巴西蘑菇多糖冲剂制备工艺研究

采用正交试验法优化了微波辅助提取巴西蘑菇多糖的条件,研究了多糖冲剂制备工艺,并用苯酚-硫酸法对巴西蘑菇多糖冲剂中多糖含量进行了测定.结果表明,以水为溶剂,巴西蘑菇多糖最佳提取条件为：提取时间30min,微波功率为80%（全功率为800w）,液料比为20：1.巴西蘑菇多糖冲剂配方为：巴西蘑菇粗多糖：可溶性淀粉：甜蜜素=1：4：0.04,冲剂中多糖含量为7.624%.     

国产巴西蘑菇多糖诱导细胞刺激因子基因表达的研究

应用RT-PCR技术,研究了国产巴西蘑菇多糖(ABPS)对几种细胞刺激因子(CSF)基因表达的影响。研究结果表明,人粒．单祖细胞克隆刺激因子(GM-CSF)和白细胞介素2(IL-2)扩增片段分别在255bp和500bp处出现明显的条带,而小鼠骨髓基质细胞表达的GM-CSF则在369bp处出现一条带。揭示国产巴西蘑菇多糖有诱导人脐血单个核细胞和小鼠骨髓基质细胞表达GM—CSF和IL-2的作用,其浓度范围为1～6μg／mL,最佳浓度为1μg／mL,从而证实了国产巴西蘑菇多糖促进粒一单祖细胞生成和调节动物机体免疫功能的分子机理。     

姬松茸多糖抗肿瘤的实验研究

应用细胞培养技术 ,观察了姬松茸多糖对K5 6 2和HL - 6 0细胞系的影响。研究结果表明 ,实验组活细胞数、集落形成明显减少 ,荧光显微镜下所见凋亡细胞显著增加。提示姬松茸多糖具有抗肿瘤效果。为了探讨姬松茸多糖抗肿瘤的机理 ,本实验还应用反义寡聚脱氧核糖核酸技术 ,进一步观察了姬松茸多糖与人端粒酶反义核酸协同诱导HL - 6 0细胞系凋亡的作用 ,实验结果表明 ,姬松茸多糖与反义寡核苷酸联合使用 ,可促进HL- 6 0细胞凋亡。     

灵芝硒多糖的制备及其分离纯化

灵芝（Ganoderma lucidum）菌丝体通过生物转化作用富集无机硒，分别经水、碱水浸提，醇析，去蛋白，脱色后，得到灵芝硒多糖粗提物。再经DEAE—Cellulose柱层析分离纯化后，收集得到的各组分在高效液相色谱上进行进一步纯化，从而得到了10个不同的硒多糖组分。以K562细胞进行的抗癌试验结果显示，SeGLP-2B-1，SeGLP-2B-2和SeGLP-2B-3三种纯化硒多糖均有抗癌活性，其中SeGLP-2B-1对K562细胞的抑制率最高，达61．58％。     

东方百合‘索邦’诱导小鳞茎发生过程中的细胞学观察

 以东方百合‘索邦’鳞片为试材,研究鳞片扦插诱导小鳞茎形成的过程及其鳞片内部糖和蛋白质的分布特点。经石蜡切片PAS和汞溴酚兰组织化学染色观察发现：（1）小鳞茎起源于近轴基部8～14层薄壁细胞,为内起源;形态发生过程依次经过了启动、生长锥形成、叶原基形成和小鳞茎形成;小鳞茎的形成与分生细胞团所在位置有关。（2）小鳞茎发生过程中多糖主要以颗粒状存在于分裂旺盛的分生组织和维管组织周围,多糖含量高时有利于诱导形成维管组织;蛋白质主要集中于细胞核周围;多糖和蛋白质在分生组织及其周围细胞之间存在着浓度梯度,由顶端分生组织细胞到周围的薄壁细胞中,蛋白质含量由高到低,多糖含量则由低到高。     

羧甲基茯苓多糖对小鼠免疫功能的影响

伏内试验证明，羧甲基茯苓多糖能明显增强荷瘤小鼠腹腔巨噬细胞的蚕噬功能，小鼠脾抗体分泌细胞数（PFC）以及特异抗原结合细胞数（SRFC）和小鼠对牛血清白蛋白（BSA）诱导的迟发型超敏反应（DTH），明显促进小鼠脾T细胞分泌白细胞介素－2（IL－2）。体内外试验均证明，羧甲基茯苓多糖能明显促进小鼠腹腔巨噬细胞分泌肿瘤坏死因子－α（TNF－α）。体外试验证明，羧甲基茯苓多糖对小鼠脾混合淋巴细胞的增殖有促进作用。     

不同提取方法对槐耳提取物的粗多糖含量及K562细胞抑制作用的影响

采用普通水提法、高温高压水提法、冷碱法、热碱法对槐耳(Trametes robiniophila)子实体进行提取,考察了水溶性提取物中粗多糖含量、粗多糖实际得率和对K562细胞的抑制率。碱提法水溶性提取物的得率高于水提法,其中热碱法提取物的得率最高为29.65%,但其粗多糖含量最低,冷碱法粗多糖的实际得率最高为6.93%。四种提取物对K562细胞均有抑制作用,热碱法提取物浓度为2mg/mL作用于K562细胞72h后,抑制率高达92.28%。     

桦褐孔菌多糖诱导人卵巢癌细胞凋亡的研究

探讨Bcl-xl、p53基因在桦褐孔菌多糖诱导人卵巢癌SKOV3细胞凋亡中的作用。不同浓度桦褐孔菌多糖作用于人卵巢癌SKOV3细胞,荧光显微镜观察人卵巢癌SKOV3细胞凋亡作用,RT-PCR法检测Bcl-xl、p53基因表达量的变化。桦褐孔菌多糖能诱导人卵巢癌细胞凋亡,并且上调p53和降低Bcl-xl表达。桦褐孔菌多糖促进SKOV3凋亡,其机制可能与激活p53、抑制Bcl-xl表达有关。     

秋栽姬松茸出菇期管理技术

姬松茸又名巴西蘑菇,属担子菌亚门,层菌纲,伞菌目,蘑菇科,蘑菇属,原产巴西、秘鲁、美国等地.它所含的甘露聚糖,对肿瘤、癌症具有明显的疗效,是一种兼具美食、保健作用的珍稀食用菌.本试验研究的是姬松茸秋季栽培技术,试验从7月中旬堆料发酵,8月中旬接种,8月下旬进人大棚栽培,于9月中旬第1次采收,12月中旬采收结束.     

茯苓多糖对非小细胞肺癌NCI-H460细胞的抑制作用

为评价茯苓(Poria cocos)多糖对非小细胞肺癌NCI-H460细胞的抑制作用,用CCK-8法测定茯苓多糖对细胞增殖的抑制率,用DAPI染色法、AOEB双荧光染色法分析茯苓多糖对细胞凋亡的影响,用细胞划伤愈合实验测定茯苓多糖对细胞迁移能力的影响,用蛋白免疫印迹实验检测茯苓多糖对细胞凋亡、迁移相关蛋白表达的影响。结果表明：茯苓多糖具有抑制NCI-H460细胞增殖、集落形成和迁移的能力,可诱导NCI-H460细胞凋亡;500、1 000μg·mL^-1的茯苓多糖可使Bax和P53蛋白表达量升高,Bcl-2和MMP-2蛋白表达量降低。     

姬松茸发酵全液多糖提取及其抗肿瘤活性

液体发酵姬松茸 ,从发酵全液中提粗多糖 ,研究其体内抗肿瘤活性。用 0 2g/kg、 0 4g/kg、 0 8g/kg三个剂量组对荷S180 小鼠进行灌胃 ,结果发现 ,姬松茸发酵全液粗多糖得率为 1 78g/ 10 0mL、多糖含量为 4 6 4 %。低中高三个剂量组对肿瘤的抑制率分别为 2 5 6 1%、 4 7 5 6 %和 5 4 34%。同时能提高荷瘤小鼠的胸腺指数和脾指数。姬松茸发酵全液多糖具有明显的抗肿瘤活性。     

Preparation of recombinant human soluble TRAIL and its inducing effect on apoptosis of tumor cells by TRAIL combined with bortezomib

AIM: To construct a prokaryotic expression plasmid to produce recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and to verify the biological activity of TRAIL. METHODS: The prokaryotic expression plasmid pET-28a (+)-TRAIL_114-281 was constructed. Human soluble TRAIL was obtained through optimized inducing protein expression and purification conditions. The biological activity of TRAIL was verified by CCK-8 assay. The apoptosis-inducing effect of TRAIL alone and/or in combination with proteasome inhibitor bortezomib (Velcade, PS-341) on the tumor cell lines H460(TRAIL-sensitive) and K562(TRAIL-resistance) for 24 h was determined. The apoptotic rates of the cells were analyzed by flow cytometry with Annexin V-FITC/PI staining. The activities of caspase-8, -9 and -3 in the cells were detected by colorimetric method. The protein expression of Bax, Bcl-2 and cFLIP was measured by Western blot. The expression of DR4 and DR5 in the H460 cells and K562 cells after treated with bortezomib for 24 h was detected by flow cytometry. RESULTS: The recombinant human soluble TRAIL protein with stable bioactivity was successfully acquired, which induced apoptosis in H460 cells and K562 cells. After treatment with different concentrations of TRAIL, the apoptotic rate of H460 cells was significantly increased with the increase in the concentration of TRAIL (P<0.05), but the apoptotic rate of K562 cells was not affected by the increasing TRAIL concentration. Apoptotic rate in combination group was obviously higher than that in single group (P<0.05). In the process of apoptosis, the activities of caspase-8, -9 and -3 in H460 cells and K562 cells were both increased. The expression of Bcl-2 and cFLIP in treatment groups (especially the combination group) was decreased compared with control group. No significant change of the Bax expression level was observed. The expression of DR4 and DR5 in the H460 cells and K562 cells was significantly up-regulated after treated with bortezomib (P<0.05). CONCLUSION: Bortezomib combined with recombinant human soluble TRAIL synergistically induces apoptosis in tumor cell lines H460 and K562 through initiating intrinsic apoptotic pathways by up-regulating death receptors DR4 and DR5, and reducing the expression of antiapoptotic proteins Bcl-2 and cFLIP.     

Inhibition of K562 cell growth by antisense drug targeting with VEGF mRNA in vitro

AIM: To investigate inhibition of K562 cell growth by antisense drug targeted VEGF mRNA. METHODS: X7, 20-mer antisense sequences were selected, synthesized and modified with phosphorothioate. The drug was transfected into K562 cells in the present of lipofection. Cell growth was assayed by trypan blue dye exclusion assay and MTT. The level of VEGF protein in the media was determined by ELISA. The morphology of apoptotic cells were observed by Giemsa staining, and the propotion of apoptotic cells was detected by flow cytometry. RESULTS: The antisense drug inhibited growth of K562 and downregulated expression of VEGF protein significantly, compared with Scrambed control group and showed dose-dependent relation. Signs of apoptosis of K562 cells were not observed. CONCLUSION: Inhibition of K562 cell proliferation, but not cells apoptosis induction is the mechanism of inhibing growth of K562 cells by antisense drug targeted VEGF mRNA. At same time, VEGF has function of promoting K562 cell proliferation, and VEGF mRNA may be a new target attached by drugs.     

Knockdown of RALA by siRNA inhibits cell proliferation and induces apoptosis in human leukemic K562 cells

AIM: To investigate the effect of siRNA-induced knockdown of v-ral simian leukemia viral oncogene homolog A(RALA) on proliferation and apoptosis of chronic myelogenous leukemia(CML) K562 cells. METHODS: The chemically synthesized siRNA targeting to RALA gene was transfected into K562 cells using Lipofectamine^TM 2000. The proliferation and viability of K562 cells were detected by MTT assay and trypan blue dye exclusion. The expression levels of RALA mRNA and protein were determined by quantitative real-time PCR and Western blotting,respectively. The cell apoptosis was analyzed using flow cytometry by double staining with annexin V and propidium iodide, and the apoptotic morphological changes were detected by Hoechst 33258 staining. RESULTS: RALA siRNA significantly down-regulated RALA mRNA and protein expression in K562 cells(P<0.05). The proliferation of K562 cells in RALA siRNA group was inhibited compared with control group(P<0.05). The apoptotic rate was much higher in RALA siRNA group than that in negative control group(P<0.05). The apoptotic morphological changes were observed in the nuclei of K562 cells transfected with RALA siRNA. CONCLUSION: The siRNA-mediated knockdown of RALA results in inhibition of proliferation and induction of apoptosis in K562 cells, indicating that RALA might be used as a potential therapeutic target in chronic myelogenous leukemia.     

姬松茸多糖对人体健康的改善作用及机制

为了验证低分子姬松茸菌对人体健康的改善作用。在探讨姬松茸多糖的药用价值的基础上,对低分子姬松茸多糖的提取和纯化方法进行了试验,通过单因素试验与正交试验确定了提取最佳条件;对低分子姬松茸多糖抗肝癌HepG-2细胞的活性进行了试验,结果显示,低分子姬松茸多糖对肝癌HepG-2细胞的抵制率要高于普通多糖,但纯化后的低分子姬松茸多糖抑制率反而会降低,证明了多糖中的某些蛋白质具有一定的抗癌效果,抗癌活性与多糖的纯化和含量没有相关性。     

Effect of decitabine on resistance of K562/A02 cells to adriamycin

AIM: To investigate the effect of decitabine (DAC) on the resistance of human chronic myeloid leukemia cell line K562/A02 to adriamycin (ADR), and to explore the possible mechanism. METHODS: The K562/A02 cell line and its parental cell line K562 were treated with different concentrations of ADR or DAC alone, or in combination. The cytotoxic effects of these 2 agents were determined by CCK-8 assay. The degree of DNA methylation was evaluated by Sequenom MassARRAY system and colorimetric method. The cell cycle distribution and the apoptotic rate were determined by flow cytometry. RESULTS: K562/A02 cells were more significantly resistant to ADR than K562 cells.The half maximal inhibitory concentration of ADR for 24 h of the K562/A02 cells was about 50 times higher than that of the K562 cells. To DAC, in the concentration range of 0.5~8 μmol/L, K562/A02 cells were more sensitive than K562 cells. As compared with the same concentrations (4.31 μmol/L and 17.24 μmol/L) of ADR alone, the combination with 1 μmol/L DAC significantly improved the sensitivity of K562/A02 cells to ADR. Both DAC and ADR affected the cell cycle progression and apoptotic rate of K562/A02 cells. DAC (1 μmol/L) treatment mainly showed S phase arrest and increased early apoptotic rate for 24 h, and G₂/M phase arrest and increased late apoptosis and necrosis for 48 h in a time-related manner. ADR treatment showed G₂/M phase arrest and increased late apoptosis and necrosis in a concentration-dependent manner. In combination with 1 μmol/L DAC, the effect of ADR on the cell cycle distribution was further enhanced, showing more obvious G₂/M phase arrest, but no significant difference of the apoptotic rate was observed. The degree of methylation in the genome had no significant difference between the 2 cells, and it before and after DAC treatment had no significant change. CONCLUSION: DAC enhances the sensitivity of K562/A02 cells to ADR, showing drug resistance-reversing potential. The mechanism may be related to regulating cell cycle progression and promoting apoptosis and necrosis of K562/A02 cells.     

Reverse effects of saikoside on multidrug resistance of human leukemic cell line K562/ADM in vitro

AIM: To study the reverse effects of saikoside (SS) on the multidrug resistance (MDR) of human leukemic cell line K562/ADM and to investigate the related mechanism. METHODS: K562 cells and K562/ADM cells in the culture were treated with SS at the concentrations of 1~100 mg/L. The inhibitory rate of the cell proliferation was measured by MTT assay. Non-cytotoxic dose of SS was determined. K562/ADM cells were treated with SS at non-cytotoxic doses of 1.25, 2.5 and 5.0 mg/L with different concentrations of adriamycin (ADM,0.05~100 mg/L). The 50% inhibitory concentration (IC₅₀) and the reversal index in all groups were determined. The cell morphology was observed after treated with SS+ADM. The effects of SS on ADM accumulation in K562/ADM cells, the cell cycle profile and apoptosis were examined by flow cytometry. RESULTS: The inhibitory rates were significantly increased in a dose-dependent manner when the cells were treated with different doses of SS (1~100 mg/L). The available reversal concentration of SS was 5.0 mg/L and the reversal index was 21.5 folds for K562/ADM cells. After treated with SS+ADM, the number of tumor cells was decreased and apoptotic cells were increased in a dose-response relationship. ADM accumulation in K562/ADM cells treated with SS was significantly higher than that in control cells (P<0.05). SS may significantly enhanced the apoptosis of K562/ADM cells treated with ADM (P<0.05). K562/ADM cells treated with SS were blocked in the stage of G₀/G₁. CONCLUSION: SS has effect on proliferation inhibition and MDR reversal in K562/ADM cell line. The reversal mechanisms of SS may be due to increasing the accumulation of chemo therapeutics in the cell, inducing the cell apoptosis and arresting the cells in G₀/G₁ phase.     

Ethyl acetate extract of Pleione bulbocodioides (Franch.) Rolfe induces apoptosis of human leukemia K562 and HL-60 cells through intrinsic mitochondrial apoptosis pathway

AIM:To investigate the effects of ethyl acetate (EtOAc) extract of Pleione bulbocodioides (Franch.) Rolfe on proliferation and apoptosis of human leukemia K562 and HL-60 cells and the possible apoptosis pathway. METHODS:Human leukemia cell lines were treated with EtOAc extract of Pleione bulbocodioides at different concentrations. XTT method was used to evaluate the viability of K562 cells and HL-60 cells. The cell growth inhibition was calculated by Trypan blue exclusion test. The percentage of apoptotic cells was determined by flow cytometry, and 4',6-diamidino-2-phenylindole (DAPI) was used to observe morphological changes of the cells. The cell cycle was observed by propidium iodide (PI) staining. The protein expression of Bcl-2, Bax, cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, cytochrome C and apoptosis-inducing factor (AIF) wase determined by Western blot. RESULTS:The cell viability and proliferation were inhibited by EtOAc extract of Pleione bulbocodioides with IC₅₀ of (42.14±2.54) mg/L for HL-60 cells and (51.28±3.12) mg/L for K562 cells at 24 h. The results of Annexin V-FITC/PI and DAPI staining showed that EtOAc extract of Pleione bulbocodioides induced cell apoptosis in a dose-dependent manner. The apoptotic rate was increased compared with control group (P<0.05). The G₂ phase increased with typical cell apoptosis-induced morphological changes. The levels of pro-apoptotic proteins Bax, cleaved PARP and cleaved caspase-3 were increased, while Bcl-2 was down-regulated (P<0.05). Cytochrome C and AIF in cytosol, characteristic proteins of intrinsic mitochondrial apoptosis pathway, also increased with the concentration of EtOAc extract of Pleione bulbocodioides increasing (P<0.05). CONCLUSION:EtOAc extract of Pleione bulbocodioides significantly inhibits cell proliferation and induces cell apoptosis in human leukemia cell lines HL-60 and K562 through intrinsic mitochondrial apoptosis pathway.