In vitro effect of p2l^(WAF-1/CIP1) gene on growth of human glioma cells mediated by EGFR targeted non-viral vector GE7 system

Objective: To construct the EGFR targeted non-viral vector GE7 system and explore the in vitro effect of p21WAF-1/CIPI gene on growth of human glioma cells mediated by the GE7 system. Methods: The EGFR targeted non-viral vector GE7 gene delivery system was constructed. The malignant human glioma cell line U251MG was transfected in vitro with β-galactosidase gene ( reporter gene) and p21WAF-1/CIPI gee (therapeutic gene) using the GE7 system. By means of X-gal staining, MTS and FACS, the transfection efficiency of exogenous gene and apoptosis rate of tumor cells were examined. The expression of p21WAF-1/ CIPI gene in transfected U251MG cell was examined by immunohistochemis-try staining. Results: The highest transfer rate of exogenous gene was 70% . After transfection with p21WAF-1/CIPI gene, the expression of WAF-1 increased remarkably and steadily; the growth of U251MG cells were inhibited evidently. FACS examination showed G1 arrest. The average apoptosis rate was 25.2%. Conclusion: GE7 system has the ability to transfer exogenous gene to targeted cells efficiently, and expression of p21WAF-1/CIPI gene can induce apoptosis of glioma cell and inhibit its growth.

A RGD-Containing Oligopeptide (K)_(16)GRGBSPC: A Novel Vector for Integrin-Mediated Targeted Gene Belivery

A 23 amino acid, bifunctional integrin-targeted synthetic oligopeptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells （BMSCs）. Synthesis of the peptide （K）16GRGDSPC was performed on a solid-phase batch peptide synthesizer. BMSCs were transfected with plasmid DNA coding for luciferase by （K）j6GRGDSPC and the transfection efficiency was assayed. The influences of chloroquine and polyethyleneimine on the transfection efficiency were also examined. The target specificity of （K）16GRGDSPC to mediate exogenous gene into BMSCs was analyzed using cell attachment test and gene delivery inhibition test. The results showed that the transfection efficiency of the oligopeptide vector was lower than that of Lipofectamine. But in the presence of endosomal buffer chloroquine or endosomal disrupting agent polyethyleneimine, the transfection efficiency of the vector was greatly enhanced. In addition, RGD-containing peptides inhibited BMSCs＇ attachment to the 96-well plates pretreated with fibronectin or vitronecfin and significantly decreased the transfection efficiency of the oligopeptide vector. These studies demonstrated that oligopeptide （K）16GRGDSPC was an ideal novel targeted non-viral gene delivery vector, which was easy to be synthesized, high efficient and low cytotoxicity. The vector could effectively deliver exogenous gene into rat BMSCs.

Construction of Myostatin Gene Targeting Vector of Mouse

[Objective] This study aimed to construct Myostatin （MSTN） gene targeting vector of mouse. [Method] Total RNA was extracted from hindlimb muscle tissues of mouse to synthesize cDNA as the template to clone the coding region of MSTN. The CDS of MSTN gene including 3 kb 5’ homologous arm and 1.4 kb 3’ homologous arm were inserted into vector pBluescript_SK ＋ to construct the targeting vector pLoxP-5N3T-M. The neo and HSV-tk gene were cloned into vector pBluescript_SK＋ as positive and negative selective gene. [Result] Restriction enzyme digestion and sequencing results showed that mouse MSTN gene was cloned into the targeting vector pLoxP-5N3T-M. [Conclusion] The mouse MSTN gene targeting vector pLoxP5N3T-M was successfully constructed.

MicroRNA-regulated viral vectors for gene therapy

Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene.Besides traditional approaches, such as transcriptional and transductional targeting, micro RNA-dependent posttranscriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. Micro RNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region(UTR) of the m RNA. To control exogenous transgene expression, tandem repeats of artificial micro RNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene m RNA in cel s expressing the corresponding micro RNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying micro RNA-regulation, highlights new developments in this field and gives an overview of applications of micro RNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases.

Automatic target recognition of moving target based on empirical mode decomposition and genetic algorithm support vector machine

In order to improve measurement accuracy of moving target signals, an automatic target recognition model of moving target signals was established based on empirical mode decomposition(EMD) and support vector machine(SVM). Automatic target recognition process on the nonlinear and non-stationary of Doppler signals of military target by using automatic target recognition model can be expressed as follows. Firstly, the nonlinearity and non-stationary of Doppler signals were decomposed into a set of intrinsic mode functions(IMFs) using EMD. After the Hilbert transform of IMF, the energy ratio of each IMF to the total IMFs can be extracted as the features of military target. Then, the SVM was trained through using the energy ratio to classify the military targets, and genetic algorithm(GA) was used to optimize SVM parameters in the solution space. The experimental results show that this algorithm can achieve the recognition accuracies of 86.15%, 87.93%, and 82.28% for tank, vehicle and soldier, respectively.

Non-viral targeted delivery system mediates transfection of thymidine kinase gene of herpes simplex virus into ovarian cancer cells:a comparison between one time and continuous mediation

Objective： To compare the transferring efficiency and killing effects of one time and continuous mediation with GE7, a non-viral targeted delivery system, in transfection of thymidine kinase gene of herpes simplex virus （HSV-tk） into ovarian cancer cells. Methods： GE7 was used to prepare recombinants with β-galactosidase （β-gal） and HSVI-tk; the recombinants were then used to transfect human ovarian cancer line CaOV3 once and continuously. β-gal staining was used to compare the efficiencies of one time and continuous mediation with GE7 system. Ganciclovior （GCV） was introduced into HSVI-tk transfected ovarian cells. Through drawing the cell growth curve and flow cytometry, the killing effects of GCV on once and continuously GE7/HSVI-tk transfected cells were observed. Results： We found that the one time and continuous exogenous gene transfer efficiencies were about 80% and 85%, respectively. When 1 μg/mL GCV was used to treat ovarian cell transfected with HSVI-tk gene, growth inhibiting rates of ovarian cells of one time and continuous transferring were 82% and 90%, respectively; their apoptosis indices were 15 and 30, respectively. Under same GCV concentration, continuous mediation of GE7/pCMV-tk transfection into ovarian cancer cells had more significant inhibitory effect than one time mediation （P 〈 0.05）. Conclusion： Compared with one time mediation, continuous mediation of transfection with GE7 gene delivery system has higher efficiency. Continuous mediation of GE7/HSVI-tk/GCV therapeutic gene system has more powerful killing effect.

Non-viral liposome-mediated transfer of brain-derived neurotrophic factor across the blood-brain barrier

Brain-derived neurotrophic factor（BDNF） plays an important role in the repair of central nervous system injury,but cannot directly traverse the blood-brain barrier.Liposomes are a new type of non-viral vector,able to carry macromolecules across the blood-brain barrier and into the brain.Here,we investigate whether BDNF could be transported across the blood-brain barrier by tail-vein injection of liposomes conjugated to transferrin（Tf） and polyethylene glycol（PEG）,and carrying BDNF modified with cytomegalovirus promoter（pC MV） or glial fibrillary acidic protein promoter（p GFAP）（Tf-p CMV-BDNF-PEG and Tf-p GFAP-BDNF-PEG,respectively）.Both liposomes were able to traverse the blood-brain barrier,and BDNF was mainly expressed in the cerebral cortex.BDNF expression in the cerebral cortex was higher in the Tf-p GFAP-BDNF-PEG group than in the Tf-p CMV-BDNF-PEG group.This study demonstrates the successful construction of a non-virus targeted liposome,Tf-p GFAP-BDNF-PEG,which crosses the blood-brain barrier and is distributed in the cerebral cortex.Our work provides an experimental basis for BDNF-related targeted drug delivery in the brain.

Passive tracking and size estimation of volume target based on acoustic vector intensity

The special sections of volume target are observed with acoustic vector intensity according to the difference among their radiated-noise characteristics, then three sections are tracked with Kalman filtering, and target size is estimated. Simulation results indicate that in ideal condition three sections of a ship can be tracked and ship's size can be estimated even though one of three sections can not be observed.