重组人可溶性凋亡素-2配体在Pichia系统中的表达

目的 :在甲醇营养型酵母 (Pichia)表达系统中表达重组人可溶性肿瘤坏死因子相关的凋亡诱导配体 (TRAIL ,凋亡素 - 2配体 )分子。 方法 :人可溶性 TRAIL编码基因片段插入 p IC 3.5酵母表达载体 ,氯化锂转化酵母 GS115株 ,甲醇诱导表达 5 d,SDS- PAGE和 Western- blotting确认表达 ,L 92 9细胞鉴定活性。结果 :发现在酵母细胞中重组表达的 TRAIL在 SDS-PAGE上占总蛋白的 5 0 %以上 ,用抗人 TRAIL多抗可以确认表达了 TRAIL重组分子 ,杀伤肿瘤细胞的比活性较原核表达后复性的 TRAIL有明显提高 ,并能诱导几株肿瘤细胞 DNA片段化 ,说明 TRAIL可能已正确折叠并形成活性必需的高级结构。结论 :在 Pichia系统中正确表达了人可溶性 TRAIL分子。

凋亡素-2配体对胰腺癌细胞PC3和Panc1作用研究

目的 了解凋亡素 2配体 (又称肿瘤坏死因子相关凋亡诱导配体 ,TRAIL)对人胰腺癌细胞株Panc1、PC3凋亡作用的影响。方法 采用MTT法、琼脂糖凝胶电泳以及流式细胞仪方法检测TRAIL对Panc1和PC3体外增殖的影响。结果 TRAIL可诱导胰腺癌细胞Panc1和PC3的凋亡 ,并呈时间和浓度依赖性 ,PC3的凋亡率明显高于Panc1。结论 TRAIL可以诱导胰腺癌细胞的凋亡 ,胰腺癌细胞系对TRAIL的敏感性有差异 ,TRAIL可能为胰腺癌的生物学治疗提供一种新途径。

凋亡素-2配体联合三氧化二砷对急性髓系白血病细胞生长和凋亡的影响

目的:探讨凋亡素-2配体(Apo2L)联合三氧化二砷(As2O3)诱导急性髓系白血病(AML)细胞凋亡的作用。方法:分离25例AML患者的骨髓单个核细胞,每例分为4组进行观察:空白对照组、Apo2L组、As2O3组、Apo2L+ As2O3组,共同培养12 h、24 h和48 h。通过MTT比色法测定细胞生长抑制率及流式细胞仪检测细胞凋亡。结果:Apo2L和As2O3可协同降低AML细胞活力,诱导细胞凋亡,其作用随培养时间延长及药物浓度增加而增强。结论:Apo2L与三氧化二砷具有协同作用,能高效杀灭白血病细胞。Apo2L是一种有望应用于临床的新型生物制剂。

Bcl-2 down modulation in WEHI-3B/CTRES cells resistant to Cholera Toxin(CT)-induced apoptosis

The very different effects of Cholera Toxin （CT） on cell growth and proliferation may depend on the type of ganglioside receptors in cell membranes and different signal transduction mechanisms triggered, but other functions related to the drug resistance mechanisms can not be excluded. The effect of CT treatment on the ＂in vitro＂ clonogenicity, the Population Doubling Time （PDT）, apoptosis, PKA activation and Bax and Bcl-2 expression was evaluated in WEHI-3B cell line and its CT-resistant subclone （WEHI-3B/CTRES）. In WEHI-3B parental cells the dramatic accumulation of cAMP induced by CT correlated well with PKA activation, increased PDT value, inhibition of clonogenicity and apoptosis. H-89 treatment inhibited PKA activation by CT but did not protect the cells from apoptosis and growth inhibition. In WEHI-3B/CTRES no significant CT-dependent accumulation of cAMP occurred with any increase of PKA activity and PDT. In CT resistant cells （WEHI-3B/CTRES）, Bcl-2 expression was down regulated by both CT or drug treatment （eg, ciprofloxacin, CPX） although these cells were protected from CT-dependent apoptosis but not from drug-induced apoptosis. Differently from other cell models described, down regulation of Bcl-2 is proved to be independent on cAMP accumulation and PKA activation. Our observations support the implication of cAMP dependent kinase （PKA） in the inhibition of WEHI-3B cells growth and suggest that, in WEHI-3B/CTRES, Bcl-2 expression could be modulated by CT in the absence of cAMP accumulation. Also in consideration of many contradictory data reported in literature, our cell models （of one sensitive parental cell strain and two clones with different uncrossed specific resistance to CT and CPX） provides a new and interesting tool for better investigating the relationship between the CT signal transduction mechanisms and Bcl-2 expression and function.

Effects of extracellular iron concentration on calcium absorption and relationship between Ca^(2+) and cell apoptosis in Caco-2 cells

AIM: To determine the method of growing small intestinal epithelial cells in short-term primary culture and to investigate the effect of extracellular iron concentration ([Fe3+]) on calcium absorption and the relationship between the rising intracellular calcium concentration ([Ca2+]i) and cell apoptosis in human intestinal epithelial Caco-2 cells. METHODS: Primary culture was used for growing small intestinal epithelial cells. [Ca2+]i was detected by a confocal laser scanning microscope. The changes in [Ca2+]i were represented by fluorescence intensity (FI). The apoptosis was evaluated by flow cytometry. RESULTS: Isolation of epithelial cells and preservation of its three-dimensional integrity were achieved using the digestion technique of a mixture of collagenase Ⅺ and dispase Ⅰ. Purification of the epithelial cells was facilitated by using a simple differential sedimentation method. The results showed that proliferation of normal gut epithelium in vitro was initially dependent upon the maintenance of structural integrity of the tissue. If 0.25% trypsin was used for digestion, the cells were severely damaged and very difficult to stick to the Petri dish for growing. The Fe3+ chelating agent desferrioxamine (100, 200 and 300 μmol/L) increased the FI of Caco-2 cells from 27.50±13.18 (control, n = 150) to 35.71±13.99 (n = 150, P<0.01), 72.19±35.40 (n = 150, P<0.01) and 211.34±29.03 (n = 150,P<0.01) in a concentration-dependent manner. There was a significant decrease in the FI of Caco-2 cells treated by ferric ammonium citrate (FAC, a Fe3+ donor; 10, 50 and 100 μmol/L). The FI value of Caco-2 cells treated by FAC was 185.85±33.77 (n = 150, P<0.01), 122.73±58.47 (n = 150, P<0.01), and 53.29±19.82 (n= 150,P<0.01), respectively, suggesting that calcium absorption was influenced by [Fe3+]. Calcium ionophore A23187(0.1,1.0 and 10 μmol/L) increased the FI of Caco-2 cells from 40.45±13.95 (control, n = 150) to 45.19±21.95 (n = 150, P<0.01), 89.87±43.29 (n = 150, P<0.01) and 104.64±51.07 (n = 150,P<0.01) in a concentration-dependent manner. The positive apoptotic cell number of the Caco-2 cells after being treated with A23187 increased from 0.32% to 0.69%, 0.90% and 1.10%, indicating that the increase in the positive apoptotic cell number was positively correlated with [Ca2+]i. CONCLUSION: Ca2+ absorbability is increased with the decrease of extracellular iron concentration Fe3+ and hindered with the increase of Fe3+ consistence out of them. Furthermore, increase of [Ca2+]i can induce apoptosis in Caco-2 cells.

Correlation between expression of gastrin, somatostatin and cell apoptosis regulation gene bcl-2/bax in large intestine carcinoma

AIM: To explore the correlation between expression of somatostatin (SS), gastrin (GAS) and cell apoptosis regulation gene bcl-2/bax in large intestine carcinoma.METHODS: Sixty-two large intestine cancer tissue samples were randomly and retrospectively selected from patients with large intestine carcinoma. Immunohistochemical staining for bcl-2, bax, GAS, SS was performed according to the standard streptavidin-biotin-peroxidase (S-P) method.According to the semi-quantitative integral evaluation, SS and GAS were divided into three groups as follows. Scores1-3 were defined as the low expression group, 4-8 as the intermediate expression group, 9-16 as the high expression group. Bax and bcl-2 protein expressions in different GAS and SS expression groups of large intestine carcinoma were assessed.RESULTS: The positive expression rate of bax had a prominent difference between SS and GAS high, intermediate and low expression groups (P＜0.05, x2ss = 9.246; P＜0.05,x2GAS = 6.981). The positive expression rate of bax in SS high (80.0%, 8/10) and intermediate (76.5%, 13/17)expression groups was higher than that in low expression group (40.0%, 14/35) (P＜0.05, x2high vs low = 5.242; P＜0.05,x2middle vs low = 6.097). The positive expression rate of bax in GAS high expression group (27.3%, 3/8) was lower than that in low expression group (69.4%, 25/36) (P＜0.05,x2 = 4.594). However, bax expression in GAS intermediate expression group (46.7%, 7/15) was lower than that in low expression group, but not statistically significant. The positive expression rate of bcl-2 had a prominent difference between SS and GAS high, intermediate and low expression groups (P＜0.05, x2ss = 7.178; P＜0.05, x2GAS = 13.831). The positive expression rate of bcl-2 in GAS high (90.9%, 10/11)and intermediate (86.7%, 13/15) expression groups was higher than that in low expression group (44.4%, 16/36)(P＜0.05,x2high vs low = 5.600; P＜0.05, x2 middle vs low = 7.695).However, the positive expression rate of bcl-2 in SS high (40.0%, 4/10) and intermediate (47.1%, 8/9) expression groups was lower than that in low expression group (77.1%, 27/35)(P＜0.05, x2 high vs low = 4.710; P＜0.05, x2 middle vs low = 4.706).There was a significant positive correlation between the integral ratio of GAS to SS and the integral of bcl-2 (P＜0.01,r=0.340). However, there was a negative correlation between the integral ratio of GAS to the SS and bax the integral of (P＜0.05, r = -0.299).CONCLUSION: The regulation and control of gastrin,somatostatin in cell apoptosis of large intestine carcinoma may be directly related to the abnormal expression of bcl-2, bax.

Effects of Geldanamycin on Expression of Bcl-2 in Human Cervical Cancer HeLa Cells

OBJECTIVE Geldanamycin,a natural product of Streptomyces geldanus,binds the heat shock protein 90(Hsp90),a cell chaperone protein that interacts with Bcl-2.In this study,we investigated whether geldanamycin(GA)inhibits proliferation of HeLa cells through induction of apoptosis by decreasing the level of Bcl-2 expression. METHODS HeLa cells,a human cervical cell line,were cultured in vitro and treated with different concentrations of GA(0,0.02,0.2, 2,10μmol/L)for 24 h.or were treated for different lengths of time at a GA concentration of 10μmol/L.Proliferation of the cells was analyzed by an MTT assay,and cell apoptosis was determined by staining the cells with annexin V.In addition,cellular mRNA levels for Bcl-2 and Hsp90 were determined by the semiquantitative polymerase chain reaction(PCR),and the levels of Bcl-2 and Hsp90 protein expression were determined by Western blots. RESULTS Treatment of cells with GA was found to inhibit HeLa cell proliferation in a concentration and time-dependent manner.The inhibition was a result of increased cellular apoptotic levels.Further analyses showed that while the mRNA and protein expression levels of Hsp90 were not affected,GA treatment significantly reduced the level of Bcl-2 mRNA and protein expression in a concentration-dependent manner that correlated with the observed inhibition of cell proliferation. CONCLUSION GA can inhibit proliferation and increase apoptosis of HeLa cells by decreasing the transcription and expression of an anti-apoptotic gene bcl-2,probably through interaction and functional inhibition of Hsp90.