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       【摘要】 
       目的研究硒化壳聚糖对急性早幼粒白血病细胞株NB4增殖和凋亡的影响，探讨这种影响与PML-RARα融合蛋白表达的关系。方法应用MTT法检测硒化壳聚糖对细胞增殖的影响，应用AO/EB荧光染色法共聚焦显微镜下观察诱导凋亡作用，应用Western blot的方法检测细胞PML-RARα融合蛋白含量的变化。结果硒化壳聚糖可对体外培养的NB4细胞产生剂量和时间依赖性的抑制作用，50 mg/L 至 100 mg/L硒化壳聚糖作用NB4细胞24 h可诱导细胞出现明显的凋亡现象，经硒化壳聚糖处理24 h后，NB4细胞内PML-RARα融合蛋白含量明显减少。结论硒化壳聚糖可通过下调PML-RARα融合蛋白含量对NB4细胞产生抑制增殖和诱导凋亡作用。
       【关键词】  硒化壳聚糖; NB4细胞；增殖 ；凋亡； PML-RARα融合蛋白
       Chitosan ［β(1,4)-2-amino-2-deoxy-D-glucose］, an almost nontoxic bio-polysaccharide in nature, which has several pharmacological effects including antitumor, antimicrobial and antioxidant properties［1,2］ by report, is often used as drug preparation materials due to its excellent biocompatibility［3］. Selenium, belonging to the same family in the periodic table of elements with arsenic which is an antitumor element (Arsenic trioxide has long been used as an anti-leukemia drug in clinical practice)has been found to increase apoptosis of Acute Promyelocytic Leukemia (APL) cell lines NB4 cell through PI3K/Akt pathway［4］ . But inorganic selenium can not be easily absorbed by cancer cells , have narrower range of activity and toxicity, and have relatively small lethal dose, and so on. Therefore, we synthesized a kind of organic compound containing selenium, i. e. Chitosan selenium ［5］, via chitosan and selenious acid in acid solution, using the mixed metal ions as the catalyst, which can effectively overcome the shortcoming of inorganic selenium, should also have anti-leukemia effect and have much smaller side effects than inorganic selenium and arsenide［6］.
       
       In this work we study the effect of Selenium chitosan on proliferation and apoptosis of human APL  cell lines NB4 cell in vitro．And the ability of Selenium chitosan to modulate PML-RARα fusion proteins that might contribute to NB4 cell survival was investigated．
       1  Materials and methods
       1.1  Drugs and antibodiesSelenium chitosan was synthesized by department of pharmaceutical Chemistry， Fujian medical University；Selenium content was 0.4％．Anti- RARα fusion proteins antibodies were purchased from Santa Cruz Biotechnology．Western blot kit was from Promega．
       1.2  Cell culture The human  leukemia cell lines NB4 cell was maintained in RPMI-l640 medium supplemented with 10% (V/V)fetal calf serum，streptomycin100 mg/L，benzylpenicillin 100 kU/Lat 37% in a humidified 5%CO2．After incubation for 24 h, exponentially growing cells(1×109/L)were treated with Selenium chitosan of different concentrations (50,l00,200 mg/L) for 24 h～48 h．MTT was used to determine the proliferative effects of drugs on tumor cells and AO/EB fluoresent staining method was used to observe the inducing apoptosis effect of drugs.
       1.3  Western blot analysisProtein was extracted from Selenium chitosan-treated cells with lysis buffer(Tris-HCl 50 mmol/L，pH 8.0，NaCl 150 mmol/L，dithiothreitol 1 mmol/L, edetic acid 0.5 mmol/L，nonidet P40 0.1％，sodium dodecylsulfate 0.1％ phenylmethylsulfonly fluoride 100 mg/L)supplemented with proteinase inhibitors：aprotinin l mg/L，leupeptin 2 mg/L，and sodium orthovanadate 100 μmol/L．Appropriate protein amounts (20 μg)were subjected to sodium dodecylsulfate-poly-acrylamide gel electrophoresis(SDS-PAGE). After electrophoresis，proteins were transferred to nitrocel-lulose membrane(150 mA;4℃)for 　1.5 h．The blots were blocked in blocking-buffer(1%BSA，Tris-HCl 20 mmo1/L，pH 7.5，NaCl 150 mmol/L，0.05％ Tween- 20)at room temperature for lh and was followed by incubation with primary Abs(anti-RARα proteins l:1 000 dilution)at room temperature for 1h and then with antirrabit peroxidase-conjugated secondary IgG antibodies and developed with substrate,resulting in a visible color reaction on the membrane． Amount of protein band was quantificated by scanning densitometry(Gel-Doc 1000 model, BIO-RAD)．
       1.4  Statistical analysisData were expressed as mean±SD．The difference between treatment and control groups was evaluated by using Student"s  t-test．
       2  Result
       2.1  Effects of Selenium chitosan on NB4 cellsTreatment with Selenium chitosan at different concentrations for 24 h inhibited the proliferation of NB4 cells in a concentration-dependent manner．Selenium chitosan 50 mg/L, 100 mg/L, 200 mg/L for 24h inhibited the proliferation of NB4 cells by 19.8%,36.7% and 59.5％,respectively(Fig 1). An exposure of NB4 cells to 200 mg/L Selenium chitosan for 0h to 48h resulted in a time-dependent inhibition curves，the highest inhibitory rate for NB4 cells was 84.5％(Fig 2). Using AO-EB staining, we could see NB4 cells were in normal structure and stained green in control group(0 mg/L), after treatment with 50mg/L to 200 mg/L selenium chitosan for 24 h, the typical apoptosis were observed such as extensive membrane blebbing, condensation of heterochromatin, cell nuclear fragmentation, apoptosis bodies and so on. Many dead cells were found 24 h later after treatment. (Fig 3).
       2.2  Effects of Selenium chitosan on PML-RARα fusion proteinsTreatment of NB4 cells with Selenium chitosan for 24h resulted in a marked reduction of PML-RARα fusion proteins in a concentration-dependent manner after correction for differences in loading,the abundance of PML-RARα fusion proteins was decreased significantly after treatment with Selenium chitosan from 0 mg/L for 24 h(P＜0.05，P＜0.01)（Fig4）.
       3  Discussion
          
       NB4 cells cultured in vitro could be induced apoptosis by Sodium selenite through oxidative stress reaction［7］. But the effect of selenium chitosan on NB4 cells and its possible  effect mechanism has not been reported so far．It has been known that PML protein exerts a negative effect on cellular growth , transformation and regulation function of transcription activity in normal cells. Therefore it may behave as a tumor suppressor［8］. PML-RARα fusion proteins is present in almost all cases of human Acute Promyelocytic Leukemia． This PML-RARα fusion proteins initiated signaling decreases the ability of a variety of stimuli to inhibit proliferation and induce apoptosis in vitro and because of this fusion protein, the PML oncogenic domain(POD) structure is disrupted ,the activity of PML is disturbed, leading to inhibition of cell differentiation and apoptosis［9］．Therefore， it is a new and attractive therapeutic strategy to target PML-RARα fusion proteins and PML-RARα fusion proteins initiated signaling.
       
       In this study ,Selenium chitosan has been found to inhibit proliferation and induce apoptosis of NB4 cells. And at same time,the results were the first to show that the abundance of PML-RARα fusion proteins were strongly deregulated in Selenium chitosan-treated PML-RARα fusion protein-positive NB4 cells. The PML-RARα fusion protein blot density in control group was 402±92. After treatment with 50,100,200mg/L selenium chitosan for 24h, the value of blot density was 297±74,126±53, 88±22,respectively, and the down-regulation rate was 26.1% (P<0.05), 68.6% (P<0.01)and 78.1%(P<0.01) ,respectively.
       
       In general，present study demonstrated that Selenium chitosan inhibited the proliferation and induced apoptosis of NB4 cells through down-regulation of the abundance of PML-RARα fusion proteins．The pharmacological functionsis of Selenium chitosan on NB4 cells is worth of further study.
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