Expression of TIM-3 in PBMCs and its relation to treg in the patients with allergic rhinitis / 解放军医学杂志

Objective To detect the expression of TIM-3 (T cell immunoglobulin domain, mucin domain) and its relationship with Treg (regulatory T) cells isolated from peripheral blood mononuclear cells (PBMCs) in the patients with allergic rhinitis (AR), and investigate the role of TIM-3 in the occurrence and development of the allergic rhinitis inflammation. Methods AR patients (AR group, n=30) and healthy subjects (HC group, n=25) were selected as experimental group and control group respectively, and 2 ml peripheral venous bloods were collected from these two groups. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood by density gradient centrifugation. The proportion of CD4+CD25+FoxP3+, reflecting expression of Treg, and TIM-3+Treg was detected by flow cytometry. The relationship between TIM-3 expression and Treg cells was analyzed. The expression of TIM-3 on the surface of CD4+CD25+FoxP3+ Treg cells was analyzed by flow cytometry. Then the TIM-3 expression was blocked, and its influence on Treg cells was observed. Results The percentage of Treg cells in peripheral blood of patients with AR (1.16%±0.13%) was lower than that of healthy controls (5.12%±0.11%) (Z=–6.339, P<0.01). The expression of TIM-3 on the surface of Treg cells in AR patients (11.76%±1.07%) was higher than that in healthy controls (3.15%±0.22%) (Z=–5.570, P<0.01). The expression of CD4+CD25+FoxP3+Treg cells in AR patients was negatively correlated with the expression of TIM-3 on the surface (r=–0.763, P<0.05); The percentage of Treg cells in peripheral blood of AR patients after the block (1.67%±0.76%) was significantly higher than that before block (1.44%±0.78%) (Z=–1.45, P=0.135). The percentage of IL-10 secreted by Treg cells in peripheral blood after block (2.33%±1.45%) was higher than that before block (1.57%±1.12%) (Z=–2.97, P=0.003) in AR patients. Conclusion The expression of TIM-3 in PBMCs may increase in patients with AR, TIM-3 may play an important role in the pathogenesis of AR, and may be related to the imbalance of Treg cells.

Genetic transformation of buckwheat ( Fagopyrum esculentum Moench ) with AtNHX1 gene and regeneration of salt-tolerant transgenic plants / 生物工程学报

The Arabidopsis thaliana tonoplast Na+ /H+ antiporter gene, AtNHX1, was transferred into buckwheat by Agrobacterium-mediated method. Transgenic buckwheat plants were regenerated and selected on MS basal medium supplemented with 2.0mg/L 6-BA, 1.0mg/L KT, 0.lmg/L IAA, 50mg/L kanamycin and 500mg/L carbenicillin. 426 seedlings from 36 resistant calli originated from 864 explants (transformed about at 4.17 percentage) exhibited resistance to kanamycin. The transformants were confirmed by PCR, Southern blotting, RT-PCR and Northern blotting analysis. After stress treatment for 6 weeks with 200mmol/L NaCl, transgenic plants survived, while wild-type plants did not. After 3 days of stress treatment through different concentrations of NaCl, transgenic plants accumulated higher concentration of Na+ and proline than the control plants. However, the K+ concentration of transgenic plants declined in comparison with the control plants. Moreover, the rutin content of the roots, stems and leaves of transgenic buckwheat increased than those of the control plants. These results showed that it could be possible to improve the salt-tolerance of crops with genetic technology.

Transgenic maize plants with low copy number of foreign genes were produced with maize Ubi-1 promoter / 生物工程学报

Direct DNA delivery procedures (include biolistics method) often resulted in multiple copies of the transgenes in transformants and certain copies of them were rearranged. Integration of multiple copies of the introduced genes was the main reason of gene silencing which meant inhibition or loss of foreign gene expression in filial generations of transformants. In the present work, we compared the influences of maize Ubi-1 promoter and other promoters on copy number of transgenes in maize transgenic plants. Immature embryos from Zea mays L. plants of sib-pollinated of A188 x H99 genotype were used as initial materials. Type- I embryonic calluses derived from preculture of immature embryos were treated on N6 medium containing 0.6 mol/L sucrose for 3 approximately 5 hours and transformed via particle bombardment with PDS1000/He delivery system (Bio-Rad). Bombarded calluses were treated with hyperosmotic N6 medium for 16 approximately 20 hours continuously. Then the cultures were transferred onto normal N6 medium and incubated at 26 degrees C in dark for two weeks and subsequently selected on N6 medium supplemented with 2 or 5 mg/L phosphinothricin (PPT) but without casamino acid for another two weeks. The calluses after selective culture were transferred onto hormone-free MS medium containing 2 or 5 mg/L PPT but without casamino acid, and incubated at 24 degrees C under 16 h illumination for plant regeneration. Regenerated plantlets over 2 cm in height were transferred to Magenta box containing 1/2 hormone-free MS medium. Plantlets over 8 cm in height were transplanted to soil. After growing for one week in greenhouse, the plants were sprayed with 250 mg/L PPT solution. Fertile transgenic maize plants were regenerated and confirmed by Southern blotting and histochemical localization of beta-glucuronidase (GUS) activity. Relations between promoter and copy number of transgenes in transformants were analyzed. Maize transgenic plants possessing an intact copy and another incomplete copy of beta-glucuronidase gene (gus) were obtained in case gus gene under the control of maize Ubi-1 promoter (pUbi:GUS). Simultaneously the co-transformed phosphinothricin acetyltransferase gene (bar) controlled by CaMV 35S promoter in another plasmid (p35S:BAR) also existed with only one copy. When pDB1 and (pUbi:in2) were cobombarded, the regenerated transgenic maize plant exhibited with only one copy of in2 gene too. It suggested that the copy number of transgenes in maize transformants was low if the transgenes controlled by maize Ubi-1 promoter. The possible reason might be that the foreign genes were integrated site-specifically via homologous recombination between Ubi-1 promoter and its endogenous sequences in maize genome, and two cotransformed plasmids had reconstructed as one intact molecule before integrating into maize chromosome. On the contrary, if p35S:BAR was cobom-barded with plasmid pAct:In1 containing rice Act-1 promoter (without maize Ubi-1 promoter), the transgenic maize plants had 4 approximately 8 copies of bar gene. These results reflected that utilization of self gene promoter could reduce the copy number of the transgenes in transgenic plants of certain species itself and avoid the occurrence of gene silencing. T2 seeds have been harvested.