簇生沿丝伞液体发酵培养及其发酵物体内抗肿瘤活性研究

采用中心组合旋转设计法确定了簇生沿丝伞液体深层培养菌丝体的最佳方案。采用H22荷瘤小鼠进行体内抗肿瘤试验,对簇生沿丝伞发酵物的抗肿瘤活性进行了探究。结果表明,当培养基配方为麦芽糖59.27g/L,蛋白胨:酵母粉=1:1为8.04g/L,磷酸二氢钾:硫酸镁:硫酸铵=1:1:1为2.27g/L时,生物量达到最高,为15.06g/L。簇生沿丝伞发酵物的抑瘤率与剂量存在明显的量效关系,200mg/kg时,抑瘤率达最高,为54.71%,且白细胞数和IL-2的含量与对照组相比均显著增高。     

簇生沿丝伞的生物学特性

文中以对采自山东省的子实体进行组织分离获取的菌株作为试验材料,对其生物学特性进行研究,对温度、pH、碳源、氮源4个因素进行单因素试验。温度组选取15,20,25,30,35℃5个梯度,pH组选取pH5,6,7,8这4个梯度,碳源组选取葡萄糖、蔗糖、麦芽糖、可溶性淀粉、乳糖、果糖6种,氮源组选取酵母膏、蛋白胨、硫酸铵、牛肉膏、黄豆粉、亚硝酸钠6种,进行单因素试验。根据菌丝在培养皿中的日均生长速度和生长势综合比较,分别选取4个因素中最优的3个,温度组选取20,25,30℃;pH组选取pH 5,6,7;碳源组选取葡萄糖、蔗糖和麦芽糖;氮源组选取蛋白胨、牛肉膏、硫酸铵。然后通过4因素3水平的正交试验,结果表明:4种因子对簇生沿丝伞的影响程度为温度>氮源>碳源>pH,各因素之间呈极显著差异,最终得出该菌菌丝生长的最佳培养条件为温度20℃,pH 5,碳源为蔗糖,氮源为牛肉膏。     

玉米芯抗肿瘤活性研究

采用MTT法检测玉米芯水提物对人结肠癌细胞株SW480、SW620、DLD1以及正常人肝细胞HL-7702的增殖活力的影响;倒置显微镜下观察细胞形态的变化;DAPI染色法检测该水提物对不同细胞株细胞核凋亡的影响.结果表明:不同浓度(1、2、4、6 mg/mL)的玉米芯水提物对人结肠癌细胞株均有抑制能力,且呈明显的量效依赖关系.用该水提物处理正常细胞HL-7702,在低浓度时没有抑制作用,在高浓度时略有抑制作用.该水提物对各细胞株作用24h,半数抑制浓度(IC50)分别为2、3、4、6 mg/mL.显微镜观察表明:经该玉米芯水提物处理后的结肠癌细胞,形态发生明显的改变,细胞形状变圆,贴壁细胞数量明显减少.DAPI染色结果显示:多数细胞核表现出染色质的不均一性,有典型的“梅花”状凋亡小体出现.     

桑黄粗多糖除蛋白及抗肿瘤活性

旨在研究Sevag法除桑黄粗多糖中蛋白的最佳工艺,并通过噻唑蓝(MTT)细胞比色法测定除蛋白前后桑黄多糖的抗肿瘤活性。在单因素试验Sevag用量、氯仿与正丁醇体积比、萃取次数、震荡时间的基础上,设计正交试验,经过方差分析,确定Sevag法除桑黄粗多糖蛋白的最佳方案;并且对除蛋白后多糖与粗多糖进行体外抗肿瘤实验。Sevag法除桑黄粗多糖蛋白的最佳工艺:Sevag用量50 mL、V(氯仿)/V(正丁醇)=3、最佳萃取次数6次、最佳振荡时间20 min,在此条件下,多糖的得率为64.8%。体外细胞试验表明除蛋白后多糖与粗多糖对肿瘤细胞EC109和SiHa的抑制率没有明显差异。     

罗汉果醇抗肿瘤活性及其作用机制研究

罗汉果醇是罗汉果皂苷的苷元,有研究报道罗汉果皂苷V具有防癌抑癌作用。该研究采用噻唑蓝实验( MTT法)检测罗汉果醇对不同肿瘤细胞增殖的抑制情况,以及不同浓度的罗汉果醇对CNE1细胞的增殖抑制率;应用细胞克隆形成实验进一步验证罗汉果醇对CNE1细胞增殖的抑制作用;采用Annexin V/PI 双染法检测罗汉果醇对CNE1细胞凋亡的影响;以实时定量PCR技术检测罗汉果醇对CNE1细胞中Caspase-3、Sur-vivin、Bax和Bcl-2基因的mRNA 表达水平的影响。结果表明：罗汉果醇能显著抑制DU145、HepG2、A549、CNE1、CNE2细胞的增殖,其中对CNE1细胞增殖的抑制作用最为显著,并呈剂量依赖性,其半数抑制浓度IC50为(81.48±4.73)μmol·L-1;通过对CNE1细胞进一步的克隆形成实验,也验证了这一点;Annexin V/PI 双染法可见随着浓度的增加,凋亡比例增加;实时定量PCR技术检测显示罗汉果醇处理后,促凋亡基因Caspase-3、Bax的表达增加,抗凋亡基因Survivin、Bcl-2的表达减少。因此,罗汉果醇可能是通过促进Caspase-3、Bax等促凋亡基因和抑制Survivin、Bcl-2等抗凋亡基因的表达,来诱导肿瘤细胞凋亡,进而发挥抗肿瘤活性。     

川芎粗多糖抗氧化和细胞毒活性研究

通过川芎粗多糖(LCP)对DPPH离子、超氧阴离子、.OH、总抗氧化能力、螯合能力、还原性和脂质过氧化能力的抗氧化效果和对肝癌细胞HepG2的抑制作用研究。结果显示:LCP具有较强的还原力,对超氧阴离子自由基、脂质过氧化产物有良好的抑制作用,其对肝癌细胞HepG2也具有一定的抑制作用。LCP有望作为天然抗氧化剂及功能性食品得到开发与应用。     

两个改造后的肿瘤抑素抗肿瘤活性肽活性研究

为了研究改造后的肿瘤抑素2个抗肿瘤活性肽的作用机制,明确其不同的抗肿瘤活性,采用基因工程技术原理,人工合成肿瘤抑素中185～203位氨基酸所对应的19肽和T7肽(74～98位氨基酸)基础上改造的21肽碱基序列,将其与融合蛋白表达载体pTYB2重组后转化到大肠杆菌BL21(DE3)中进行诱导表达,用几丁质亲和层析柱一步纯化,直接获得19肽和21肽,利用MTT法、细胞生长曲线、TUNEL法、流式细胞仪早期细胞凋亡检测和细胞周期检测,小鼠H22腹水型转移型肝癌实体瘤抑瘤实验并结合组织病理学切片,来研究19肽和21肽单独应用或联合应用对肿瘤细胞和内皮细胞生长和凋亡的影响以及对体内肿瘤的抑制情况.体内外实验表明:获得的19肽抗肿瘤活性以直接作用肿瘤细胞为主,也有抑制新生血管生成的作用.基因重组21肽抗肿瘤作用是通过抑制肿瘤组织新生血管生成实现的.19肽、21肽联合应用对肿瘤细胞、内皮细胞生长抑制和促凋亡作用明显增强,抗肿瘤活性大大提高.联合用药弥补了单独用药不足,产生协同抗肿瘤作用,可能会成为今后肿瘤治疗的一个主要方向.     

鳞盖肉齿菌的抗肿瘤细胞增殖活性及抗肿瘤细胞增殖机制的研究

为了从鳞盖肉齿菌(Sarcodon scabrosuskarst)的二氯甲烷提取物中分离纯化得到Sarcodonin G,对Sarc-odonin G进行抗肿瘤细胞增殖活性及其抗肿瘤细胞增殖机制的研究。本文利用MTT实验法测定Sarcodonin G对HeLa细胞株增殖的抑制率,检测其抗肿瘤细胞增殖的效果;利用流式细胞术检测Sarcodonin G对HeLa细胞的凋亡的影响;利用电镜技术观察Sarcodonin G对HeLa细胞形态学改变。结果发现,Sarcodonin G对体外培养的HeLa细胞的增殖具有明显抑制作用,其IC50为7.19μmol/L,并有较好的剂量依赖关系;流式细胞学检查发现Sarcodonin G处理后的HeLa细胞出现凋亡现象;超微结构发现Sarcodonin G处理后的HeLa细胞的胞核染色质浓缩,边集,核固缩及形成新月体,线粒体肿胀,空泡样变,胞浆出现大量空泡等凋亡的形态学改变。结果表明,鳞盖肉齿菌的纯化物Sarcodonin G能抑制体外培养的HeLa细胞的增殖,并初步推测Sarcodonin G有可能是通过诱导HeLa细胞的凋亡来实现其抗肿瘤增殖作用的。     

四种多孔菌子实体粗多糖抗肿瘤活性的比较研究

对粗毛纤孔菌、椭圆嗜蓝孢孔菌、火木层孔菌、木蹄层孔菌4种多孔菌子实体粗多糖成分的含量及其体内抗肿瘤活性进行了比较研究。结果表明粗毛纤孔菌子实体中的粗多糖含量为4.1%,高于其他3种多孔菌;同时,4种多孔菌子实体粗多糖对H22荷瘤小鼠均显示出一定的抗肿瘤活性,除木蹄层孔菌外,其他3种多孔菌给药剂量为500mg/mL和1 000mg/mL时抑瘤率均大于40%,其中粗毛纤孔菌子实体粗多糖抑瘤率最高,低剂量组（500mg/kg）为58.12%,高剂量组（1 000mg/kg）为47.75%。     

姬松茸多糖抑制肿瘤细胞增殖的研究

姬松茸(Agaricus blazei)又名巴西蘑菇,是一种药食兼用的珍稀食用菌,具有显著的防癌、抗癌活性^[1]。本实验用水提法提取姬松茸菌丝体(深层发酵制备)及子实体多糖,测定姬松茸多糖体外对小鼠s-180癌细胞及人结肠癌细胞抑制杀伤作用。     

沉默Bmi-1基因表达稳定细胞株的建立及其生物学特性的研究

B细胞特异性莫洛尼鼠白血病病毒插入位点1(B-cell-specific moloney murine leukemia virus insertionsite 1,Bmi-1)基因是多梳基因家族成员,参与细胞增殖调控.研究发现Bmi-1基因可能参与肿瘤的形成,可能成为肿瘤潜在的治疗靶点.用RNA干扰(RNA interference,RNAi)沉默Bmi-1基因表达观察其对乳腺癌细胞株MCF-7侵袭和转移等生物学特性的影响,以探讨Bmi-1在乳腺癌发生发展中的作用.PT67细胞包装质粒后产生的逆转录病毒感染MCF-7细胞,嘌呤霉素筛选建立稳定细胞株,稳定抑制Bmi-1的细胞株命名为MCF-7/Bmi-1si.通过RT-PCR和Western blot分别从mRNA和蛋白水平检测Bmi-1的表达量;平板克隆形成实验检测细胞克隆形成能力;Transwell侵袭小室模型检测细胞体外侵袭和转移能力.MCF-7/Bmi-1si组与MCF-7和MCF-7/GFPsi组相比,Bmi-1 mRNA和蛋白表达量明显减少,克隆形成数及形成率也明显减少(P<0.05).侵袭和转移实验表明:与MCF-7和MCF-7/GFPsi组相比,MCF-7/Bmi-1si组细胞在Transwell侵袭小室中24 h穿膜细胞数明显减少(P<0.05).结果表明沉默Bmi-1基因表达稳定细胞株构建成功,Bmi-1基因表达的沉默能显著降低MCF-7细胞的体外增殖及侵袭转移能力.     

Methionine cytotoxicity in the human breast cancer cell line MCF-7

Summary Among the first nutrients to be linked to cancer were methyl group containing nutrients including methionine. Methionine and its metabolic derivatives are essential components in several indispensable biological reactions including protein synthesis, polyamine synthesis, and many transmethylation reactions. The purpose of this study was to determine the extent to which methionine excess affects the proliferation and gene expression of the human breast cancer cell line MCF-7. Cells were first grown in control medium; the medium was then replaced with either control or methionine-supplemented treatment media. We found that 5 and 10 g/L methionine significantly suppressed cell growth on day 1, and no further growth was detected after 3 d of treatment. Cell, proliferation in the methionine treated group was significantly lower than that of the control group. Northern analysis revealed that expression of p53 in methionine-treated MCF-7 cells was approximately 70% lower than that of control cells. p53 is a key cell cycle regulatory, protein that has been implicated in tumorigenesis and cancer progression. Alteration of the p53 tumor suppressor gene is the most common genetic change found in a wide variety of malignancies, including cancer. This study shows that excess methionine (5 g/L) inhibited proliferation of MCF-7 breast cancer cells, and down regulation of p53 is correlated with this inhibition. These findings may aid in the development of nutritional strategies for breast cancer therapy.     

Synergistic proliferative action of insulin-like growth factor I and 17 beta-estradiol in MCF-7S breast tumor cells.

We have analyzed the mechanism by which the combination of insulin-like growth factor I (IGF-I) and 17 beta-estradiol (E2) induces cell cycle progression in MCF-7S cells. This cell line differs from many other breast cancer-derived cell lines in that E2 (1 nM) does not induce cell cycle progression, whereas the combination of submitogenic concentrations of IGF-I (2 ng/ml) and E2 does. We find that addition of IGF-I to MCF-7S cells leads to a dose-dependent activation of the IGF type I receptor and of the MAP kinase and PI3-kinase signaling pathways. No synergy of IGF-I and E2 was detected in the activation of these signaling cascades. In terms of cell cycle-related molecules, we find that IGF-I dose-dependently raises cyclin D1 levels in serum-starved cells. Subsequent activation of cyclin E/CDK2, hyperphosphorylation of pRb, and DNA synthesis are only induced by mitogenic concentrations of IGF-I (> or =20 ng/ml). Treatment of the cells with E2 also results in the induction of cyclin D1, but in the absence of IGF-I the cells remain arrested in G1 phase. We conclude that in MCF-7S cells, the synergistic action of E2 and IGF-I derives from the ability of both hormones to induce cyclin D1 expression. The action of IGF-I is required in these cells to induce activity of the cyclin D1/CDK4 complex, which triggers progression through the cell cycle.     

人乳腺癌多药耐药细胞系MCF-7/MDRa的建立及其生物学特性的初步研究

实验以MCF-7细胞株为亲本细胞,采用阿霉素(ADM)低浓度持续加量诱导法建立了多药耐药的人乳腺癌细胞系MCF-7/MDRa,并对其耐药谱、动力学周期分布、表型变化、药物的蓄积量等生物学特性进行了初步分析评价。结果表明,MCF-7/MDRa细胞较亲本细胞的ADM半数致死浓度(IC50)高500倍,撤药培养150天后耐药指数仍维持在200倍以上,并对多种化疗药物产生交叉耐药性;耐药细胞分化程度低于同步传代的MCF-7细胞,细胞倍增时间与亲本细胞接近,S期细胞显著增加,G1期细胞减少;随着撤药时间的延长,细胞的增殖速度加快;耐药细胞P-gP、LRP和GSTπ的表达水平较亲本细胞有显著增加,ER阳性表达丢失;在稳定生长的撤药培养6天的细胞中仍有ADM蓄积。建立的MCF-7/MDRa模型具有多药耐药细胞的基本生物学特性,可用于肿瘤多药耐药机制的研究。     

积雪草甙对乳腺癌MCF-7细胞凋亡及VEGF、bFGF蛋白表达水平的影响

目的:探讨积雪草甙对乳腺癌MCF-7细胞凋亡及VEGF、bFGF蛋白表达水平的影响。方法:选取人乳腺癌细胞MCF-7细胞系进行体外培养后,根据是否进行积雪草甙干预而分为两组,应用积雪草苷进行干预后,HE染色后用光学显微镜法观察细胞形态学变化,干预后的24h、48h以及72h时,应用TUNEL技术对对细胞凋亡情况进行检测,同时应用免疫荧光法检测血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)的表达。结果:(1)与对照组相比较,积雪草甙干预的乳腺癌MCF-7细胞出现空泡、胞质外溢以及胞核皱缩等细胞凋亡现象,大量癌MCF-7细胞发生破碎死亡;(2)TUNEL技术法检测结果证实积雪草甙能够提高人乳腺癌MCF-7细胞凋亡率,与对照组比较差异具有统计学意义(P0.05),且呈时间依赖性;(3)积雪草甙干预的MCF-7细胞VEGF阳性表达和bFGF阳性表达显著低于对照组,差异具有统计学意义(P0.05),积雪草甙的抑制作用且呈时间依赖性。结论:积雪草甙不仅能够促进乳腺癌MCF-7细胞凋亡,而且能够降低VEGF和bFGF表达。     

Role of mitochondria in tamoxifen-induced rapid death of MCF-7 breast cancer cells

Tamoxifen (Tam) is widely used in chemotherapy of estrogen receptor-positive breast cancer. It inhibits proliferation and induces apoptosis of breast cancer cells by estrogen receptor-dependent modulation of gene expression, but recent reports have shown that Tam (especially at pharmacological concentrations) has also rapid nongenomic effects. Here we studied the mechanisms by which Tam exerts rapid effects on breast cancer cell viability. In serum-free medium 5–7 μM Tam induced death of MCF-7 and MDA-MB-231 cells in a time-dependent manner in less than 60 min. This was associated with release of mitochondrial cytochrome c, a decrease of mitochondrial membrane potential and an increase in production of reactive oxygen species (ROS). This suggests that disruption of mitochondrial function has a primary role in the acute death response of the cells. Accordingly, bongkrekic acid, an inhibitor of mitochondrial permeability transition, was able to protect MCF-7 cells against Tam. Rapid cell death induction by Tam was not associated with immediate activation of caspase-9 or cleavage of poly (ADP-ribose) polymerase. It was not blocked by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone either. Diphenylene ionodium (DPI), an inhibitor of NADPH oxidase, was able to prevent Tam-induced cell death but not cytochrome c release, which suggests that ROS act distal to cytochrome c. The pure antiestrogen ICI 182780 (1 μM) could partly oppose the effect of Tam in estrogen receptor positive MCF-7 cells, but not in estrogen receptor negative MDA-MB-231 cells. Pre-culturing MCF-7 cells in the absence of 17β-estradiol (E₂) or in the presence of a low Tam concentration (1 μM) made the cells even more susceptible to rapid death induction by 5 or 7 μM Tam. This effect was associated with decreased levels of the anti-apoptotic proteins Bcl-X_L and Bcl-2. In conclusion, our results demonstrate induction of a rapid mitochondrial cell death program in breast cancer cells at pharmacological concentrations of Tam, which are achievable in tumor tissue of Tam-treated breast cancer patients. These mechanisms may contribute to the ability of Tam therapy to induce death of breast cancer cells.     

川芎嗪逆转人乳腺癌MCF-7/ADM细胞对阿霉素的耐药性

目的 探讨川芎嗪（tetramethylpyrazine,TMP）逆转人乳腺癌MCF-7/ADM细胞对阿霉素（ADM）的耐药性.方法 MTT法测定细胞的药敏性,荧光分光光度法检测细胞内阿霉素浓度的变化,流式细胞术检测耐药细胞凋亡百分率的变化.结果 非细胞毒性剂量（320 mg/L）及低毒剂量（1250 mg/L）川芎嗪均能显著降低MCF-7/ADM的IC50（P＜0.01）,逆转倍数分别为2.13倍和2.82倍;均能显著增加耐药细胞内ADM的浓度（P＜0.01）.320 mg/L川芎嗪能显著增加耐药细胞的凋亡百分率（P＜0.01）.结论 川芎嗪具有部分逆转人乳腺癌MCF-7/ADM细胞对阿霉素的耐药性,其逆转机制与增加细胞内ADM浓度有关.