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Objective To investigate the constituents and analgesic and anti‐inflammatory effects of leaves essential oil in Schef fleraoctophylla .Methods SOLEO was dilated by water vapor .The mouse acetic acid‐induced twisting test and mouse auricle swelling test induced by xylene were employed to evaluate the anti‐ inflammation and analgesia effects of SOLEO . Results 55 constituents of SOLEO were identified by GC‐MS .The major constituents ,4‐terpenol ,(‐)‐spthulenol ,caryo‐phllene oxide andβ‐linalool with total percent of 51 .86% were found .The twisting number and relieve swollen auricle of mice induced by xylene were significantly reduced by SOLEO .Conclusion SOLEO was the major active components of anti‐inflam‐mation and analgesia .
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Kallistatin, which protects organs and cells against inflammation, fibrosis and oxidative stress, is mainly synthesized and secreted in liver. However, its relationship to human liver disease remains unclear. The purpose of this study was to explore the relationship between serum kallistatin and clinical evidence of both cirrhosis and hepatocellular carcinoma (HCC), and to determine if serum kallistatin levels could be used as a diagnostic indicator of hepatic health status, especially human liver cirrhosis (LC). Our cohort consisted of 115 patients with clinically proven liver fibrosis (LF), LC, or HCC by liver biopsies, and 31 healthy controls (CON). Serum kallistatin levels were quantified by ELISA. Results of the present study demonstrated that irrespective of the underlying etiology, serum kallistatin levels were significantly lower in the LF/LC group when compared with the CON group. A decrease in serum kallistatin levels appeared to reflect the extent of cirrhosis, with the lowest levels associated with higher grades of cirrhosis. Patients with LC had a noticeable correlation between serum kallistatin levels and other serum biochemical indicators. The area under the curve (AUC) for LC, viral liver cirrhosis (VLC) and alcoholic liver cirrhosis (ALC) was 0.845, 0.757 and 0.931, respectively. In conclusion, our findings demonstrated that kallistatin, a plasma protein produced by the liver, can be a useful and reliable diagnostic indicator of hepatic health status, especially for LC.
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Hepatits B virus( HBV) infection is a global epidemic which seriously harms the public health. In spite of the great progress in hepatits B prevention and treatment, there is few ef-fective medicine. Research findings show that liver damage and degree of liver failure are sophisticatedly related to the interaction between HBV and the immune response of host. All these make it important to know the replication mechanism and the contrac-tion process, in order to lay a preliminary solid foundation for studying HBV drug targets and making a new ant-virus strategy. This article aims to summarize HBV viral replication process, while focusing on the latest research findings about drug targets, to find a new kind of anti-HBV drugs, and to explore the under-lying mechanism of effective drugs.
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To explore the regulation of eIF4E, we screened the protein interacting with eIF4E from human cDNA library by using yeast two-hybrid system. Several clones interacting with eIF4E were identified. One of them was homologous with HUWE1 (HECT, UBA and WWE domain containing 1, also named as ARF-BP1, HECTH9 or HUWE1). Cell co-immunoprecipitation showed that eIF4E could bind to HUWE1 in mammalian cells. We also found that HUWE1 bearing the HECT domain is necessary for its association with eIF4E.
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Human tissue kallikrein-binding protein (Kallistatin, KAL), a secretory protein that participates in the regulation of multiple signaling pathways by binding to the extracellular receptor, however, at present has not been reported about the intracellular activity, and whether it has the similar biological activity with extracellular activity. Here we constructed no signal peptide KAL (NSK) into the adeno-associated virus vector to explore the intracellular activity of KAL. Both the endothelial cell and lung cancer cells could express KAL, but not secreted after rAAV2-NSK transfection. The proliferation and migration of human umbilical vein endothelial cells (HUVECs) were inhibited, but the apoptosis rate was not affected. The proliferation rates, mobility and tubule formation of all the three tested lung cancer cells, such as NCI-H446, NCI-H460 and A549, were inhibited to different extents. This cellular study not only confirmed the intracellular activity, but also suggested it may serve as a kind of "balance factor" in multi-targeted controlling, which may provide a new train of thoughts to explain the regulatory contradiction in PI3K-Akt signaling pathways by KAL.
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How to reduce immune response is an unprecedented challenge for rAAV gene medicine. Recent studies suggested that lowering dosage of the vector used could reduce immune response caused by rAAV gene medicine. Nevertheless, it would also decrease the transgene expression, leading to failure of gene treatment. It is therefore important to take appropriate steps to maintain high gene expression level and pharmacodynamic, while the dosage of rAAV used is reduced. Here, steps to enhancing gene therapy, such as optimization of the administration, reconstruction of the viral vector and selection of the promoter, are discussed in order to achieve maximum outcome.
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Cell adhesion mediated by cell adhesion molecules (CAMs) constitutes essential life phenomenon. In inflammation, immunity, infection, thrombosis, tumor metastasis and wound healing, cell adhesion comes into being the basic physiological and pathological process. Intervening with cell adhesion has been the important therapeutic and prophylactic strategies for diseases. Accumulated evidence has indicated that plant polysaccharides especially those exacted from Chinese traditional and herbal drugs displayed various pharmacological effects such as anti-inflammation, anti-cancer, anti-infection, immunomodulation, cardiovascular protective effects and so on. In this paper, the research progress of plant polysaccharides on cell adhesion is reviewed.
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High mobility group A2 protein (HMGA2), an architectural factor, is highly expressed in various cancer types including lung cancers. It is a candidate target for cancer therapy. RNAi is an effective gene silencing method with low cost and less time-consuming. It is possible to exploit this technology in therapy. Here, 5 siRNAs targeting Hmga2 gene (HMGA2 siRNA1-5) were designed and synthesized. MTT assay, colony formation assay, transwell assay and flow cytometry were used to evaluate the effects of these siRNAs on lung cancer cell lines (NCI-H446 and A549). Results from cell proliferation, clone formation, migration and apoptosis showed that HMGA2 siRNA1, 3, 5 could affect these aspects for both lung cancer cell lines. Among the five siRNAs, HMGA2 siRNA5 showed the greatest inhibition effects. The inhibition effects of HMGA2 siRNA5 are sequence specific and are not due to the induction of interferon response. Taken together, siRNAs targeting Hmga2 gene are potential candidates for lung cancer gene therapy.
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Gene medicine based on recombinant adeno-associated virus (rAAV) vector has rapidly become the prior-choose reagent for gene therapy, since it had been shown that the rAAV was able to stably express many genes in vivo without detectable side-effect. However, recent findings of CTL immune responses to AAV capsid in a clinical trial highlighted a new issue regarding safety that previously was not identified in animal studies. Obviously it is so important to understand the interaction of rAAV with the immune system in details for the safety and success of rAAV gene medicine. In this review we evaluate several current hypotheses aiming to explain the cellular immunotoxicity, also analysis the current findings including the presentation kinetics of the capsid antigen and the activation of CTL. Focusing on the key steps of the immune response several solutions are proposed, including immunosuppression, optimization of vector and improvement of purity, in order to insure clinical safety and efficacy of rAAV.
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Equipping tumor-targeting carrier with cell-penetrating peptide with high transduction efficacy has become a trend. According to the fusion modes and tumor-targeting mechanisms, cell-penetrating peptides can be divided into five categories: first, self-targeting cell-penetrating peptides; second, fusion carriers made of targeting ligands and cell-penetrating peptides; third, cell-penetrating peptide-modified nano-carrier; fourth, tumor-microenvironment-targeting cell-penetrating peptides; fifth, other special cell-penetrating peptides. Fusion carriers, which can not be effectively released into the cytoplasm after transducted into target cells, can be further modified to increase their efficacy. In all, cell-penetrating peptide introduced into tumor-targeting carrier should provide a new era for anti-tumor pharmacy research and cancer therapy.
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In order to research the bioactivity of kallistatin (Kal), we obtained the recombinant Kal using Pichia pastoris expression system. Kal cDNA was amplified from pAAV-Kal and inserted into pPIC9 vector to generate a recombinant vector of pPIC9-Kal. Then, pPIC9-Kal was linearized and transformed into Pichia pastoris strain GS115 (His4) by electroporation. The positive transformants were selected by MD plate and confirmed by PCR. High level of Kal was obtained in BMMY medium (pH 7.0) after 96 hours induction of 29 degrees C and 2% methanol, with the highest yield of 14 mg/L in shake flask culture. Kal protein was purified from the supernatant with Phenyl Superose and Heparin Sepharose FF chromatograph. The recombinant Kal had a molecular weight of 58 kDa with 98% purity, showing by SDS-PAGE. Moreover, it had a high peroxidase activity (163+/-4) U/(mgmin), which could protect LX-2 cell against oxidation of H2O2. Recombinant Kal also effectively inhibited HUVEC proliferation. In this report, we successfully established the expression system using Pichia pastoris and obtained the bioactive recombinant human Kal. It lays a foundation for its further anti-cancer therapy.
Subject(s)
Humans , Antioxidants , Pharmacology , DNA, Complementary , Electroporation , Genetic Vectors , Genetics , Pichia , Genetics , Metabolism , Recombinant Proteins , Genetics , Pharmacology , Serpins , GeneticsABSTRACT
Purpose To investigate human soluble TRAIL(sTRAIL)protein expression and purification and its potential anti-tumor activity on hepatocellular carcinoma(HepG2).Methods Soluble TRAIL gene ligated with expression vector pPIC9 was transfected into GS115(his4)and the recombination strain expressing sTRAIL was screened by MD plate.The effects of different media,methanol inducement period,methanol concentration,and pH were investigated and optimized using shaking flask.The anti-tumor activity of sTRAIL with HepG2 cells Was analyzed after purification.Results The highest expression of sTRAIL was obtained at pH 6.0,1% methanol in BMMY medium,with the concentration of(58.7±2.4)mg/L at 48 h.Recombinant sTRAIL protein could induce HepG2 cells apoptosis and inhibit HepG2 cells proliferation effectively.Conclusion The optimized condition of human sTRAIL expression and purification Was developed and the obtained recombinant sTRAIL protein may be a promising therapeutic agent for hepatocellular carcinoma.
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A safe and effective targeting viral vector is the key factor for successful clinical gene therapy. microRNA, a class of small, single-stranded endogenous RNAs, act as post-transcriptional regulators of gene expression. The discovery of these kind regulatory elements provides a new approach to regulate gene expression more accurately. In this review, we elucidated the principle of microRNA in regulation of targeting viral vector. The applications of microRNA in the fields of elimination contamination from replication competent virus, reduction of transgene-specific immunity, promotion of cancer-targeted gene therapy and development of live attenuated vaccines were also discussed.
Subject(s)
Humans , Gene Expression Regulation , Genetic Therapy , Genetic Vectors , Genetics , MicroRNAs , Genetics , Viruses , Genetics , MetabolismABSTRACT
Recombinant adeno-associated virus has been proven to be a promising gene delivery vector for human gene therapy with many advantages. Successful applications of recombinant adeno-associated virus vectors in preclinical and clinical human gene therapies make it become a demanded product. A well-established and large-scale production system is therefore required. Since wild type of adeno-associated virus was well characterized in 1989, progress has been made. Particularly, package system of recombinant adeno-associated virus has been developed to use insect cell instead of human cell. These advances in adeno-associated virus production will allow it to meet the demands of basic research and clinical applications. This review will focus on trends in packaging systems and development on a large scale of recombinant adeno-associated virus production.
Subject(s)
Animals , Humans , Biotechnology , Methods , Dependovirus , Genetics , Metabolism , Physiology , Genetic Therapy , Genetic Vectors , Genetics , Insecta , Cell Biology , Recombinant Proteins , Genetics , Virus AssemblyABSTRACT
Objective:To construct K-ras-targeted siRNAs (K-ras siRNA) and to investigate the inhibitory effects of K-ras siRNAs on the growth and migration of lung cancer A549 cells (containing mutant K-ras gene) and NCI-H446 cells (containing wild-type K-ras gene). Methods: Four K-ras siRNAs (K-ras siRNA1～K-ras siRNA3 targeting wild-type K-ras and K-ras siRNA4 targeting mutant K-ras) were designed and artificially synthesized; they were used to transfect A549 cells and NCI-H446 cells. The expressions of Ras mRNA and protein were examined by RT-PCR and Western blot-ting. The inhibitory effects of K-ras siRNAs on the proliferations of A549 and NCI-H446 cells were determined by MTT assay. The effects of K-ras siRNAs on the cell migration and apoptosis were observed by Transwell assay and Hoechst 33258 staining, respectively. Results: Mutant K-ras-targeted siRNA (K-ras siRNA4) specifically inhibited the K-ras ex-pression but had no influence on H-ras and N-ras expression in A549 cells. K-ras siRNA4 inhibited the proliferation of A549 cells but did not inhibit that of NCI-H446 cells, which contained wild type K-ras gene. K-ras siRNA4 also induced apoptosis and inhibited migration of A549 cells. Conclusion: Mutant K-ras-targeted siRNA4 can inhibit the proliferation, migration and induce apoptosis of A549 cells. It may be a potential and personalized drug for the treatment against lung cancer containing mutant K-ras gene.
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Vasostatin, a 180-amino acid fragment from the N-terminal domain of calreticulin, is a potent endogenous angiogenesis inhibitor, which can inhibit the growth of many kinds of experimental tumor. But a recent report that vasostatin can enhance the malignant behavior of neuroendocrine tumor reminds us to be cautious to develop it as an anti-tumor medicine. VAS cDNA was cloned into pAAV-2 expression vector; recombinant virus rAAV-VAS was generated by a three plasmids, helper free packaging method. MS1 mouse pancreatic endothelial cell and human colon tumor HCT-116 cell were infected with rAAV-VAS. Transgene expression was analyzed by Western blotting analysis; cell proliferation was determined by MTT assay. The therapeutic potential of rAAV-VAS was evaluated in subcutaneous HCT-116 xenograft mouse model. rAAV-VAS inhibited the proliferation of MS1 but not HCT-116 cell. HCT-116 cell infected with rAAV-VAS secreted VAS protein into the supernatant effectively. The intratumoral delivery of rAAV-VAS inhibited the xenograft growth and microvessel density in tumors significantly. Our results show the effectiveness of rAAV-VAS as an angiogenesis inhibitor in suppressing tumor growth, validating the application of rAAV-VAS gene therapy in treatment against colon cancer.
Subject(s)
Animals , Male , Mice , Angiogenesis Inhibitors , Genetics , Pharmacology , Calreticulin , Genetics , Cell Proliferation , Colonic Neoplasms , Pathology , Dependovirus , Genetics , Metabolism , Genetic Vectors , Mice, Inbred BALB C , Mice, Nude , Peptide Fragments , Genetics , Recombinant Proteins , Genetics , Pharmacology , Tumor Cells, CulturedABSTRACT
Human gene therapy needs to express exogenous DNA at the targeting cells,producing a practical and efficient therapeutic dosage at an approp-riate time(quantitative pharmacology)with a safe man-ner.Recombinant adeno-associated virus(rAAV)Vec-tom possess a number of properties and recent progress in rAAV production made it rapidly become the reagent of choice for therapeutic gene tmasfer.Over 60 clinical trials of gene therapy based on rAAV have been carried out.The dose response reaction between rAAV vectors and gene expression activity or clinical outcome is one of major aspects of these trials.Most studies showed that vector genomes(vg)and gene expression had a concentration-dependent relationship during a certain scope.However,gene expr~sion Can be afffected by viral serotypes,tissue tropisms,cell targeting,drug regulation,injection route,age and sex,etc.Thus,these aspects should be carefully comidered by scienti-sts,pharmacologisis and physicians during animal ex-periments or clinical trails.KEY WORDS gene therapy;viral vector;dose-re-sponse;quantitative pharmacology;clinical thempy
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AIM: Pancreatic and duodenal homeobox gene 1(pdx-1) is a crucial transcription factor in pancreatic islet development and differentiation. This study was conducted to evaluate whether pdx-1 delivered by adeno-associated virus (AAV) could induce liver cells to differentiate into insulin-producing cells in diabetic rats and thus provide more information for cell replacement therapy for diabetes. METHODS: Recombinant AAV vector was employed to deliver pdx-1to STZ-induced diabetic rats via portal vein (4×1011). Blood glucose and body weight were monitored. Gene expression of pdx-1 and insulin were determined by RT-PCR and immunocytotochemistry (ICC) at the 6th week after the injection. RESULTS: AAV-pdx-1 group showed obvious gene expression of pdx-1 and insulin by RT-PCR analysis and the presence of more insulin-positive cells by ICC. Hyperglycemia was partially ameliorated and body weight was also increased in AAV-pdx-1 treated diabetic rats, though still significantly different from those in the non-diabetic group. CONCLUSION: The data indicate that AAV-pdx-1 can induce more rat liver cells into insulin-producing cells in vivo, thereby ameliorate hyperglycemia. Further experiments are needed to explore which subpopulation of liver cells responds to this development shift and the mechanism of this development shift induced by pdx-1 in order to improve the differentiation efficiency.