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.

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.

Ex vivo non-viral vector-mediated neurotrophin-3 gene transfer to olfactory ensheathing glia: effects on axonal regeneration and functional recovery after implantation in rats with spinal cord injury

Objective Combine olfactory ensheathing glia （OEG） implantation with ex vivo non-viral vector-based neurotrophin- 3 （NT-3） gene therapy in attempting to enhance regeneration after thoracic spinal cord injury （SCI）. Methods Primary OEG were transfected with cationic liposome-mediated recombinant plasmid pcDNA3.1 （＋）-NT3 and subsequently implanted into adult Wistar rats directly after the thoracic spinal cord （T9） contusion by the New York University impactor. The animals in 3 different groups received 4x 1050EG transfected with pcDNA3.1 （＋）-NT3 or pcDNA3.1 （＋） plasmids, or the OEGs without any plasmid transfection, respectively; the fourth group was untreated group, in which no OEG was implanted. Results NT-3 production was seen increased both ex vivo and in vivo in pcDNA3.1 （＋）-NT3 transfected OEGs. Three months after implantation of NT-3-transfected OEGs, behavioral analysis revealed that the hindlimb function of SCI rats was improved. All spinal cords were filled with regenerated neurofilament-positive axons. Retrograde tracing revealed enhanced regenerative axonal sprouting. Conclusion Non-viral vector-mediated genetic engineering of OEG was safe and more effective in producing NT- 3 and promoting axonal outgrowth followed by enhancing SCI recovery in rats.

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.

Surface Modification of Biomimetic PLGA-(ASP-PEG) Matrix with RGD-Containing Peptide:a New Non-Viral Vector for Gene Transfer and Tissue Engineering

RGD-containing peptide （ K16-GRGDSPC） , characterized as non-viral gene vectors, was fabricated to modify the surface of PLGA-[ASP- PEG] matrix, which offered the foundation for gene transfer with porous matrix of gene activated later. Peptide was synthesized and matrix was executed into chips A, B and chip C. Chip C was regarded as control. Chips A and B were reacted with cross-linker. Then chip A was reacted with peptide. MS and HPLC were ased to detect the .14W and purity of peptide. Sulphur, existing on the surface of biomaterials, was detected by XPS. The purity of un-reacted peptide in residual solution was detected by a spectrophotometer. HPLC shows that the peptide purity was 94%- 95% , and MS shows that the MW was 2 741. 3307. XPS reveals that the binding energy of sulphur was 164 eV and the ratio of carbon to sulphur （C/S） was 99. 746 ：0. 1014 in reacted chip A. The binding energy of sulphur in reacted chip B was 164 eV and 162 eV, C/ S was 99.574：0.4255, aM there was no sulphur in chip C. Peptide was manufactured and linked to the surface of biomimetic and 3-D matrix, which offered the possibilities for gene transfer and tissue engineering with this new kind of non-viral gene vector.

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.

Targeting lentiviral vectors to primordial germ cells(PGCs):An efficient strategy for generating transgenic chickens

Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission efficiency still limit the broad usage of this method in creating transgenic chickens. In this study, we implemented a simple strategy using modified lentiviral vectors targeted to chicken primordial germ cells(PGCs) to generate transgenic chickens. The lentiviral vectors were pseudotyped with a modified Sindbis virus envelope protein(termed M168) and conjugated with an antibody specific to PGC membrane proteins. We demonstrated that these optimized M168-pseudotyped lentiviral vectors conjugated with SSEA4 antibodies successfully targeted transduction of PGCs in vitro and in vivo. Compared with the control, 50.0%–66.7% of chicken embryos expressed green fluorescent protein(GFP) in gonads transduced by the M168-pseudotyped lentivirus. This improved the targeted transduction efficiency by 30.0%–46.7%. Efficient chimerism of exogenous genes was also observed. This targeting technology could improve the efficiency of germ line transmission and provide greater opportunities for transgenic poultry studies.

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.

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.

Hepatitis B virus envelope as a targeting gene transfer vector for hepatic cancer cells

Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the supematant fluid of HepG 2.2.15 cells was mixed with a PEG8000 solution for concentration and was inactivated by β-propiolactone. The acquired HBVE was used to pack plRES2-EGFP to test its package ability. Then, we examined its quantity and quality with ELISA, PCR, SDS-PAGE and electron microscopy. The plRES2-EGFP was packed with HBVE and obtained the product HBVE-GFP. The plRES2-EGFP was packed with liposome and obtained the product liposome-GFP. HBVE-GFP and liposome-GFP were used to transfer HepG 2 cells to study the transfection efficiency. HBVE-GFP was used to transfer HepG 2, A549, HeLa and FB cells to study the targeting ability. The green fluorescent protein （GFP） expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometry. Results： 1. The acquired HBVE could retain the surface protein HBsAg ＋ pre S1 ＋ pre S2 and had no virus DNA. It had good package ability for plRES2-EGFP. 2. Transfection efficiency： The GFP could be observed in both the liposome group and HBVE group under the fluorescent microscope. But the HBVE group had a higher fluorescent intensity than liposome group. The transfection rate of liposome group was 49.97% ＋ 2.37% while the HBVE group was 70.65% ＋ 3.15% and the fluorescent intensity of the HBVE group was 3-4 times （P = 0.000） for liposome group with the determination of flow cytometry. 3. Targeting ability： The GFP could be observed in the four groups under the fluorescent microscope. The HepG 2 group had the highest fluorescent intensity among the four groups. The transfection rate of HepG 2 group was 71.35% ＋ 0.03% which was highly expressed than other groups （P = 0.000） and the fluorescent intensity of the HepG 2 group was 2-3 times （P = 0.000） for the other 3 groups with the determination of flow cytometry. Conclusion： HBVE can be constructed successfully with the methods of PEG8000 and β-propiolactone from the supernatant fluid of HepG 22.15 cells. The HBVE can be a candidate gene transfer vector for liver cancer cells.

Support vector regression model for complex target RCS predicting

The electromagnetic scattering computation has developed rapidly for many years; some computing problems for complex and coated targets cannot be solved by using the existing theory and computing models. A computing model based on data is established for making up the insufficiency of theoretic models. Based on the ＂support vector regression method＂, which is formulated on the principle of minimizing a structural risk, a data model to predicate the unknown radar cross section of some appointed targets is given. Comparison between the actual data and the results of this predicting model based on support vector regression method proved that the support vector regression method is workable and with a comparative precision.

Construction and application of gene targeting replacement vector formouse coagulation factor IX gene

Objective: To construct and apply a replacement targeting vector for mouse coagulation factor IX(mFIX) gene in embryonic stem (ES) cells. Methods: Based on the cloning and structural analysis of the genomic DNA fragment of coagulation factor IX gene from 129Sv mouse genomic DNA A phage library, PMFIXDEL plasmid was designed with positive-negative-selection (PNS) strategy, and constructed with commonmolecular cloning techniques. Structure of PMFIXDEL was identified by PCR and restriction analysis. Afterelectroporation with the linearized PMFIXDEL DNA, transfected ES cells were cultured in G418/GANC drugselection medium. The recombination efficacy of this vector was tested. Results: The main components ofPMFIXDEL were two copies of negative selection gene (HSV-tk expression cassette), a positive selectiongene (Neo expression cassette), long and short homologous fragments and plasmid backbone. The introduction of negative selection gene (HSV-tk ) into the construct resulted in 24-fold increase of selection.Conclusion: An effective replacement vector for mFIX gene targeting was constructed and applied in ES cell.

基于状态可观测性和多模态数据PF的移动目标跟踪

为了实现对移动目标的跟踪,提出了一种新颖的基于目标状态可观测性和多模态数据粒子滤波(Particle Filter,PF)的跟踪方案。通过部署在目标移动区域中的传感器获得跟踪目标的距离和到达方向测量值,对接收到的数据进行预处理来计算PF的观测值,以形成一个临时距离图像。通过利用状态更新函数和形成的候选图像模板确定目标状态向量;在PF器中加入额外的加权阶段,使得PF器可自适应地同步多模态数据流,以实现鲁棒的目标跟踪。仿真实验结果验证了所提方案能够有效地跟踪移动目标。

点云多特征分类构建的车辆目标智能识别方法

激光雷达是智能车环境感知的重要传感器之一。针对目前激光点云感知和识别车辆效果较差的问题,提出了一种基于点云多特征分类构建的车辆目标智能识别方法。首先,通过地面分割、空间聚类等方法对点云数据进行预处理,得到若干待识别目标,其次,从车辆尺寸、车身材料、点云特征3个方面,对待识别目标进行多特征分类构建,最后,制作训练集对支持向量机进行训练,利用训练好的分类器对待识别目标进行识别。利用百度Apollo数据集中的6个场景进行实验,本文方法的识别准确率均在90%以上,识别效果较现有方法有所提高。

基于SVM算法的虚假航迹识别

针对云雨杂波和主被动干扰导致多雷达传感器产生虚假目标航迹的问题,利用支持向量机(SVM)算法的自主学习能力,通过构建基于数据驱动的判别模型进行虚假航迹识别。针对航迹起始得到的目标潜在航迹,利用人工智能数据驱动、自学习的特点,设计了SVM算法。通过对已标记真假的目标航迹样本进行离线学习,形成虚假航迹识别的SVM分类器,实现了基于数据驱动的判别模型代替先验知识规则约束的固定模型,并在工程应用中,利用SVM分类器在线识别虚假航迹,完成实时剔除。通过实测雷达数据实验验证,该算法的目标虚假航迹准确率高达95%以上,完全满足实际的工程应用需求。相比基于阈值或规则进行硬性判断的传统虚假航迹识别方法,所提出的算法不仅提高了准确率,还具有较高的实时性,能够适应复杂多变的杂波环境,在实际应用中具有更强的适应性和实用性。因此,提出的基于SVM算法的虚假航迹识别方法对于密集杂波场景下的虚假航迹剔除问题具有显著的实际应用价值。

基于卷积神经网络的红外弱小车辆目标检测方法

传统方法无法获得理想的红外弱小车辆目标检测结果,导致检测误差大,无法满足实际应用要求,为了解决传统红外弱小车辆目标检测方法存在的局限性,及时检测红外图像中的弱小车辆,提高车辆检测精度,设计了基于卷积神经网络的红外弱小车辆目标检测方法。首先对弱小车辆目标检测需要的红外图像进行采集,并对红外图像噪声进行处理,消除噪声对弱小车辆目标检测的干扰,然后采用卷积神经网络建立弱小车辆目标检测模型,最后通过具体仿真实验测试弱小车辆目标检测方法的性能。结果表明,该方法的弱小车辆目标检测精度超过了90%,大幅度减少了弱小车辆目标的误检率,同时弱小车辆目标检测时间控制在5 s内,可以满足弱小车辆目标检测的实时性要求,具有较高的实际应用价值。

基于三维激光扫描技术的智能制造生产线目标检测研究

智能制造生产线目标图像数据采集过程受到噪声、光照变化等问题,导致输入图像的质量不佳,进而影响目标检测的准确性,对此,设计一种基于三维激光扫描技术的智能制造生产线目标检测方法。首先,采用三维激光扫描仪获取待检测目标点云数据,通过点云变换准则计算数据之间的拓扑关联,生成完整三维激光图像。然后,利用杂交小波变换对三维激光目标图像进行去噪处理。最后,使用能量、熵、对比度、相关性4种参数提取图像纹理特征并采取归一化处理,创建最优分类函数,并运用支持向量机算法划分生产线目标样本图像数据,完成智能制造生产线目标检测工作。实验结果表明,所提方法的交并比值最高时达到0.97,F1值最高时达到0.96,平均检测耗时仅为0.53 s,说明所提方法的检测精度高、效率快,鲁棒性强,在实际操作中具备相当的可用性,为智能制造产业提供技术支持。

加权有向关联网络构建与表征的水中目标远距离检测

水中目标的远距离检测是海洋防御体系的关键技术之一,对国防及民用领域均具有十分重要的作用。然而,目前尚缺乏行之有效的水中目标远距离检测方法,特别是目标先验信息未知的情况下变的愈加困难。为解决这一问题,提出一种新的方法—加权有向关联网络。通过矢量声信号到加权有向关联网络的映射,将信号检测问题转化为网络拓扑的表征,并通过对网络拓扑的特性分析及特征提取,实现无目标先验信息下的水中目标远距离检测。并通过仿真与实测数据对所提出的方法进行验证。研究结果表明:与现有的窄带互谱检测、冒泡熵等方法相比,所提方法能够检测到更低信噪比的水中目标,实现了无需目标先验信息的水中目标远距离检测;该方法的应用具有一定的实际意义和应用前景,可以为海洋防御和民用领域的水下目标检测提供有效的技术支撑。

复杂海洋环境下线性调频信号多目标探测研究

将不同长度线性调频(Linear Frequency Modulation, LFM)信号装入矢量信号源,其载波频率设置为中频,分别采样其输出的不同长度的LFM信号,对输出的LFM信号进行脉冲压缩Matlab仿真处理。实验表明,在采样频率及输出幅度相同的条件下,102.00μs脉冲内LFM信号仿真处理后的信噪比和旁瓣抑制比,相较3.00μs的指标提高了13.35 dB和4.61dB。进一步说明了由组合脉冲组成的LFM发射波形信号,其脉压体制雷达存在近距离回波比远距离回波差问题。提出了一种复杂海洋环境下多目标探测方法,对海用脉压体制雷达真实回波性能进行了改善和提升。

基于MEMS矢量水听器的水下目标方位探测系统设计

本文设计了一种结构、尺寸均适配于微型浮标的小型化水下目标方位探测系统,针对水下固定频率的声目标方位获取进行研究,主要适用于水下目标主、被动双基地探测。测试系统利用微机电系统(MEMS)矢量水听器作为核心传感器,设计现场可编程门阵列(FPGA)读写控制电路,一方面,采集的数据存储于SD卡中;另一方面,则将数据暂存在随机存取存储器(RAM)中,以备数据回传需求。通过驻波管实验证明:搭建的小型化目标方位探测系统稳定可靠,根据其所采数据在有效范围内可以获得目标的方位估计,驻波管内角度误差估计小于2°,误差小且定向准确。