Artesunate negatively controls expression of cyclin D1 and activity of AP-1 by inhibiting the activation of ERK1/2 protein in HSC-T6 cells

AIM: To investigate the effects of artesunate(Art) on the expression of ERK1/2, AP-1 and cyclin D1 in rat hepatic stellate cells (HSCs), and to elucidate the molecular mechanism of Art against hepatic fibrosis. METHODS: HSC-T6 cells were treated with platelet-derived growth factor BB(PDGF-BB) to induce cell proliferation. The cells were divided into control group, PDGF-BB group, PDGF-BB+Art groups (with 6.25 mg稬^-1, 25 mg稬^-1or 50 mg稬^-1 of Art) and PDGF-BB+PD98059 group. The level of collagen type I in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of ERK1/2 and cyclin D1 were measured by RT-PCR. The protein levels of p-ERK1/2 and cyclin D1 in HSC-T6 cells were detected by Western blotting. The activity of AP-1 was analyzed by electrophoretic mobility shift assay. RESULTS: The concentration of collagen type I was significantly higher in PDGF-BB group than that in control group (P<0.05), and decreased in PDGF-BB+Art group and PDGF-BB+PD98059 group in comparison with that in PDGF-BB group (P<0.05, P<0.01). The protein level of ERK1/2 in PDGF-BB+Art group (50 mg稬^-1) was lower than that in PDGF-BB group (P<0.05), and was even lower in PDGF-BB+PD98059 group (P<0.01). The mRNA expression of cyclin D1 in PDGF-BB+Art groups (25 mg稬^-1and 50 mg稬^-1) and PDGF-BB+PD98059 group were significantly lower than that in PDGF-BB group (P<0.05). The protein levels of p-ERK1/2 and cyclinD1 were the highest in PDGF-BB group, and significantly lower in PDGF-BB+Art groups (6.25 mg稬^-1, 25 mg稬^-1 and 50 mg稬^-1) and PDGF-BB+PD98059 group (P<0.05, P<0.01). The AP-1 binding activity in HSC-T6 cells was down-regulated by Art. CONCLUSION: Artesunate inhibits the proliferation of HSC-T6 cells in vitro by inhibiting the activation of ERK1/2, thus down-regulating the activity of AP-1 and expression of cyclin D1.     

Strontium ranelate promotes differentiation of bone marrow mesenchymal stem cells to osteoblasts by increasing expression of bone morphogenetic protein 2

AIM: To explore whether strontium ranelate (Sr) promotes differentiation of rat bone marrow mesenchymal stem cells (BMSCs) to osteoblasts by increasing the expression of bone morphogenetic protein 2 (BMP-2). METHODS: Rat BMSCs were isolated, purified and cultured, then were induced to differentiate into osteoblasts. The cells were treated with different concentrations of Sr or noggin (an inhibitor of BMP-2) according to the experimental purposes. The activity of alkaline phosphatase (ALP) was measured by colorimetry. Mineralized nodules were measured by alizarin red staining. The expression of BMP-2 was detected by Western blotting. RESULTS: Treatment with Sr at concentrations of 0.1 mmol/L to 7 mmol/L for 7 d obviously increased the activity of ALP,and Sr at concentration of 3 mmol/L produced the maximum effect. Exposure of the cells to Sr at concentration of 3 mmol/L for 21 d significantly increased mineralized nodules. Exposure of the cells to Sr at concentrations of 0.1 mmol/L to 7 mmol/L for 7 d markedly increased the expression of BMP-2. Preconditioning with noggin at concentration of 100 μg/L for 2 h not only inhibited Sr-induced expression of BMP-2, but also antagonized the increase in the activity of ALP and mineralization induced by Sr in BMSCs. CONCLUSION: Up-regulation of the expression of BMP-2 may be one of the mechanisms by which Sr promotes differentiation of rat BMSCs to osteoblasts.     

Identification of specific serum biomarkers in gastric adenocarcinoma patients

AIM: To screen the possible serum biomarkers for the diagnosis of gastric adenocarcinoma. METHODS: The surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was used to screen the serum samples from 109 cases of gastric adenocarcinoma and 106 control subjects (60 healthy subjects, 30 patients with chronic superficial gastritis and 16 cases of chronic atrophic gastritis). The differentially-expressed protein peaks were selected and isolated with high-performance liquid chromatography (HPLC), and processed with enzyme before liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS/MS) analysis. The data mining was performed with software Xcalibur program component Bioworks 3.2. RESULTS: Three differentially-expressed protein peaks were selected as potential serum biomarkers of gastric adenocarcinoma patients.The m/z peak at 5 906.5 showed the increase (8.53±4.33 in cancer group, and 0.88±0.31 in control group). The m/z peaks at 6 635.7 and 8 716.3 showed the decrease (6.54±2.44 and 0.93 ± 0.29, respectively, in cancer group and 17.56±4.43 and 2.16±0.98, respectively, in control group, P<0.01). The 3 m/z peaks were identified as fibrinogen α-chain, apolipoprotein A-II and apolipoprotein CI,respectively. The combined use of the 3 biomarkers distinguished the samples in the cancer patients out of the controls at a sensitivity of 93.85% (61/65) and a specificity of 94.34% (50/53). CONCLUSION: The fibrinogen α-chain, apolipoprotein A-II and apolipoprotein CI identified as potential markers for gastric adenocarcinoma show diagnostic values in clinical application.     

Effects of cyclic tensile strain on expression of p38 MAPK and phospho-p38 MAPK in osteoarthritis chondrocytes

AIM: To observe the effect of cyclic tensile strain (CTS) on the expression of p38 MAPK and phospho-p38 MAPK in rabbit osteoarthritis (OA) chondrocytes in vitro. METHODS: The animal model of OA was induced by anterior cruciate ligament transection in New Zealand white rabbits. The animals in all groups were evaluated 10 weeks later. The rabbits in OA group were randomly divided into 3 groups, low CTS (0.5 Hz, sin10%, 6 h/d) group, high CTS (1.0 Hz, sin10%, 6 h/d) group and control group. Both CTS groups were stimulated by a Flexercell-4000 tension system. The expression of p38 MAPK and phospho-p38 MAPK of the chondrocytes was analyzed by RT-PCR and Western blotting at the time points of 24 h, 1 week and 2 weeks. RESULTS: The knee joints of the rabbits in OA group had obvious degeneration of articular cartilage. The expression of p38 MAPK in normal group was significantly lower than that in control group (P<0.01), and the difference between low CTS group and high CTS group 1 week after stimulation (P<0.05) was observed. Meanwhile, significant difference was found between low CTS group and control group 2 weeks after CTS treatment (P<0.01). The expression of phospho-p38 MAPK was decreased at different time points in low CTS group. CONCLUSION: Different cyclic tensile strains lead to different effects on the expression of p38 MAPK and phospho-p38 MAPK in the chondrocytes. p38 MAPK signaling pathway plays an important role in the development of osteoarthritis in chondrocytes.     

Circadian protein Cry2 is a prognostic factor of colorectal cancer and a predictor of chemotherapy

AIM:To assess the association between the expression of Cry2 and the prognosis of colorectal cancer for determining the role of Cry2 in predicting the outcome of chemotherapy. METHODS:Sixteen primary colorectal cancer patients who consecutively underwent neoadjuvant chemotherapy with 5-fluorouracil were enrolled in the present study. The tumor specimens were obtained by colonscopy prior to treatment. The tumor response was evaluated by the response evaluation criteria in solid tumors(RECIST). Accordingly, the patients were divided into 2 groups:complete response(CR)/partial response(PR) and stable disease(SD)/progress disease(PD). Two-tail ² test was applied to analyze the data. In parallel, we assessed 307 patients, who underwent tumor resection between 2001 and 2005. Survival time was calculated from the date of surgery to the date of death or the last follow-up date. Survival curves were obtained by Kaplan-Meier method. Log-rank test was applied for univariate analysis. The Cox proportional hazards regression model was used to identify the independent prognostic factors. RESULTS:The expression of Cry2 was high in CR/PR patients and was low in SD/PD patients, with significant difference(P<0.05). The average survival time of the 84 patients with high expression of Cry2 was 83.458 months, while the average survival time of 223 patients with low expression of Cry2 was 100.10 months, also with significant difference(P<0.05). The patients with low expression of Cry2 lived longer. The multivariate Cox regression analysis indicated that the expression of Cry2 was an independent prognostic factor for colorectal cancer patients(HR was 1.70, 95% CI was 1.09 to 2.66, P<0.05). Multivariate Cox regression showed that high Cry2 expression predicted a worse prognosis(HR was 2.88, 95%CI was 1.03 to 8.06, P<0.05) in colorectal cancer patients with neoadjuvant chemotherapy. CONCLUSION:Expression of Cry2 is an independent prognostic factor for colorectal cancer patients. Lower expression of Cry2 indicates good response to neoadjuvant chemotherapy.     

鸭DCN基因编码区扩增、序列分析及其在大肠杆菌中的表达、蛋白纯化

采用RT-PCR方法从鸭腿肌总RNA中扩增了鸭核心蛋白聚糖基因编码区序列,进行序列分析,并将编码鸭核心蛋白聚糖成熟蛋白的核酸片段连入原核表达载体pET-32a(+)中,加入IPTG诱导其表达后,亲和层析纯化表达的蛋白。采用SDS-PAGE检测,用质谱方法对表达的蛋白进行鉴定。研究成功克隆出鸭核心蛋白聚糖基因编码区序列,序列分析表明,鸭DCN编码区序列由1074个碱基组成,编码357个氨基酸,其中信号肽有16个氨基酸。将鸭DCN蛋白划分为9个结构域(LRR1-9),结合人DCN序列进行序列分析推测结构域中LRR4、5与TGF-β可能存在一定关系。SDS-PAGE检测和质谱鉴定结果表明获得了分子量为54.7ku的鸭DCN融合蛋白。     

消化道上皮细胞中乳酸杆菌黏附配体蛋白含量的变化

利用兔抗鸡乳酸杆菌黏附配体蛋白血清,以不连续活性-PAGE电泳技术和间接ELISA方法,对不同日龄、健康和患球虫病鸡消化道不同部位乳酸杆菌黏附配体蛋白含量进行了检测。结果表明,2日龄鸡嗉囊部位产生乳酸杆菌黏附配体蛋白成分,D450nm值为0.181;1日龄鸡小肠部位产生乳酸杆菌黏附配体蛋白成分,D450nm值为0.168;5日龄乳酸杆菌黏附配体蛋白成分达到稳定,D450nm值分别为0.200和0.123。健康鸡体内嗉囊与小肠部位乳酸杆菌黏附配体蛋白含量比患球虫病鸡明显增多,D450nm值分别为：嗉囊,0.143和0.132;小肠,0.148和0.134。     

山羊痘病毒抗凋亡蛋白基因的表达与细胞凋亡的相关性

以贵州本地分离纯化的山羊痘病毒为试验材料,感染Vero细胞,通过台盼蓝、吖啶橙/溴乙锭荧光染色分析细胞的死亡及凋亡比例;经特异性PCR从山羊痘病毒基因组中分离出山羊痘病毒抗凋亡蛋白样基因,基因长531bp,含有完整的编码框,与绵羊痘病毒抗凋亡蛋白基因的相似性为97%,因此确定为山羊痘病毒抗凋亡蛋白基因。进一步研究山羊痘病毒抗凋亡蛋白基因在Vero细胞中的表达,结果显示,病毒感染Vero细胞24h时没有检测到凋亡现象,山羊痘病毒抗凋亡蛋白基因的mRNA水平最高;感染48h时,Vero细胞的凋亡率上升到13%,山羊痘病毒抗凋亡蛋白基因的表达量随之下降。结果表明,山羊痘病毒抗凋亡蛋白基因的表达量与Vero细胞的凋亡呈负相关,有助于病毒在细胞内的生存和侵染等过程。     

棉籽粕源发酵蛋白质饲料的代谢产物研究

本试验旨在通过液相色谱串联质谱(LC-MS)代谢组学分析平台对不同微生物棉籽粕源发酵蛋白质饲料代谢产物进行研究.采用LC-MS法对对照组、假丝酵母组、酿酒酵母组、复合组的发酵代谢产物进行分析,结合偏最小二乘法-判别分析(PLS-DA)方法对数据进行模式识别和小分子差异代谢产物寻找.结果表明:各试验组代谢产物相对含量与对照组相比差异显著(P＜0.05),且在PLS-DA分析中得到了很好的分离,主要表现在甘露醇、琥珀酸等糖类代谢产物;磷酸胆碱、L-肉碱、甘油磷酸、磷脂酰乙醇胺(PE)和磷脂酰胆碱(PC)等脂类代谢产物;二肽、三肽、甜菜醛、同型半胱氨酸等蛋白质与氨基酸的代谢产物;以及烟酸、阿魏酸、尿嘧啶、乙醇醛等其他途径代谢产物.除与脂类代谢有关的甘油磷酸、PE、PC相对含量显著降低之外(P＜0.05),其他试验组小分子代谢产物相对含量均比对照组显著增加(P＜0.05).由此可见,不同菌种发酵饲料中含有大量糖类、脂类、蛋白质等代谢途径产生的小分子代谢产物,含量因发酵菌种的不同而不同.     

CHD9:一个新的与流感病毒PA蛋白相互作用的宿主因子

PA蛋白是禽流感病毒(AⅣ)聚合酶的组成部分,对AIV的复制具有重要作用.为鉴定家鸭体内与AIV PA蛋白相互作用的宿主蛋白,本研究利用酵母双杂交系统,以表达A/goose/Hubei/65/05(H5N1)AIV PA蛋白的重组质粒为诱饵,筛选家鸭脑组织cDNA文库,鉴定得到一个cDNA片段,经测序分析表明,该cDNA片段编码蛋白为色素域解旋酶DNA结合蛋白9(CHD9).将重组诱饵质粒pDEST32-PA与捕获的鸭cDNA重组质粒共转化MaV203受体菌,进行X-gal显色反应检测,分析结果显示,菌落可以迅速变蓝,并且菌落在SC-Leu-TrpHis+3AT 3种营养缺陷培养基平板上生长良好.证明CHD9与PA蛋白在酵母双杂交系统中具有较强的相互作用.CHD9的初步鉴定为研究AIV在机体中的复制过程奠定了基础.