行Y-迷宫实验,麻醉后断头取脑,分离海马组织。应用尼氏染色和电镜观察海马神经元的形态学改变,通过Western blot检测海马神经元synapsin和PSD-95的表达水平。结果: 与Normoxia组相比,CIH组小鼠水迷宫和Y-迷宫的成绩显著下降(P＜0.01,P＜0.01),海马神经元尼氏小体的数量和突触后致密物质的厚度均减少,PSD-95蛋白表达下调(P＜0.01),而synapsin表达无明显改变。与CIH组小鼠比较,消痰化瘀利窍方干预可显著提高小鼠水迷宫和Y-迷宫的成绩(P＜0.01),增加海马神经元尼氏小体的数量和突触后致密物质的厚度,上调PSD-95蛋白表达水平(P＜0.01)。结论: 消痰化瘀利窍方可改善由CIH诱导的突触后致密区的结构和功能受损,进而对认知功能障碍起到保护作用。     

Azure a-Schiff, Alcian Blue, HIO4-Schiff, Naphthol Yellow S—A Sequential Staining Method for Paraffin Sections

Paraffin sections from tissue fixed 4-12 hr in 10% formalin containing 0.5% cetyl pyridinium chloride, and washed 2 hr, were stained as follows: (1) Hydrolyze in 5 N HCl at room temperature for 8.5-9 min, or use standard Feulgen hydrolysis at 60 C. (2) Stain in azure A-Schiff, 0.5% in bisulfite bleach (1 N HCl, 5; 10% Na₂S₂O₅, 5; and distilled water 90—parts by volume) for 10 min. (3) Place in bisulfite bleach 2 changes, 2 min each; wash in water, 1-2 min. (4) Stain in Alcian blue (0.1% in 0.01 2V HCl, pH 2.0) for 10 min. (5) Place in 0.01 N HCl for 2-3 min; wash in water for 1-2 min. (6) Oxidize in 0.5% HIO₄ for 5 min; wash in water, 1-2 min. (7) Stain in Schiff's leucofuchsiu, 10 min. (8) Treat with bisulfite bleach as in step 3; wash in running water, 10 min. (9) Stain in naphthol yellow S (0.01% in 1% acetic acid) for 1-2 min. (10) Place in 1% acetic acid for 2 min, dehydrate in tertiary butanol, clear and cover. Result: DNA is deep blue; acidic mucins are light blue; neutral polysaccharides, red to magenta; and proteins, yellow. Proper timing of the hydrolysis for the Feulgen reaction is the most critical step. Overhydrolysis results in green nuclei (staining by naphthol yellow S) whereas purplish nuclei are the results of insufficient hydrolysis.     

Expression of the A12 form of acetylcholinesterase by developing avian leg muscle cells in vivo and during differentiation in primary cell cultures

The accumulation of the molecular forms of acetylcholinesterase (AChE) has been studied in leg muscles during embryonic chick development and in cell cultures initiated with myoblasts obtained from embryos at different stages of development. The collagen-tailed, A₁₂ form appears in leg muscles as soon as day 5 in ovo. An early excision of the lumbar zone of the neural tube at day 2 1/2 in ovo severely delayed the morphological development. In leg muscles dissected at day 12 in ovo from operated embryos, we found that the total amount of AChE activity and particularly the proportion of A₁₂ form were dramatically reduced.

Muscle cells were grown in vitro in a medium supplemented with fetal calf serum. In these conditions, chick muscle cells unequivocally synthesize the A₁₂ form when they originated from muscles which accumulated this form in vivo. In contrast, myoblasts obtained from 5-day old embryo leg muscles did not produce the A₁₂ form either in aneural cultures or in the presence of nerve cells. In relation with previous observations concerning chick myogenesis, we discuss the possibility that this difference reflects the existence of two types of myoblasts. This hypothesis would also explain the results of cocultures performed with nerve cells and normal or demedullated leg muscle myoblasts.     

Adaptation of the Millon, Sudan Black B, and Periodic Acid-Schiff Technics for Block Staining of Insect Tissues

Spermatophores and reproductive systems of the beetle, Lytta nuttalli Say, fixed in Bouin's aqueous picroformol or buffered 10% neutral formol were stained in toto by the Millon, Sudan black B and periodic acid-Schiff reactions as follows. Millon: after excess fixative is removed in 70% ethanol, specimens are brought to water, stained in Millon's reagent at 60 C for 1 hr, rinsed in 2% aqueous nitric acid at 40-50 C, dehydrated rapidly, cleared, embedded and sectioned as usual. Sudan black B: specimens are taken to absolute ethanol, stained in a saturated solution of Sudan black B in absolute ethanol at room temperature for 24-48 hr, rinsed and cleared in xylene, embedded and sectioned. PAS: specimens are brought to water, oxidized in 0.5 aqueous HIO₄ at 37 C for 30 min, washed in 2 changes of water, stained in Schiif reagent at room temperature for 1 hr, rinsed in 3 changes of 0.5% aqueous potassium metabisulfite, washed in running water for 10-15 min, dehydrated, cleared, embedded and sectioned. All 3 methods produced their characteristic staining in specimens up to 3 mm thick     

MCC950对脑出血大鼠神经损伤的作用*

目的: 研究NLRP3炎性小体抑制剂MCC950对脑出血(ICH)大鼠神经损伤的作用。方法: 72只SD大鼠随机分为3组(n=24):sham组、ICH组和MCC950组。ICH组和MCC950组采用自体非抗凝血注射方法建立大鼠脑出血模型后,给予MCC950组大鼠腹腔注射MCC950 10 mg/kg(2 mg/ml),连续给药3 d。模型建立72 h后,进行前肢放置实验、转角实验和mNSS评分,观察ICH大鼠神经功能情况;新鲜脑组织切片,观察血肿体积变化情况;HE染色,观察脑组织病理学改变情况;干湿比重法,观察脑组织水肿变化情况;FJC染色,观察神经元退化的情况;TUNEL染色,观察神经元凋亡情况;Western blot,观察NLRP3、ASC、caspase-1、IL-1β、IL-18、GSDMD蛋白表达及激活水平的情况。结果: 与sham组比较,ICH组大鼠左前肢放置成功百分比和左侧转身百分比显著下降(P＜0.01,P＜0.05),mNSS评分显著升高(P＜0.01),右侧脑内血肿体积显著增大,血肿周围脑组织中小胶质细胞数量增加,神经元数量减少,神经细胞肿胀,排布不均,部分细胞固缩性坏死,染色加深,右侧基底部含水量显著增多(P＜0.05),血肿周围脑组织中FJC阳性和TUNEL阳性细胞数量显著增加(P＜0.05),NLRP3、ASC、caspase-1、pro-caspase-1、caspase-1/pro-caspase-1比值、GSDMD-N、GSDMD、GSDMD-N/GSDMD比值、IL-1β和IL-18水平显著升高(P＜0.01,P＜0.05)。与ICH组比较,MCC950组大鼠左前肢放置成功百分比和左侧转身百分比显著升高(P＜0.05),mNSS评分显著降低(P＜0.01),右侧脑内血肿体积显著减小,血肿周围脑组织中神经细胞肿胀显著减轻,固缩坏死细胞数量减少,右侧基底含水量显著减少(P＜0.05),血肿周围脑组织中FJC阳性和TUNEL阳性细胞数量显著减少(P＜ 0.05),NLRP3、ASC、caspase-1、pro-caspase-1、caspase-1/pro-caspase-1比值、GSDMD-N、GSDMD、GSDMD-N/GSDMD比值、IL-1β和IL-18水平显著降低(P＜0.05)。结论: MCC950可以通过抑制NLRP3炎性小体介导的炎症反应和细胞焦亡改善ICH后的神经损伤。     

柔木丹改善小鼠肝纤维化的TGF-β1/Smad4信号通路机制*

目的:探讨柔木丹(RMD)对改善CCl₄诱导的小鼠肝纤维化的TGF-β1/果蝇抗生物皮肤生长因子蛋白家族4号因子(Smad4)信号通路机制。方法:雄性BALB/c小鼠随机分为空白对照组、模型组、RMD治疗组(n=11)。腹腔注射CCl₄诱导小鼠肝纤维化模型,模型及RMD治疗组小鼠腹腔注射20 % CCl₄(CCl₄∶橄榄油=1∶4),注射量为2.5 ml/kg,空白对照组以同样方法注射等量橄榄油,每周2次;第2周起调整模型及RMD治疗组小鼠CCl₄腹腔注射量为5 ml/kg(空白对照组注射等量橄榄油),每周2次。成模后,RMD治疗组小鼠使用RMD灌胃给药(6.2 g/(kg·d);空白对照组、模型组使用等量的水灌胃),模型及RMD治疗组小鼠继续腹腔注射20 % CCl₄,注射量为1.5 ml/kg(空白对照组注射等量橄榄油),每周1次,持续3 周。采取各组小鼠血清样本检测谷丙转氨酶(ALT)、谷草转氨酶(AST)活性;采取各组小鼠肝组织样本使用HE、Masson、原位杂交、免疫组织化学染色、Western blot、Q-PCR等方法进行检测。结果:与正常组相比,CCl₄造模5 周后,模型组小鼠肝脏纤维化病理特征明显。与模型组相比,RMD治疗3 周,治疗组小鼠肝组织病理学改变减轻,小鼠的肝脏指数(P＜0.01)、血清中的ALT(P＜ 0.01)、AST(P＜0.01)活性、肝组织中羟脯氨酸的含量(P＜0.05)均降低;Ⅰ型胶原(Collagen Ⅰ,P＜0.01)、Ⅲ型胶原(Collagen Ⅲ,P＜0.01)表达减少,胶原沉积减少;肝组织中TGF-β1(P＜0.05)和α-SMA(P＜0.05)表达均降低;肝组织中Smad4阳性表达区域缩小、表达强度降低。结论:RMD通过抑制TGF-β1/Smad4通路信号转导,减少胶原沉积,进而发挥抗小鼠肝纤维化的作用。     

蒙药绍沙-7味丸防治大鼠心肌缺血/再灌注损伤的作用

目的:探讨蒙药绍沙-7味丸对心肌缺血/再灌注损伤大鼠的防治作用及机制.方法:60只大鼠随机分成6组:假手术组、模型组、蒙药绍沙-7味丸低、中、高剂量组以及阳性药对照组,每组10只;蒙药绍沙-7味丸低、中、高剂量组分别灌胃0.4 g/kg、0.8 g/kg、1.6 g/kg蒙药绍沙-7味丸,阳性药对照组灌胃0.3 g/k...     

辣木叶对糖尿病大鼠认知功能及海马神经细胞凋亡的影响*

目的:探讨辣木叶对链脲佐菌素(STZ)诱导的糖尿病模型大鼠认知功能障碍及海马神经细胞凋亡的影响。方法:选取健康SD雄性大鼠50只,随机选取10只作为对照组,40只大鼠给予腹腔注射25 mg/kg STZ构建糖尿病大鼠模型。40只大鼠随机分为模型组、辣木高、中、低剂量组,分别每日灌胃给药2.0 g/kg、4.0 g/kg、8.0 g/kg辣木叶,对照组和模型组每日灌胃给药等量生理盐水,1次/日,持续给药8周。Morris水迷宫实验评价大鼠学习记忆能力;HE染色及免疫组化染色对大鼠海马神经元切片进行染色,观察各组大鼠海马神经元病理改变的情况及Bax、caspase-3及Bcl-2蛋白表达情况;酶联免疫法(ELISA)检测大鼠海马组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)。结果:与对照组相比,模型组大鼠血糖值明显提高(P＜0.01),血胰岛素水平明显降低(P＜0.05);与模型组相比,辣木组血糖值显著降低(P＜0.05,P＜0.01),辣木中、高剂量组血胰岛素水平显著升高(P＜0.05,P＜0.01)。不同剂量组辣木组间比较,给药后3组大鼠FBG及INS无统计学差异(P＞0.05);Morris水迷宫实验的第4-5日,与模型组相比较,辣木各剂量组潜伏期均明显缩短(P＜0.05);辣木不同剂量组目标象限停留时间明显延长(P＜0.05)。炎症因子变化结果,与模型组相比较,辣木低、中、高剂量组TNF-α、IL-6含量及蛋白表达水平显著降低(P＜0.05)。HE染色及免疫组化染色结果显示,与模型组相比较,辣木中剂量组中阳性表达,呈棕黄色,细颗粒状。辣木叶中剂量组(53.21±7.19)能显著减少神经元细胞凋亡的数目(P＜0.01);辣木组中剂量组大鼠海马组织中Bax、caspase-3的蛋白表达及Bax/Bcl-2比例显著降低(P＜0.05)。结论:辣木叶能改善糖尿病大鼠认知功能障碍其作用机制可能与调节Bax、Bcl-2、caspase-3的蛋白表达,降低炎症因子TNF-α及IL-6含量相关。     

有氧运动改善自发性高血压大鼠肾纤维化的作用*

目的: 研究有氧运动训练对自发性高血压大鼠(SHR)肾脏纤维化影响, 探讨有氧运动对高血压肾损害的保护作用。方法: 8周龄雄性SHR和同龄Wistar京都大鼠(WKY)随机分为4组(n=6):安静WKY对照组(WKY-S)、安静SHR对照组(SHR-S)、低强度运动组(SHR-L)和中强度运动组(SHR-M)。SHR-L组、SHR-M组分别以14 m/min(最大有氧速度的35%)、20 m/min(最大有氧速度的50%)在0°坡度的运动跑步机上跑步,共运动14周,每周5次,每次60 min,WKY-S和SHR-S组安静饲养。14周后,运动训练结束72 h后检测大鼠血压;之后取血和肾脏检测血清肌酐SCr和尿素氮BUN含量,苏木精与伊红(HE)染色观察肾组织形态,Masson染色观察肾组织胶原沉积情况,计算肾脏胶原容积分数(CVF),检测肾脏 AngⅡ、AT1R、TGF-β、α-SMA、CTGF蛋白表达。结果: 与WKY-S组相比,SHR-S组的血压和血清SCr、BUN含量、肾脏CVF水平和AngⅡ、AT1R、TGF-β、α-SMA、CTGF蛋白表达均明显升高(P＜0.05);与SHR-S组相比,SHR-L组和SHR-M组血压和血清SCr、BUN含量、肾脏CVF水平和AngⅡ、AT1R、TGF-β、α-SMA、CTGF蛋白表达均明显下降(P＜0.05)且SHR-M组下降趋势更明显(P＜0.05)。结论: 有氧运动可通过抑制肾脏AngⅡ-AT1R-TGF-β通路,改善自发性高血压大鼠的肾纤维化与肾功能。     

Staining Mitochondria in Saccharomyces cerevisiae

After testing various procedures (amidoblack 10B, acid fuchsin-methyl blue, Luxol fast blue MBS-phloxine, toluidine blue O, Jams green B and pinacyanol), three stains can be recommended for staining both types of mitochondria (globose and threadlike) in the cells of Saccharomyces cerevisiae: (1) 0.1% solution of amidoblack 10B in citrate buffer (pH 3.0) for 10 min; (2) 0.01% solution of toluidine blue O in phosphate buffer (pH 6.0) for 30 min; (3) 0.01% solution of Janus green B in distilled water (pH 5.6) for 30 min. The latter stain is most specific because its staining reaction depends upon the action of the mitochondrial enzyme cytochrome c oxidase. Yet, low concentrations and short incubation periods must be applied to avoid poisoning of the cell metabolism.     

Book Review

LEGGETT, W. F. Ancient and Medieval Dyes. 5 × 8 in. 96 pp. Cloth. Chemical Publishing Co., Brooklyn, N. Y. 1944. $2.25.

Microtechnic In General. McCARTNEY, J. E. A new immersion oil “polyric”. J. Path. & Bact., 56, 265-6. 1944.

NICKERSON, MARK. A dry ice freezing unit for rotary microtomes. Science, 100, 177-8. 1944.

Dyes And Thedx Biological Uses. BERGEIM, FRANK H., and BRAKER, WILLIAM. Homosulfanilamides. J. Amer. Chem. Soc., 66, 1459. 1944.

CALDWELL, W. T., TYSON, F. T., and LAUER, LOTHAR. Substituted 2-sulfonamido-5-aminopyridines. II. J. Amer. Chem. Soc., 66, 1479. 1944.

JOHNS, C. K. Dye concentration in resazurin tablets. Amer. J. Pub. Health, 34, 955-8. 1944.

SMITH, WINSLOW WHITNEY. Relative sensitivity of different phases of growth curve of Bact. salmonicida to alkaline acriflavine. Proc. Soc. Exp. Biol. & Med., 56, 240-2. 1844.

VAN ARENDONK, A. M., and SHOULE, H. A. Dialkylaminoalkyl derivatives of substituted quinolines and quinaldines. J. Amer. Chem. Soc., 66, 1284. 1944.

WHEELER, KEITH, and DEGERING, E. F. Preparation and properties of certain derivatives of sulfamide. J. Amer. Chem. Soc., 66, 1242. 1944.

Animal Microtechnic. BOARDMAN, EDWARD T. Methods for collecting ticks for study and delineation. J. Parasitology, 30, 57-9. 1944.

DICKIE, MARGARET M. A new differential stain for mouse pituitary. Science, 100, 297-8. 1944.

GOVAN, A. D. TELFORD. Fat staining by Sudan dyes suspended in watery media. J. Path. & Bact., 56, 262-4. 1944.

LILLIE, R. D., and ASHBURN, L. L. Supersaturated solutions of fat stains in dilute isopropanol for demonstration of acute fatty degenerations not shown by Herxheimer technic. Arch. Path., 36, 432. 1943.

MULLEN, J. P. A convenient and rapid method for staining glycogen in paraffin sections with Best's carmine stain. Amer. J. Clin. Path., Tech. Sect., 8, 9-10. 1944.

NYKA, W. A method for staining the rickettsiae of typhns in histological sections. J. Path. & Bact., 56, 264. 1944.

POPPER, HANS, GYORGY, PAUL, and GOLDBLATT, H. Fluorescent material (ceroid) in experimental nutritional cirrhosis. Arch. Path., 37, 161. 1944.

SMALL, C. S., and SCHULTZ, M. A. Sustaining faded tissue sections. Amer. J. Clin. Path., Tech. Sect., 7, 66-7. 1943.

YOFFEY, J. M., and PARNELL, J. The lymphocyte content of rabbit bone marrow. J. Anat., 78, 109-12. 1944.

ZIEGLER, E. E. Hematoxylin-eosin tissue stain. An improved, rapid, and uniform technic. Arch. Path., 37, 68. 1044.

Plant Microtechnic. HAASIS, FERDINAND W. Staining rubber in ground or milled plant tissues. Ind. and Eng. Chem., Anal. Ed., 16, 480. 1944.

PARRIS, G. K. A simple nuclear stain and staining technique for Helminthosporia. Phytopathology, 34, 700. 1944.

Microorganisms. DARZINS, E. Rickettsienstudien. Zentbl. Bakl., Abt. I, Orig., 151, 18-20. 1943.

GOHAR, M. A. A staining method for Corynebacterium diphtheriae. J. Bact., 47, 575. 1944.

GRAY, P. H. H. Two-stain method for direct bacteria count. J. Milk Techn., 6, 76. 1943.     

Mercuric Bromphenol Blue Staining of Precipitin Lines in Agar

Lines formed by antibody-organ antigen reactions are stained particularly well by a modification utilizing the mercuric bromphenol blue (MBB) mixture of Mazia et al. (Biol. Bull., 104: 57-67, 1953). The agar covered slides are placed overnight in 0.85% NaCI at 4 C, followed by washing for 2 hr in 0.85% NaCI at 25 C. They are then rinsed for 10 min in distilled water, and dried overnight at 37 C. The precipitin lines are fixed by immersing the slides for 25 min in 95% alcohol, followed by 5 min hydration in distilled water. They are stained for 25 min in MBB mixture (HgCI₂, 10 gm; bromphenol blue, 0.1 gm; 95% ethanol, 100 ml). Excess stain is removed by immersing in acidified alcohol (95% ethanol, 98 ml; glacial acetic acid, 2 ml). Finally, the slides are passed through alcohol and xylene, and resin-mounted under coverslips.     

改良法分离培养SD新生乳鼠原代心肌细胞*

目的:建立一种稳定、快速的SD新生乳鼠原代心肌细胞分离培养改良方法。 方法:取SD新生乳鼠心室,0.12%Ⅱ型胶原酶消化,Percoll密度梯度离心结合5-溴脱氧尿嘧啶(5-BrdU)化学抑制法纯化心肌细胞,体外培养于含5%马血清的改良DMEM/F12中,次日更换为普通含10%胎牛血清的高糖DMEM继续培养,并比较此改良方法与传统差速贴壁法的差异。 结果:改良法获得的心肌细胞生长良好,接种24 h 后几乎全部贴壁生长,细胞呈三角形、梭形或不规则形,个别细胞出现自主搏动,频率为10～30 beats/min不等。48 h后心肌细胞变长伸出伪足,部分细胞呈现同步搏动, 频率接近50～80 beats/min。72 h后心肌细胞成菊花样交织成网,自发搏动趋于同步,频率加快至80～100 beats/min;96 h后细胞聚集成簇,呈岛屿样,同步搏动频率在100～120 beats/min左右,一周内细胞状态良好。改良法纯化原代心肌细胞的得率((1.17±0.15)×10⁶ vs (1.21±0.22)×10⁶,P＞0.05)和存活率与传统差速贴壁法相当(93.3%±1.4% vs 92.2%±0.7%, P＞0.05 ),但是改良方法获得的原代心肌细胞纯度更高 (94.7%±2.1% vs 89.5%±1.3%, P＜0.05),且用时较短((3.1±0.4)h vs (4.3±0.3)h, P＜0.01)。 结论:改良法获得心肌细胞耗时短、纯度高、结构功能保存完整,且实验重复和稳定性好,是一种理想且简单易行的的原代心肌细胞分离培养方法。     

Enhancement of the Affinity of Nucleoli and Chromosomes for Zinc by Treatment with Chromic Acid

Cultured mammalian cells and wet touch preparations from human organs were fixed for 10 min in 5:85:10 acetic-alcohol-formalin; placed in 5% aqueous CrO₃ for 30 min at 22-25 C; washed in running water 1 min; placed in 2 mM zinc acetate in 0.14 M veronal-acetate buffer, pH 6.5, at 37 C, 30 mm; rinsed 5 sec in 50% acetone; and stained 10 min in a solution dithizone. This results in selective staining of the nucleoli of interphase cells, and of the chromosomes of mitotic cells.     

小鼠及猕猴胚胎MECP2基因T158M单碱基突变体系的建立

目的:利用单碱基编辑技术定点修饰MECP2基因T158M位点,获得单一定点突变类型的啮齿类及非人灵长类胚胎,为建立模拟临床上Rett综合征的疾病动物模型奠定基础。方法:利用单碱基编辑技术构建3对T158M-sgRNA质粒,并分别与BE系统(BE3或BE4max)质粒共转染293T细胞筛选高效的sgRNA,通过显微注射将体外转录的mRNA以不同的浓度组合注射到ICR小鼠和猕猴受精卵中,检测小鼠和猕猴胚胎发育率和编辑效率,评估BE3和BE4max的工作效率及最优浓度组合。结果:小鼠胚胎上BE4max(ng/μl)∶sgRNA(ng/μl)=100∶50浓度组合时,小鼠胚胎发育率和编辑效率最佳,同时该浓度组合在猕猴胚胎上也获得了有效编辑效率。结论:成功在小鼠及猕猴胚胎上实现了MECP2基因T158M位点上C→T的转变,为后期建立T158M突变的RTT动物模型建立了稳定的胚胎编辑体系。     

依达拉奉对毒死蜱所致大鼠脑损伤的保护作用及其机制

目的:探讨依达拉奉在毒死蜱诱导的神经元凋亡中的保护作用及其线粒体机制。方法:在随机双盲的原则下,将大鼠分为对照组、毒死蜱组、依达拉奉组(n=6);以毒死蜱(18 mg/0.7 ml/kg, sc.)造模,在注射毒死蜱1 h后用依达拉奉(10 mg/1.6 ml/kg, ip.)治疗。连续注射毒死蜱及依达拉奉28 d后,通过旷场和水迷宫试验测试大鼠学习记忆能力。在心脏灌流后取大鼠脑组织,通过HE染色检测大脑海马区的神经元损伤情况以及透射电子显微镜观察线粒体和细胞核损伤情况。测定Na⁺-K⁺-ATP酶、ATP含量判断线粒体损伤情况。用免疫组织化学和免疫印迹法测定线粒体分裂蛋白DRP1及DRP1的Ser 637位点磷酸化的表达。结果:与对照组相比,毒死蜱组大鼠在旷场实验的3 min内的总运动距离和平均速度明显减小(P<0.01),在水迷宫试验中的1 min内的逃避潜伏期明显延长、平台穿越次数明显减少(P<0.01),脑组织的ATP酶活性明显降低(P<0.01)且ATP含量下降(P<0.05)、线粒体DRP1的Ser637位点...     

糖尿病诱导硫氧还蛋白相互作用蛋白(TXNIP)对小鼠胰岛β细胞衰老的影响*

目的: 本研究旨在观察糖尿病中硫氧还蛋白相互作用蛋白(TXNIP)的表达是否影响胰岛β细胞衰老。方法: 正常小鼠(db/m)、糖尿病小鼠(db/db)随机各取6只,血糖仪检测其空腹血糖值,Western blot检测胰腺组织TXNIP蛋白表达、免疫化学染色检测胰腺组织中衰老相关β-半乳糖苷酶活性,Western blot检测胰腺组织衰老相关指标p16、p21、Rb表达的变化。INS-1胰岛β细胞随机分7组(n=6),用各组慢病毒(30 μl)转染4~6 h后,嘌呤霉素(PM, 3 μg/m)筛选7 d,构建Normal组(正常组)、Scramble ShRNA组(干扰空病毒组)、TXNIP-ShRNA-1(TXNIP沉默一组)组、TXNIP-ShRNA-2(TXNIP沉默二组)组、TXNIP-ShRNA-3组(TXNIP沉默三组)、Ad-GFP组(过表达空病毒组)、Ad-TXNIP-GFP组(TXNIP过表达组)稳转INS-1胰岛β细胞株,检测其TXNIP蛋白表达、衰老相关β -半乳糖苷酶活性、衰老相关指标。结果: 与正常小鼠相比,db/db组小鼠空腹血糖显著上升(P＜0.01),胰腺组织TXNIP蛋白表达显著升高(P＜0.05),胰腺组织β -半乳糖苷酶阳性染色率增加,p16、p21、Rb蛋白表达显著升高(P＜ 0.05)。与Ad-GFP组相比,Ad-TXNIP-GFP组β -半乳糖苷酶阳性染色率增加,p16、p21、Rb蛋白表达均显著增加(P＜0.01)。与Scramble ShRNA组相比,TXNIP-ShRNA组β -半乳糖苷酶阳性染色率降低,p16、p21以及Rb蛋白表达均降低(P＜0.05)。结论: 糖尿病可通过上调TXNIP表达,诱导胰岛β细胞衰老。     

Regioselective acylation of several polyhydroxylated natural compounds by Candida antarctica lipase B

Regioselective acylation of four polyhydroxylated natural compounds, deacetyl asperulosidic acid (1), asperulosidic acid (2), puerarin (3) and resveratrol (4) by Candida antarctica Lipase B in the presence of various acyl donors (vinyl acetate, vinyl decanoate or vinyl cinnamoate) was studied. Compounds 1, 2 and 4 were regioselectively acetylated with vinyl acetate to afford products, 3'-O-acetyl-10-O-deacetylasperulosidic acid (1a), 3',6'-O-diacetyl-10-O-deacetylasperulosidic acid (1b), 3'-O-acetylasperulosidic acid (2a), 3',6'-O-diacetylasperulosidic acid (2b), 4'-O-acetylresveratrol (4a), respectively, with yields of 22 to 50%, while reactions with vinyl decanoate and vinyl cinnamoate were slow with lower yields. Compound 3 was readily acylated with all three acyl donors and quantitatively converted to products 6'-O-acetylpuerarin (3a), 6'-O-decanoylpuerarin (3b), 6'-O-cinnamoylpuerarin (3c), respectively. The structures of these acylated products were determined by spectroscopic methods (MS and NMR).     

Correlation between vein density and water use efficiency of Salix matsudana in Zhangye Wetland,China

Aims The correlation between vein density and water use efficiency (WUE) affects the balance between water supply and demand of plant leaves, which is significant for comprehending the ecological adaptation strategies of plants. The objective of this study was to study how Salix matsudana modulated vein density and WUE along a soil moisture gradient in Zhangye Wetland, China. Methods The study was conducted in floodplain wetland near Heihe River in Zhangye City, Gansu Province, China. Three sample plots, at a spatial interval of 70 m, were set up along a soil moisture gradient ordinally from the area near the water body to the wetland edge, plot I (69.23%), spot II (48.38%) and spot III (35.27%). Community traits were investigated by using diagonal method, and all individuals of S. matsudana were used for measurements of height and canopy. At each plot, 5 individuals of S. matsudana at 4 vertices and diagonal intersection were selected for measurements of vein density, WUE, net photosynthetic rate (P_n), transpiration rate (T_r), photosynthetically active radiation (PAR), saturated vapor pressure differences (VPD), specific leaf area, stomatal conductance (G_s) and intercellular CO₂ concentration (C_i). We used mathematical methods of correlation analysis and standardized major axis to investigate relationships between vein density and WUE. Important findings With decreasing soil moisture, the height, canopy, specific leaf area, G_s and C_i of S. matsudana decreased gradually, while the vein density, WUE, P_n, T_r, PAR and VPD increased gradually. The correlation between vein density and WUE was positive in all the three plots, but the relationship varied along the soil moisture plots gradient. There was a highly significant positive correlation (p < 0.01) between the vein density and WUE at plot I and III, whereas the correlation only reached a significant level (p < 0.05) at plot II; The correlation coefficient between vein density and WUE is significantly smaller than 1 at plot I (p < 0.05), while the correlation coefficient is significant greater than 1 at plot II and III (p < 0.05). We can conclude that varied relationships between vein density and WUE of S. matsudana along a soil moisture gradient could reflect plant acclimation.     

苍术素对大鼠心脏正性肌力的作用及其机制*

目的: 研究中药苍术的有效成分苍术素(Atractylodin)的正性肌力作用及其机理。方法: 随机选取6只雄性SD大鼠进行在体压力-容积环 (P-V loop)实验,加药前为Control组,经腹腔注射苍术素(3 mg/kg)后为苍术素组(自身对照),分析6只雄性大鼠加药后对大鼠左心室心输出量、容积及动脉压的作用;大鼠离体心脏灌流实验中,依次灌流给药:第一部分为Control→ 0.1→1→10 μmol/L苍术素浓度梯度灌流,第二部分为Control→200 nmol/L H89 (PKA抑制剂)→200 nmol/L H89+10 μmol/L 苍术素,第三部分为Control→500 nmol/L KN-93 (CaMKII抑制剂)→500 nmol/L KN-93+10 μmol/L 苍术素,第四部分为Control→10 nmol/L Calyculin A (PP1, PP2A抑制剂)→10 nmol/L Calyculin A+10 μmol/L 苍术素,加药前的正常空白组为Control组,分析每部分各6只雄性大鼠的组间左心室发展压的变化;在大鼠心肌细胞钙释放实验中,分组、给药的方法和浓度同离体心脏实验,分析来源于6个雄性大鼠6个左心室心肌细胞组间钙释放幅值的变化。结果: ①P-V loop实验表明:与Control组相比,苍术素组(3 mg/kg)腹腔注射30 min后,显著增加大鼠心输出量、搏出功及心率(P＜0.05),降低动脉舒张压(P＜0.05),而对收缩压无明显影响;②离体心脏实验表明:与Control组相比,苍术素组(0.1, 1, 10 μmol/L)灌流10 min后,能显著增加大鼠离体心脏左心室发展压(LVDP) (P＜0.05),其作用能被H89组(200 nmol/L)所阻断;③心肌细胞钙释放实验表明:与Control组相比,苍术素组(10 μmol/L)灌流10 min后,基于肌浆网钙泵(SERCA2a)显著增加大鼠心肌细胞钙释放幅值(P＜0.05),其作用能被H89组(200 nmol/L)所阻断。结论: 苍术素具有正性肌力作用,其血流动力学特点表现为降低在体大鼠动脉舒张压而不增加收缩压,苍术素的正性肌力作用机制是通过PKA-SERCA2a通路发挥的。