RNA干扰靶向沉默COX 2基因对MG 63骨肉瘤细胞生物学特性的研究

目的 通过构建小干扰RNA(small interfering RNA,siRNA)降低MG 63骨肉瘤细胞环氧合酶 2(COX 2)基因的表达,并进一步研究其对MG 63骨肉瘤细胞增值、侵袭、迁移能力的影响及分子机制。方法 设计靶向干扰COX 2基因的siRNA,通过脂质体转染MG 63骨肉瘤细胞,使其抑制MG 63骨肉瘤细胞COX 2基因的表达,后采用噻唑蓝(MTT)比色法、Transwell小室实验研究其对MG 63骨肉瘤细胞增殖、侵袭、迁移能力的影响,采用RFQ PCR和Western blot分别从基因和蛋白的水平检测MG 63骨肉瘤细胞侵袭性相关因子基质金属酶(MMP 9)的表达及血管内皮生长因子(VEGF)的表达。结果 转染MG 63骨肉瘤细胞后,实验组与阴性对照组和空白对照组比较,通过RFQ PCR和Western blot检测COX 2基因表达降低约90%(P0.05),MTT检测MG 63骨肉瘤细胞增值能力明显受到抑制(P0.05),Transwell实验检测MG 63骨肉瘤细胞侵袭、迁移能力明显下降(P0.05),经RFQ PCR、Western blot检测侵袭性相关因子MMP 9和血管内皮生长因子VEGF的mRNA及蛋白表达降低(P0.05)。空白对照组和阴性对照组比较无明显变化,差异无统计学意义(P0.05)。结论 人MG 63骨肉瘤细胞COX 2基因被抑制后,MG 63骨肉瘤细胞增值、侵袭、迁移能力明显下降。     

两种新生大鼠皮质发育障碍模型的差异基因表达谱研究

目的研究两种不同的方法构建皮质发育障碍(DCDs)动物模型中的共同差异表达基因,为进一步研究DCDs形成机制提供基因水平筛选的研究平台。方法用两种方法制备DCDs模型:①射线损伤模型(射线组):采用剂量为1.45 Gy的γ射线照射妊娠15 d的SD大鼠制作子代大鼠DCDs模型;②卡莫司汀(BCNU)药物损伤模型(药物组):妊娠15 d的SD大鼠,腹腔注射BCNU制作子代大鼠DCDs模型。同时设正常对照组。对两种DCDs模型的子代新生鼠(P0)全脑做基因芯片扫描,结果与正常对照组比较,获得两种模型共有的差异基因。结果射线组大鼠与正常比较得到170个差异基因,其中25个上调,145个下调;药物组大鼠与正常比较得到259个差异基因,其中67个上调,192个下调。两组重合的基因共54个,其中3个上调,51个下调。结论皮质发育障碍是一个复杂的病理过程,本实验运用基因芯片技术,对两种不同方法构建的DCDs模型进行了研究,获得两种DCDs模型共有的基因差异表达谱,为进一步研究DCDs形成机制提供了分子生物学平台。     

基因组研究离诠释生命奥妙还有多远

基因组研究表明，人类与黑猩猩和老鼠的基因序列98％以上是相同的，是否是这1％～2％的基因的差异，就决定了人与黑猩猩和老鼠在表现型方面很大的差异?本文通过大量分析表明，由碱基(5个)→核酸[DNA→RNA(mRNA、rRNA、tRNA)]→氨基酸(22种)→蛋白质(数百千种)→染色体→基因组→性状，其遗传变异信息有逐级放大的过程。这是生物间基因序列虽然有98％甚至99％的相同，但表现型差异却较大的原因所在。基因序列完全相同，并不能说明基因功能相同，基因组研究的结果不能完全诠释基因功能表达的复杂过程。今后必将由序列基因组学→结构基因组学→功能基因组学→蛋白质组学深入发展，必须从网络层次研究基因表达，才能揭示生命的奥妙。另外我们使用的各种基因组研究技术和软件还有待改进，才能真正反映生物基因的差异。     

慢病毒介导的α-烯醇化酶基因沉默对宫颈癌细胞化疗药物敏感性的影响

目的探讨沉默α-烯醇化酶(ENO1)基因对宫颈癌SiHa细胞化疗药物敏感性的影响。方法采用慢病毒介导的RNA干扰技术,建立ENO1基因稳定沉默SiHa细胞系;通过western blot方法验证ENO1的表达水平;ENO1基因沉默后运用细胞增殖实验检测其对细胞增殖能力的影响;采用MTT的方法评价沉默ENO1基因的SiHa细胞对紫杉醇和顺铂的敏感性。结果成功建立ENO1基因沉默的SiHa细胞系;沉默ENO1基因表达后,SiHa细胞增殖能力下降;沉默ENO1基因能够增强SiHa细胞的药物敏感性。结论:ENO1能够影响宫颈癌细胞的增殖及其对化疗药物的敏感性。     

脑组织特异性血管生成抑制因子1(BAI1)在肾癌中的表达及临床相关性研究

目的观察脑组织特异性血管生成抑制因子1(BAI1)基因在不同病理类型肾癌组织及不同分级分期肾癌中的表达情况,探讨BAI1在肾癌发生、发展中的作用。方法采用免疫组织化学PV9001二步法检测27例肾脏癌周组织、60例透明细胞癌、15例乳头状细胞癌、13例嫌色细胞癌中BAI1基因的表达情况,结合病理学分级进行统计学分析。结果 1)肾癌细胞中BAI1基因高表达率显著低于癌周组织,分别为31.8%(28/88),70.4%(19/27);肾透明细胞癌、乳头状细胞癌及嫌色细胞癌较癌周组织BAI1基因表达均降低,分别为35.0%(21/60),26.7%(4/15),23.1%(3/13),70.4%(19/27)。2)BAI1基因在透明细胞癌不同分期的高表达率为Ⅰ期63.6%(7/11),Ⅱ期47.4%(9/19),Ⅲ期18.2%(4/22),Ⅳ期12.5%(1/8),BAI1基因表达率与肾透明细胞癌分期成负相关,有显著统计学差异(P0.05)。结论 BAI1基因在肾癌中的表达与病理分级、临床分期和淋巴转移相关,可能与肾癌的发生、发展有重要关系。     

基于DNA技术的对称加密方法

DNA密码是伴随着DNA计算的研究而出现的密码学前沿领域．文中结合现代基因工程技术和密码学技术设计了一个对称加密系统——DNASC．在DNASC中，加密钥和解密钥是DNA探针，密文是特殊设计的DNA芯片．系统的安全性主要基于生物学困难问题而不是传统的计算问题，因而对未来的量子计算机的攻击免疫．加密过程是制作特殊设计的DNA芯片（微阵列），解密过程是进行芯片杂交．在DNASC中，数以万亿计的DNA探针被方便地同时进行杂交并识别出来，从一定程度上体现了DNA在超大规模并行计算和超高容量数据存储方面的巨大潜力．     

遗传病分子诊断：新的进展和可预见的将来

随着分子生物学的进步，遗传病分子诊断在许多方面取得了显著进展，策略上的改善使我们有可能在分子水平上对多基因遗传病进行诊断；着床前诊断使有遗传病风险的夫妇有望获得完全正常的孩子；RNA诊断适用于检测基因表达的缺陷；着色FISH使染色体病的诊断更加准确可靠。得益于人基因组计划的成果，遗传病分子诊断将进入一个全新的时代。     

携SiRNA的聚乳酸羟基乙酸微球对Bel-7402/5-Fu细胞MDR1基因沉默作用的研究

目的 探讨携SiRNA的聚乳酸羟基乙酸(PLGA)微球通过沉默肝癌耐药细胞多药耐药基因(MDR1)基因,实现肝癌耐药的逆转.方法 合成针对MDR1的RNA干扰小片段(SiRNA),通过超声复乳法制备携药微球,转染肝癌耐药细胞Bel-7402/5 -Fu.运用Real-time PCR与Western blot方法,通过检测其mRNA及P糖蛋白表达水平,评价RNA干扰对MDR1表达的影响.MTT法检测RNA干扰逆转Bel-7402/5 Fu细胞对药性的效果,通过实验组细胞和对照组细胞之间的半数抑制剂量(IC50),计算RNA干扰组细胞的耐药逆转倍数.结果 在肝癌耐药细胞Bel-7402/5-Fu中,RNA干扰明显抑制了MDR1 mRNA和蛋白产物P糖蛋白的表达水平,且出现了P糖蛋白的持续低表达.MTT法显示,相同药物浓度下,实验组细胞生长明显受到抑制,表明其耐药性明显下降,其对药逆转倍数为11.56.结论 携SiRNA的PLGA微球能够有效减少肝癌耐药细胞Bel-7402/5-Fu的MDR1mRNA及P糖蛋白的表达水平,降低其耐药性.     

胃癌侧群细胞耐药性研究及干细胞相关基因检测

目的研究胃癌侧群（Side Population,sP）细胞对化疗药物5-Fu（氟尿嘧啶）的耐药性及可能机制,并检测干细胞相关基因Nanog、Musashi-1及cD44的表达情况。方法选择人胃癌细胞株sGc-7901,以荧光染料H0echst 33342染色,维拉帕米桔抗对照,应用流式细胞仪分选sP细胞和nonsP细胞。细胞耐药实验比较sP细胞与nonsP细胞对化疗药物5．Fu的耐药性差异;westem_h10t检测ABcG2和bcl-2蛋白表达情况;流式细胞仪分析细胞周期;荧光定量PcR检测两组细胞中干细胞相关基因Nanog、Musashi-1及cD44mRNA的表达差异。结果胃癌细胞株sGc．790l中sP细胞的比例为2．8％,sP细胞对5-Fu的耐药存活率明显高于non-sP细胞（P〈0．05）,与nonsP细胞相比,sP细胞高表达耐药蛋白ABcG2和抗凋亡蛋白bcl-2,有更多的细胞处于c0／G1期（P〈0．05）,并高表达干细胞相关基因Musashi-1和cD44。结论胃癌sGC_7901细胞株中sP细胞对化疗药物5．Fu的耐药性明显高于nonsP细胞,其耐药机制可能与sP细胞高表达耐药蛋白ABcG2和抗凋亡蛋白bcl-2,有更多细胞处于G0／Gl期有关;Musashi-1和cD44可能是相对特异性的胃癌干细胞标志物。     

地震动随机场的POD降维表达

本文从本征正交分解(POD)的基本理论出发,推导了非平稳随机向量过程的两类POD表达,即基于正交随机变量的POD表达和基于随机相位角的POD表达.澄清了基于随机相位角的POD表达是基于正交随机变量的POD表达的一个特例,并从基本随机变量的数量和约束条件两个方面剖析了它们之间的差异,发现通过施加约束条件可以有效地降低随机向量过程模拟的随机度(基本随机变量的数量).为此,通过对基于正交随机变量的POD表达施加更强的约束条件,实现了仅用2个基本随机变量即可对非平稳随机向量过程进行高效降维模拟,进而可在全概率信息上精细地表达非平稳随机向量过程.通过对地震动随机场模拟的数值分析,得到了非平稳地震动加速度向量过程的均值、标准差、演变功率谱及相关函数等均与目标值拟合一致的良好结果,说明了本文方法的有效性.同时,为了进一步从地震动的工程特性方面验证本文方法的适用性,根据《中国地震动参数区划图》中划分的5种场地类别,选取920条实测强震动记录,从地震动加速度的反应谱和傅里叶幅值谱两个方面进行了对比分析.研究表明,本文方法模拟结果与实测强震动记录具有很好的一致性,这为本文方法的工程应用奠定了坚实基础.     

Effect of air ionization of blood serotonin in vitro.

The effect of negative and positive air ionisation on siliconized blood serotonin was studied in vitro. The experiments showed that within 10 min positive ionisation increased serotonin levels in total blood (+40%), plasma (+90%), erythrocytes (+50%) and thrombocytes (+240%). On the other hand, negative ionization (10 min) lowered the serotonin content of total blood (-30%), plasma (-42.5%), erythrocytes (-41.7%) and thrombocytes (-72.3%), thus confirming the 'Krueger Effect' in vitro.     

Molecular Physiology of Mammalian Glucokinase

The glucokinase (GCK) gene was one of the first candidate genes to be identified as a human “diabetes gene". Subsequently, important advances were made in understanding the impact of GCK in the regulation of glucose metabolism. Structure elucidation by crystallography provided insight into the kinetic properties of GCK. Protein interaction partners of GCK were discovered. Gene expression studies revealed new facets of the tissue distribution of GCK, including in the brain, and its regulation by insulin in the liver. Metabolic control analysis coupled to gene overexpression and knockout experiments highlighted the unique impact of GCK as a regulator of glucose metabolism. Human GCK mutants were studied biochemically to understand disease mechanisms. Drug development programs identified small molecule activators of GCK as potential antidiabetics. These advances are summarized here, with the aim of offering an integrated view of the role of GCK in the molecular physiology and medicine of glucose homeostasis. Received 11 June 2008; received after revision 18 July 2008; accepted 30 July 2008     

GSK3 and β-catenin determines functional expression of sodium channels at the axon initial segment

Neuronal action potentials are generated through voltage-gated sodium channels, which are tethered by ankyrinG at the membrane of the axon initial segment (AIS). Despite the importance of the AIS in the control of neuronal excitability, the cellular and molecular mechanisms regulating sodium channel expression at the AIS remain elusive. Our results show that GSK3α/β and β-catenin phosphorylated by GSK3 (S33/37/T41) are localized at the AIS and are new components of this essential neuronal domain. Pharmacological inhibition of GSK3 or β-catenin knockdown with shRNAs decreased the levels of phosphorylated-β-catenin, ankyrinG, and voltage-gated sodium channels at the AIS, both “in vitro” and “in vivo”, therefore diminishing neuronal excitability as evaluated via sodium current amplitude and action potential number. Thus, our results suggest a mechanism for the modulation of neuronal excitability through the control of sodium channel density by GSK3 and β-catenin at the AIS.     

PD-L1在人子宫颈癌细胞异常表达并促T细胞凋亡

目的 探讨人子宫颈癌中负性协同刺激分子PD-L1的表达和它与肿瘤浸润淋巴细胞的关系以及PD-L1融合蛋白促宫颈癌患者外周血活化T细胞凋亡的作用.方法 采用免疫组化S-P法检测67例宫颈癌组织及20例正常宫颈组织中PD-L1的表达,分析PD-L1同临床病理特征的相关性,免疫荧光观察肿瘤浸润淋巴细胞数量,TUNEL法检测T细胞凋亡,体外实验将PD-L1融合蛋白细胞加入PHA刺激活化的宫颈癌患者外周血T细胞中共同培养,流式细胞术分析T细胞凋亡率和CD8+/CD4+T细胞比例.结论 正常子宫颈组织不表达PD-L1;宫颈癌组织中PD-L1的表达率为70％.宫颈癌PD-L1的表达与宫颈癌浸润深度相关(P＜0.05).PD-L1阳性病例肿瘤局部浸润淋巴细胞存在凋亡且CD8+T细胞数量明显减少;PD-L1融合蛋白组T细胞凋亡率明显高于抗PD-1组和空白对照组T细胞,分别为32.7％、18.3％和17.9％;CD8+T/CD4+T细胞的比值低于加入抗PD-1组和空白对照组,分别为0.864、0.894和0 907.结论 PD-L1在子宫颈癌中高表达且与肿瘤浸润程度及肿瘤浸润淋巴细胞数量减少有关,PD-L1能促进活化的T细胞尤其是CD8+T细胞的凋亡.     

Molecular mechanisms in male determination and germ cell differentiation

Sex determination and gametogenesis are key processes in human reproduction, and any defect can lead to infertility. We describe here the molecular mechanisms of male sex determination and testis formation; defects in sex determination lead to a female phenotype despite the presence of a Y chromosome, more rarely to a male phenotype with XX chromosomes, or to intersex phenotypes. Interestingly, these phenotypes are often associated with other developmental malformations. In testis, spermatozoa are produced from renewable stem cells in a complex differentiation process called spermatogenesis. Gene expression during spermatogenesis differs to a surprising degree from gene expression in somatic cells, and we discuss here mechanistic differences and their effect on the differentiation process and male fertility.Received 23 January 2004; received after revision 30 March 2004; accepted 6 April 2004     

Role of Chk1 in the differentiation program of hematopoietic stem cells

Hematopoietic stem cells (HSC) isolated from umbilical cord blood (UCB) were treated with ionizing radiation (IR) and sensitivity and IR induced checkpoints activation were investigated. No difference in the sensitivity and in the activation of DNA damage pathways was observed between CD133+ HSC and cells derived from them after ex vivo expansion. Chk1 protein was very low in freshly isolated CD133+ cells, and undetectable in ex vivo expanded UCB CD133+ cells. Chk1 was expressed only on day 3 of the ex vivo expansion. This pattern of Chk1 expression was corroborated in CD133+ cells isolated from peripheral blood apheresis collected from an healthy donor. Treatment with a specific Chk1 inhibitor resulted in a strong reduction in the percentage of myeloid precursors (CD33+) and an increase in the percentage of lymphoid precursors (CD38+) compared to untreated cells, suggesting a possible role for Chk1 in the differentiation program of UCB CD133+ HSC.     

Folic acid levels in blood and seminal plasma of normo- and oligospermic patients prior and following folic acid treatment

Folic acid was estimated in blood and seminal plasma of normo- and oligospermic men. Following folic acid administration (10 mg TID for 30 days), the levels in blood and semen increased. However, sperm counts, motility and DNA content of spermatozoa were not affected.