全反式维甲酸前体脂质体的制备及体外评价

目的:制备维甲酸前体脂质体,并对其体外性质进行考察。方法:采用乙醇注入结合冷冻干燥法制备前体脂质体;微柱离心-高效液相色谱法测定脂质体的包封率;并进一步对其粒径、Zeta电位、血浆释放率及乙醇残留量进行测定。结果:所制备的前体脂质体包封率为95.2%,Zeta电位为-(28.4±17.5)mV,粒径为(170±29)nm,乙醇残留量为3.98%。结论:乙醇注入结合冷冻干燥法制备的维甲酸前体脂质体包封率高,粒径均匀,稳定性好。     

Preparation and characterization of chitosan-treated alginate microparticles incorporating all-trans retinoic acid

Chitosan treated alginate microparticles were prepared with the purpose of incorporating all-trans retinoic acid (ATRA) using an inexpensive, simple and fast method, enhancing dermal localization and sustaining the release of ATRA into the skin. Microparticles characterization, drug–polymer interaction, release profile and in vitro skin retention were investigated. Microparticles presented spherical shape and drug loading capacity of 47%. The drug content of these microparticles was affected by ATRA concentration and by the solvent used and it was more weakly affected by chitosan concentration. The release of ATRA was also affected by chitosan concentration. Microparticles prepared with 0.4% chitosan (w/w) resulted in drug release with a more sustained profile. The results of in vitro retention studies showed that chitosan treated alginate microparticles decreased the drug retention in the stratum corneum (SC), where occur the skin irritation, but maintained the ATRA concentration in the deeper skin layers, where occur the pathologies treated with ATRA. Then, the microparticles developed in this work can be a good candidate to improve the topical therapy with retinoid.     

HPLC-MS/MS法测定NB4细胞培养液中的全反式维甲酸

目的 建立HPLC-MS/MS法测定NB4细胞培养液中全反式维甲酸的浓度。方法 采用ACQUITY UPLCTM BEH C₁₈（50 mm×2.1 mm,1.7 μm）色谱柱,以布洛芬为内标,流动相为乙腈-水（含0.1%乙酸）,体积流量为0.2 mL/min,梯度洗脱方式分离全反式维甲酸,同时采用ESI源负离子检测方式,定量分析时的离子反应分别为m/z 299.2→m/z 255.2（全反式维甲酸）和m/z 205.4→m/z 161.3（内标布洛芬）。结果 全反式维甲酸在2.55~255 ng/mL线性关系良好,定量限为2.55 ng/mL,日内精密度RSD ≤ 10.3%,日间精密度RSD ≤ 12.9%。结论 建立的HPLC-MS/MS法可用于测定NB4细胞含维甲酸的培养液中全反式维甲酸的浓度,以及在加入维甲酸代谢阻断剂时全反式维甲酸浓度的变化。     

Induction of cell-cycle arrest by all-trans retinoic acid in mouse embryonic palatal mesenchymal (MEPM) cells.

all-trans retinoic acid (atRA), the oxidative metabolite of vitamin A, is essential for normal embryonic development. Also, high levels of atRA are teratogenic in many species and can effectively induce cleft palate in the mouse. Most cleft palate resulted from the failed fusion of secondary palate shelves, and maintenance of the normal cell proliferation is important in this process of shelf growth. To clarify the mechanism by which atRA causes cleft palate, we investigated the effect of atRA on proliferation activity and cell cycle distribution in mouse embryonic palatal mesenchymal (MEPM) cells. atRA inhibited the growth of MEPM cells by inducing apoptosis in a dose-dependent manner. atRA also caused a G1 block in the cell cycle with an increase in the proportion of cells in G0/G1 and a decrease in the proportion of cells in S phase, as determined by flow cytometry. We next investigated the effects of atRA on molecules that regulate the G1 to S phase transition. These studies demonstrated that atRA inhibited expression of cyclins D and E at the protein level. Furthermore, atRA treatment reduced phosphorylated Rb and decreased cdk2 and cdk4 kinase activity. These data suggest that atRA had antiproliferative activity by modulating G1/S cell cycle regulators and by inhibition of Rb phosphorylation in MEPM cells, which might account for the pathogenesis of cleft palate induced by retinoic acid.     

全反式视黄酸促进胚胎神经干细胞诱导分化的研究

目的本研究观察全反式视黄酸（atRA）对于体外分离培养的胚胎神经干细胞（ENSCs）生长增殖和诱导分化的作用及其可能的分子机制。方法分离培养孕15dSD大鼠（E15 d SD Rattus）海马回ENSCs,采用不同组合成分的神经干细胞培养基培养ENSCs,利用MTF法检测其对于ENSCs存活和增殖的影响;采用不同组合成分的神经细胞诱导分化培养基培养ENSCs,利用细胞免疫荧光染色法鉴定ENSCs及atRA对于其诱导分化的影响。结果MTT实验结果表明,atRA^＋组、atRA^-组和DMSO组均出现ENSCs的增殖效果,而对照组则没有明显的增殖现象,其中atRA^-组最明显,DMSO组次之,atRA^＋组最弱。AtRA^＋组与atRA^-的ENSCs经过诱导分化后产生的神经细胞类型有较明显差异,并且atRA组处理产生的神经元是对照组的3倍左右。结论atRA能够抑制ENSCs的增殖,并且抵消神经生长因子对于ENSCs的促进有丝分裂作用,atRA还可以促进ENSCs定向诱导分化为神经元。     

All-trans retinoic acid induced differentiation of rat mammary epithelial cells cultured in serum-free medium

Retinoids are applied to not only cancer prevention but also cancer chemotherapy by stimulating differentiation of cells. We studied differentiation inducing effect of all-trans retinoic acid (ATRA) by studying proportion of high dense fractions of stem-like cells and the size of S phase fraction in cell cycle. From mammary organoids obtained from 7- to 8-week old F344 female rat mammary gland, we cultured rat mammary epithelial cells (RMEC) and treated physiological doses of 10⁻⁶, 10⁻⁷, and 10⁻⁸ M ATRA from the first day and then cultured for 4, 7, and 14 days. After that, immunostaining was performed using peanut agglutinin (PNA) and anti-Thy-1.1 monoclonal antibody (Thy-1.1) that can be used as markers of differentiation. We separated four different cell subpopulations by flow cytometry: cells negative to both reagents (B-), PNA-positive cells (PNA+), Thy-1.1-positive cells (Thy-1.1+), and cells positive to both reagents (B+). We observed continuous decreases of high dense fractions of stem-like cells (PNA+ subpopulations) for 14 days and as much decreases as high doses of ATRA, which were thought to be proportional to doses of ATRA. We labeled RMEC with bromodeoxyuridine and investigated cell cycle fractions that went through S phase. We observed a tendency of decrease of S phase fraction with time in culture, which is though to be related to continuous decreases of PNA+ subpopulations and inhibitory role of ATRA on cell cycle. These results suggest that physiological doses of ATRA could stimulate differentiation of RMEC and convert stem-like RMEC to differentiated cells in SFM for a relatively long period of 14 days.     

全反式维甲酸联合猪胆酸钠对人卵巢癌细胞生长抑制作用的影响

目的 探讨维甲酸（ATRA）及猪胆酸钠（SBANa)联合反应对人卵巢癌细胞生长的抑制作用。方法 实验共分为三组：单用ATRA组、单用SBANa组及ATRA＋SBANa联合用药组。应用MTT比色法、流式细胞仪对COC1、COC2、CAOV3三种卵巢癌细胞系在不同浓度的ATRA及SBANa下进行检测。结果 ATRA及SBANa联合应用后，能增强抑制卵巢癌细胞生长作用，当达到ATRA10μmol/L SBANa 100μg/ml浓度时，G0、G1期细胞比例增加，G2M、S期细胞比例下降，细胞增殖速度较单独用药组减缓。MTT检测提示在联合用药浓度达到ATRA 10μmol/L SBANa 100μg/ml时，癌细胞抑制率最佳。结论 ATRA及SBANa联合应用可增强对卵巢癌细胞增殖的抑制作用，并在ATRA 30μmol/L SBANa 100μg/ml浓度时，促进癌细胞凋亡。     

Uptake and metabolism of all-trans retinoic acid by three native north American ranids.

Retinoids, which are Vitamin A derivatives, are important signaling molecules that regulate processes critical for development in all vertebrates. The objective of our study was to examine uptake and metabolism of the model retinoid, all-trans retinoic acid (all-trans RA), by three native North American anurans, Rana sylvatica, R. pipiens, and R. clamitans. Limb-bud stage tadpoles (stages 26-28) were exposed to all-trans RA concentrations of 0, 250, 500, 750, 1000, and 1250 ng/ml for 24 h. Water and tissue samples, collected at 0, 4, 12, and 24 h, were analyzed by high pressure liquid chromatography with diode-array detection (HPLC-DAD) to characterize aqueous exposure and all-trans RA uptake and metabolism. All-trans RA degraded rapidly in exposure water (i.e., with organisms), with over 70% of the parent compound gone in 4 h and none detected by 24 h. Consistent with this result, tadpoles from the three species showed the greatest accumulation of all-trans RA at 4 h followed by decreasing tissue concentrations at 12 and 24 h. In addition to all-trans RA, several other chromatographic peaks were observed in the tissue extracts indicating metabolism of the retinoid by the tadpoles. Identification of potential metabolites of all-trans RA and endogenous retinoids was conducted by comparing retention times and absorption spectra of available standards (i.e., 4-oxo-all-trans RA, 4-oxo-13-cis RA, 13-cis RA, 9-cis RA, all-trans retinol, all-trans retinal) to those in the tissue extracts. In all three species, all-trans RA was metabolized to 4-oxo-all trans RA and 13-cis RA. The RA isomer, 9-cis RA, was detected in two species,R. sylvatica and R. pipiens. All three species also had measurable levels of vitamin A (all-trans retinol), while the aldehyde form (all-trans retinal) was detected only in R. clamitans. Our results indicate that all-trans RA is rapidly metabolized by these Ranid species to a variety of retinoid derivatives, several of which are known ligands for RA and retinoid receptors, and are capable of activating this signaling transduction pathway.     

全反式维甲酸与苯那普利对肾硬化大鼠肾脏组织α-SMA表达的影响

目的研究全反式维甲酸(ATRA)与苯那普利对肾硬化大鼠肾脏组织α-SMA表达的影响。方法80只Wistar♂大鼠随机分为假手术组、模型组、苯那普利组和ATRA组,每组均为20只。采用单侧肾切除加1wk后尾静脉注射阿霉素(5mg·kg-1)的方案建立大鼠肾小球硬化(GS)模型,造模后12wk末处死。HE染色观察肾脏病理改变,计算肾小球硬化指数(GSI)。RT-PCR结合免疫组织化学方法检测肾组织α-平滑肌肌动蛋白(α-SMA)的表达情况。结果与模型组相比,ATRA组和苯那普利组GSI、α-SMA mRNA相对表达量和α-SMA阳性面积比均明显下降(P<0.05),但两组间各项指标比较无差异(P>0.05)。结论ATRA与苯那普利对大鼠肾硬化均有明显的延缓效应,且疗效一致。     

全反式维甲酸对肝星状细胞活化及氧化应激的作用和机制探索

目的 探讨全反式维甲酸（ATRA）对肝星状细胞（HSCs）活化及氧化应激的作用和潜在机制。方法 应用10 ng/ml的血小板源性生长因子（PDGF-bb）诱导HSCs活化,以5 μmol/L剂量的ATRA处理48 h。检测细胞生长活力和表型标志物表达的变化,评价ATRA对HSCs活化的影响;检测细胞内活性氧（ROS）、还原型谷胱甘肽（GSH）和丙二醛（MDA）、抗氧化基因表达的变化,评价ATRA对HSCs氧化应激的影响;检测自噬标志物表达和自噬流的变化,评价ATRA对HSCs自噬活性的影响。结果 与PDGF-bb组相比,ATRA处理的HSCs生长活力显著降低（P<0.01）,α-SMA和Collagen I蛋白的表达明显减少（P<0.01）,细胞内ROS和MDA显著减少（P<0.01）,GSH显著增加（P<0.01）,抗氧化基因NRF2、HO-1和ATF4的表达明显增加（P<0.01）;同时自噬标志物Beclin 1和LC3 II/I的表达明显减少（P<0.01）,自噬流信号显著降低。结论 ATRA显著抑制PDGF-bb诱导的HSCs活化,降低HSCs的氧化应激水平和自噬活性,对肝纤维化的防治具有潜在应用价值。     

全反式维甲酸对糖尿病大鼠肾脏组织BMP-7表达及细胞外基质代谢的影响

目的探讨全反式维甲酸（atRA）对糖尿病大鼠肾脏组织骨形成蛋白-7（BMP-7）、转化生长因子-β1（TGF-β1）表达和细胞外基质（E（M）Ⅳ、Ⅲ型胶原代谢的影响。方法随机选择24只舍SD大鼠制作糖尿病模型,其中12只入糖尿病模型（DM）组,另12只入糖尿病模型＋atRA（DM＋atRA）组,此外再随机选择12只正常大鼠作为空白对照（NC）组。分别于第8、12周末各组处死6只大鼠,通过光镜、电镜观察各组大鼠肾脏病理结果;通过免疫组织化学方法观察BMP-7,TGF-β1,Ⅳ、Ⅲ型胶原表达,并进行半定量分析;通过实时荧光定量PCR方法对TGF-β1 mRNA、BMP-7mRNA,进行相对定量分析。结果DM＋atRA组大鼠肾组织BMP-7表达较同时期DM组增加（P〈0.01）,主要表达于大鼠肾小管上皮细胞,而TGF-β1表达较同时期DM组减少（P〈0．01）：且DM＋atRA组大鼠肾组织ECM重要成分Ⅳ、Ⅲ型胶原表达与DM组相比较显著减少（P〈0．01）。同时观察到BMP-7蛋白与Ⅳ型胶原蛋白表达呈负相关（r=-0．75,P〈0．01）,与Ⅲ型胶原蛋白表达呈负相关（r=-0．71,P〈0．01）,与TGF-β1蛋白表达呈负相关（r=-0．86,P〈O．01）。荧光定量PCR结果表明,DM＋atRA组BMP-7mRNA水平升高,TGF-β1 mRNA水平下降,且两者星显著负相关（r=-0．83,P〈0．01）。结论atRA能减轻糖尿病大鼠肾脏病理损伤,延缓肾脏纤维化,这一防治作用可能与其增加BMP-7表达,下调TGF-β1表达从而减少肾组织ECM沉积有关。     

三氧化二砷联合全反式维甲酸治疗急性早幼粒细胞白血病29例

目的观察三氧化二砷（As2O3）联合全反式维甲酸（ATRA）治疗急性早幼粒细胞白血病（APL）的疗效及毒副反应。方法患者使用三氧化二砷联合全反式维甲酸双诱导治疗,对高白细胞患者加用单一化疗药物高三尖衫酯碱（H）或柔红霉素（DNR）,合并弥漫性血管内凝血（DIC）患者予以输注血浆／纤维蛋白原。结果29例患者中26例完全缓解,完全缓解（CR）率89．66％;3例死于颅内出血。结论三氧化二砷联合全反式维甲酸治疗急性早幼粒细胞白血病的疗效确切,患者耐受性较好。     

全反式维甲酸抑制兔颈动脉内皮损伤后内膜增殖和聚集素表达的研究

目的观察全反式维甲酸(atRA)对兔颈动脉内皮损伤后内膜增生、聚集素表达的影响,探讨atRA抗血管再狭窄的作用及机制。方法新西兰雄性大白兔36只,随机分为6组:治疗组(A、B)、对照组(A、B)、假手术组(A、B),每组6只。治疗组和对照组给予高脂饮食,2周后行颈动脉内膜空气干燥术,假手术组正常饮食,手术但不损伤内膜,治疗组术前3d开始atRA灌胃。分别于术后7、28d处死A、B组动物,取病变血管进行形态学观察,免疫组化检测聚集素、PCNA表达水平。结果对照组术后7d内膜开始增生,28d增生明显,管腔狭窄,有粥样斑块形成。治疗组内膜增生较轻,28d时内膜面积、内膜/中膜面积比、管腔狭窄度均低于对照组(P<0.01),聚集素、PCNA阳性表达低于对照组(P<0.05或<0.01)。结论 atRA抑制动脉内皮损伤后新生内膜增殖及动脉粥样硬化病变形成,抑制聚集素表达是机制之一。     

CRABP2对肺癌细胞应答全反式视黄酸及细胞增殖的影响

目的：探讨细胞视黄酸结合蛋白2（CRABP2）对肺癌细胞应答全反式视黄酸（ATRA）及细胞增殖的影响。方法观察两株肺癌细胞95‐D和A549对ATRA 的敏感性。应用小干扰RNA （siRNA）敲低CRABP2的表达,采用CCK‐8和流式细胞术分别检测细胞增殖和周期变化,Western blot检测NF‐κB和丝裂原活化蛋白激酶（MAPKs）信号通路分子的表达。结果 ATRA抑制95‐D和A549细胞增殖,其中95‐D细胞的抑制作用更为明显;而干扰CRABP2的表达则显著降低ATRA对95‐D细胞的抑制作用。干扰CRABP2表达在一定程度上能直接抑制95‐D细胞增殖,增加G1期的细胞比例,减少S期的细胞,降低NF‐κB、应激活化蛋白激酶／c‐JUN氨基末端激酶（SAPK／JNK ）和c‐JUN的磷酸化水平。结论 CRABP2能增强肺癌细胞对 ATRA的敏感性,并且还可能通过调控NF‐κB和SAPK／JNK信号通路促进细胞增殖,提示CRABP2在肿瘤细胞中的双重作用。     

维甲酸对晶状体上皮细胞的作用

目的通过观察维甲酸(RA)对传代培养的人晶状体上皮细胞(hLECs)生长特性的影响,探讨RA抑制晶状体后囊混浊形成(PCO)的细胞学机制。方法选取第2~5代hLECs,利用倒置显微镜观察RA对细胞形态的影响;MTT法观察RA对细胞增殖的影响,计算RA对细胞增殖的抑制率;间接免疫荧光法观察RA对整合素β1表达阳性率和波形蛋白形态的影响。结果倒置显微镜下,1×10-8~1×10-6mol.L-1组上皮细胞增多,成纤维细胞减少,1×10-5mol.L-1组2种细胞都减少;MTT法显示,1×10-7~1×10-5mol.L-1组细胞增殖受抑制,抑制作用呈剂量、时间依赖性(F检验,P<0.05),抑制率在10.8%~32.0%。1×10-6~1×10-5mol.L-1组整合素β1表达明显增高(x2检验,P<0.05),1×10-7~1×10-5mol.L-1组波形蛋白在细胞内铺展较均匀。结论RA具有抑制传代细胞增殖的作用,但表现为抑制成纤维细胞和促进上皮细胞二者作用的总和,只是抑制作用远远大于促进作用。RA抑制PCO的功能,可能是通过整合素途径来实现的。     

全反式维甲酸对高糖诱导肾小管上皮细胞增殖及凋亡的影响

摘要： 目的 探讨全反式维甲酸 （ATRA） 对高糖诱导人 HK-2 细胞增殖及凋亡的影响, 以期延缓糖尿病肾病（DN）。方法 体外培养 HK-2 细胞, 随机分为 6 组： 空白组 （未加任何刺激物）、 高糖组 （加 D-葡萄糖 30 mmol/L）、 高渗组 （加入甘露醇 24.5 mmol/L）、 低浓度 ATRA 组 （加入 ATRA 1×10-7 mol/L+D-葡萄糖 30 mmol/L）、 中浓度 ATRA 组（加入 ATRA 1×10-6 mol/L+D-葡萄糖 30 mmol/L）、 高浓度 ATRA 组 （加入 ATRA 1×10-5 mol/L+D-葡萄糖 30 mmol/L）。各组细胞培养 48 h。MTT 法检测各组细胞增殖情况, 流式细胞术检测各组细胞凋亡情况。结果 空白组与高渗组细胞光密度 （OD） 值和凋亡率差异无统计学意义。高糖组较空白组、 高渗组细胞增殖减少, 凋亡率增加; 低浓度、 中浓度、 高浓度 ATRA 组细胞增殖均较高糖组增加, 且低、 中及高浓度 ATRA 组依次增加, 凋亡率较高糖组减少, 且低、 中及高浓度 ATRA 组依次减少 （均 P＜0.05）。结论 ATRA 可促进高糖诱导的 HK-2 细胞增殖, 抑制其凋亡, 并与 ATRA 浓度可能具有一定的依赖性。     

Bupivacaine causes cytotoxicity in mouse C2C12 myoblast cells： involvement of ERK and Akt signaling pathways

Aim:

The adverse effects of local anesthetics (LAs) on wound healing at surgical sites have been suggested, and may be related to their cytotoxicity. This study was aimed to compare the cellular toxicity of bupivacaine and lidocaine (two well-known LAs), and to explore the molecular mechanism(s).

Methods:

Toxicity of bupivacaine and lidocaine was assessed in cultured mouse C2C12 myoblasts by cell viability and apoptosis assays. Effects of LAs on extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) activation, which are essential for cell proliferation and survival, were evaluated by immunoblotting.

Results:

Both LAs, especially bupivacaine, prevented cell growth and caused cell death in a dose-dependent manner. The half maximal inhibitory concentrations (IC₅₀) for bupivacaine and lidocaine were 0.49±0.04 and 3.37±0.53 mmol/L, respectively. When applied at the same dilutions of commercially available preparations, the apoptotic effect induced by bupivacaine was more severe than that of lidocaine in C2C12 cells. Furthermore, bupivacaine significantly diminished the ERK activation, which may underlie its anti-proliferative actions. Both LAs suppressed Akt activation, which correlated with their effects on apoptosis.

Conclusion:

Our study demonstrated that, when used at the same dilutions from clinically relevant concentrations, bupivacaine is more cytotoxic than lidocaine in vitro. Anti-proliferation and cell death with concomitant apoptosis mediated by bupivacaine may offer an explanation for its adverse effects in vivo (eg slowing wound healing at the surgical sites). A less toxic, long-acting anesthetic may be needed.     

新型维甲酸衍生物诱导NB4细胞分化的初步研究

目的:本研究探讨10种新型维甲酸衍生物对白血病细胞株NB4的抑制增殖和诱导分化活性。方法:维甲酸类衍生物作用于NB4细胞后,通过MTT法检测细胞的增殖。瑞氏染色法在倒置相差显微镜下观察加药处理前后细胞形态学变化。NBT还原实验法分析细胞的分化指标。FCM检测分析细胞周期和细胞表面分化抗原变化。结果:维甲酸衍生物作用3 d后,抑制细胞增殖作用呈剂量依赖效应。10种维甲酸衍生物(10-5mol/L)的诱导分化活性表现在油镜下观察NB4细胞向粒系分化成熟的改变,NBT阳性细胞率增加,细胞表面分化抗原CD11b表达量增加,CD13表达则减少。通过对细胞周期的分析,发现G0/G1期细胞表达量增加,呈G1期阻滞。结论:维甲酸衍生物2a-03,4a-02和5a-02显示有较强的诱导NB4细胞分化的活性。