猪IL-2真核表达质粒构建及对PRRSV-ORF5基因疫苗免疫佐剂作用的研究

本研究应用真核表达载体pcDNA3.1（＋）和猪白细胞介素2（IL-2）基因成功构建了IL-2的真核表达质粒（pcDNA-IL-2）,探讨了pcDNA-IL-2作为分子免疫佐剂对猪繁殖与呼吸综合征（PRRS）ORF5基因疫苗（pcDNA-PRRSV-SC2-ORF5）免疫猪的增强作用。经间接ELISA法、MTT比色法及流式细胞仪分别对pcDNA-IL-2与pcDNA-PRRSV-SC2-ORF5共同免疫猪、pcDNA-PRRSV-SC2-ORF5单独免疫猪、pcDNA3.1（＋）空载体和灭菌水免疫对照猪的血清抗PRRSV抗体IgG、外周血T淋巴细胞的增殖活性、CD4^＋和CD8^＋细胞比例进行检测,结果表明pcDNA-IL-2与pcDNA-PRRSV-SC2-ORF5共同免疫猪的IgG含量、外周血T淋巴细胞的增殖活性、CD4^＋和CD8^＋细胞比例与pcDNA-PRRSV-SC2-ORF5单独免疫猪相比有显著差异（P〈0.05）。说明pcDNA-IL-2能显著增强pcDNA-PRRSV-SC2-ORF5基因疫苗免疫猪的体液和细胞免疫应答,可作为PRRSV基因疫苗的良好佐剂。     

AMOS PCR在布鲁氏菌种型鉴定中应用的研究

AMOSPCR是近年来在布鲁氏菌上尝试使用的一种布鲁氏菌检测方法,可作为布鲁氏菌诊断及菌种类型鉴定的PCR方法,可区分牛种布鲁氏菌1、2、4型,羊种布鲁氏菌、猪种布鲁氏菌、绵羊附睾种布鲁氏菌。本试验用来自国内的疫苗S2,M5及国际上常用的布鲁氏菌疫苗S19,104M试验AMOSPCR的效果,同时对AMOSPCR扩增条带进行测序。结果表明,除国内布鲁氏菌S19检测结果与国外菌株不同外,其余的几种疫苗株都得到典型的特征性条带。同时测序结果也表明各种属条带无交叉反应,为今后国内诊断布鲁氏菌病诊断方法研究指出一个方向。     

F-2毒素致体外培养大鼠睾丸支持细胞DNA损伤

为探讨F-2毒素对雄性生殖机能的影响,取大鼠睾丸支持细胞进行体外培养,运用单细胞凝胶电泳技术检测51、0、20和40 mg/L的F-2毒素攻毒后24 h支持细胞DNA的损伤情况。结果显示,F-2毒素在一定浓度范围内对体外培养的支持细胞DNA产生损伤作用,且受损程度随着F-2毒素攻毒剂量而升高,具有明显量效关系。除5 mg/L剂量组外,其余各组的细胞受损率、彗星尾长和DNA损伤程度与阴性对照组相比差异极显著（P〈0.01）,表明F-2毒素对体外培养的大鼠睾丸支持细胞有毒性作用,能够损伤细胞DNA。     

精子载体法获得转人her2基因猪

将带有外源基因的载体酶切纯化后与去精清的猪新鲜精液按比例混合，17℃静置处理使外源DNA进入精子头部，通过人工授精使受体母猪怀孕后产下20头仔猪，经PCR特异性片段扩增，特异片段序列测定和比对，共4头PCR结果为阳性。初步证明人her2基因已在猪染色体上整合，其整合率约为20％。本研究利用精子载体法成功获得了转基因阳性猪，为大动物的转基因研究提供了理论与实践的参考。     

Higher than optimum temperature under CO2 enrichment influences stomata anatomical characters in rose (Rosa hybrida)

There is little available information on the effects of temperature and CO₂ enrichment on stomata anatomical characteristics of plants. Effect of these two microclimates was studied on five rose (Rosa spp.) cultivars, viz. ‘First Red’ (used as check), ‘Arjun’, ‘Raktima’, ‘Raktagandha’ and ‘Pusa Pitamber’. Budded, single-stemmed rose cultivars having five lateral buds were grown in controlled environment growth cabinets under enriched CO₂ (1000 μmol mol⁻¹) and optimum (28/18 °C, T₀) or high (35/25 °C, T₁) temperature for 50 days. All observations were made on the abaxial leaf surface. Significant increases in stomatal density (68.7%), index (29.6%) and epidermal cell density (37.3%) were recorded in plants grown at high temperature over control with CO₂ enrichment. The cultivars responded differently in terms of length and width of guard cell and stoma (pore) under high temperature, however, the values averaged over treatments showed a significant reduction in these parameters. Further, number of stomata per leaf was higher (28.3%) in plants grown at high temperature, except First Red. A reduction in mean leaf area (26.7%) and dry mass (32.0%) was recorded at high rather than optimum temperature. The specific leaf area was maximum in Arjun (87%) while in First Red, a 14% reduction was noted at high temperature.     

Cryopreservation of carnation (Dianthus caryophyllus L.) shoot tips by encapsulation-vitrification

Shoot tips excised from in vitro cultured plants of Dianthus caryophyllus L. (cv. Pallas, cv. Pink Candy and cv. Wanessa) were successfully cryopreserved using an encapsulation-vitrification method. Shoot tips (2–3 mm in length) were encapsulated in sodium alginate, precultured on liquid Murashige and Skoog (1962) medium supplemented with various sucrose concentrations (0.25, 0.5, 0.75, 1.0 M) for 24 h or 48 h and dehydrated with the vitrification solution PVS2 (up to 4 h) at 24 °C or 0 °C prior to direct immersion in liquid nitrogen (−196 °C). A maximum of shoot regeneration from cryopreserved shoot tips was obtained with the following combinations: preculture in 0.5 M sucrose and 180 min dehydration treatment at 0 °C for cv. Pallas (60% shoot formation), or preculture in 0.75 M and 200 min dehydration at the same temperature for cv. Pink Candy (66.6% shoot formation) and cv. Wanessa (73% shoot formation).     

Biological characteristics of JAK2 transduced CD34+ cells from cord blood during ex vivo expansion

AIM: To explore the feasibility and biological characterization of long-term regulated expansion of JAK2 transduced human CD34⁺ cord blood cells in vitro.METHODS: A retrovirus (RV) vector which contains JAK2 catalytic domain and two binding sites for a chemical inducer,dimerization (AP20187),was cloned (designated MGI-F₂JAK2).CD34⁺cells were enriched from cord blood with a MiniMACS system.The purified CD34⁺ cells were transfected with supernatant from the retrovirus packaging cell line that expressed JAK2.Following transduction,cells were expanded into four groups: AP20187 alone,FL alone,TPO,alone,AP20187+FL+TPO,respectively.The expanded cells were monitored by GFP expression,immunophenotyping,progenitor colony assay,karyotype analysis as well as tumorigenesis in nude mice.RESULTS: The purity of selected CD34⁺ cells was over 91% and gene transfer rate was 49.32%±6.21%.Only the group of AP20187 +FL+ TPO was obtained a significant sustained outgrowth of the transduced CD34⁺ cord blood cells.The percentage of GFP+ cells consistently produced a rise to the 90% peak level by the end of 8th week of culture.Flow cytometry analysis showed that the phenotype of the expanded cells was CD33⁺,CD61⁺ and Gly-A⁺ partial positive;CD38⁺ and HLA-DR⁺ strong positive,while CD2,CD7 and CD19 were almost negative.Colony assays performed in methycelluos,which can give rise to BFU-E,CFU-GM and CFU-Mix,the CFU-GM was predominantly in all colonies.The tumor was not observed in nude mice and the karyotype analysis was normal from expanded cells.CONCLUSION: The results demonstrate that AP20187-mediated activation of JAK2 signaling is capable of stimulating expansion JAK2 transduced CB CD34⁺ cells in combination with FL and TPO.This system may have applications for studies in signaling transduction,hematopoiesis,and for gene and cell therapy.     

Mechanisms for protection of berberine against LPS-induced acute lung injury in mice

AIM:To investigate the mechanisms by which berberine attenuates LPS-induced acute lung injury, and provide a new strategy for the treatment of the lung injury due to LPS. METHODS:BALB/c mice were randomly assigned into three groups (control, LPS group, and berberine treatment group). Mice were administered intragastrically with distilled water (0.1 mL/10 g) or neutral sulfate berberine (50 mg/kg) once a day for 3 days, 1 h after intragastrical treatment on day 3, LPS (20 mg/kg) or normal saline was injected intraperitoneally (ip). All animals were sacrificed 12 h after LPS injection, the left lung tissue sections were prepared for histology analysis and the right lung were used to determine the ratio of wet to dry lung tissue weight (W/D). In another experiment, bronchoalveolar lavage fluid (BALF) was collected, and then the total protein content, and the amounts of white blood cells (WBC) and polymorphonuclear neutrophils (PMN) in BALF were determined. Furthermore, the phosphorylation of cytosolic phospholipase A₂ (cPLA₂) was detected with immunohistochemical analysis by using phospho-cPLA2(Ser505) antibody, and the contents of thromboxane B₂ (TXB₂) in BALF, malondialdehyde (MDA) in the lungs, and activity of superoxide dismutase (SOD) in lung tissues were also determined.RESULTS:LPS induced acute lung injury, activated cPLA₂, and increased TXB₂ content in the BALF and MDA level in the lung tissue. The pretreatment with berberine significantly attenuated lung injury, lung edema and protein leakage induced by intraperitoneal injection of LPS. The expression of phospho-cPLA₂ in the lung tissues and TXB₂ content in the BALF in the berberine treatment group were lower than those in LPS group (P<0.05). In addition, the content of MDA in the lung tissue was lower in the berberine treatment group than LPS group (P<0.05), but there was no significant difference in activity of lung SOD between the berberine treatment and LPS group (P>0.05). CONCLUSION:Pretreatment with berberine remarkably reduces the LPS-induced lung injury, which is, at least in part, through inhibiting phosphorylation of cPLA₂ and decreasing lipid peroxidation. These findings provide a new strategy for the prevention and treatment of LPS-induced acute lung injury.     

Effect of rhEGF activating extracellular signal-regulated kinase on the expression of Bcl-2 and p53 protein in human amniotic cells

AIM: To explore the effect of recombinant human epidermal growth factor (rhEGF) on the ERK1/2 pathway and its safety in use when the proliferation of cultured FL cells (human amnion epithelial cells) were promoted by rhEGF.METHODS: FL cells were stimulated by rhEGF at various concentrations.The proliferation rate of FL cells was evaluated by MTT assay.Western blotting was used to detect phosphorylation of ERK1/2 (p-ERK1/2) and the change of Bcl-2 and P53 protein.RESULTS: The proliferation rate FL cells reached the maximum when the concentration of rhEGF was 10 μg/L (42.4％，P<0.01).Western blotting showed that the activity of p-ERK1/2 increased significantly when rhEGF ranged in 10 μg/L to 60 μg/L.Level of Bcl-2 was unchanged in each groups.Level of P53 decreased significantly when the concentration of rhEGF was 60 μg/L.CONCLUSION: It is safe when rhEGF presents in optimization concentration of 10 μg/L.The capability of apoptosis may be depressed when rhEGF is in larger dose.     

喷施磷酸二氢钾对桃叶片和果实性状的影响

以不同成熟期的桃品种豫甜、豫香、秋甜、秋蜜红为试材,研究了喷施磷酸二氢钾对其叶片叶绿素含量、干鲜质量比及果实的可溶性固形物含量、可溶性糖含量、可滴定酸含量、维生素C含量等品质特性的影响。结果表明,喷施磷酸二氢钾能提高叶片中叶绿素含量、干鲜质量比和果实可溶性固形物含量、可溶性糖含量;对果实的可滴定酸含量、维生素C含量影响不明显;叶片喷施不同浓度和次数的磷酸二氢钾均可推迟桃果实成熟期;喷施清水和对照处理间叶片性状和果实品质差异不明显;喷施磷酸二氢钾浓度和次数以500mg/L3次效果最显著,以300mg/L3次最经济有效。