雌性激素对骺生长板闭合过程的影响

目的:了解雌性激素对骺生长板闭合过程的影响。方法:实验于2003-07/2004-03在重庆医科大学动物实验中心完成。选用性发育前(12周龄)的新西兰雌兔40只,切除双侧卵巢后随机分为雌二醇组和对照组各20只,4周后(16周龄)雌二醇组肌肉注射雌二醇(140 μg/kg,1次/周),对照组肌肉注射等体积不含雌二醇的灭菌棉籽油。从16周龄开始2组中各抽取5只动物,每两周1次进行跟踪股骨X射线拍片,直至29周龄,测定股骨生长率。血清雌二醇测定采用ELISA法。两组分别于20,23,26周龄时各处死5只,取股骨远端、胫骨近端和胫骨远端骺生长板作组织病理学分析及组织计量学测定,了解雌性激素对骺生长板生理性闭合时间、骺生长板组织形态结构和软骨细胞增殖率的影响。结果:雌兔40只全部进入结果分析。①血清雌二醇水平:干预前2组比较差异不显著,肌肉注射雌二醇后各时期雌二醇组均高于对照组(P<0.05)。②股骨生长率:两组均随年龄增长逐渐降低,至性成熟后(28周龄)股骨生长基本趋于停止。雌二醇组性发育早期(18周龄)快于对照组[(2.5±0.20),(2.2±0.05)mm/周,P<0.05],但20周龄后增长逐渐减慢,低于对照组。③骺生长板闭合时间:两组动物在20周时胫骨远端生长板均已发生闭合,雌二醇组胫骨近端和股骨远端生长板在23～26周龄闭合,而对照组闭合的时间延长或实验结束时仍未发生闭合。④骺生长板组织形态结构:雌二醇组雌兔骺生长板厚度和细胞柱密度显著低于对照组(P<0.05),对照组骺生长板出现“病理性”增厚。⑤生长板软骨细胞增殖率:雌二醇组股骨远端和胫骨近端生长板软骨细胞增殖率在20和23周龄均高于对照组(P<0.05),26周龄时,雌二醇组大部分雌兔骺生长板已经闭合,对照组仍可检出。结论:雌性激素早期可促进骺生长板软骨细胞增殖,提高长骨生长率,作用中、后期引起骺生长板组织结构发生衰退性改变促进骺生长板生理性闭合过程。     

大鼠抗组胺药敏感性细胞色素P450基因的克隆及其反转录病毒载体的构建和鉴定

目的:构建反转录病毒表达载体pLXSN-HP450。方法:实验于2004-12/2006-05在广西医科大学生化药理实验室完成。提取正常的Wistar大鼠肝组织的总RNA,反转录-聚合酶链反应技术扩增出抗组胺药敏感性细胞色素P450(HP450)基因。并克隆到pMD18-T载体,经蓝白斑筛选后进行聚合酶链反应,酶切,测序鉴定。BamHI,EcoRI双酶切构建好的重组质粒和反转录病毒载体pLXSN在16℃下经T4连接酶连接过夜,并转化到感受态细胞DH5α,以菌液为模板做聚合酶链反应,结合酶切筛选及鉴定阳性重组反转录病毒载体pLXSN-HP450。结果:反转录-聚合酶链反应扩增出HP450基因。重组质粒pMD18-HP450经双酶切后得到1461bp的酶切产物。测序的结果用ClustalW在线与Genebank对比,此目的基因与基因库中的H1基因100%同源。阳性重组载体pLXSN用其菌液聚合酶链反应扩增出目的条带。对重组体pLXSN-HP450进行双酶切得到1461bp和5900bp两条特异性条带。结论:克隆了HP450基因,并成功构建了重组反转录病毒载体pLXSN-HP450,为进一步研究肝癌的基因治疗奠定了基础。     

自体骨髓干细胞移植治疗糖尿病足17例

目的:观察自体骨髓干细胞下肢局部肌肉注射移植对糖尿病足的治疗效果。方法:①选择2004-03/2005-10郑州市中心医院内分泌内科住院确诊的糖尿病足患者17例(共17条患肢,左下肢10条,右下肢7条),男11例,女6例;年龄62～78岁,平均69岁;足部坏疽6例,静息痛或/和间歇性跛行11例。②纳入标准:不同程度的趾端坏疽、缺血性溃疡或静息痛;生活方式或职业必须解决的间歇性跛行;强烈要求缓解间歇性跛行者;血管造影证明存在周围动脉闭塞性病变,病变远端流出道差。排除标准:并发严重心、肺、肾、脑等脏器功能不全或不能耐受手术者。③分别采集患者自体骨髓,从双侧髂后上棘穿刺,每例约采集骨髓250～300mL,Ficoll液分离,配制成干细胞混悬液50mL,细胞计数为4×108～9×108。静脉麻醉下进行小腿肌肉及足部局部注射,每个位点注射0.3～0.5mL,两个位点间距约3cm,进针深度1.5～2.0cm/下肢,0.5～1.0cm/足部。④分别于术前及术后12周对患者进行肢体疼痛、冷感、间歇跛行状况评估,并采用SDW数字点温仪检测患者足背第3趾根后2cm皮肤温度,以多普勒血流探测仪及臂踝指数检查套件测量踝肱指数。观察溃疡的外观、范围、深度,有无异常症状或体症出现;血、尿、粪常规以及肝、肾功能和出、凝血时间的改变。结果:17例糖尿病足患者全部完成1年随访而进入结果分析。①基本体征指标的改变:骨髓干细胞移植后患者肢体疼痛、患肢冷感、间歇性跛行、皮肤温度及踝肱指数均得到明显缓解,与术前比较差异显著(t=-9.644～7.750,P均<0.01)。②术后足部坏疽病例创面愈合情况:5例糖尿病足患者于术后14～55d足部坏疽创面感染被控制,局部有新鲜肉芽出现,创面明显缩小并基本愈合;1例患者在接受骨髓干细胞移植的同时对足部坏疽进行彻底清创,切除坏死的左足第2趾关节远端,术后14d伤口完全愈合。③不良事件和副反应:17例糖尿病足患者骨髓干细胞移植后均未出现异常症状或体症,血、尿、粪常规及肝、肾功能和出、凝血时间均正常。随访1年,无不良反应发生。结论:自体骨髓干细胞下肢局部肌肉注射移植治疗糖尿病足患者,能够明显改善肢体疼痛、冷感、间歇跛行、踝肱指数等指标,安全有效。     

重庆市两所大学学生营养知识、态度及行为的基线调查

目的:了解重庆市某两所大学学生的营养知识、态度和行为现况,为开展营养教育效果评价提供基线数据。方法:选取重庆渝中区某学院为干预学校,文理科各一个系,整群抽取一年级9个班的学生,共289人;选取九龙坡区某学院为对照学校,选择与干预学校相匹配的文理科各一个系,整群抽取9个班的学生,共344人。于2005-03采用自编调查问卷,对以上633名在校学生进行营养知识、态度和行为的调查。问卷内容包括一般人口统计学资料、营养知识、态度、行为情况,营养知识获取途径等,其中知识问题满分16分,有关态度问题满分9分,行为问题满分40分,得分越高越好。结果:发放问卷650份,回收有效问卷633份,有效率97.4%。①两组学生一般人口学特征和家庭背景均有可比性(P>0.05)。②干预学校学生营养知识、态度和行为得分分别为(9.04±2.43),(7.13±1.20),(16.27±3.53)分,对照学校得分分别为(9.23±2.22),(6.96±1.28),(16.38±3.39)分,两组比较无差异。③只有30.33%的同学每天吃早餐,只有37.91%的学生在选择食物时考虑了营养价值或自己的营养需要,不常吃水果,常吃零食现象严重。大部分学生都有良好的求知欲望,希望通过多种途径获得营养知识。结论:①大学生对营养知识缺乏全面深入的了解,大部分学生营养态度端正,行为良好,但部分学生也存在一些问题。②两所学校营养知识、态度与行为无显著差异。     

Monocytoid differentiation of freshly isolated human myeloid leukemia cells and HL-60 cells induced by the glutamine antagonist acivicin

Previously we showed that starvation of HL-60 promyelocytic leukemia cells for a single essential amino acid induced irreversible differentiation into more mature monocyte-like cells. Although not an essential amino acid, glutamine is important in the growth of normal and neoplastic cells. The glutamine analogue, alpha S,5S-alpha-amino-3- chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) inhibits several glutamine-utilizing enzymes and therefore depletes cells of certain metabolic end products. The current study was designed to examine in vitro the effects of acivicin on growth and differentiation of several established human myeloid leukemia cell lines, including the HL-60 cell line, and of freshly isolated cells from patients with acute nonlymphocytic leukemia (ANLL). Four-day culture of HL-60 cells with acivicin at concentrations of 0.1 to 10.0 micrograms/mL (0.56 to 56 nmol/L) decreased cell growth by 33% to 88% as compared with untreated control cells. Viability of cells was greater than 92% for untreated cells and 93% to 41% for acivicin-treated cells. Cells treated with acivicin differentiated along a monocytic pathway as shown by increased H2O2 production and alpha-naphthyl butyrate esterase (NSE) content. Differentiation was time and dose dependent, and was irreversible. Changes in H2O2 production and NSE content were partially abrogated by co-culture with 10 mmol/L exogenous cytidine and guanosine but not by co-culture with other nucleosides or glutamine. At these concentrations of acivicin, differentiation was associated with expression of the N- formyl-methyl-leucyl-phenylalanine-receptor (FMLP-R) on 8% to 29% of cells as compared with 8% for control cells. Acivicin potentiated the differentiating effects of interferon-gamma, tumor necrosis factor, dihydroxyvitamin D3, dimethylsulfoxide, and retinoic acid. Culture of cells from the U937 (monoblastic), K562 (erythroleukemia), and KG-1 (myeloblastic) cell lines resulted in decreased growth and viability, but not consistently in differentiation. Acivicin decreased survival of freshly isolated ANLL cells and increased their H2O2 production and NSE content. These results suggest that the glutamine analogue acivicin may be useful as a differentiating agent with antileukemia activity in patients with ANLL.     

Nonrandom inactivation of the X chromosome in early lineage hematopoietic cells in carriers of Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked (Xp11.22) recessive immunodeficiency syndrome characterized by susceptibility to opportunistic and pyogenic infections, thrombocytopenia, and eczema. Previous studies of obligate carriers of WAS documented that nonrandom inactivation of the X chromosome carrying the defective gene is observed in all peripheral blood cells. The existence of both abnormal platelets and lymphocytes is consistent with a defect that affects early hematopoietic precursors. We isolated CD34+ hematopoietic progenitor cells collected from obligate carriers of WAS by apheresis and used polymerase chain reaction analysis of a polymorphic variable number of repeats (VNTR) within the X-linked androgen receptor to document nonrandom inactivation. These data show that nonrandom inactivation of the X-chromosome in WAS-obligate carriers occurs early during hematopoietic differentiation.     

Alpha 4 beta 1 and alpha 5 beta 1 are differentially expressed during myelopoiesis and mediate the adherence of human CD34+ cells to fibronectin in an activation-dependent way

To study the receptors involved in the interaction between extracellular matrix proteins and hematopoietic progenitor cells, we analyzed the expression of beta 1 integrins on CD34+ bone marrow cells by means of immunoflowcytometry. Alpha 4 beta 1 and alpha 5 beta 1 were expressed, whereas alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, and alpha v beta 1 were virtually absent. Furthermore, we assessed the alpha 4 and alpha 5 expression on committed myeloid progenitor cells. These colony-forming cells were detected in the alpha 4 dull fraction and the alpha 5 dull fraction. During myeloid differentiation, both in vivo and in vitro, a differential expression of alpha 4 beta 1 and alpha 5 beta 1 was observed. alpha 5 beta 1 was found to be lost at the myelocytic-metamyelocytic stage, before the loss of alpha 4 beta 1, at the band stage. Functional studies showed no binding of erythroid progenitor-depleted, CD34+ bone marrow cells to fibronectin. However, protein kinase C activation strongly induced fibronectin binding (68% of the cells). Inhibition experiments with specific antibodies and peptides showed the binding to be mediated by both alpha 4 beta 1 and alpha 5 beta 1. Also, colony-forming cells of granulocytes and macrophages were demonstrated to adhere to fibronectin in an activation-dependent way. During granulocyte colony-stimulating factor-induced in vitro maturation, the activation-dependent fibronectin binding capacity is gradually lost. We conclude that: (1) CD34+ bone marrow cells express alpha 4 beta 1 and alpha 5 beta 1; (2) the expression of alpha 4 beta 1 and alpha 5 beta 1 is differentially expressed during myeloid differentiation; and (3) binding of CD34+ bone marrow cells to fibronectin is activation dependent.     

Myeloperoxidase: an enzyme involved in intrinsic vincristine resistance in human myeloblastic leukemia

One of the differences between acute myeloblastic leukemia (AML) and acute lymphoblastic leukemia (ALL) is their sensitivity to vincristine. Although vincristine plays an important role in chemotherapeutic regimens for ALL, it does not possess clinically significant activity in AML. Horseradish peroxidase, a heme-centered peroxidase, oxidatively degrades Vinca derivatives and thereby abrogates their cytotoxic activity. This finding suggested that myeloperoxidase (MPO), a heme- centered peroxidase characteristically found in AML and not in ALL, might also degrade vincristine. We first examined the effects of MPO on vincristine in a cell-free system and demonstrated that this enzyme is capable of catalyzing vincristine's oxidative breakdown. We also observed that vincristine is more rapidly degraded in tissue culture by MPO-positive HL-60 cells than by a MPO-negative HL-60 subclone. The degree of MPO activity in these cell lines correlated in a positive manner with their degree of resistance to vincristine's cytotoxic activity. Moreover, the differential resistance to vincristine observed between these cell lines could be increased by increasing the concentration of H2O2 available to the enzyme. These data support the hypothesis that MPO-mediated oxidation of vincristine accounts in part for this drug's lack of activity in AML.     

胎儿和新生儿同种异体免疫性血小板减少症:进展与持续性争议

胎儿和新生儿同种异体免疫性血小板减少症(AIT)是引起胎儿和新生儿严重血小板减少的最常见原因.母亲针对源自父亲的胎儿血小板抗原的IgG抗体,在妊娠早期就可通过胎盘,通常导致胎儿严重血小板减少.由于一些血小板减少症临界值(50、100或150×109/L)的不同,他们的发生率亦各不相同.但在多数未经选择的人群中,AIT影响1/1 000到1/2 000活产数.在新生儿病房,临床确诊的重症AIT很罕见,可能只有1:10 000分娩数.     

How to obtain excellent response rates when surveying physicians

This paper outlines ways to maximize response rates to surveysby summarizing the most relevant literature to date and demonstratinghow these techniques have resulted in consistently high ratesof return in family practice research. We describe the methodologyused in recent surveys of physicians conducted by the Centrefor Studies in Family Medicine through its Thames Valley FamilyPractice Research Unit, located in London, Ontario, Canada andfunded by the Ontario Ministry of Health and Long-Term Care.The identification and implementation of these techniques tomaximize response rates is critical, as primary health careresearchers often rely on information gathered through questionnairesto study physicians' practice profiles, experiences and attitudes.Four separate and distinct mailed surveys of physicians usinga modified Dillman approach were conducted from 2001 to 2004.The sampling strategies, topics, types of questions and responseformats of these surveys varied. The first survey did not useany incentives or recorded delivery/registered mail and receiveda response rate of 48%. In sharp contrast, the other three surveysobtained responses rates of 76%, 74%, 74%, respectively, achievedthrough the use of gift certificates and recorded delivery/registeredmail. Sending a survey by recorded delivery/registered mailtends to result in the survey package being given priority inthe physicians' incoming mail at the practice. Gift certificatespartially compensate physicians for time spent completing thesurvey and recognition of the time required is appreciated.The response rates achieved provide strong evidence to supportthe use of monetary incentives and recorded delivery/registeredmail (along with the Dillman approach) in survey research. Itis anticipated that this evidence will be used by other researchersto justify requests for funding to cover the costs associatedwith incentives and recorded delivery/registered mail. We recommendthe use of these strategies to maximize response rates and improvethe quality of this type of primary health care research. Keywords. Response rates, surveys, physicians.