Effect of sodium fluoride on myocardial damage by regulating apoptosis through JNK signaling pathway / 环境与职业医学

Background It has been found that fluoride may cause cardiomyocyte damage. c-Jun N-terminal kinases (JNK) signaling pathway plays an important role in apoptosis, but its role in fluorosis-induced cardiomyocyte damage is still unknown yet. Objective To explore the toxic effect of sodium fluoride (NaF) on H9c2 cardiomyocytes of rats and whether NaF affects cardiomyocyte apoptosis through the JNK signaling pathway. Methods According to the concentrations of sodium fluoride and whether sp600125 (JNK inhibitor) was added, cardiomyocytes of rats were divided into six groups, including control group, SP600125 group (SP group), 0.24, 0.48, and 0.96 mmol·L−1 NaF groups, and 0.96 mmol·L−1 NaF+SP600125 group (NaF+SP group). Cardiomyocytes exposed to NaF for 24 h were observed using a fluorescence inverted microscope. The changes of cell viability at 24, 48, and 72 h after the treatment were detected by CCK-8 method. The levels of reactive oxygen species (ROS) at 24 h after the treatment in H9c2 cardiomyocytes were determined by fluorescent probe method. The expression levels of Bcl-2, Bax, Caspase-3, and JNK mRNA at 24 h after the treatment were detected by real-time PCR. The protein expression levels of Bcl-2, Bax, Caspase-3, and p-JNK at 24 h after the treatment were detected by Western blotting. Results Compared with the control group, after being exposed to 0.48 and 0.96 mmol·L−1 NaF for 24 h, the cell growth density decreased. With the increase of NaF concentration, rounded cells and some suspended dead cells appeared. At 24h after exposure to NaF, the cell viability of the 0.48 and 0.96 mmol·L−1 NaF groups decreased compared with the control group (P<0.05). At 48h and 72h after exposure to NaF, the cell viability levels of the NaF treated groups were significantly lower than that of the control group (P<0.05). After NaF exposure for 24 h, compared with the control group, the intracellular ROS levels were increased (P<0.05); the mRNA expression levels of Bcl-2 were decreased to varying degrees, especially in the 0.48 and 0.96 mmol·L−1 NaF groups (P<0.05); the mRNA expression levels of Bax, Caspase-3, and JNK were increased (P<0.05); the protein expression levels of Bcl-2 were reduced (P<0.05); the protein expression levels of Bax, Caspase-3, and p-JNK were elevated (P<0.05). Compared with the 0.96 mmol·L−1 NaF group, the cell viability of the NaF+SP group was increased, the intracellular ROS level was decreased, the mRNA expression levels of Bax, Caspase-3, and JNK were decreased, the protein expression level of Bcl-2 was increased, and the protein expression levels of Bax, Caspase-3, and p-JNK were decreased (P<0.05); the expression level of Bcl-2 mRNA had a rising trend but showed no statistical significance (P>0.05). Conclusion Cardiomyocyte damage after excessive fluoride exposure may result from fluoride inducing excessive ROS production in cardiomyocytes, which may activate the JNK signaling pathway and induce cardiomyocyte apoptosis.

Construction of a replicative expression vector based on the porcine circovirus 2 replicon / 生物工程学报

The antigen gene expression level of a DNA vaccine is the key factor influencing the efficacy of the DNA vaccine. Accordingly, one of the ways to improve the antigen gene expression level of a DNA vaccine is to utilize a plasmid vector that is replicable in eukaryotic cells. A replicative DNA vaccine vector pCMVori was constructed based on the non-replicative pcDNA3.1 and the replicon of porcine circovirus 2 (PCV2) in this study. An EGFP gene was cloned into pCMVori and the control plasmid pcDNA3.1. The two recombinant vectors were transfected into PK-15 cell, and the plasmid DNA and RNA were extracted from the transfected cells. Real-time PCR was used to determine the plasmid replication efficiency of the two plasmids using plasmid before and after Bcl Ⅰ digestion as templates, and the transcription level of the Rep gene in PCV2 replicon was detected by RT-PCR. The average fluorescence intensity of cells transfected with the two plasmids was analyzed with software Image J, and the transcription level of EGFP was determined by means of real-time RT-PCR. The results showed that the replication efficiency of pCMVori in PK-15 cells incubated for 48 h was 136%, and the transcriptions of Rep and Rep' were verified by RT-PCR. The average fluorescence intensity of the cells transfected with pCMVori-EGFP was 39.14% higher than that of pcDNA3.1-EGFP, and the transcription level of EGFP in the former was also 40% higher than that in the latter. In conclusion, the DNA vaccine vector pCMVori constructed in this study can independently replicate in eukaryotic cells. As a result, the expression level of cloned target gene was elevated, providing a basis for developing the pCMVori-based DNA vaccine.

Unnatural amino acid orthogonal translation: a genetic engineering technology for the development of new-type live viral vaccine / 生物工程学报

Unnatural amino acid orthogonal translation machinery can insert unnatural amino acids at desired sites of protein through stop codon by means of foreign orthogonal translation system composed of aminoacyl-tRNA synthetase and orthogonal tRNA genes. This new genetic engineering technology is not only a new tool for biochemical researches of proteins, but also an epoch-making technology for the development of new-type live viral vaccines. The mutated virus containing premature termination codon in genes necessary for replication can be propagated in transgenic cells harboring unnatural amino acid orthogonal translation machinery in media with corresponding unnatural amino acid, but it cannot replicate in conventional host cells. This replication-deficient virus is a new-type of live viral vaccine that possesses advantages of high efficacy of traditional attenuated vaccine and high safety of killed vaccine. This article reviews the application and prospect of unnatural amino acid orthogonal translation machinery in the development of novel replication-deficient virus vaccines.

Assessment of fingertip's microcirculation changes in patients with systemic sclerosis by E-flow imaging / 中华超声影像学杂志

Objective To investigate the clinical value of high frequency ultrasonography with E-flow imaging in the evaluation of fingertip's microcirculation changes in patients with systemic sclerosis(SSc).Methods Twenty-four SSc patients and 29 healthy subjects were involved.High frequency ultrasonography with E-flow imaging was used to observe the configuration and distribution of digital arteries in the last segment of left and right middle finger.Peak systolic velocity (PSV),end diastolic velocity (EDV),mean velocity(MV),vascular resistance index (RI) and pulsatility index(PI) of digital palmar propria arteries,nail bed arteries and finger ventral arteries were measured.Results In control group,rich blood supply was revealed within the fingertips.Digital palmar propria arteries,nail bed arteries and finger ventral arteries and their small branches were displayed clearly and continuously by E-flow imaging.While in SSc patients,the definition and continuity of fingertip's small vascular flow images were not as good as that in the control group,with the distribution of blood flow markedly reduced.compared with the control group,PSV,EDV and MV of digital palmar propria arteries,nail bed arteries,finger ventral arteries were decreased in SSc group(P＜0.01),but both RI and PI were increased(P＜0.05).There were no statistically significant differences between left and right fingertip's arteries index in normal control group (P＞0.05).But PSV,EDV and MV of left digital palmar propria arteries in SSc group were higher than that of the right(P＜0.05),whose differences bear statistic significance.Conclusions High frequency ultrasonography with E-flow imaging is sensitive and reliable to reflect fingertip's microcirculation changes and provide a new method to assess microvascular changes in SSc patients.

Pulsed wave Doppler ultrasonic manifestations of acute experimental incomplete testicular torsion / 中国医学影像技术

Objective To explore the pulsed wave Doppler (PWD) manifestations of acute experimental incomplete testicular torsion. Methods Eight healthy dogs were surgically modeled to obtain unilateral acute testis torsion from 180° to 630°, respectively. The peak systole velocity (PSV) and resistance index (RI) of intratesticular artery, capsular artery and arteria spermatica interna were measured with PWD before torsion, 2 h, 4 h and 6 h after torsion. Testes were examined pathologically after the experiment. Results PSV and RI of capsular artery and intratesticular artery decreased gradually with the extension of torsional time (P<0.05). PSV and RI of superior, torsional and inferior segments of arteria spermatica interna varied greatly and showed no strong regularity (P＞0.05) . The pathological results of testes showed no distinct changes or just mild interstitial hyperemia and edema. Conclusion The decrease of PSV and RI of capsular artery and intratesticular artery, especially RI have referential value in the judgement of acute experimental incomplete testicular torsion, whereas changes of PSV and RI of superior, torsional and inferior segments of arteria spermatica interna are not reliable.