浮体与载重体分离的不会沉没的救生船——上海市闵行中学创造教育科学技术研究性学习课题

研究课题的由来在航运史上,全世界每年都会发生客轮沉没、渡船倾覆等海难事件。判断失误、气象预报不准确、导航与驾驶装置失控等人为与技术因素往往会引发轮船触礁、撞冰山等事故,而恶劣气候下的狂风巨浪是造成海难事件中人员惨重伤亡的主要原因。在海洋上一般无风也三尺浪,在陆地上刮二、三级风时,人们的感觉只是微风拂脸撩发,而海洋上的波浪已有一。

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.

Construction of Recombinant Retroviral Vector Containing HIV-1 Tat Gene and Functional Detection of Expressed Tat in Target Cells

Objective: To construct recombinant retroviral vector containing HIV-1 Tatgene and evaluate the junction of the expressed Tat in target cells. Methods: HIV-1 Tat_(101) genewas recovered from pEV plasmid by Hind Ⅲ digestion and cloned into expression plasmid LZESpBMN-Z toconstruct recombinant retroviral expression plasmid named LZRS-Tat_(101). Using the method ofcalcium phosphate, the construct of LZRS-Tat_(101) was then transfected into packaging cell linesPhoenix (ΦNX) which contained env and gal genes encoding structural proteins and pol gene codingfor 3 enzymes ( reverse transcriptase, protease and integrate) essential for retroviral integrationand replication . The stable transfected cell lines was obtained using puromycin to screen for morethan 3 days. Then, immunohistochemical (IHC ) staining was carried out to detect the expressionlevel of Tat_(101) protein in both transiently and stably trancfected ΦNX, respectively. Thesupematants containing recombinant virus collected from transient and stable transfected cells wereemployed to infect 293 cells, respectively, and the expressed Tat in 293 cells was tested by Westernblot. Meantime, the supematants of infected 293 cells was further added to HL3T1 cells which wereHela cell lines containing an HIV-1-LTR/CAT reporter construct to establish a co-culture system.After co-culture for 72 hours, the protein was extracted from HL3T1 cells and used for CAT activityassay. Results: After LZRS- Tat_(101) was transfected into ΦNX, the amount of expressed Tat intransient transfection cells was significantly higher than that in stable transfection cells; Tatcould be detected not only in 293 cells but also in the supematants from 293 cells culture, and Tatin the supematants could activate HIV-1 LTR promoter in HL3T1, resulting in high 'expression of CATlocated at the downstream of LTR. Conclusion: The construct of recombinant retrovirus LZRS-Tat_(101) could express Tat protein in target cells and the expressed Tat was functionally activeand can really exhibit the ability to activate transcription.

Enhanced Chemotherapy Sensitivity of Human Colon Cancer Cells to 5-Fluorouracil by siRNA Recombinant Expression Vector Targeting Survivin Gene

Objective To investigate the effects of small interfering RNA （siRNA） recombinant expression vector targeting survivin gene on chemotherapy sensitivity of human colon cancer cells to 5-fluorouracil. Methods siRNA recombinant expression vector targeting survivin gene was constructed and transfected into human colon cancer cell lines LOVO. After 48 hours of transfection, cells were harvested for analysis of survivin mRNA and protein expressions using RT-PCR and Western blot. In addition, after human colon cancer cell lines were treated with Survivin siRNA and/or 5-fluorouracil, MTT assay and flow cytometry were used to analyze cell proliferation and apoptosis. Results Restriction endonuclease analysis confirmed that siRNA recombinant expression vector targeting survivin gene was successfully constructed. Inhibitory ratios of survivin mRNA and protein expressions by Survivin siRNA were 36.33% and 44.65%, respectively. Survivin siRNA combined with 5-fluorouracil significantly increased the cell proliferation inhibitory ratio and apoptosis ratio compared with 5-fluorouracil treatin~ alone （P〈0.05）. Conclusion The siRNA recombinant expression vector targeting survivin gene can inhibit the expression of survivin gene, and enhance chemotherapy sensitivity of human colon cancer cells to 5- fluorouracil.

CORAL AS A CARRIER FOR RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-2

By combining coral with recombinant human bone morphogenetic protein-2 (rhBMP-2), rhBMP-2/coral composite was obtained in this study. Following implantation of the composite into the muscle pouches of mice, cartilage growth was induced in the pores or on the surface of the implants at one week, woven bone at three week and lamellar bone with bone marrow at six week, and coral was absorbed partially. The induced formation of endochondral bone was time-related and rhBMP-2 dose-related. The results of this study indicate that the composite possesses a superior ability of osteogenesis, and coral acts as one of the most suitable rhBMP-2 slowrelease carriers currently available. The composite will be a new type of bone substitute to be used in orthopaedics and maxillofacial surgery.

Gene therapy for allergic rhinitis with recombinant adenovirus vector containing CTLA4Ig in mice

Objective: To examine the role of recombinant adenovirus vector containing CTLA4Ig gene(Ad-CTLA4Ig) in the treatment of induced allergic rhinitis in mice.Methods: Allergic rhinitis was induced by sensitizing and challenging with ovalbumin(OVA).Ad-CTLA4Ig was intraperitoneally injected 30 min before OVA challenge.Adenovirus vector without inserted CTLA4Ig cDNA served as the control.The symptoms and morphological changes of nasal mucosa of each group were observed, and the serum levels of IgE against OVA were detected with ELISA.Results: There were no obvious symptoms and pathological changes in Ad-CTLA4Ig treated group, in which the serum OVA-specific IgE levels were significantly lower than that in control groups(P< 0.05).Conclusion: Ad-CTLA4Ig prevents and treats allergic rhinitis of mice,implying the possibility of the usage of Ad-CTLA4Ig against allergic rhinitis in clinic in future.

Construction of Fusion Expression Vector Carrying GFP and ZmCIPK

[Objective] The aim was to isolate the CBL-interacting protein kinases(CIPK)from maize(Zea mays L.)and construct the fusion gene expression vector which consisted the ZmCIPK8 and GFP.[Method] The ZmCIPK8 cDNA was successfully cloned by using RT-PCR method.And then,it was connected to the pBlueScript SK(pSK)plasmid,which contained the GFP gene.So that the fusion gene vector pSK-CIPK-GFP was obtained.Then,the fusion gene was connected into the efficient plant expression vector PBI121 to construct the fusion gene expression vector PBI-CIPK-GFP.At last,the recombined expression vector was transformed to Agrobacterium tumefaciems LBA4404 to produce the engineering strain LBA4404-PBI-CIPK-GFP.[Result] The fusion gene expression vector which consisted of GFP and ZmCIPK8 gene and engineering strain LBA4404-PBI-CIPK-GFP were successfully constructed.[Conclusion] The results lays a foundation for further study of subcellular localization of ZmCIPK8,which can help to clarify the molecular mechanism of regulation serious stresses,and also provides an important basis for the research on resistance stress engineering of maize.

Expression of Rice Gall Dwarf Virus Outer Coat Protein Gene (S8) in Insect Cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.

Optimization of the construction of recombinant plasmids PPARγ-pSUPER-EGFP for RNA interference

Objective：To construct and identify the recombinant plasmids PPARγ-pSUPER-EGFP for RNA interference. Methods： The pSUPER-EGFP vectors were used to transcribe functional short interfering RNA （siRNA）. Four pairs of 64 nt PPARγ siRNA encoding sequences were inserted into the downstream of the H1 promoter. The recombinant plasmids were confirmed by double digestion with the enzymes and sequencing. Western blotting was used to examine the silencing effect of PPARγ gene in RAW264.7 cells. Following procedures were used to optimize the experiments： the oligonucleotides were incubated 5 min at 95 C and cooled automatically in boiled water bath to anneal, and then phosphorylated oligonucleotides, pSUPER-EGFP plasmids was digested with Bgl Ⅱ and Hind Ⅲ , and the product was ligated into digested pSUPER-EGFP plasmids, and transforming the ligation products followed by screening and identifying positive clones. Results ：Four kinds of positive clones producing 285 bp fragments were selected. Sequencing further proved their correctness. Four recombinant plasmids containing corresponding PPARγ gene-specific target sequences induced the silencing of its target gene more or less. Conclusion： The optimizing method in constructing these recombinant plasmids serves other plasmid-based RNA interference research. The final plasmids PPARγ-pSUPER-EGFP established the basis for research on the function of PPARγ gene.

cDNA cloning and expression of a collectin from red-spotted grouper(Epinephelus akaara)

Lectins play a crucial role in the innate immunity of invertebrates and vertebrates by recognizing and disposing of pathogens. We obtained the complete cDNA of a C-type lectin （EALecl） from Epinephelus akaara using RACE. The complete EALecl cDNA sequence was 827 bp. The 5-UTR and 3-UTR were 28 bp and 151 bp, respectively, in length. The sequence also contained a polyadenylation signal AATAAA and a poly（A） tail. The EALecl cDNA encodes polypeptides with 215 amino acids, including a signal peptide of 31 amino acids. The protein has a cysteine-rich region at the N terminal, a collagenous region characterized by G-X-Y repeats, a neck region, and a typical carbohydrate-recognition domain （CRD）, indicating that EALecl is a collectin. The key recognition positions of this CRD are EPD, isolated for the first time in fish. These are likely the interim types, between mannan-binding lectin and galactose-binding lectin. We evaluated the expression pattern of EALecl in 12 different tissues using RT-PCR. EALecl was expressed in all tissues, though at different levels. In addition, we inserted EALecl into an expression vector （pET-28a） for transformation into the BL21 engineering bacteria. Based on enzyme digestion and sequencing of the positive clone, we successfully constructed the EALecl recombinant expression vector.

Adenoviral vector mediated-expression of caspase-3 siRNA on apoptosis induced by lipopolysaccharide

Objective： To construct the recombinant adenovirus expressing small RNA of rats caspase-3 and observe the down-regulation effect of caspase-3 in neurons induced by lipopolysaccharide（LPS） in vitro. Methods： pShuttleHl-siCas3 containing Oligo DNA of the targeting sequences and pEGFPC1-Cas3 containing caspase-3 and EGFP sequences were constructed respectively, pShuttleH 1-siCas3 and pEGFPC 1-Cas3 were co-transfected to the 293 cells by liposomes to determine interfering efficacy by flow cytometry, pShuttleHl-siCas3 was linearized and transformed into E. coli B J5183 cells containing backbone plasmid pAdEasy-1. The recombinant plasmid was transfected into 293 cells to package the adenovirus Ad-siCas3. The titers of adenovirus were determined by the specific 50% tissue culture infection dosage method. After virus infected the cultured hippocampus neurons, LPS-induced apoptosis and caspase-3 mRNA expression were observed. Results： It was identified that the sequence of target gene was correctly inserted into the genome of virus. The expression of green fluorescence protein was reduced by pShuttleHl-siCas3 in 293 cells. The titer of recombinant adenovirus was 1.06×10^10pfu/ml. After virus infection, caspase-3 mRNA was greatly reduced and neurons apoptosis was suppressed. Conclusion： The recombinant adenovirus expressing rats caspase-3 siRNA were successfully constructed, which may probably be further used in pain therapy by its anti-apoptosis effect.

A Viral Expression Vector from Foxtail mosaic virus to Express Green Fluorescent Protein

[Objective]Foxtail mosaic virus(FoMV)infects gramineous and dicotyledonous plants.In this study,we sought to construct a viral vector based on FoMV to express exogenous proteins in plants.[Method]A recombinant viral expression vector was constructed by inserting the promotor of Potato virus X(PVX)and exogenous gene sequences into the 3’non-coding region of the FoMV coat protein gene.[Results]The plasmid pCB301-FoMV-CP-PVXprom-GFP expressed green fluorescent protein in inoculated Nicotiana benthamiana leaves.[Conclusion]A recombinant viral expression vector was constructed successfully.

Application Study on Elastomer Expansion Joint in Heavy Haul Railway

The article summarizes related research results and achievements of elastomer expansion device in railway bridge and puts forward a new idea of using polyurethane elastomer material to seal concrete bridge joints between adjacent spans in heavy haul railways. The new type expansion device is composed of polyurethane elastomer material and named TTXF （elastomer expansion joint）. In theory, researchers find out expansion joint deformation regularity between adjacent bridge spans through theoretical analysis and detection in heavy haul railways, such as Datong-Qinhuangdao Railway and Shenchi-Huanghua Port Railway. Fatigue tests prove that TTXF can adapt to permanent and dynamic deformation. On the other hand, it has been successfully applied in the test section of Central South of Shanxi Railway Passage and continuous monitoring has been conducted in extreme weather for over one year. The expansion joint has a good effect practically.

A Rapid Estimation Method of Structural Fatigue Analysis for a 17k Ton DWT Oil Tanker

Fatigue cracks and fatigue damage have been important issues for ships and offshore structures for a long time.However,in the last decade,with the introduction of higher tensile steel in hull structures and increasingly large ship dimensions,the greater attention should be paid to fatigue problems.Most research focuses on how to more easily access the fatigue strength of ships.Also,the major classification societies have already released their fatigue assessment notes.However,due to the complexity of factors influencing fatigue performances,such as wave load and pressure from cargo,the combination of different stress components,stress on concentration of local structure details,means stress,and the corrosive environments,there are different specifications with varying classification societies,leading to the different results from different fatigue assessment methods.This paper established the Det Norske Veritas（DNV） classification notes ＂fatigue assessment of ship structures＂ that explains the process of fatigue assessment and simplified methods.Finally,a fatigue analysis was performed by use data of a real ship and the reliability of the result was assessed.

Immunogenicity of the recombinant adenovirus type 5 vector with type 35 fiber containing HIV-1 gag gene

The immune efficiency of a recombinant adenovirus type 5 with type 35 fiber containing HIV-1 gag gene （rAd5/F35-mod. gag） was investigated in BALB/c mice, in which the rAd5/F35-mod, gag was firstly identified with PCR, then transfected to 293 cells and the in vitro expression level of Gag protein was determined by Western blotting and indirect immuno-fluorescent assay. Mice were immunized with intramuscular injections of rAd5/F35-mod, gag, rAd5-mod, gag or DNA and were boosted after 3 weeks. To test the effect of pre-existing anti-viral immunity on immunization, mice were also injected with Ad5- GFP vector and then immunized 4 and 7 weeks later with Ad5/F35-mod. gag vector. The P24-specific IgG antibody in sera of immunized mice was determined by ELISA and the specific cytotoxic T lymphocyte （CTL） response was assayed by intracellular cytokine staining. It was demonstrated that the rAd5/F35- rood. gag vector could express efficiently the HIV Gag protein in 293 cells in vitro and induce strong HIV- specific immune responses in vivo. The strongest CTL and serum IgG response occurred when mice were immunized twice with injection of rAd5/F35 alone, but the anti-Ad5 antibody after primary infection with adenovirus could inhibit the specific immune responses induced by rAd5/F35 vector. It is concluded that single immunization with recombinant adenovirus rAd5/F35-mod, gag can induce specific CTL and serum IgG antibody responses in mice, but the immunogenicity of rAd5/F35 is comparably weaker than that of rAd5.

Recombinant adenovirus-mediated expression of GHS-R1a in HEK 293 cells

Objective To construct the recombinant adenovirus vector carrying human growth hormone secretagogue receptor type 1a （GHS-R1a） ,for genetic transfection.Methods The full-length human GHS-R1a gene was obtained by PCR amplification and then cloned into the shuttle plasmid pAdTrack-CMV.The linearized plasmid pAdTrack-CMV-GHS-R1a was co-transformed into Escherichia coli （E.coli） BJ5183 cells along with an adenoviral backbone plasmid pAdEasy1.The HEK293 cells were then infected with adenoviruses.The expression of GHS-R1a was indicated by green fluorescent protein （GFP） ,and confirmed by Reverse Transcription Polymerase Chain Reaction （RT-PCR） and Western blot.Results Enzymatic digestion of pAdGHS-R1a yielded a large fragment （approximately 30 kb） and a small fragment （4.5 kb） ,indicating the success-ful construction of recombinant adenovirus expression vector.Expression of GFP was observed by confocal laser scanning microscopy at 24 h after infection.RT-PCR and Western blot further confirmed that GHS-R1a was efficiently expressed in 293 cells.Conclusion Recombinant adenovirus （AdGHS-R1a） is successfully constructed,and the target gene can be expressed efficiently in 293 cells,which provide a valuable tool for further studying the function of GHS-R1a.

Expression of recombinant adeno-associated virus in the brain of rats with a focal embolic stroke via carotid artery

OBJECTIVE: To study whether recombinant adeno-associated virus (rAAV) mediated foreign gene, LacZ, could pass the blood brain barrier by intra-carotid artery delivery and express in vivo in ischemic brain of the focal embolic stroke rats to investigate a possibility of delivering foreign gene through carotid artery to treat acute ischemic stroke. METHODS: The carotid artery territory in 41 rats was embolized with or without arterial-like fibrin rich clots to make a model of focal embolic stroke rat. rAAV containing LacZ gene (rAAV-LacZ) was constructed in 293 cells by calcium phosphate cotransfection. The rats were assigned to one of the following treatments: 1 control (without embolism) groups, including PBS treated (n = 6), pLacZ treated (n = 6 ) and rAAV-LacZ treated (n = 6): 2 embolic groups, including embolism + PBS (n =7),embolism + pLacZ (n = 8) and embolism + rAAV-LacZ (n = 8). Brains were cryosectioned and kappa-Gal stain was performed at 2, 4, and 8 weeks, respectively, after transfection, and then infarct volume was measured and the percentage of LacZ staining-positive cells was calculated. RESULTS: In all the control groups and embolism + PBS treated animal, no kappa-Gal staining-positive cells were found, but in embolism + pLacZ (n = 8) and embolism+rAAV-LacZ groups a lot of kappa-Gal staining-positive cells were found. The expression cells were in the tissues around the infarction. The gene expression persisted only nearly four weeks in embolic group with pLacZ. In the embolic group with rAAV-LacZ the expression was very stable during the experiment course (eight weeks) and the percentage of the expressed cells was significantly higher than that of its contralateral areas at the same time points, respectively. CONCLUSIONS: The plasmid vector and rAAV could enter the brain through the ischemia-damaged blood barrier and foreign gene can be expressed in brain. The positive gene expression is mainly in the peripheral areas of the infarction. rAAV as a permanent expression vector may ultimately be used for gene therapy of human ischemia cerebravascular diseases.

Specific cellular immune responses in mice immunized with DNA,adeno-associated virus and adenoviral vaccines of Epstein-Barr virus-LMP2 alone or in combination

Cellular immune responses,particularly those associated with CD3+CD8+ cytotoxic T lymphocytes (CTL),are critical factors in controlling viral infection.Nasopharyngeal carcinoma (NPC) is closely associated with persistent Epstein-Barr virus (EBV) infection.NPC vaccine studies have focused on enhancing specific antiviral CTL responses.In this study,three vaccines capable of expressing the EBV-latent membrane protein 2 (LMP2) (a DNA vector,an adeno-associated virus (AAV) vector,and a replication-defective adenovirus serotype 5 (Ad5) vector) were respectively used to immunize female Balb/c mice (4-6 weeks old) at weeks 0,2 and 4,either alone or in combination.Our results suggest that combined immunization with DNA,AAV,and adenovirus vector vaccines induced specific cellular immunity more effectively than any of these vectors alone or a combination of two of the three,constituting a sound vaccine strategy for the prevention and treatment of NPC.

A histological and biomechanical study of bone stress and bone remodeling around immediately loaded implants

Immediate loading(IL)increases the risk of marginal bone loss.The present study investigated the biomechanical response of peri-implant bone in rabbits after IL,aiming at optimizing load management.Ninety-six implants were installed bilaterally into femurs of 48 rabbits.Test implants on the left side created the maximal initial stress of 6.9 and 13.4 MPa in peri-implant bone and unloaded implants on the contralateral side were controls.Bone morphology and bone-implant interface strength were measured with histological examination and push-out testing during a 12-week observation period.Additionally,the animal data were incorporated into finite element(FE)models to calculate the bone stress distribution at different levels of osseointegration.Results showed that the stress was concentrated in the bone margin and the bone stress gradually decreased as osseointegration proceeded.A stress of about 2.0 MPa in peri-implant bone had a positive effect on new bone formation,osseointegration and bone-implant interface strength.Bone loss was observed in some specimens with stress exceeding 4.0 MPa.Data indicate that IL significantly increases bone stress during the early postoperative period,but the load-bearing capacity of peri-implant bone increases rapidly with an increase of bone-implant contact.Favorable bone responses may be continually promoted when the stress in peri-implant bone is maintained at a definite level.Accordingly,the progressive loading mode is recommended for IL implants.