Objective To cultivate glioblastoma U87 stem-like cells(SLCs)and to detect the level of stemness bio-markers,mitochondrial respiratory capacity and the capacity of in vivo tumorigenesis.Methods B-27,growth factors EGF and bFGF was added into DMEM/F-12 culture in serum-free stem cell culture medium for U87 SLCs.Suspended culture of U87 SLCs was suspended using the neuro-sphere formation assay,while adherent culture of U87 SLCs was achieved by coating Matrigel matrix on the culture surface.The mRNA and protein level of stemness biomarkers in culture were detected using real-time quantitative PCR and Western blot.The proportion of CD133+cells in culture was detected by flow cytometry.The changes of cell oxygen consumption rate were detected by Seahorse cell metabo-lism analysis.Cell tumorigenesis ability was verified by subcutaneous tumor transplantation in animals.Results U87 SLCs in stem cell culture medium would grow into typical sphere morphology within one week,and the spheres would continue to grow as the culture process prolongs.At the appropriate concentration of adhesive,U87 SLCs adhered to and grow well in stem cell culture medium.The mRNA transcription of stemness biomarkers such as CD133,nes-tin,OLIG2,CD44,CD15,and integrin α6(ITGA6)was significantly increased as found in both culture methods,and the protein levels of CD133 and nestin were also increased under both methods(P＜0.05).U87 SLCs showed higher mitochondrial reserve respiratory capacity(P＜0.05).U87 SLCs could form larger subcutaneous tumors with fewer inoculated cells(P＜0.05),and grew faster in vivo with stronger tumorigenic ability.Conclusions U87 SLCs have typical stemness characteristics and may function as tumor cell model with higher stemness properties.

Objective To study the effect of carboxyamidotriazole-orotate(CTO)on the proliferation and fatty acid anabolism regulation of human pancreatic cancer cells.Methods Human pancreatic cancer cell lines AsPC-1,AsPC-1/GEM(AR),PANC-1 and MiaPaCa-2 were used as the study subjects;cell survival rate was detected by sulfo-nylrhodamine B(SRB);the mRNA level of key genes for fatty acid synthesis was detected by qPCR;the protein level of the AMPK/ACC pathway was detected by Western blot;intracellular lipid metabolites were examined by liquid chromatography-mass spectrometry(LC-MS).Results Comparing to control group,CTO significantly de-creased the cell viability of AsPC-1,AR,PANC-1,and MiaPaCa-2(P＜0.05).CTO down-regulated the mRNA level of key fatty acid synthesis genes(P＜0.05).CTO significantly reduced the protein expression of AMPK,ACC and c-Myc(P＜0.05),while increasing the protein expression of p-AMPK and p-ACC(P＜0.05).CTO decreased lipid metabolite content in AR cells(P＜0.05).Conclusions CTO attenuates cellular fatty acid anabolism by inhibition of oncogene c-Myc expression and AMPK/ACC pathway,down-regulates the expression of fatty acid synthesis-related genes,and then inhibits proliferation of the human pancreatic cancer cell lines AsPC-1,AR,PANC-1 and MiaPaCa-2.

Objective To explore and analyze the feasibility of using indocyanine green(ICG)near-infrared fluores-cence(NIF)imaging technology for the early diagnosis of oral potential malignant disorders and oral squamous cell car-cinoma.Methods 7,12-Dimethylbenz[a]anthracene in acetone solution was used to induce various pathological models of buccal mucosal lesions(mild/moderate dysplasia,severe dysplasia,squamous cell carcinoma)in golden hamster.ICG-NIF was conducted for the quantitative analysis of the fluorescence signal of lesion tissue,and evaluation of the diagnos-tic and discriminative capabilities of the ICG-NIF technology for mucosal lesions in various pathological states.Immuno-histochemical staining was perform to examine the mi-crovessel density(MVD)and microlymphatic vessel den-sity(MLVD)of mucosa in various pathological states and explore the histological reasons underlying the differ-ences in fluorescence signals.Results The results of ICG-NIF fluorescence quantitative analysis reveal the higher fluorescence intensity of mucosal lesions in the experimental group compared with that of the normal mucosa on the control side,with statistical differences(P＜0.05).Moreover,the more severe the malignancy of mucosal lesions in the experimental group,the higher the fluorescence intensity.According to histopathological analysis,the malignant pro-gression of mucosal lesions in golden hamsters was accompanied with an increase in MVD(P＜0.05)and a decrease in MLVD(P＜0.05).Conclusion The abnormal proliferation of mucosal lesions in golden hamsters exhibits a difference in ICG-NIF fluorescence signal compared with normal mucosal tissue.Fluorescence quantitative analysis methods can provide assistance in differentiation and show potential for clinical applications.

Objective To analyze three-dimensional(3D)radiographic characterizations of bone island(BI)in jaw using cone-beam computed tomography(CBCT).Methods CBCT data from four thousand patients were selected,reconstructed and analyzed using NNT 10.0 software.The sagittal,coronal and axial planes were used to analyze the 3D radiographic characteristics of BIs,including the localization,shape,density,boundary,the relationship between BIs and tooth and bone cortex,diameter and anatomical structures and complications involved.Their relationship with gender were analyzed.Results A total of 803 people had BIs,with the prevalence rate of 20.08%,including 338 males with 389 BIs and 465 females with 526 BIs.Both males and females had a dominant BI,and the ratio between male and female was 1∶1.38,but the difference was not statistically significant(P＞0.05).The BIs of both male and female mostly occurred in the mandibular premolars and molars area,and appeared irregular in shape,dense and contact with lingual bone cortex.Mostly BIs were apical type and with unclear boundary.The mean maximum diameter of mesial/distal direction was greater than buccal/lingual direction(P＜0.05).The most commonly involved anatomy structure was the inferior alveolar neural canal,cortical infil-tration and mental foramen.Conclusion There are no significant differences between males and females in the three-dimensional image characteristics of BIs in Chinese populations.CBCT can accurately and comprehensively analyze the 3D radiographic characteris-tics of BI and its relationship with the surrounding teeth and bone.

Therapeutic drug monitoring(TDM)has played an important role in clinical medicine for precise dosing.Currently,chromatographic technology and immunoassay detection are widely used in TDM and have met most of the needs of clinical drug therapy.However,some problems still exist in practical appli-cations,such as complicated operation and the influence of endogenous substances.Surface plasmon resonance(SPR)has been applied to detect the concentrations of small molecules,including pesticide residues in crops and antibiotics in milk,which indicates its potential for in vivo drug detection.In this study,a new SPR-based biosensor for detecting chloramphenicol(CAP)in blood samples was developed and validated using methodological verification,including precision,accuracy,matrix effect,and extraction recovery rate,and compared with the classic ultra-performance liquid chromatography-ultraviolet(UPLC-UV)method.The detection range of SPR was 0.1-50 ng/mL and the limit of detec-tion was 0.099±0.023 ng/mL,which was lower than that of UPLC-UV.The intra-day and inter-day ac-curacies of SPR were 98％-114％and 110％-122％,which met the analysis requirement.The results show that the SPR biosensor is identical to UPLC-UV in the detection of CAP in rat blood samples;moreover,the SPR biosensor has better sensitivity.Therefore,the present study shows that SPR technology can be used for the detection of small molecules in the blood samples and has the potential to become a method for therapeutic drug monitoring.

To assess the () recombinant gingivalis gingipain R2 (rRgpB)-induced Ca mobilization in human gingival fibroblast (HGF) mediated by protease-activated receptor (PAR) and its downstream signal transduction pathways. : Flow cytometry was used to detect the expression of PAR in HGF. The proliferation of HGF was measured by CCK-8. The dynamic changes of intracellular Ca concentration in HGF induced by rRgpB and the blocking effect of PAR-1 antagonist were observed by laser confocal microscopy. Western blot was performed to determine the phosphorylation levels of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (p38 MAPK) and p65 in HGF. : PAR-1 and PAR-3 were expressed in HGF, and the rRgpB could promote the proliferation of HGF. rRgpB caused a transient increase in [Ca], which could be completely suppressed by vorapaxar, a PAR-1 antagonist. The phosphorylation levels of JNK, ERK1/2 and p65 were significantly up-regulated after the induction of rRgpB for and (all <0.05), which was completely inhibited by vorapaxar. However, the phosphorylation level of p38 MAPK had no significant change after rRgpB stimulation. : rRgpB causes an increase in [Ca] in HGF mediated by PAR-1. JNK, ERK1/2 and nuclear factor-κB may be involved in intracellular signal transduction after PAR-1 activation.
Fibroblasts ; Humans ; JNK Mitogen-Activated Protein Kinases/metabolism* ; MAP Kinase Signaling System ; Phosphorylation ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases/metabolism*

Objective:To research the mitochondrial cytochrome c oxidase subunit I (MT-COI) gene methylation levels in patients with occupational chronic benzene poisoning, and to explore effective molec μlar biomarkers in patients with occupational chronic benzene poisoning.Methods:38 confirmed cases of occupational chronic benzene poisoning were selected in the case group. 46 healthy people who underwent physical in our hospital were selected in the control group. Pyrosequencing was used to detect the methylation sites of methylation sites, flow cytometry was used to detect peripheral blood cell count levels, and non-parametric statistical methods were used to analyze the differences in detection results between the two groups.Results:The methylation level of mitochondrial MT-COI site 1 (2.21±0.81) % in the case group was less than that in the control group, and the difference was statistically significant ( P<0.05) . The methylation level of mitochondrial MT-COI site 2 (2.31±0.96%) in the case group was less than that in the control group, and the difference was statistically significant ( P<0.05) . The methylation average level of mitochondrial MT-COI (2.26±0.75) % in the case group was less than that in the control group, and the difference was statistically significant ( P<0.05) . Analysis of the average level of methylation found that the methylation level of mitochondrial MT-COI was correlated with WBC ( P<0.05) . Analysis of the average level of methylation found that the methylation level of mitochondrial MT-COI was correlated with platelets ( r=0.254、0.280, P<0.05) . Conclusion:The level of mitochondrial MT-COI gene methylation in patients with occupational chronic benzene poisoning may be related to the sensitivity to benzene exposure. Mitochondrial MT-COI gene methylation may serve as a potential predictive biomarker for benzene poisoning.

Objective:To research the mitochondrial cytochrome c oxidase subunit I (MT-COI) gene methylation levels in patients with occupational chronic benzene poisoning, and to explore effective molec μlar biomarkers in patients with occupational chronic benzene poisoning.Methods:38 confirmed cases of occupational chronic benzene poisoning were selected in the case group. 46 healthy people who underwent physical in our hospital were selected in the control group. Pyrosequencing was used to detect the methylation sites of methylation sites, flow cytometry was used to detect peripheral blood cell count levels, and non-parametric statistical methods were used to analyze the differences in detection results between the two groups.Results:The methylation level of mitochondrial MT-COI site 1 (2.21±0.81) % in the case group was less than that in the control group, and the difference was statistically significant ( P<0.05) . The methylation level of mitochondrial MT-COI site 2 (2.31±0.96%) in the case group was less than that in the control group, and the difference was statistically significant ( P<0.05) . The methylation average level of mitochondrial MT-COI (2.26±0.75) % in the case group was less than that in the control group, and the difference was statistically significant ( P<0.05) . Analysis of the average level of methylation found that the methylation level of mitochondrial MT-COI was correlated with WBC ( P<0.05) . Analysis of the average level of methylation found that the methylation level of mitochondrial MT-COI was correlated with platelets ( r=0.254、0.280, P<0.05) . Conclusion:The level of mitochondrial MT-COI gene methylation in patients with occupational chronic benzene poisoning may be related to the sensitivity to benzene exposure. Mitochondrial MT-COI gene methylation may serve as a potential predictive biomarker for benzene poisoning.

Objective To investigate the rules of medication and combinations of anti-tumor formulas containing marine Chinese medicinals. Methods The anti-tumor formulas containing marine Chinese medicinal were collected from Marine Chinese Medicinal and Effective formulas, Dictionary of Chinese Marine Medicinals, Marine Chinese Materia Medica, and databases including CNKI. A database was established for data mining by using V2. 5 software of TCM inheritance support system (TCMISS). Results There were totally 251 anti-tumor formulas containing marine Chinese medicinals involved 417 Chinese medicinal including 36 marine Chinese medicinals. The nature of medicinals in these formulas were mainly cold, neutral and warm, flavors of them were mainly bitterness, pungency, sweetness and saltiness,and they mainly entered into meridians of liver,lung,stomach,spleen and heart. The analysis of association rules showed that core combination was Muli (Oyster Shell, Concha Ostreae)-Haizao (Seaweed, Sargassum)-Kunbu (Kelp, Thallus Laminariae), which was commonly combined with medicinals with effects of clearing heart,tonifying,resolving phlegm,dissipating binds,activating blood and resolving stasis in clinical practice. Entropy clustering analysis showed that there were 12 combinations with core medicinals,and they were Haizao-Haigeqiao(Clam Shell,Goncha Meretricis seu Cyclinae)-Kunbu, Zaojiaoci (Chinese Honeylocust Spine, Spina Gleditsiae)-Chuanshanjia (Pangolin Scales,Squama Manitis)-Jiangcan (Batryticated Silkworm, Bombyx Batryticatus), Muxiang (Common Aucklandia Root,Radix Aucklandiae)-Walengzi (Ark Shell, Concha Arcae)-Puhuang (Cattail Pollen, Pollen Typhae), Chansu (Toad Venom, Venenum Bufonis)-Xionghuang (Realgar, Realgar)-Zhenzhu (Pearl,Pernulo),Huangbai(Amur Cork-Tree,Cortex Phellodendri)-Yunaoshi(Fish Otolith,Asteriscus Pseudosciaenae)-Bingpian (Bomeol, Bomeolum Syntheticum), Haima (Sea Horse, Hippocampus)-Qingpi (Green Tangerine Peel, Pericarpium Citri Reticulatae Viride)-Qianniuzi (Pharbitis Seed, Semen Pharbitidis), Haizao-Kunbu-Gancao (Liquorice Root, Radix Glycyrrhizae), Wangbuliuxing (Vaccaria Seed,Semen Vaccariae)-Chuanshanjia-Muli, Fengfang (Hornet Nest, Nidus Vespae)-Walengzi-Qianxie (Scorpion,Scorpio),Shexiang (Musk,Moschus)-Xuejie (Dragon's Blood,Risina Draconis)-Zhenzhu, Huangbai (Amur Cork-Tree, Cortex Phellodendri)-Ruxiang (Frankincense, Olibanum)-Bingpian, and Qingpi-Wulingzhi (Trogopterus Dung, Faeces Trogopterorum)-Dafupi (Shell of Arecanut, Pericarpium Arecae). There were 6 new combinations, and they were Haizao-Haigeqiao-Kunbu-Gancao, Zaojiaoci-Chuanshanjia-Jiangcan-Wangbuliuxing-Muli, Muxiang-Walengzi-Puhuang-Fengfang-Qianxie, Chansu-Xionghuang-Zhenzhu-Shexiang-Xuejie, Huangbai-Yunaoshi-Bingpian-Ruxiang, and Haima-Qingpi-Qianniuzi-Wulingzhi-Dafupi. The high-frequency tumor diseases, totally 47, treated with anti-tumor for-mulas containing marine Chinese medicinals,were successively thyroid tumor,lung cancer,liver cancer, uterine fibroids, nasopharyngeal carcinoma, esophageal cancer, gastric cancer and mammary cancer. Conclusion There are rules to follow in treatment of tumors with marine Chinese medicinal. The different therapeutic effects can be achieved by different medicinal combinations,which has a very important value in guiding clinical medication and new medicinal development.

According to the Chinese Pharmacopoeia 2015, only processed Aconitum tubers can be clinically applied, and the effect of processing is unclear. This research aimed to explore the effect of processing on cardiac efficacy of alkaloids in Aconitum tubers. First, the chemical ingredients in unprocessed and processed Aconitum tubers were identified and compared by using high performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF/MS) and multivariate pattern recognition methods. Then the representative alkaloids in Aconitum tubers, aconitine, benzoylaconine, and aconine, which belong to diester-diterpenoid alkaloids, monoester-diterpenoid alkaloids, and amine-diterpenoid alkaloids, respectively, were selected for further validation of attenuated mechanism. Subsequent pharmacological experiments with aconitine, benzoylaconine, and aconine in SD rats were used to validate the effect of processing on cardiac functions. After processing the Aconitum tubers, it was found that the contents of diester-diterpenoid alkaloids were reduced, and those of monoester-diterpenoid alkaloids and amine-diterpenoid alkaloids were increased, suggesting that diesterditerpenoid alkaloids were transformed into monoester-diterpenoid alkaloids and amine-diterpenoid alkaloids. Through further decocting the aconitine in boiling water, it was confirmed that the three alkaloids could be progressively transformed. Pharmacological experiments with aconitine, benzoylaconine, and aconine in SD rats showed that aconitine at a dose of 0.01 mg/kg and aconine at a dose of 10 mg/kg enhanced the cardiac function, while benzoylaconine at a dose of 2 mg/kg weakened the cardiac function. The effect of processing is attributed to the transformation of the most toxic diester-diterpenoid alkaloids into less toxic monoesterditerpenoid alkaloids and amine-diterpenoid alkaloids.