miR-9靶向调控PTEN在HPV16+及HPV18+宫颈癌细胞凋亡中的作用

研究微小RNA-9(microRNA-9,miR-9)靶向调控人第10号染色体缺失的磷酸酶及张力蛋白同源蛋白（Recombinantphosphataseandtensinhomolog,PTEN）在人乳头瘤病毒（Humanpapillomavirus,HPV）16+及HPV18+宫颈癌细胞凋亡中的作用。收集宫颈癌组织及正常宫颈组织，培养HPV16+SiHa细胞、HPV18+HeLa细胞、HPV阴性C33A细胞及正常宫颈上皮H8细胞，检测miR-9、PTEN的表达水平。SiHa细胞和HeLa细胞进行分组转染后检测增殖抑制率、凋亡率、PTEN及miR-9表达水平。结果显示高危型HPV阳性及阴性的宫颈癌组织中miR-9的表达水平高于正常宫颈组织、PTEN的表达水平低于正常宫颈组织（P<0.05）且高危型HPV阳性宫颈癌组织中miR-9、PTEN表达的变化更显著；SiHa细胞、HeLa细胞中miR-9的表达水平高于C33A细胞和H8细胞、PTEN的表达水平低于C33A细胞和H8细胞（P<0.05）；敲低miR-9表达后，SiHa细胞、HeLa细胞的PTEN表达水平、增殖抑制率、凋...     

PI3K抑制剂LY294002在HPV感染宫颈癌细胞的调节作用

高危型人乳头瘤病毒(Human papiloma virus,HPV)感染是宫颈癌的危险因素,HPV16是常见的高危型HPV,与宫颈癌的发病有关,但具体机制未明确.有临床研究报道,宫颈癌组织中HPV16感染与磷脂酰肌醇-3激酶(PI3K)、蛋白激酶B(AKT)表达增加有关,为了阐明PI3K/AKT信号通路及其抑制剂LY294002在HPV感染宫颈癌细胞增殖中的调控作用,本实验培养了 HPV16感染的宫颈癌细胞株SiHa、HPV阴性的宫颈癌细胞株C33A、正常宫颈上皮细胞株H8,检测了 p-PI3K、p-AKT的表达水平;SiHa细胞分为对照组、50 μmol/L及100 μmol/L LY294002组,药物干预后检测细胞增殖活力A490值及p-PI3K、p-AKT、c-myc、B淋巴细胞瘤-2基因(Bcl-2)的表达水平;皮下注射SiHa细胞建立移植瘤小鼠模型,分为对照组、25mg/kg、50mg/kg LY294002组,药物干预后取移植瘤称重.结果显示,SiHa细胞中p-PI3K、p-AKT的表达水平均高于C33A、H8细胞;50 μmol/L及100 μmol/L LY294002组 SiHa 细胞的 A490值及 p-PI3K、p-AKT、c-myc、bcl-2的表达水平均低于对照组;25 mg/kg、50 mg/kg LY294002组移植瘤小鼠的移植瘤质量均低于对照组(P＜0.05).以上结果表明HPV16感染的宫颈癌细胞中PI3K/AKT信号通路过度激活具有促增殖作用.本实验阐明了 PI3K/AKT通路及其抑制剂LY294002在HPV16感染的宫颈癌细胞增殖中的调控作用,PI3K/AKT通路的激活能够促进HPV16感染的宫颈癌细胞增殖及移植瘤的生长,使用信号通路抑制剂能够抑制细胞增殖及移植瘤生长,未来PI3K/AKT通路可能成为HPV16感染引起宫颈癌的防治靶点.     

宫颈癌中高危型HPV感染与miR-362-3p、Nemo样激酶表达的相关性研究

高危型人乳头瘤病毒(High-risk human papillomavirus,HPV)感染是宫颈癌发病的重要因素,但高危型HPV感染引起宫颈上皮恶变的机制尚不清楚.微小RNA (micro RNA,miR)-362-3p是具有抑癌活性的miR,在宫颈癌中表达降低;Nemo样激酶(Nemo-like kinase,NLK)是生物信息学预测得到的miR-362-3p靶基因,在宫颈癌中表达增加.但宫颈癌发病过程中高危型HPV感染与miR-362-3p、NLK的关系尚不清楚.本研究检测了miR-362-3p、NLK在宫颈癌组织及宫颈癌细胞株中的变化,并通过转染miR-362-3p模拟物、NLK表达质粒的方式验证了miR-362-3p靶向NLK调节高危型HPV阳性宫颈癌细胞增殖的作用.结果 显示:与癌旁组织比较,宫颈癌组织中miR-362-3p的表达明显降低、NLK的表达明显增加且与高危型HPV阴性的宫颈癌组织比较,高危型HPV阳性的宫颈癌组织中miR-362-3p的表达明显降低、NLK的表达明显增加;与正常宫颈上皮细胞比较,HPV16感染的SiHa细胞、HPV18感染的HeLa细胞及HPV阴性的C33A细胞中miR-362-3p的表达明显降低、NLK的表达明显增加且SiHa细胞中miR-362-3p表达降低、NLK表达增加最明显;在SiHa细胞中,过表达miR-362-3p能够降低细胞活力及NLK的表达,同时也使NLK双荧光素酶报告基因的荧光活力降低;过表达NLK能够逆转miR-362-3p降低细胞活力及NLK表达的作用.以上结果表明宫颈癌中高危型HPV感染与miR-362-3p低表达、NLK高表达有关,过表达miR-362-3p通过靶向抑制NLK降低高危型HPV感染宫颈癌细胞的增殖活力.本研究的创新点在于初步探索了高危型HPV感染促进宫颈癌细胞增殖的作用及机制,在高危型HPV感染的宫颈癌中miR-362-3p表达减少并靶向引起NLK表达增加,进而促进了宫颈癌细胞的增殖,这为将来深入认识高危型HPV引起宫颈癌发病的机制提供依据.     

HPV16E7的表达对宫颈癌变过程中CD163+巨噬细胞的浸润及上皮间质转化的影响

目的 研究人乳头瘤病毒16E7(HPV16E7)、上皮间质转化(epithelial-mesenchymal transition,EMT)“cadherin switch”标记物E-cadherin、N-cadherin,及肿瘤相关巨噬细胞(tumor-associated macrophages,TAMs)标记CD163在宫颈癌变过程中的表达情况,探讨在不同的HPV16E7表达情况下,上皮间质转化程度及肿瘤相关巨噬细胞浸润的差异及二者之间的关系。方法 通过免疫组化法检测宫颈鳞状细胞癌、高级别上皮内瘤变(CINII-III)、低级别上皮内瘤变(CINI)及慢性宫颈炎患者的HPV16E7、E-cadherin、N-cadherin、CD163的表达情况。结果 E-cadherin的表达随着宫颈癌变的进展逐渐降低,N-cadherin的表达以及CD163+巨噬细胞数量随着宫颈病变的进展逐渐升高。慢性宫颈炎、CINI、CINII-III及宫颈鳞状细胞癌的HPV16E7阳性组和阴性组比较中,E-cadherin的表达在CINII-III及宫颈鳞状细胞癌中HPV16E7阳性组明显低于阴性组;N-cadherin的表达在宫颈鳞状细胞癌中HPV16E7阳性组明显高于阴性组;CD163+巨噬细胞数目在CINI、CINII-III及宫颈鳞状细胞癌中HPV16E7阳性组明显高于阴性组。进一步统计CD163+巨噬细胞与上皮间质转化间关系后发现,宫颈癌变过程中,随着CD163+巨噬细胞数目的增多,E-cadherin的表达逐渐降低,无论HPV16E7的表达如何,二者均呈负相关关系;宫颈鳞状细胞癌中,随着CD163+巨噬细胞数目的增多,N-cadherin的表达逐渐增加,HPV16E7阳性组二者仍呈正相关关系,而HPV16E7阴性组无明显相关关系。结论 HPV16E7可能通过肿瘤相关巨噬细胞来调节上皮间质转化的“cadherin switch”,从而促进宫颈癌的癌变。     

TLR4在宫颈癌与不同HPV亚型宫颈癌细胞株中表达及意义

目的 探讨TLR4在正常宫颈组织、宫颈上皮不典型增生、宫颈癌组织以及不同HPV亚型宫颈癌细胞株中表达与意义.方法 采用SP免疫组织化（IHC）检测TLR4在12例正常宫颈组织、30例宫颈轻度不典型增生（cervical intraepithelial neoplasia Ⅰ,CINⅠ）、30例宫颈中-重度不典型增生（cervical intraepithelial neoplasiaⅡ-Ⅲ,CIN Ⅱ-Ⅲ）以及49例宫颈癌（invasive cervical carcinoma,ICC）组织中的表达;应用细胞免疫荧光法、逆转录聚合酶链反应（RT-PCR）检测TLR4在不同HPV亚型宫颈癌细胞株HPV18（＋）HeLa、HPV16（＋）Siha以及HPV（-）C33a宫颈癌细胞株上的表达.结果 TLR4的表达随着宫颈病变程度的增高逐渐增强,在正常宫颈组织、CINⅠ、CIN Ⅱ～Ⅲ、ICC中阳性表达率分别为8.33 %（1/12）、20.00 %（6/30）、26.67 %（8/30）和55.10 %（27/49）,ICC与正常宫颈组织、CINⅠ、CINⅡ～Ⅲ之间均有显著性差异（P＜0.01）.TLR4表达与HPV感染有关,在HPV阳性宫颈癌细胞株上表达强于HPV阴性细胞株.结论 TLR4可能参与宫颈癌起始、发展,TLR4的表达与功能与HPV感染有关.     

HPV16 E6对HeLa细胞中Rap1GAP蛋白水平下调的研究

目的探讨HPV16E6对细胞中Rap1GAP蛋白水平的影响,为阐明宫颈癌发生发展的分子机制提供实验依据。本研究组前期实验显示,宫颈癌石蜡切片组织中Rap1GAP蛋白水平下降,且与高危型HPV16/18感染相关。本文将进一步探讨HPV16 E6是否导致Rap1GAP蛋白下调的原因。方法通过将HPV16 E6基因插入pGEX-KG的BamHI和Hind Ⅲ酶切位点构建GST标记的HPVE6质粒,采用脂质体转染法将其转染入HeLa细胞中,Westernblot方法观察GST-tagged-HPV16 E6在HeLa细胞中的表达,并观察其对HeLa细胞中内源性Rap1GAP蛋白水平的影响。结果测序表明成功构建GST标记的HPV16E6质粒;Western blot检测表明HPV16E6在HeLa细胞中成功表达;并且发现HeLa细胞中过表达HPV16 E6后,Rap1GAP蛋白相对含量(0.602±0.205)明显低于未转染组(1.130±0.163),差异具有统计学意义(P0.05)。结论HPV16 E6下调Rap1GAP蛋白水平。     

端粒酶RNA和端粒酶活性在人生殖道癌细胞株中的表达

本文采用反转录套式PCR(nested RT-PCR)和PCR端粒重复扩增(TRAP)方法分别检测了5种人生殖道癌细胞株端粒酶RNA(hTR)基因及端粒酶活性的表达。结果表明,宫颈癌HeLa和SiHa细胞株,绒毛膜癌JAR和BeWo细胞株及卵巢癌OCCI细胞株hTR及端粒酶活性均呈强阳性。但正常卵巢、宫颈和胎盘组织hTR和端粒酶呈阴性或弱阳性。提示反转录套式PCR和端粒重复扩增法可以简便而有效地检测端粒酶RNA及端粒酶活性。端粒酶的激活与肿瘤细胞增殖密切相关,并可能成为一项具有临床价值的肿瘤标志物。     

HLA-I表达与维吾尔族妇女宫颈癌前病变及HPV16感染的关系研究

目的：观察人白细胞相关抗原I（human leukocyte antigen class I,HLA-I）表达与维吾尔族妇女宫颈癌前病变进程及高危型HPV16的关系。方法：收集维吾尔族妇女宫颈炎、宫颈内上皮瘤样病变（cervical intraepithelial neoplasia,CIN）和宫颈鳞癌患者的石蜡包埋组织标本共148例,提取组织DNA,应用PCR的方法检测HPV阳性及HPV16型别;同时采用免疫组织化学SP法检测HLA-I蛋白表达水平。结果：（1）在维吾尔族妇女中HLA-I抗原在宫颈炎、CINI-II、CINIII、SCC组中阳性表达逐渐减少,差异有统计学意义（P〈0.001）。（2）HLA-I的阳性表达下降趋势与宫颈癌临床分期、组织分化程度和淋巴结转移密切相关。（3）HPV在宫颈炎、CINI-II、CINIII、宫颈癌中的感染率分别为13%、46%、82%、95%,差异有统计学（P〈0.001）。（4）HPV16在宫颈炎、CINI-II、CINIII、宫颈癌中的感染率分别为4%、30%、68%、85%,差异有统计学（P〈0.001）。（5）在HPV16阳性标本中,存在HLA-I表达缺失的占71%（58/82）,HPV16感染与HLA-I表达呈负相关（r=-0.625,P〈0.001）。结论：（1）HLA-I表达缺陷可能是宫颈病变进展的重要标志,对宫颈癌的预测预警提供依据。（2）HPV16感染在宫颈病变的发展过程中起到了极大的促进作用,是一个很强的致癌因素。（3）HPV16感染与HLA-I表达之间的关系对揭示宫颈癌发病机制提供了客观依据。     

E-cadherin 在宫颈上皮内瘤变及宫颈癌中的表达及其与高危型HPV感染的相关性

目的:研究各级宫颈上皮内瘤变(CIN)及宫颈癌组织中E-cadherin的表达及其与高危型人乳头瘤病毒(high risk human papillomavirus,HR-HPV)感染的相关性探讨其在宫颈疾病发生、发展中的意义。方法:选取2008年1月至2014年12月我院收治的150例患者标本并将其分为CINⅠ级组、CINⅡ-Ⅲ级组及宫颈癌组用免疫组化法对E-cadherin的表达情况进行检测并于术前采用PCR-反向点杂交法检测患者高危型HPV感染情况所得结果:进行统计学分析。结果:(1)E-cadherin在CINⅠ级、CINⅡ-Ⅲ级及宫颈癌中的阳性表达率分别为40/50(80.0%),24/50(48.0%)、17/50(34.0%),随疾病的进展E-cadherin的表达明显减少,各组间差异有统计学意义(P0.05)。(2)高危型HPV在CINI级、CINⅡ-Ⅲ级及宫颈癌中的阳性感染率分别为21/50(42.0%)、38/50(76.0%),48/50(96.0%),各组间差异有统计学意义(P0.05)。(3)在CIN和宫颈癌中,HR-HPV阳性组中E-cadherin阳性率39.3%(42/107)低于HR-HPV阴性组中E-cadherin阳性率90.7%(39/43)(P0.05)。结论:E-cadherin的表达下降或缺失可能是HR-HPV导致宫颈癌发生、发展的机制之一。     

白蚁与动物及微生物的共生类型及其机制

综述了白蚁螱客的主要种类、共生关系及相关机制的研究进展。白蚁螱客中,已报道的动物种类达170种。在与动物的共生关系中存在偏利共生（宾主共栖和异种共栖）、互利共生和无关共生三种;在与微生物的共生关系中,存在与内生菌（原生动物、细菌、真菌和放线菌）和外生菌（蚁巢伞菌等）间的互利关系。指出了白蚁与螱客研究中存在的问题,给出了解决方案,并提出了今后可能的研究热点或方向,为白蚁的综合利用（如纤维素酶）及今后研究物种间的协同进化提供了基础资料。     

Relative and combined effects of ethanol and protein deficiency on strontium and barium bone content and fecal and urinary excretion

To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women, ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined to date. Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in women than in men, whereas their content in the external iliac artery was lower in women than in men.     

New amino-dithiaphospholanes and phosphoramidodithioites and their rhodium and iridium complexes

New sulfur derivatives of phosphoramidite ligands were synthesized and the impact of the sulfur unit on the spectroscopic properties of their rhodium and iridium complexes was investigated. The new ligands Bn₂NPSCH₂CH₂S^a(P-S^a) (Bn = benzyl, 4), Bn₂NPSCHCHS^a(CH₂)₃C^aH₂(P-S^a)(C^a-S^a) (6) and Bn₂NP(4-XC₆H₄OMe)₂ (X = S, 7a; X = O, 7b) were converted to the rhodium and iridium complexes trans-[Rh(CO)Cl(L)₂] (L = 4, 6, 7), [RhCl(COD)(L)] (L = 4, 6, 7), [IrCl(COD)(7a)] and [IrCl₂Cp∗(6)]. For comparison, some phosphoramidite complexes of these formulations also were synthesized. The new metal complexes were spectroscopically analyzed. For the carbonyl complexes, the ν_CO IR stretching frequencies were lower than for the corresponding phosphite and phosphoramidite ligands. The ¹J_PRh coupling constants for the rhodium complexes with the new ligands were also smaller than for the respective phosphoramidite and phosphite complexes. Finally, the ¹J_PSe coupling constants of the selenides of the new ligands were lower than those of the phosphoramidite ligands but higher than for PPh₃. The spectroscopic data reveal that the new thio ligands 4, 6 and 7a are more electron donating than phosphites and phosphoramidites but less electron donating than PPh₃.     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

Rapporteur report: Basics and technology and metrology and standards

The first and second sessions of the Workshop focussed on the basics of ultrasound and infrasound, their applications in both industry and medicine, and metrology and protection standards for ultrasound applications.     

Prostaglandin and thromboxane production by human and guinea-pig macrophages and leucocytes

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection.The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemic leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE₂, PGD₂, PGF_2α, TXB₂ and 6-keto-PGF_1α. In all types studied PGE₂ and TXB₂ were the major products formed. The identification of PGE₂ and TXB₂ was confirmed by GC/MS with multiple ion monitoring.The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.     

Cytoskeleton and mitochondrial morphology and function

It has been well established that the cytoskeleton is an essential modulator of cell morphology and motility, intracytoplasmic transport and mitosis, however cytoskeletal linkage to the organelles has not been unequivocally demonstrated. Indeed, cytoskeleton appears to be essential in determining and modulating gene phenotype as a function of cellular environment. According to recent studies, the organization of the cytoskeleton network together with associated protein(s) could be essential in regulating mitochondrial function and particularly the permeability of the mitochondrial outer membrane to ADP. The aim of this chapter is to summarize the main properties of the cytoskeletal environment of mitochondria and the possible role(s) of this network in mitochondrial function in myocytes.     

人血管能抑素基因的克隆、表达及其表达产物的纯化和生物活性测定

以人胎盘脐带组织为材料,提取组织总RNA,用netRTPCR方法合成人血管能抑素cDNA基因,将该cDNA克隆进pSP72载体获得重组质粒pSP72C, DNA序列分析结果与预期序列一致。用BamHⅠ和NdeⅠ双酶切,切下pSP72C上的血管能抑素cDNA,插入pET3c载体的相应位点获得重组表达质粒pETC, 转化E. coli BL21(DE3), SDSPAGE分析显示：在IPTG诱导下,血管能抑素基因获得了高效表达,表达量约占菌体总蛋白的 27.9 %,主要以包涵体形式存在。包涵体经过洗涤、裂解、蛋白复性以及Sephadex G75凝胶过滤层析等步骤后,获得了纯度达91.4 %的人血管能抑素。CAM实验证明10 μg纯化蛋白就能显著抑制鸡胚新生血管生成。