2-甲氧基雌二醇对骨肉瘤MG63细胞增殖和凋亡的影响及其机制研究

目的探讨2-甲氧基雌二醇(2-methoxyestradiol,2-ME)对骨肉瘤MG63细胞增殖和凋亡的影响及其分子机制。方法不同浓度(0、10、20、40μmol/L)的2-ME作用于肿瘤细胞后,通过显微镜观察细胞形态变化、MTT实验检测增殖抑制、流式细胞术检测细胞周期和凋亡、Western Blot和qRT-PCR实验检测相关分子表达情况。结果随着2-ME浓度的增加,细胞形态学观察显示肿瘤细胞数量逐渐减少、变形;MTT实验表明2-ME对肿瘤细胞增殖抑制作用逐渐增强;流式细胞术结果显示,2-ME能剂量依赖性的诱导肿瘤细胞凋亡,使肿瘤细胞周期阻滞在G_0/G_1期;Western Blot和qRT-PCR实验结果表明,细胞内Bcl-2、VEGF的表达逐渐下降,而Caspase-3的表达则逐渐升高。结论2-ME能抑制骨肉瘤MG63细胞增殖、诱导凋亡,其作用机制可能与Bcl-2、VEGF、Caspase-3相关。     

脑胶质瘤间质内化疗联合用药的敏感性研究

目的 观察卡铂（CBP）、威猛（Vm-26）、甲氨喋呤（MTX）及尼莫地平（NIM）联合应用对脑腔质瘤的抑制作用，探讨脑间质内联合用药与服质瘤细胞敏感性的关系，为临床脑胶质瘤间质内化疗提供有效的化疗依据。方法 40倒脑胶质瘤标母体外细胞培养，采用噻唑兰（MTT）比色法测定体外用药对脑肢质瘤的增殖抑制作用。光镜和电镜下观察药物作用的细胞形态学改变，并采用流式细胞术（FCM）测定药物在脑腔质瘤细胞周期中的分布厦细胞凋亡情况，比较脑胶质瘤细胞对CBP、Vm-26、MTX及NIM单独使用、联合使用的敏感性。结果CBP、Vm-26、MTX、NIM联合用药对肿瘤细胞的抑制率可连06．64％；明显高于CBP＋NIM（69.03％）、Vm-26＋NIM（71.53％）、MTX＋NIM（52.75％）及CBP＋Vm-26＋MTX（78．59％）的抑制效果（P〈0．01），同时CBP、Vm-26、MTX的浓度降低为单独用药的1／10～I／100；光镜和电镜检查发现，在药物作用下肢质瘤细胞形态发生明显改变；FCM检查结果表明，MTX＋CBP＋Vm-26＋NIM联合应用能显著诱导细胞发生凋亡，且主要作用于G2期、S期细胞。结论 联合应用CBP、Vm～26、MTX及NIM对肢质瘤细胞的敏感性明显优于单用药，具有抑制率高、用药浓度低的特点。药物对胶质瘤细胞各个周期均有杀伤力，以G2期和S细胞为主。     

SEC对胶质瘤的体内杀伤及其联合放射治疗的研究

目的：观察SEC活化的淋巴细胞对胶质瘤的体内杀伤作用，并观察SEC联合手术、放疗、化疗对胶质瘤的治疗效果。方法：选用T、B淋巴细胞联合免疫缺陷的SCID小鼠，通过建立荷瘤动物模型及HuPBL-SCID免疫嵌合模型，观察肿瘤生长曲线。荷瘤小鼠生存曲线。30例胶质瘤患者，均经手术切除、放疗、化疗，随机分对照组，处理1组（全身应用SEC），处理2组（局部应用经SEC活化的淋巴细胞）。结合MRI片观察疗效。结果：生长曲线显示：A组、C组、B组对胶质瘤的抑制率分别为58．5％、46．2%和36．3％。生存期曲线显示：对照组（D组）小鼠于第27天开始出现死亡，至第40天全部死亡，而A组小鼠在40天才开始出现死亡，其中有50％的小鼠到观察的最后时期（60天）仍生存良好。临床资料显示对照组有效率为40％，处理1组及处理2组有效率为50％和62．5%。结论：SEC在胶质瘤移植后的人源化免疫嵌合模型（SCID／HuPBL／SHG44）中显示出强大的抗胶质瘤作用。SEC与手术、放疗、化疗结合，能明显提高临床病人的疗效。     

替莫唑胺对胶质瘤侵袭性的影响及可能机制

目的：探讨肿瘤坏死因子-α( tumor necrosis factor-α, TNF-α)在替莫唑胺( temozolomide, TMZ)降低胶质瘤侵袭性过程中的作用及机制。方法对数生长期的C6胶质瘤细胞随机分为TMZ处理10、30、60、120、180和240 min组,每组各15例,动态监测培养液中TNF-α的含量(放射免疫法)、C6细胞内 p53蛋白的表达( Western blot法)和细胞凋亡水平( AnnexinV-FITC法)。制备体外胶质瘤的侵袭模型,利用结晶紫染色法检测胶质瘤的侵袭性。结果 TMZ作用于C6细胞后,培养液中TNF-α的含量明显增加,于120 min时含量最多(P<0．01),其后开始减少。 C6细胞内p53蛋白的表达于处理后120 min达高峰(P<0．01),其后逐渐减少。 TMZ作用于体外胶质瘤侵袭模型后,胶质瘤细胞的侵袭性降低。结论 TNF-α介导TMZ降低胶质瘤侵袭性,此作用可能是由于TMZ促进C6细胞释放TNF-α,增加的TNF-α又促进胶质瘤细胞凋亡所致。     

胶质瘤细胞对卡氮芥、威猛联合的敏感性实验研究

目的:观察卡氮芥(BCNU)、威猛(Vm-26)及尼莫地平(NIM)联合应用对胶质瘤细胞的抑制作用,探讨联合用药剂量与胶质瘤细胞敏感性的关系,为临床提供有效的治疗依据。方法:采用MTT法进行活细胞数测定及-TdR掺入法观察药物对肿瘤细胞增殖率的影响,比较胶质瘤细胞对BCNU、Vm26及NIM单独使用、联合使用的敏感性。结果:BCNU、Vm-26和NIM联合使用对肿瘤细胞的抑制率可达97.04%,明显高于BCNU加NIM的抑制效果(P＜0.01),也明显高于Vm-26、NIM联合的抑瘤效果(P＜0.01)。同时BCNU、Vm-26的浓度降低至单独使用的1/10-1/100。结论:BCNU、Vm-26及NIM联合用药对肿瘤细胞的疗效明显优于单独用药,且具有抑制率高,用药浓度低的特点。     

FM19G11体外增敏替莫唑胺对MGMT阳性恶性胶质瘤细胞系的作用机制

目的 观察FM19G11是否影响人源性恶性胶质瘤细胞系T98G对替莫唑胺(TMZ)的敏感性以及相关的分子机制.方法 以0、0.5、1和2μmol/L的FM19G11和TMZ分别处理以及二者联合处理T98G细胞,用CCK-8法和细胞克隆形成实验检测T98G细胞增殖;用Hoechst 33258染色法检测T98G细胞的凋亡...     

2-氨基嘌呤对apoE~(-/-)小鼠肝功能、肝组织形态及动脉粥样硬化的影响

目的:研究强效真核细胞翻译起始因子2α(eIF-2α)激酶抑制剂2-氨基嘌呤(2-AP)对载脂蛋白E缺陷(apoE-/-)小鼠肝功能、肝组织形态和动脉粥样硬化(AS)的影响。方法:肝脏功能、形态研究:将apoE-/-小鼠随机分为3组,即对照组、200mg/Kg和300mg/Kg 2-AP两药物干预组,30天内每两天用2-AP灌胃一次,分析各组血浆谷丙转氨酶(ALT)和谷草转氨酶(AST)水平、肝组织形态和肝脏重量(干重、湿重、干重/湿重比、湿重/体重比)变化。AS研究:将apoE-/-小鼠随机分为对照组和200mg/Kg 2-AP干预组,后者84天内每两天灌胃一次2-AP。观测两组AS病变面积、血浆总胆固醇(TC)和甘油三酯(TG)含量。结果:200、300mg/Kg 2-AP组apoE-/-小鼠血浆ALT、AST、肝脏重量各指标、肝组织形态与空白对照组比较差异均无统计学意义(P0.05)。200mg/Kg 2-AP组apoE-/-小鼠AS病变面积明显小于对照组,差异有统计学意义(P0.05);血浆TC、TG水平与对照组比较,差异无统计学意义(P0.05)。结论:实验剂量2-AP不影响apoE-/-小鼠肝脏组织形态及功能,其防治AS作用不依赖降低血脂水平。     

2-甲氧雌二醇对人肺癌细胞的放射增敏作用

目的:探讨2-甲氧雌二醇(2-ME)对肺癌细胞A549和GLC-82的放射增敏作用及其对细胞周期的影响,并从分子角度初步探讨2-ME可能的增敏机制。方法:将体外培养的人肺癌细胞A549和GLC-82分为实验组和对照组,其中实验组加入不同浓度的2-ME,对照组不含2-ME。通过MTT法定量检测2-ME对2株细胞增殖的抑制作用,集落形成实验测定2-ME对这2株细胞的放射增敏作用,流式细胞术检测细胞周期分布的变化,通过免疫沉淀法检测细胞周期素依赖性蛋白激酶2(CDK2)活性的改变。结果:分别以2-ME对人肺癌细胞GLC-82和A549的最小有效浓度(0.15625×10-6mol/L和1.25×10-6mol/L)作为放射增敏浓度,均可增加细胞对X线的敏感性,2株细胞存活曲线均见2-ME增敏组比单纯照射组整体下移,D0、Dq值均降低,增敏比:GLC-82细胞为1.98;A549细胞为2.06。细胞周期的检测表明2-ME使细胞周期阻滞于G2/M期,并呈剂量依赖性。2-ME作用后2株细胞CDK2活性均无明显改变。结论:2-ME可通过对细胞周期的调节增加非小细胞肺癌(NSCLC)细胞GLC-82和A549对射线的敏感性。     

2-甲氧基雌二醇对骨髓瘤细胞增殖及c-myc基因表达改变的影响

探讨2-甲氧基雌二醇(2ME2)对骨髓瘤细胞增殖及c-myc基因表达改变的影响。用2ME2分别处理两种骨髓瘤细胞系CZ-1、LP-1 72 h,用锥蓝拒染法检测细胞活力、流式细胞术检测细胞周期分布、RT-PCR和Western blot方法检测c-myc基因mRNA和蛋白的表达改变情况。结果显示,经2ME2处理72 h后,两种骨髓瘤细胞系细胞周期均被阻滞在G1期,细胞的增殖明显受到抑制,c-myc基因mRNA及其蛋白的表达均下调。2ME2可下调c-myc基因mRNA和蛋白的表达并可抑制骨髓瘤细胞的增殖。     

2-乙氧基乙醇对小鼠睾丸毒性作用的病理学研究

2-乙氧基乙醇（2－Ethoxyethanol，EE）属亚乙基二醇烷化酯类，工业上用于合成感光液、生产SP版等。高星等曾对SP版生产线男工进行调查，发现接触EE越多，其精子数量下降越多，并且精子存活率下降、平均精子畸形率上升。本实验观察染毒小鼠睾丸的病理形态学变化，探讨其形态与功能影响的关系。1材料和方法使用北京化工厂生产的EE。取健康昆明种雄性小白鼠40只，体重18～22g。实验动物分为高（485mg·kg-1）、中（243mg·kg-1）、低（12mg·kg-1）3个不同浓度的染毒组及生理盐水阴性对照组。每天腹腔注射1次，连续5天。30天后将小鼠处死…     

反义hTERT/PTEN联合基因治疗恶性胶质瘤的体内外研究

目的探讨腺病毒介导的反义hTERT和野生型PTEN联合基因转染对恶性胶质瘤细胞生长的影响。方法用细菌内高效同源重组系统构建的含有hTERT反义序列及野生型PTEN的腺病毒在体内外单独或联合转染恶性胶质瘤细胞系U251，检测肿瘤细胞生长情况、端粒酶活性、蛋白表达、细胞周期变化等。结果在体外试验中单独或联合转染都能明显抑制肿瘤细胞的生长，以联合转染效果最明显，转染后第6天联合转染组的细胞生存率为37．6％，端粒酶活性水平和hTERT蛋白表达水平明显下降，分别为28．8TPG、0．2106，PIEN蛋白表达水平上升为0．9630；在体内试验中肿瘤生长也明显减慢。结论腺病毒介导的反义hTERT和野生型PTEN联合转染能明显抑制恶性胶质瘤细胞的生长。     

Effect and mechanism of pirfenidone combined with 2-methoxy-estradiol perfusion through portal vein on hepatic artery hypoxia-induced hepatic fibrosis

PurposeThe aim of this study was to explore the effect and mechanism of pirfenidone (PFD) combined with 2-methoxyestradiol (2-ME2) perfusion through portal vein on hepatic artery hypoxia-induced hepatic fibrosis.Materials and methodsSprague-Dawley rats were divided into 5 groups (n ?= ?3/group): control group, hepatic artery ligation (HAL) group, HAL ?+ ?PFD (portal vein perfusion of PFD) group, HAL+2-ME2 (portal vein perfusion of 2-ME2) group and HAL ?+ ?PFD+2-ME2 group depending on whether they received HAL and/or portal vein perfusion (PFD and/or 2-ME2). Livers were harvested for pathology, western blotting (WB), and quantitative real-time PCR (qRT-PCR).ResultsSirius red staining showed that portal vein perfusion of drugs resulted in degradation of liver fibrosis. Immunohistochemistry showed decreased hypoxia-inducible factor-1 α (HIF-1α) and α-smooth muscle actin (α-SMA) after portal intravenous drugs infusion compared with HAL group (P ?< ?0.05). WB analysis showed increased Smad7 in HAL ?+ ?PFD group compared with HAL group (P ?< ?0.05). qRT-PCR analysis showed decreased matrix metallo-proteinase 2 (MMP2), transforming growth factor β1 (TGF-β1), monocyte chemoattractant protein-1 (MCP-1), and Collagen I mRNA in HAL ?+ ?PFD group except for tissue inhibitor of metalloproteinase-1 (TIMP-1) compared with HAL group (P ?< ?0.05). Compared with HAL ?+ ?PFD group, the addition of 2-ME2 did not lead to better results in qRT-PCR analysis.ConclusionsThe portal vein perfusion of PFD significantly reduced the hepatic artery hypoxia-induced fibrosis degree in treated rats by down-regulating the expression of HIF-1α, α-SMA, MMP2, TGF-β1, MCP-1, and Collagen I, as well as up-regulating the TIMP-1 expression and Smad7 protein level. Combined 2-ME2 infusion was not better than PFD alone.     

Cytogenetic effects of cis-platinum(II)diamminedichloride in vivo

The chemotherapeutic agent cis-platinum(II)diamminedichloride (cis-PDD) has been shown to be mutagenic, teratogenic, and carcinogenic. We determined the cytogenetic effects of cis-PDD on human and rabbit lymphocytes in vitro and on rabbit marrow cells, lymph node cells, and lymphocytes in vivo. Lymphocyte cultures from two humans and one rabbit were treated in vitro with cis-PDD. For in vivo studies, five New Zealand white rabbits were given iv injections of cis-PDD. Posttreatment blood samples were withdrawn for analysis and rabbits were sacrificed at either 6 or 24 hr for cytogenetic analysis of marrow and node cells. Sister chromatid exchange (SCE) analysis of human and rabbit metaphases from lymphocytes treated in vitro showed that rabbit lymphocytes are more sensitive to SCE induction by cis-PDD. Significant increases in SCE were observed in lymphocyte cultures obtained as early as 1 hr post treatment from injected rabbits. Analysis of node, marrow, and lymphocyte metaphases from injected rabbits showed a high number of chromosome aberrations in these cells with bone marrow showing a delayed response to treatment. These results indicate that cis-PDD is clastogenic in hematopoietic tissues in vivo and that SCE methodology may be useful in monitoring patients receiving cis-PDD therapy.     

联合输注Flt3-L和GM-CSF表达质粒体内诱导小鼠DC的研究

目的:探讨联合输注Flt3-L和GM-CSF真核表达质粒体内诱导小鼠DC的方法并检测其抗原提呈功能。方法:采用尾静脉注射法分别输注Flt3-L及GM-CSF质粒,15d后收获小鼠脾脏;采用流式细胞术检测小鼠脾细胞CD11c、CD11b、B220、CD8α和NK1.1等表面标志以鉴定DC的比例及亚型;将体内诱导DC与HBsAg共孵育,然后刺激HBsAg预免疫小鼠的脾细胞并检测其分泌IFN-的水平。结果:联合输注Flt3-L和GM-CSF质粒小鼠的脾细胞达到4×108个细胞/脾脏、其中CD11c+细胞占20%以上,CD11c+细胞中CD11b+、CD11b-、B220+、CD8+、NK1.1+细胞的比例分别达17%、10%、26%、16%、7%左右;体内诱导DC负载HB-sAg后刺激HBsAg特异性淋巴细胞分泌IFN-γ的水平明显高于HBsAg单独刺激组。结论:采用小鼠尾静脉注射技术输注Flt3-L及GM-CSF质粒能够诱导CD11c+ DC的产生并包含多种亚型,体内诱导DC具备有效的抗原提呈功能。     

Inhibition of non-quantal acetylcholine leakage by 2(4-phenylpiperidine)cyclohexanol in the mouse diaphragm

The drug 2(4-phenylpiperidine)cyclohexanol (AH 5183) caused hyperpolarization by 1.8 +/- 0.6 mV in an end-plate zone of mouse diaphragm fibers without any change in the amplitude of miniature end-plate potentials. This supports the idea that the drug inhibits the non-quantal leakage from motor nerve terminals, probably at those parts of the nerve terminals which were incorporated into the terminal membrane after vesicle exocytosis.     

Lupus manifestations in severe combined immunodeficient (SCID) mice and in human/mouse radiation chimeras

The objective of this study was to develop a human lupus model. To this end we have established and compared two models: (1) severe combined immunodeficient (SCID) mice reconstituted with peripheral blood lymphocytes (PBL) of either systemic lupus erythematosus (SLE) patients or healthy controls and (2) lethally irradiated BALB/c mice radioprotected with bone marrow of SCID mice, to which human PBL were transferred (human/mouse chimera). Engraftment was successful in most (78.4%) recipient mice as determined by the levels of human IgG measured. In about 50% of either SCID mice or human/mouse chimeras that were successfully engrafted with PBL of SLE patients, significant anti-dsDNA autoantibodies, mostly of the IgG1 and IgG2 isotypes, were determined. Interestingly, in a significant number (84.5%) of recipients of PBL of the healthy controls, anti-dsDNA antibodies were observed as well, suggesting that PBL of at least some of the healthy controls have the potential to develop SLE-associated autoantibodies under the appropriate stimulatory conditions. Glomerular immune deposits (human IgG, mouse C3) were detected in 70–80% of SCID mice with human DNA specific antibodies and in a third of the human/ mouse chimeras. Thus, SLE serology and glomerular pathology were reproducibly demonstrated in two models of human SLE. These models should allow the evaluation of potential therapies for the treatment of lupus patients.     

Tissue-specific expression of B-cell translocation gene 2 (BTG2) and its function in T-cell immune responses in a transgenic mouse model

B-cell translocation gene 2 (BTG2) belongs to the anti-proliferativegene family. According to previous in vitro studies, BTG2 overexpressionleads to delayed cell cycling. We investigated BTG2 expressionduring mouse ontogeny and its immune and circadian functionsin this study. In situ hybridization showed that BTG2 was expressedat high levels in the central nervous system, liver, stomach,thymus, spleen, skin, adrenal gland, pituitary gland and salivaryglands during embryonic days (E10–E17), postnatal days(P1 and P10) and adult stages. Expression was observed in organsand tissues from adult mice with and without a robust proliferationprogram. Thus, the gene might have important functions thatare both related and unrelated to proliferation. BTG2 expressionwas induced after in vitro T-cell receptor stimulation in Tcells using anti-CD3 antibodies. However, transgenic (Tg) micewith actin promoter-driven expression of BTG2 showed normalin vitro and in vivo T-cell responses, such as thymus development,T-cell activation marker expression, T-cell proliferation andmigration, as well as in vivo delayed-type hypersensitivityreactions. Although BTG2 was expressed in the suprachiasmaticnucleus and pineal gland in the brain, BTG2 Tg mice had no abnormalcircadian behavior. Our data on BTG2 expression during ontogenyprovide useful clues for the further investigation of BTG2 function.Additional studies are warranted to examine its role in immuneand other systems.     

中国野生小鼠1号染色体遗传结构研究

目的 对我国25个不同地区野生小鼠遗传多样性进行检测.方法 本研究利用1号染色体上20对微卫星标记,采用多重PCR技术对我国25个不同地区野生小鼠遗传多样性进行检测,统计了野生小鼠群体等位基因数、期望杂合度、等位基因范围、G-W统计量以及群体间的遗传距离.利用MEGA软件进行系统发生分析.结果 野生小鼠群体在20个STR位点上平均等位基因数为(15.4±3.5),而实验小鼠群体在20个STR位点上平均等位基因数为(5.3±1.0).而对于两个群体之间的平均期望杂合度来说,野生小鼠群体(0.886±0.04)明显高于实验小鼠群体(0.727±0.112)(t=-6.7025,P=1.04×10-6).在野生小鼠群体中20个STR位点的G-W统计量为(0.781±0.132),明显高于实验小鼠群体的G-W统计量(0.377±0.184)(t=-8.8744,P=1.76×10-8).结论 这些指标都说明野生小鼠具有更丰富的遗传多样性.根据不同野生小鼠群体与实验小鼠群体间的聚类分析,显示野生小鼠群体与实验小鼠群体明显聚为两个类别.

Abstract：

Objective Recent studies have shown that the genetic diversity of wild mouse (Mus musculus )is far more extensive than the laboratory mice, and these diversities are useful for genetic studies of complex traits and can be important genetic resources. This sutdy is to detect the diversity of wild mice captured in 25 regions of China. Methods In this study, 20 STR loci of Chr1 were selected and analyzed, using multiplexed tandem PCR ( MT-PCR ) technology to detect the diversity of wild mice captured. The allele number, expected heterozygosity, allele range, G-W index of wild mice, and genetic distance between wild mouse populations were calculated by Alequin software. MEGA software is used for phylogenetic analysis. Results Wild mice had number of alleles of ( 15.4±3.5 ) for 20 STR loci,while the laboratory mcie had (5.3 ± 1.0 ) alleles. The wild had higher level of average expected heterozygosity (0. 886 ±0.04 ) for 20 STR loci than the laboratory derived hybrids (0. 727 ± 0. 112 ),(t= -6.7025, P=1.04 × 10-6). The G-W stat of 20 STRloci were (0.781 ± 0. 132) in wild population, significantly higher than those of laboratory mice whose G-W stat were (0.377± 0. 184 )(t= -8.8744, P=1.76×10-8). Conclusion Wild mouse (Mus musculus) has more extensive genetic diversity. According to the cluster analysis between wild mice and laboratory mice, they were clearly clustered into two categories.