Factors influencing induction and in vitro culture of hairy roots in Phytolacca americana L / 生物工程学报

To use hairy roots for producing medicinal ingredients of Phytolacca americana L. we studied the factors influencing the induction and in vitro culture. Hairy roots could be incited from the veins of cut surface (morphological lower) of P. americana L. leaf explants around 18 days after infection with the strain of Agrobacterium rhizogenes ATCC15834. The highest rooting rate, 70%, was obtained when leaf explants were pre-cultured for 1 day, infected for 20 min, and co-cultured for 4 days. The transformation was confirmed by PCR amplification of rolC of Ri plasmid and silica gel thin-layer chromatography of opines from P. americana L. hairy roots. All the hairy root lines could grow rapidly on solid exogenous phytohormone-free MS medium. Among the 9 hairy root lines, the hairy root line 2 had most rapid growth, most branched lateral roots and most intensive root hair; the root surface of some hairy root lines seemed purple or red, while that of the other hairy root line appeared white. Among liquid media MS, 1/2MS, B5 and 6,7-V tested, the best growth for hairy root lines was attained in liquid exogenous phytohormone-free MS medium. Compared with exogenous phytohormone-free MS medium, 6,7-V medium was better for synthesis and accumulation of esculento side A in hairy roots. The established optimal conditions for induction and in vitro culture of P. americana hairy roots had laid an experimental and technological foundation for production of medicinal constituents esculento side A from large scale culture of hairy roots.

Role of AMPK signaling pathway in regulating antibacterial autophagy in murine bone marrow derived macro-phages / 局解手术学杂志

Objective To investigate the regulatory effects and possible mechanisms of autophagy induction and bactericidal activity in muring bone marrow derived macrophages ( BMDMs) . Methods Murine BMDMs were isolated and cultured in vitro. Then standard strain of E. coli was used to infect BMDMs and cellular autophagy levels and AMPK activation were detected. We next modulated functions of AMPK by pretreating cells with the specific agonist or antagonist of AMPK. Then cellular AMPK activation, autophagy levels and bactericidal activity were observed after E. coli infection. Results In E. coli infected BMDMs,autophagy related molecules like LC3B and Beclin1 were upregu-lated,accompanied with elevated LC3Ⅱ/Ⅰratio. Phosphorylation of AMPK was also upregulated by E. coli treatment. Enhanced AMPK activi-ty by its agonist leads to increased cellular autophagy and bactericidal activity,whereas inhibition of AMPK by its suppressor downregulated autophagy and dampened bactericidal activity. Conclusion AMPK and its related signaling pathway is required for anti-bacterial response in macrophage,which is dependent on its function in upregulating autophagy related molecules and inducing autophagy.

Induction and in vitro culture of hairy roots of Dianthus caryophyllus and its plant regeneration / 生物工程学报

To use Agrobacterium rhizogenes-induced hairy roots to create new germplasm of Dianthus caryophyllus, we transformed D. caryophyllus with A. rhizogenes by leaf disc for plant regeneration from hairy roots. The white hairy roots could be induced from the basal surface of leaf explants of D. caryophyllus 12 days after inoculation with A. rhizogenes ATCC15834. The percentage of the rooting leaf explants was about 90% 21 days after inoculation. The hairy roots could grow rapidly and autonomously in liquid or solid phytohormone-free MS medium. The transformation was confirmed by PCR amplification of rol gene of Ri plasmid and silica gel thin-layer chromatography of opines from D. caryophyllus hairy roots. Hairy roots could form light green callus after cultured on MS+6-BA 1.0-3.0 mg/L + NAA 0.1-0.2 mg/L for 15 days. The optimum medium for adventitious shoots formation was MS + 6-BA 2.0 mg/L + NAA 0.02 mg/L, where the rate of adventitious shoot induction was 100% after cultured for 6 weeks. The mean number of adventitious shoot per callus was 30-40. The adventitious shoots can form roots when cultured on phytohormone-free 1/2 MS or 1/2 MS +0.5 mg/L NAA for 10 days. When the rooted plantlets transplanted in the substrate mixed with perlite sand and peat (volume ratio of 1:2), the survival rate was above 95%.

Gene therapy efficacy of CTLA4Ig on experimental autoimmune thyroiditis / 中国综合临床

Objective To investigate the therapeutic effect of CTLA4Ig gene on experimental autoimmune thyroiditis (EAT) by the use of portable synthetic costimulatory molecules of cytotoxic T lymphocyte antigen 4 (CTLA-4) antagonist (CTLA4Ig) eukaryotic expression vector.Methods Thirty C57BL/6J female mice were divided into three groups,named EAT model group (EAT,n =10),CTLA4Ig-treatment group (CTLA4Ig-EAT,n =10) and control group(n =10).At 28 day after first immunization,plasmids mixture with pCI or pCI/CTLA4Ig were injected into thyroid tissues of EAT and CTLA4Ig-EAT by surgery,respectively.Serum,thyroid tissues and spleens were collected as samples.Thyroid autoantibody and expression of interleukin (Th)1,Th2 related cytokinesby were measured by real-time quantitative PCR and ELISA.Results Compared with EAT group,the expression of CTLA-4 in thyroid of CTLA4Ig-EAT group was elevated double folds (P =0.038),and the expression of Th1 cytokine interferon γ and intercellular adhesion molecule-1 decreased significantly (P =0.016,0.042).Meanwhile,Th2 cytokine IL-4 was increased after CTLA4Ig treatment (P =0.044).The same changes were seen in spleen tissues and serum.There was no significant difference in terms of TPOAb between EAT and treated group.Conclusion Local thyroid injection of CTLA4Ig gene shows the therapeutic effect to same degree on EAT through adjusting the underlying Th1/Th2 imbalance.

The expression of CXCR5 and CD57 in autoimmune thyroid diseases / 中国综合临床

Objective To investigate the potential role of T follicular helper cells (Tfh) in the pathogenesis of autoimmune thyroid diseases by comparing the expression of C-X-C chemokine teceptor type 5 (CXCR5) and CD57 in Hashimoto's thyroiditis (HT) and Graves' disease (GD) thyroid tissues.Methods The expression of CXCR5 and CD57 proteins was determined by immunohistochemical analysis in 15 HT thyroid samples,18 GD samples and 10 normal thyroid samples.Results Immunohistochemical staining showed that CXCR5 and CD57 were mainly positive in cytomembrane and cytoplasm of the infiltrated lymphocytes both in HT and GD tissues,with much higher levels than that of normal thyroid tissues ( P ＜ 0.05 ).Both CXCR5 and CD57 were not significantly different between the HT and GD tissues.Conclusion CXCR5 and CD57 expressions were increased with a similar expression pattern in both of the two main autoimmune thyroid diseases( AITD),indicating that Tfh may participate inthe development and progression of AITD.