BRCA1在人乳腺癌细胞MCF-7辐射抗性中作用的初步研究

<正>MCF-7细胞是被广泛用以研究乳腺癌的一株模式细胞,该细胞拥有野生型p53基因,但其辐射敏感性与p53基因表达状态无关,这提示可能存在其他基因参与调节其辐

potential mechanisms involved in resistant phenotype of MCF-7breast carcinoma cells to ionizing radiation induced apoptosis

In the present study, we investigated the mechanisms of apoptosis resistance and the roles of the phosphorylation of BRCA1, p21, the Bax/Bcl-2 protein ratio and cell cycle arrest in IR-induced apoptosis in MCF-7 cells. X-irradiation, in particular at low dose (1 Gy), but not carbon ion irradiation, had a significant antiproliferative effect on the growth of MCF-7 cells. 1 Gy X-irradiation resulted in G1 and G2 phase arrest, but 4 Gy induced a significant G1 block. In contrast, carbon ion irradiation resulted in a significant accumulation in the G2 phase. Concomitant with the phosphorylation of H2AX induced by DNA damage,carbon ion irradiation resulted in an approximately 1.9–2.8-fold increase in the phosphorylation of BRCA1 on serine residue 1524, significantly greater than that detected for X-irradiation. Carbon ion irradiation caused a dramatic increase in p21 expression and drastic decrease in Bax expression compared with X-irradiation. The data implicated that phosphorylation of BRCA1 on serine residue 1524 might,at least partially, induce p21 expression but repress Bax expression. Together, our results suggested that the phosphorylation of BRCA1 at Ser-1524 might contribute to the G2 phase arrest and might be an upstream signal involved in preventing apoptosis signal via upregulation of p21 and downregulation of the Bax/Bcl-2 ratio.

紧密连接分子Symplekin 在肝脏疾病中表达及调控机制的研究

肝细胞癌是世界上多发的肿瘤之一，在中国及东南亚地区尤为多见，其死亡率高且预后差。肝癌具有多种发病原因且伴有多种肿瘤相关基因的分子突变。细胞连接分子（紧密连接、粘着连接、桥粒）在维护细胞极性及上皮细胞屏障方面起着重要作用，其表达异常与恶性肿瘤发生、发展有很大相关性。Symplekin 是新近发现的紧密连接相关分子，紧密连接分子 Symplekin 是多定位与多功能的蛋白，除参与上皮细胞紧密连接的形成外，Symplekin 还参与RNA 3’端腺苷酸化的过程，并且具有调节细胞增殖的作用。我们前期工作发现Symplekin 在癌前病变、恶性病变的肝细胞中明显降低，可能参与肝细胞的恶性转化。研究紧密连接分子Symplekin 在肝脏疾病中表达及调控机制对于阐明肝癌发生的机理及对于肝癌的预防和治疗具有十分重要的意义。多种分子调控机制导致基因表达水平的降低，如：基因启动子区域的超甲基化现象，基因核心启动子区域的碱基缺失，炎症相关因子TNF-alpha 和/或 INF-gamma导致基因表达水平的下降以及microRNAs对于靶基因的下调作用。因此，本研究利用Bisulfite restriction PCR、半定量PCR、q-RT-PCR、Western-blot等方法检测Symplekin在肝硬化、肝癌及多种癌细胞系中表达水平改变，及其在肝癌及肝癌细胞系中表达降低的机理——启动子区域发生 CpG岛的甲基化；启动子区域缺失；细胞因子TNF-alpha 和 IFN-gamma 对Symplekin 表达水平的影响；MicroRNAs在癌细胞系中与Symplekin的相对表达情况。实验结果显示（1）Symplekin 在肝硬化和肝癌组织中mRNA 表达水平呈下降趋势， Symplekin 在癌细胞系如肝癌细胞系（ HepG2 、HuH-7 ）、肺癌细胞系（GLC,Spca-1,Ncih446,801D）、宫颈癌细胞系（Hela）、乳腺癌细胞系（Mcf-7）中表达均下降。（2）利用细胞因子TNF-alpha、INF-gamma 同时处理HepG2 细胞系，Symplekin mRNA、蛋白均表达下降。（3）应用q-RT-PCR 检测5 个细胞系中Symplekin、Mir-124 的相对表达量，发现Mir-124 和Symplekin 表达量变化有相反趋势。（4）应用bisulfite restriction PCR 对13 例肝癌组织、10 例肝硬化组织、4 例正常肝组织以及肝癌细胞系HepG2 、Huh7 启动子区域甲基化状态进行检测，发现Symplekin 启动子区域都无甲基化现象；（5）同时，对8 例肝癌组织、10 例正常肝组织、5 例上皮细胞系及6 例白血病细胞系启动子区域缺失进行检测，发现Symplekin 启动子区域确实有碱基缺失，但其在肝癌组织、肝硬化组织、正常肝组织间没有统计学意义。实验结果提示Symplekin 很可能在肝细胞的恶性转化中起着重要的作用，此外 Symplekin 表达下降可能不仅参与肝癌发生且与其它肿瘤的发生具有相关性。推测在肝炎、肝硬化中，Symplekin 的下降可能会导致紧密连接功能下降，肝胆管上皮屏障功能降低， CB(结合胆红素)返流入血中，可能也是造成黄疸形成的原因之一。在肝脏疾病炎症反应过程中，细胞因子可能会协同作用影响Symplekin 的表达。Mir-124 有可能直接负调控Symplekin 的表达从而导致其表达降低。而Symplekin 启动子区域甲基化或缺失与肝癌发生无相关性。结论：（1）Symplekin 在大部分肝炎、肝硬化、肝癌组织中mRNA 表达水平呈下降趋势，这表明Symplekin 很可能在肝细胞的恶性转化中起着重要的作用。（2）Symplekin 在癌细胞系如肝癌细胞系（HepG2,HuH-7）肺癌细胞系（GLC,Spca-1,Ncih446,801D）、宫颈癌细胞系（Hela）、乳腺癌细胞系（Mcf-7）中表达均下降，这提示Symplekin 表达下降可能不仅参与肝癌发生而且参与其它肿瘤的发生。（3）Symplekin 启动子区域甲基化或缺失在肝癌、肝硬化及正常肝组织之间无显著性差异，表明在肝癌发生时Symplekin 的表达下降可能与启动子DNA 甲基化和缺失无关。（4）体外实验表明炎症细胞因子TNF-alpha 与INF-gamma 的协同参可能是体内Symplekin 表达及调控的机制之一。（5）Mir-124 对于Symplekin 的负调控作用也可能是体内Symplekin 表达及调控的机制之一。炎症细胞因子TNF-alpha 与INF-gamma 及Mir-124 可能在肝脏疾病及肝癌发生过程中起着重要作用。

两种抗HER2重组免疫毒素的构建与活性研究

HER2／neu基因在肿瘤中的过度表达使其成为许多肿瘤的标志分子。人肿瘤坏死因子（TNF-α）和肿瘤坏死因子相关的凋亡诱导配体（Trail）对肿瘤细胞的杀伤作用使其成为前景看好的抗肿瘤药物，对它们的细胞杀伤机制研究日渐深入。但临床研究发现HER2／neu过度表达的肿瘤细胞抵制TNF-α和Trail的肿瘤杀伤作用，因此经常产生耐药现象。为了增加过度表达HER2／neu的肿瘤细胞对TNF-a的敏感性和提高HER2／neu抗体的肿瘤杀伤效应，我们将抗HER2／neu人源化单链抗体scFvC6.5与人翔F-a融合，构建了免疫毒素scFvC6.5-TNF-α，完成了该重组蛋白在大肠杆菌中的表达，产率为800μg／L菌液。经过亲和层析和柱复性，融合蛋白的纯度达95％以上。ELISA试验表明scFvC6.5-TNF-a能够特异结合HER2／neu阳性卵巢癌细胞SKO从3和乳腺癌细胞MCF-7，而不结合HERZ／neu阴性的黑色素瘤细胞A-375。MTT试验表明scFvC6.5-TNF-a能够选择性的杀伤SKOV-3和MCF-7细胞，而不影响A-375细胞的生长。同时为了增加过度表达HER2／neu的肿瘤细胞对人可溶性肿瘤坏死因子相关的凋亡诱导配体（sTrail）的敏感性和提高HER2／neu抗体的肿瘤杀伤效应，我们构建了scFvC6．5与人sTrail的融合蛋白scFvC6.5-sTrail。重组子经酶切及测序证明序列正确后，在大肠杆菌BL21（DE3）中进行诱导表达。经SDS-PAGE及westem一blot鉴定，获得高水平包含体表达菌株，产率为700雌／L菌液。对表达产物进行变性、复性及纯化，SDS-PAGE结果显示纯度达95％以上。用ELISA法检测纯化后蛋白的结合活性表明融合蛋白scFvC6.5-sTrail能够特异结合HERZ／neu阳性卵巢癌细胞SKO从3、乳腺癌细胞McF-7和Trail敏感菌株MDA-MB-231，而不结合HER2／neu阴性和Trail受体阴性的黑色素瘤细胞A-375。MTT法检测其生物活性显示纯化后的scFvC6.5-sTrail蛋白对SKO从3、MCF-7、MDA-MB-231均具有细胞毒活性，并存在剂量依赖性，但对A-375细胞没有作用。细胞凋亡流式分析表明这两种免疫毒素对SKO从3靶细胞的杀伤作用是通过诱导细胞凋亡所致。提示这两种免疫毒素在抗肿瘤靶向治疗中具有潜在的应用价值。

Egonol龙胆三糖苷及Egonol衍生物促雌二醇生成活性研究

对Egonol龙胆三糖苷及以Egonol衍生物对雌二醇生成活性及其相关机制进行了研究。发现Egonol龙胆三糖苷促雌二醇最高生成率在MCF-7、HepG2、ROS1728中分别为157% 、182.4%、226.8%（以空白组200μg/ml睾酮转换成E2值作为100%生成率）。活性的强弱可能与芳香化酶的组织特异性表达情况一致，说明Egonol龙胆三糖苷促雌二醇活性可能与芳香化酶有关。芳香化酶的组织特异性表达与特异性启动子有关系，Egonol龙胆三糖苷在各组织中皆有促雌二醇活性，说明该化合物不是通过调节该酶的基因表达而起作用。 在探究Egonol龙胆三糖苷及其衍生物是否介导cAMP-PKA途径从而影响芳香化酶的表达中，发现该系列化合物在HEK-293T细胞中对cAMP的影响非常弱小。在人HepG2细胞中显示了极强的提高cAMP的作用。而化合物对cAMP的作用与其促雌二醇活性强弱不呈正相关关系，对c AMP-PKA途径的激活可能与胞内雌激素有关。 Egonol龙胆三糖苷及其衍生物对HepG2细胞增殖影响显示，该系列化合物同雌二醇一样有相似的较弱促HepG2细胞增殖作用。而且存在一定剂量依赖性。在瞬时转染有ERE（雌激素作用元件）的HepG2中，Egonol龙胆三糖苷及其衍生物也显示了类似于雌二醇与ERE结合的作用，进一步提示Egonol龙胆三糖苷及其衍生物在HepG2细胞中具备雌激素样作用。 为研究Egonol龙胆三糖苷及其衍生物是否可能直接提高芳香化酶的活性，我们计划将芳香化酶从芳香化酶阳性细胞中克隆后表达到芳香化酶阴性的细胞中。在MCF-7细胞中以Oligo dT为引物合成的cDNA模板，和在ROS1728细胞中以Oligo dT及大鼠引物F链为引物合成的cDNA模板能成功扩增出与芳香化酶全长编码序列大小一致的片段。 Egonol衍生物在HepG2、ROS1728细胞中促雌二醇活性的实验表明，Egonol苯环上引入其它基团可以提高Egonol的活性。 从雌激素经典的基因组效应和非基因组效应两方面对雌激素信号转导研究进展进行了简单的综述。 The promoting effects of egonol gentiotrioside and egonol derivatives on the synthesis of estrogen E2 were studied. In vitro test, egonol gentiotrioside promoted the synthesis of estrogen E2 in MCF-7, HepG2,ROS1728 cell lines with mean yields of estrogen E2 57%,82.4% and 126.8%, higher than those of blank control at a concentration of 100 mg/ml. The difference of estrogen E2 synthesis promoting effects among the cell lines suggested tissue specificity. It is in accordance with tissue specific character of aromatase expression. The evidence implied that effect of egonol gentiotrioside on promoting the synthesis of estrogen E2 was related to the aromatase. Different expression levels of aromatase in different tissues are attributed to their specific promoters, but egonol gentiotrioside can promote the synthesis of estrogen E2, in many tissues,so the fact is controversary to the estimation that this compound regulates the aromatase on gene level. In order to investigate whether egonol gentiotrioside and its synthetic derivatives regulates aromatase activity through the cAMP-PKA signal pathway,we transfected the p CRE-Luc luciferase reporter gene into the HEK-293T cells and HepG2 cells. These compounds had weak activity in promoting the cAMP activity in HEK-293T cells but strong in HepG2 cells.The compounds’effect of promoting the cAMP may be related to their estrogenic activity in cells. The modified HepG2 cell proliferation assay was used to evaluate the estrogenic activity of egonol gentiotrioside and its derivatives. The weak estrogenic activity of egonol gentiotrioside and its derivatives at various concentrations expressed as proliferative effect relative to that of blank control was examined. We transfected the pERE-Luc luciferase reporter gene into the HepG2 cells. These compounds possessed significant activity on estrogen response element compared with the one treated with 10 n M estrogen E2. This evidence indicated that the estrogenic activity of egonol gentiotrioside and its derivatives. In order to investigate whether the egonol gentiotrioside and its derivatives can upregulate the activity of aromatase directly, The full-length of P450 aromatase cDNA encoding aromatase were amplified by using primer Oligo dT in MCF-7,and specific primer in ROS1728,respectively. The structure-activity relationship of Egonol in promoting the synthesis of E2 in HepG2 and ROS1728 cells indicated that introduction of some group on the basic sketon of egonol could improve the effect. The progress in research of signal pathway of estrogen in recent years was summarized.

瓦山安息香中苯并呋喃促雌激素合成的机理研究

雌激素是人体内重要的激素之一，具有广泛的生理功能。雌激素缺乏与许多疾病相关，如卵巢功能低下，更年期综合征以及骨质疏松等；雌激素过剩也将导致某些疾病，如乳腺癌、卵巢癌、子宫内膜癌等。目前，如何降低肿瘤组织中的雌激素水平而达到治疗肿瘤的目的，已经得到广泛的研究，但促雌激素生成或调节卵巢功能药物或其相关研究则很少。 本实验室前期的研究发现，瓦山安息香属植物果实中的乙醇提取物具有促雌激素生成作用，通过活性追踪和结构鉴定，确认促E2 生成的主要成分为苯并呋喃类化合物。苯并呋喃类化合物的作用与芳香酶有关，但其确切的作用机理有待证实和深入研究。 为了探讨安息香苯并呋喃类化合物的促雌激素合成的作用机理，拟采用如下的实验方案： 1、细胞学方面，对小鼠3T3-L1 前脂肪细胞、人乳腺癌细胞MCF-7、MDA-MB-231 以及人卵巢癌细胞OVCAR-3、OVCAR-4、OVCAR-5、OVCAR-8、IGROV1 等细胞株，采用RT-PCR 和ELISA 方法研究芳香酶Aro基因的表达和雌二醇E2 的生成，芳香酶抑制剂Formestane 作为阳性对照，研究时效曲线和量效曲线，确定安息香苯并呋喃类化合物SP25 的有效浓度和作用时间。 2、RNAi 方面，设计合成了针对人芳香酶Aro基因的3 对RNAi 序列，转染入细胞，芳香酶促进剂Forskolin 和地塞米松、芳香酶抑制剂Formestane 作为阳性对照，采用实时定量PCR 技术，研究RNA 干扰后，安息香苯并呋喃类化合物SP25 对人芳香酶Aro基因表达水平瓦山安息香苯并呋喃促雌激素合成的机理研究的影响。 3、雌激素受体方面，设计一段ERE 的雌激素调控元件，构建重组荧光素酶报告基因载体，瞬时转染人乳腺癌细胞株MDA-MB-231，建立针对雌激素受体的报告基因筛选模型，观察安息香苯并呋喃类化合物SP25 对雌激素受体的选择性和亲和力，从受体水平考察安息香苯并呋喃类化合物SP25 促进雌激素生成的药理学机理。 实验结果显示： 1、分化后的小鼠3T3-L1 前脂肪细胞、人乳腺癌细胞MCF-7 、MDA-MB-231 以及人卵巢癌细胞OVCAR-3、OVCAR-4、OVCAR-8 等细胞株具有芳香酶基因的表达。睾酮向雌二醇的转化能够被芳香酶抑制剂Formestane 所阻断，其中OVCAR-3 最适合进行下一步的RNAi研究。 2、RNAi 实验结果显示，设计的3 对RNAi 序列中R2 的干扰效果最强，相应的阴性对照C2 与R2 的表达量相差118 倍（24 小时）和19 倍（48 小时），显示R2/C2 这组序列可用于进一步的RNAi 试验。以R2 干扰OVCAR-3 细胞株，药物作用24、48 小时后，芳香酶抑制剂Formestane 与R2 相对表达量相比分别为0.83 倍和0.04 倍；芳香酶促进剂Forskolin 与R2 相对表达量相比分别为3.61 和1.84 倍；芳香酶促进剂地塞米松与R2 相对表达量相比分别为5.76 倍和3.49倍；苯并呋喃类化合物SP25 与R2 相对表达量相比分别为8.13 倍和4.59 倍。实验证实安息香苯并呋喃类化合物SP25 能够促进因RNAi 而发生基因沉默的人芳香酶Aro表达水平的上调。 3、雌激素受体实验结果显示，构建成功重组pERE-pGL3-promoter 荧光素酶报告基因载体和基于报告基因系统的雌激素受体激动剂或拮抗剂的细胞筛选模型。实验结果表明安息香苯并呋喃类化合物SP25 与雌激素受体ERα和ERβ亲和力选择性之比约为3：1 ，SP25通过与雌激素受体ERα结合作用其受体，刺激芳香酶的表达。 本课题通过RNA 干扰、ELISA、荧光实时定量PCR、报告基因筛选模型等技术手段，从细胞水平、蛋白酶水平和基因表达水平、雌激素受体水平等方面系统地研究了从瓦山安息香属植物果实中提取的苯并呋喃SP25 促进促雌激素生成的机理研究。试验结果显示苯并呋喃类化合物SP25 促雌激素生成的主要作用机制是直接促进芳香酶基因表达水平，以及与雌激素受体a 结合，刺激芳香酶活性。 Estrogen is an important hormone that has versatile physiologicalfunctions. Lack of estrogen will lead to many diseases such as lower ovarianfunction, climacteric syndrome and osteoporosis. Excessive estrogen alsoinduces breast carcinoma, oophoroma and endometrial carcinoma and otherdiseases. To depress the estrogen level in tumor tissue to cure carcinomawas widely studied, but there is only few studies reported on the induction ofestrogen and on the regulation of ovary function. We found that the extracts from seeds of Styrax perkinsiae couldpromote the synthesis of estrogen. The active compounds benzofurans wereidentified. Effect of benzofurans may be related to aromatase, but the mechanism was not clear. To reveal the mechanism of these benzofurans to promote estrogensynthesis, the following protocols were adopted: 1 Cytology: 3T3-L1 preadipocytes,human ovary carcinoma celllines OVCAR-3,OVCAR-4,OVCAR-5,OVCAR-8,IGROV1 andbreast carcinoma cell lines MCF-7 and MDA-MB-231 were usedto determine Aro gene expression and estrogen production withRT-PCR AND ELISA methods. Formestane, an aromataseinhibitor, was used as positive control. And dose-curve,time-curve and the effective concentration of SP25 were also studied. 2 Designed 3 pairs of RNAi for human aromatase gene, andtransfected into cell. Aromatase inducer Forskolin andDexamethasone, and aromatase inhibitor Formestane were usedas positive controls. We studied the change of Aro expressionlevel with SP25 by using real-time PCR after RNA interfering. 3 Estrogen Receptor: We constructed the recombined Luciferasereport vector and establish a screening system for estrogenagonist and antagon. With this system, we studied the affinity ofSP25 and estrogen receptor. Results: 1 Differentiated 3T3-L1 preadipocytes¡¢human ovary carcinomacell lines:OVCAR-3, OVCAR-4, OVCAR-8 and breast carcinomacell lines MCF-7, MDA-MB-231 had detected aromatase geneexpression.And OVCAR-3 is more suitable for further aromatasegene function research. 2 In RNAi assay, R2 has a strong interfering effcet in OVCAR-3 cellline, and ratio of C2 (the negative control) to R2 were 118 times(24 hours) and 19 times (48 hours). This means sucessful inRNA interfering. After R2 acted on OVCAR-3 cell line, the ratiosof formestane to R2 were 0.83 and 0.04 times, 5.76 and 3.49times (Dex), 3.61 and 1.84 times (forskolin) and 8.13 and 4.59times (sp25) after drug treated 24 or 48 hours respectively.These results indicated that SP25 can directly induce aromatasegene up-regulation. 3 We had constructed pERE-pGL3-promoter recombined vectorand the Luciferase report gene screening system. Luciferasereport gene assay showed that sp25 had a higher affinity with strogen receptor alpha than estrogen receptor beta, this indicated that SP25 can act on estrogen receptor and induce aromatase. Our results revealed that the mechanisms of benzofuran to promoteestrogen were the upregulation aromatase gene expression and promotion ofaromatase activity and have partially elective affinity with estrogen receptoralpha.

生物还原药物AQ4N在肿瘤治疗增敏中的研究进展

目的观察尼美舒利(nimesulide)对人乳腺癌细胞MCF-7辐射敏感性的影响,并探讨其可能机制。方法实验分为对照组、加药组、单纯照射组(radiation treatment,RT)及加药照射组,用噻唑蓝(MTT)法检测尼美舒利对MCF-7细胞生长抑制率的影响,并选择用药后72h测得的IC20-IC30(抑制浓度,inhibiting concentration,IC)对应的药物浓度作为辐射增敏工作浓度;克隆形成实验检测尼美舒利对MCF-7的放射增敏作用;通过流式细胞仪检测细胞周期分布的变化。Western印迹法检测与凋亡相关蛋白Bcl-2和Bax的表达水平;并用单细胞凝胶电泳技术检测DNA的损伤及修复。结果尼美舒利对MCF-7细胞的生长抑制作用呈时间依赖性和剂量依赖性,但对周期分布无明显变化。尼美舒利加药照射组与单纯照射组比较,克隆存活曲线的肩区变窄,bcl-2表达下调,但对Bax表达没有明显影响。尼美舒利本身不明显增加MCF-7的DNA损伤,但可明显延缓放射引起的SSB的修复。结论提示尼美舒利对人乳腺癌MCF-7细胞株具有辐射增敏作用,其放射增敏机制可能通过抑制DNA损伤修复并下调bcl-2来得以实现。

BRCA1 and its phosphorylation involved in caffeine-inhibitable event upstream of G2 checkpoint

Caffeine, which specifically inhibits ATM/ATR kinases, efficiently abrogates the ionizing radiation (IR)-induced G2 arrest and increases the sensitivity of various tumor cells to IR. Mechanisms for the effect of caffeine remain to be elucidated. As a target of ATM/ATR kinases, BRCA1 becomes activated and phosphorylated in response to IR. Thus, in this work, we investigated the possible role of BRCA1 in the effect of caffeine on G2 checkpoint and observed how BRCA1 phosphorylation was regulated in this process. For these purposes, the BRCA1 protein level and the phosphorylation states were analyzed by Western blotting by using an antibody against BRCA1 and phospho-specific antibodies against Ser-1423 and Ser-1524 residues in cells exposed to a combination of IR and caffeine. The results showed that caffeine down-regulated IR-induced BRCA1 expression and specifically abolished BRCA1 phosphorylation of Ser-1524, which was followed by an override of G2 arrest by caffeine. In addition, the ability of BRCA1 to transactivate p21 may be required for MCF-7 but not necessary for Hela response to caffeine. These data suggest that BRCA1 may be a potential target of caffeine. BRCA1 and its phosphorylation are most likely to be involved in the caffeine-inhibitable event upstream of G2 arrest.

BRCA1对人乳腺癌细胞辐射敏感性的影响及其作用机制研究

目的：研究肿瘤抑制子BRCA1在人乳腺癌细胞辐射抗性中的作用，并探讨其作用机制。材料和方法：利用实时细胞分析系统检测辐射对细胞存活的影响；流式细胞术检测辐射对细胞周期分布的影响；RT-PCR检测辐射导致的BRCA1、Bax和Bcl-2在 mRNA水平变化；Western blot方法检测辐射诱导的蛋白表达水平的变化； In-cell western定量检测辐射引起的蛋白表达水平的变化； AO/EB染色法检测辐射导致的细胞死亡情况。结果：第一，分别用1Gy和4Gy x射线和碳离子束辐照人乳腺癌细胞MCF-7，研究MCF-7对不同LET射线的辐射敏感性差异。结果显示，x射线组1Gy辐射导致细胞存活显著下降，4Gy辐射对细胞存活影响不明显；而碳离子束辐射对细胞生长无抑制作用。与x射线组比较，碳离子辐射诱导了更低的亚“G1”期细胞百分数和更显著的G2期阻滞现象。同时碳离子束辐射诱导BRCA1磷酸化水平和p21表达上调，Bax表达下调。以上结果表明MCF-7对辐射的耐受性与凋亡功能相关，而BRCA1 Ser-1524磷酸化作用可能参与细胞周期和凋亡的信号调控。第二，研究BRCA1在咖啡因诱导的辐射增敏效应中作用。当2mM咖啡因联合4Gy的x射线或碳离子束辐射处理MCF-7细胞后，观察到细胞存活显著下降，辐射诱导的G2期阻滞被废除，BRCA1和p21蛋白表达被抑制，而p53表达水平无明显变化。结果表明咖啡因诱导的MCF-7细胞的辐射增敏作用可能与G2期阻滞被废除相关，BRCA1可能参与该过程的信号调节。第三， 利用BRCA1功能正常的MCF-7细胞和BRCA1功能缺失的HCC1937细胞进一步研究BRCA1对细胞辐射敏感性的影响。辐射显著抑制了HCC1937细胞存活，但对MCF-7细胞存活无明显影响。与HCC1937细胞相比，辐射诱导MCF-7细胞发生显著的G2期阻滞。辐射诱导HCC1937细胞发生晚期凋亡，而MCF-7细胞则多发生早期凋亡，且MCF-7细胞凋亡数明显少于HCC1937细胞。RT-PCR检测结果显示，辐射增强了MCF-7细胞中BRCA1的mRNA 水平，抑制了Bax的mRNA 水平，对Bcl-2的影响不明显；而HCC1937细胞中Bax的mRNA表达水平则被增强。同时辐射诱导MCF-7细胞中BRCA1和p21蛋白表达增强，Bax表达下降，Bcl-2水平略有增高。而HCC1937细胞Bax表达水平增强，但p21和Bcl-2的表达水平则检测不到。这些结果表明，正常的BRCA1功能对Bcl-2的转录表达是必须的，BRCA1通过上调p21水平，下调Bax/Bcl-2影响细胞的辐射敏感性。结论： BRCA1在人乳腺癌细胞的辐射抗性发生中发挥重要作用，BRCA1通过上调p21水平诱导G2期阻滞，下调Bax/Bcl-2抑制凋亡信号，使得细胞对辐射诱导的凋亡产生抗性，最终导致细胞对辐射产生耐受性

抗HER2-hTNF-α新型免疫毒素的制备及功能研究

HER2/neu基因在肿瘤中的过度表达使其成为许多肿瘤的标志分子.为了增加过度表达HER2/neu的肿瘤细胞对肿瘤坏死因子(TNF)的敏感性和提高HER2/neu抗体的肿瘤杀伤效应,将抗HER2/neu单链抗体C6.5与人肿瘤坏死因子hTNF-α融合,构建了scFvC6.5-hTNF-α融合蛋白,完成了重组蛋白在大肠杆菌中的表达,产率为400μg/L菌液.经过亲和层析和柱复性,融合蛋白的纯度达95%以上.ELISA试验表明,scFvC6.5-hTNF-α能够特异结合HER2/neu阳性卵巢癌细胞SKOV-3和乳腺癌细胞MCF-7,而不结合HER2/neu阴性的黑色素瘤细胞A375.MTT试验表明,scFvC6.5-hTNF-α能够选择性地杀伤SKOV-3和MCF-7细胞,而不影响A375细胞的生长.这种肿瘤细胞特异性杀伤作用提示该免疫毒素具有肿瘤靶向治疗的潜在应用价值.

A novel anti-tumor protein extracted from Meretrix meretrix Linnaeus induces cell death by increasing cell permeability and inhibiting tubulin polymerization

Discovery and development of new pharmaceuticals from marine organisms are attracting increasing interest. Several agents derived from marine organisms are under preclinical and clinical evaluation as potential anticancer drugs. We extracted and purified a novel anti-tumor protein from the coelomic fluid of Meretrix meretrix Linnaeus by ammonium sulphate fractionation, ion exchange and hydrophobic interaction chromatography. The molecular weight of the highly purified protein, designated MML, was 40 kDa as determined by SDS-PAGE analysis. MML exhibited significant cytotoxicity to several cancer cell types, including human hepatoma BEL-7402, human breast cancer MCF-7 and human colon cancer HCT116 cells. However, no inhibitory effect was found when treating murine normal fibroblasts NIH3T3 and benign human breast MCF-10A cells with MML. The cell death induced by MML was characterized by cell morphological changes. The induction of apoptosis of BEL-7402 cells by MML was weak by DNA ladder assay. The possible mechanisms of its anti-tumor effect might be the changes in cell membrane permeability and inhibition of tubulin polymerization. MML may be developed as a novel, highly selective and effective anti-cancer drug.

两种海藻提取物的化学成分和生物活性的研究及其应用

本论文的研究工作由两部分组成，第一部分研究了海带（Laminaria japonica）水提取物中的活性物质，并研究了提取物对蔬菜促生长的影响及其作用机制。第二部分对三列凹顶藻(Laurencia tristicha)乙醇提取物的乙酸乙酯相进行了活性筛选和化学成分研究，并对其中分离得到的单体化合物进行了生物活性筛选。 第一部分主要以中国人工养殖的海带为原料，使用与海藻多糖生产相结合的提取技术并浓缩其中的有效成分。对浓缩提取物进行了蔬菜的农田效果实验，并对作物抗旱性能的增加、作物硝酸盐积累的减少、作物品质的改善、以及作物抵抗病毒病的能力等影响进行了作用机制方面的研究。海藻浓缩提取物进行的农田效果实验表明：作物抗旱型相对含水量RWC值在92%~94%之间；病毒病的防治效果最高可达到91%；作物的品质有明显的改善，最重要的是首次发现海藻提取物有降低蔬菜中硝酸盐的含量（硝酸盐的含量是与有机蔬菜区别的重要指标之一）的作用。该部分研究工作的创新性主要体现在：（1）首次在国内外提出和采用与海藻多糖生产相结合的提取技术。该技术的应用不但减少了提取成本，使工业化生产成为可能,更重要的是使我国的海藻工业生产可能实现高值化和开辟综合利用的新途径。（2）首次发现海藻中的小分子海藻多糖具有和细胞激动素、甜菜碱、植物生长素等活性物质同样的生物活性。 第二部分的研究是在查阅了大量的近20年来国内外有关红藻凹顶藻中化学成分研究的相关文献的基础上，对凹顶藻中的次生代谢产物进行了综述。该论文主要是通过对红藻三列凹顶藻的95%乙醇提取物的乙酸乙酯相进行化学成分分析和生物活性筛选以期能够发现具有药用前景的活性先导化合物。 为了寻找具有生物活性的化合物，我们对采自我国南海硇洲岛海域的红藻三列凹顶藻的95%乙醇提取物的乙酸乙酯相进行了活性筛选。采用MTT法对其在KB细胞株、Bel-7402细胞株、PC-3M细胞株、MCF-7细胞株、Ketr-3细胞株模型上进行了细胞毒活性测试；采用酶模型对其进行了Na+,K+-ATPase的抑制活性测试；采用MTT法对其在犬主动脉血管模型上进行了血管平滑肌细胞增殖抑制活性测试；结果表明，三列凹顶藻的95%乙醇提取物的乙酸乙酯相对Na+,K+-ATPase和犬血管平滑肌细胞增殖具有一定的抑制活性。 利用正相和反相色谱、Sephadex LH-20色谱以及反相HPLC等手段进行分离纯化，从我国南海海域的红藻三列凹顶藻中分离得到33种化学成分，通过波谱学方法（IR、MS、NMR）以及X-ray单晶衍射试验对其化学结构进行了确证，其中化合物L1~L8为新结构化合物，化合物L5为具有新骨架的全新结构化合物，化合物L9~L13为新天然产物，化合物L18和L22系首次从海洋生物中获得，所有化合物均为首次从该属海藻中得到。新化合物L1~L8均为倍半萜类化合物，命名分别为：(1R,3R)-(-)-3-(3-hydroxy-4-methylphenyl)- 1,3-dimethyl–2-methylidene cyclopentanol (L1), (1R,3R)-(-)-3-(4-methylphenyl)-1,3-dimethyl-2-methylidenecyclopentanol (L2)， (1R, 3R)-(-)-3-(2-hydroxy-4-methylphenyl)-1,3-dimethyl–2–methylidenecyclopentanol (L3)，(+)-(1S,2R)–2-(3–hydroxy–4–methylphenyl)-1,2-[3.1.0]bicy-clohexane (L4)，()-(1S,2R) -5-hydroxy–6–methyl-spiro-dihydrobenzofuran-2(3H),2-{1-methyl-[3.1.0]bicyclohexane} (L5), (+)-6-methyl-2-(p-tolyl)hept-4-en-2,6-diol (L6)，(3R,3aS,8bS)-(-)-2,3,3a,8b–tetrahydro–7-bromo – 3 a– hydroxymethyl - 3, 6, 8b - trimethyl-1H- cyclopenta[b] benzofuran (L7 )，(3R, 3aS, 8bS) - (-) - 2,3,3a,8b–tetrahydro–3 a–hydroxymethyl-3,6,8b -trimethyl -1H – cyclopenta [b] benzofuran (L8)。25个已知结构化合物确定为：(+)-(1R,2R)-4-bromo-1,5, 9–trimethyl–12– methylidene–8–oxa-tricyclo[7.2.1.02]dodeca-2,4,6-triene (L9)，(3S,3aR,8bS)-(-)-2,3,3a, 8b– tetra -hydro–7-bromo–3–hydroxy-3,3a,6,8b-tetramethyl-1H-cyclopenta[b]benzofu- ran (L10 )，(3R, 3aR, 8bS) - (-) - 2, 3, 3a, 8b – tetrahydro – 7 - bromo – 3 – hydroxy - 3,3a,6,8b - tetramethyl - 1H - cyclopenta [b] benzofuran (L11 )，(3S,3aR,8bS) - (-) - 2, 3, 3a, 8b – tetrahydro –3–hydroxy -3, 3a, 6, 8b - tetramethyl-1H-cyclopenta[b]benzofuran (L12 )， ( 3aR, 8bS) - (-) - 3a,8b –dihydro–7 - bromo – 3, 3a, 6, 8b - tetramethyl - 1H - cyclopenta[b]benzofuran (L13 )，aplysinol (L14 ) ，aplysin (L15)，laurebiphenyl (L16)，johnstonol (L17)，gossonorol (L18)，7,10-epoxy-ar- bisabol-11-ol (L19)，10-epi-7,10-epoxyarbisabol-11-ol (L20) 3β-hydroxy- 5α, 6α-epoxy- β- ionone (L21 )，3β-hydroxy-5β,6β-epoxy-β-ionone (L22 )，胆甾醇 (L23 )，胆甾-5-烯-3β,7α二醇胆甾-5-烯-3β,7α二醇 (L24)，β-谷甾醇 (L25)，叶绿醇 (L26 )，玉米黄素 (L27 )，对羟基苯甲醛 (L28 )，3-吲哚甲醛 (L29 )，1-O-十六烷酰基-3-O-β-D-吡喃半乳糖基-丙三醇(L30 )，1-O-十八烷酰基-3-O-β-D-吡喃半乳糖基-丙三醇 (L31 )，丙三醇-1-软脂酸单酯 (L32 )，正十六碳酸 (L33 )。 采用MTT法对其中23个单体化合物在Bel-7402细胞株、BGC-823细胞株、A549细胞株、A2780细胞株、HCT-8细胞株和HELL细胞株模型上进行了细胞毒活性测试；采用MTT法对其中13个单体化合物在犬主动脉血管模型上进行了血管平滑肌细胞增殖抑制活性测试；结果表明，部分单体化合物显示出一定的生物活性。

Cadinane Sesquiterpenes from the brown alga Dictyopteris divaricata

Seven new cadinane sesquiterpenes, (-)-(1R,6S,7S,10R)-1-hydroxycadinan-3-en-5-one (1), (+)-(1R,5S,6R,7S, 10R)-cadinan-3-ene-1,5-diol (2), (+)-(1R,5R,6R,7S,10R)-cadinan-3-ene-1,5-diol (3), (+)-(1R,5S,6R,7S,10R)-cadinan-4(11)-ene-1,5-diol (4), (+)-(1R,5R,6R,7R,10R)-cadinan-4(11)-ene-1,5,12-triol (5), (-)-(1R,4R,5S,6R,7S, 10R)-cadinan-1,4,5-triol (6), and (-)-(1R,6R,7S,10R)-11-oxocadinan-4-en-1-ol (7), together with nine known compounds were isolated from the brown alga Dictyopteris divaricata. The structures of the new natural products, as well as their absolute configuration, were established by means of spectroscopic data including IR, HRMS, 1D and 2D NMR, single-crystal X-ray diffraction, and CD. All compounds were inactive against several human cancer cell lines including lung adenocarcinoma (A549), stomach cancer (BGC-823), breast cancer (MCF-7), hepatoma (Bel7402), and colon cancer (HCT-8) cell lines.

Norsesquiterpenes from the brown alga Dictyopteris divaricata

Three bisnorsesquiterpenes (1-3) with novel carbon skeletons and a norsesquiterpene (4) have been isolated from the brown alga Dictyopteris divaricata. By means of spectroscopic data including IR, HRMS, 1D and 2D NMR techniques, single-crystal X-ray diffraction, and CD, their structures including absolute configurations were proposed as (+)-1R,6S,9R)-1-hydroxyl-6-isopropyl-9-methylbicyclo[4.3.0]non-4-en3-one (1), (-)-(1S,6S,9R)-1-hydroxyl-6-isopropyl-9-methylbicyclo[4.3.0] non-4-en-3-one (2), (+)-(5S,6R,9S)5-hydroxyl-6-isopropyl-9-methylbicyclo [4.3.01 non-1-en-3-one (3), and (-)-(1R,7S,10R)-1-hydroxy-1lnorcadinan-5-en-4-one (4). Biogenetically, the carbon skeleton of 1-3 may be derived from the co-occurring cadinane skeleton by ring contraction and loss of two carbon units, and compound 4 from the oxidation of cadinane derivatives. Compounds 1-4 were inactive (IC50 > 10 mu g/mL) against several human cancer cell lines including lung adenocarcinoma (A549), stomach cancer (BGC-823), breast cancer (MCF-7), hepatoma (Bel7402), and colon cancer (HCT-8) cell lines.

The antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo

To investigate the antitumor effect of bromophenol derivatives in vitro and Leathesia nana extract in vivo, six bromophenol derivatives 6-(2,3-dibromo-4,5-dihydroxybenzyl)-2,3-dibromo-4,5-dihydroxy benzyl methyl ether (1), (+)-3-(2,3-dibromo-4,5-dihydroxyphenyl)-4-bromo-5,6-dihydroxy-1,3-dihydroisobenzofuran (2), 3-bromo-4-(2,3-dibromo-4,5-dihydroxybenzyl)-5-methoxymethyl-pyrocatechol (3), 2,2',3,3'-tetrabromo-4,4',5,5'-tetrahydroxy-diphenylmethane (4), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (5), 2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-ethyloxymethyldiphenylmethane (6) were isolated from brown alga Leathesia nana, and their cytotoxicity were tested by MTT assays in human cancer cell lines A549, BGC-823, MCF-7, B16-BL6, HT-1080, A2780, Bel7402 and HCT-8. Their inhibitory activity against protein tyrosine kinase (PTK) with over-expression of c-kit was analyzed also by ELISA. The antitumor activity of ethanolic extraction of Leathesia nana (EELN) was evaluated on S-180-bearing mice. All compounds showed very potent cytotoxicity against all of the eight cancer cell lines with IC50 below 10 mu g/mL. In PTK inhibition study, all bromophenol derivatives showed moderate inhibitory activity and compounds 2, 5 and 6 showed significant bioactivity with the inhibition ratio of 77.5%, 80.1% and 71.4%, respectively. Pharmacological studies reveal that EELN could inhibit the growth of Sarcoma 180 tumor and increase the indices of thymus and spleen to improve the immune system remarkably in vivo. Results indicated that the bromophenol derivatives and EELN can be used as potent antitumor agents for PTK over-expression of c-kit and considered in a new therapeutic strategy for treatment of cancer.