CYP3A29稳定表达HepG_2细胞株的建立及其硝苯地平代谢活性鉴定

目的建立稳定表达Bama小型猪CYP3A29的HepG2细胞株。方法通过总RNA提取、经RT-PCR得到Bama小型猪CYP3A29的基因,并将此基因克隆至亚克隆载体PMD18-T上得到重组质粒pMD-3A29;以测序正确的重组质粒pMD-3A29为模板,采用PCR扩增CYP3A29基因并在其3′添加组氨酸标签,扩增产物经BamHI/XhoI双酶切,将带有组氨酸标签的CYP3A29基因定向克隆至pcDNA3.1(+)中,并测序验证;将测序正确的CYP3A29基因通过脂质体转染至HepG2细胞,G418筛选10代,经RT-PCR及Western-blot分析及探针药物硝苯地平对重组细胞进行活性鉴定。结果与HepG2相比,HepG2-CYP3A29细胞株具有极显著的硝苯地平氧化活性。结论成功建立了稳定表达CYP3A29的HepG2细胞株,可用于相关药物代谢研究。

肝细胞癌cathepsin L表达分析

目的探讨cathepsin L蛋白在肝细胞癌中的表达及其与肿瘤临床病理特征的关系。方法58例肝细胞癌中病理分级高分化18例、中分化18例、低分化22例;临床分期Ⅰ+Ⅱ期26例、Ⅲ+Ⅳ期32例。采用免疫组织化学染色、RT-PCR法检测cathepsin L表达,结合患者临床病理指标及预后进行分析。结果cathepsin L表达阳性率为72.4%(42/58),不同病理分级、临床分期及转移分组cathepsin L表达阳性率差异显著。cathepsin L mRNA检出阳性率65.5%(38/58),cathepsin L mRNA的表达与肝细胞癌的病理分级、临床分期及淋巴转移有相关性。结论cathepsin L在肝细胞癌的生长浸润、侵袭转移中发挥重要作用,可作为肝细胞癌预后判断的客观指标之一。

两种试剂检测抗-HCV在丙型肝炎诊断中的意义

目的提高丙型肝炎病毒抗体(抗-HCV)检出率和检测结果的准确率。方法应用两个厂家抗-HCV ELISA试剂(试剂1、2)检测本院2006年1～12月间2516份输血、手术前住院患者血清标本。再将试剂1、2均阳性的血清标本28份、仅试剂1阳性的15份和仅试剂2阳性的17份血清标本进行HCV RNA RT-PCR荧光定量检测。结果试剂1、2抗-HCV均阳性28份标本的RT-PCR结果均阳性,仅试剂1抗-HCV阳性的15份标本中2份阳性,试剂2抗-HCV阳性的17份标本中3份阳性;仅试剂1或2抗-HCV阳性的32份血清标本中,HCV RNA RT-PCR检测阳性率为15.63%。结论采用双试剂检测能提高抗-HCV的检出率和结果的准确率。

Influence of paeonol on expression of COX-2 and p27 in HT-29 cells

AIM： To investigate the effect of paeonol on controlling the proliferation of colorectal cancer cell line HT-29 and to discuss its possible mechanism. METHODS： The inhibitory effect of paeonol on proliferation of HT-29 cells was detected by M-I-I- assay. The results of apoptosis were measured by flow cytometry. Immunocytochemical staining was performed to detect the expression of cyclooxygenase-2 （COX-2） and protein p27 in HT-29 cells treated with paeonol at different concentrations. Reverse transcription-polymerase chain reaction （RT- PCR） was used for mRNA analysis. RESULTS： From the data of both MTT and flow cytometry, we observed that cell proliferation was inhibited by different concentrations of paeonol. By immunocytochemical staining, we found that HT-29 cells treated with paeonol （0.024-1.504 mmol/L） reflected reduced expression of COX-2 and increased expression of p27 in a dose-dependent manner. RT-PCR showed that paeonol down-regulated COX-2 and up-regulated p27 in a dose- and time-dependent manner in HT-29 cells. CONCLUSION： One of the apoptotic mechanisms of paeonol is down-regulation of COX-2. p27 is upregulated simultaneously and plays an important part in controlling cell proliferation and is a crucial factor in the Fas/FasL apoptosis pathway.

Polo-like kinase 1,a new therapeutic target in hepatocellular carcinoma

AIM： To investigate the role of polo-like kinase 1 （PLK1） as a therapeutic target for hepatocellular carcinoma （HCC）. METHODS： PLK1 gene expression was evaluated in HCC tissue and HCC cell lines. Gene knockdown with short-interfering RNA （siRNA） was used to study PLK1 gene and protein expression using real-time reverse transcription polymerase chain reaction （RT-PCR） and Western blotting, and cell proliferation using 3-（4,5-dim ethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2（4-sulf ophenyl）-2H-tetrazolium （MTS） and bromodeoxyuridine （BrdU） assays. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end label- ing （TUNEL） assay, and caspase-inhibition assay. Huh-7 cells were transplanted into nude mice and co-cultured with PLK1 siRNA or control siRNA, and tumor progres- sion was compared with controls. RESULTS： RT-PCR showed that PLK1 was overexpre- ssed 12-fold in tumor samples compared with controls, and also was overexpressed in Huh-7 cells, siRNA against PLK1 showed a reduction in PLK1 gene and protein expression of up to 96% in Huh-7 cells, and a reduction in cell proliferation by 68% and 92% in MTS and BrdU cell proliferation assays, respectively. There was a 3-fold increase in apoptosis events, and TUNEL staining and caspase-3 assays suggested that this was caspase-independent. The pan-caspase inhibitor Z-VAD-FMK was unable to rescue the apoptotic cells. Immnofluorescence co-localized endonuclease-G to fragmented chromosomes, implicating it in apoptosis. Huh-7 cells transplanted subcutaneously into nude mice showed tumor regression in siPLKl-treated mice, but not in controls. CONCLUSION： Knockdown of PLK1 overexpression in HCC was shown to be a potential therapeutic target, leading to apoptosis through the endonuclease-G path- way.

15-PGDH is reduced and induces apoptosis and cell cycle arrest in gastric carcinoma

AIM: To investigate the expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in human gastric cancer and it's mechanism in apoptosis and cell cycle arrest. METHODS: Expression of 15-PGDH mRNA and protein was examined by immunohistochemistry, immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting in tissue from human gastric cancer, gastric precancerous state (gastric polyps and atrophic gastritis), normal stomach, and gastric cancer cell lines. The relationship between gastric cancer, gastric precancerous state and 15-PGDH expression was determined. The association between expression of 15-PGDH and various clinicopathological parameters in gastric cancer was evaluated. Human gastric cancer cell line SGC-7901 was transfected with 15-PGDH expression plasmids. The effect of 15-PGDH on the cell cycle was examined by flow cytometry. The effect of 15-PGDH on apoptosis was examined by transmission electron microscopy, flow cytometry and transferasemediated nick end labeling (TUNEL) assay. Expression of cell cycle (p21, p27, p16 and p53) and apoptosis (Survivin, BCL-2, BCL-XL, BAK and BAX) genes was analyzed by RT-PCR. RESULTS: Expression of 15-PGDH mRNA and protein in human gastric cancer tissues was significantly lower than in normal gastric tissues (P < 0.01). Expression in human gastric cancer cell lines MKN-28 and MKN-45 was reduced, and absent in SGC-7901 cells (P < 0.05). Reduction of 15-PGDH expression was also found in precancerous tissues, such as gastric polyps and atrophic gastritis (P < 0.01). There was a significant difference in expression of 15-PGDH among various gastric cancer pathological types (P < 0.05), with or without distant metastasis (P < 0.05) and different TNM stage (P < 0.01). Flow cytometry demonstrated a significant increase in apoptotic cells in SGC-7901 cells transfected with pcDNA3/15-PGDH plasmid for 24 and 48 h (P < 0.01), and an increased fraction of sub-G1 phase after transfection (P < 0.05). TUNEL assay showed an increased Apoptotic Index in cells overexpressing 15-PGDH (P < 0.01). After transfection, expression of proapoptotic genes, such as BAK (P < 0.05), BAX and p53 (P < 0.01), was increased. Expression of antiapoptotic genes was decreased, such as Survivin, BCL-2 and BCL-XL (P < 0.01). Expression of cyclin-dependent kinase inhibitors p21 and p16 (P < 0.01) was significantly upregulated in cells overexpressing 15-PGDH. CONCLUSION: Reduction of 15-PGDH is associated with carcinogenesis and development of gastric carcinoma. 15-PGDH induces apoptosis and cell cycle arrest in SGC-7901 cells.

Na^+/K^+-ATPase α-subunit in swimming crab Portunus trituberculatus: molecular cloning, characterization, and expression under low salinity stress

Na^＋/K^＋-ATPases are membrane-associated enzymes responsible for the active transport of Na^＋ and K^＋ ions across cell membranes, generating chemical and electrical gradients. These enzymes＇ α-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na^＋/K^＋-ATPase α-subunit eDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction （RT-PCR） and rapid amplification of eDNA end methods. Analysis of the nucleotide sequence revealed that the eDNA had a full-length of 3 833 base pairs （bp）, with an open reading frame of 3 120 bp, 5＇ untranslated region （UTR） of 317 bp, and 3＇ UTR of 396 bp. The sequence encoded a 1 039 amino acid protein with a predicted molecular weight of 115.57 kDa and with estimated pI of 5.21. It was predicted here to possess all expected features of Na^＋/K^＋-ATPase members, including eight transmembrane domains, putative ATP-binding site, and phosphorylation site. Comparison of arnino acid sequences showed that the P. tritubereulatus α-subunit possessed an overall identity of 75%-99% to that of other organisms. Phylogenetic analysis revealed that this α-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this α-subunit＇s transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that α-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, α-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab＇s Na^＋/K^＋-ATPase α-subunit was potentially involved in the observed acute response to low salinity stress.

Cell proliferation of esophageal squamous epithelium in erosive and non-erosive reflux disease

AIM： To elucidate cell proliferation in erosive reflux disease （ERD） and non-erosive reflux disease （NERD）, we evaluated markers in squamous epithelial cells.METHODS： Thirty-four consecutive patients with gas- troesophageal-reflux-disease-related symptoms （21 NERD and 13 ERD） were evaluated for the enrolment into the study. All patients underwent 24-h pH moni- toring, standard endoscopy, and biopsy for histological evaluation. The expression of cyclins D and A was eval- uated by real-time reverse transcription polymerase chain reaction （RT-PCR） from isolated epithelial cells. In all samples, analysis of the isolated cell population revealed the presence of epithelial cells only.RESULTS： Real-time RT-PCR showed that, in patientswith ERD, the relative expression of cyclin D1 mRNA in esophageal epithelium was strongly decreased in comparison with NERD patients. The mean value of relative expression of cyclin D1 mRNA in NERD patients was 3.44 ± 1.9, whereas in ERD patients, it was 1.32 ± 0.87 （P = 0.011）. Real-time RT-PCR showed that, in patients with ERD, relative expression of cyclin A mRNA in esophageal epithelium was decreased in comparison with that in NERD patients （2.31 ± 2.87 vs 0.66 ± 1.11）. The mean bromodeoxyuridine labeling index in the NERD patients was 5.42% ± 1.68%, whereas in ERD patients, it was 4.3% ± 1.59%.

Differential expression of Rab27A/B correlates with clinical outcome in hepatocellular carcinoma

AIM:To investigate the association of Rab27A and Rab27B expression with clinicopathological characteristics and prognosis of hepatocellular carcinoma(HCC).METHODS:We used reverse transcription polymerase chain reaction(RT-PCR),real-time PCR,and Western blotting to detect Rab27A and Rab27B mRNA and protein expression in 5 human HCC lines and the immortalized hepatic HL-7702 cell line.We further examined 148 primary HCC samples matched with adjacent normal tissue and 80 non-HCC specimens by immunohistochemistry to evaluate the correlation of Rab27A and Rab27B expression with clinicopathological features and prognosis.RESULTS:Our data showed that Rab27A and Rab27Bwere differentially expressed in cell lines and primary HCC tumors.Rab27A mRNA and protein were detected in 67%(4/6)of human cell lines and 80%(4/5)of HCC cell lines,while Rab27B was found in 50%(3/6)of human lines and 40%(2/5)of HCC lines.Rab27A expression was higher in primary HCC(46.2%,66/143)than in matched adjacent tissue(24.3%,33/136,P<0.001),whereas immunopositivity for Rab27B was lower in primary HCC(57.4%,81/141)than in matched adjacent tissue(87.5%,119/136,P<0.001).Analysis of clinicopathological characteristics of 148 HCC specimens revealed significant correlations between Rab27A and Rab27B expression and tumor tumor-node-metastasis(TNM)classification(P=0.046 and P=0.027,respectively),and between strong Rab27A expression and tumor differentiation grade(P=0.008).Survival analyses revealed that patients with Rab27A+or Rab27B+tumors had significantly reduced overall survival compared with that of patients with Rab27A-or Rab27B-tumors(P= 0.015 and P=0.005,respectively).Risk analyses revealed that Rab27B+and TNMⅢ-Ⅳwere independent poor prognosis factors associated with a 3.36-and 3.37fold higher relative risk of death,respectively.CONCLUSION:Rab27A and Rab27B expression were closely correlated with tumor progression and can be valuable prognostic indicators for HCC patients.

Notch2 regulates matrix metallopeptidase 9 via PI3K/AKT signaling in human gastric carcinoma cell MKN-45

AIM：To clarify the role of activated Notch2 in the invasiveness of gastric cancer.METHODS：To investigate the invasiveness of silencing Notch2 gene expression,we established a Notch2small interfering RNA（siRNA） transfected cell line using the MKN-45 gastric cancer cell line.After the successful transfection confirmed by real-time reverse transcription-polymerase chain reaction（RT-PCR） and Western blotting,migration and invasion assays were employed to evaluate the aggressiveness of the gastric cancer.RT-PCR and Western blottings were employed to confirm the down-regulation of Notch2 and to evaluate the expression of epithelial mesenchymal transition-related gene matrix metallopeptidase 9（MMP9）,Akt,p-Akt.To confirm the relationship between PI3KAkt and MMP9,the PI3K inhibitor LY294002 was used to treat MKN-45 cells.RESULTS：Notch2 expression was dramatically decreased after Notch2 siRNA transfection（100.00% ± 9.74% vs 11.61% ± 3.85%,P 〈 0.01 by qRT-PCR）.There was also a marked reduction of Notch target gene Hes1（100.00% ± 4.74% vs 61.61% ± 3.58%,P 〈 0.05） at the mRNA,indicating an inhibition of Notch signaling.Inhibition of Notch signaling was also confirmed by the marked reduction of Notch2 intracellular domain at the protein levels（100.00% ± 9.74% vs 65.61% ± 7.58%,P 〈 0.05）.Down-regulation of Notch2 by siRNA enhanced tumor cell invasion（100.00% ± 21.64% vs 162.22% ± 16.84%,P 〈 0.05） and expression of MMP9（1.56 fold,P 〈 0.05）,and activated the pro-MMP9 protein to its active form（1.48 fold,P 〈 0.05）.There was no significant difference in the protein levels of Akt between the two groups（100.00% ± 10.87% vs 96.61% ± 7.33%,P 〉 0.05）,while down-regulation of Notch2 elevated p-Akt expression（100.00% ± 9.87% vs 154.61% ± 13.10%,P 〈 0.05）.Furthermore,p-Akt and MMP9 was down-regulated in response to the inhibitor LY294002（p-Akt 100.00% ± 8.87% vs 58.27% ± 5.01%,P 〈 0.05;MMP9 100.00% ± 9.17% vs 50.03% ± 4.88%,P 〈 0.05）.CONCLUSION：Notch2 may negatively regulate cell invasion by inhibiting the PI3K-Akt signaling pathway

Aression of TLR_9 in human pulmonary adenocarcinoma cell line A549

Objective:Being considered as a bridge between the innate immunity and acquired immunity,Toll-like receptors(TRLs) are very important innate immunity moleculars.Recent researchs on the innate immunity have focused on the relationship between TLRs and human tumor.This paper investgated the expression and significance of TLR9 in human pulmonary adenocarcinoma cell(A549 cell) and human bronchial epithelial cell(HBE cell).Methods:After culturing A549 cell and HBE cell in vitro,the expression of TLR9 mRNA and protein in both cells were detected by immunocytochemistry,Real-time Quantitative Reverse Transcriptase-Polymerase Chain Reaction(Real-time Quantitative PCR) and Western blot,respectively.Results:By immunocytochemistry staining,TLR9 was mainly expressed in both cells' cell membrane and endochylema as brown-yellow material.It showed that the expressions of TLR9 mRNA and protein in A549 cell were stronger than those in HBE cell(P < 0.01).Conclusion:The results suggest TLR9 might cause the progression of human pulmonary adenocarcinoma,and the mechanism needs to be further investgatied.

Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay

A quantitative real time reverse-transcription polymerase chain reaction （qRT-PCR） assay with specific primers recommended by the World Health Organization （WHO） has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin （HA） genes of the pandemic HlN1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic HIN1/2009 virus infection.

Effect of Maternal Antibodies on the Pathogenesis of Avian Reovirus Infections in Broiler Chickens Using Real-Time Reverse Transcriptase Polymerase Chain Reaction

The effect of maternal antibodies on the pathogenesis of avian reovirus （ARV） was studied in commercial and specific pathogen free broilers （SPF） using a real-time reverse transcriptase （RT）-polymerase chain reaction （PCR） assay, along with the incidence and severity of morbidity, mortality, and gross lesions. ARV RNA was detected in cloacal swabs in both bird groups from the first day throughout the 21 days experiment. Commercial broiler chickens, which had high maternal antibodies against ARV, showed minimum clinical signs, gross lesions, and lower numbers of birds with viral RNA excretion, whereas specific pathogen free （SPF） broiler chickens, which did not have antibody against ARVs, had 30% mortality, more severe signs, and higher numbers of birds excreting viral RNA. The highest peak of SPF birds excreting viral RNA occurred during the time of maximum mortality. The protective effect of maternal antibody on ARV pathogenesis in broiler chickens correlated with the detection of ARV RNA using the real-time RT-PCR.

宁夏及其周边地区博尔纳病病毒感染的分子流行病学研究

目的探讨博尔纳病病毒(BDV)在宁夏及其周边地区的流行状况。方法采用巢式逆转录酶聚合酶链反应结合荧光定量PCR检测了52例病毒性脑炎(VE)患者和32名健康人外周血液(PBMC)和脑脊液有核细胞(CSFMC)、53例抑郁症(DD)患者和360只绵羊PBMC中BDV p24基因片段,对阳性产物进行基因序列测定、同源性和氨基酸顺序分析;并绘制系统发生树,分析BDV感染的分子流行病学特点。结果BDV p24基因片段阳性率在VE患者CSFMC为11．54％、DD患者PBMC 11．32％明显高于健康人0％(P<0．05);绵羊的PBMC为7．78％与健康人对比差异无统计学意义(P>0．05);系统发生树图形表明,绵羊PBMC目的基因片段与人类VE和DD核苷酸序列亲缘关系最近,来源于一个分支,并与德国H1766病毒株核苷酸序列亲缘关系最近,其变异性较小。结论宁夏及其周边地区部分VE、DD可能与BDV感染有关,健康绵羊存在BDV的自然感染,其流行可能具有一定的地域局限性;人类VE、DD的BDV感染可能存在潜在动物源性,有待于进一步研究。

Screening for differentially expressed genes in Anoectochilus roxburghii (Orchidaceae) during symbiosis with the mycorrhizal fungus Epulorhiza sp.

Mycorrhizal fungi promote the growth and development of plants, including medicinal plants. The mechanisms by which this growth promotion occurs are of theoretical interest and practical importance to agriculture. Here, an endophytic fungus （AR-18） was isolated from roots of the orchid Anoectochilus roxburghii growing in the wild, and identified as Epulorhiza sp. Tissue-cultured seedlings of A. roxburghii were inoculated with AR-l 8 and co-cultured for 60 d. Endotrophic mycorrhiza formed and the growth of A. roxburghii was markedly promoted by the fungus. To identify genes in A. roxburghii that were differentially expressed during the symbiosis with AR-18, we used the differential display reverse transcription polymerase chain reac- tion （DDRT-PCR） method to compare the transcriptomes between seedlings inoculated with the fungus and control seedlings. We amplified 52 DDRT-PCR bands using 15 primer combinations of three anchor primers and five arbitrary primers, and nine bands were re-amplified by double primers. Reverse Northern blot analyses were used to further screen the bands. Five clones were up-regulated in the symbiotic interaction, including genes encoding a uracil phosphoribosyltransferase （UPRTs; EC 2.4.2.9） and a hypothetical protein. One gene encoding an amino acid transmembrane transporter was down-regulated, and one gene encoding a tRNA-Lys （trnK） and a maturase K （matK） pseudogene were expressed only in the inoculated seedlings. The possible roles of the above genes, especially the UPRTs and marK genes, are discussed in relation to the fungal interaction. This study is the first of its type in A. roxburghii.

Characterization of cancer stem-like cells in the side population cells of human gastric cancer cell line MKN-45

Objective:Side population(SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer.Many kinds of cell lines and tissues have demonstrated the presence of SP cells,including several gastric cancer cell lines.This study is aimed to identify the cancer stem-like cells in the SP of gastric cancer cell line MKN-45.Methods:We used fluorescence activated cell sorting(FACS) to sort SP cells in the human gastric carcinoma cell line MKN-45(cells labeled with Hoechst 33342) and then characterized the cancer stem-like properties of SP cells.Results:This study found that the SP cells had higher clone formation efficiency than major population(MP) cells.Five stemness-related gene expression profiles,including OCT-4,SOX-2,NANOG,CD44,and adenosine triphosphate(ATP)-binding cassette transporters gene ABCG2,were tested in SP and MP cells using quantitative real-time reverse transcription polymerase chain reaction(RT-PCR).Western blot was used to show the difference of protein expression between SP and MP cells.Both results show that there was significantly higher protein expression in SP cells than in MP cells.When inoculated into non-obese diabetic/severe combined immunodeficiency(NOD/SCID) mice,SP cells show higher tumorigenesis tendency than MP cells.Conclusions:These results indicate that SP cells possess cancer stem cell properties and prove that SP cells from MKN-45 are gastric cancer stem-like cells.

Tetrahydroxy stilbene glucoside improved the behavioral disorders of APP695V717l transgenic mice by inhibiting the expression of Beclin-1 and LC3-Ⅱ

OBJECTIVE:To observe the effect of tetrahydroxy stilbene glucoside(TSG) on the behavior of APP695V717 I transgenic mouse models and the expression of autophagy-associated proteins Beclin-1and LC3-Ⅱ.METHODS:Forty 3-month-old APP695V717 I transgenic mice were randomized equally into either a TSG group(n = 20) or a model group(n = 20).A normal control group consisted of C57BL/6J mice of the same age and background(n = 20).The TSG group received TSG intragastric administration for1 month.Behavior was measured using the Morris water maze and the Y-maze tests.Changes in protein expression and mRNA of autophagy-associated Beclin-1 and LC3-Ⅱ in mice hippocampus were detected by western blot and Reverse Transcription-Polymerase Chain Reaction(RT-PCR) analyses.RESULTS:The number of electric-stimulus escapes significantly increased and the Morris water maze test showed prolonged escape latency,greater swimming distance,less time taken to cross the exact former platform location in the model group,and increased mRNA and protein expressions of Beclin-1 and LC3-Ⅱ compared with the control group(P < 0.05).The TSG group showed a decrease in the number of electric-stimulus escapes,shorter escape latency and swimming distance,greater time taken to cross the exact former platform location,and decreased mRNA and protein expressions of Beclin-1 and LC3-Ⅱ compared with the model group(P< 0.05).CONCLUSION:These results indicate that tetrahydroxy stilbene glucoside can decrease expressions of Beclin-1 and LC3-Ⅱ in the autophagy pathway.It can attenuate injury to endoplasmic reticulum functions caused by Ab neurotoxicity,improving learning,memorizing,and spatial orientation behavior in mice.

Substance P stimulates differentiation of mice osteoblast through up-regulating Osterix expression

Objective： To investigate the molecular pathway of substance P （SP） to induce osteoblastic differentiation. Methods： Mesenchymal stem cells were isolated and cultured. The cultures were divided into four groups with Group A （control group） cultured without any factors, Group B cultured with SP, Group C cultured with SP and SP receptor neurokinin-1 （NK1） antagonist, and Group D cultured with SP NK1 antagonist respectively to induce osteoblastic cells differentiation. Osterix gene expression was detected by reverse transcription-polymerase chain reaction （RT-PCR） for three times after 1-2 weeks of cultivation and the results were analyzed by one-way analysis of variance （ANOVA）. Results： The log phase of bone marrow stromal cells appeared at 4-6 days. ALP staining revealed that the majority of cells, more than 95%, were positive and small bluepurple granules were found in the cytoplasm. And Group B, treated with SP, showed a higher level of ALP activity than the other three groups. Meanwhile, RT-PCR found that Osterix expression in Group B was obviously up-regulated, compared with other groups. But Osterix expression in Group D had no remarkable differences, compared with the controls. Conclusions： SP can up-regulate Osterix gene expression to stimulate differentiation of mesenchymal stem cells into osteoblastic cells at the final stage. The regulatory effect of SP on Osterix expression was dependant on SP NK1 receptors.

Effect of quercetin on secretion and gene expression of leptin in breast cancer

OBJECTIVE: To investigate the possible inhibitory action of pure quercetin on secretion and gene expression of leptin in the T47 D breast cancer cell line.METHODS: In this experimental study, T47 D cells were cultured in monolayers in RPMI 1640. IC50 was determined by MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay after 24 h treatment at different concentrations of quercetin.The levels of leptin gene expression were measured by reverse-transcription real-time polymerase chain reaction(PCR). Secreted leptin was measured in the supernatant of cells by an enzyme-linked immuno sorbent assay.RESULTS: Analysis of MTT assay data showed that quercetin has a cytotoxic effect on T47 D breast cancer cells with 40 μM IC50 after 24 h exposure. Dataanalysis of real-time PCR showed that with increases in quercetin concentration, a decreasing trend was seen in m RNA levels of leptin of treated cells compared with the control cells(P < 0.05). Also,measurement of secreted leptin in the culture media showed a similar decreasing trend in the amount of leptin protein in the treated cells compared with the control cells(P < 0.05).CONCLUSION: Quercetin significantly inhibits the growth of T47 D cells through inhibition of leptin secretion and gene expression in T47 D breast cancer cells. Therefore, it might be an alternative approach to breast cancer therapy through leptin targeting.