皮肤黑素细胞酪氨酸酶基因高表达模型的研究

目的　细胞黑素的产生与色素基因表达量有关。本研究旨在建立体外黑素细胞色素基因高表达模型 ,为基因调控、基因治疗、化学治疗的效果和机理的研究提供实验依据。方法　应用黑素细胞培养技术培养人皮肤黑素细胞 ,行Dopa染色和Fantana银染法。黑素细胞经紫外线和内皮素处理后 ,检测黑素细胞的黑素含量和酪氨酸酶mRNA表达状态。结果　黑素细胞经紫外线照射后 ,细胞黑素含量由 (11± 1.5 ) pg/cell上升至 (5 4± 2 .3)pg/cell (P<0 .0 1) ,而酪氨酸酶mRNA的表达相对值由 (6 4± 1.2 ) %上升至 (14 2± 2 .8) % (P <0 .0 1) ;内皮素 (ET - 1)处理黑素细胞后 ,黑素的含量也上升至 (6 8± 1.9) pg/cell (P <0 .0 1) ,而酪氨酸酶mRNA的表达量上升至 (187± 3.4 ) %。结论　紫外线和内皮素可以增加黑素细胞的黑素含量和增加酪氨酸酶mRNA的表达量。     

川芎嗪对体外培养的黑素细胞的抑制作用

目的观察川芎嗪(TMP)对体外培养的黑素细胞的抑制作用.方法采用MTT法测定细胞增殖情况;NaOH裂解法测定黑素合成;Takahashi法测定酪氨酸酶含量;用透射电镜观察黑素小体的生成.结果川芎嗪对黑素细胞的增殖有抑制作用,能使细胞数明显减少(P＜0.05),并能使黑素合成显著下降(P＜0.01),使酪氨酸酶活性逐渐减弱(P＜0.01);透射电镜观察显示,加川芎嗪后黑素小体明显减少.结论川芎嗪能抑制黑素细胞增殖、黑素合成、酪氨酸酶活性,这可能是临床应用中药川芎治疗皮肤色素性疾病的机制.     

反义寡聚脱氧核苷酸对黑素细胞TYR基因和黑素产量的调控作用

目的 设计并合成针对人酪氨酸酶的反义寡核苷酸，从基因表达水平调控细胞黑素的合成，为色素沉着性疾病的治疗寻找新途径。方法 将培养的黑素细胞分为内皮素处理组、紫外线处理组和对照组，并分别加入5′端反义核酸、3′端反义核酸、混合反义核酸或单纯脂质体，分别检测黑色素含量和酪氨酸酶mRNA的表达。结果 反义核酸对内皮素及紫外线照射引起的黑素细胞色素含量的增加以及TYR基因表达的增强有明显的抑制作用，其中3′端反义核酸作用最强。结论 反义寡聚脱氧核苷酸对黑素细胞黑素产量和TYR的基因表达有显著的调控作用，3′端反义核酸的调控作用优于5′端反义核酸。     

原花青素对紫外线辐射后人表皮黑素细胞光保护机制的研究

目的:研究葡萄籽提取物原花青素(OPC)对紫外线辐射后人表皮黑素细胞内活性氧自由基(ROS)清除、细胞周期变化以及黑素合成关键酶表达的影响,以探讨OPC对黑素细胞的光保护机制.方法:培养的正常表皮黑素细胞经15mJ/cm2紫外线辐射后分别以高、中、低(10、50、100μg/m1)三种浓度的OPC作用24 h后,以四甲基偶氮唑蓝(MTT)比色法测定细胞存活率;采用二氯荧光素二酯(DCFH-DA)标记法测定细胞内ROS水平;碘化丙啶(PI)、溴脱氧尿苷(BrdU)双标记结合流式细胞仪检测细胞周期;免疫印迹法测定细胞酪氨酸酶(TYR)、酪氨酸酶相关蛋白1(TRP1)、酪氨酸酶相关蛋白2(TRP2)蛋白含量.结果:OPC可呈浓度依赖性地保护受紫外线辐射的黑素细胞存活率、清除紫外线诱导的黑素细胞内活性氧自由基的产生;≥50μg/ml OPC可减少黑素细胞内TYR,TRP1的蛋白表达量、修复紫外线辐射后下降的TRP2蛋白表达;减少紫外线辐射后G1期细胞比例,增加s期细胞比例.结论:天然寡聚体OPC可能通过抑制细胞内ROS的产生、减少黑素合成关键酶的表达、修复停滞于细胞分裂前期状态的细胞来发挥对黑素细胞的光保护作用.     

两种培养状态下黑素细胞的生物学特征比较

目的:比较角质形成细胞无血清培养基(KC-SFM)和含血清的黑素细胞培养基培养的人类黑素细胞的生物学特征的差异,寻找适合黑素细胞移植治疗的黑素细胞培养方法。方法:以KC-SFM和含血清的黑素细胞培养基培养黑素细胞,在培养7d内倒置显微镜观察黑素细胞形态,MTT法观察生长曲线,并测定酪氨酸酶活性和黑素含量。结果:KC-SFM培养的黑素细胞具有较多树突且树突明显延长,增殖活性低于含血清的黑素细胞培养基培养的黑素细胞,而酪氨酸酶活性、黑素含量测定吸光值分别为0.385±0.036、0.276±0.022,和含血清的黑素细胞培养基培养黑素细胞比较均有显著差异。结论:KC-SFM可促进黑素细胞树突增多和树突延长,提高酪氨酸酶活性和黑素含量,适合用于移植治疗的黑素细胞培养。     

氨甲环酸对中波紫外线照射后黑素细胞影响的研究

目的:探讨氨甲环酸对中波紫外线照射后黑素细胞增殖及酪氨酸酶代谢的影响。方法:设立紫外线照射组和非照射组,以不同浓度的氨甲环酸作用于黑素细胞,cck-8法测定黑素细胞增殖;多巴氧化方法测定酪氨酸酶活性,RT-PCR方法测定酪氨酸酶mRNA表达水平。结果:氨甲环酸对两组黑素细胞的增殖均无明显抑制作用(P0.05);而对酪氨酸酶活性和酪氨酸酶的mRNA表达有抑制作用(P0.05),两组结果比较无明显差异;结论:氨甲环酸对黑素细胞的酪氨酸酶的抑制作用与中波紫外线照射无直接关系。     

紫草素抑制紫外线辐射后黑素合成的研究

目的:研究紫草素对正常人表皮黑素细胞黑素合成的影响,及对紫外线辐射后黑素细胞黑素合成的影响。方法:用体外培养正常人表皮黑素细胞供给实验,黑素细胞经10m J/cm2紫外线辐射后,分别以选定的几个浓度(主要为0.25μg/L、0.5μg/L、1μg/L三种浓度)的紫草素培养72h,测定细胞增殖率、黑素含量及酪氨酸酶活性。结果:紫草素可减轻紫外线辐射对细胞增殖的抑制作用,并可抑制紫外线辐射后的表皮黑素细胞的黑素合成;较高浓度紫草素(大于0.5μg/L)对未经照射的表皮黑素细胞的增殖有抑制作用,但对黑素合成无明显影响。结论:紫草素可以明显抑制紫外线辐射后表皮黑素细胞的黑素合成,推测其对紫外线辐射的黑素细胞有光保护作用。     

芦荟苦素对色素化皮肤类似物模型中黑素细胞的影响

目的 观察芦荟苦素、熊果苷及茶多酚对色素化皮肤类似物模型中黑素细胞的影响.方法 体外构建色素化皮肤类似物模型,检测芦荟苦素、熊果苷及茶多酚作用于此模型后对细胞形态、黑素细胞酪氨酸酶活性以及黑素合成的影响.结果 在色素化皮肤类似物模型中3种退色剂对黑素细胞酪氨酸酶活性有明显抑制作用,可显著减少黑素生成,其中以茶多酚的抑制作用最强,芦荟苦素次之.但茶多酚对细胞的毒性最大,熊果苷和芦荟苦素毒性均较小.结论 3种退色剂对色素化皮肤类似物中黑素细胞的酪氨酸酶活性和黑素合成均呈浓度依赖性抑制,而茶多酚则显示出较大的细胞毒性(P＜0.05).芦荟苦素所表现出的对酪氨酸酶和黑素的合成有较强的抑制作用及对细胞较低的毒性作用,有可能成为一种比较安全的退色剂.     

维A酸在细胞和基因水平调控黑素表达的研究

目的探讨维A酸治疗皮肤色斑的作用机制。方法应用不同浓度的维A酸作用于B16F10黑素瘤细胞及正常人黑素细胞,观察对酪氨酸酶mRNA的表达、酪氨酸酶活性、黑素含量及黑素细胞增殖率的影响。结果维A酸的作用浓度为100μmol／L时,黑素细胞酪氨酸酶mRNA表达下调均值为30．13％;当浓度为500μmol／L以上时,可使酪氨酸酶活性降低82．79％,并随着浓度的增加作用越明显;而对黑素含量的降低则需要更高的浓度（1000μmol／L）;同时,还有促进黑素细胞增殖的作用。对照组氢醌对酪氨酸酶mRNA的表达无明显影响,对色素细胞有抑制和毒性作用。结论维A酸能够抑制黑素产生,是通过下调酪氨酸酶的表达和活性来完成。     

复方中药对肝硬化大鼠肝组织内皮素-1mRNA表达的影响

目的　研究复方中药对肝硬化大鼠血浆及肝组织内皮素 1(endothelin 1,ET 1)和肝组织ET 1mRNA表达的影响。方法　采用放射免疫法测定血浆和肝组织ET 1水平 ,逆转录多聚酶链反应 (RT PCR)测定大鼠肝组织ET 1mRNA表达水平。结果　模型组血浆 (171 99± 2 7 86pg/ml)和肝组织ET 1(80 4 8± 2 1 4 5 pg/ml)较对照组 (114 33± 17 34pg/ml,5 3 2 5± 13 6 4pg/ml,P <0 0 5 )均显著升高 ,肝组织ET 1mRNA表达也较对照组 (0 4 6 74± 0 10 4 5vs.0 14 13± 0 0 2 97,P <0 0 5 )显著升高。治疗组血浆ET 1(12 9 80± 13 15 pg/ml)和肝组织ET 1mRNA表达 (0 30 4 0±0 0 813)均显著低于模型组 (171 99± 2 7 86 pg/ml,0 4 6 74± 0 10 4 5 ,P <0 0 5 )。而治疗组肝组织ET 1(77 36± 18 2 7pg/ml)与模型组 (80 4 8± 2 1 4 5 pg/ml)相比无显著差异。 结论　复方中药能显著降低肝硬化大鼠肝组织ET 1mRNA的表达及血浆ET 1水平。     

Hazards and complications of endoscopic transurethral ureterolithotripsy and lithoextraction and means of prevention

Complications related to ureterolithotomy and ultrasonic ureterolithotripsy performed under the control of visual endoscope were analyzed in 86 ureterolithiasis patients, methods of their prevention discussed. All the aforementioned complications were distributed into three groups: inapplicability of surgery due to anatomic and functional defects of lower and upper urinary tracts, intraoperative, and postoperative complications. The commonest ones were ureteral abruption and perforation, acute pyelonephritis, temporary vesicoureteral reflux. Their control measures were considered as relative methods of treatment: immediate surgical intervention in case of ureteral abruption, renal catheterization in patients with insignificant ureteral perforation or acute pyelonephritis. Adequate ureteroscopy, careful consideration of pro- and contraindications, catheterization of renal pelvis and urinary bladder performed within 2-3 days after the surgery and adequate antibacterial therapy are the most decisive steps in the control of aforementioned complications.     

牙体牙弓颌骨阻力中心及其临床意义

牙体、牙弓及颌骨的阻力中心在正畸矫治力系统中具有重要的意义,也是正畸学领域争论较多的一个问题。Dermaut等研究表明,当力作用于物体阻力中心时,物体将发生平动,否则将发生平动和转动的复合运动。目前,国内外多数学者认为牙体、牙弓及颌骨存在阻力中心,但其位置存在争议。本文就牙体、牙弓及颌骨的阻力中心及其临床意义作一综述。     

Characterisation and differentiation of chondroblastomas and chondromyxoidfibromas - presence and expression of collagen types I and II

AIM: Chondroblastomas and chondromyxoidfiibromas are rare benign skeletal neoplasms with reported overlapping histology. Aim of this study was to analyse the biochemical composition of the matrix of these tumour entities in order to further characterise the cellular phenotypes of these neoplasms using typical cell biological marker genes. METHODS: The matrix compositions of chondroblastomas and chondromyxoidfibromas were analyzed by HE-histology, histochemistry, and immunolocalization techniques. Cellular gene expression patterns were detected by mRNA in situ hybridization. RESULTS: Chondroblastomas are rich in collagen type I and show foci of an osteoid-like matrix, whereas collagen type II as a typical marker of chondrocytic differentiation was not detected in any of the specimens. Chondromyxoidfiibromas had foci of chondroid appearance with chondroblastic cellular differentiation characterised by collagen type II expression. CONCLUSION: These results characterise chondroblastomas and chondromyxoidfiibromas as skeletal neoplasms that have a different biology and which can be distinguished by matrix protein expression products: collagen type II, the typical marker of chondroblast differentiation, could only be detected in chondromyxoidfibromas, but not in chondroblastomas. Thus, both neoplasms are clearly different on the cell biological level.     

Histochemical and contractile properties of striated muscles of urethra and levator ani of dogs and sheep

AIMS: To understand their possible importance in long- and short-term control of continence, some properties of the striated muscles of the urethra and pelvic floor (levator ani) of dogs and sheep were investigated, especially fiber types and contractile characteristics. MATERIALS AND METHODS: Striated muscles of urethra and levator ani of 29 male and 6 female dogs and 11 male and 6 female sheep were removed and cut into strips. Some strips were frozen and stained for ATPase at pH 9.4 and 4.3 for fiber typing; others were set up in an organ bath to study contractile responses to nerve stimulation. RESULTS: All muscles contained both type I (slow) and type II fibers, ranging from 97% type II in female greyhound urethra to 60% in female sheep levator ani. For each muscle, there were fewer type II muscles in sheep than in dog. The diameters of the urethral fibers were about 60% of the levator ani in dogs and 34% in sheep. Contraction of the urethral muscle was faster than for levator ani and declined to about 80% of the peak, 500 msec after the beginning of stimulation at 20 Hz. The levator ani contraction rose to a steady level as long as stimulation continued. CONCLUSIONS: Both the levator ani and urethral striated muscles contain slow and fast fiber types. The levator ani muscles are capable of sustained contraction with rapid onset which will produce long-term closure of the urethra. The circular urethral muscle contraction was faster but less well maintained.     

Incorporation and release of85Sr and14C-proline-hydroxyproline in mineral and collagen of bone and incisor teeth of growing rats

The extent to which exchange and reutilization processes of mineral tracers affect skeletal mineral accretion and resorption measurements was evaluated by comparing the rates of appearance and disappearance of⁸⁵Sr and¹⁴C-proline-hydroxyproline in bones and teeth in growing rats for 12 days following simultaneous parenteral injection of these tracers. Expressions for the relative rates of collagen synthesis and breakdown, which unlike mineral metabolism are considered not to be complicated by exchange phenomena, were based on¹⁴C-proline conversion to¹⁴C-hydroxyproline; the specific activity of the latter was determined. Both the mineral and the collagen specific activities reflected the rates and patterns of growth of the samples assayed; rapid growth and a short interval of time between formation and resorption of tissue in themetaphyseal bone which contains the cartilagineous growth plate, slow growth and an interval of time between formation and resorption of tissue indiaphyseal bone and incisor teeth which is longer than the 12 days of the experiment. However, in metaphyseal bone the specific activity collagen/mineral ratio dropped by one half during the 4–12 day interval in contrast to diaphyseal bone and incisor teeth in which no change in this ratio was observed during this period of time. The data indicate that collagen in the metaphyseal growth zone is removed by resorption before it has become fully mineralized, and that exchange is a relatively unimportant factor in the long term kinetics of bone mineral.

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Zusammenfassung Das Ausmaß, bis zu welchem Austausch- und Wiederverwendungsprozesse der mineralen Tracer die Messungen des mineralen Skelett-Auf- und Abbaues beeinflussen können, wurde ausgewertet; zu diesem Zweck wurde die Geschwindigkeit des Auftretens und Verschwindens von⁸⁵Sr und von¹⁴C-Prolin-Hydroxyprolin in Knochen und Zähnen von wachsenden Ratten während der 12 auf die simultane parenterale Injektion dieser Tracer folgenden Tage verglichen.Der Ausdruck für die relative Geschwindigkeit des Kollagen-Auf- und Abbaues, bei welchem im Gegensatz zum Mineralmetabolismus kein Mitwirken des Austauschphänomens vermutet wird, basiert auf der Umwandlung von¹⁴C-Prolin zu¹⁴C-Hydroxyprolin; die spezifische Aktivität des letzteren wurde bestimmt.Aus der spezifischen Aktivität des Minerals sowie jener des Kollagens konnten die Geschwindigkeit und die Art des Wachstums der untersuchten Proben ersehen werden, d.h.schnelles Wachstum und ein kurzes Zeitintervall zwischen Bildung und Resorption des Gewebes imKnochen der Metaphyse, die auch die knorpelige Wachstumsplatte enthält, und andererseitslangsames Wachstum und längeres Zeitintervall (länger als die 12 Tage des Experimentes) zwischen Bildung und Resorption des Gewebes imKnochen der Diaphyse und in den Schneidezähnen. Immerhin fiel die spezifische Aktivität des Kollagen/Mineral-Anteils im Knochen der Metaphyse während dem 4–12tägigen Zeitintervall auf die Hälfte, im Gegensatz zum Knochen der Diaphyse und der Schneidezähne, bei welchen während dieser Zeitspanne kein Unterschied in diesem Verhältnis beobachtet wurde.Diese Ergebnisse zeigen, daß Kollagen in der Wachstumszone der Metaphyse durch Resorption verschwindet, bevor es ganz mineralisiert ist, und daß der Austausch ein relativ unwichtiger Faktor in der Kinetik auf lange Sicht des Knochenminerals ist.