中国人肠道产甲酸草酸杆菌甲酰辅酶A转移酶基因的分离、克隆和鉴定

目的 克隆产甲酸草酸杆菌甲酰辅酶A转移酶基因（frc），检测其在真核细胞中的表达。方法 用聚合酶链式反应技术从产甲酸草酸杆菌基因组DNA中扩增frc基因片段并插入真核表达载体获取重组质粒pEGFP-frc，通过限制性内切酶酶切电泳和测序鉴定重组质粒。重组质粒转染人胚。肾293细胞，检测frc基因在mRNA和蛋白水平上的表达。被转染细胞在含草酸培养液中培养，检测0～72h培养液中草酸浓度的变化。结果 frc基因的真核表达载体构建成功。转染293细胞后24～72h，可观察到明亮的绿色荧光，在mRNA和蛋白水平上可检测到frc基因在真核细胞中的表达。转染pEGFP-frc细胞12～72h培养液中草酸浓度明显低于转染空载体的细胞（P〈0．05）。结论 中国人肠道产甲酸草酸杆菌中可分离出frc基因；frc基因可在真核细胞293细胞中表达并使293细胞获得代谢草酸的能力。     

可分解草酸小鼠肠干细胞群的构建

目的 通过提取草酸分解菌的分解草酸功能基因frc和oxc,转染小鼠肠干细胞群使之获得草酸分解功能.方法 (1)构建能同时表达oxc和frc基因的双表达载体质粒pIRES-oxc-frc.(2)分离培养小鼠肠干细胞群,并了解其生长分化功能.(3)将pIRES-oxc-frc通过脂质体转染至小鼠肠干细胞群中,经过G418筛选后,了解验证转基因肠干细胞群生长、分化及基因表达状况.(4)用离子色谱法测定转基因后细胞培养液中草酸浓度,鉴定其草酸分解功能.结果 oxc和frc基因片段与GenBank提供的序列比较有极高的同源性.带有oxc和fre基因片段的重组质粒能顺利转染小鼠肠干细胞群,并能在后者中表达.转基因小鼠肠十细胞群培养液中草酸浓度[(2.48±0.03)g/L]低于普通小鼠肠干细胞群和空白对照组[(2.69±0.01)、(2.69±0.01)g/L,P<0.01].结论 产甲酸草酸杆菌分解草酸的重要功能基因oxc和frc基因能在体外转入小鼠肠十细胞群,并使后者具有草酸分解功能;原核细胞的多个基因能在一定条件下通过基因工程技术转入真核细胞.     

Apoptosis of human trabecular meshwork cells induced by transforming growth factor-β<Subscript>2</Subscript><Emphasis Type="Italic">in vitro</Emphasis><Emphasis Type="Italic">in vitro</Emphasis>

Summary Whether transforming growth factor-β₂ (TGF-β₂) induces apoptosis of human trabecular meshwork cells was investigatedin vitro. Cultured 3–5 passage human trabecular meshwork cells were treated with 0 (control). 0. 32. 1, 3. 2 ng/ml TGF-β₂ for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmisson electron microscopy. TUNEL technique and flow cytometry. The results showed character istic morphologic changes of apoptotic cells were observed under transmission electron microscopy. DNA fragmentation of human trabecular meshowork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79±0.44)%. (4.43±1.17)% and (9.60±2.05)% respectively with different concentrations [1 ng/ml (P<0.05), 3.2 ng/ml (P<0.01)] of TGF-β₂ with the difference being significant between experimental group and control group [(1.41±0.34)%]. It was concluded that TGF-β₂ can induce apoptosis of human trabecular meshwork, cellsin vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people. CAO Yang, male, born in 1972, M. D., Ph. D., Associate Professor This project was supported by a grant from the National Natural Sciences Foundation of China (No. 38970758).     

In vitro Recombination and Identification of Mutated Fragment Corresponding to Regulation Region of mtrR Gene of Neisseria Gonorrhoeae

A site-directed mutant DNA fragment was synthesized and transfected into clinical Neisseria Gonorrhoeae(NG) stains to construct the transformants that contained the corresponding mutagenesis of regulation region of mtrR gene.According to the technique of gene splicing by overlap extension(SOEing),a DNA segment with specific mutagenesis was constructed by two-step polymerase chain reaction(PCR).The mutation fragments EF could be used for the next experiment in which the mutation NG strains were induced.By comparing the recombinant EF fragments to the corresponding DNA fragments of clinical NG strains,2 of these were not compatible completely.The results of sequencing revealed that there was a 9 bp deletion between the 45 to 54 inverted repeat sequence localized within the mtrR promoter.It can be confirmed that the fragments EF are the specifically designed mutant fragments.     

Superparamagnetic Iron Oxide Labeling of Neural Stem Cells and 4.7T MRI Tracking in vivo and in vitro

Neural stem cells were labeled with superparamagnetic iron oxide (SPIO) and tracked by MRI in vitro and in vivo after implantation. Rat neural stem cells were labeled with SPIO combined with PLL by the means of receptor-mediated endocytosis. Prussian blue staining and electron mi-croscopy were conducted to identify the iron particles in these neural stem cells. SPIO-labeled cells were tracked by 4.7T MRI in vivo and in vitro after implantation. The subjects were divided into 5 groups, including 5×105 labeled cells cultured for one day after labeling, 5×105 same phase unla-beled cells, cell culture medium with 25 μg Fe/mL SPIO, cell culture medium without SPIO and dis-tilled water. MRI scanning sequences included T1WI, T2WI and T2WI. R2 and R2 of labeled cells were calculated. The results showed: (1) Neural stem cells could be labeled with SPIO and labeling efficiency was 100%. Prussian blue staining showed numerous blue-stained iron particles in the cyto-plasm; (2) The average percentage change of signal intensity of labeled cells on T1WI in 4.7T MRI was 24.06%, T2WI 50.66% and T2WI 53.70% respectively; (3) T2 of labeled cells and unlabeled cells in 4.7T MRI was 516 ms and 77 ms respectively, R2 was 1.94 s-1 and 12.98 s-1 respectively, and T2 was 109 ms and 22.9 ms, R2 was 9.17 s-1 and 43.67 s-1 respectively; (4) Remarkable low signal area on T2WI and T2WI could exist for nearly 7 weeks and then disappeared gradually in the left brain transplanted with labeled cells, however no signal change in the right brain implanted with unlabeled cells. It was concluded that neural stem cells could be labeled effectively with SPIO. R2 and R2 of labeled cells were increased obviously. MRI can be used to track labeled cells in vitro and in vivo.     

Construction of Prokaryotic Expressing Vector of Antisense Nucleic Acid of lasR and Its Effect on the Virulence of Pseudomonas Aeruginosus

To construct a pUCP18/lasRantisense plasmid carrying the reversed gene and analyze its effect on the virulence of Pseudomonas aeruginosus,LasR gene was amplified from the genome of Pseudomonas aeruginosus by PCR and reversely recombined with plasmid pUCP18.The recombi-nant pUCP18/lasRantisense was verified by enzyme digestion,PCR and sequencing.The biological ef-fects of pUCP18/lasRantisense were examined by using RT-PCR,NAD method and the assay of pyo-cyanin.Our results showed that the expected full length lasR fragment(721 bp) was extended from Pseudomonas aeruginosus gene with PCR.And it is consistent with LasR gene of Pseudomonas aeruginosa in GenBank(No.NC-002516).The recombinant plasmid was successfully constructed and transferred into Pseudomonas aeruginosus.The antisense nucleic acid of LasR gene could reduce the virulence of Pseudomonas aeruginosus and might serve as a new target site for treatment purpose.     

Ultrasound and Microbubbles： Their Functions in Gene Transfer In Vitro

To examine the role of ultrasound in gene delivery in vitro, three cells lines were exposed to the low-frequency ultrasound of varying intensities and for different durations to evaluate their effect on gene transfection and cell viability of the cells. Microbubble （MB）, Optison （10%）, was also used to observe the role of the microbubbles in gene transfection. The results demonstrated that as the ultrasound intensity and the exposure time increased, the gene transfer rate increased and the cell viability decreased, but at high energy intensities, the cell viability decreased dramatically, which caused the transfer rate to decrease. The most efficient ultrasound intensity for inducing gene transfer was 1 W/cm^2 with duration being 20 s. At the same energy intensity, higher ultrasound intensity could achieve maximal gene transfer rate earlier. Microbubbles could increase ultrasound-induced cell gene transfer rate by about 2 to 3 times mainly at lower energy intensities. Moreover, microbubbles could raise the maximum gene transfer rate mediated by ultrasound. It is concluded that the low-frequency ultrasound can induce cell gene transfer and the cell gene transfer rate and viability are correlated with not only the ultrasound energy intensity but also the ultrasound intensity, the higher ultrasound intensity achieves its maximal transfer rate more quickly and the ultrasound intensity that can induce optimal gene transfer is 1 W/cm^2 with duration being 20 s, and microbubbles can significantly increase the maximal gene transfer rate in vitro.     

HBsAg/HBsAb double positive hepatitis B virus infection model <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

The pathogenesis of HBsAg （＋）/HBsAb （＋） double positive hepatitis B virus infection was investigated by simulating HBsAg/HBsAb coexistence in vitro and establishing HBsAg/HBsAb double positive model in vivo. Eukaryotic expression plasmids PCI-SY, PCI-adw, PCI-adr, PCI-ayw, which expressed S gene product of different serotypes, were constructed and transfected into HepG2 cells. Recombinant proteins were purified from the transfected cells. At the same time, HBsAg mouse antiserum was obtained by immunizing mice with PCI-SY plasmid. HBsAg/HBsAb coexistence was simulated using these antigens and antiserum. Furthermore, the expression plasmids expressing different serotypes of S gene product including PCI-adw, PCI-adr, and PCI-ayw were injected into mice via tail vein. HBsAg and HBsAb in mice sera were tested at the first and 7th day respectively after antigen plasmids injection. Both in vitro simulation and in vivo animal models demonstrated that HBsAg antigen and HBsAb of the same serotypes Could not coexist, but HBsAg antigen and HBsAb of different serotype could coexist. HBsAg/HBsAb double positive hepatitis B virus infection could be due to infection of viruses of different serotypes.     

Role of Caspase-3 Inhibitor in Induced Anoikis of Mesenchymal Stem Cells In Vitro

By preventing mesenchymal stem cells (MSCs) from adhering to precoated agarose to create a model of MSC suspension in vitro, we investigated anoikis in MSCs and the role of cas- pase-3 in the anoikis. The cultured MSCs were randomly divided into 3 groups: the anoikis group, caspase-3 inhibitor group and control group. Before experiment, we coated dishes with 1.5 % agarose; in the anoikis group, MSCs were put into the precoated dishes; and in the inhibitor group, caspase-3 inhibitor and MSCs were also put into the precoated dishes; but there were not intervention in the control group. MSCs were collected at 2 h, 6 h, 12 h and 24 h. The alteration of caspase-3 activity was evaluated by caspase-3 fluorometric assay and western blot analysis. The apoptosis rates were detected by flow cytometry. MSCs were round and suspended sufficiently in the anoikis and inhibitor groups. Caspase-3 fluorometric assay showed that there were significant differences in statistics be- tween the anoikis group and the others (P<0.05). Western blot analysis discovered that caspase-3 ex- pression in the anoikis group was more than that in the control and inhibitor groups (P<0.05). Flow cytometry showed that the apoptosis peak appeared in all the three groups, but it increased dramati- cally in the anoikis group. The apoptosis rates in the inhibitor and control groups were low and stable. And there were significant differences in statistics between the anoikis group and the others (P<0.05). MSCs will undergo anoikis in suspended condition if they are separated from the extracellular matrix. Caspase-3 inhibitors can suppress caspase-3 activity and reduce the apoptosis rate significantly. Cas- pase-3 plays a vital part in induced MSC anoikis in vitro. MSCs suspension culture system might be set up with argorose and caspase-3 inhibitor.     

A Decision Support System for Preventing <Emphasis Type="Italic">Legionella</Emphasis> Disease

Information systems plays an important role in medicine because it helps process more data more efficiently while providing access to more people in different parts of the world. In this research we analyzed the data of legionella pneumophila and other legionella species collected by the public hygiene center (PHC). PHC collected 7,211 water samples from different sources of different locations in different cities in Turkey from year 1995 to 2008. The main goal of this research is to develop a conceptual framework for preventing disease and to design a medical decision support system to help administration assessing the risk of Legionnaires’ disease and preventing the outbreaks of the disease. The DSS involves SOM software which was programmed with C# to search for patterns and similarities in data sets by producing SOM risk maps. Thus administrators can decide where to monitor cautiously to prevent the disease.     

Cloning of chicken anemia virus vp3 gene and apoptosis inductive effect of vp3 gene<Emphasis Type="Italic">in vitro</Emphasis>

Summary Using PCR technique, the vp3 gene of chicken anemia virus (CAV) was cloned into the eukaryotic expression vector pcDNA3 to construct a recombinant pcDNA-vp3. Restriction enzyme digestion and sequencing analysis revealed that CAV vp3 gene was correctly inserted into the blank vector pcDNA3. After LipofectAMINE^TM-mediated transfectionin vitro with pcDNA-vp3 and pcDNA3 respectively, the total mRNA was extracted from liver carcinoma cell lines HepG2 and diploid cell line L-02, and RT-PCR was performed afterward. The results of RT-PCR suggested that vp3 gene was expressed in these two cell lines. At the same time, usingin situ apoptotic detection assay, TUNEL kits, the apoptotic cells were found in pcDNA-vp3 transfected HepG2, but not in mock transfected cell lines. VP3 could induce cell death by apoptosis in cancer cell lines, but not in diploid cell lines. All the results indicated that CAV vp3 gene, a potential therapeutic agents, has the potential of being used for cancer treatment. Sun Jun, male, born in 1970, Graduate Student This project was supported by a grant from the Hubei Natural Science Foundation (No. 2002ABA004).     

Study of <Emphasis Type="Italic">Candida albicans</Emphasis> vaginitis model in Kunming mice

Summary The model of vaginal candidiasis in Kunming mice was constructed in order to search for the optima construction conditions and provide an economic animal model of Candida albicans (C. albicans) vaginitis. Estrogen benzoate (E₂) was given to mice at different concentrations ranging from 0.0 to 0.05 mg/mouse (4 levels) beginning 72 h prior to vaginal inoculation, then mice were inoculated intravaginally with various concentrations of stationary-phase C. albicans blastoconidia (ATCC90028) (5 levels) in 20 μL of phosphate-buffered saline (PBS) in each E₂ level. General state, scores of genital pathology, the hyphae and vaginal fungal burden (CFU) in vaginal lavage fluid, the hydrops rate of uterus and vaginal tissues for pathological section in mice were observed and obtained at day 2, 4, 7, 14 and 21 after inoculation. The results showed the infection rate in mice was related to the dosage of E₂ and concentration of C. albicans blastoconidia. Additionally there was better cross-effect between the two treated factors. The infection rate was about 80% on the day 4, and could reach 100% on the day 7 until the end of experiment after inoculated intravaginally in groups of E2I3, E₂ 0.025 mg/mouse injected hypodermically and inoculated intravaginally with 5 × 10⁴ C. albicans blastoconidia, and large amount of hyphae and blastoconidia could be observe in superficial layer tissue and canal of vaginal by PAS. From the results in our experiment it was concluded that E2I3 was the optima construction condition in kunming mice. CHEN Zhuo, female, born in 1965, Associate Professor This project was supported by the National “10th Five-Year” Key Technologies R&D (No.2004BA709B13-02)     

Antiviral Activity of Nano Carbon Fullerene Lipidosome against Influenza Virus In Vitro

The activity of nano carbon fullerene lipidosome(NCFL) against influenza virus H1N1 in vitro was studied by observing the cytotoxicities and its activity rendered by different intensities of lighting with various periods of time.Rimantadine hydrochloride was used as the positive control drug.By using microcultural technique,the morphological changes of cells were observed and by using the gentian violet staining,antiviral activity of the NCFL against influenza virus was assayed.The results showed that:(1) The maximal concentration of the NCFL was 7 μg/mL and the 50% toxic concentration(TC50) was 13.54 μg/mL respectively;(2) NCFL had a significant activity of directly killing the influenza virus,while the activities in antiadsorption and antireplication were not obvious;(3) There was a dose-activity relationship between the dosages of NCFL and the direct killing effect against the influenza virus,and the periods of lighting-time could influence the activity partly.It was concluded that NCFL had a significant activity of directly killing the influenza virus.     

Comparison of fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization and immunohistochemistry for assessment of HER-2 status in breast cancer patients

The accurate assessment ofa proto-oncogene, human epidermal growth factor receptor-2 gene （HER-2）, is extremely important for the therapy and prognosis of breast cancer. Currently, immunohistochemistry （IHC） is the method widely used for the detection of HER-2 protein. Fluorescence in situ hybridization （FISH） has been suggested to be a golden standard assay for HER-2 amplification. This study examined the expression and amplification of HER-2 in paraffin-embedded sections of breast cancer tissues, and compared the two methods on the measurement of HER-2 status. HER-2 gene and protein were determined in breast cancer samples from 52 Chinese women by FISH and IHC respectively. The findings indicated that the HER-2 gene amplification was found in 18 cases （34.6%） by FISH and the HER-2 protein over-expression （score 3＋） in 15 cases （28.8%） by IHC. hnmunohistochemically, 28.6% of the cases scored as 2＋ and 93.3% of the cases scored as 3＋ were HER-2-positive by FISH. There was a significant correlation between the HER-2 gene amplification and HER-2 protein over-expression in breast cancer （P〈0.005）. No correlation was noted between the HER-2 gene amplification and any of the clinicopathological parameters examined, including age, menopausal status, menarche age, tumor size, histological tumor type, histological grade, lymph node status, and the expression of ER and PR. It was concluded that the detection of HER-2 gene amplification in breast cancer by FISH is valuable and can compare with HER-2 protein detection by IHC.     

Effect of Laser in situ Keratomileusis on Accommodation

The accommodative function before and after laser in situ keratomileusis （LASIK） was observed, and the effect of LASIK on accommodation was investigated. In a prospective clinical trial, 48 myopic patients （96 eyes） subject to bilateral LASIK in Refractive Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology （China） from March 2006 to June 2006 were selected and studied. Refractions, accommodative range, amplitude of accommodative response and high frequency component （HFC） of accommodative microfluctuations were measured with NEDIK-730A before and one week and 30 days after operation. Dominant and non-dominant eyes were determined by hole-in-card method. It was found that all of the operative eyes showed an uncorrected visual acuity of 0.8 or better one week postoperatively, and 1.0 or better 30 days postoperatively. Compared with those preoperatively, accommodative range and HFC had no significant difference at first week and 30th day after operation in both dominant eyes and non-dominant eyes （P〉0.05）, but there was a significant difference in the amplitude of accommodative response/accommodative stimulus ratio （A/S） after operation （P〈0.01）, and no significant difference was found in accommodation between one week and 30 days postoperation. No ocular dominance＇s change was noted. There was no significant difference in accommodative function between dominant eyes and non-dominant eyes. It was suggested that LASIK produced no significant effect on accommodation.     

Effect of Anti-KDR Antibody on the Proliferation of Hemangioma Vascular Endothelial Cells in vitro

The suppressive effect of anti-KDR antibody against VEGF on proliferation of hemangioma-derived vascular endothelial cells(HVECs) was investigated.HVECs from one case of hemangioma in proliferative phase were cultured.Both primary culture and sub-culture were conducted in M199 medium.The HVECs of passage 3 were divided into 4 groups based on the concentrations of anti-KDR antibody.Cell count was performed and inhibitory rate of HVECs was measured before and 9 days after interference.The results showed that the number of HVECs in the anti-KDR antibody-treated groups was significantly decreased and the inhibitory rate of HVECs by anti-KDR antibody(50,10 and 2 μg/mL) was 84%,63% and 39% respectively at 9th day after interference,with the difference being significant.In the control group,the number of HVECs was increased significantly.In was concluded that the anti-KDR antibody could suppress the activity of VEGF through blocking the KDR,indicating the potential clinical applications of anti-KDR antibody in the treatment of hemangioma.     

Anti-endotoxic effects of syringic acid of<Emphasis Type="Italic">Radix Isatidis</Emphasis>

Summary The anti-endotoxic effect of syringic acid (SA) isolated fromRadix Isatidis (Banlangen, BLG) was studied. SA was extracted and isolated from BLG and diluted into 1% solution. The content of SA-pretreated endotoxin (ET) was quantitatively determined using Limulus test. The ability of fever induction of ET pretreated with SA was measured using endotoxin-induced fever test in rabbits. The LPS-induced death in mice pretreated with and without SA was compared. Results showed that after pretreatment with SA, 83.16% of ET was destroyed, the ET-induced fever in rabbits relieved markedly and the LPS-induced death rate in mice dropped from 68 % to 20 %. It was concluded that SA isolated from BLG had anti-endotoxic effects. LIU Yunhai, male, born in 1942, Pharmacist This project was supported by a grant from National Natural Sciences Foundation (No. 39870872) and Foundation of Public Healthy Ministry of China (No. 98-2-110).     

Reversal of multi-drug resistance by vector-based-ShRNA-Mdr1 <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis>

In order to investigate the effects of vector-based hairpin small interference RNA （shRNA） on the reversal of multi-drug resistance （mdr） of A2780/Taxol cells, a novel vector pEGFP-HI/mdrl containing mdrl-shRNA targeting at position 2943-2963 of mdrl was designed and synthesized. Subsequently, A2780/Taxol cells were transfected with pEGFP-H1/rndrl, and the expression ofmdrl mRNA and P-gp was detected by using RT-PCR and Western blot respectively. MTT was used to measure the 50% inhibition concentration （IC50） of Taxol to A2780/Taxol cells. The results showed that at the 24th and 48th h after transfection, the expression of mdrl mRNA was decreased to （52.1±1.0）% and （0.01±1.7）%, and that of P-gp decreased to （88.3±2.1）% and 0%, respectively. At the 48th h after transfection, the relative reversal rate of A2780/Taxol cells to Taxol was 69.54%. In vivo, the nude mice xenografts were injected with pEGFP-H1/mdrl, and then administrated Taxol. The tumor volume in pEGFP-H1/mdrl-transfected group was significantly reduced as compared with that in blank control group or pEGFP-Hl-transfected group （807.20±103.16 vs 1563.78±210.54 or 1480.78±241.24 mm^3, both P〈0.01）. These results suggested that transfection of pEGFP-HI/mdrl could efficiently down-regulate the expression of mdrl mRNA and P-gp in A2780/Taxol cells, and effectively restore the sensitivity of A2780/Taxol ceils to Taxol both in vitro and in vivo.     

Construction of Prokaryotic Expression Plasmid of Fusion Protein Including Porin A and Porin B of Neisseria Gonorrhoeae and Its Expression in E. coli

In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a ( ) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E. coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a ( ) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E. coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0. 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.