OA对人肝细胞HL-7702和肝癌细胞Bel-7402增殖的影响和对细胞凋亡的诱导作用研究

 以人肝细胞HL-7702和肝癌细胞Bel-7402为研究对象,通过MTT法、细胞外部形态观察、Annexin V.FITC&;PI双染法及TUNEL法等方法,研究了腹泻性贝毒的主要组分大田软海绵酸（Okadaic acid, OA）对其增殖的影响及对细胞凋亡的诱导作用。结果表明：OA对人两株细胞的增殖均有显著的抑制作用,24 h IC₅₀分别为65 ng/mL和60 ng/mL,且这种抑制作用呈剂量依赖性。OA导致两株细胞的形态均发生明显变化：细胞变圆,脱壁,细胞膜形成泡状突起,最终形成膜包裹的凋亡小体。Annexin V.FITC和PI双染法及TUNEL法检测均发现：OA能够诱导两株细胞发生不同程度的凋亡,且凋亡细胞的比例与OA的浓度相关。以上结果表明：OA能够通过抑制细胞增殖和诱导细胞凋亡而对人肝细胞和肝癌细胞造成影响,且这种影响的程度与OA的浓度和作用时间密切相关。     

敬钊缨毛蛛蛛毒对人肝癌细胞BEL-7402细胞增殖、细胞周期和C-myc蛋白的影响

通过MTT方法可知敬钊缨毛蛛蛛毒对BEL 7402细胞增殖有较强的抑制作用(p<0.05),时效和量效关系良好.IC50为18mg/L.敬钊缨毛蛛蛛毒可以抑制BEL 7402细胞DNA的合成.流式细胞仪检测发现经过蛛毒作用的BEL 7402细胞凋亡率增加,细胞周期阻滞在G0/G1期.WesternBlot方法进一步检测到蛛毒作用BEL 7402细胞72h后,C myc蛋白表达减弱.实验结果表明,敬钊缨毛蛛蛛毒可以抑制人肝癌细胞BEL 7402的增殖和DNA的合成,其药理作用机理可能除了诱导凋亡外主要是使BEL 7402细胞周期相关蛋白C myc表达减弱,导致细胞周期的变化.     

加热对血管平滑肌细胞的影响实验研究

 为支架磁感应热疗预防和治疗PTCA术后血管再狭窄提供基础研究数据,对临床常用的316L型不锈钢冠脉支架进行磁感应诱导升温,探讨加热对兔血管平滑肌细胞（VSMCs）的增殖、迁移、凋亡及周期的影响。将平滑肌细胞置于恒温水浴槽中,待其达到预定温度（43、47℃）后持续10min,观察细胞形态变化,采用MTT法检测加热对血管平滑肌细胞增殖活性的影响,流式细胞术检测VSMC细胞周期和细胞凋亡,划痕实验检测加热对细胞迁移能力的影响,免疫细胞化学法检测不同温度作用后血管平滑肌细胞中增殖细胞核抗原（PCNA）的表达。结果发现,支架在交变磁场下可以升温至热疗所需温度,加热后细胞增殖受到了显著抑制,43℃组细胞存活率为（83.23±2.87）%,47℃组细胞存活率仅为（37.58±0.78）%。细胞的凋亡率显著增加,47℃组细胞凋亡率为（87.37±2.95）%,与对照组相比具有极显著差异,而43℃组的凋亡率为（6.00±0.26）%,与对照组相比无统计学差异。细胞周期也受到抑制,被阻滞在S期。加热后随着时间的推移,47℃组细胞的迁移能力受到显著抑制,而加热对43℃组细胞的迁移能力无显著影响。免疫细胞化学检测显示,随着温度的升高,PCNA的表达受到明显抑制。研究表明,临床常用冠脉支架在交变磁场下升温可行。加热显著抑制了细胞的增殖并促进了细胞凋亡,使细胞周期阻滞在S期,且抑制了细胞的迁移。加热能显著抑制细胞PCNA的表达,这些可能是加热抑制细胞增殖和迁移、促进凋亡的机制之一。     

端粒酶转染防治角膜内皮失代偿的研究

目的评价端粒酶转染对角膜内皮细胞形态和功能的影响。方法将体外培养的猫角膜内皮细胞随机分为3组,兔端粒酶基因转染组、正常对照组和表皮生长因子组。培养30代后观察传代细胞形态和细胞周期各时相细胞比例的变化,检测端粒酶逆转录酶蛋白和Ⅳ型胶原的表达。结果兔端粒酶基因转染组细胞数目较正常对照组和表皮生长因子组增多,伸展贴壁能力增强,表型正常,G2～M期和S期细胞比例显著增加,端粒酶逆转录酶蛋白和Ⅳ型胶原的表达也较正常对照组增多。结论兔端粒酶转染可以促进猫角膜内皮细胞增殖且表型正常,可以增强其伸展粘附能力和分泌功能,有利于防治角膜内皮失代偿。     

新型姜黄素结构衍生物抗肝癌细胞增殖作用的研究

采用MTT法对姜黄素结构衍生物（CCM系列化合物）进行抗人肝癌细胞Bel-7402和SMMC-7721活性筛选;利用流式细胞技术和荧光显微镜检测SMMC-7721细胞凋亡及周期分布;采用Western-Blot方法检测SMMC-7721中蛋白caspase-3和剪切后p17的表达.结果表明,化合物CCM-5和CCM-14抗肿瘤活性较好,其对SMMC-7721细胞的凋亡作用呈剂量依赖性,且凋亡率与阴性对照组相比有显著差异（P<0.01）;化合物浓度增高时,G0/G1期细胞减少,S期以及G2/M期细胞增加,凋亡峰SubG1峰增大;两个化合物均可增强caspase-3的表达,随着浓度的提高,caspase-3的表达趋势减弱,而剪切形式p17亚基表达量逐渐提高.因此,姜黄素结构衍生物CCM-5和CCM-14能抑制人肝癌细胞SMMC-7721细胞的增殖,促进凋亡,其作用机制可能与化合物诱导caspase-3活性的增强有关.     

依地福新抑制Jurkat细胞生长机制的研究

目的观察依地福新对体外培养的Jurkat细胞的生长抑制作用,并进一步探讨其作用机制。方法应用MTT法检测细胞增殖活性;流式细胞仪检测细胞周期分布;Annexin V—FITC染色法检测细胞凋亡率。结果不同浓度依地福新处理Jurkat细胞24～96h后,细胞增殖显著受到抑制,并呈现浓度及时间依赖性;1．0μmol／L、5．0ymol／L、10．0μmol／L依地福新处理72h后,Jurkat细胞G0／G1期细胞数量显著增加,S期细胞数量显著降低（P〈0．01）;各浓度组细胞凋亡率均显著增加（P〈0．01）。结论依地福新对Jurkat细胞具有生长抑制作用,其机制与阻滞细胞周期及诱导凋亡有关。     

Behavior of MC3T3-E1 Osteoblast Cultured on Chitosan Modified with Polyvinylpyrrolidone

The physical and chemical properties of four kinds of modified chitosan materials made by blending chitosan with polyvinylpyrrolidone （PVP） were investigated. All four of these modified chitosan materials were hydrophilic with water contact angles ranging from 59°to 69°. Fourier transform-infrared spectra of the modified materials showed a new band at 1288 cm^-1, implying formation of a surface physical interpenetrating network structure. Enzyme linked immunosorbent assay results indicated that much less fibronectin was adsorbed on the modified materials than on only chitosan. The viability of MC3T3-E1 osteoblasts cultured on the materials was assessed by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl- 2H-tetrazolium bromide assay. The results show that adding PVP10000 into the chitosan promotes adhesion of MC3T3-E1 osteoblasts on the modified materials, but has no effect on cell growth and proliferation; while adding PVP40000 reduces cell adhesion, growth, and proliferation. The results suggest that the increased hydrophilicity of the material surface does not always improve its biocompatibility, which will influence the selection and design of biomaterials.     

白花蛇舌草提取物体外抗肿瘤作用及机制研究

目的　研究中药白花蛇舌草提取物对Bel 7402细胞抗肿瘤作用及其作用机制.方法　采用MTT法和集落形成实验检测白花蛇舌草提取物对Bel 7402细胞生长的抑制作用;采用淋巴细胞转化实验检测白花蛇舌草提取物对小鼠脾脏T淋巴细胞增殖的刺激作用,并筛选出药物的安全剂量范围;采用透射电镜初步观察药物作用后Bel 7402细胞的形态变化.结果　1)MTT比色实验、集落形成实验的结果表明,白花蛇舌草提取物对Bel 7402细胞的生长具有明显的抑制作用,且这种作用是剂量依赖关系,IC50为1.6g/L;2)透射电镜观察表明,药物作用后的Bel 7402肿瘤细胞的体积变小,核分裂像显著减少,没有明显的凋亡现象;细胞核固缩,异染色质块状聚集浓染,线粒体变大变圆,基质变淡,线粒体嵴变短变少甚至消失,在极度肿胀时,线粒体转化为小空泡状结构,并有细胞膜破损现象.结论　1)白花蛇舌草提取物可能通过直接影响肿瘤细胞能量代谢,对Bel 7402细胞生长具有明显的抑制作用,且呈剂量依赖关系;2)白花蛇舌草提取物具有促进小鼠脾淋巴细胞增殖的作用.     

应用SCID鼠筛选肝癌转移性亚克隆及M-H7402亚细胞系的建立

本研究应用人肝癌细胞SCID鼠转移模型，筛选和建立肝癌转移细胞系，旨在为肝癌转移研究提供实验材料．采用人肝癌细胞H7402，腋后背部皮下接种SCID鼠，接种后66天时，在荷瘤鼠赢弱时引颈处死，取其肺组织，进行原代细胞培养和传代培养，获得肝癌细胞H7402的亚细胞克隆，命名为M—H7402．然后，对M—H7402细胞进行了生物学鉴定，检测了细胞形态、染色体、细胞动力学、细胞周期、甲胎蛋白表达、癌基因表达和转移相关基因表达等指标．结果显示，M—H7402细胞的生长、增殖和形态等与亲本细胞H7402十分相近，其染色体形态仍为人类核型，众数维持在72～80之间，占75．0％．RT—PCR检测结果显示，M—H7402细胞甲胎蛋白表达为阳性．Western blot检测结果显示，与亲本细胞H—7402相比，癌基因C—myc表达水平较高，转移抑制基因nm23的表达水平明显下调．从肺组织中获得的肝癌转移细胞系M—H7402，在体外可连续传代培养，细胞形态不变．应用SCID鼠筛选获得的亚细胞克隆，与亲本细胞形成配对关系，可为肝癌细胞转移的研究提供新的实验材料。     

丁酸钠对重组CHO细胞生长及产物EPO表达的影响

在CHO－S－SFM II无血清培养基中维持培养重组CHO细胞,考查添加丁酸钠对细胞生长,糖代谢及产物EPO表达的影响,丁酸钠浓度达到2mmol/L时完全抑制了细胞的生长,同时,丁酸钠的添加也降低了葡萄糖比消耗速率（30％左右）,在本培养系统中,丁酸钠并不能促进产物的表达,但有助于维持EPO表达的稳定性。     

Growth potential of human hepatocarcinoma cells in the liver of neonatal immunocompetent mice and its relation to immunological tolerance

To determine the pathological behavior of human hepatocarcinoma cells in the liver microenvironment of neonatal non-immunode-ficient mice, three human hepatocarcinoma cell lines (Bel7402, HepG2, and SK-Hep-1), traced by DiI, were transplanted into the intrahepatic or subcutaneous tissue of neonatal and adult Kunming mice. Histopathological observations showed that cells in the adult liver induced a severe immune response as early as the second day after the implantation, while the subcutaneous neoplasm underwent extensive necrosis by the end of the study. Only the cells injected into the neonatal liver underwent a delayed immunologic rejection in the organ microenvironment. These cells retained recognizable tumor features over the first seven days, and displayed an intrahepatic invasive pattern. The expression of tumor markers including alpha-fetoprotein and survivin was maintained. The quantitative ELISA for the expression patterns of IL-2 and IL-10 also confirmed that the intrahepatic immunity was non-susceptive during this period. The high serum alpha-fetoprotein level was inversely correlated with the change in immune response. Our study provided a bio-system for the research of immune responses to xenografts in the liver.     

清热解毒药对血管平滑肌细胞增殖、细胞周期的影响

目的 :观察清热解毒药双花、公英、虎杖、连翘对血管平滑肌细胞生长、贴壁和增殖抑制作用 ,并从对细胞周期的影响角度分析它们的作用机制。方法 :采用细胞计数、MTT法和流式细胞技术。结果 :双花、公英、虎杖、连翘对h PDGF B/B刺激下的平滑肌细胞生长和贴壁均有抑制作用。对平滑肌细胞增殖抑制率分别达 9.2 6%～ 77.35% ,7.78%～ 62 .65% ,5.88%～ 47.81 % ,3.98%～ 33.35% ,其增殖抑制作用呈量效时效关系。通过对细胞周期分析知道 ,双花、公英、虎杖均可以减少 S期 ,增加 G0 /G1期细胞数目。结论 :清热解毒药双花、公英、虎杖、连翘对平滑肌细胞生长及增殖有抑制作用 ,其作用机制可能是通过抑制 DNA合成 ,减少进入 S期细胞数目 ,使细胞增殖停留在 G0 /G1期来实现的     

Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase.

The macrolide rapamycin induces cell cycle G1 arrest in yeast and in mammalian cells, which suggests that an evolutionarily conserved, rapamycin-sensitive pathway may regulate entry into S phase. In mammals, rapamycin inhibits interleukin-2 receptor-induced S phase entry and subsequent T-cell proliferation, resulting in immunosuppression. Here we show that interleukin-2 selectively stimulates the phosphorylation and activation of p70 S6 kinase but not the erk-encoded MAP kinases and rsk-encoded S6 kinases. Rapamycin completely and rapidly inhibits interleukin-2-induced phosphorylation and activation of p70 S6 kinase at concentrations comparable to those blocking S phase entry of T cells (0.05-0.2 nM). The structurally related macrolide FK506 competitively antagonizes the actions of rapamycin, indicating that these effects are mediated by FKBP, which binds the transition-state mimic structure common to both rapamycin and FK506 (refs 4, 6, 9-11). The selective blockade of the p70 S6 kinase activation cascade by the rapamycin-FKBP complex implicates this signalling pathway in the regulation of T cell entry into S phase.     

新型TiO2纳米管透析膜与传统透析膜对细胞生长状态的影响

 研究采用阳极氧化法制备新型高强度的TiO₂纳米管阵列薄膜, 通过对纳米管底部进行腐蚀获得两端通透的TiO₂纳米管阵列薄膜.在纳米管阵列薄膜表面和高分子透析膜表面种植HK-2细胞和HUVEC细胞, 成功制备具有生理功能的生物膜材料.采用MTT方法对比研究了TiO₂纳米管阵列薄膜、聚醚砜(PES)、混合纤维素以及再生纤维素4种薄膜材料黏附细胞的活性;利用荧光显微镜观察4种材料对两种细胞黏附的影响;同时使用扫描电镜观察细胞在4种薄膜材料上的生长形态.结果表明, TiO₂纳米管阵列薄膜最有利于细胞的黏附及增殖, 且细胞活性最高, PES薄膜的效果次之, 再生纤维素薄膜最不适合细胞的增殖及黏附;荧光显微镜观察证实TiO₂纳米管阵列薄膜相比高分子薄膜更能促进细胞的黏附及生长, 证实所制备的TiO₂纳米管阵列薄膜能够很好地与两种细胞相容, 克服了传统透析材料的不足, 改善了细胞与材料的黏附, 是用于生物人工肾研究较为理想的候选材料.     

皮蛋模拟胃肠道消化物对HepG2细胞的影响

皮蛋是中国传统食品,其水解物具有抗氧化、抗炎和抗癌等功效,具有良好的医学前景,但目前皮蛋发挥抗癌功效的途径及作用机制尚不清楚。探究了皮蛋模拟胃肠道消化物(preserved eggs simulated gastrointestinal digests,PESD)对人肝癌HepG2细胞增殖和细胞周期的影响。将皮蛋经体外模拟胃肠道消化处理后,进行细胞实验,用CCK-8法进行细胞增殖分析,研究PESD对HepG2细胞存活率的影响,采用碘化丙啶(propidium iodide, PI)染色标记的方法,通过流式细胞术测定PESD对HepG2细胞周期的影响,用western blot法测定细胞周期相关调控蛋白的表达。研究发现:PESD以剂量依赖性方式对HepG2细胞增殖进行抑制,IC₅₀为4.17mg/mL;PESD处理显著增加了S期细胞的占比(P<0.05),使HepG2细胞阻滞于S期;4mg/mL的PESD处理HepG2细胞24h后,细胞中cyclin A、cyclin E、ATM、chk2和CDC25A的蛋白表达水平上升(P<0.05),而CDK2蛋白表达水平下降(P<0.05)。研究认为,皮蛋模拟胃肠道消化物是以剂量依赖性的方式来抑制人肝癌HepG2细胞的增殖,主要是通过ATM-chk2-CDC25A信号通路,上调cyclin A和cyclin E蛋白的表达,下调CDK2蛋白的表达来实现人肝癌HepG2细胞阻滞于S期,影响人肝癌HepG2细胞周期进程,抑制癌细胞的增殖。研究结果旨在为皮蛋抗癌作用机制的阐明提供一定的理论基础。     

Myofibrillogenesis regulator-1 promotes cell adhesion and migration in human hepatoma cells

Myofibrillogenesis regulator-1 (MR-1) is a gene overexpressed usually in many human cancers. However, the effects of MR-1 on cell proliferation, adhesion, migration and genome-wide gene regulation are still unclear. In this study, a human hepatoma cell line that highly overexpresses MR-1, BEL-7402/MR-1 cells was established. While the high expression of MR-1 did not promote cell proliferation, it significantly increased cell spreading, adhesion and migration compared with control cells. A total of 147 genes were regulated by MR-1 expression, 46 genes were down-regulated and 101 genes were up-regulated by MR-1 overexpression. Many of these genes were related to cell adhesion, cytoskeletal regulation, MAPK signaling, and cell cycle related pathways. Western blot analysis further confirmed the regulation of pathways associated with migration by MR-1. These results suggest that MR-1 is involved in the regulation of cancer cell adhesion, migration and related gene expression.     

Ca~(2 )对酿酒酵母与粟酒裂殖酵母细胞增殖的影响

SD培养基中外加不同浓度的Ca2 可促进酿酒酵母与粟酒裂殖酵母细胞的增殖 ,但在促进增殖方式上存在明显差异 .随SD培养基中外Ca2 浓度增加 ,酿酒酵母到达稳定期的细胞终浓度也越高 ;而粟酒裂殖酵母生长到达稳定期细胞终浓度随Ca2 浓度增加而增加的效应不明显 .同时SD培养基中外加Ca2 对酿酒酵母的促进作用主要是通过加快生长对数期细胞分裂速度 ;对粟酒裂殖酵母主要是靠缩短生长延滞期来促进增殖 .     

银杏叶提取物对大鼠真皮FB增殖及胶原蛋白分泌的影响

原代培养大鼠真皮成纤维细胞（Fibroblast,FB）传代培养后,以不同浓度的银杏叶提取物（EGb761,20、50、100mg／L）对其生长进行干预,四甲基偶氮唑盐（MTT）法检测药物干预后24、48、72h时FB细胞活力变化,碱水解法测定药物干预后3、6d时培养液中羟脯氨酸含量变化,以分别反映EGb761对大鼠真皮FB增殖及胶原蛋白分泌的影响。研究表明EGB761在一定范围内能促进大鼠真皮FB增殖及及胶原蛋白分泌,且呈一定的剂量、时间相关性。     

Inhibition Mechanism of Emodin on Rabbit Vascular Smooth Muscle Cells Proliferation

ＩｎｔｒｏｄｕｃｔｉｏｎＥｍｏｄｉｎｉｓｏｎｅｏｆｔｈｅａｎｔｈｒａｑｕｉｎｏｎｅｃｏｍｐｏｕｎｄｓ[1] ,ｗｈｉｃｈｅｘｉｓｔｓｉｎｍａｎｙｍｅｄｉｃａｌｐｌａｎｔｓｓｕｃｈａｓｒａｄｉｘ ｐｏｌｙｇｏｎｉｍｕｌｔｉｆｌｏｒｉ,ｒｈｉｚｏｍａｐｏｌｙｇｏｎｉ,ｅｔｃ .Ｅｍｏｄｉｎｉｓａｎｉｍｐｏｒｔａｎｔｃｏｍｐｏｕｎｄｉｎｍｅｄｉｃｉｎｅ .Ｉｔｈａｓｍａｎｙｂｉｏｌｏｇｉｃａｌａｃｔｉｖｉｔｉｅｓｓｕｃｈａｓａｎｔｉ ｉｎｆｅｃｔｉｏｎ ,ｄｅｃｒｅａｓｉｎｇｂｌｏｏｄｌｉｐｉｄｓｃｏｎｔｅｎｔ ,ｅ…     

Regulation of activin A in cell proliferation and hormone secretion by human normal trophoblast cells

Regulation of activin A in cell proliferation as well as hCG and progesterone secretion was investigated using primary cultured cytotrophoblast cells and normal placenta origin cytotrophoblast cell line-NPC cells in serum-free system. It was shown that activin A promoted hCG and progesterone secretion in primary cultured cytotrophoblast cells as well as progesterone secretion in NPC cells, while it had no effect on cell proliferation and hCG secretion in NPC cells. Important evidence is provided for the autocrine regulatory mechanism of activin A on hormone secretion in placental trophoblast cells at early pregnancy.