麝香、麝香酮对体外培养大鼠视网膜神经细胞存活影响的比较研究

为比较麝香、麝香酮对大鼠视网膜神经细胞存活的影响,分别单独将麝香、麝香酮加入大鼠视网膜神经细胞混悬液中,用MTT法评价麝香、麝香酮对大鼠视网膜神经细胞存活的影响;另分别在麝香和麝香酮中加入定量的m-NGF,采用同法评价麝香(或麝香酮) m-NGF对大鼠视网膜神经细胞存活的影响。结果表明,单独应用麝香、麝香酮对体外培养大鼠视网膜神经细胞的存活无明显影响(P>0.05);在加入定量m-NGF的条件下,麝香、麝香酮分别在50～800μg/mL和100～800μg/mL的浓度范围明显促进大鼠视网膜神经细胞的存活,且呈一定的量效关系。说明麝香、麝香酮无直接促进视网膜神经细胞存活的作用;麝香、麝香酮均与NGF协同增进视网膜神经细胞的存活;麝香酮是麝香中增强NGF生物效应的有效成分之一。     

利用气相色谱/质谱联用分析林麝麝香中麝香酮和甾类成分

采用气相色谱/质谱(GC/MS)联用的方法,对林麝麝香中的甾体成分进行分析,确定了林麝麝香样品含有多种甾体成分的结构,并分析了不同来源的林麝麝香的麝香酮及甾体成分.通过检索NIST05质谱库,进一步确定了麝香中含有16种甾体成分.利用外标法、标准曲线法同时测定了麝香样品中3种甾类成分(胆固醇、苯胆烷醇酮及麝香酮)的含量,麝香酮的定量分析显示所有样品麝香中麝香酮含量均较高(30.1～45.2 mg/g),但甾类成分含量波动较大.聚类分析显示,9个麝香样品聚为两支.利用GC/MS技术检测麝香成分的方法,可以提供麝香较为全面的甾类信息,可高效准确地对麝香进行质量分析.     

麝香多肽分离纯化及其抗炎作用机制研究CSCD

从天然麝香中提取、纯化麝香多肽,利用体外细胞模型筛选有抗炎活性的麝香多肽流分,并研究麝香多肽对小鼠急性肺损伤的保护作用及作用机制。采用冰浴超声、离子交换色谱法从天然麝香中提取、纯化获得5个流分,即为SXP1、SXP2、SXP3、SXP4、SXP5。在LPS诱导的THP-1巨噬细胞炎症模型中,SXP4(100μg/mL)可以显著抑制TNF-α和IL-1β产生,表现出明显的抗炎活性。SDS-PAGE初步检测SXP4主要分布在10~26 kDa范围内。在LPS诱导的急性肺损伤小鼠模型中,SXP4(5、15、50 mg/kg)可减少小鼠血清TNF-α和IL-6的含量(P<0.05或P<0.01);改善小鼠肺组织中炎症细胞浸润和改善肺泡壁增厚情况,减轻小鼠肺组织病理损伤;减少小鼠肺组织中IL-1β、IL-18产生,抑制NLRP3、ASC、Caspase-1、Gasdermin D蛋白表达(P<0.01)。综上,麝香多肽SXP4具有较强的抗炎活性,可减轻急性肺损伤小鼠的肺组织病理损伤,其机制可能与SXP4抑制了小鼠体内NLRP3/Caspase-1介导的细胞焦亡有关。     

青蒿鞣质抗病毒活性研究

从青蒿水提物中分离得到一种缩合鞣质(CTA),在体外进行了抗HSV-2和HBV活性研究。结果表明,CTA具有显著抗HSV-2活性,与阳性对照药物阿昔洛韦(ACV)比较,OC50分别为6．84mg／mL和3．69mg／mL,IG50分别为0．162mg／mL和0．138mg／mL,表明CTA的细胞毒性比ACV小,最低抗病毒活性浓度相当。用含CTA的培养基培养HepC2．2．1．5细胞,结果表明在浓度为2．5mg／mL时有轻微细胞毒性,在浓度为2．5～0．156mg／mL对HepG2．2．1．5细胞表达的HBeAg具有显著的抑制作用,0．625mg／mL浓度档培养第12d对HBeAg抑制率达到90．45％,提示CTA具有潜在的抗HBV活性。     

NGF 对大鼠胚胎中脑神经细胞体外增殖和分化的影响

目的:探讨神经生长因子(nerve growth factor,NGF)对大鼠胚胎中脑神经细胞体外增殖和分化的影响。方法:在体外分离培养大鼠胚胎中脑神经细胞的培养液中加入不同浓度(10、50、100、200ng/ml)的NGF,培养不同时间,以不加神经营养因子的细胞为对照组,通过MTT法检测细胞活性,神经元特异性烯醇化酶免疫细胞荧光技术鉴定神经细胞,光镜下形态学观察各组大鼠中脑神经细胞体外增殖和分化情况。结果:胚胎中脑神经细胞胞体增大、突起延长且有丰富的神经纤维连结成网络状,细胞集落数增加,显示出剂量-效应关系。结论:一定剂量的NGF能促进大鼠中脑神经细胞分化和增殖,增强其活性。     

佳乐麝香和镉复合污染对土壤中放线菌数量的影响

佳乐麝香是目前备受关注的PPCPs污染物之一,重金属Cd是土壤中常见的重要污染物,两者在土壤生态系统中存在联合暴露的潜在可能性;放线菌是土壤中分布最广数量最多的一类微生物。本研究在实验室模拟条件下,研究了佳乐麝香(HHCB)和Cd单一、复合污染对持续培养10周土壤中放线菌数量的影响,结果发现,HHCB和Cd单一、复合处理对土壤放线菌数量抑制作用显著(P0.01),随着HHCB浓度的增加,对土壤放线菌数量的抑制作用增加,其浓度-效应关系都呈对数关系,第3周的EC50值最低,前5周的EC50值均不超过10 mg·kg-1;土壤HHCB和Cd复合污染的联合毒性效应为拮抗作用。本研究表明,在实际风险评价中,应考虑到HHCB输入土壤的特征选择特定的EC50值范围。     

不同激素配比对麝香百合鳞茎芽诱导的影响

采用麝香百合的鳞茎片作为外植体进行组织培养,结果表明,不同激素浓度配比对百合鳞茎芽的诱导效果不同,供试的几个组合中,以MS+6-BA 1.0mg/L+NAA 0.5mg/L效果最好;在继代培养中,以MS+6-BA 2.0mg/L+NAA 0.1mg/L对百合鳞茎的增殖效果最好;在生根培养中,附加活性炭可促进根的生长。     

麝香水溶物对大鼠神经干细胞的生长、分化和电转染率的影响

目的：研究麝香水溶物对培养的大鼠神经干细胞的生长、分化和电转染率的影响。方法：在培养基中加入不同浓度的麝香水溶性提取物后,观察大鼠神经干细胞的生长分化情况;利用表达增强型绿色荧光蛋白的质粒pEGFP-C1,对麝香水溶物处理的神经干细胞进行电穿孔转染,调查电转染率。结果：麝香水溶物处理后的大鼠神经干细胞的细胞团分散,神经突起增多、变长,贴壁细胞增加,细胞形态呈多样性。在0.3‰浓度下,神经干细胞有向神经胶质样细胞分化的趋势。对于麝香处理后变化的细胞,再转到正常培养基中后,细胞基本都能恢复到正常的神经干细胞形态,浓度较高（3‰）时,细胞的恢复能力下降,部分细胞因细胞膜受损严重而死亡。电转染结果表明,麝香处理后发生变化的细胞对pEGFP-C1的电转染率明显提高。结论：麝香水溶物能促进大鼠神经干细胞团的分散和细胞贴壁、变形,并有向神经胶质样细胞分化的趋势。同时可以提高神经干细胞对pEGFP-C1的电转染率。     

桑根酮D对肿瘤细胞及移植瘤生长作用研究

研究桑根酮D的体内外抗肿瘤作用。采用的体外实验,桑根酮D溶液体外共培养三种癌细胞(人肝癌HepG2细胞、小鼠黑色素瘤B16F0细胞、小鼠黑色素瘤B16F10细胞),采用SRB法(Sulforhodamine B)检测桑根酮D对肿瘤细胞增殖的影响,计算IC_(50)。对黑色素瘤细胞(B16F0和B16F10细胞)给予不同浓度的桑根酮D,考察其对黑色素含量的影响。采用的体内实验,建立小鼠H22肿瘤模型并分组,三个剂量的桑根酮D分别为12.5、25、50 mg/kg,考察其对肿瘤生长的抑制作用。结果显示,桑根酮D抑制B16F0、B16F10、HepG2细胞增殖,HepG2细胞对桑根酮D最敏感,得IC_(50)为22.61±0.26μmol/L。随着桑根酮D浓度的增加,抑制细胞的作用增强且均有显著性差异(P0.01)。桑根酮D促进B16F0细胞内黑色素生成,促进细胞分化。12.5、25、50 mg/kg剂量的桑根酮D对小鼠肝癌细胞H22移植瘤抑制率为32.51%～45.72%。给药组小鼠的体重及各脏器指数变化与空白对照组和模型组比较均无显著性差异。得出结论,桑根酮D具有体内外抗肝癌作用和促进肿瘤细胞分化的作用。     

脱氧胆酸钠对人脐静脉内皮细胞凋亡的影响

目的:探讨脱氧胆酸钠(SD)对体外培养的人脐静脉内皮细胞(HUVEC)凋亡的影响。方法:(1)以不同终浓度(0、0.015mg/mL、0.05 mg/mL、0.15 mg/mL、0.5 mg/mL、1.0 mg/mL)的脱氧胆酸钠分别作用于人脐静脉血管内皮细胞,使用CCK-8检测细胞活力、TUNEL荧光染色检测细胞凋亡;(2)以终浓度为0.15 mg/mL的SD作用于HUVEC4、8、12 h后用Western blot检测Caspase-3、7、9蛋白及PARP活化情况;(3)观察Caspase-3抑制剂Z-DEVD-FMK对0.15 mg/mL脱氧胆酸钠组的影响。结果:CCK8结果显示随SD浓度(0~1.0 mg/mL)及作用时间(0~12 h)增加,HUVEC活力降低,0.15 mg/mL时活力为80%,1.0 mg/mL时细胞活力仅不到10%;Tunel检测示随着SD浓度的增加HUVEC凋亡明显增多;Western Blot结果示SD作用于HUVEC后Caspase-3、7、9蛋白及PARP活化明显增加;Z-DEVD-FMK明显抑制了0.15 mg/mLSD引起的PARP活化。结论:脱氧胆酸钠(SD)通过启动Caspase级联反应介导了人脐静脉内皮细胞的凋亡。     

Synthesis, analysis, in vitro characterization, and in vivo disposition of a lamivudine-dextran conjugate for selective antiviral delivery to the liver

A liver-selective prodrug (3TCSD) of the antiviral drug lamivudine (3TC) was developed and characterized. 3TC was coupled to dextran ( approximately 25 kDa) using a succinate linker, and the in vitro and in vivo behavior of the conjugate was studied using newly developed size-exclusion and reversed-phase analytical methods. Synthesized 3TCSD had a purity of >99% with a degree of substitution of 6.5 mg of 3TC per 100 mg of the conjugate. Furthermore, the developed assays were precise and accurate in the concentration ranges of 0.125-20, 0.36-18, and 1-50 microg/mL for 3TC, 3TC succinate (3TCS), and 3TCSD, respectively. In vitro, the conjugate slowly released 3TC in the presence of rat liver lysosomes, whereas it was stable in the corresponding buffer. In vivo in rats, conjugation of 3TC to dextran resulted in 40- and 7-fold decreases in the clearance and volume of distribution of the drug, respectively. However, the accumulation of the conjugated 3TC in the liver was 50-fold higher than that of the parent drug. The high accumulation of the conjugate in the liver was associated with a gradual and sustained release of 3TC in the liver. These studies indicate the feasibility of the synthesis of 3TCS-dextran and its potential use for the selective delivery of 3TC to the liver.     

Evidence for genistein mediated cytotoxicity and apoptosis in rat brain

The effects of chronic treatment with high doses of genistein, a major isoflavone of soybeans and soy-based products, have yet to be determined and what is known remains controversial. The present study was undertaken to investigate the cytotoxic effects of chronic ingestion of genistein on rat brain in vivo and the observations were compared with results from in vitro studies with primary cultures of cortical neurons. Sprague-Dawley rats were given 2 or 20 mg/day genistein (p.o.) for four weeks. The high dose of genistein (20 mg/day) significantly increased lactate dehydrogenase (LDH) in rat brain tissue homogenates, whereas the low dose of genistein (2 mg/day) decreased LDH. In addition, DNA fragmentation was detected in homogenates of brain tissue from rats receiving either dose of genistein. These results are consistent with those of in vitro studies indicating that high concentrations of genistein caused cytotoxicity and DNA ladder formation in primary cultures of cortical neurons. Genistein decreased the expression of the 32 kDa caspase-3 precursor and increased the levels of cleaved caspase-3 (18 kDa) in both rat brain tissue homogenates and in primary cultures of cortical neurons. Furthermore, expression of poly (ADP-ribose) polymerase (PARP) was also decreased in both experimental systems. These results suggest that chronic administration of genistein at high doses may induce cytotoxicity and apoptosis in the rat brain.     

抗菌肽对种植体周围炎主要致病菌生长抑制作用的研究

目的研究抗菌肽KSL及其衍生物KSL—W对种植体周围炎主要致病菌的体外抑菌效果。方法应用二倍稀释法检测KSL和KSL—W对血链球菌、具梭核杆菌和牙龈卟啉单胞菌的最小抑菌浓度（MIC）和最小杀菌浓度（MBC）;MTT法检测KSL和KSL—W对成骨样细胞MG-63的细胞毒性。结果KSL和KSL—W对具梭核杆菌的MIC和MBC分别为0．0156mg／mL和0．0313mg／mL,对牙龈卟啉单胞菌的MIC和MBC分别为0．125mg／mL和0．5mg／mL,在0．5mg／mL的浓度范围内对血链球菌没有抑制作用;KSL和KSL-W在0．5mg／mL的浓度范围内没有细胞毒性。结论KSL和KSL—W没有细胞毒性,对具梭核杆菌和牙龈卟啉单胞菌具有抑制作用。     

千金子不同极性部位对酪氨酸酶活性的影响

测定千金子醇提物不同极性部位对酪氨酸酶活性的影响。以L-酪氨酸为底物,采用比色法测定千金子不同极性部位对酪氨酸酶的抑制率。得到了千金子醇提物的乙酸乙酯相、正丁醇相和水相的半数抑制率(IC50值)分别为0.150 mg/mL,0.813 mg/mL,7.570 mg/mL,并对乙酸乙酯相进一步分离得到七叶内酯,其IC50值0.103 mg/mL。结果表明千金子中起到酪氨酸酶抑制性的物质为七叶内酯,主要分布在乙酸乙酯相中。为千金子中酪氨酸酶抑制性物质的筛选提供科学依据。     

Sympathetic neuronal survival induced by retinal trophic factors.

Neuronal survival in the vertebrate peripheral nervous system depends on neurotrophic factors available from target tissues. In an attempt to identify novel survival factors, we have studied the effect of secreted factors from retinal cells on the survival of chick sympathetic ganglion neurons. Embryonic day 10 sympathetic neurons undergo programmed cell death after 48 h without appropriate levels of nerve growth factor (NGF). Retina Conditioned Media (RCM) from explants of embryonic day 11 retinas maintained for 4 days in vitro supported 90% of E10 chick sympathetic neurons after 48 h. Conditioned medium from purified chick retinal Muller glial cells supported nearly 100% of E10 chick sympathetic neurons. Anti-NGF (1 microg/mL) blocked the survival effect of NGF, but did not block the trophic effect of RCM. Neither BDNF nor NT4 (0.1-50 ng/mL) supported E10 sympathetic neuron survival. Incubation of chimeric immunoglobulin-receptors TrkA, TrkB, or TrkC had no effect on RCM-induced sympathetic neuron survival. The survival effects were not blocked by anti-GDNF, anti-TGFbeta, and anti-CNTF and were not mimicked by FGFb (0.1-10 nM). LY294002 at 50 microM, but not PD098059 blocked sympathetic survival induced by RCM. Further, the combination of RCM and NGF did not result in an increase in neuronal survival compared with NGF alone (82% survival after 48 h). The secreted factor in RCM is retained in subfractions with a molecular weight above 100 kDa, binds to heparin, and is unaffected by dialysis, but is heat sensitive. Our results indicate the presence of a high-molecular weight retinal secreted factor that supports sympathetic neurons in culture.     

Oxysterol-induced toxicity in R28 and ARPE-19 cells

Studies have shown an intimate relationship between cholesterol and retinal diseases; we examined the effects of cholesterol oxides on cultured cells. Using the rat retinal precursor cell line R28 and the human RPE cell line ARPE-19, we investigated the potential cytotoxicity of cholesterol oxides. Cultured R28 and ARPE-19 cells were treated with either 25-hydroxycholesterol and 7-ketocholesterol (0–50 µg/ml). Cell viability was determined by the WST-1 colorimetric assay. Production of reactive oxygen intermediate (ROI) was assessed by a fluorescent probe–based assay (2,7-dichlorodihydrofluorescein diacetate [H₂DCFDA]). To detect the presence of apoptosis, DNA fragmentation gel analysis and Hoescht nuclear staining were performed. Both cholesterol oxides tested were toxic in a time- and dose-dependent fashion to the two cell lines used in this study. Treatment of R28 cells with either 25-hydroxycholesterol or 7-ketocholesterol at a concentration of 25 µg/ml resulted in greater than 50% loss of cell viability after 24 h. ARPE-19 cells were slightly less affected, with a loss of cell viability of approximately 20% and 40% after 24 h-exposure of 25-hydroxycholesterol and 7-ketocholesterol, respectively. DNA fragmentation and chromatin condensation demonstrated apoptotic events occurring in 7-ketocholesterol–treated cells. The fluorescent assay for ROI production showed that after an hour of exposure to 7-ketocholesterol, R28 cells responded with increased levels of ROIs, whereas no immediate production of ROIs were detected with treated ARPE-19 cells. These in vitro findings provide evidence that cholesterol oxides can directly damage cultured retinal and RPE cells. The oxysterol-induced oxidative stress in these cells may be a factor in the pathology of retinal degenerative diseases.     

Cytotoxicity and genotoxicity of chitooligosaccharides upon lymphocytes

Two COS mixtures and a low molecular weight chitosan (LMWC) were tested for potential cytotoxicity and genotoxicity upon human lymphocytes. Genotoxicity was evaluated in vitro by cytokinesis-blocked micronucleus and alkaline comet assays, while cytotoxicity was assessed by flow cytometry analysis. Our results suggest that COS do not exhibit any genotoxicity upon human lymphocytes, independently of MW or concentration. However, above 0.07 mg/mL COS induced strong cytotoxic effects. According to the concentration used, such cytotoxicity will induce cell death, essentially by necrosis (>0.10 mg/mL) and/or apoptosis (<0.10 mg/mL). The level of necrosis/apoptosis induced by high COS concentrations, suggests a promising use as apoptosis inducers in specific cancer situations.     

Antitumor agents 200. Cytotoxicity of naturally occurring resveratrol oligomers and their acetate derivatives

Eleven resveratrol oligomers and six acetylated derivatives were evaluated for in vitro cytotoxicity against a panel of human tumor cell lines. The acetate of (-)-ampelopsin A (12) showed potent and selective cytotoxic activity with ED50 values of 0.6, 0.7 and 2.0 microg/mL against KB, 1A9 and MCF-7 cells, respectively. Hopeaphenol (10) and pallidol hexaacetate (13) also showed significant cytotoxicity against KB cells with ED50 values of 1.2 and 1.6 microg/mL, respectively.