Gene therapy for liver regeneration:Experimental studies and prospects for clinical trials

The liver is an exceptional organ,not only because of its unique anatomical and physiological characteristics,but also because of its unlimited regenerative capacity.Unfolding of the molecular mechanisms that govern liver regeneration has allowed researchers to exploit them to augment liver regeneration.Dramatic progress in the field,however,was made by the introduction of the powerful tool of gene therapy.Transfer of genetic materials,such as hepatocyte growth factor,using both viral and non-viral vectors has proved to be successful in augmenting liver regeneration in various animal models.For future clinical studies,ongoing research aims at eliminating toxicity of viral vectors and increasing transduction efficiency of non-viral vectors,which are the main drawbacks of these systems.Another goal of current research is to develop gene therapy that targets specific liver cells using receptors that are unique to and highly expressed by different liver cell types.The outcome of such investigations will,undoubtedly,pave the way for future successful clinical trials.

Construction, Expression and Identification of a Recombinant BCG Vaccine Encoding Human Mycobacterium Tuberculosis Heat Shock Protein 65

Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection. The signal sequence of antigen 85B and the whole HSP65 DNA sequence of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR). These two sequences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M. tuberculosis, yielding the prokaryotic shuttle expression plasmid pBCG-SP-HSP65. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained. The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating. The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69 % in total bacterial protein and 74.09 % in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M. tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.

架空线路改造工程造价的组合预测方法

架空线路改造工程造价预测是项目管控的重要环节,为提高其预测精度,提出一种组合预测方法。首先,将架空线路改造工程分解为一些子工程,分别预测各子工程造价,之后进行集成。接着,利用主成分分析法并结合专家经验,筛选出影响子工程造价的关键因素,之后,采用基于遗传算法优化的支持向量机和极限梯度提升算法对子工程造价分别进行预测。然后,借鉴博弈论中的Shapley值理论确定组合预测模型中的权重,得到组合预测模型。最后,用实际项目数据对所提出的组合预测方法进行验证,仿真结果表明,与采用单个预测模型相比,所构造的组合预测模型获得了更高的预测精度。

Combined forecast method of HMM and LS-SVM about electronic equipment state based on MAGA

For the deficiency that the traditional single forecast methods could not forecast electronic equipment states, a combined forecast method based on the hidden Markov model（HMM） and least square support vector machine（LS-SVM） is presented. The multi-agent genetic algorithm（MAGA） is used to estimate parameters of HMM to overcome the problem that the Baum-Welch algorithm is easy to fall into local optimal solution. The state condition probability is introduced into the HMM modeling process to reduce the effect of uncertain factors. MAGA is used to estimate parameters of LS-SVM. Moreover, pruning algorithms are used to estimate parameters to get the sparse approximation of LS-SVM so as to increase the ranging performance. On the basis of these, the combined forecast model of electronic equipment states is established. The example results show the superiority of the combined forecast model in terms of forecast precision,calculation speed and stability.

A bicistronic retroviral vector to introduce drug resistance genes into human umbilical cord blood CD34^+ cells to improve combination chemotherapy tolerance

OBJECTIVE: To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamide (4-HC) and P-glycoprotein effluxed drugs. METHODS: A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed and used to transfect the packaging cell lines GP + E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC agents for cloning selection and ping-ponging supernatant infection between the ecotropic producer clone and the amphotropic producer clone, we obtained high titer amphotropic PA317 producing cells with high titers up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopoietic growth factors. RESULTS: Bicistronic retroviral vector construction was verified by restriction endonuclease analysis. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell sorting (FACS) method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4-fold stronger resistance to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduced cells. CONCLUSION: The bicistronic retroviral vector-mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in tumor clinical trial.

Inhibitory effect of retroviral vector containing anti-sense Smad_4 gene on Ito cell line, LI90

Background Transforming growth factor-β1 (TGF-β1) exerts strong fibrogenic potential in culture-activated HSCs Smad 4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors The aim of this study was to assess the effects of the antisense Smad 4 gene on Ito cell line, LI90 Methods The recombinant retroviral vector pLXSN-Smad 4 was constructed by cloning the rat antisense Smad 4 cDNA into the retroviral vector pLXSN Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad 4 and pLXSN vectors into PA317 cells Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418 The expression of Smad 4 was detected by Northern and Western blots Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed Results mRNA and protein expressions of Smad 4 in LI90 cells transfected with retrovirus containing the antisense Smad 4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells Cells hypoexpressing the Smad 4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline ( P <0 01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus Conclusions The antisense Smad 4 gene can suppress the expression of the Smad 4 gene, reduce endogenous production of Smad 4 mRNA and protein, block TGF-β1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM) Our results may provide a basis for the development of antifibrotic gene therapy

Retroviral endostatin gene transfer inhibits human colon cancer cell growth in vivo

Objective To investigate the therapeutic effect of retroviral endostatin gene transfer on the human colon cancer cell line,LoVo.Methods A retroviral vector pLESSN expressing secretable endostatin was constructed and packaged with a titer of 8.2×10 5 CFU/ml. A LoVo cell line was subjected to retrovirus-mediated endostatin gene transfer. The proviral integration of endostatin was analyzed with PCR. The function of endostatin was tested by MTT assay in vitro and a mouse xenograft model in vivo.Results After transfection and superinfection,amphotropic retrovirus was collected,and transduction with amphotropic retroviruses resulted in endostatin proviral integration. The endostatin secreted by transduced LoVo cells markedly inhibited endothelial cell growth up to 67% ( P <0.001),compared with the control cells. The gene expression of endostatin in LoVo colon tumor cells significantly inhibited tumor growth in vivo. There was an 86% reduction in tumor size in the endostatin-transduced group,accompanied by a reduction in vessels,compared with the control group ( P <0.01). Conclusion Retroviruses can allow functional expression of the endostatin gene in human colon tumors,showing promise for an antitumor strategy using antiangiogenesis.

Modification of pGH cDNA using the first intron and adenovirus-mediated expression in CHO cells

Objective This study was conducted to investigate the function of the first intron of porcine growth hormone (pGH) gene in the gene expression.Methods PCR method was used to amplify the first intron from pig genomic DNA. The intron was then inserted into pGH cDNA to construct pGH cDNA-intron (pGH cDNA-in). The recombinant adenoviruses containing pGH cDNA and pGH cDNA-in genes under control of CMV promoter were generated by homologous recombination method in HEK 293 cells respectively. The effect of the first intron on gene expression was evaluated by comparing the expression levels of pGH cDNA-in and pGH cDNA mediated by adenovirus vectors in CHO cells.Results The expression level of pGH cDNA containing the first intron increased by 117%,which was significantly higher than that of pGH cDNA without the intron ( P <0.001). Conclusion The first intron of pGH gene has the function to improve pGH gene expression.

Effects of podocin transfection on CD2AP distribution in HEK293 cells

The aim of this paper is to construct a podocin fluorescence expression vector and observe the effects of podocin transfection on CD2AP distribution in HEK293 cells.The pGEMT-easy vector containing the full-length cDNA encoding human podocin was cloned and digested with BamHI and XhoI.The digested full-length podocin was subcloned into pEGFP-C2.The constructed plas-mids were transfected into HEK293 cells and its effects on CD2AP distribution were observed by immunofluo-rescence.The pEGFP-NPHS2 expression vector was successfully constructed and podocin exclusively located on the HEK293 cell membrane.After podocin transfec-tion,CD2AP redistributed from the perinucleus to the cytoplasm in HEK293 cells.It can be concluded that podocin can recruit CD2AP to redistribute from the perinucleus to the cytoplasm in HEK293 cells.

Simultaneous characterization of multiple properties of solid and liquid phases in crystallization processes using NIR

Near infrared spectroscopy （NIR） is now probably the most popular process analytical technology （PAT） for pharmaceutical and some other industries. However, unlike mid-IR, NIR is known to have difficulties in monitoring crystallization or precipitation processes because the existence of solids could cause distortion of the spectra. This phenomenon, seen as unfavorable previously, is however an indication that NIR spectra contain rich information about both solids and liquids, giving the possibility of using the same instrument for multiple property characterization. In this study, transflectance NIR calibration data was obtained using solutions and slurries of varied solution concentration, particle size, solid concentration and temperature. The data was used to build calibration models for prediction of the multiple properties of both phases. Predictive models were developed for this challenging application using an approach that combines genetic algorithm （GA） and support vector machine （SVM）. GA is used for wavelength selection and SVM for mode building. The new GA-SVM approach is shown to outperform other methods including GA-PLS （partial least squares） and traditional SVM. NIR is thus successfully applied to monitoring seeded and unseeded cooling crystallization processes of L-glutamic acid.