MRP1/CD9蛋白在人肝细胞癌中的表达及其意义

目的　探讨MRP1 /CD9蛋白在人肝细胞癌 (HCC)中的表达及其与癌侵袭转移的关系。方法　构建肝癌组织芯片。样本包括肝细胞癌及癌旁肝组织 1 5 2例,癌栓 2 2例,肝内转移癌 4例,肝外转移癌 1 7例。正常对照肝组织 5例。应用免疫组织化学 (免疫组化 )方法检测肝癌组织芯片中样本MRP1 /CD9蛋白的表达。结果　 2 7% ( 4 1 /1 5 2 )肝细胞癌原发灶表达MRP1 /CD9蛋白。伴癌栓形成HCC中MRP1 /CD9蛋白表达率低于无癌栓形成者 (分别为 2 1. 8 2%和 4 0. 4 8% ; P < 0. 0 5 )。巨块型肝癌中MRP1 /CD9 蛋白表达率亦低于直径在 1 0cm以下者 (分别为 5%和 3 4. 8 2% ; P <0. 01 )。MRP1 /CD9蛋白表达尚与HCC病理分级及血清AFP水平有关:病理分级 2级的阳性表达率高于 3 ~ 4级 (分别为 3 9. 0 2%和 2 2. 5 2% ; P = 0. 0 4 3 ),血清AFP≤ 2 0μg/L者阳性表达率高于 >2 0μg/L者 (分别为 4 1. 9 4%和 2 2. 5 0% ; P = 0. 0 2 9 )。结论　肝细胞癌MRP1 /CD9蛋白表达水平低下可能与癌组织侵袭转移有关。     

血瘀证兔模型血管内皮细胞中一氧化氮合酶 的基因表达及其分泌一氧化氮的变化

【目的】了解实验性血瘀证动物模型血管内皮细胞中一氧化氮合酶(NOS)的基因表达及其分泌NO的变化。【方法】用半定量RT-PCR方法检测模型组体内血管内皮细胞及体外培养的细胞中组成型一氧化氮合酶mRNA的表达,并相应测定分泌的NO水平。【结果】与对照组比较,模型组兔体内血管内皮细胞中组成型NOS(cNOS)基因表达以及血浆和原代培养液中NO水平皆明显下降(P＜0.01),同期血浆中NO含量与内皮细胞中cNOSmRNA的表达水平呈正相关(r=0.739,P＜0.01)。两组间体外培养的传代细胞中cNOS基因表达及培养液上清中NO含量无明显差异(P＞0.05),但NO水平都比各自原代培养液中的明显升高(P＜0.01,P＜0.05)。【结论】短期内血瘀证兔模型体内内源性NO水平降低主要是cNOS基因表达下降导致的,随时间的延长,不排除诱导型NOS(iNOS)及体内其他因素对分泌NO的综合影响。     

血瘀证兔血清对培养的血管内皮细胞活性因子基因表达的影响

【目的】探索血瘀证形成和发展过程中血管内皮细胞活性因子内皮素和一氧化氮 (NO)改变的分子机理。【方法】用半定量RT -PCR方法 ,观察血瘀证兔模型血清对体外培养的血管内皮细胞内皮素 (ET)和组成型NO合成酶 (constitutivenitricoxidesynthase ,cNOS)基因表达的影响。【结果】血瘀证动物模型血清导致体外培养的内皮细胞中ET - 1mRNA表达升高 (P <0 .0 1) ,cNOSmRNA表达下降 (P <0 .0 1) ,两者呈显著负相关 (r=- 0 .857,P <0 .0 1)。【结论】血管内皮细胞中ET基因高表达及cNOS基因低表达导致的二者平衡失调在血瘀证形成和发展过程中起着重要作用。     

豚鼠内淋巴囊上皮细胞Na+,K+-ATP酶不同β亚基的mRNA表达

目的　进一步研究内淋巴囊上皮细胞Na ,K -ATP酶不同 β亚基的表达。方法　采用豚鼠内淋巴囊冰冻切片、原位杂交的方法检测Na ,K -ATP酶不同 β亚基mRNA在内淋巴囊上皮细胞中的表达。结果　在内淋巴囊上皮细胞胞浆内可见Na ,K -ATP酶不同 β亚基的阳性颗粒 ,β1亚基表达较弱 ,β2 亚基表达较强。结论　结合其他报道 ,内淋巴囊上皮细胞具有多种亚基异构体组成的Na ,K -ATP酶的表达 ,而结肠上皮、肾小管上皮的Na ,K -ATP酶几乎仅有α1β1型 ,提示其离子转运过程可能不同。     

Transfection of Immortalized Keratinocytes by Low Toxic Poly(2-(Dimethylamino)Ethyl Methacrylate)-Based Polymers

Skin carcinoma are among the most spread diagnosed tumours in the world. In this study, we investigated the transfection of immortalized keratinocytes, used as an in vitro model for skin carcinoma, using antisense technology and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based polymers, with original architecture and functionalities. We tested PDMAEMA polymers with different structures: linear, with two (DEA-PDMAEMA) or three (TEA-PDMAEMA) arms. The cytotoxicity of these polymers was assessed over a wide range of apparent M _n (from 7600 to 64 600). At a N/P ratio of 7.38, cytotoxicity increases with the M _n. Keratinocytes were transfected with a fluorescent oligonucleotide and then analyzed by flow cytometry. For the three architectures tested, the percentage of transfected cells and abundance of internalized oligonucleotide were closely related to the M _n of the polymer. Confocal microscopy and FACS analyses showed a wide spread fine granular distribution of the oligonucleotide up to 3 days post-transfection. Then, we assessed the silencing efficiency of the polymers, targeting GFP in GFP expressing keratinocytes. The maximal silencing effect (±40%) was obtained using a DEA-PDMAEMA polymer (M _n = 30 300). These results suggest that PDMAEMA-based polymers can be efficiently used to transfect immortalized keratinocytes and, thus, open new perspectives in the therapy of skin carcinoma.     

A Comparative Evaluation of Disulfide-Linked and Hydrophobically-Modified PEI for Plasmid Delivery

Non-viral gene therapy has become an important approach for treatment of hereditary and acquired diseases as a result of better understanding of molecular mechanisms involved in disease development. To design more effective gene carriers, plasmid DNA (pDNA) delivery to 293T cells was investigated by using two types of polymeric carriers; polymer constructed with disulfide (–S–S–) linkages and polymers modified with hydrophobic moieties. The base polymer used for this study was 2-kDa poly(ethylene imine) (PEI2), a relatively cell-compatible but ineffective gene carrier. The –S–S– linking was achieved via Michael addition reaction using cystamine bisacrylamide (CBA), whereas hydrophobic modification by N-acylation of PEI2 amines with palmitoyl chloride (PA). The cytotoxicity of the polymers was found to be lower than that of the 25-kDa branched PEI, but both types of modifications increased the toxicity of PEI2 to some extent. The polymers were able to form polyplexes with pDNA with variable hydrodynamic sizes (130–600 nm) and ζ-potential (3.6–20.9 mV). Based on the expression of the reporter gene Enhanced Green Fluorescent Protein (EGFP), disulfide linking significantly increased the efficiency of native PEI2, which was not effective on its own. The PA-modified PEI2 was also effective for gene delivery, but disulfide linkage of this polymer did not increase its efficiency any further. Our results showed that hydrophobic modification of 2-kDa PEI significantly improved its transfection efficiency but improvements in transfection efficiency as a result of disulfide linking was dependent on the nature of the polymeric building blocks.     

PEG- and PDMAEG-Graft-Modified Branched PEI as Novel Gene Vector: Synthesis,Characterization and Gene Transfection

The cytotoxicity of polyethylenimine (PEI) was a dominating obstacle to its application. Introduction of poly(ethylene glycol) (PEG) blocks to PEI is one of the strategies to alleviate the cytotoxocity of PEI. However, it is well known that the transfection efficiency of PEGylated PEI is decreased to some extent compared to the corresponding PEI. Thus, the aim of our study was to enhance the transfection efficiency of PEGylated PEI. A series of tri-block co-polymers, PEG-g-PEI-g-poly(dimethylaminoethyl L-glutamine) (PEG-g-PEI-g-PDMAEG), as novel vectors for gene therapy was synthesized and evaluated. PEG-g-PEI was first obtained by linking PEG and PEI using isophorone diisocyanate (IPDI) as coupling reagent. The anionic co-polymerization of γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) using PEG-g-PEI as a macro-initiator was carried out, followed by aminolysis with 2-dimethylaminoethylamine to obtain the target water-soluble tri-block co-polymer. The structures of the polymers were confirmed by FT-IR and ¹H-NMR. The influence of the molecular weight of PEI and the length of the PDMAEG chain on the physicochemical properties and transfection activity of polymer/DNA was evaluated. All PEI derivates were revealed to compact plasmid DNA effectively to give polyplexes with suitable size (approx. 100 nm) and moderate zeta potentials (10–15 mV) at N/P ratios over 10. The PEG-g-PEI-g-PDMAEG tri-block co-polymers displayed particularly low cytotoxicity, even at high concentration, reflecting an improved safety profile compared to PEI 25k. Gene transfection efficiency of PEG-g-PEI-g-PDMAEG on HeLa in the presence and absence of serum was determined. Remarkably, the transfection activity of PEG-g-PEI (10k)-g-PDMAEG (PPP-4)/DNA polyplex formulations was nearly twofold higher than PEI 25k/DNA formulations in vitro, and the transfection efficiency was less affected by the presence of serum. These results indicated that the synthesized PEG-g-PEI-g-PDMAEG tri-block co-polymers are promising candidates as carriers for gene delivery.     

Novel Quaternized Chitosan and Polymeric Micelles With Cross-Linked Ionic Cores for Prolonged Release of Minocycline

Novel multifunctional octadecyl quaternized carboxymethyl chitosans (OQCMCs) with varying degree of quaternary substitution (DS) and molecular mass were prepared and compared with quaternized chitosan. OQCMCs exhibited excellent solubility both in water and organic solvents. Nanoparticles of OQCMCs offered many advantages, such as easier fabrication and modulation of their size and degree of positive charge, and a lower cytotoxic effect compared with PEI (25 kDa). DNA can be successfully adsorbed on its surface. Electrostatic attraction of carboxymethyl and quaternary groups in OQCMCs was utilized as micellar template for the synthesis of cross-linked micelles. Formation and characteristics of OQCMC polymeric micelles were studied by fluorescence spectroscopy, tensiometry, SEM, TEM and particle size analysis. Self-assembled OQCMC micelles were evaluated as carrier of the lipophilic drug, minocycline hydrochloride (MH). MH was incorporated into cross-linked ionic cores of micelles with remarkably high efficiency (22.7%, w/w). MH-loaded OQCMC polymeric micelles exhibited a slow steady release profile over a 1-week period at 37°C. The OQCMC micelles are potentially useful for gene and lipophilic drug delivery applications.     

Preparation of novel silica-coated alginate gel beads for efficient encapsulation of yeast alcohol dehydrogenase

Biomimetic formation has undoubtedly inspired the preparation of novel organic–inorganic hybrid composites. In this study, silica-coated alginate gel beads were prepared by coating the surface of alginate gel beads with silica film derived from tetramethoxysilane (TMOS). The composition and structure of the silica film were characterized by FT-IR and SEM equipped with EDX. The swelling behavior of silica-coated alginate gel beads was studied to be more stable against swelling than that of alginate gel beads. The results showed that silica-coated alginate gel beads exhibited appropriate diffusion property. The effective diffusion coefficient (D _e) of NADH in silica-coated alginate beads was 1.76 × 10^?10 m²/s, while the effective diffusion coefficient in alginate beads was 1.84 × 10^?10 m²/s. The model enzyme yeast alcohol dehydrogenase (YADH) was encapsulated in silica-coated alginate and pure alginate beads, respectively. Enzyme leakage of YADH in alginate gel beads was determined to be 32%, while the enzyme leakage in silica-coated alginate gel beads was as low as 11%. Furthermore, the relative activity of YADH in alginate gel beads decreased almost to zero after 10 recycles, while the relative activity of YADH in silica-coated alginate gel beads was 81.3%. The recycling stability of YADH in silica-coated alginate gel beads was found to be increased significantly mainly due to the effective inhibition of enzyme leakage by compact silica film.     

Study of cellular responses to polymeric biomaterials using the differential display method

In this study, we attempted to detect altered gene expressions in the cells that had adhered to various surfaces using the differential display method. Thioglycollate-elicited peritoneal exudate cells (PEC) and mouse fibroblast (L929) cells were cultured on the polymer films. After a predetermined time, the total RNA was isolated from cells and the differential mRNA expressions were evaluated by RT-PCR method. As a result, in the differential display of amplified cDNA from PEC, the different patterns of cDNA fragments among the samples were obtained. This indicates that there were many different mRNA expressions depending on the polymer surfaces. The use of differential method was proven to be useful for studying cell-polymer interaction.