Beclin-1 shRNA对低氧诱导的SH-SY5Y细胞自噬的影响

目的：构建Beclin-1基因短发夹干扰RNA（shRNA）慢病毒载体,感染人SH-SY5Y细胞,观察沉默Beclin-1基因后低氧对SH-SY5Y细胞自噬的影响。方法：构建特异性靶向Beclin-1基因的shRNA慢病毒表达载体和阴性对照序列慢病毒载体;再将载体转染入SH-SY5Y细胞;RT-PCR检测Beclin-1的mRNA表达;Western blot检测Beclin-1蛋白表达;CCK-8法测定Beclin-1 shRNA对SH-SY5Y细胞活力的影响。再将空白对照、阴性对照、转染型三种细胞分别以21%常氧及5%低氧培养,Western blot检测各组细胞LC3蛋白表达;电镜观察自噬小体。结果：Beclin-1 shRNA能明显抑制SH-SY5Y细胞Beclin-1的mRNA及蛋白的表达;沉默Beclin-1基因后,Beclin-1 shRNA组细胞存活率与阴性对照组相比无差异;成功建立了稳定表达Beclin-1 shRNA的SH-SY5Y细胞。5%低氧处理后,与阴性对照组相比较,Beclin-1 shRNA组细胞中LC3Ⅱ/LC3Ⅰ比值下调,细胞内自噬小体数量减少。结论：慢病毒介导的Beclin-1shRNA对SH-SY5Y细胞的活力无影响,但可以抑制低氧诱导的自噬。     

Enhancement of Growth Characteristics and Biological Activities in <Emphasis Type="Italic">Astragalus membranaceus</Emphasis> Using Artificial Light Sources

We investigated the effects of light emitting plasma (LEP) and red, blue, green, and white light emitting diode (LED) artificial light sources on plant growth characteristics and antioxidant activities in Astragalus membranaceus B. The highest germination rate (91.33 ± 2.08%) was produced by growth under red LED light. Root growth under LEP light was superior to that under the other light treatments. We measured antioxidant activity using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and determined the total phenol and flavonoid contents and tyrosinase inhibitory activity in response to the various light treatments. Antioxidant activities were 53.6% as the highest level of activity under LEP light. Growth under LEP light also produced the highest total phenolic and flavonoid contents of 36.05 and 5.94 mg/mL, respectively. Extracts from plants grown under LEP light caused the highest inhibition of tyrosinase activity with inhibition of 35.37, 61.87 and 65.49%, respectively, for extract concentrations of 100, 500, and 1000 μg/mL compared with other artificial light treatments. These results suggest that LEP light may be an important artificial light source for the enhancement of plant growth and biological activity and indicate the importance of further study of the use of LEP light sources for agricultural production.     

Influence of various medium environment to in vitro human T cell culture

Nowadays, adoptive T cell immunotherapy is emerging as a novel and potent treatment for cancer. To prepare enough effective T cells for treatment use, their rapid expansion is favorable. Our study compared 6 commonly used cultural media for human T cells, including serum-containing media and serum-free media, namely RPMI 1640, IMDM, Gibco OpTmizer CTS T Cell Expansion SFM, Gibco AIM-V Medium CTS, LONZA X-VIVO 15, and StemSpan SFEM with or without Dynabeads Human T-Activator CD3/CD28, on in vitro T cell expansion, apoptosis, and immune phenotype. Our study results suggest that serum-free media provide better proliferation environment for T cells. Among the 3 serum-free media, we identify OpTmizer and AIM-V as better T cell culture environments compared with X-VIVO as T cells are proved to have higher viability in the first two media. Besides, we found that in vitro human T cells keep relatively resting status among non-CD3/CD28 groups, since they have weak proliferation and apoptosis abilities. The phenotypes of T cells in different cultural environments over time indicate T cells maturation during culture duration. These results provide a firm foundation of adoptive T cell immunotherapy.     

Quantitative analysis of mitral valve morphology in atrial functional mitral regurgitation using real-time 3-dimensional echocardiography atrial functional mitral regurgitation

Background

Atrial fibrillation (AF) can result in atrial functional mitral regurgitation (MR), but the mechanism remains controversial. Few data about the relationship between the 3-dimensional morphology of the MV and the degree of MR in AF exist.

Methods

Real-time 3-dimensional transesophageal echocardiography (3D-TEE) of the MV was acquired in 168 patients with AF (57.7% persistent AF), including 25 (14.9%) patients with moderate to severe MR (the MR+ group) and 25 patients without AF as controls. The 3-dimensional geometry of the MV apparatus was acquired using dedicated quantification software.

Results

Compared with the group of patients with no or mild MR (the MR- group) and the controls, the MR+ group had a larger left atrium (LA), a more dilated mitral annulus (MA), a reduced annular height to commissural width ratio (AHCWR), indicating flattening of the annular saddle shape, and greater leaflet surfaces and tethering. MR severity was correlated with the MA area (r²?=?0.43, P?<?0.01) and the annulus circumference (r²?=?0.38, P?<?0.01). A logistic regression analysis indicated that the MA area (OR: 1.02, 95% CI: 1.01–1.03, P?<?0.01), AHCWR (OR: 0.24, 95% CI: 0.14–0.35, P?=?0.04) and MV tenting volume (OR: 3.24, 95% CI: 1.16–9.08, P?=?0.03) were independent predictors of MR severity in AF patients.

Conclusions

The mechanisms of “atrial functional MR” are complex and include dilation of the MA, flattening of the annular saddle shape and greater leaflet tethering.