Establishment and application of a multiplex genetic mutation-detection method of lung cancer based on MassARRAY platform

Objective: This study aims to establish a method for highly parallel multiplexed detection of genetic mutations in Chinese lung cancer samples through Agena i PLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on Mass ARRAY mass spectrometry platform.Methods: We reviewed the related literature and data on lung cancer treatments. We also identified 99 mutation hot spots in 13 target genes closely related to the pathogenesis, drug resistance, and metastasis of lung cancer. A total of 297 primers, composed of99 paired forward and reverse amplification primers and 99 matched extension primers, were designed using Assay Design software. The detection method was established by analyzing eight cell lines and six lung cancer specimens. The proposed method was then validated through comparisons by using a Lung Carta^(TM) kit. The sensitivity and specificity of the proposed method were evaluated by directly sequencing EGFR and KRAS genes in 100 lung cancer cases.Results: The proposed method was able to detect multiplex genetic mutations in lung cancer cell lines. This finding was consistent with the observations on previously reported mutations. The proposed method can also detect such mutations in clinical lung cancer specimens. This result was consistent with the observations with Lung Carta^(TM) kit. However, an FGFR2 mutation was detected only through the proposed method. The measured sensitivity and specificity were 100% and 96.3%, respectively.Conclusions: The proposed Mass ARRAY technology-based multiplex method can detect genetic mutations in Chinese lung cancer patients. Therefore, the proposed method can be applied to detect mutations in other cancer tissues.

Protection by the gross saponins of Tribulus terrestris against cerebral ischemic injury in rats involves the NF-κB pathway

The aim of this study was to investigate whether the gross saponins of Tribulus terrestris(GSTT),a traditional Chinese herbal medicine,have neuroprotective effects on rats subjected to middle cerebral artery occlusion(MCAO),through nuclear factor-κB(NF-κB)pathway and inflammatory mediators.Cerebral ischemia was produced by MCAO in either untreated(control)or GSTT-pretreated rats,and the animals were examined for infarct volume,cerebral edema,neuro-behavioral abnormality and pathological changes.Meanwhile,the expression of NF-kB protein in brain tissue was analyzed on Western blots and the serum levels of TNF-α and IL-1 were determined by ELISA.The experimental results demonstrated that,compared with the control MCAO group,GSTT-pretreated MCAO group had significantly reduced infarct volume,brain edema and neuro-behavioral abnormality,and lesser degree of pathologic changes in the brain,as well as had lower levels of serum TNF-α and IL-1β,and higher levels of brain NF-κB(Po0.05).Furthermore,treatment with an NF-κB inhibitor pyrrolidine dithiocarbamate(PDTC)abolished the protective effects of GSTT against MCAO-induced cerebral ischemic injury.These results indicated that GSTT’s ability to protect against cerebral ischemic injury was mediated through the NF-κB signaling pathway,and that GSTT may act through inhibition of the production of inflammatory mediators.