Objective To construct eukaryotic expressing vector of human melanin-concentrating hormone receptor1(MCHR1) and transfect HEK293 cells so as to establish stable HEK293 cell line.Methods The full-length MCHR1 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and was inserted into pcDNA3.1(+).After the identification of digestion and sequencing on the recombinant pcDNA3.1(+)/MCHR1,we transfected the recombinant into HEK293 cell by lipofectamineTM2000.After screening culture by G418,stable transfected HEK293 cell line was established,and the expression of MCHR1 was identified by RT-PCR and Western blotting.Results The eukaryotic expression vector pcDNA3.1(+)/MCHR1 was constructed,stable transfected HEK293 cell line was established,and MCHR1 protein was expressed successfully.Conclusion The construction of the eukaryotic expression vector pcDNA3.1(+)/MCHR1 and the establishment of stable transfected HEK293 cell line have provided solid experiment foundation for further studies on the function of MCHR1.

Objective To construct eukaryotic expressing vector of human melanin-concentrating hormone receptor 1(MCHR1), then to transfect CHO cells with the vector for establishment of stable CHO cell line. Methods The full-length MCHR1 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and then inserted into eukaryotic expression vector pcDNA3.1(+).The recombinant was transfected into CHO cells by lipofectamine TM 2000 after identification of digestion and sequencing on the recombinant eukaryotic expression vector pcDNA3.1(+)/MCHR1. The stable transfected CHO cell line was then established by screening cultures with G418, and the transcription and expression of MCHR1 were identified by RT-PCR, Western blot and immunofluorescence. Results The eukaryotic expression vector pcDNA3.1(+)/MCHR1 was constructed successfully, stable transfected CHO cell line was established, the MCHR1 protein was expressed successfully. Conclusion The construction of eukaryotic expression vector pcDNA3.1(+)/MCHR1 and the establishment of stable transfected CHO cell line provided a solid experimental foundation for further studies on the function of MCHR1.

Objective To construct eukaryotic expression vectors expressing short hairpin RNA(shRNA)sections targeting human MCHR2 and to observe their effects on MCHR2 gene expression in HEK293 cell line.Methods According to the sequence of human MCHR2 gene,the oligonucleotides of shRNA were designed and synthesized and directionally cloned into plasmid pGenesil-1 with enhancing green fluorescence protein(EGFP)gene and Kan gene.The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing.The recombinant vectors were transfected into HEK293 cell line by LipofectamineTM2000,the effects on MCHR2 at mRNA and protein levels were observed.Results Four shRNA expressing recombinants and the corresponding negative control vector were constructed and transfected into HEK293 cell successfully.MCHR2 transcript was reduced by about 45.8%-66.4%,the protein of MCHR2 was reduced by about 44.2%-81.0% in four transfectants respectively.Conclusion The construction of eukaryotic expression vectors expressing shRNA sections targeting human MCHR2 and identification successfully established a favourable foundation for further study on the function of MCHR2.

Objective To establish a drug resistant human hepatoma cell line (QGY/DDP) induced by cisplatin (DDP) in vitro, and to investigate its biological features and mechanisms of resistance. Methods The drug resistant cell line (QGY/DDP) of hepatoma was established by intermittent administration of stepwise increas of the dosage of DDP. Drug sensitivity was evaluated by MTT assay. Cell counting was employed to graph the growth curve and to calculate the doubling time. Flow cytometry (FCM) was used to study the cell cycle distribution. In addition, the intracellular platinum accumulation was detected by atomic absorption spectrometry, and the expressions of P-glycoprotein (P-gp) and glutathione S-transferase-? (GST-?) were analyzed by FCM. Results QGY/DDP cell line was established successfully after 3 months with stable resistance to DDP, and the resistance index (RI) was 10.81. The established cell line exhibited obvious cross-resistance to 5-FU, VCR, MMC, EPI and HCPT. Compared with parental cell line, the QGY/DDP cell line grew slower and its doubling time became longer. The proportion of G0/G1 phase cells of the resistant cells was significantly higher than that of their parental cells, whereas the proportion of S and G2/M phase cells was decreased. The cellular platinum accumulation was obviously decreased in the QGY/DDP cell line, and the expression of GST-? in QGY/DDP cells was higher than that of their parental cells, but no over expression of P-gp was found in the resistant cell line. Conclusions QGY/DDP cell line shows the typical and stable resistant phenotype and possesses the basic biological features of resistant cells. The resistance of the cell line to DDP may be due to the reduction of intracellular platinum accumulation and the over expression of GST-?, but may not be related to the expression of P-gp.

Aim To study the effects of total saponins of Panax japonicus(TSPJ) on liver inflammation of natural aging rats.Methods The experimental rats were allocated into seven groups (twelve rats in each group): three months group, nine months group, fifteen months group, twenty-four months group, and TSPJ low-, mid-and high-dose groups(10, 30, 60 mg·kg-1).When the rats were eighteen months old, the TSPJ low-, mid-and high-dose groups of rats were given lavage treatments with TSPJ 10,30, 60 mg·kg-1 respectively until twenty-four months.During lavage, we stopped a day every week for six consecutive months.HE staining was used to observe the pathological morphological changes.Western blot was utilized to test IL-1β and TNF-α protein expressions.RT-PCR method was adopted to test IL-1β, IL-6, IL-12, IL-17α, TNF-α, IFN-γ mRNA expressions.Results HE staining observation showed that as the rats grew older the hepatic cord and sinusoid were arranged in more severe disorder, and the fat vacuole and inflammatory cells were increased significantly.While every dose group of TSPJ could improve these pathological changes distinctly.The IL-1β, TNF-α protein and IL-1β, IL-6, IL-12, IL-17α, TNF-α, IFN-γ mRNA expressions were increased gradually as the rats grew older, and every dose group of TSPJ could reduce their expressions to some extent.Conclusion TSPJ could protect the aging rat liver to some extent by inhibiting the liver inflammation.

Objective To prepare polyclonal antibody of transactivated protein 5 of hepatitis C virus nonstructural 5A (NA5ATP5) and to explore its expression in the liver tissues. Methods In Escherichia coil BL21, the prokaryotic expression vector pET32a(+)-NS5ATP5 was induced by isopropyl-β-D-thiogalactoside (IPTG), and it was analyzed with sodium dodecyl sulfate-polyaerylamide gel electrophoresis (SDS-PAGE) and Western blotting. And the purified protein was used to immunize the rabbit to prepare polyelonai antibody, with which we studied the function of NSSATP5 by determining the different liver tissues with the streptavidin-perosidase (SP) immunohistochemistry method. Results Recombinant NS5ATP5 (molecular weight: 65 kD) and polyclonal antibody were successfully prepared. NS5ATP5 expression in the liver of patients with chronic HCV infection was much higher than that of a normal person, and it was detected mainly in the cytoplasm. Conclusion The findings of the expression difference between HCV patients and normal people led to a novel diagnostic marker to detect HCV infection.

Aim To construct a eukaryotic expression vector of human MCHR2 and to analyze the stable expression and signal transduction pathways of MCHR2 in CHO cells. Methods The full-length MCHR2 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and inserted into eukaryotic expres-sion vector pcDNA3.1(+), resulted in the recombinant expression vector pcDNA3.1-MCHR2. The recombinant plasmid was confirmed by the restriction enzymatic digestion and DNA sequencing analysis, and then transfected into CHO cells by lipofectamine. A stably-transfected cell line was obtained by the dominant G418 selection, and the expression of the MCHR2 gene on transcription and translation levels were identified by RT-PCR and Western blot. Signal transduction pathways mediated by MCHR2 were analyzed by measurement of intracellular cAMP and calcium. Results The eukaryotic expression vector pcDNA3.1-MCHR2 was successfully constructed and the MCHR2 gene was stably transfected into CHO cells. A stably-transfected cell line was established and the MCHR2 gene was efficiently transcribed and translated. MCH stimulation had no effect on the production of cAMP, however, it could induce a clear and transient increase of intracellular calcium, suggesting that MCHR2 was only coupled to Gq protein. Conclusion The stable expression of MCHR2 and analysis of its signal transduction pathways in CHO cell line provided a solid experimental foundation for further studies on the function of the MCHR2 gene in vitro.

Objective:To investigate whether long non-coding RNA(lncRNA) AW112010 can improve insulin resistance in aging adipocytes through the miR-204/POU2F2 signaling pathway.Methods:In vivo experiment: C57BL/6 mice were divided into young control group(4 months old) and aging model group(18 months old) based on body weight. The expression levels of AW112010, miR-204-5p, POU2F2, aging related indicators(p16, p21), and insulin signaling pathway genes [insulin receptor(INSR), insulin receptor substrate 1(IRS1), phosphatidylinositol kinase(PI3K), protein kinase B(AKT)] in epididymal adipose tissue were detected using real-time fluorescence quantitative PCR(RT-qPCR) and Western blotting. In vitro experiment: Using adriamycin(ADR) to induce 3T3-L1 aging adipocyte model, β-gal staining was used to observe cellular senescence, and miR-204 inhibitor and miR-204 mimic small interfering RNA were successfully constructed and transfected into 3T3-L1 adipocytes. Results:RT-qPCR and Western blot results showed that compared with the young group, the expression of AW112010 in the adipose tissue of aging mice was increased, while the expression of miR-204-5p was decreased. The expressions of POU2F2, p16, and p21 in the adipose tissue of aging mice were increased, while the expressions of INSR, IRS1, PI3K, GLUT4 mRNA and protein were decreased. The β-gal stainging results showed that the number of 3T3-L1 senescent adipocytes induced by ADR was significantly increased, and the expression levels of AW112010, POU2F2, p16, and p21 in ADR-induced senescent adipocytes were increased compared with the control group, while the expression levels of miR-204-5p, INSR, IRS1, PI3K, GLUT4 were decreased, and remaining glucose in the culture medium was increased. Compared with control, overexpression of miR-204 resulted in decreased expressions of aging indicators p16, p21, and target gene POU2F2 while the expressions of INSR and GLUT4 were increased.Conclusion:Upregulation of lncRNA AW112010 in adipocytes of aging mice may induce insulin resistance by targeting miR-204-5p/POU2F2/IRS1.

ObjectiveTo investigate the effect and mechanism of total saponins from Panax japonicus （TSPJ） on white adipose tissue （WAT） browning/brown adipose tissue （BAT） activation in C57BL6/J male mice fed on a high-fat diet （HFD）. MethodThirty-two C57BL6/J male mice （8-week-old） were randomly divided into a normal group， a model group， a low-dose TSPJ group， and a high-dose TSPJ group. The mice in the low-dose and high-dose TSPJ groups were given TSPJ for four months by gavage at 25， 75 mg·kg^-1·d^-1， respectively， and those in the other groups were given 0.5% sodium carboxymethyl cellulose （CMC-Na） accordingly. After four months of feeding， all mice were placed at 4 ℃ for acute cold exposure， and the core body temperature was monitored. Subsequently， all mice were sacrificed， and BAT and inguinal WAT （iWAT） were separated rapidly to detect the corresponding indexes. Hematoxylin-eosin （HE） staining was used to observe the morphological changes in each group. The effect of TSPJ on the mRNA expression of uncoupling protein 1 （UCP1）， fatty acid-binding protein 4 （FABP4）， cytochrome C （CytC）， PR domain-containing protein 16 （PRDM16）， elongation of very long chain fatty acids protein 3 （ELOVL3）， peroxisome proliferator-activated receptor γ （PPARγ）， and peroxisome proliferator-activated receptor-γ coactivator-1α （PGC-1α） in iWAT and BAT was detected by Real-time polymerase chain reaction （Real-time PCR）. Western blot was also used to detect the protein expression of UCP1， PRDM16， PPARγ， and PGC-1α in BAT and iWAT of each group. The effect of TSPJ on UCP1 expression in BAT and iWAT was detected by immunohistochemistry. Result① Compared with the model group， TSPJ could decrease the body weight and proportions of iWAT and BAT in the HFD-induced mice （P<0.05， P<0.01）. ② The body temperature of mice in the model group decreased compared with that in the normal group in the acute cold exposure tolerance test （P<0.05）. The body temperature in the high-dose TSPJ group increased compared with that in the model group （P<0.01）. ③ Compared with the normal group， the model group showed increased adipocyte diameter in iWAT and BAT and decreased number of adipocytes per unit area. Compared with the model group， the TSPJ groups showed significantly reduced cell diameter and increased number of cells per unit area， especially in the high-dose TSPJ group. ④ Compared with the normal group， the model group showed decreased mRNA expression of FABP4， UCP1， CytC， PRDM16， ELOVL3， PGC-1α， and PPARγ in adipose tissues of mice （P<0.05， P<0.01）. Compared with the model group， after intervention with TSPJ， the mRNA expression was significantly up-regulated （P<0.05， P<0.01）. ⑤ Compared with the normal group， the model group showed decreased protein expression of UCP1， PRDM16， PPARγ， and PGC-1α in adipose tissues of mice （P<0.05， P<0.01）. Compared with the model group， after intervention with TSPJ， the protein expression increased significantly （P<0.05， P<0.01）. ConclusionTSPJ could induce the browning of iWAT/BAT activation and enhance adaptive thermogenesis in obese mice induced by HFD. The underlying mechanism may be attributed to the activation of the PPARγ/PGC-1α signaling pathway.