Effect of lentiviral vector-mediated overexpression of hypoxia-inducible factor 1 alpha delivered by pluronic F-127 hydrogel on brachial plexus avulsion in rats

Brachial plexus avulsion often results in massive motor neuron death and severe functional deficits of target muscles. However, no satisfactory treatment is currently available. Hypoxia-inducible factor 1α is a critical molecule targeting several genes associated with ischemia-hypoxia damage and angiogenesis. In this study, a rat model of brachial plexus avulsion-reimplantation was established, in which C5–7 ventral nerve roots were avulsed and only the C6 root reimplanted. Different implants were immediately injected using a microsyringe into the avulsion-reimplantation site of the C6 root post-brachial plexus avulsion. Rats were randomly divided into five groups: phosphate-buffered saline, negative control of lentivirus, hypoxia-inducible factor 1α(hypoxia-inducible factor 1α overexpression lentivirus), gel(pluronic F-127 hydrogel), and gel + hypoxia-inducible factor 1α(pluronic F-127 hydrogel + hypoxia-inducible factor 1α overexpression lentivirus). The Terzis grooming test was performed to assess recovery of motor function. Scores were higher in the hypoxia-inducible factor 1α and gel +hypoxia-inducible factor 1α groups(in particular the gel + hypoxia-inducible factor 1α group) compared with the phosphate-buffered saline group. Electrophysiology, fluorogold retrograde tracing, and immunofluorescent staining were further performed to investigate neural pathway reconstruction and changes of neurons, motor endplates, and angiogenesis. Compared with the phosphate-buffered saline group, action potential latency of musculocutaneous nerves was markedly shortened in the hypoxia-inducible factor 1α and gel + hypoxia-inducible factor1α groups. Meanwhile, the number of fluorogold-positive cells and ChAT-positive neurons, neovascular area(labeled by CD31 around av ulsed sites in ipsilateral spinal cord segments), and the number of motor endplates in biceps brachii(identified by α-bungarotoxin) were all visibly increased, as well as the morphology of motor endplate in biceps brachil was clear in the hypoxia-inducible factor 1α and gel + hypoxia-inducible factor 1α groups. Taken together, delivery of hypoxia-inducible factor 1α overexpression lentiviral vectors mediated by pluronic F-127 effectively promotes spinal root regeneration and functional recovery post-brachial plexus avulsion. All animal procedures were approved by the Institutional Animal Care and Use Committee of Guangdong Medical University, China.

A Bird’s Eye View of Quantitative Proteome of Tumor Tissues from Lung Cancer Patients by a High Precision Mass Spectrometry Method

Lung cancer produces a high incidence of malignant tumors.There have been many studies on lung cancer using mass spectrometry(MS)technologies.However,most studies have focused on humoral samples.In this work,26 pairs of tissue samples(tumor vs.para-tumor)from patients with lung cancer were analyzed using liquid chromatography-tandem MS(LC-MS/MS)with data-independent acquisition mode.In total,3152 proteins were quantified from tissue samples with high confidence,including 189 up-regulated and 522 down-regulated proteins(tumor vs.para-tumor).In addition,79 and 690 proteins were identified only in para-cancerous samples and cancerous samples,respectively.The results from bio-informatics tools indicated that altered proteins like PEBP1,HRG and LYZ could be ideal reservoirs for screening the potential biomarkers for lung cancer.It is believed these tissue-specific proteomics results will assist in the studies of lung cancer.

Inhibition of the Hedgehog Signaling Pathway Depresses the Cigarette Smoke-Induced Malignant Transformation of 16HBE Cells on a Microfluidic Chip

Background：The hedgehog signaling system （HHS） plays an important role in the regulation of cell proliferation and differentiation during the embryonic phases.However,little is known about the involvement of HHS in the malignant transformation of cells.This study aimed to detect the role of HHS in the malignant transformation of human bronchial epithelial （16HBE） cells.Methods：In this study,two microfluidic chips were designed to investigate cigarette smoke extract （CSE）-induced malignant transformation of cells.Chip A contained a concentration gradient generator,while chip B had four cell chambers with a central channel.The 16HBE cells cultured in chip A were used to determine the optimal concentration of CSE for inducing malignant transformation.The 16HBE cells in chip B were cultured with 12.25% CSE （Group A）,12.25% CSE ± 5 μmol/L cyclopamine （Group B）,or normal complete medium as control for 8 months （Group C）,to establish the in vitro lung inflammatory-cancer transformation model.The transformed cells were inoculated into 20 nude mice as cells alone （Group 1） or cells with cyclopamine （Group 2） for tumorigenesis testing.Expression of HHS proteins was detected by Western blot.Data were expressed as mean ± standard deviation.The t-test was used for paired samples,and the difference among groups was analyzed using a one-way analysis of variance.Results：The optimal concentration of CSE was 12.25%.Expression of HHS proteins increased during the process of malignant transformation （Group B vs.Group A,F =7.65,P 〈 0.05）.After CSE exposure for 8 months,there were significant changes in cellular morphology,which allowed the transformed cells to grow into tumors in 40 days after being inoculated into nude mice.Cyclopamine could effectively depress the expression of HHS proteins （Group C vs.Group B,F =6.47,P 〈 0.05） and prevent tumor growth in nude mice （Group 2 vs.Group 1,t=31.59,P〈 0.01）.Conclusions：The activity of HHS is upregulated during the CSE-induced malignant transformation of 16HBE cells.Cyclopamine can effectively depress expression of HHS proteins in vitro and prevent tumor growth of the transformed cells in vivo.