mPEG-PCL-g-PEI聚合物纳米粒介导的小干扰RNA递送

本文旨在合成小干扰核糖核酸(siRNA)递送载体聚合物聚乙二醇-聚己内酯-聚乙烯亚胺(mPEG-PCL-g-PEI),并探讨其体外siRNA递送性能。通过开环聚合反应制备二嵌段共聚物聚乙二醇-聚己内酯(mPEG-PCL-OH),将mPEG-PCL-OH的羟基末端依次化学转化为羧基(-COOH)和N-羟基琥珀酰亚胺(-NHS)生成mPEG-PCL-NHS,再将mPEG-PCL-NHS同枝化聚乙烯亚胺(PEI)反应生成三元共聚物mPEG-PCL-g-PEI。应用傅里叶变换红外光谱(FTIR)、核磁共振(NMR)和凝胶渗透色谱(GPC)对聚合物mPEG-PCL-g-PEI进行结构表征;通过复凝聚法制备mPEG-PCL-g-PEI/siRNA纳米粒,并测定其粒径和zeta电位;通过体外细胞MTT测试,比较mPEG-PCL-g-PEI/siRNA纳米粒和PEI/siRNA纳米粒的细胞毒性;通过体外细胞转染实验,考察不同N/P比的mPEG-PCL-g-PEI/siRNA纳米粒对萤火虫荧光素酶基因表达的抑制效率。结果表明,合成的三元聚合物(mPEG5k-PCL1.2k)1.4-g-PEI10k能压缩siRNA形成粒径为50...     

1,8-二乙酰基大黄酸-(2-溴)-乙酯的合成及对骨肉瘤MG-63细胞的作用研究

目的设计并合成新的大黄酸衍生物1,8-二乙酰基大黄酸-(2-溴)-乙酯(大黄酸衍生物B),探讨其对骨肉瘤MG-63细胞的作用和机制。方法以大黄酸为原料合成了1,8-二乙酰基大黄酸-(2-溴)-乙酯,经UV、IR、NMR确定合成产物的结构,并利用HPLC测定其纯度。采用MTT法测定大黄酸和大黄酸衍生物B对骨肉瘤MG-63细胞的体外生长抑制作用;流式细胞仪检测细胞凋亡和细胞周期分布。结果经UV、IR、~1H NMR、¹³C NMR进行分子结构表征,确证合成的目标化合物为1,8-二乙酰基大黄酸-(2-溴)-乙酯,纯度>98%。MTT结果表明,大黄酸和大黄酸衍生物B对骨肉瘤MG-63细胞的IC₅₀值分别为110.60、25.78μmol·L^-1。流式细胞仪检测细胞凋亡和周期结果表明,80μmol·L^-1的大黄酸和大黄酸衍生物B对骨肉瘤MG-63细胞的凋亡率分别为(6.87±0.53)%、(48.84±2.20)%,且主要将细胞周期阻滞在S期。结论合成化合物1,8-二乙酰基大黄酸-(2-溴)-乙酯的体外抗肿瘤活性明显优于大黄酸,且具有阻滞骨肉瘤MG-63细胞周期进程和促进其凋亡的作用。     

三氧化二砷体外对人EGFR T790M肺腺癌细胞抑制作用的研究

目的观察三氧化二砷对在体外对EGFR T790M突变非小细胞肺癌H1975细胞的生长抑制作用。方法运用MTT法检测三氧化二砷对H1975细胞增殖抑制,流式细胞仪检测细胞周期变化。倒置显微镜观察三氧化二砷作用下细胞形态变化。结果不同浓度的三氧化二砷在体外均能明显抑制H1975细胞的增殖,且具有时间和浓度依赖性,与浓度和时间呈正比。倒置显微镜观察发现三氧化二砷具有诱导H1975细胞出现核固缩、染色质凝集等凋亡形态改变。流式细胞仪检测提示细胞周期停留在G0/G1期。结论三氧化二砷在体外能抑制EGFRT790M突变肺癌细胞增殖、诱导调亡和改变细胞周期的作用。     

新型维甲酸衍生物ATPR对肿瘤细胞株SKOV3增殖及分化的影响

目的：探讨新型维甲酸衍生物4-氨基-2-三氟甲基苯基维甲酸酯（4-amino-2-trifluorometh—yl—phenylretinate,ATPR）对卵巢癌SKOV3细胞的增殖及分化作用。方法：ATPR作用于细胞株SKOV3后,MTT法检测细胞的增殖;吉姆萨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;ELISA法检测卵巢癌标志物糖链抗原125（CAl25）;流式细胞术检测细胞周期的变化情况。结果：ATPR呈浓度依赖性抑制SK—OV3细胞增殖,明显抑制作用出现在药物作用48h,但在72h后则更显著;倒置显微镜下观察发现细胞形态学趋于成熟分化;SKOV3中CAl25水平下降;流式细胞仪测定G0／G1期细胞表达量增加,S期细胞表达量减少,细胞周期进程受影响,细胞阻滞在G0／G1期。结论：ATPR对SKOV3细胞具有抑制增殖和-定的诱导分化作用。     

4-氨基-2-三氟甲基苯基维甲酸酯诱导神经母细胞瘤SH-SY5Y分化的研究

目的 该实验探讨一种新型的维甲酸衍生物4-氨基-2-三氟甲基苯基维甲酸酯(4-amino-2-Trifluoromethyl-Phenyl Reti-nate,ATPR)对神经母细胞瘤SH-SY5Y体外增殖及分化的作用.方法 不同浓度的ATPR作用SH-SY5Y细胞后,用MTT法检测细胞增殖状况;吉姆萨染色法于倒置相差显微镜下观察细胞形态学变化;免疫细胞化学法检测神经元特异性分化标志物——神经元特异性烯醇化酶(NSE);流式细胞仪测定细胞周期的变化;采用RT-PCR检测ATPR作用后RARβ mRNA的表达情况.结果 ATPR呈浓度依赖性抑制SH-SY5Y细胞增殖;倒置显微镜下观察细胞形态趋于成熟分化;NSE水平上升;细胞S期表达减少,G0/G1期表达量有所上升,细胞周期进程受到影响.维甲酸受体RARβ表达量增加.结论 ATPR对SH-SY5Y 细胞具有抑制增殖和一定的诱导分化作用,其作用机制可能与上调RARβ mRNA表达水平有关.     

Artesunate对K562细胞增殖作用及细胞周期的影响

目的:观察Artesunate对人白血病细胞株K562细胞增殖及细胞周期的影响.方法:用Artesunate处理K562细胞,细胞计数法绘制生长曲线;MTT比色法检测细胞增殖抑制的效果;倒置光显微镜观察细胞形态学变化;流式细胞术(flow cytometry,FCM)进行细胞周期分析.结果:Artesunate的浓度为1×10-4、1×10-5、1×10-6mol/L时,细胞生长受到显著抑制,并呈剂量依赖性;对K562细胞的半数抑制浓度为3.75×10-5mol/L;倒置光显微镜观察细胞出现不同程度的皱缩,核分裂相减少,内可见空泡,培养基中可见较多分泌颗粒;流式细胞仪检测G2期细胞比例增高.结论:Artesunate对K562细胞生长及增殖有抑制作用,其作用机制可能是通过抑制DNA合成,使细胞增殖停留在G2/M期来实现的.     

青蒿琥酯对人胃癌BGC803细胞力学特性增殖迁移影响的研究

目的利用不同生物学检测方法观察青蒿琥酯(Art)对胃癌细胞在增殖、细胞周期、迁移黏附、细胞形态结构及细胞力学特性等方面的影响。方法采用不同浓度的Art作用于人胃癌细胞株BGC803,用药一定时间后,细胞增殖实验(MTT)检测细胞增殖情况;流式细胞仪检测细胞周期变化情况;利用划痕实验检测细胞迁移能力的变化;使用原子力显微镜观察细胞黏弹性、膜表面形貌等细胞力学。结果对照组(0 mg/L Art)细胞增殖、迁移能力显著高于Art实验组(30 mg/L Art);而对照组G1期细胞比例则显著低于实验组;实验组细胞黏附力显著高于对照组细胞,而细胞的弹力则显著低于对照组细胞;对细胞表面形态检测发现用药后的实验组细胞膜表面不再光滑,有多处凹陷出现,并且细胞表面粗糙度大幅上升。结论实验结果表明Art是有效的抗胃癌药物,可以通过抑制细胞增殖、阻滞细胞周期、改变细胞力学及细胞表面微观形态结构等方面起到抑癌作用。     

地钱素C衍生物F41对子宫颈癌HeLa细胞凋亡的影响

目的对地钱素C(MC)羟基衍生物进行筛选,寻找具有高抗肿瘤活性的MC衍生物,并初步探讨其抗肿瘤的细胞与分子生物学机制。方法用MTT法观察56种MC羟基衍生物对子宫颈癌HeLa细胞的毒性作用,筛选其中细胞毒作用最强的MC衍生物,并比较其与MC对HeLa细胞存活的抑制作用。用倒置显微镜、DAPI染色和DNA梯带检测其对细胞凋亡的影响。用流式细胞术检测其对细胞周期的影响。用Western印迹法检测其对细胞周期和凋亡相关蛋白表达的影响。结果在56种MC衍生物中,F41对HeLa细胞毒性最强,抑制HeLa细胞存活的IC50为(11.31±2.13)μmol·L-1,明显低于MC〔IC50为(17.19±3.28)μmol·L-1〕(P<0.01)。F41和MC与HeLa细胞作用24,48和72h,F41对HeLa细胞存活的抑制作用均明显强于MC(P<0.05)。形态学和DNA梯带检测显示,F41处理后,HeLa细胞皱缩变小,有空泡和凋亡小体出现,胞核浓缩变小,DNA电泳呈梯状条带。流式细胞分析显示,F41 15μmol·L-1处理HeLa细胞24h,G2/M期细胞占总细胞的比例为(43.8±3.0)%,明显高于对照组的(13.1±1.6)%(P<0.01);G1期细胞比例为(34.8±3.8)%,明显低于对照组的(63.6±5.5)%(P<0.01)。Western免疫印迹结果表明,F41可使HeLa细胞内磷酸化细胞周期蛋白依赖性蛋白激酶1水平降低,细胞周期蛋白B1和P53蛋白表达增多。结论 F41可诱导HeLa细胞凋亡,抑制HeLa细胞分裂,其抗肿瘤活性可能强于MC。     

红花多糖对人结肠癌 LoVo 细胞增殖、凋亡、侵袭作用机制研究

目的 红花多糖对人结肠癌 LoVo 细胞增殖、凋亡、侵袭作用机制研究。方法 体外培养人结肠癌 LoVo 细胞,在不同浓度(0.5、1.0、1.5 g·L-1)的红花多糖处理后,分别在干预24、48、72 h后采用 MTT 比色法测定红花多糖对LoVo细胞的增殖抑制作用,利用倒置显微镜观察细胞的形态,采用PI单标流式细胞仪测定细胞周期分布,Annexin V-FITC /PI 双标法检测细胞凋亡率;采用RT-PCR检测Bax、Bcl-2、Caspase-3 mRNA表达水平及检测Caspase-3活性,Western-blot检测Bax、Bcl-2、Caspase-3蛋白表达情况;应用Transwell细胞侵袭试验检测红花多糖对结肠癌LoVo细胞侵袭能力的影响。结果 MTT 检测显示红花多糖能显著抑制人结肠癌 LoVo 细胞的生长、増殖,呈现浓度和时间效应关系;倒置显微镜显示红花多糖能使结肠癌LoVo 细胞出现典型凋亡特征;流式细胞仪分析结果提示红花多糖具有调节人结肠癌LoVo细胞周期的功能。RT-PCR实验和Western-blot实验结果显示随着红花多糖浓度增加可促进LoVo细胞中Bax、Caspase-3 mRNA表达,并抑制 Bcl-2 mRNA表达。Transwell侵袭实验示随着红花多糖浓度增加,LoVo 细胞侵袭能力降低。结论 红花多糖能显著促进人结肠癌 LoVo 细胞凋亡,可使结肠癌LoVo 细胞增殖受到抑制,侵袭能力减弱,调节细胞周期,红花多糖的抗肿瘤的生物学效应可能与Bax、Bcl-2、Caspase-3 的信号通路有关。     

光叶番荔枝中二萜类化合物对人肝癌细胞株SMMC-7721生长的抑制

目的研究光叶番荔枝中二萜类化合物对人肝癌细胞株SMMC-7721生长的抑制作用。方法以四唑盐比色试验(MTT)法检测二萜类化合物对SMMC-7721细胞生长的抑制率,用相差倒置显微镜观察细胞形态变化,用流式细胞术检测细胞周期、凋亡率及多药耐药基因1(MDR1)的表达水平。结果5～25／2M的二萜类化合物对SMMC-7721细胞有较强的抑制作用,与浓度呈线性相关,并呈时间依赖性,72h半数抑制浓度(IC50)为19．41／2M,20／2M的药物作用于SMMC-7721细胞48h,流式细胞术检测细胞周期阻滞于G0～G1期,细胞凋亡率为24．95％;在相差倒置显微镜下观察到细胞凋亡的特征形态改变,MDR1表达明显下调,且随药物浓度的增加MDR1表达水平逐渐下降。结论光叶番荔枝中二萜类化合物对人肝癌细胞株SMMC-7721有显著的抑制作用,可诱导癌细胞凋亡,明显下调MDR1的表达,具有逆转多药耐药性的作用。     

泽泻汤对油酸诱导HepG2细胞脂肪堆积的抑制作用

目的评价泽泻汤95%乙醇、50%乙醇和水提取物抑制HepG2肝细胞脂肪累积能力。方法采用油酸诱导HepG2细胞脂肪累积建立模型,以噻唑兰染色和油红O染色法吸光度值为指标,结合细胞内脂滴形态,评价泽泻汤对油酸诱导HepG2细胞脂肪累积的干预作用。结果泽泻汤提取物作用HepG2细胞后,经染色后均发现细胞内脂滴明显减少,且质量浓度越高脂类含量越少,50%乙醇提取物和水提物在终质量浓度160、320μg/m L时,细胞内脂滴与模型组相比明显降低(P0.01),95%乙醇提物80μg/m L作用细胞24 h后,细胞内脂滴较模型组有减少(P0.05)。结论 3种泽泻汤提取物均能够减少细胞内脂肪的堆积,其中以50%乙醇提取物对HepG2细胞脂肪堆积抑制能力最强。     

Polyacrylamide-Chitosan Hydrogels: In Vitro Biocompatibility and Sustained Antibiotic Release Studies

Controlled drug delivery is gaining importance over the conventional methods of drug administration because of its inherent benefits. Self-regulated release from the delivery vehicle may enhance drug potency with a sustained action. The present study describes a novel hydrogel blend of polyacrylamide with chitosan for controlled delivery of antibiotics. Hydrogel was synthesized by cross-linking acrylamide-chitosan mixture (8:2 v/v) with N,N' methylene bisacrylamide. Hydrogel was characterized for surface morphology, hydrophilicity, pH-dependent swelling properties, cytotoxicity, and control release properties. Scanning electron microscopy (SEM) revealed the macroporous surface morphology of the matrix with average pore size at 104n plus/minus 7.61 mu. Hydrogel was found to be highly hydrophilic as assessed by octane contact angle (154.5 + 0.572) measurement. Hydrogel showed no cytotoxic effects on NIH3T3 and HeLa cells up to 40% of extract concentrations as determined by MTT and neutral red assay. This showed hydrogel biocompatibility and thus absence of deleterious effects of the hydrogel on cell viability and functionality. Hydrogels did not show any pH-dependent swelling profile, and they swelled considerably to achieve a swelling ratio of approximately 16.0 at the end of 24 hr. Amoxicillin was incorporated in the hydrogel matrix as a candidate antibiotic for release studies. In vitro release studies of amoxicillin revealed the sustained nature of delivery and matrix released 56.47 + 1.12% and 77.096 + 1.72% of amoxicillin at the end of 24 and 75 hr, respectively. Although in vivo studies are awaited, the present study provides enough documentation to consider polyacrylamidechiotsan hydrogel as a possible candidate for controlled delivery of antibiotics.     

Polyacrylamide-chitosan hydrogels: in vitro biocompatibility and sustained antibiotic release studies

Controlled drug delivery is gaining importance over the conventional methods of drug administration because of its inherent benefits. Self-regulated release from the delivery vehicle may enhance drug potency with a sustained action. The present study describes a novel hydrogel blend of polyacrylamide with chitosan for controlled delivery of antibiotics. Hydrogel was synthesized by cross-linking acrylamide-chitosan mixture (8:2 v/v) with N,N' methylene bisacrylamide. Hydrogel was characterized for surface morphology, hydrophilicity, pH-dependent swelling properties, cytotoxicity, and control release properties. Scanning electron microscopy (SEM) revealed the macroporous surface morphology of the matrix with average pore size at 104n plus/minus 7.61 mu. Hydrogel was found to be highly hydrophilic as assessed by octane contact angle (154.5 + 0.572) measurement. Hydrogel showed no cytotoxic effects on NIH3T3 and HeLa cells up to 40% of extract concentrations as determined by MTT and neutral red assay. This showed hydrogel biocompatibility and thus absence of deleterious effects of the hydrogel on cell viability and functionality. Hydrogels did not show any pH-dependent swelling profile, and they swelled considerably to achieve a swelling ratio of approximately 16.0 at the end of 24 hr. Amoxicillin was incorporated in the hydrogel matrix as a candidate antibiotic for release studies. In vitro release studies of amoxicillin revealed the sustained nature of delivery and matrix released 56.47 + 1.12% and 77.096 + 1.72% of amoxicillin at the end of 24 and 75 hr, respectively. Although in vivo studies are awaited, the present study provides enough documentation to consider polyacrylamidechiotsan hydrogel as a possible candidate for controlled delivery of antibiotics.     

骨化三醇体外诱导人肝癌细胞IGFBP-3的表达及细胞凋亡的实验研究

目的:探讨骨化三醇[2β-(3一hydro-)-calcitriol]对人肝癌细胞HepG2增殖和凋亡的影响及其作用机制。方法:应用MTT比色法观察骨化三醇对HepG2的生长抑制作用,以荧光显微镜和流式细胞仪观察HepGz的凋亡,RT-PCR检测骨化三醇处理前后HepG2细胞中胰岛素样生长因子受体结合蛋白-3(IGFBP- 3)mRNA表达水平的变化。结果:骨化三醇显著抑制HepG2细胞的体外增殖,并诱导细胞凋亡。不同浓度的骨化三醇(10-8,10-7,10-6,10-5mol·L-1)作用HepG2 48 h细胞凋亡率分别达(27.3±s 2．3)％,(33±6)％,(35±5)％,(48±7)％,明显高于对照组(8±4)％(P<0．05)。RT-PCR结果显示骨化三醇处理HepG2 24 h后IGFBP-3 mRNA表达水平明显升高。结论:骨化三醇体外显著抑制HepG2增殖并促进其凋亡,其作用机制可能与通过激活细胞内IGFBP-3 mRNA的表达有关。     

3-O-(3-甲氧基肉桂酰)-甘草次酸酯对HepG2细胞体外抑制作用研究

目的探讨3-O-(3-甲氧基肉桂酰)-甘草次酸酯对Hep G2细胞体外抑制作用及其作用机制。方法采用MTT法考察药物对肝癌细胞Hep G2、宫颈癌细胞He La、结肠癌细胞HT-29、心肌细胞H9C2、犬肾上皮细胞MDCK和血管内皮细胞HY926的细胞毒性,利用细胞染色法观察不同浓度3-O-(3-甲氧基肉桂酰)-甘草次酸酯对Hep G2细胞形态的影响,流式细胞术评价该化合物诱导Hep G2细胞的凋亡。结果 3-O-(3-甲氧基肉桂酰)-甘草次酸酯具有较好的抑制Hep G2细胞增殖活性作用,其半数有效抑制浓度(IC50)为8.28μmol/L,明显强于甘草次酸的活性(IC5050μmol/L)。3-O-(3-甲氧基肉桂酰)-甘草次酸酯及其合成原料甘草次酸、3-甲氧基肉桂酸对非肿瘤细胞MDCK、HY926、H9C2细胞毒性较弱(IC5024μmol/L)。3-O-(3-甲氧基肉桂酰)-甘草次酸酯浓度在12.5μmol/L时,对MDCK、HY926、H9C2细胞抑制率分别为17.05%、16.08%、4.66%,与对Hep G2细胞的抑制效果相比差异明显。不同浓度3-O-(3-甲氧基肉桂酰)-甘草次酸酯对Hep G2细胞Giemsa染色、H33342染色的细胞形态有明显差异。随着3-O-(3-甲氧基肉桂酰)-甘草次酸酯浓度的升高,早期凋亡率逐渐增加,而晚期凋亡率无明显变化,提示3-O-(3-甲氧基肉桂酰)-甘草次酸酯可以有效地诱导细胞早期凋亡,并呈现一定的量效关系。结论 3-O-(3-甲氧基肉桂酰)-甘草次酸酯具有良好的抗肿瘤作用,对Hep G2细胞抑制效果最好,对非癌细胞MDCK、HY926、H9C2没有明显抑制效果,该作用主要通过诱导Hep G2细胞凋亡,且呈现一定的量效关系。     

Immunosuppressant deoxyspergualin-induced inhibition of cell proliferation is accompanied with an enhanced reduction of tetrazolium salt

Deoxyspergualin (DSG) has both antitumor and immunosuppressive activities. We explored the mechanism of DSG activities using an aqueous soluble analogue, methyldeoxyspergualin (MeDSG) for in vitro culture studies. It is known that DSG has inhibitory effects on cell proliferation, and we also observed that MeDSG inhibited [3H]-thymidine incorporation by rapidly dividing murine T cell hybridomas. However, when tetrazolium (MTT) colorimetric assay was adopted to evaluate its inhibitory effects on cell proliferation, MeDSG induced an enhanced MTT reduction. When we examined whether these results were applicable to the actively dividing cells of other origins than T cells, similar effects were seen with Raji cells, J774.1 cells and NIH3T3 cells. N-30, another analogue which was capable of suppressing anti-SRBC antibody production in vivo, also induced inhibition of cell growth and an enhanced MTT reduction. In contrast, the analogue which failed to prevent the antibody production, neither enhanced MTT reduction nor inhibited cell proliferation. Our results demonstrated that the ability to generate MTT formazan in dividing cells is a common property among, DSG analogue with the immunosuppressive and antiproliferative activities.     

Resistance of HepG2 cells against the adverse effects of ethanol related to neutral lipid and phospholipid metabolism

The influence of both short- and long-term ethanol exposure on the lipid metabolism was determined in the human hepatoma cell line HepG2. Ethanol did not cause any cytotoxicity or lipid peroxidation even after 7 days of 100 mM ethanol treatment of HepG2 cells. Incubation of cells in the presence of [1-(14)C]ethanol demonstrated that these cells actively metabolize ethanol to acetyl CoA, incorporating the radioactive label into neutral lipids and phospholipids. [1,2,3-(3)H]glycerol was efficiently used in phospholipid and neutral lipid biosynthesis, showing higher radioactivity in phosphatidylcholine, phosphatidylethanolamine and triacylglycerols. Exposure of HepG2 cells to 100 mM ethanol for 24 hr did not significantly modify the incorporation of glycerol into newly synthesized phospholipids and neutral lipids, nor was lipid degradation affected by the presence of ethanol. When the alcohol treatment was prolonged for 7 days, incorporation of [1,2,3-(3)H]glycerol into triacylglycerols and diacylglycerols showed a slight increase concomitantly with decreased radioactivity in the major phospholipids, phosphatidylcholine and phosphatidylethanolamine. In addition, these changes were associated with a greater release of radiolabeled triacylglycerols into the culture medium. These results indicate that ethanol does not cause in HepG2 cells the marked lipogenic stimulation widely shown in hepatocytes, and demonstrate that HepG2 cells strongly resist the adverse effects of ethanol. Since these cells lack the isoenzymatic form of cytochrome P(450) mainly involved in the ethanol metabolism (namely cytochrome P(450)2E1) and also are devoid of alcohol dehydrogenase activity, we propose that the toxic actions of ethanol on liver must be linked to the activity of one or both of these systems.     

Uptake characteristics of galactosylated emulsion by HepG2 hepatoma cells

Galactosylated (Gal) emulsions containing various molar ratios of cholesten-5-yloxy-N-(4-((1-imino-2-D-thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol) as a ligand for asialoglycoprotein receptors were prepared to study the effect of the galactose content of Gal-emulsions labeled with [3H]cholesteryl hexadecyl ether on their targeted delivery to hepatocytes. The uptake characteristics of Gal-emulsions having Gal-C4-Chol of 1, 3, 4, 6, and 9 mol% were evaluated in HepG2 cells which possess asialoglycoprotein receptors and NIH3T3 cells which are lack of asialoglycoprotein receptors. The uptake and internalization by HepG2 cells was enhanced by the addition of Gal-C4-Chol to the Gal-emulsions whereas the uptake of Gal-emulsions by NIH3T3 cells was not much and was comparable with that of bare-emulsions. In the presence of excess Gal-BSA, the uptake of Gal-emulsions having Gal-C4-Chol of 4, 6, and 9% was inhibited suggesting asialoglycoprotein receptor mediated uptake. Moreover, Gal-emulsions having Gal-C4-Chol of 4, 6, and 9% showed a slight increase in surface binding and exhibited extensive uptake and internalization into HepG2 cells. The present study strongly suggested that the Gal-emulsions are taken up by the asialoglycoprotein receptor-mediated endocytosis and galactose density of Gal-emulsions is important for effective recognition and cell internalization.     

舒芬太尼对体外培养HepG 2 细胞增殖及细胞凋亡的影响

目的了解阿片类镇痛药舒芬太尼对体外培养肝癌细胞株HepG2细胞增殖、细胞周期及细胞凋亡的影响。方法体外培养人肝癌HepG2细胞,实验组在RPMI-1640培养液中加入不同浓度舒芬太尼（0.0001,0.001,0.01,0.1,1,10,20μmol·L-）1,对照组RPMI-1640培养液中不加舒芬太尼,分别孵育48小时后,采用MTT比色法检测HepG2细胞增殖活性,采用流式细胞技术检测舒芬太尼对HepG2细胞周期的影响,用Hoechst33258染色方法分析鉴定舒芬太尼对HepG2细胞凋亡的影响。结果舒芬太尼浓度≥1μmol·L-1时,细胞增殖活性较对照组显著下降（P〈0.05）;随着舒芬太尼浓度增加,G1期细胞比例逐渐增高,S期细胞比例逐渐降低。用Hoechst33258方法染色后在表面荧光显微镜下发现实验组部分HepG2细胞发生凋亡,当舒芬太尼浓度≥0.1μmol·L-1时,荧光显微镜下一个视野内凋亡细胞占总细胞数的比例较对照组显著增加（P〈0.05）。结论舒芬太尼在临床常用剂量范围内对肝癌细胞株HepG2细胞增殖和凋亡无明显影响,当浓度≥1μmol·L-1时可以抑制人肝癌细胞株HepG2细胞增殖,主要将细胞周期阻滞在G1期,当舒芬太尼浓度≥0.1μmol·L-1时可引起肝癌HepG2细胞凋亡。     

Short multi-armed polylysine-graft-polyamidoamine copolymer as efficient gene vectors

Polyamidoamine-polylysine graft copolymers (PAMAM-g-PLL) were prepared by ring-opening polymerization of benzyloxycarbonyl lysine N-carboxyanhydride (Lys(Z)-NCA) initiated with primary amine of generation 4 polyamidoamine (PAMAM G4) and subsequent deprotection of polyamidoamine-poly-(benzyloxycarbonyl lysine) copolymer (PAMAM-PLL(Z)). The chemical structure and composition of the PAMAM-g-PLL with varying length of PLL arms were characterized by Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy (¹H NMR). Agarose gel electrophoresis test revealed that the PAMAM-g-PLL could completely combine DNA to form complexes. The scanning electronic microscopy (SEM) and atomic force microscopy (AFM) observation showed that the morphology of these complexes was spherical. Dynamic light scattering (DLS) measurement illustrated that the sizes of complexes were in range of 100-200 nm. The MTT assay demonstrated that cytotoxicity of PAMAM-g-PLL were lower than the either PAMAM G4 or the poly-l-lysine-15k (PLL-15k). The in vitro transfection test indicated that the PAMAM-g-PLL with 3.8 average polymerization degrees of PLL arms (PAMAM-PLL-3.8) displayed significantly higher transfection efficiency than that of PAMAM G4 and PLL-15k at the same N/P ratio, Furthermore, PAMAM-PLL-3.8 at the N/P of 40 or 80 displayed better serum-resistant capability than that of PEI-25k and Lipofectamine 2000. The DNA local delivery test in rabbit vessel exhibited that the restenosis was inhibited to a significant extent. The above facts revealed that PAMAM-PLL-3.8 is a promising gene vector with low cytotoxicity, high transfection efficiency and serum-resistant ability.