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。     

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.     

Cardiomyocyte apoptosis and expression of Bcl-2, Bax protein after acute myocardial infarction and late reperfusion in dogs

AIM: To study the effect of late reperfusion on apoptotic cardiomyocytes in the risk area of acute myocardial infarctin in dogs. METHODS: The experiment was divided into three groups: sham operation group, acute myocardial infarction (AMI) group, and late reperfusion (LR) group. Apart from sham operation group, the other two groups were subjected to left anterior descending branch of coronary artery ligation. The acute myocardial infarction group was only subjected to ligation for 12 hours, late reperfusion group was subjected to ligation for 6 hours following by 6 hours of reperfusion. The cardiomyocyte apoptosis was measured by TUNEL assay. Immunohistochemistry and Western blotting analysis were used to detect the expression of Bcl-2 and Bax protein. RESULTS: The number of apoptotic cardiomyocytes in late reperfusion group was much less than acute myocardial infarction group (P<0.05), and increased significantily as compared with sham operation group (P<0.01). The expression of Bcl-2 protein was enhanced gently in late reperfusion group in contrast to acute myocardial infarction group, but no significant difference in the two groups (P>0.05) was observed, although it was much more in the two groups than that in sham operation group (P<0.01). The expression of Bax protein in late reperfusion group was much higher than that in sham operation group (P<0.01), and was lower than that in acute myocardial infarction group (P<0.05). CONCLUSION: Late reperfusion reduces cardiomyocyte apoptosis in the risk area of acute myocardial infarction. The mechanism may be that late reperfusion can decrease the expression of Bax protein.     

Serine protease HtrA2/Omi and cell death or survival

The serine protease HtrA2/Omi is released from the mitochondrial intermembrane space following apoptotic stimuli.Once in the cytosol,HtrA2/Omi has been implicated in promoting cell death in caspase-dependent or caspase-independent pathway.On the other hand,HtrA2/Omi can refold and degrade misfolded proteins in the mitochondria during cell stress.