BACKGROUND:The liver,as the main target organ for arsenic toxicity,has become the focus of studies related to the mechanism of action of arsenic toxicity.OBJECTIVE:To investigate the effects of sodium arsenite(NaAsO2)on lipid metabolism,cell proliferation,apoptosis,and expression of related regulatory factors in human normal hepatocytes.METHODS:MIHA normal human hepatocyte cell lines were exposed to 0,10,20,and 30 μmol/L NaAsO2 for 48 hours.Cell morphology changes were observed by light microscopy.Cell viability was detected by CCK-8 assay.The cell serum total cholesterol,triacylglycerol,and total bile acids were detected by single-agent COD-PAP assay,single-agent GPO-PAP assay,and enzyme microplate assay.The intracellular lipid content was detected by oil red O staining.Cell proliferation was detected by Edu-488 infiltration.Cell cycle and apoptosis were detected by PI staining and Annexin V-FITC/PI dual-labeling combined with flow cytometry.The mRNA and protein expression levels of hepatocyte nuclear factor 4 alpha,cholesterol 7α-hydroxylase,and farnesoid X receptor were detected by real-time fluorescence quantitative PCR and western blot assay,respectively.RESULTS AND CONCLUSION:(1)Compared with the control group(0 μmol/L NaAsO2),with the increase of NaAsO2 concentration:MIHA cell viability decreased gradually.The content of total cholesterol and triacylglycerol in cell supernatant increased gradually,while the contents of total bile acids decreased gradually.The content of intracellular lipid increased gradually.The proportion of cells stagnating in S phase and G2/M phase gradually increased,and the apoptosis rate gradually increased.The expression level of hepatocyte nuclear factor 4 alpha mRNA did not show significant changes,while cholesterol 7α-hydroxylase and farnesoid X receptor mRNA expression levels decreased.The protein expression levels of hepatocyte nuclear factor 4 alpha,cholesterol 7α-hydroxylase,and farnesoid X receptor decreased gradually.(2)NaAsO2 has cytotoxicity,significantly reduces MIHA cell viability,induces cell steatosis,inhibits cell proliferation,and induces cell apoptosis.NaAsO2 down-regulates hepatocyte nuclear factor 4 alpha protein expression and the transcription and expression of cholesterol 7α-hydroxylase and farnesoid X receptor,which further induces lipid metabolism disorders in hepatocytes.

BACKGROUND:The liver,as the main target organ for arsenic toxicity,has become the focus of studies related to the mechanism of action of arsenic toxicity.OBJECTIVE:To investigate the effects of sodium arsenite(NaAsO2)on lipid metabolism,cell proliferation,apoptosis,and expression of related regulatory factors in human normal hepatocytes.METHODS:MIHA normal human hepatocyte cell lines were exposed to 0,10,20,and 30 μmol/L NaAsO2 for 48 hours.Cell morphology changes were observed by light microscopy.Cell viability was detected by CCK-8 assay.The cell serum total cholesterol,triacylglycerol,and total bile acids were detected by single-agent COD-PAP assay,single-agent GPO-PAP assay,and enzyme microplate assay.The intracellular lipid content was detected by oil red O staining.Cell proliferation was detected by Edu-488 infiltration.Cell cycle and apoptosis were detected by PI staining and Annexin V-FITC/PI dual-labeling combined with flow cytometry.The mRNA and protein expression levels of hepatocyte nuclear factor 4 alpha,cholesterol 7α-hydroxylase,and farnesoid X receptor were detected by real-time fluorescence quantitative PCR and western blot assay,respectively.RESULTS AND CONCLUSION:(1)Compared with the control group(0 μmol/L NaAsO2),with the increase of NaAsO2 concentration:MIHA cell viability decreased gradually.The content of total cholesterol and triacylglycerol in cell supernatant increased gradually,while the contents of total bile acids decreased gradually.The content of intracellular lipid increased gradually.The proportion of cells stagnating in S phase and G2/M phase gradually increased,and the apoptosis rate gradually increased.The expression level of hepatocyte nuclear factor 4 alpha mRNA did not show significant changes,while cholesterol 7α-hydroxylase and farnesoid X receptor mRNA expression levels decreased.The protein expression levels of hepatocyte nuclear factor 4 alpha,cholesterol 7α-hydroxylase,and farnesoid X receptor decreased gradually.(2)NaAsO2 has cytotoxicity,significantly reduces MIHA cell viability,induces cell steatosis,inhibits cell proliferation,and induces cell apoptosis.NaAsO2 down-regulates hepatocyte nuclear factor 4 alpha protein expression and the transcription and expression of cholesterol 7α-hydroxylase and farnesoid X receptor,which further induces lipid metabolism disorders in hepatocytes.

Objective： ：To explore a novel chimeric antigen receptor (CAR)-T cell treatment to treat Multiple Myeloma (MM) via target B cell maturation antigen (BCMA). Methods： ：A CAR-BCMA molecular was constructed based on mouse originated BCMA scFv, and was packaged into lentiviral vector and transfected into T cells from healthy donors to construct CAR-BCMA-T cells. The BCMApositive cell lines A549-BCMA, A549-BCMAOFP and K562-BCMA were constructed as target cells. Then, the CAR-BCMA-T cells were co-incubated with the constructed target cells and human myeloma U266 cells, and the cytotoxic effects of CAR-BCMA-T cells were evaluated via CCK-8 and FACS. Finally, the CAR-BCMA-T cells originated from MM patients were constructed, and its cytotoxicity against A549-BCMA were examined; in addition, the IFN-γ release level in CAR-BCMA-T cells was evaluated by ELISA and FACS. Results: After 11 days’incubation, the CAR-BCMA-T cells originated from healthy donors amplified 300 times with a positive rate of 43%. The BCMApositive target cell lines were constructed successfully. Under an effector : target ratio of 5:1, the killing rates of CARBCMA-T cells against A549-BCMA, K562-BCMA and U266 were about 80%, 60%, and 80%, respectively, which were significantly higher than those against BCMA negative cells; and the cytotoxicity was related to the BCMA expression level in target cells. What’ s more, at the effector : target ratio of 20:1, the CAR-BCMA-T cells originated from MM patients were demonstrated to exhibit a killing rate of more than 95% againstA549-BCMApositive cells, and produced large amount of IFN-γ. Conclusion: CAR-BCMA-T cells originated from both healthy and MM donors were successfully constructed, and they can effectively and specifically kill BCMA positive tumor cells.

Xanthogranulomatous cholecystitis (XGC) is a rare type of chronic cholecystitis characterized by severe proliferative fibrosis with infihration of macrophages and foamy cells in the gallbladder wall.Since XGC and gallbladder carcinoma have similar clinical manifestations and radiological features,XGC is often misdiagnosed as gallbladder carcinoma in clinical practice,which leads to unnecessary extensive surgical resection and has an adverse effect on patients.At present,the preoperative diagnosis of XGC is still based on imaging results (ultrasound,computed tomography,and magnetic resonance imaging),and a definite diagnosis of this disease relies on intraoperative frozen biopsy or postoperative pathological examination.Meanwhile,XGC should be differentiated from gallbladder adenomyomatosis,gallbladder carcinoma,and gallbladder actinomycosis.Laparotomy or laparoscopic cholecystectomy is the major method for the treatment of XGC,but laparoscopic cholecystectomy is associated with a longer time of operation,more complications,and a higher rate of conversion to laparotomy.Therefore,surgeons are facing difficulties in preoperative diagnosis and intraoperative decision-making process of XGC.

Objective To construct a cDNA expression library from unfed female tick Haemaphysalis longicornis for screening and cloning potential antigenic genes.Methods Total RNA was isolated from unfed female ticks,mRNA was purified and a library of oligo(dT)-primed cDNA with added directional EcoR Ⅰ/Hind Ⅲ linkers was constructed from the purified mRNA.The constructed cDNA was ligated to the EcoRⅠ/HindⅢ arms of the ?SCREEN vector.Pure phage stocks were harvested by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8.Recombinant plasmids that were subcloned to E.coli BM25.8 were isolated and transformed into E.coli JM109.Recombinant plasmids abstracted from JM109 were identified by PCR and sequencing.Rusults The recombinant phage DNA was packaged by using phage-marker packaging extracts,resulting in a primary cDNA library with a size of 1.8?106 pfu.Data showed 100% of the library were recombinant and the titer of the amplified library was 2.4?109 pfu/ml.Forty-two clones of encoding immunodominant antigens were obtained from the cDNA library.Sequence analysis revealed 12 unique cDNA sequences and the encoded putative proteins showed similarities to H.longicornis tropomyosin mRNA,Rhipicephalus annulatus unknown larval protein mRNA,chromosome 2R of Drosophila melanogaster,mitochondrial DNA of H.flava,clones HqL09 unkown mRNA and Hq05 mRNA of H.qinghaiensis,and myosin alkali light chain protein mRNA.Conclusion The cDNA expression library from unfed female H.longicornis was successfully constructed and screening of protective genes may provide candidate antigens of the tick.