宫颈癌组织人乳头瘤病毒的荧光偏振基因分型

采用荧光偏振人乳头瘤病毒(human papillomavirus,HPV)分型新方法探讨了8种常见型别HPV在陕西宫颈癌患者中的流行情况。首先,用HPV GP5 ／GP6 通用引物PCR扩增65例早期宫颈癌(Ⅱa期内)和72例慢性宫颈炎病变组织DNA粗提物,继之将模板指导的末端延伸反应与荧光偏振检测技术结合(TDI-FP),用GP5 ／GP6 扩增区内的HPV6、11、16、18、31、33、35和58型特异性探针与PCR产物杂交后,荧光素标记的特异碱基(TAMRA-ddTTP或R110-ddGTP)在GP5 ／GP6 产物中相应的模板指导下,掺入延伸至相应探针末端,致使对应的TAMRA或R110 FP值升高,从而对扩增的HPV阳性产物进行HPV分型。65例宫颈癌患者中检出HPV57例,阳性率87．69％,72例慢性宫颈炎患者中检出HPV28例,阳性率38．89％,两组间HPV阳性率有显著性差异。宫颈癌与慢性宫颈炎患者中4种最常见的HPV型别分别是HPV 16(45．6％)、HPV 18(22．8％)、HPV 58(17．5％)、HPV 31(7．02％)和HPV 16(35．7％)、HPV 11(32．1％)、HPV 6(21．4％)、HPV 18(10．7％)。慢性宫颈炎患者中检出的HPV型别57．14％属高危型。HPV 16在两组中均最为多见。中国陕西宫颈疾病患者中HPV感染有其特点,世界范围内少见的HPV 58在陕西宫颈癌与慢性宫颈炎患者中均较为多见,在进行HPV新诊断方法及疫苗研制时应考虑到这种特点。     

1235例妇科门诊患者高危型HPV感染状况及亚型分布调查研究

目的了解本地区妇科门诊患者宫颈高危型HPV感染状况及亚型分布,为今后的宫颈癌前病变、宫颈癌防治提供临床依据。方法采用基因芯片技术对1 235例妇科门诊患者进行HPV筛查,筛查出的阳性患者应用流式荧光杂交法进行高危型HPV亚型检测,分析比较宫颈炎、宫颈鳞癌及宫颈腺癌患者高危型HPV感染情况及亚型分布差异。结果六安市金安区妇幼保健院妇科门诊患者HPV感染率高达60%,其中高危型HPV感染率为43. 2%,主要以HPV16、HPV18为主;低危型HPV感染率为30.0%,主要以HPV11为主;单一感染阳性率为34. 1%,而混合型感染高达65. 9%,且两者均以HPV16型和HPV18型为主。宫颈炎患者HPV16型、HPV18型及HPV16 ＋ HPV18型的检出率明显低于宫颈鳞癌和宫颈腺癌患者,其中宫颈腺癌患者HPV16 ＋ HPV18型混合感染率最高。结论妇科门诊患者HPV感染率较高,宫颈癌患者HPV16及18型检出率显著高于宫颈炎患者,加强HPV高危基因型的监测有助于预警宫颈癌尤其是宫颈腺癌的发生。     

妇科临床病例人乳头瘤病毒检测结果分析

目的 观察妇科临床病例人乳头状瘤病毒(HPV)感染现状,为预防宫颈癌提供参考.方法 以有性生活史的妇科病例作为检测对象;由妇产科医生采样,以反向杂交膜技术基因芯片进行HPV DNA检测;采用x2检验和趋势分析(Z)作检出率显著性检验.结果 共检测病例4347例,HPV检出率为24.64％;检出亚型23种,检出率以16型(8.10％)居高(X2＞6.63,P＜0.01),其次为43(3.93％)、58(3.52％)和52型(3.31％),显著高于其他19种亚型(x2＞6.63,P＜0.01),＞1％＜3％的有6、11、18、33、56型,其余14个亚型＜1％;随着年龄增长检出率呈上升趋势(Z=3.32,P＜0.01);阴道分泌物增多、不明原因阴道出血、月经紊乱等4类症状检出率为20.68％～25.59％,阴道分泌物增多加不明原因出血病例检出率为30.76％,宫颈鳞状上皮癌80例均检出HPV,高危型占97.50％ (78/80),16型占70.00％ (56/80),仅为低危型感染占2.50％(11型,2/80);炎性疾病检出率为24.75％,宫颈炎、阴道炎、宫颈炎合并症检出率为23.03％ ～ 28.38％,外阴炎为16.81％.结论 妇科临床病例HPV检出率以16型居首,其次为43、58和52型;宫颈癌检出率100％,阴道分泌物增多加阴道不明原因出血和宫颈炎为主的病例检出率较高;年龄越大,检出率越高,显示高年龄宫颈炎性疾病或阴道分泌物增多加不明原因阴道出血病例,是HPV和宫颈鳞状上皮癌防治的首要对象.     

尖锐湿疣样本中HPV病毒的分子检测

调查男性和女性尖锐湿疣样本中人乳头瘤病毒（HPV）的检出率及病毒类型,为研发相关防治疫苗提供依据,以HPV 外壳蛋白DNA序列为模板设计特异引物,SSP－PCR扩增检测样本中HPV的感染率和病毒类型。收集了北京及邯郸市医院门诊尖锐湿疣样本22例,其中男性13例,女性9例。检测发现所有样本中存在着高浓度的HPV病毒DNA。男性样本中有5例感染HPV6型,6例感染HPV11型,2例为HPV6＋HPV11混合感染。女性样本中有3例感染HPV6型,2例感染HPV11型,4例为 HPV6＋HPV11混合感染。被诊断为宫颈湿疣的4位女性还在其含宫颈粘膜脱落细胞的样本中检出了HPV16、HPV18、HPV33、HPV35、HPV45、HPV54、HPV56或HPV58等高危险型病毒类型。所有检测到的HPV病毒DNA片段均TA克隆并将测定的DNA序列存入了国际基因数据库GenBank（DQ003066－DQ003079）。调查没有在单纯的男女尖锐湿疣组织块中检测到除HPV6和HPV11以外的其他HPV类型。该研究建立了灵敏可靠的HPV分子检测及分型方法,尖锐湿疣中HPV的检出率达100％。 本研究初步结果显示导致男女尖锐湿疣的HPV病毒类型没有显著差异,主要为HPV6及HPV11型。     

兰州地区女性感染HPV及其基因类型分析

目的:了解兰州地区成年女性感染人乳头瘤病毒(humanpapillomavirus,HPV)及其基因类型分布状况,为本地区HPV分子流行病学研究提供理论依据。方法:利用PCR技术分别对100例妇科门诊就诊者进行HPV基因亚型检测。结果:100例样品中,HPV DNA检出率为19%(19/100),其中HPV16 DNA感染率15%(15/100),HPV58 DNA感染率3%(3/100),HPV18 DNA感染率2%(2/100),HPV 16与HPV18双重感染1例。结论:本地区成年女性HPV感染主要以HPV16多见,而HPV16与恶性肿瘤密切相关,因此对HPVDNA阳性者定期随访,有利于宫颈癌的防治。     

我国人乳头瘤病毒（HPV16Z）基因克隆和鉴定

本文作者采用基因克隆手段,以pAt153为载体,从一份山西襄坦宫颈癌高发区宫颈癌患者的手术标本中,成功地克隆到2株与HPV16同源的基因片段。经PstI、KpnI、TaqvI、PvuII等16种限制性内切酶酶谱分析及其部分基因序列的鉴定,证明这是在国内首次克姓到一株分子量约为8．0kb完整的HPV16型全序列DNA及一株分子量为5．4kb的HPV6基因片段。经实验证明：该基因片段E6、E7及部分LI基因丢失,在750单核苷酸处发生变异,产生一新的BamHI酶切位点。该完整的HPV16基因组被命名为HPV16Z,HPV16基因片段被命名为HPV16F。用新分离到的HPV16Z作分子探针,检测襄坦337份宫颈癌及阴道活检标本的HPV16型同源序列的结果显示,慢性阴道炎阳性率为17．28％（14／81）;宫颈炎为11．89％（17／143）;宫颈癌前病变46．81％（22／47）;宫颈癌为72．73％（48／66）。证明山西宫颈癌高发区宫颈癌前病变及宫颈癌组织中主要为HPV16Z感染。     

外阴皮肤营养不良组织中人乳头瘤病毒DNA的检测

采用Southern Blot分子杂交方法检测了9例外阴皮肤营养不良组织中人乳头瘤病毒DNA的存在情况。结果显示8例增生型营养不良组织中,2例HPV-16阳性,1例HPV33阳性,在3例组织中探测到HPV16相关序列,在1例混合型中也检测到HPV16相关序例,HPV DNA的总检出率为77％。结果提示外阴皮肤营养不良的发生可能与HPV感染有关。     

HPV基因分型检测在宫颈癌筛查中的应用

目的:探讨人乳头瘤病毒(human papilloma virus,HPV)基因分型技术在宫颈癌筛查中的临床应用价值。方法:采用反向斑点杂交法对HPV基因型进行分析,包括低危型(如6、11、42、43、44型等)和高危型(如16、18、31、33、35、MM4型等)共23个型别。结果:98例样本中检出HPV阳性者38例,23种基因型中共检出15种HPV型别,HPV总感染率38.8%(38/98)。慢性宫颈炎患者中HPV感染率30.4%(14/46),检测到的HPV亚型主要是HPV43,11,6,42,31和51(其中HPV6,42,31,51检出率一致)。CINI患者HPV感染率15.4%(4/26),最常见的HPV亚型为HPV35,其次为HPV16,52,53和59(其中16,52,53,59检出率一致)。CINⅡ患者HPV感染率71.4%(10/14),以HPV16,6,11,51等亚型最常见。CINⅢ患者HPV感染率75%(6/8),HPV亚型以HPV16,18,31,53为主。SCC患者HPV阳性率为100%(4/4),HPV16检测率最高,其次为HPV52,18,33,58,59和66。结论:持续的HPV感染与宫颈疾病有着密切的关系。HPV基因型的检测为临床进行宫颈疾病的筛查、诊断、治疗以及预后观察提供了较大的帮助。     

宫颈癌患者人乳头瘤病毒(HPV)主要型别及其感染研究

本文探讨了江西省和广东省宫颈癌患者人乳头瘤病毒(Human papillomavirus,HPV)感染及其型别分布,分析了高危型HPV对各种宫颈病变的感染情况,为宫颈癌的早期发现和临床诊治提供科学依据。首先采用细胞学、HPV DNA检测(第二代杂交捕获法,HC2)、电子阴道镜和宫颈化学着色方法筛查宫颈癌患者,经病理镜检确诊,然后用GP PCR-SBT法对宫颈癌患者进行HPV基因分型。江西省溪口镇、古市镇及修水县城宫颈癌癌前病变发生率为5．7‰。HC2方法发现宫颈癌患者13种高危型HPV DNA阳性率为89．9％,宫颈上皮内瘤样病变的为84．8％,对照组为24．5％。采用GP PCR-SBT方法进行基因分型发现,江西省宫颈癌患者存在HPV16、58、31、33、18、66、6、11、56和81十种型别,其中HPV81型在国内外鲜有报道。据此提出生殖道高危型HPV感染是妇女宫颈癌发病的重要因素。并发现江西省宫颈癌高发区妇女高危型HPV感染率为24．5％。建立了HPV基因分型的方法,对HPV致宫颈病变的分子机制进行了分析。     

中国东北地区人乳头瘤病毒16型全基因组克隆及HPV16.HaCaT重组细胞模型的构建及初步鉴定

为构建含东北地区人乳头瘤病毒16型(HPV16)全基因组的HPV16.HaCaT细胞模型,收集中国东北地区HPV16单一感染患者宫颈脱落细胞,提取DNA,将HPV16全基因组分成4个区段,通过4对特异性引物对HPV16全基因组进行分段扩增,测序后进行序列拼接及核酸序列分析,克隆HPV16全基因组序列;通过细胞转染,构建含HPV16全基因组的HPV16.HaCaT重组细胞模型;利用聚合酶链式反应(PCR)和细胞免疫荧光法检测重组细胞内HPV16早期基因的表达.成功克隆出中国东北地区HPV16全基因组序列(GenBank登录号:MW320358);构建了东北地区HPV16全基因组的重组质粒及HPV16.HaCaT重组细胞模型;证明了 HPV16早期基因E1-E4、E5、E6和E7在重组细胞模型内均有表达,从而获得中国东北地区HPV16全基因组序列及含有HPV16全基因组的HPV16.HaCaT重组细胞模型.     

Purification and properties of aldehyde reductases from human placenta

Aldehyde reductases (alcohol: NADP⁺-oxidoreductases, EC 1.1.1.2) I and II from human placenta have been purified to homogeneity. Aldehyde reductase I, molecular weight about 74 000, is a dimer of two nonidentical subunits of molecular weigths of about 32 500 and 39 000, whereas aldehyde erductase II is a monomer of about 32 500. Aldehyde reductase I can be dissociated into subunits under high ionic concentrations. The isoelectric pH for aldehyde reductases I and II are 5.76 and 5.20, respectively. Amino acid compositions of the two enzymes are significantly different. Placenta aldehyde reductase I can utilize glucose with a lower affinity, whereas aldehyde reductase II is not capable to reducing aldo-sugars. Similarly, aldehyde reductase I does not catalyse the reduction of glucuronate while aldehyde reductase II has a high affinity for glucuronate. Both enzymes, however, exhibit strong affinity towards various other aldehydes such as glyceraldehyde, propionaldehyde, and pyridine-3-aldehyde. The pH optima for aldehyde reductases I and II are 6.0 and 7.0, respectively. Aldehyde reductaase I can use both NADH and NADPH as cofactors, whereas aldehyde reductase II activity is dependent on NADPH only. Both enzymes are susceptible to inhibition by sulfhydryl group reagents, aldose reductase inhibitors, lithium sulfate, and sodium chloride to varying degrees.     

冷冻对视网膜色素上皮细胞分泌肝细胞生长因子的影响

本研究观察冷冻处理后的人视网膜色素上皮细胞(human retinal pigment epithelium cells,hRPE)在体外培养和体内玻璃体环境中分泌肝细胞生长因子(hepatocyte growth factor,HGF)水平的变化,调查变化后的玻璃体对正常RPE细胞的促增生作用。体外培养的RPE细胞在-80℃下进行冷冻,冷冻时间分为0s、15s和60s,随后继续体外培养和注入正常兔眼玻璃体,冷冻后的第3d和6d收集细胞培养液和玻璃体样本,ELISA法测定hHGF含量;进一步,用MTT法测定正常RPE细胞加入玻璃体样本后的生长状态。结果显示冷冻刺激RPE细胞hHGF的分泌增多,并可促进RPE细胞增生。     

Effects of valinomycin on lymphocytes independent of potassium permeability

10^?7 M valinomycin affects human lymphocytes in the following manner: (1) it is non-toxic; (2) it inhibits mitogenesis; (3) it causes a reduction in cell ATP; and (4) it causes a marked increase in steady-state Na⁺ exchange. However, it has a minimal effect on cell ion (K⁺, Na⁺, Ca²⁺, Mg²⁺) contents and no effect whatever on K⁺ exchange. Neither the fast nor the slow fraction of steady-state K⁺ exchange is affected by 10^?7 M valinomycin. The various reported effects of valinomycin on lymphocyte functions cannot be assumed to be due to changes in plasma membrane K⁺ permeability. The mechanism of the increase in steady-state Na⁺ exchange, and whether or not it is related to inhibition of mitogenesis, are unsettled issues.     

Characterization of α2-adrenergic receptors in human platelets by binding of a radioactive ligand [3H]yohimbine

[³H]Yohimbine, a potent α₂-adrenergic antagonist, was used to label the α₂-adrenergic receptors in membranes isolated from human platelets. Binding of [³H]yohimbine to platelet membranes appears to have all the characteristics of binding to α₂-adrenergic receptors. Binding reached a steady state in 2–3 min at 37°C and was completely reversible upon the addition of excess phentolamine or yohimbine (both at 10^?5 M;$\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.37}\text{min}$). [³H]Yohimbine bound to a single class of noncooperative sites with a dissociation constant of 1.74 nM. At saturation, the total number of binding sites was calculated to be 191 fmol/mg protein. [³H]Yohimbine binding was stereo-specifically inhibited by epinephrine: the (?) isomer was 11-times more potent than the (+) isomer. Cathecholamine agonists competed for the occupancy of the [³H]yohimbine-binding sites with an order of potency: clonidine > (?)-epinephrine > (?)-norepinephrine >> (?)-isoproterenol. The potent α₂-adrenergic antagonist, phentolamine, competed for the sites whereas the β-antagonist, (±)-propanolol, was a very weak inhibitor. 0.1 mM GTP reduced the bindng affinity of the agonists, while producing no change in antagonist-binding affinity. Dopamine and serotonine competed only at very high concentrations. Similarly, muscarinic cholinergic ligands were also poor inhibitors of [³H]yohimbine binding. These results suggest tht [³H]yohimbine binding to human platelet membranes is specific, rapid, saturable, reversible and, therefore, can be successfully used to label α₂-adrenergic receptors.     

Ca2+ ionophores and the activation of human blood platelets: The effects of ionomycin,beauvericin, lysocellin,virginiamycin S,lasalocid-derivatives and McN 4308

Platelet activation is linked to an increase in the cytoplasmic Ca²⁺ concentration and consequently can also be induced by ionophores which mobilize Ca²⁺ from intracellular storage sites or transport it through the plasma membrane. The ionophores mostly used in studies on platelet activation are A 23187 and lasalocid (X-537A). The effects of eight compounds with known Ca²⁺-ionophoric activity in synthetic or natural membrane systems were studied in order to investigate the relationship between transport of Ca²⁺ and activation of platelets.Ionomycin acts as a true Ca²⁺ ionophore: it elicits rapid shape change, aggregation, the release reaction (secretion) and clot retraction (contraction). Beauvericin activates platelets too, but probably not by increasing the cytoplasmic Ca²⁺ concentration. Lysocellin does not activate platelets but induces a passive loss of serotonin. Virginiamycin S has no effect on platelets. Bromolasalocid and one epimer of dihydrolasalocid, like lasalocid, activate platelets by increasing the cytoplasmic Ca²⁺ concentration, and also induce a passive loss of serotonin. McN 4308 does not activate platelets but induces a slow uptake of ⁴⁵Ca²⁺.     

Aldose and aldehyde reductases in human tissues

Immunochemical characterizations of aldose reductase and aldehyde reductases I and II, partially purified by DEAE-cellulose (DE-52) column chromatography from human tissues, were carried out by immunotitration, using antisera raised against the homogenous preparations of human and bovine lens aldose reductase and human placenta aldehyde reductase I and aldehyde reductase II. Anti-aldose reductase antiserum cross-reacted with aldehyde reductase I, anti-aldehyde reductase I antiserum cross-reacted with aldose reductase and anti-aldehyde reductase II antiserum precipitated aldehyde reductase II, but did not cross-react with aldose reductase or aldehyde reductase I from all the tissues examined. DE-52 elution profiles, substrate specificity and immunochemical characterization indicate that aldose reductase is present in human aorta, brain, erythrocyte and muscle; aldehyde reductase I is present in human kidney, liver and placenta; and aldehyde reductase II is present in human brain, erythrocyte, kidney, liver, lung and placenta. Monospecific anti-α and anti-β antisera were purified from placenta anti-aldehyde reductase I antiserum, using immunoaffinity techniques. Anti-α antiserum precipitated both aldehyde reductase I and aldose reductase, whereas anti-β antibodies cross-reacted with only aldehyde reductase I. Based on these studies, a three gene loci model is proposed to explain the genetic interrelationships among these enzymes. Aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α and β subunits and aldehyde reductase II is a monomer of δ subunits.     

l-DOPA (l-3,4-dihydroxyphenylalanine) uptake by human red blood cells

The uptake of l-DOPA (l-3,4-dihydroxyphenylalanine) was studied in normal human red blood cells in vitro using l-[3-¹⁴C]DOPA. Uptake was slow, tending towards a distribution ratio close to unity with a half-time to equilibrium of one hour. Uptake was not Na⁺-dependent. Concentration dependence studies showed both saturable and non-saturable components of uptake, and inhibition studies using l-leucine and l-tryptophan suggest that the L and T systems of red cell amino acid uptake are involved. A powerful inhibitor of both systems, 3,4-dihydroxy-2-methylpropriophenone (U-0521), is described. It is concluded that uptake is by carrier-mediated facilitated diffusion via the L and T systems for which l-DOPA has low affinity.     

Simultaneous net accumulation of both K+ and Na+ by lymphocytes at 0°C

Human lymphocytes at 0°C in low Na⁺ medium accumulate both K⁺ and Na⁺ to levels higher than in the external medium. This is not due to an impermeable compartment or a Donnan equilibrium, and is incompatible with the membrane Na⁺-pump concept. In contrast, it supports prior evidence that ion exchange in lymphocytes is mediated by adsorption onto and desorption from fixed anionic sites within the cell. Additional aspects of ion and water contents of cells in low Na⁺ medium are described and are explained by this concept.     

Kinetics of histone hyperacetylation and deacetylation in human diploid fibroblasts

We have utilized sodium butyrate-treated normal human diploid fibroblasts to study core histone hyperacetylation kinetics. We report a small, distinct population of core histone characterized by a very rapid rate of hyperacetylation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈10–15}\text{min}$ for monoacetylated histone H4) compared to the slower rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈140–200}\text{min}$ for monoacetylated H4) observed for bulk histone. Two rates of core histone deacetylation were also detected and we demonstrated that the rapidly hypermodified histone H4 population was also rapidly deacetylated. The kinetics of histone H4 hyperacetylation and deacetylation in these cells were not significantly altered, regardless of whether cultures were exponentially growing, confluent or arrested in an essentially non-mitotic state.     

Interrelationships among human aldo-keto reductase: immunochemical, kinetic and structural properties

We have propsed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α, β subunits, and aldehyde reductase II is a monomer of δ subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (α and β) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (α subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li₂SO₄(0.4 M) for the expression of maximum enzyme activity, and its K_m for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li₂SO₄ and its K_m for glucose is more than 200 mM. The β subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The β subunits hybridized with the α subunits of placenta aldehyde I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristics properties of placenta aldehyde reductase I.