【作者】 张曙萱；

【导师】 王海琦；

【作者基本信息】 南京医科大学 ， 围产医学， 2004， 硕士

【摘要】 前言 计划生育是我国的一项基本国策，抗生育药物的研制则是计划生育工作的重要组成部分。目前临床抗生育药物主要是甾体合成药，作用位点高且多系抗排卵药物，对机体内分泌有一定的干扰。而中草药则具有特有的天然疗效，作用位点低，副作用小等特点；对马鞭草有效部位进行提取分离，筛选出其抗生育的有效组分或有效成分，对于开发新型计划生育药物具有非常重要的意义。 蜕膜是孕卵着床和胚胎发育所必须的组织，用药物干扰蜕膜的发育应是理想的抗早孕手段。体外细胞培养以其直观、便利展现了其独特的优越性。马鞭草有破血通经、利尿消肿等作用，孕妇忌用。我院曾对76例停经33-42天健康早孕妇女口服马鞭草粉剂，流产成功率达83.4％。孕早期人绒毛组织体外培养显示，马鞭草能明显抑制绒毛生长及滋养层细胞分泌HCG的功能；动物实验表明，马鞭草也能明显抑制胚胎生长而使其固缩死亡，破坏滋养层细胞，对小鼠有明显的抗旱孕作用。但对马鞭草是否能抑制蜕膜细胞的生长还不甚清楚，以及对于马鞭草终止早孕的有效部位的确定国内也尚未有报道。 中药抗生育效果良好，副作用小；服用方便，已成为各国当前日益关注的课题。目前，应用中草药抗孕方面的研究虽然有些报道，但缺乏基础细胞等方面深入的研究。南京人学硕上学位沦文 本研究目的首先是进行蜕膜基质细胞的体外培养，并对其培养方法做了一定的改进，然后观察经不同提取途径获得的马鞭草提取液A、B、C、D及米非司酮对蜕膜细胞形态，增殖，细胞凋亡及细胞周期动力学的影响。从而了解马鞭草抗生育的机制及有效抗生育作用组分及作用位点。第一部分人类蜕膜基质细胞体外培养的研究目的:建立良好的人类蜕膜机质细胞培养方法。方法: 实验选用早孕人工流产蜕膜组织进行体外培养，配制FD完全培养液及0.25%胰蛋白酶和0.02%EDTA(1:1)消化液，进行全组分蜕膜细胞培养，24小时后换液，清除未贴壁的蜕膜细胞及红细胞。视细胞生长情况，2饱天换培养液一次。待细胞融合达800/011寸，用0.25%胰蛋白酶与0.OZEDTA(1:1)消化贴壁细胞，传代。矛，」用传三代的方法对蜕膜基质细胞进行分离提纯。传三代时部分细胞用盖片法培养，制作细胞爬片，用泌乳素(prolaCtin PRL)免疫组化试剂盒检测培养细胞成分，鉴定细胞纯度。结果二 人体蜕膜基质细胞悬浮时呈典型的卵圆形，单层培养时为纤维母细胞状。接种后30分钟开始贴壁，逐渐伸长成为纤维母细胞状，呈南京大学硕卜学位论文较长的梭形和不规贝!}星形，核呈卵圆形，位于细胞中央，核仁清晰，有丰富的胞质颗粒。24小时细胞大多贴壁，6一了天可贴壁汇合。原代培养的蜕膜细胞在20cy0FCS的培养液中可增生数周，直至细胞汇合后才保持不变。其中约750/0为蜕膜基质细胞，细胞之间相互连接，胞间界限模糊。传代后的蜕膜细胞生长迅速，2一3天即可铺满瓶底，镜下细胞形态均一，呈梭形或不规则星形。传三代后其形态无明显变化，而传代培养极性渐不明显。免疫组化显示体外培养传代后97ry0蜕膜细胞为蜕膜基质细胞。结论: 该实验方法经济实用、简单，简化了蜕膜细胞的提纯程序，获得的蜕膜基质细胞纯度高。第二部分马鞭草提取液及米非司酮对体外培养人早孕蜕膜细胞的的影 响 目的:l.探讨马鞭草及米非司酮抗早孕的细胞学作用机理;2.初步确定马鞭草杭生育的有效部位。方法: 1.取正常妊振6一9周妇女人工流产的蜕膜组织，进行体外细胞培养，传三代后接种于培养瓶中。培养24小时后，换液，分为含25mg/ml(含马鞭草生药)的A、B、C、D组(A、B、C、D分别为马鞭南京大学硕士学位论文草石油醚、氯仿、乙酸乙醋、甲醇提取液，每毫升含生药1克)及SOug/nll的米非司酮组及未加药组。继续培养48，J、时，在倒置显微镜下观察。 2.将传三代后的蜕膜基质细胞接种与三块%孔培养板，设三复孔。24，J、时后对照组加入培养液20Oul，加药组分另，{加含A、B、C、D及米非司酮培养液ZO0ul，A、B、C、D的终浓度为12.5mg/ml，25mg/ml，50mg/ml(含马鞭草生药).米非司酮的终浓度为80ug/ml。加药后分另，J继续培养24，J、时，48，J、时，72，J、时。MTT显色法测各孔的吸光度0D值。 3.培养蜕膜基质细胞，传三代后接种于培养瓶中(共30瓶，平分为6组)。培养24，J、时后，分为加入25mg/ml(含马鞭草生药)的A、B、C、D及25mg/ml的米非司酮组及未加药组。继续培养48小时后FACScan流式细胞仪测定荧光强度。对细胞凋亡及细胞周期动力学进行分析。结果: 1.高于12.smg/ml的A、B、C及80ug/ml的米非司同酮均可明显改变蜕膜基质细胞的形态，使细胞数明显减少，细胞体积缩小，漂浮细胞较多，细胞变圆，缩小，细胞固缩，空泡化，有的细胞破碎。D药无明显作用。 2.MTT显色法示A、B、C及米非司酮组对细胞生长有明显的抑制作用，并呈剂量依赖性抑制。在一定的范围内，有良好的量效关系。D药无明显作用。 3.25。lg/ml的A、B、C及米非司酮(8 Oug/ml)均促进细胞凋亡，A、B、C组蜕膜细?更多还原

【Abstract】 IntroductionBirth control is an basic policy in our contry ,so the sutudy of the anti-pregnancy drugs is an important part in birth control programme.By now mainly clinical anti-pregnancy drugs are steroid-compound ones which having high position effect and always belonging to inhibiting-ovulation durgs.This kind of durgs have certain interference to organism.Traditional Chinese medicine have character of special nature effect ,low-position effect and little side effect.Extracting the effective portion of Verbena of offcinalis L ,preparating the anti-pregnancy portion have significant meaning to develop new drugs.Decidua is the necessary tissue to embedding of ovum and growth of embryo.Interfering the developing of deciduas should be the good anti-early pregnancy activity.Cell culture in vitro has the vitue of direction and Traditional Chinese medicine. Verbena of offcinalis L have urging menstruation and diuresis effect and be avoiding to pregnancy women . The successful abortion rate was 83.4% in 76 healthy early pregnancy(33~42day) women who had taken Verbena of offcinalis L power.Cluture of the trophoblast in vitro had shown thatVerbena of offcinalis L could inhibit the growth of the trophoblast and HCG secretion obviously.Animal experiment had shown Verbena of offcinalis L also could inhibit the developing of embryo and make it wither ,die;destroy the syncytiatrophoblast cells;could terminate the early pregnancy in pregnant mouse. However ,until now whether Verbena of offcinalis L can inhibit the growth of the decidual cell is obscure and there was no report of the effective portion of Verbena of offcinalis L in terminating early pregnancy internal.Traditional Chinese medicine have good anti-preganancy effect with little side effct and can be taken convinently.lt is being paid attention to in the world. Now-Adays, although some study of traditional Chinese medicine used in terminating pregnancy had been reported ,but few studies about its activity were on cell level.In this experiment,by means of culture of decidual stromal cells , the effect of diffirent extraction of Verbena of offcinalis L (A, B, C, D)and mefepristone on the morphology and viability, proliferation , apoptosis, cellcycle proliferation was be observed to explore machanism of Verbena of offcinalis L and mefepristone with anti-early pregnancy activity on cell level and investigate the active portion in the Verbena of offcinalis on anti-early pregnancy activity .PART 1Study on Culture of Human Decidul Stromal Cell in Vitro Objective To explore the simple and valuable method to culture human decidual stromal cell.Methods In this experiment ,abortion decidual tissues of earlypregnant healthy women were cultured in vitro. Compounding FD(F-12/DMEM, 1:1) compelete culture fluid , 0.25% trypsin and0.02%EDTA (1:1) .All components of decidual cells were culturedtogether. After culturing for 24 hours ,the culture fluid were changedand unadhesive decidual cells and red blood cells were got ridof.According to the growth of cells,changed the culture fluid every 2-3days. When merged to 80%,the cells were digested by 0.25%trypsin and 0.02%EDTA (1:1) . After transferring three times the DSCcould be isolated and purified. Made cell climbing flake.Then theDSC were identified by immunohischemistry (prolactin PRL).Result Human decidual stromal cells present typical elliptic shape andpresent shape like fiberblast when cultured in singlelayer. Afterinoculating ,30 minitue later the cells began to adhere to the wall andelongate to fiberblast shape gradually,presenting the longer shuttle shapeand irregular star shape. The cell nucleus presenting elliptic shape andwere in the center of the cells.The cell nucleous were distinct withabundant cytoplasm pellet.24 hours later cells mostly adhere to the walland 6~7 days later could merge .These cells could multiplicate for someweeks in 20%FCS culture fluid and kept unchanged state until merging.In these cell更多还原