ABSTRACT
Objective Apolipoprotein E (APOE) is mainly involved in lipid metabolism and cholesterol transport, which is important for health. To establish a pyrosequencing based method for detection of 112T>C and 158C>T single nucleotide polymorphisms (SNPs) in Apolipoprotein E gene. Methods Three amplification systems including Taq enzyme buffer from TaKaRa company (T buffer), La Taq enzyme buffer specified for G-C rich regions (L buffer) and Trans Taq enzyme buffer from TransGen company (TT buffer) were chosen to optimize PCR system and temperature by annealing at 60 °C and gradient annealing from 62 to 68 °C respectively. The specificity of this method was evaluated by comparing its results with those of Sanger sequencing. The sensitivity of this method was evaluated by gradient diluting human genomic DNA as detection template. Results According to the concentration and specificity of the products, the optimum condition was L buffer with 60℃ annealing programs. Pyrosequencing results of 20 samples were completely consistent with those of Sanger sequencing. The sensitivity of this method could be as low as 0.16 ng genomic DNA. Conclusion A method based on pyrosequencing detecting 112 and 158 polymorphisms in APOE gene was established, which can be applied in clinical personalized medicine.
ABSTRACT
Preimplantation genetic diagnosis (PGD) was developed to play a supporting role in assisted reproductive technology. With this kind of detection method, embryos with copy number variations, chromosome translocations or single mutations were excluded and the normal embryos were chosen and implanted. Theoretically, the application of these procedures could improve the implantation and pregnancy rate and help to delivery healthy offspring. PGD was considered to be more precise, higher specific and non-invasive with the appearance of microarray hybridization technology, the next generation sequencing and time-lapse monitoring technology. This paper presented a review of new Methods used in PGD, including fluorescence in situ hybridization, array comparative genomic hybridization, SNP array, next generation sequencing, MicroSeq-PGD, MaReCs, time-lapse monitoring and cfDNA-based method, and their advantages and disadvantages as well as efficacy in the procedures in which they are used.
ABSTRACT
AIM To establish the quality standard for Compound Tougucao Solution [Impatiens balsamina L.Zanthoxylum bungeanum Maxim.,Gleditsia sinensis Lam and Momordica cochinchinensis (Lour.) Spreng.].METHODS The fingerprints of ten batches of samples were established,after which TLC was used for the qualitative identification of I.balsamina and Z.bungeanum,HPLC was adopted in the content determination of quercetin.RESULTS There were twenty-seven common peaks in the fingerprints with the similarities of 0.978-0.986,four of which were sodium benzoate,rutin,hyperoside and quercetin,respectively.The clear TLC spots were free from negative interference.Quercetin showed a good linear relationship within the range of 1.68-107.6 μg/mL (R2 =0.999 9),whose average recovery was 104.3％ with the RSD of 2.2％.CONCLUSION This stable and reproducible method can be used for the quality control of Compound Tougucao Solution.
ABSTRACT
BACKGROUND: Preliminary experimental studies have shown that the supernatant of human placental fetal mesenchymal stem cells (fPMSCs) has a certain ability to scavenge reactive oxygen species and itself has a certain antioxidant enzyme activity. OBJECTIVE: To investigate the protective role and mechanism of fPMSCs supernatant in serum-free culture on oxidative stress-injured lung epithelial cells. METHODS: Different concentrations of hydrogen peroxide produced oxygen stimulation to lung epithelial cell lines A549 for 6, 12, 24 hours, and the survival rate of lung epithelial cells was detected using cell counting kit-8 method. When the survival rate of lung epithelial cells was 50%, the concentration of hydrogen peroxide was most suitable to make an oxidative damage model. The validity of the model was verified using Hocheest33258 staining and western blot. fPMSCs were cultured in serum-free culture medium, and the supernatant of passage 3 cells was collected. Afterwards, the injured lung epithelial cells were cultured in the fPMSCs cell supernatant for 24 hours. Meanwhile, injury group (oxidative damage only) and vitamin C group (100 μmol/L vitamin C was added in the medium) were established. In the three groups, cell apoptosis was detected by flow cytometry; and western blot was used to detect apoptosis-related proteins and proteins related to the Nrf2-Keap1-ARE signaling pathway. RESULTS AND CONCLUSION: After oxygen stimulation by 600 μmol/L hydrogen peroxide for 24 hours, the survival rate of A549 cells was (56.41±3.31)% as ascertained by the cell counting kit-8 assay. Findings from Hocheest33258 staining and western blot further confirmed the reliability of this model. Flow cytometry results showed that the apoptosis rate in the vitamin C group and the supernatant group decreased to some extent compared with the injury group, and the difference between the supernatant group and the injury group was statistically significant (P < 0.05). In addition, the expression of Bax significantly decreased and the expression of Bcl-2 significantly increased in the vitamin C group and the supernatant group as detected by western blot assay, in comparison with the injury group (P < 0.05). Compared with the injury group, the expression of Nrf2 protein increased and the expression of Keap1 decreased in the vitamin C group and the supernatant group (P < 0.05). These findings suggest that fPMSCs supernatant has a certain antioxidant capacity, and may attenuat oxidative damage and inhibit apoptosis in A549 cells. The mechanism is probably related to the Nrf2-Keap1-ARE signaling pathway.
ABSTRACT
To evaluate the effect of Tongxie Yaofang on cardiac endogenous metabolism in irritable bowel syndrome(IBS) rats by using metabolomics method, find its potential biomarkers, analyze the metabolic pathways, and explore the pharmacological effects, mechanisms of action and syndrome essence of syndrome model. Forty Wistar rats were used to establish IBS models, and then randomly divided into four groups： model control group and Tongxie Yaofang treatment groups (high, medium, low dose). Another 10 rats were used as normal group. The rats in Tongxie Yaofang-treated(low, medium and high dose) groups were orally administrated with Tongxie Yaofang extracts once a day for 2 weeks, respondingly with the doses of 0.203,0.406,0.812 g•mL⁻¹. The rats in normal group and model control group were given with equal volume of saline once a day for 2 weeks. On the 0 and 15th days, serum was collected and each sample extract was analyzed by UPLC-Q-TOF-MS. Eight potential biomarkers were identified and 8 major metabolic pathways were found to be related with IBS diseases neurotransmitter metabolism, inflammatory immunity, brain function and energy metabolism, etc. Tongxie Yaofang had certain pharmacological effects on IBS, and its mechanism may be related to serotonergic synapse, tryptophan metabolism, cysteine and methionine metabolism, glycerophospholipid metabolism, nicotinate and nicotinamide metabolism and so on, which might be the biological basis of IBS liver-spleen deficiency syndrome.
ABSTRACT
<p><b>OBJECTIVE</b>To explore the pathogenesis of tumors by blocking the normal differentiation process of stem cells.</p><p><b>METHODS</b>Bone marrow mesenchymal stem cells (BMSCs) from rats were isolated, cultured and purified by whole bone marrow adherence method. The rat BMSCs were induced to differentiate into adipocytes with dexamethasone, insulin and indomethacin. Blockage of the differentiation process was induced by 3-methylcholanthrene (3-MC).</p><p><b>RESULTS</b>The differentiation experiment showed that at 30 days after the induction, oil red O staining-positive cells occurred with increased intracytolasmic lipid droplets, characteristic for adipocytes. The differentiation blockage experiment showed that at 30 days after induction, the deposits of oil red O staining-cytoplasmic lipid droplets was significantly reduced, indicating that the blocked cells were adipocytes, but not fully differentiated. Morphological identification showed that cell contact inhibition disappeared, abnormal cell nuclei, increased number of micronucleus aberration and karyotype abnormalities, indicating that malignant transformation of the stem cells occurred after the differentiation blockage.</p><p><b>CONCLUSIONS</b>The results of this study show a blockage of the differentiation of that stem cells at the intermediate phase, and a tendency of malignant transformation of the stem cells. The results of our study provide new evidence that cancer stem cells may be originated by suppression of stem cell differentiation.</p>
Subject(s)
Animals , Female , Rats , Adipocytes , Cell Biology , Bone Marrow Cells , Cell Biology , Cell Differentiation , Cell Transformation, Neoplastic , Cells, Cultured , Dexamethasone , Pharmacology , Drug Combinations , Indomethacin , Pharmacology , Insulin , Pharmacology , Mesenchymal Stem Cells , Cell Biology , Methylcholanthrene , Pharmacology , Rats, WistarABSTRACT
<p><b>OBJECTIVE</b>To assess the therapeutic effect of acupuncture on depression.</p><p><b>METHODS</b>A systematic evaluation of all relevant randomized controlled trials (RCT) about acupuncture and moxibustion treatment of depression was carried out by the study methods of evidence-based medicine. The data were statistically analyzed with a special analysis software RevMan 4.2.</p><p><b>RESULTS</b>Fourteen papers of RCT met the enrolled criteria. Four of the trials used double-blind method. Meta-analysis indicated that the effective rate was no significant difference between the acupuncture treatment and medication, and acupuncture treatment is better than Amitriptyline in improvement of HAMD scores, but no significant differences as compared with other drugs.</p><p><b>CONCLUSION</b>Both acupuncture and medication possibly are effective for depression with good safety. However, because of lower methodological quality of the trials, this conclusion needs further be confirmed.</p>