AIM: To investigate the synergistic effect of resveratrol and 5-fluorouracil on osteosarcoma CD133+ cell subset.METHODS: Human osteosarcoma cell line MG-63 CD133+ cell subset and the corresponding CD133-cell subset were treated with resveratrol and 5-fluorouracil.After treatment, the viability of MG-63 cells was measured by MTT assay.The apoptosis of MG-63 cells was analyzed by flow cytometry.Activation of caspase-9 and caspase-3, the expression of Apaf-1, and the release of cytochrome C were evaluated by Western blot.The interaction between Apaf-1 and pro-caspase-9 was detected by co-immunoprecipitation.RESULTS: The cell death and apoptosis of MG-63 CD133+ cell subset induced by 5-fluorouracil were significantly weaker than those in the corresponding MG-63 CD133-cell subset.However, co-treatment with resveratrol significantly enhanced the effect of 5-fluorouracil on inhibiting the viability of MG-63 CD133+ cell subset.Mechanically, treatment with resveratrol upregulated the expression of Apaf-1.Transfection with Apaf-1 siRNA abolished the synergistic effect of resveratrol and 5-fluorouracil in MG-63 CD133+ cell subset.In addition, the results of co-immunoprecipitation indicated that the combination of resveratrol and 5-fluorouracil significantly induced the formation of Apaf-1/pro-caspase-9 complex, leading to the activation of caspase-9 in MG-63 CD133+ cell subset.CONCLUSION: Resveratrol enhances 5-fluorouracil-induced apoptosis of osteosarcoma CD133+ cell subset by promoting the formation of Apaf-1/caspase-9 complex.

Objective:To evaluate the vomer development of cleft palate patients.Methods:38 patients over 1 4 years(averaged 23.4 years)of age with cleft palate and 76 controls of normal people(aged 22.8 year on average)were included.The 3D computed tomo-graphy reconstruction images of the bony nasal septum were measured.The development of the vomer was evaluated by comparing the L1 (the length of the lower edge of the vomer),L2 (the length from nasal spine to the point of the sella)and S (the approximate area of vomer)among deferent groups.Results:The L1 ,L2,and S of cleft palate patients were smaller than those of the controls(P <0. 05).Compared with the postoperative cleft cases,the S and L2 of preoperative cases were bigger(P <0.05).Conclusion:The vomer development is adversely affected by cleft palate.Not only the vomer-palate fusion is lower,but also the sutura between vomer and na-sal septum cartilage and ethmoid bone are short.And the latter is greatly influenced by surgical trauma.

Ebola virus (EBOV) and Marburg virus (MARV) are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates and there is currently no licensed vaccine or therapeutics.To date,there is no specific laboratory diagnostic test in China,while there is a national need to provide differential diagnosis during outbreaks and for instituting acceptable quarantine procedures.In this study,the TaqMan RT-PCR assays targeting the nucleoprotein genes of the Zaire Ebolavirus (ZEBOV) and MARV were developed and their sensitivities and specificities were investigated.Our results indicated that the assays were able to make reliable diagnosis over a wide range of virus copies from 103 to 109,corresponding to the threshold of a standard RNA transcript.The results showed that there were about 1010 RNA copies per milliliter of virus culture supernatant,equivalent to 10,000 RNA molecules per infectious virion,suggesting the presence of many non-infectious particles.These data indicated that the TaqMan RT-PCR assays developed in this study will be suitable for future surveillance and specific diagnosis of ZEBOV and MARV in China.

Objective To explore the relationship between the expression of XRCC1 and glioma. Methods Total of 26 samples of glioma were divided into 4 groups:gradeⅠ,gradeⅡ,gradeⅢand gradeⅣ. The expression of XRCC1 in 26 Gliomas tissues were examined using SP immunohistochemical staining.Results The positive staining of XRCC1 protein was localized in nucleus of tumor cells in Glioma. There was no correlation among them. The difference of XRCC1 expression among gradeⅠ~Ⅳ was not significant ( >0.05) .Conclusion The difference of XRCC1 expression among gradeⅠ~Ⅳ was not significant. The expression of XRCC1 was closely correlated with the effect of radiotherapy.

Objective To investigate the molecular mechanism of retinoic acid in targeted treatment of craniopharyngioma by detecting the expression of RARαand PPARβ/δin craniopharyngioma cells and analyzing the correlation between the expression and effect of retinoic acid. Methods The expression of RARα and PPARβ/δ in craniopharyngioma cells from 31 patients cultured in vitro was quantified by reverse transcription-PCR. The inhibition rates of RA on craniopharyngioma with different expression of RARα and PPARβ/δ were detected by using MTT assay, and the correlation between the expression of RARα and PPARβ/δand the effect of RA was analyzed. Results 1. The RT-PCR results showed that the expression levels of PPARβ/δand RARα mRNA were different. Craniopharyngioma cells from 31 patients in primary culture were divided into three groups according the expression levels of nuclear receptor: PPARβ/δ>RARα group, RARα>PPARβ/δ group and RARα>>PPARβ/δ group. 2.MTT results showed that the inhibition rate of RARα>>PPARβ/δgroup was significantly higher than the other groups under same drug, the differences had statistical significance ( <0.01) . Conclusions The expression of PPARβ/δ, RARα can be used to evaluate the effect of RA in treatment of craniopharyngioma. The craniopharyngioma with low-expression of PPARβ/δ is more sensitive to RA. Targeting higher RARα or targeting lower PPARβ/δ is beneficial to the treatment of craniopharyngiomas.

AIM: To prepare and identify mouse polyclonal antibody against protein Hlb, which is the variant of major subunit of human ASGPR. METHODS: Hlb specific peptide was synthesized and coupled with keyhole limpet hemocyanin (KLH) for immunization. Then H1b-KLH conjugation was injected into mouse subcutaneously to produce polyclonal antibody. ELISA assay was used to detect the titer of the antibody. Antibody was also identified by Western blot and immunohistochemistry assays. RESULTS: Mouse antibody against Hlb was prepared after injection of H1bKLH conjugation. The titer of H1b antibody was about 1:10~5.Western blot confirmed its high specificity. This antibody could also be used for immunohistochemistry analysis. CONCLUSION: The successful preparation of the polyclonal antibody against protein H1b, which can discriminate the two variants of the major subunit of ASGPR with high specificity, will provide an efficient reagent for further study of the physiologic functions of H1b and its role in the pathogenesis of human disease.

To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR H1b) on ASGPR ligands' binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH1a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector pIRES2EGFP, pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRH1a and H2c in 4-1-6 were confirmed by RT-PCR, Western blotting and immunofluorescence. The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H1b/pCDNA3.1 (neo) was transfected into cell line 4-1-6, H1b did not down-regulate the ligand binding ability of ASGPR. The eukaryotic expression plasmid H1b/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single H1b nor H1b and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both H1a and H2c stably was established. The new split variant H1b has no effect on ASGPR binding to ASOR. ASGPRH1b alone can't bind to ASOR, it yet can't form functional complex with ASGPRH2c.

Objective UPLC-Q-TOF-MS integrated molecular network strategy was used to rapidly analyze and identify the chemical components of Yuye detoxification Particle. Methods The secondary mass spectrometry data of compounds were obtained using mass spectrometry scanning in both positive and negative ion mode. The similarity of MS/MS fragmentation patterns was calculated to create the global natural product social molecular networking (GNPS) platform. The major components in Yuye detoxification Particle were quickly identified according to the molecular clusters with similar structures in GNPS. Manual analysis and identification of other compounds were performed according to the mass fragment ion information of the primary and secondary mass spectrum data and related references by using the molecular feature extraction (MFE) function of Agilent Masshunter Qualitative Analysis workstation and traditional Chinese Medicine composition database (TCM-DATA). Results A total of 89 compounds in Yuye detoxification Particle were identified by LC-MS,including 22 phenoliacids,21 flavonoids and their glycosides,6 iridoid glycosides,28 triterpenoid saponins and 12 other types of ingredients. Conclusion UPLC-Q-TOF-MS/MS integrated molecular network technology can be used for rapid and systematic identification of chemical components of Yuye detoxification Particle,which provides theoretical basis for its quality control and clinical application. The established molecular network can provide reference for rapid qualitative analysis of components of traditional Chinese medicine compound.

Objective: To explore the effects of transient receptor potential vanilloid 1 (TRPV1) on autophagy in early hypoxic mouse cardiomyocytes and the mechanism in vitro. Methods: The hearts of 120 C57BL/6 mice aged 1-2 days, no matter male or female, were isolated, and then primary cardiomyocytes were cultured and used for the following experiments, the random number table was used for grouping. (1) The cells were divided into normoxia group and hypoxia 3, 6, and 9 h groups, with one well in each group. The cells in normoxia group were routinely cultured (the same below), the cells in hypoxia 3, 6, and 9 h groups were treated with fetal bovine serum-free and glucose-free Dulbecco′ s modified Eagle medium under low oxygen condition in a volume fraction of 1% oxygen, 5% carbon dioxide, and 94% nitrogen for 3, 6, and 9 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, TRPV1 were determined with Western botting. (2) The cells were divided into normoxia group and hypoxia group, with two coverslips in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above. The positive expression of TRPV1 was detected by immunofluorescence assay. (3) The cells were divided into 4 groups, with one well in each group. The cells in simple hypoxia group were treated with hypoxia for 6 h as above, and the cells in hypoxia+ 0.1 μmol/L capsaicin group, hypoxia+ 1.0 μmol/L capsaicin group, and hypoxia+ 10.0 μmol/L capsaicin group were respectively treated with 0.1, 1.0, 10.0 μmol/L capsaicin for 30 min before hypoxia for 6 h. The protein expressions of LC3, Beclin-1, and TRPV1 were detected by Western blotting. (4) The cells were divided into 5 groups, with 5 wells in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ chloroquine group, hypoxia+ capsaicin group, and hypoxia+ capsaicin+ chloroquine group were treated with hypoxia for 6 h after being cultured with 50 μmol/L chloroquine, 10.0 μmol/L capsaicin, and 50 μmol/L chloroquine+ 10.0 μmol/L capsaicin for 30 min, respectively. Viability of cells was detected by cell counting kit 8 assay. (5) The cells were divided into simple hypoxia group and hypoxia+ 10.0 μmol/L capsaicin group, with one well in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ 10.0 μmol/L capsaicin group were treated with 10.0 μmol/L capsaicin for 30 minutes and then with hypoxia for 6 h. The protein expressions of lysosomal associated membrane protein 1 (LAMP-1) and LAMP-2 were detected by Western blotting. Each experiment was repeated for 3 or 5 times. Data were processed with one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: (1) Compared with those of normoxia group, the protein expressions of LC3, Beclin-1, and TRPV1 were significantly increased in cardiomyocytes of hypoxia 3, 6, and 9 h groups (t_{3 h}=4.891, 5.890, 4.928; t_{6 h}=9.790, 6.750, 10.590; t_{9 h}=6.948, 6.764, 5.049, P<0.05 or P<0.01), which of hypoxia 6 h group were the highest (1.08±0.05, 1.12±0.10, 0.953±0.071, respectively). (2) The density of TRPV1 in cell membrane and inside the cardiomyocytes in hypoxia group was significantly increased with lump-like distribution, and the expression of TRPV1 was higher than that in normoxia group. (3) Compared with those of simple hypoxia group, the protein expression of Beclin-1 in cardiomyocytes of hypoxia+ 0.1 μmol/L capsaicin group was increased (t=10.488, P<0.01), while the protein expressions of LC3 and TRPV1 were increased without statistically significant differences (t=4.372, 3.026, P>0.05); the protein expressions of LC3, TRPV1, and Beclin-1 in cardiomyocytes of hypoxia+ 1.0 μmol/L capsaicin group and hypoxia+ 10.0 μmol/L capsaicin group were significantly increased (t=15.505, 5.773, 13.430; 20.915, 8.054, 16.384; P<0.05 or P<0.01), which of hypoxia+ 10.0 μmol/L capsaicin group were the highest (2.33±0.09, 1.34±0.07, 1.246±0.053, respectively). (4) Compared with 0.585±0.045 in normoxia group, the cardiomyocyte viability in hypoxia group was significantly decreased (0.471±0.037, t=4.365, P<0.05). Compared with that in hypoxia group, the cardiomyocyte viability in hypoxia+ chloroquine group was further decreased (0.350±0.023, t=6.216, P<0.01), while 0.564±0.047 in hypoxia+ capsaicin group was significantly increased (t=3.489, P<0.05). Compared with that in hypoxia+ chloroquine group, the cardiomyocyte viability in hypoxia+ capsaicin+ chloroquine group did not significantly change (0.364±0.050, t=0.545, P>0.05). (5) Compared with 0.99±0.04 and 0.54±0.04 in simple hypoxia group, the protein expressions of LAMP-1 and LAMP-2 in hypoxia+ 10.0 μmol/L capsaicin group were significantly increased (1.49±0.06, 0.81±0.05, t=12.550, 7.442, P<0.01). Conclusions TRPV1 can further promote the expression of autophagy-related proteins in hypoxic cardiomyocytes through autophagy-lysosomal pathway, enhance autophagy activity, and improve autophagic flow for alleviating early hypoxic cardiomyocyte injury.

To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR Hlb) on ASGPR ligands' binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH 1 a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector plRES2EGFP,pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRHla and H2c in 4-1-6 were confirmed by RT-PCR,Western blotting and immunofluorescence. The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H lb/pCDNA3.1 (neo)was transfected into cell line 4-1-6, Hlb did not down-regulate the ligand binding ability of ASGPR.The eukaryotic expression plasmid Hlb/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single Hlb nor Hlb and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both Hla and H2c stably was established. The new split variant Hlb has no effect on ASGPR binding to ASOR. ASGPRHlb alone can't bind to ASOR, it yet can't form functional complex with ASGPRH2c.