Persistent gene expression after retroviral gene transfer into liver cells in vivo.

The development of liver-directed gene therapy protocols depends upon the ability to transfer genes into a large number of liver cells such that the genes are expressed persistently. We used a retroviral vector to transfer the gene for neomycin phosphotransferase (neo) into mouse liver cells in vivo. Direct injection of the retrovirus preparation into mitotically active (regenerating) liver parenchyma resulted in efficient gene transfer, with neo sequences detectable in the livers of every animal tested 10 weeks to 6 months later. The neo gene was expressed for at least 3 months. This methodology may eventually be applicable to the treatment of human disease.     

Age-dependent expression of the erythropoietin gene in rat liver and kidneys.

Using RNAse protection, we have made quantitative measurements of erythropoietin (EPO) mRNA in liver and kidneys of developing rats (days 1-54), to determine the relative contribution of both organs to the total EPO mRNA, to monitor changes which occur with development, and to compare the hypoxia-induced accumulation of EPO mRNA with the changes in serum EPO concentrations. To determine whether developmental and organ-specific responsiveness is related to the type of hypoxic stimulus, normobaric hypoxia was compared with exposure to carbon monoxide (functional anemia). Under both stimuli EPO mRNA concentration in liver was maximal on day 7 and declined during development. In contrast, EPO mRNA concentration in kidney increased during development from day 1 when it was 30-65% the hepatic concentration to day 54 when it was 12-fold higher than in liver. When organ weight was considered the liver was found to contain the majority of EPO mRNA in the first three to four weeks of life, and although, in stimulated animals, the hepatic proportion declined from 85-91% on day 1, it remained approximately 33% at day 54 and was similar for the two types of stimuli. When normalized for body weight the sum of renal and hepatic EPO mRNA in animals of a particular age was related linearly to serum hormone concentrations. However, the slope of this regression increased progressively with development, suggesting age-dependent alterations in translational efficiency or EPO metabolism.     

Advance in polyamidoamine dendrimers as gene delivery agents

Ｇｅｎｅｔｈｅｒａｐｙｒｅｃｅｎｔｌｙｈａｓｂｅｃｏｍｅａｎｉｍｐｏｒｔａｎｔａｒｅａｏｆｒｅｓｅａｒｃｈａｓａｎｅｗｔｈｅｒａｐｅｕｔｉｃｍｅｔｈｏｄ .Ｉｎｖｉｖｏａｎｄｉｎｖｉｔｒｏｇｅｎｅｔｈｅｒａｐｉｅｓｒｅｑｕｉｒｅｅｆｆｉｃｉｅｎｔｄｅｌｉｖｅｒｙｏｆｇｅｎｅｔｉｃｍａｔｅｒｉａｌｉｎｔｏａｃｅｌｌａｎｄｐｒｅｆｅｒａｂｌｙｈｉｇｈｌｅｖｅｌｓｏｆｅｘｐｒｅｓ ｓｉｏｎｏｆｔｒａｎｓｆｅｒｒｅｄｇｅｎｅ.Ｔｒａｄｉｔｉｏｎａｌｌｙ ,ｇｅｎｅｄｅｌｉｖｅｒｙｓｙｓｔｅｍｓａｒｅｃｌａｓｓ…     

Coagulation-dependent gene expression and liver injury in rats given lipopolysaccharide with ranitidine but not with famotidine

In an animal model of drug idiosyncrasy, rats cotreated with nonhepatotoxic doses of lipopolysaccharide (LPS) and ranitidine (RAN) develop hepatocellular injury, whereas rats treated with LPS and famotidine (FAM) do not. The coagulation system and neutrophils (PMNs) are requisite mediators of LPS/RAN-induced liver injury. We tested the hypothesis that unique gene expression in LPS/RAN-treated rats requires coagulation system activation and that these changes are absent in rats given LPS and FAM. Rats were treated with a nonhepatotoxic dose of LPS (44.4 x 10(6) endotoxin units/kg i.v.) or its vehicle, and then 1 h later, they were treated with heparin (3000 U/kg) or its vehicle. One hour thereafter, they were given RAN (30 mg/kg), FAM (6 mg/kg, a pharmacologically equiefficacious dose, or 28.8 mg/kg, an equimolar dose), or vehicle (i.v.). They were killed 2 or 6 h after drug treatment for evaluation of hepatotoxicity, coagulation system activation, and liver gene expression (2 h only). Statistical filtering of gene array results and real-time polymerase chain reaction identified groups of genes expressed in LPS/RAN-treated rats but not LPS/FAM-treated rats that were either changed or unchanged by heparin administration. For example, LPS/RAN-induced mRNA expression of the inflammatory mediators interleukin-6, cyclooxygenase-2, and macrophage inflammatory protein-2 (MIP-2) was reduced by anticoagulation. Enhancement of serum MIP-2 and plasminogen activator inhibitor-1 concentrations in LPS/RAN-treated rats was prevented by anticoagulation. The results suggest cross-talk between hemostasis-induced gene expression and inflammation (e.g., PMN function) in the genesis of hepatocellular injury in LPS/RAN-treated rats. In contrast, neither the expression of such genes nor hepatocellular necrosis occurred in rats treated with LPS/FAM.     

Activated polyamidoamine dendrimers, a non-viral vector for gene transfer to the corneal endothelium.

We investigated the efficiency of activated polyamidoamine dendrimers, a new class of nonviral vectors, to transfect rabbit and human corneas in ex vivo culture. In addition to assessing the expression of a marker gene we have demonstrated that this approach can be used to induce the production of TNF receptor fusion protein (TNFR-Ig), a protein with therapeutic potential. Whole thickness rabbit or human corneas were transfected ex vivo with complexes consisting of dendrimers and plasmids containing lacZ or TNFR-Ig genes. Following optimisation 6-10% of the corneal endothelial cells expressed the marker gene. Expression was restricted to the endothelium and was maximal after transfection with 18:1 (w/w) activated dendrimer:plasmid DNA ratio and culture for 3 days. The supernatant of corneas transfected with TNFR-Ig plasmid contained TNFR-Ig protein which was able to inhibit TNF-mediated cytotoxicity in a bioassay. We have therefore shown that activated dendrimers are an efficient nonviral vector capable of transducing corneal endothelial cells ex vivo. They may have applications in gene-based approaches aimed at prevention of corneal allograft rejection or in treatment of other disorders of corneal endothelium.     

HOXB7基因沉默表达对大鼠氧化应激及肝脏Nrf2表达的影响

目的探讨同源盒基因(HOX)B7基因沉默表达对大鼠氧化应激及肝脏NF-E2相关因子2(Nrf2)表达的影响。方法C57野生型雄性大鼠20只(对照组)与HOXB7基因沉默C57雄性大鼠20只(沉默组)分别采用皮下多点注射50%四氯化碳法建立肝硬化模型。记录两组建模后第4周、第8周大鼠体重;采用速率法检测两组建模后第4周、第8周血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、γ-谷氨酰转肽酶(GGT)含量;采用化学比色法检测建模后第4周、第8周肝脏组织丙二醛(MDA)、总超氧化物歧化酶(T-SOD)和谷胱甘肽还原酶(GSH)含量;免疫组化分析SOD表达;记录建模后第8周的肝脏Nrf2 mRNA与蛋白表达水平。结果两组大鼠建模后主要表现为毛发不整、反应变迟缓,病理显示所有大鼠均建模成功。建模后第4周、第8周沉默组大鼠体重显著高于对照组(P<0.05)、血清AST、ALT、GGT值以及肝脏组织MDA含量均显著低于对照组(P<0.05),肝脏组织T-SOD和GSH含量显著高于对照组(P<0.05);沉默组建模后第8周的肝脏SOD表达阳性率显著高于对照组(P<0.05)。沉默组建模后第8周的肝脏Nrf2 mRNA与蛋白表达水平显著高于对照组(P<0.05)。结论HOXB7基因沉默表达能抑制大鼠氧化应激反应,提高肝脏SOD表达水平,有利于肝脏Nrf2的表达,从而能促进肝硬化大鼠恢复体重,降低肝功能损伤。     

Ultrasound with microbubbles enhances gene expression of plasmid DNA in the liver via intraportal delivery

Current ultrasound (US)-mediated gene delivery methods are inefficient due, in part, to a lack of US optimization. We systematically explored the use of microbubbles (MBs), US parameters and plasmid delivery routes to improve gene transfer into the mouse liver. Co-presentation of plasmid DNA (pDNA), 10% Optison MBs and pulsed 1-MHz US at a peak negative pressure of 4.3 MPa significantly increased luciferase gene expression with pDNA delivered by intrahepatic injection to the left liver lobe. Intraportal injection delivered pDNA and MBs to the whole liver; with insonation, all lobes expressed the transgene, thus increasing total gene expression. Gene expression was also dependent on acoustic pressure over the range of 0-4.3 MPa, with a peak effect at 3 MPa. An average of 85-fold enhancement in gene delivery was achieved. No enhancement was observed below 0.25 MPa. Increasing pulse length while decreasing pulse repetition frequency and exposure time to maintain a constant total energy during exposure did not further improve transfection efficiency, nor did extend the US exposure pre- or postinjection of pDNA. The results indicate that coupled with MBs, US can more efficiently and dose-dependently enhance gene expression from pDNA delivered via portal vein injection by an acoustic mechanism of inertial cavitation.     

Dual pH-sensitive and UCST-type thermosensitive dendrimers: phenylalanine-modified polyamidoamine dendrimers with carboxyl termini

Dendrimers are unique polymers with well-defined structures, and are useful as functional unimolecular nanoparticles. Previous reports have shown that polyamidoamine (PAMAM) dendrimers modified with hydrophobic molecules, such as amino-terminal phenylalanine (Phe), are thermosensitive at high pH. In the present study, we designed carboxyl-terminal Phe-modified PAMAM dendrimers that are thermosensitive under acidic conditions. We reacted an amino-terminal PAMAM dendrimer with various acid anhydrides, such as succinic anhydride, cyclohexanedicarboxylic anhydride, and phthalic anhydride, prior to the reaction with Phe. Interestingly, the amino-terminal Phe-modified PAMAM dendrimers exhibited LCST (lower critical solution temperature)-type thermosensitivity at approximately pH 7, but the carboxyl-terminal Phe-modified dendrimers exhibited UCST (upper critical solution temperature)-type thermosensitivity in acidic solutions. Temperature sensitivity was dependent on both pH and the anhydride modifier. We were able to separate rose bengal (a model compound) from aqueous solutions of the carboxyl-terminal Phe-modified dendrimer at low pH.

We designed carboxyl-terminal Phe-modified PAMAM dendrimers by using various acid anhydrides. These dendrimers exhibited UCST-type thermosensitivity, which was dependent on both pH and the anhydride modifier.

There are many reports of smart materials that are capable of responding to some stimuli such as temperature, pH, or light.^1–3 pH-responsive polymers can be designed by adding ionic groups to polymers. Polymers comprising weak acids such as poly(methacrylic acid) respond at low pH, and polymers consisting of weak bases such as poly[(2-dimethylamino)ethyl methacrylate] respond at high pH.² Thermosensitive (temperature-responsive) polymers undergo dramatic changes in solubility at the phase transition temperature. Such polymers can be applied to green chemistry and biomedical applications, e.g., chromatography, drug delivery systems, gene therapy, thermally switchable optical devices, and separation of substances.^1,3 Thermosensitive polymers can be divided into two types: lower critical solution temperature (LCST)-type polymers and upper critical solution temperature (UCST)-type polymers.^4,5 There have been many reports on LCST-type thermosensitive polymers such as poly(N-isopropyl acrylamide) (PNIPAm).^4,5 Studies on UCST-type thermosensitive polymers, however, are much less common, although poly(N-acryloyl glycinamide) (PNAGA), ureido polymers and poly(sulfobetaine)s have been reported.^4,5 Dual stimuli-responsive polymers have been developed to construct more sophisticated smart systems. Because pH and temperature are easily controlled, dual pH-sensitive and thermosensitive materials are receiving considerable attention.^6,7Dendrimers are polymers with a unique branched structure. They are synthesized in step-wise reactions, so their molecular weights, shapes, and sizes can be precisely defined. Furthermore, dendrimers can encapsulate and/or modify various kinds of molecules. Many researchers have used dendrimers as nanocarriers or platforms for smart materials, and there are many review articles about dendrimers.^8–14 Among a number of nanocarrier, such as dendrimers, liposomes, polymeric micelles and linear polymers, much attention has been paid to dendrimers because of their advantages for biomedical applications. It is because dendrimers have the ability to maintain drug levels in a therapeutically desirable range, the capability to deliver a variety of bioactive molecules and targeting ability. Dendrimers also increase half-life, solubility, stability, and permeability of drugs, improve delivery efficiency, and reduce macrophage uptake and side effects.¹⁴ Polyamidoamine (PAMAM) dendrimers were one of classical dendrimers, and have been widely studied.^8,9 In previous studies, PAMAM dendrimers modified with various hydrophobic acids, oligo-(ethylene glycols), and an N-isopropyl group exhibited LCST-type thermosensitivity.^12,15–18 LCST-type thermosensitive dendrimers have also been prepared by attaching hydrophobic amino acids such as phenylalanine (Phe) and/or leucine (Leu) to PAMAM polymers.¹⁹ Such PAMAM dendrimers also respond to pH owing to their terminal and inner tertiary amines. However, they are not thermosensitive in acidic solutions because these amines are protonated and become much hydrophilic. pH-sensitive polymers that respond between acid and neutral conditions are more useful for biomedical applications. It is because the physiological pH is 7.4 and the pH in various internal organs and organelles is lower: stomach (pH 1–3), lysosomes (pH 4.5–5), endosomes (pH 5–6.5), and tumor (pH 6.5–7.2).³In the present study, we designed Phe-modified PAMAM dendrimers with carboxyl termini that were thermosensitive in acidic solutions. We reacted amino-terminal PAMAM dendrimers with acid anhydrides prior to the reaction with Phe. We used succinic anhydride (Suc), cyclohexanedicarboxylic anhydride (CHex), and phthalic anhydride (Ph) to compare thermosensitivity and pH-sensitivity in the dendrimers. Finally, we demonstrated the recovery of a useful compound (rose bengal, a model compound) from aqueous solution using a carboxyl-terminal Phe-Ph-modified dendrimer.The carboxyl-terminal Phe-modified dendrimers were synthesized according to Fig. 1. First, we reacted a fourth-generation (G4) amine-terminal PAMAM dendrimer with each of the three different acid anhydrides (Suc, CHex, and Ph) to produce carboxyl-terminal dendrimers. We then reacted the carboxyl groups of the PAMAM dendrimers with carboxyl group-protected Phe. Phenylalanine benzyl ester (Phe-OBzl) was used for the Suc- and CHex-reacted dendrimers. Phenylalanine methyl ester (Phe-OMe) was used for the Ph-reacted dendrimer, owing to the reduced steric hindrance. After removing the protecting groups from Phe in an alkaline solution, we obtained G4-Suc-Phe, G4-CHex-Phe, and G4-Ph-Phe. The numbers of acid anhydride and Phe residues bound to the dendrimers were estimated from the proton nuclear magnetic resonance (¹H NMR) spectra (Fig. S1^†). In the spectrum of G4-Suc-Phe (Fig. S1A^†), the bound numbers of Phe and Suc to the dendrimer were evaluated from the integral ratios of the signals at 4.3 ppm (Phe) and 2.2 ppm (Suc) to 2.6 ppm (dendrimer), respectively. Because the signal at 2.2 ppm for Suc was overlapped with the dendrimer signal, the integral ratio for Suc was obtained by subtracting the dendrimer signal. In the spectrum of G4-CHex-Phe (Fig. S1B^†), the bound numbers of Phe and CHex to the dendrimer were evaluated from the integral ratios of the signals at 4.2–4.3 ppm (Phe) and 1.1–1.9 ppm (CHex) to 2.2 ppm (dendrimer), respectively. In the spectrum of G4-Ph-Phe (Fig. S1C^†), the bound numbers of Phe and Ph to the dendrimer were evaluated from the integral ratios of the signals at 4.4 ppm (Phe) and 7.0–7.4 ppm (Ph) to 2.2 ppm (dendrimer), respectively. Because the signal at 7.0–7.4 ppm for Ph was overlapped with the Phe signal, the integral ratio for Ph was obtained by subtracting the Phe signal. The bound numbers of acid anhydride and Phe to the dendrimer were listed in Table S1.^† These shows that almost all the termini of the dendrimers were modified with both the acid anhydride and Phe.Open in a separate windowFig. 1Preparation of PAMAM dendrimers modified with Phe via linkers.We investigated the temperature-dependent transmittance of synthesized dendrimers at various pH values (Fig. 2). The G4-Suc-Phe solution was clear at pH 6, but turbid at pH 4. The pH sensitivity is caused by the protonation behavior of the inner tertiary amines and terminal carboxyl groups. The dendrimer was highly soluble at pH 6 owing to the deprotonated terminal carboxyl groups. However, it is likely that the carboxyl group became protonated and the negative charge disappeared at pH 4. At pH 5, the dendrimer exhibited UCST-type thermosensitivity. G4-CHex-Phe and G4-Ph-Phe behaved in a similar way to G4-Suc-Phe: solutions of these polymers were clear at high pH and turbid at low pH, and the dendrimers exhibited UCST-type thermosensitivity at the intermediate pH. Carboxyl-terminal dendrimers without Phe, such as G4-Suc, G4-CHex, and G4-Ph, did not exhibit UCST-type thermosensitivity, even at low pH (Fig. S2^†). Therefore, the Phe modification induced UCST-type thermosensitivity. These results indicate that the carboxyl-terminal Phe-modified dendrimers were dual pH-sensitive and UCST-type thermosensitive materials.Open in a separate windowFig. 2Temperature-dependent transmittance curves of (A) G4-Suc-Phe, (B) G4-CHex-Phe, and (C) G4-Ph-Phe at various pH values.The phase transition temperature was defined as the temperature at which the light transmittance was 50%. †¹⁹ However, the carboxyl-terminal Phe-modified dendrimers had UCST-type thermosensitivity at acidic pH. Because PAMAM dendrimers have numerous inner tertiary amines, amino-terminal Phe-modified dendrimers are classified as polycationic polymers. However, carboxyl-terminal Phe-modified dendrimers can become zwitterionic, when the pH is adjusted. Some zwitterionic polymers exhibit UCST-type thermosensitivity.^4,5 It is likely that the UCST-type thermosensitivity of carboxyl-terminal Phe-modified dendrimers is based on the zwitterionic model. The carboxyl-terminal Phe-modified dendrimers aggregated at low temperature, owing to the interaction between the terminal carboxyl anions of Phe and the ammonium cations of PAMAM, which were dissociated by heating to induce UCST-type thermosensitivity. Therefore, we think that the type of thermosensitivity of the amino- and carboxyl-terminal Phe-modified dendrimers depends on the difference in the ionic state. To investigate the UCST-type phase transition mechanism, we examined the dendrimer solutions with the UCST-type thermosensitivity by using an optical microscope. Spherical droplets were observed at low temperature, and they decreased with increasing temperature (Fig. 3). This suggests that coacervation, that is liquid–liquid phase separation, occurred at low temperature in our dendrimers. Previously, ureido polymers exhibited UCST-type thermosensitivity, which resulted from a coacervate formation. These ureido polymers showed the salt sensitivity, at which the phase transition temperature showed a rise of more than 10 °C in the presence of salt.²⁰ The phase transition behavior of our dendrimer was compared in the absence and presence of 150 mM NaCl. The temperature-dependent transmittance curves were almost similar, although the phase transition temperature slightly increased from 47 °C to 53 °C (Fig. S4^†). Thus, the salt effect was limited in our dendrimer. The detailed phase transition mechanisms remain to be investigated.Thermoresponsive behavior of various dendrimers modified with Phe via linkers

Synthesis and characterization of guanidinylated poly(propylene imine) dendrimers as gene transfection agents.

Fourth generation poly(propylene imine) dendrimer has been completely or partially functionalized with guanidinium groups. In the second case, the remaining toxic primary amino groups of the dendrimers were reacted with propylene oxide affording the corresponding hydroxylated derivatives. Five derivatives have been prepared bearing 0, 6, 12, 24 or 32 guanidinium groups. These guanidinylated dendrimers were interacted with plasmid DNA affording the corresponding dendriplexes. The complexes were physicochemically characterized by dynamic light scattering, zeta-potential measurements and AFM, while the extent of complexation was evaluated by agarose gel electrophoresis. Furthermore, their transfection efficiency was assessed employing HEK 293 and COS-7 cell lines, while the serum effect was studied in HEK 293 cells. It was found that complete replacement of primary amino groups with the hydroxylated moieties resulted in complete loss of transfection efficiency. On the contrary, guanidinylation of the parent dendrimer resulted to significant enhancement of its transfection efficiency, this enhancement being dependent on the number of guanidinium groups per dendrimer, the cell line used and the presence or absence of FBS. The fully guanidinylated dendrimer exhibited the best transfection efficiency under all the conditions studied. This efficiency has been attributed to the enhanced penetrating ability of the guanidinylated dendrimers due to the accumulation of the guanidinium group at the dendrimeric surface. It was also found that the derivative with 12 guanidinium groups exhibited the lowest toxicity. The reduction of toxicity was apparently attributed to the decrease of the external primary amino groups coupled with the presence of hydroxylated moieties located at the dendrimeric surface. The functionalization strategy employed leads to dendrimeric derivatives that combine satisfactory transfection efficiency and cytotoxicity.     

Imaging of hypoxia-driven gene expression in an orthotopic liver tumor model

The purpose of this study was to monitor hypoxia in an orthotopic liver tumor model using a hypoxia-sensitive reporter imaging system and to image enhanced gene expression after clamping the hepatic artery. C6 and RH7777 Morris hepatoma cells were transduced with a triple reporter gene (HSV1-tk/green fluorescent protein/firefly luciferase-triple fusion), placed under the control of a HIF-1-inducible hypoxia responsive element (HRE). The cells showed inducible luciferase activity and green fluorescent protein expression in vitro. Isolated reporter-transduced Morris hepatoma cells were used to produce tumors in livers of nude rats, and the effect of hepatic artery clamping was evaluated. Tumor hypoxia was shown by immunofluorescence microscopy with the hypoxia marker EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl acetamide)] and the fluorescent perfusion marker Hoechst 33342, and by pO(2) electrode measurements. For tumor hypoxia imaging with the HRE-responsive reporter, both luciferase bioluminescence and [(18)F]2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil positron emission tomography was done, and the presence of hypoxia in Morris hepatoma tumors were successfully imaged by both techniques. Transient clamping of the hepatic artery caused cessation of tumor perfusion and severe hypoxia in liver tumors, but not in adjacent liver tissue. These results show that the orthotopic reporter-transduced RH7777 Morris hepatomas are natively hypoxic and poorly perfused in this animal model, and that the magnitude of hypoxia can be monitored using a HRE-responsive reporter system for both bioluminescence and positron emission tomography imaging. However, the severity of tumor ischemia after permanent ligation of the hepatic artery limits our ability to image severe hypoxia in this animal model.     

Immune response gene function correlates with the expression of an Ia antigen. I. Preferential association of certain Ae and E alpha chains results in a quantitative deficiency in expression of an Ae:E alpha complex

These studies were stimulated by the observation, reported in the accompanying paper (19), that IEu failed to interact with I-Ak or I-As in F1 mice to allow a response to the antigen, pigeon cytochrome c, unlike I-E subregions derived from other Ia.7+ haplotypes. Serological and biochemical analyses were performed to determine whether or not cells from these F1 mice express the Ak,se:E alpha complexes that should function as restriction elements for T cell recognition of pigeon cytochrome c on antigen-presenting cells. Using the Y-17 monoclonal antibody, which recognizes the combinatorial or conformational determinant Ia.m44 on certain Ae:E alpha complexes, we were able to distinguish between Aue:Eu alpha and Ab,k,se:Eu alpha complexes on cell surfaces. Although complement-dependent microcytotoxicity with Y-17 failed to detect Ab,k,se:Eu alpha complexes on cells from appropriate F1 mice, these molecules were detected by both quantitative absorption and quantitative immunofluorescence studies. However, Ab,k,se:Eu alpha complexes were found to be present at levels only one-seventh to one-eighth the levels expressed by homozygous I-Ab, I-Ek; I-Ak, I-Ek; and I-As, I-Ek cells. The results of two-dimensional polyacrylamide gel electrophoresis analyses suggest that the low levels of expression of Ab,k,se:Eu alpha complexes are a consequence of the preferential association of Aue and Eu alpha chains with each other in the F1 cells. As will be shown in the following paper (19), the quantitative deficiency in the expression of Ake:Eu alpha and Ase:Eu alpha complexes results in a corresponding defect in antigen-presenting cell function, thus providing strong evidence that Ia antigens represent products of Ir genes.     

Leptin受体在实验性脂肪肝及肝硬化中的基因表达情况

目的观察Leptin受体(OB-R)在实验性脂肪肝与肝硬化大鼠中肝脏mRNA的表达情况,探讨OB-R在实验性脂肪肝与肝硬化发生和发展中的作用。方法 (1)建立实时荧光定量逆转录聚合酶链反应(FQ RT-PCR)技术检测OB-R的mRNA表达水平的方法及标准曲线;(2)采用FQ RT-PC检测非酒精性脂肪肝组、肝硬化组及对照组中OB-R的mRNA表达水平,从定量的角度研究OB-R及OB-R mRNA与非酒精性脂肪肝和肝硬化的关系以及它们二者的关系,以探讨表达与非酒精性脂肪肝、肝硬化的相关性及其在实验性脂肪肝与肝硬化发生和发展中的作用。结果非酒精性脂肪肝组、肝硬化组和对照组均有OB-R的mRNA表达,三者比较差异无统计学意义(P>0.05)。结论 OB-R的mRNA在非酒精性脂肪肝、肝硬化和对照组中均有表达,但其可能在脂肪肝和肝硬化的发生和发展中并没有起到直接作用,可能还有其他因子的共同参与。提示OB-R可能参与了肝脂肪变性形成,促进肝纤维化的发生。     

Tumor targeting of gene expression through metal-coordinated conjugation with dextran.

Tumor targeting of plasmid DNA was achieved through the conjugation of dextran derivatives with chelate residues based on metal coordination. Diethylenetriamine pentaacetic acid (DTPA), spermidine (Sd), and spermine (Sm) were chemically introduced to the hydroxyl groups of dextran to obtain dextran-DTPA, dextran-Sd and dextran-Sm derivatives. Conjugation of the dextran derivative by Zn(2+) coordination decreased the apparent size of the plasmid DNA, depending on the derivative type. The negative zeta potential of plasmid DNA became almost 0 mV after Zn(2+)-coordinated conjugation with dextran-Sm. When the dextran derivative-plasmid DNA conjugates with Zn(2+) coordination were intravenously injected subcutaneously into mice bearing Meth-AR-1 fibrosarcoma, the dextran-Sm-plasmid DNA conjugate significantly enhanced the level of gene expression in the tumor, in contrast to the conjugate of other dextran derivatives and free plasmid DNA. The enhanced gene expression produced by the Zn(2+)-coordinated dextran-Sm-plasmid DNA conjugate was specific to the tumor, whereas a simple mixture of dextran-Sm and plasmid DNA was not effective. The level of gene expression depended on the percentage of chelate residues introduced, the mixing weight ratio of the plasmid DNA/Sm residue used for conjugate preparation, and the plasmid DNA dose. A fluorescent microscopic study revealed that localization of plasmid DNA in the tumor tissue was observed only after injection of the dextran-Sm-plasmid DNA conjugate with Zn(2+) coordination. In addition, the gene expression induced by the conjugate lasted for more than 10 days after the injection. We conclude that Zn(2+)-coordinated dextran-Sm conjugation is a promising way to enable plasmid DNA to target the tumor in gene expression as well as to prolong the duration of gene expression.     

Gene expression control by temperature with thermo-responsive polymeric gene carriers.

A thermo-responsive copolymer, poly(N-isopropylacrylamide (IPAAm)-co-2-(dimethylamino)ethyl methacrylate (DMAEMA)-co-butylmethacrylate (BMA)), was synthesized and its in vitro gene transfection efficiency at different incubation temperatures was evaluated. A copolymer containing 8 mol% DMAEMA and 11 mol% BMA (P(IP-8DA-11BM)) had a lower critical solution temperature (LCST) at 21 degrees C, therefore the copolymer was insoluble above 21 degrees C and soluble below 21 degrees C. The LCST of P(IP-8DA-11BM) solution was not affected by the presence of salmon DNA. This copolymer was complexed with plasmid DNA, and the stability of the complex was analyzed by gel electrophoresis. DNA was completely retained in the complex, which was observed in the gel loading slot at 37 degrees C. At 20 degrees C, DNA was found to be partially dissociated from the complex by the appearance of the same band as DNA in the control experiment. These results clearly show that complex formation/dissociation was modulated by temperature alteration. The transfection efficiency of polymer-plasmid complexes was evaluated in COS-1 cells using pCMV-lacZ plasmid, encoding for beta-galactosidase as a reporter gene. The transfection efficiency of PDMAEMA homopolymer incubated at 37 degrees C for 48 h was greater than that incubated at 20 degrees C for 3 h and 37 degrees C for 45 h. In contrast, the transfection efficiency of P(IP-8DA-11BM) incubated at 20 degrees C for 3 h and 37 degrees C for 45 h was much higher than that incubated at 37 degrees C for 48 h. Such an increased transfection efficiency on lowering the temperature is considered to be due to appropriate formation/dissociation control of P(IP-8DA-11BM)-DNA complexes.     

乌司他丁对脓毒症大鼠肝组织基因表达的影响

目的 采用基因芯片技术检测乌司他丁(ulinastatin,UTI)预处理对脓毒症大鼠肝组织基因表达的影响,并从基因水平上探讨其作用机制.方法 将45只雄性Wistar大鼠随机等分为对照组、脓毒症组和UTI组.采用盲肠结扎穿孔术(cecal ligation and puncture,CLP)复制脓毒症模型;UTI组于制模前1 h肌肉注射UTI 100 kU/kg;脓毒症组及对照组肌肉注射平衡液5 mL/kg.采用RatRef-12大鼠表达谱基因芯片进行检测,用计算机软件筛选并分析比较对照组、脓毒症组及UTI组大鼠肝组织基因表达的差异.结果 在22 523条基因中,与对照组比较,脓毒症组大鼠肝组织差异表达基因共284条,其中表达上调者149条,已知功能基因74条;表达下调者135条,已知功能基因63条.与对照组比较,UTI组大鼠肝组织差异表达基因共100条,其中表达上调者51条,已知功能基因26条;表达下调者49条,已知功能基因22条.脓毒症组和UTI组与对照组比较,同样表达的基因有21条,其中上调10条,下调11条.结论 UTI预处理可部分纠正脓毒症大鼠过度炎症反应及免疫抑制所致肝组织基因表达异常,从基因水平上对肝组织起到保护作用.