EHEG O157△ler/stx(pBR322∷stx 1/2B∷eae)的构建与生物学特性分析

为了获得安全性好,对出血性大肠杆菌具有良好免疫保护作用的疫苗株,本实验以构建的O157△ler/stx基因缺失突变株为载体,将决定特异性粘附的eae全基因插入到质粒pBR322∷stx1/2B中,构建了基因工程菌O157△ler/stx(pBR322∷stx1/2B∷eae),对该基因工程菌进行生物学特性分析,实验结果表明,该菌正常培养可表达eae;具有粘附HEp-2细胞的特性;该菌培养上清对Vero细胞没有毒性作用;给小鼠口服接种该工程菌后可在肠道中定居至少10 d;14日龄小鼠口服活菌量为5×109CFU/只,小鼠生长正常,没有出现任何临床症状;该菌通过口服免疫小鼠可以诱导肠道粘膜免疫应答和体液免疫应答.这些结果表明,该工程菌安全性好,免疫原性强,是有价值的预防EHEC O157感染的基因工程疫苗候选株.     

Antioxidant and stomatal responses of grapevine (Vitis vinifera L.) to boron toxicity

Although of considerable agronomic importance, our understanding of B toxicity mechanism in plants is still not completely understood, and remains an open question. Therefore, we investigated the effect of increasing levels of B (0, 10, 20 and 30 mg kg⁻¹) on the growth, boron (B) concentrations, stomatal resistance, lipid peroxidation (MDA), membrane permeability (MP), lypoxygenase activity (LOX), proline (PRO) and H₂O₂ accumulation, and the activities of major antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT and ascorbate peroxidase, APX) of grapevine (Vitis vinifera L. cv. Kalecik Karasi) grafted on 5BB rootstock (V. berlandieri × V. riparia) was investigated. Applied toxic levels of B significantly reduced leaf and root growth and increased the B concentration of the leaf, and stem, bark and root of rootstock. In the all B levels leaf tissues of grapevine accumulated more B than that of the other plant parts. In order to restrict excessive uptake of B, stomatal resistance of the leaves increased especially at high B treatments (20 and 30 mg kg⁻¹). The concentrations of H₂O₂, MDA and membrane permeability were increased as the result of B toxicity while proline and the activity of lypoxygenase were decreased. Compared with control plants, the activities of SOD and CAT were increased by B treatments while the activity of APX was decreased. To our knowledge, this is the first report that B toxicity elevated the antioxidant enzymes to protect the membrane functions from reactive oxygen species (ROS) injury in grapevine and it was hoped that this study would provide a basis for developing strategies for reducing the risks associated with B toxicity.     

转rolA、B、C基因枳橙快繁技术

通过对转rol基因枳橙B、D、E系及对照的高接植株春梢茎段进行萌芽诱导、增殖及试管苗生根成苗的试验,摸索出适宜枳橙快繁各种培养基配方及培养条件。结果表明,以MS+6-BA1mg/L的培养基适合于芽萌发,以MS+6-BA0.5mg/L+NAA0.05mg/L的培养基适合于芽增殖,生根则以1/2MS+NAA0.5mg/L+0.2%活性炭的组合最佳,转基因各系生根率均在93.3%以上,显著高于对照生根率(66.7%)。快繁苗移栽成活率可达90%,目前生长良好。     

犬瘟热病毒自然感染犬淋巴组织中T、B细胞变化的研究

为了调查患犬瘟热病犬淋巴组织中T、B细胞变化的特点及淋巴细胞减少的发病机制,试验通过免疫组织化学的方法观察了T细胞(用CD3和CD45RO检测T细胞)、B细胞(用IgG、IgM抗血清检测B细胞)和犬瘟热病毒(抗犬瘟热病毒抗体)在病犬淋巴组织中的分布。结果表明:在淋巴组织中的淋巴细胞、淋巴小结中树突状细胞和巨噬细胞中均检出了抗病毒阳性反应细胞。在骨髓组织的前髓细胞中也发现抗病毒阳性反应细胞和嗜酸性胞浆内及核内包涵体的存在。与对照组相比,CD3和CD45RO阳性细胞主要存在于T细胞的分布域;但CD3和CD45RO阳性T细胞的数量较少。位于淋巴组织中的巨噬细胞有的被CD45RO染成阳性。在B细胞分布的区域中,IgG、IgM阳性细胞的数量明显减少;一些位于淋巴组织的浆细胞也被IgG或IgM染成阳性。在淋巴组织中淋巴细胞减少的顺序为:IgG阳性细胞减少最明显,其次为IgM和CD45RO阳性细胞,再次为CD3阳性细胞。依据试验结果,作者认为病犬淋巴组织中淋巴细胞减少主要是由B细胞缺乏所引起的;淋巴细胞的增殖能力减弱是引起淋巴组织中淋巴细胞减少的重要原因。     

Effect of Ganoderma lucidum spores powder on the expression of IGF-1, NF-κB and apoptosis of nerve cells in the brain from epileptic rat

AIM: To investigate the effect of Ganoderma lucidum spores powder on the expression of insulin-like growth factor-1 (IGF-1), nuclear factor-κB (NF-κB) and apoptosis of nerve cells in rats with epilepsy established by pentetrazole. METHODS: The sub-eclampsia dosage of pentetrazole (PTZ) was used to make epilepsy model. Ganoderma lucidum spores powder group was given from stomach. The enduring time and latent period were recorded. The immune reactivity of IGF-1, NF-κB/P65 and apoptosis of nerve cells were measured with immunohistochemical staining and TUNEL method. RESULTS: In high power sight (×400), there were much more apoptosis cells in hippocampus and brain cortex of model group (18.80±2.13, 16.87±2.00) than those in control group (0.97±0.52, 0.58±0.25). The expressions of IGF-1, NF-κB in model group were higher than those in control group. Compared with model group, the latent period of Ganoderma lucidum spores powder group at the 17th, 21th, 25th days were longer (P<0.05, P<0.05, P<0.01, respectively).There were less apoptosis cells in hippocampus and brain cortex of Ganoderma lucidum spores powder group (12.30±2.46, 10.48±1.33) than those in model group. The expression of NF-κB/P65 in Ganoderma lucidum spores powder group was lower than that in model group, but the immune reactivity of IGF-1 increased more distinctly in Ganoderma lucidum spores powder group than that in model group. CONCLUSION: IGF-1, NF-κB and apoptosis of nerve cells may have play a role in occurrence and development of PTZ-induced epilepsy. Ganoderma lucidum spores powder can suppress expression of NF-κB strongly, and facilitate the immune reactivity of IGF-1, which may be one of the mechanisms by which Ganoderma lucidum spores powder restrains the apoptosis of nerve cells caused by epilepsy to prevent the damages of nerve cells.     

uclear factor κB in LPS stimulated rat alveolar macrophages promotes TNF-α secretion

AIM: To investigate the activation of nuclear factor κB (NF-κB) induced by lipopolysaccharides (LPS) in rat alveolar macrophages (AMs) and its regulatory role in tumor necrosis factor (TNF-α) secretion. METHODS: The dynamic activity changes of NF-κB induced by LPS were determined with electrophoretic mobility shift assay (EMSA). Antisense oligonucleotides of NF-κB subunit (p65) were transfected into AMs prior to LPS stimulation. The effect of antisense oligonucleotide transfection on expressions of p65 and TNF-α in supernatant were measured with Western blotting and enzyme linked immunosorbent assay (ELISA), respectively.RESULTS: NF-κB activity increased markedly and reached its peak level at 4 h after LPS stimulation. After transfected with antisense oligonucleotides of NF-κB subunit (p65), expression of p65 and TNF-α in supernatant decreased markedly.CONCLUSION: NF-κB activity has a positive effect on regulating secretion of TNF-α in AMs induced by LPS.     

Caspase 3 inhibitor Ac-DEVD-CHO attenuates 10-hydroxycamptothecin-induced activation of NF-κB in Bcap37 cells

AIM:To investigate the role of caspase 3 inhibitor Ac-DEVD-CHO in caspase 3 signaling pathway and NF-κB activation induced by 10-hydroxycamptothecin (HCPT) in human breast carcinoma cells. METHODS:The cell growth inhibition was measured by MTT assay. Agarose gel electrophoresis was performed for detecting cell apoptosis. Western blotting was used for determining protein expression. DIG-EMSA was conducted to measure the DNA-binding activation of NF-κB. RESULTS:Caspase 3 inhibitor Ac-DEVD-CHO attenuated HCPT-induced apoptosis in human breast carcinoma. Ac-DEVD-CHO also suppressed the degradation of caspase 3 and IκBα,and arrested the activation of NF-κB. CONCLUSION:Caspase 3 inhibitor Ac-DEVD-CHO regulates the activation of caspase 3 and NF-κB,and attenuates apoptosis in Bcap37 cell line induced by HCPT.     

Effects of PDS on rat brain cortical nuclear factor kappa B in LPS shock

AIM:To explore the molecular mechanism of brain tissue injury induced by lipopolysaccharide (LPS),the effects of panaxadiol (PDS) on the expression of nuclear factor kappa B (NF-κB) in cerebral cortex of rat with LPS shock were studied. METHODS:Rats were randomly divided into LPS roup,LPS+dexamethasone group,LPS+PDS group and control group. The DNA binding activity and protein expression of NF-κB were observed. These indices were assayed at 1 h and 4 h after intravenous injection of LPS (4 mg·kg^-1). RESULTS:EMSA showed that PDS inhibited NF-κB DNA-binding activity in nuclear extracts at both 1 h and 4 h after LPS injection,compared with the LPS group (P<0.01). Western blotting showed that PDS down-regulated the expression of p65 and p50 protein in the nuclear extracts compared with the LPS group. However,the expression of p65 and p50 protein from cytosolic extracts did not show any significant change. CONCLUSION:PDS may alleviate brain injury by inhibiting NF-κB activation.     

The levels of 2′, 5′ oligoadenylate synthetase,interleukin 2 and interleukin 12 in hepatitis B virus infection

AIM: In order to study the effect of endogenous interferon system and Th1 response modes on hepatitis B virus infection, the 2′, 5′ oligoadenylate synthetase (2-5OAS), IL-2 and IL-12 were selected as the research parameters. METHODS: The activity of 2-5OAS in peripheral blood mononeuclear cells was determined by sensitive radioenzymatic assay. IL-2 and IL-12 were determined by ELISA. RESULTS: Compared to normal control, the 2-5OAS, IL-2 or IL-12 were not significantly changed (P>0.05) in the asymptomatic HBsAg carricer group. The 2-5OAS, IL-2 and IL-12 were significantly up-regulated (P<0.01) in the group of acute hepatitis, but in the groups of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, the 2-5OAS, IL-2, IL-12 were significantly down-regulated (P<0.05). Moreover, with the progression of patient′s conditions and with the complications of liver cirrhosis and hepatocellular carcinoma, the 2-5OAS, IL-2 and IL-12 decreased progressively, the 2-5OAS, IL-2, IL-12 were the lowest in guoups of liver cirrhosis and hepatocellular carcinoma (vs each groups of chronic hepatitis, P<0.05). CONCLUSION: The endogenous interferon system and Th1 response are significantly alterable in the different period of hepatitis B virus infection and among the different clinical types. The cellular immunity plays an important role in recovery from HBV infection.     

印楝素A和印楝素B生物活性比较

对印楝素A和印楝素B的生物活性进行了研究。在3μg/mL的剂量下处理后24 h,印楝素A和印楝素B对小菜蛾3龄幼虫的拒食率分别为85.3%和80.4%;在5μg/mL的剂量下处理后24 h,对棉铃虫3龄幼虫的拒食率分别为94.9%和80.2%。处理后72 h,印楝素A和印楝素B对斜纹夜蛾3龄幼虫的LC₅₀分别为1.0μg/mL和3.3μg/mL。印楝素A和印楝素B在3μg/mL的剂量下处理后72 h,小菜蛾3龄幼虫的死亡率分别为90.42%和84.11%;在5μg/mL的剂量下处理后72 h,棉铃虫3龄幼虫的死亡率分别为99.1%和100.0%;在0.5μg/mL的剂量下处理后72 h,斜纹夜蛾4龄幼虫体重降低率分别为57.5%和72.73%;在0.8μg/mL的剂量下处理后72 h,棉铃虫3龄幼虫体重降低率分别为84.1%和80.9%。