顺铂通过p66Shc-线粒体信号通路诱导人肾小管上皮细胞凋亡

 目的：探讨p66Shc-线粒体信号通路在顺铂诱导的人肾小管上皮细胞凋亡中的作用。方法：体外培养人肾小管上皮细胞,Western blotting法检测顺铂对p66Shc及磷酸化p66Shc(Ser36)蛋白表达的影响,然后将细胞分为对照组、顺铂组及顺铂+p66ShcS36A（第36位Ser突变为Ala的p66Shc）组,用激光共聚焦显微镜观察p66Shc对顺铂诱导的细胞活性氧簇、线粒体活性氧簇及细胞凋亡的影响,Western blotting法检测线粒体凋亡信号通路相关蛋白的表达。结果：顺铂促进p66Shc蛋白磷酸化,对p66Shc蛋白表达无影响;顺铂诱导细胞凋亡,细胞及线粒体活性氧簇产生增加,细胞色素C释放,caspase-9表达增加,p66ShcS36A可以抑制顺铂诱导的细胞氧化损伤及凋亡。结论：顺铂通过p66Shc-线粒体信号通路诱导人肾小管上皮细胞凋亡。     

重组腺病毒Ad-IL-12感染淋巴细胞对人卵巢癌细胞SKOV3增殖和凋亡的影响

目的 探讨重组腺病毒Ad-IL-12感染人外周血淋巴细胞后对人卵巢癌细胞SKOV3周期、增殖和凋亡的影响.方法 采用重组人IL-12腺病毒(Ad-IL-12)感染人外周血淋巴细胞,感染48 h后与人卵巢癌细胞SKOV3共培养(SKOV3/Ad-IL-12),同时设未感染组(SKOV3)和Ad-GFP空载感染组(SKOV3/Ad-GFP)作为对照.RT-PCR检测IL-12双亚基P35和P40的mRNA表达,ELISA检测细胞培养上清中IL-12 P70蛋白的分泌,CCK8法检测卵巢癌细胞增殖,流式细胞术分析卵巢癌细胞周期和凋亡,RT-PCR和Western blot检测抗凋亡蛋白BCL-2、survivin 的表达.结果 重组腺病毒Ad-IL-12可成功感染人外周血淋巴细胞,分泌较高水平的IL-12 P70蛋白;淋巴细胞过表达IL-12可抑制SKOV3细胞的增殖;与未感染组和空载组相比,SKOV3/Ad-IL-12组G1期细胞显著增加,凋亡率显著升高(P＜0.05),SKOV3细胞中抗凋亡蛋白BCL-2和survivin显著下调(P＜0.05).结论 人IL-12基因重组腺病毒可以成功感染人外周血淋巴细胞,分泌IL-12 P70蛋白,并可以通过阻滞细胞周期进程和促进细胞凋亡抑制人卵巢癌细胞SKOV3的增殖.     

重组腺相关病毒介导黑色素瘤分化相关基因7对人肝癌细胞体内外抑制效应的研究

目的 观察PEG启动子调控腺相关病毒介导的黑色素瘤分化相关基因7(MDA-7)在体内外抑制肝癌细胞的生物学活性,探讨重组腺相关病毒介导MDA-7基因用于肝癌基因治疗的应用前景.方法 构建重组腺相关病毒rAAV-PEG-MDA-7表达系统,体外感染人肝癌细胞株HepG2细胞.Western印迹检测转染细胞内MDA-7蛋白;MTT法检测细胞增殖抑制率;流式细胞术分析细胞周期和细胞凋亡变化.构建肝癌细胞HepG2裸鼠皮下移植瘤模型,尾静脉注射rAAV-PEG-MDA-7,观察其对肝癌生长的抑制作用;ELISA方法检测血浆MDA-7蛋白浓度;TUNEL法分析MDA-7对肿瘤细胞的凋亡诱导情况;免疫组织化学分析MDA-7在肿瘤组织中的表达.结果 重组腺相关病毒rAAV-PEG-MDA-7可特异性转染HepG2细胞,MDA-7蛋白在HepG2细胞中高效表达,并呈时间依赖性.重组腺相关病毒rAAV-PEG-MDA-7可抑制HepG2细胞增殖并诱导其凋亡,GO/G1期细胞百分比明显增多,G2/M期的细胞显著减少(P＜0.05).全身系统性给予rAAV-PEG-MDA-7后,血清中可持续检测到MDA-7蛋白,且注射后2周浓度达高峰(200 ng/m1);肿瘤生长受抑制,抑瘤率为62%(P＜0.05);免疫组化结果显示MDA-7在肿瘤组织中表达;TUNEL结果显示rAAV-PEG-MDA-7可诱导肿瘤细胞凋亡.结论 构建出的重组腺相关病毒rAAV-PEG-MDA-7表达系统具有良好的肿瘤靶向性,通过抑制肝癌细胞增殖和诱导其凋亡发挥抗肝癌作用.     

膀胱癌中衔接蛋白p66Shc的表达及临床意义

目的观察p66Shc蛋白在膀胱癌中的表达,分析其表达与膀胱癌临床分期、病理类型、转移等的关系,以及p66Shc影响膀胱癌细胞增殖、凋亡的分子机制。方法应用免疫组化法和Western blot技术检测32例膀胱癌中p66Shc蛋白的表达,分析p66Shc蛋白表达和临床病理特征的关系。Western blot技术检测T24细胞中PKCβ和p66Shc蛋白及其磷酸化水平,以及p53、Bax和BCL-2蛋白水平。结果膀胱癌组织中p66Shc阳性率为65.6%(21/32),其在T3+T4期膀胱癌中的表达显著高于T1+T2期膀胱癌(P0.05);p66Shc表达与患者年龄、性别、分化程度无关(P0.05)。LY333531下调T24细胞中PKCβ和p66Shc蛋白及其磷酸化水平,以及下调p53、Bax和上调BCL-2的蛋白水平。结论 p66Shc蛋白在膀胱癌中的表达与膀胱癌临床分期相关,随着pTNM分期增加而表达增多。p66Shc磷酸化促进肿瘤细胞的增殖和凋亡。     

p53正向凋亡调节因子重组腺病毒载体的构建及对胰腺癌细胞增殖的抑制作用

目的 构建p53正向凋亡调节因子重组腺病毒(Ad-PUMA)载体,探讨Ad-PUMA对人体内外胰腺癌的治疗作用.方法 利用Ad-Easy系统在大肠杆菌同源重组,构建Ad-PUMA腺病毒载体,在293细胞内成功包装并鉴定后,以Ad-PUMA转染人转移胰腺癌细胞株AsPC-1.用MTT法检测转染前后存活细胞,观察Ad-PUMA的体外抑瘤作用;用Western blot方法鉴定转染前后AsPC-1细胞内PUMA蛋白表达.通过裸鼠皮下胰腺癌移植瘤模型观察Ad-PUMA的体内抑瘤效果;Westem blot方法鉴定Ad-PUMA转染后肿瘤组织内PUMA蛋白表达,TUNEL(terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling)法检测肿瘤组织细胞凋亡.结果 在体外,随着Ad-PUMA感染剂量增加,细胞内PUMA蛋白表达逐渐增加,细胞存活率逐渐下降;在体内,Ad-PUMA可显著抑制裸鼠皮下肿瘤的生长,其抑瘤率为44.2%,瘤体组织PUMA表达水平及凋亡指数明显升高.结论 PUMA抑制体内外胰腺癌细胞增殖,可能是一种潜在的肿瘤生物治疗手段.     

腺病毒介导的PML(NLS-)过表达对白血病NB4细胞增殖和凋亡的影响

目的 利用腺病毒介导PML(NLS-)的过表达,探讨PML(NLS-)对NB4白血病细胞增殖凋亡的影响.方法 重组腺病毒质粒Ad-PML(NLS-)经Pac Ⅰ酶切线性化后转染AD293细胞,获得重组腺病毒Ad-PML(NLS-),经4轮扩增后,测定重组腺病毒滴度;重组腺病毒感染NB4细胞,荧光显微成像和流式细胞术检测感染效率,RT-PCR法和Western blot法检测NB4细胞中PML(NLS-)的转录及表达水平,MTT实验观察细胞增殖,流式细胞术分析细胞周期及凋亡.结果 重组腺病毒质粒Ad-PML(NLS-)经酶切和测序鉴定正确,经4轮扩增病毒滴度为1 ×1010pfu/mL;重组腺病毒Ad-PML(NLS-)感染NB4细胞效率达75％,PML(NLS-)基因可在NB4细胞中高效表达;NB4细胞增殖活力明显高于未感染组和空病毒载体感染组(P＜0.05),S期细胞比例明显增高(P＜0.05),G2期细胞比例明显减少(P＜0.05);实验组细胞凋亡率明显低于空病毒感染组(P＜0.05).结论 PML(NLS-)过表达能够促进白血病NB4细胞的体外增殖能力,抑制其凋亡.     

rBTI蛋白对人肝癌HepG2细胞增殖、迁移及ROS产生的影响

目的:研究重组荞麦胰蛋白酶抑制剂(rBTI)对HepG2细胞增殖、凋亡、迁移及胞内活性氧(ROS)的影响。方法:以不同浓度的rBTI蛋白(1.25 10μmol/L)作用肝癌HepG2细胞,通过MTT比色法检测rBTI蛋白对HepG2细胞增殖抑制作用;DAPI荧光染色法观察凋亡细胞的发生及形态学变化,流式细胞术(FCM)检测细胞内活性氧变化、划痕擦伤实验检测rBTI蛋白对HepG2细胞迁移的影响。结果:rBTI蛋白对HepG2细胞的生长具有明显的抑制作用,并呈剂量依赖效应,但对正常细胞HL-7702影响很小;DAPI染色发现细胞产生凋亡小体;细胞内活性氧水平检测结果表明rBTI可以诱导HepG2细胞内ROS水平显著升高;划痕擦伤实验显示rBTI在一定浓度范围内可以抑制HepG2细胞迁移。结论:rBTI蛋白能够诱导HepG2细胞发生凋亡并抑制细胞增殖和迁移。     

p16和p27表达对前列腺癌细胞系的作用

目的 探讨p16和p27Kip1 基因蛋白对抑制前列腺癌细胞增殖和调控其凋亡的作用.方法 构建携带人P16和p27基因的腺病毒载体,转染体外培养的前列腺癌细胞系PC-3,采用RT-PCR、Western blot检测目的 基因的表达.通过细胞生长试验、流式细胞仪检测PC-3转染前后细胞增殖和凋亡的变化.结果 病毒滴度Ad-p16为115×10^8 pfuPml 、Ad-p27为112×10^9 pfuPml ,RT-PCR 检测可见p16-mRNA（520bp）和p27Kip12mRNA （320bp）表达,Western blot检测有p16 蛋白（65KD）和P27蛋白（27KD） 特异表达,并可明显抑制PC-3细胞的增殖,诱导其凋亡,联合基因治疗组与单基因组相比差异显著（P＜0.01）.结论 p16和p27可明显抑制前列腺癌细胞株PC-3的增殖,增加细胞的凋亡,转染携带p16和p27的重组腺病毒载体有望成为治疗前列腺癌的有效方法.     

Ad-siRNA-FoxO1腺病毒载体的构建及对人肝癌细胞系增殖的影响

目的 利用小干扰RNA(small interfering RNA,siRNA)抑制肝癌细胞系中FoxO1基因的表达,观察其受抑制后对细胞增殖能力的影响.方法 设计针对FoxO1基因的siRNA,构建于腺病毒穿梭载体pAdTrack-CMV中,与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组,转染包装细胞293细胞,获得含Ad-siRNA-FoxO1的重组腺病毒.体外感染人肝癌细胞系HepG2,Western印迹检测Ad-siRNA-FoxO1对FoxO1蛋白的抑制效率.与PEPCK.luc共转染HepG2细胞,检测FoxO1表达水平对PEPCK启动子活性的影响,并观察FoxO1表达水平的改变对肝癌细胞系增殖的影响.结果 成功构建3个针对FoxO1的siR-NA重组腺病毒载体,重组腺病毒均能显著抑制HepG2细胞中FoxO1蛋白的表达,并抑制PEPCK启动子的活性和显著促进细胞增殖.结论 肝癌细胞系中FoxO1参与了细胞增殖的调节.     

Pre-miR-10a重组腺病毒载体对K562细胞增殖的抑制作用

目的:观察在K562细胞高表达miR-10a对细胞增殖及凋亡的影响。方法:用pAd-pre-miR-10a重组腺病毒载体感染K562细胞,采用RT-PCR检测感染后K562细胞中miR-10a和bcr/abl融合基因的表达水平,Western blot法检测BCR/ABL融合蛋白表达水平,MTT、流式细胞术(FCM)分别检测感染后K562细胞的增殖及凋亡。结果:与对照组相比,K562细胞在转染pAd-pre-miR-10a重组腺病毒载体后,其miR-10a表达水平明显升高、bcr/abl融合基因水平显著降低;BCR/ABL融合蛋白表达明显下调、K562细胞增殖活力被抑制、细胞凋亡增加,差异均具有统计学意义(P<0.05)。结论:pAd-pre-miR-10a重组腺病毒载体能明显抑制K562细胞的增殖、促进细胞凋亡。     

Immunoregulatory function of mesenchymal stem cells

Mesenchymal stem cells (MSC) are a rare subset of stem cells residing in the bone marrow where they closely interact with hematopoietic stem cells and support their growth and differentiation. MSC can differentiate into multiple mesenchymal and non-mesenchymal lineages, providing a promising tool for tissue repair. In addition, MSC suppress many T cell, B cell and NK cell functions and may affect also dendritic cell activities. Due to their limited immunogenicity, MSC are poorly recognized by HLA-incompatible hosts. Based on these unique properties, MSC are currently under investigation for their possible use to treat immuno-mediated diseases. However, both their condition of immunoprivilege and their immunosuppressive function have recently been challenged when analyzed under particular experimental conditions. Thus, it is likely that MSC effects on the immune system may be deeply influenced not only by cell-to-cell interactions, but also by environmental factors shaping their phenotype and functions.     

Influences of the pia mater on the precursors of nerve cells

Summary In the developing cerebellum of 8-day old rats surgical lesions were made. During regeneration of the cerebellum the pia mater was found to penetrate inside the neural tissue. Partially differentiated Purkinje cells and granule cells, that were in close contact with the pial cells, were found atrophied. When the profilerative cells of external granular layer came into contact with the pial cells, they were reduced to a primitive type of epitheloid cells. In this instance epithelio-mesenchymal interaction was found deleterious to the precursors of neurons. However, when the epithelioid cells were freed from the contact with the pial cells by intervening basement membrane, they differentiated into ependymal cells. Such ependymal cells gave rise to small as well as large new ventriculer structures, and structures resembling chorioid plexus.This research was supported by NIH Research Grant NS08817-03. Acknowledgements are due to Sheila Anderson for histology and to Donna Whitehurst for photographic work.     

Genetic aspects of the immunomodulationg action of interferon

Laboratory of Antiviral Immunity, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. D. Solov'ev.) Translated from Byulleten' Éksperimentl'noioi Biologii i Meditsiny, Vol. 105, No. 4, pp. 459–461, April, 1988.     

干细胞若干定义的相关探讨

背景：近10年来干细胞研究所取得的巨大进展,不仅影响和促进了生物学及其相关基础科学,而且还在医学、药物开发、农业等许多领域得到广泛的应用,目前已成为研究的热点问题。 目的：在干细胞的定义上还存在一些需要探讨的问题,明确定义将有利于干细胞研究的快速发展。 方法：回顾干细胞的发展和概念提出,特别是通过中英文权威定义的比较,提出作者的看法。 结果与结论：干细胞的定义上还存在一些问题值得探讨,特别是一些中英文表述不同影响了理解。例如,“多能干细胞”对应译成两个单词：Pluripotent Stem Cell和Multipotent Stem Cell,然而Pluripotent和Multipotent两个单词的英文意义不同。为了学术交流和沟通,作者提出将Pluripotent Stem Cell称为“万能干细胞”,而保留Multipotent Stem Cell为“多能干细胞”的定义。以上结论供广大同仁探讨,以利于抛砖引玉。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Identification of a CD8α Dendritic Cell Subpopulation in Rat Spleen and Evaluation of Its OX-62 Expression

Rat spleen DC and bone marrow-derived DC were isolated and characterized by morphology and flow cytometry. We found a CD8α⁺ DC subpopulation representing 19–48% (27.4 ± 12.0) of total spleen DC. The OX-62 expression on total spleen DC was 41–59% (51.8 ± 7.5). Myeloid bone marrow-derived DC were negative for CD8α and OX-62. We demonstrated the coexpression of CD8α and OX-62 molecules, at least in a portion CD8α⁺ spleen DC. Both CD8α⁺ and CD8α⁻ spleen DC subpopulations separated by MACS were able to induce an in vivo primary immune response to OVA. The immune response induced by the CD8α⁻ DC subpopulation was higher (P < 0.05). We identified a CD8α⁺ DC subpopulation in rat spleen less effective in inducing an immune response than CD8α⁻ DC. Moreover, our results suggest the presence of DC subpopulations with different lineages in DC preparations based on OX-62 expression.     

Accessory phenotype and function of macrophages induced by cyclic adenosine monophosphate

Mature macrophages (Mph) differentiated in culture from normal human peripheral blood monocytes (Mo) exhibit low activity as accessory cells (antigen-presenting cells) in T lymphocyte stimulation. A test system was established based on mitogenicity to quantitate the accessory activity of Mph-derived cells and to follow its changes for several days. The system used accessory cells treated with the oxidative mitogen, sodium periodate. The cells were subsequently co-cultured with pooled human lymphocytes from a cryopreserved stock. DNA synthesis in these cells was used as an indicator of accessory activity. Mph could be converted within 5-6 days into highly active accessory cells if a continuous stimulus of exogenously added dibutyryl cyclic AMP (db-cAMP) was provided. Mph treated by db-cAMP retained a high degree of HLA-DR expression but typical Mph markers such as non-specific esterase, phagocytosis, and expression of Fc-receptors were down-regulated. Acid phosphatase and myeloperoxidase underwent only slight changes, while the monocyte marker 5'-nucleotidase remained undetectable. Morphologically, the cells rounded up and developed veils and dendritiform elongations. In contrast to dendritic cells, Mph-derived accessory cells retained the CD14 antigen characteristic of monocytes and Mph. It is concluded that Mph are able to respond to exogenous stimuli and to convert into a highly active accessory cell. This contrasts to the well-known state of the 'activated Mph' with respect to markers and function. Both states appear to be antagonistically controlled by intracellular second messengers, as the accessory cell phenotype is positively correlated with intracellular cyclic AMP increase, whereas Mph activation correlates with cyclic GMP increase.     

Human Osteogenic Sarcoma: Fine Structure of the Osteoblastic Type

The fine structure of representative regions of 13 osteoblastic osteogenic sarcomas was studied. These regions contained four morphologically distinguishable subtypes of osteoblastlike cells. In addition, fibroblastlike and chondroblastlike cells were present, along with multinucleated giant cells, leukocytes, macrophagelike cells, and small populations of histogenetically unclassifiable (but probably neoplastic) cells.

The morphologic evidence was compatible with the view that the variations in appearance among the subgroups of osteobl astlike cells reflected differences in maturation and differentiation of these cells. In at least one subgroup, the morphologic findings suggested that the ceils were capable of manufacturing a secretory product. The multinucleated giant cells occurring in genuine tumor areas appeared to be closely related to neoplastic osteoblasts.

The presence of chondroblastlike cells in the tissues illustrates that cells with a diverging differentiation can occur in an osteoblast-dominated cell population. This agrees with the view that the neoplastic cells originate from a mesenchymal stem cell with potential for multifaceted differentiation.     

Trypanosoma cruzi calreticulin: In vitro modulation of key immunogenic markers of both canine tumors and relevant immune competent cells

Recombinant calreticulin from Trypanosoma cruzi (rTcCalr), the parasite responsible for Chagas’ disease, binds to Canine Transmissible Venereal Tumor (CTVT) cells from primary cultures and to a canine mammary carcinoma cell line. A Complement-binding assay indicated that interaction of the first component C1q with these tumor cells operated independently of the rTcCalr-presence. This apparent independence could be explained by the important structural similarities that exist among rTcCarl, endogenous normal canine and/or mutated calreticulins present in several types of cancer. In phagocytosis assays, tumor cells treated with rTcCalr were readily engulfed by macrophages and, co-cultured with DCs, accelerated their maturation. In addition, DCs maturation, induced by tumor cells co-cultured with rTcCalr, activated T cells more efficiently than DCs, treated or not with LPS. In an apparent paradox, a decrease in MHC Class I expression was observed when these tumor cells were co-cultivated with rTcCalr. This decrease may be related to a down regulation signaling promoting the rescue of MHC I. Possibly, these in vitro assays may be valid correlates of in vivo sceneries. Based on these results, we propose that rTcCalr improves in vitro the immunogenicity of two widely different tumor cell lines, thus suggesting that the interesting properties of rTcCalr to boost immune responses warrant future studies.     

含噻唑烷-4-酮的糖类衍生小分子免疫调节剂对人淋巴细胞分化的影响

目的 研究含噻唑烷-4-酮的糖类衍生小分子免疫调节剂对人外周血淋巴细胞活化与分化的影响.方法 从健康成年人分离外周血单个核淋巴细胞,经ConA+免疫调节剂CH1a、CH2a、CH1b CH2b和匹多莫德(pidotimod)刺激48 h后,收集培养上清,ELISA法检测IL-2、IL-4和IFN-γ含量；72 h后MTT法检测细胞增殖率；经ConA+免疫调节剂CH1a、CH2a、CH1b CH2b和pidotimod刺激,孵育72 h后收获细胞,利用流式细胞技术检测细胞表面黏附分子.结果 所有样品均可促进T细胞增殖；免疫调节剂CH1a、CH2a和pidotimod上调CD3、CD4、CD19和CD16CD56的表达并促进IL-2和IFN-γ分泌.免疫调节剂CH1b、CH2b上调CD3、CD4、CD19和CD16CD56的表达并促进IL-2和IL-4分泌.结论 免疫调节剂CH1a、CH2a和pidotimod有可能诱导Th0向Th1分化并可提高B细胞和NK细胞增殖活化水平.而CH1b、CH2b则有可能诱导Th0向Th2分化并提高B细胞和NK细胞的增殖活化水平.