【作者】 楼湘莹；

【导师】 王自能；

【作者基本信息】 暨南大学 ， 妇产科， 2008， 博士

【摘要】 目的:1.研究米非司酮对人早孕绒毛滋养细胞血管生成因子的影响,探讨其与绒毛间质血管形成的可能关系;2.研究米非司酮对体内人早孕绒毛滋养细胞增殖与凋亡的影响;3.探讨米非司酮对体外培养的绒癌细胞系BeWo、JEG-3的凋亡作用。方法:1.应用免疫组织化学技术标记早孕人流组和米非司酮药流组绒毛组织中CD34,并行血管体视学观察;2.应用免疫组织化学方法检测早孕人流组和米非司酮药流组绒毛组织中VEGFAb7、VEGF JH121、Ang-1和TSP蛋白的表达,并进行图像分析;3.免疫组化与TUNEL技术,检测滋养层细胞增殖与凋亡指数;免疫组化与图像分析法,检测Caspase-3在人早孕绒毛滋养层的表达强度;Western blotting技术检测绒毛组织中凋亡下游和上游蛋白Caspase-3、-7和Caspase-9的表达;4.体外培养绒癌细胞系BeWo和JEG-3,分别以不同浓度米非司酮0、0.25、1、2.5、10、25μmol/L对两细胞系进行干预,分为对照组和米非司酮处理组。通过Annexin V-FITC/PI双染流式细胞术测定凋亡和坏死细胞比率;5.采用透射电子显微镜技术观察早孕人流组与米非司酮药流组绒毛组织的超微结构特征。结果:1.米非司酮对人早孕绒毛间质血管影响的体视学观察米非司酮药流组微血管长度密度(L_V)较人流组降低(P=0.032,P＜0.05);微血管体积密度(V_V)显著降低(P=0.002,P＜0.01)。2.米非司酮对早孕绒毛滋养层血管生成因子分泌的影响早孕人流组绒毛滋养层细胞VEGF Ab-7和Ang-1的表达强度高于米非司酮药流组,差异有统计学意义(P＜0.05);而VEGF JH121和TSP在药流组早孕绒毛滋养层细胞的表达强度高于人流组,差异均有统计学意义(P＜0.05)。各类血管生成因子分别与绒毛间质微血管L_L和V_V作相关性分析,VEGFAb-7和Ang-1与血管新生成正相关(P＜0.05),而VEGF JH121和TSP与血管新生负相关(P＜0.05)。3.米非司酮对绒毛滋养层细胞增殖与凋亡的影响早孕人流组与米非司酮药流组均有Ki67和TUNEL阳性表达,前者主要定位于细胞滋养细胞,后者主要在合体滋养细胞表达。采用“单位面积阳性核数”来评价增殖与凋亡状况,经图像分析,米非司酮作用后,绒毛滋养层细胞增殖能力降低,凋亡增多,差异具有统计学意义(P＜0.05)。4.米非司酮对凋亡上、下游凋亡蛋白Caspase的影响采用免疫组化检测两组绒毛滋养层细胞Caspase-3的表达,结果经图像分析定量处理,在米非司酮药流组的表达强度显著高于早孕人流组,差异具有统计学意义(P＜0.01)。利用Western blotting技术检测早孕人流组(n=8)和药物流产组(n=8)绒毛组织中凋亡下游蛋白Caspase-3,-7和凋亡上游蛋白Caspase-9表达。16例标本均检测到Caspase-3,它在药流组表达高于早孕人流组。Caspase-7在药流组的检出率(75%)高于早孕人流组(37.5%),Caspase-9在药流组的检出率(50%)亦高于早孕人流组(12.5%),可见Caspase-7和Caspase-9在药流组表达均较早孕人流组强。5.米非司酮对体外绒癌细胞系BeWo和JEG-3凋亡的影响米非司酮作用于BeWo细胞24h后,随米非司酮浓度增高BeWo存活细胞百分数略呈下降趋势;空白组凋亡比率0.4%,各浓度组凋亡比率较空白组增高,在米非司酮浓度为2.5μmol/L时,细胞凋亡率最高,为8.03%。不同浓度米非司酮作用JEG-3细胞系24h后,其存活细胞百分数随浓度增高逐渐降低,而凋亡比率逐渐升高,空白组凋亡比率为4.69%,10μmol/L米非司酮作用下,凋亡比率最高,为14.96%。6.早孕人流组及米非司酮药流组绒毛组织的超微结构观察正常早孕绒毛组织超微结构特征:(1)合体滋养细胞具丰富的微绒毛,胞浆中粗面内质网、线粒体发达,可见高电子密度的分泌颗粒,游离核糖体、脂滴多见;(2)细胞滋养细胞胞浆内游离核糖体丰富,线粒体多见,周围有粗面内质网包绕;(3)绒毛间质血管内皮细胞(EC)具丰富胞质突,与血管周细胞、间质细胞多发生细胞-细胞接触。米非司酮药流组绒毛组织超微结构特征:(1)合体滋养细胞部分发生脱落,表面微绒毛疏密不均、甚发生缺如,胞浆中粗面内质网扩张,线粒体发生空泡化,近游离缘胞核染色质发生浓集,似凋亡改变;(2)细胞滋养细胞胞浆中可见大块无膜包绕的糖原颗粒密集,线粒体部分空泡样变;(3)绒毛间质EC胞核染色质浓集、功能欠活跃,胞浆中粗面内质网不发达,线粒体部分空泡化,胞质突减少,与血管周细胞、间质细胞少发生细胞-细胞接触。结论:1.米非司酮作用下,人早孕绒毛间质血管新生受到一定程度抑制,可能不利于胚胎生存。2.在米非司酮作用下,滋养细胞分泌功能受到损伤,血管生成因子动态平衡被打破,血管新生被破坏。3.米非司酮作用下,细胞滋养细胞增殖受阻,凋亡通路上游及下游Caspases家族蛋白被上调,增殖与凋亡的动态平衡被打破,不利于胚胎生存。4.米非司酮在体外可诱导绒癌BeWo和JEG-3细胞发生凋亡,推测它可直接促使滋养细胞凋亡。5.米非司酮引起的绒毛滋养细胞超微结构的变化反应其分泌、增殖、凋亡等功能的改变更多还原

【Abstract】 Objective:1.To clarify the effects of mifepristone on the secretion of angiogenesis factor of human first trimester trophoblast,and to discuss the relationship of mifepristone and angiogenesis in villous stroma.2.To study the effects of mifepristone on proliferation and apoptosis of human first trimester trophoblastic cells.3.To study the effects of mifepristone on the induction of apoptosis of in vitro cultured BeWo and JEG-3 cell lines.Methods:1.The villous capillaries in the early pregnancy were stained with anti-CD34 and evaluated sterologically.2.Using immunohistochemistry and image analysis technique to study the expression of VEGF Ab-7,VEGF JH121,Ang-1 and TSP in the villous trophoblasts of mifepristone group and control group.3.The proliferative rate and apoptotic rate were evaluated by immunohistochemistry and TUNEL technique.The staining intensity and distribution of Caspase-3 was evaluated by image analysis technique.The expression of apoptotic related proteins, such as caspase-3,-7,-9 were detected by Western blotting technique.4.The BeWo and JEG-3 cell lines were cultured in vitro,the experimental group is divided into following 2 subgroups,(1) control group:BeWo and JEG-3 cell lines without mifepristone;(2) mifepristone group:cells were treated by 0,0.25,1,2.5,10 and 25μmol/L mifepristone respectively.Detecting the apoptotic and necrotic cell ratios by Annexin V-FITC/PI double staining flow cytometry.5.The ultrastructure of the villous tissue in mifepristone group(n=3) and control group(n=3) was investigated by transmitted electron microscope(TEM).Results:1.Effects of mifepristone on the stereological examination of the first trimester placental villiAlmost all villous capillaries of the first trimester villi were stained by CD34.In mifepristone group,the length density and volume density of villous capillaries were statistically significantly decreased than that in control group(P＜0.05).2.Effects of mifepristone on the secretion of angiogenesis factors in the first trimester trophoblastic cellsIn trophoblastic cells,the staining intensity of VEGF Ab-7 and Ang-1 in control group were statistically significantly higher than that in mifepristone group(P＜0.05). While the expressions of VEGF JH121 and TSP were predominantly decreased in control group than that in mifepristone group(P＜0.05).3.Influence of mifepristone on the proliferation and apoptosis in trophoblastic cellsAll villous specimen showed Ki67 and TUNEL positive expression.Ki67 mainly situated in cytotrophoblast,while TUNEL were mainly expressed in syncytotropho- blast.Using the number of positive nucleus in an unit area,the states of trophoblastic proliferation and apoptosis in the first trimester were examined.Compared with the control group,the trophoblastic proliferation in mifepristone group was decreased, while apoptosis was enhanced.4.Effects of mifepristone on the expression of apoptotic-related protein caspase-3,-7,and -9 in trophoblastic cellsIn trophoblastic cells,the staining intensity of caspase-3 in mifepristone group was significantly higher than that in control group(P＜0.05).Using western blotting technique,all the villous tissues expressed caspase-3.The detection rate of caspase-7 was higher in mifepristone than that in control group(75%vs 37.5%).And the detection rate of caspase-9 had the same outcome(50%vs 12.5%).5.Effects of mifepristone on the apoptotic state in vitro cultured BeWo and JEG-3 cell linesBeWo cells were treated with mifepristone for 24h,the number of apoptotic cells trended upwards obviously as mifepristone concentration increased.The ratios of cell apoptosis in different mifepristone group were higher than that in control group.The highest apoptotic rate of BeWo cells was found in 2.5μmol/L mifepristone group (8.03%vs 0.4%).JEG-3 cells were also treated with mifepristone for 24h.The number of live cells were trended downwards while the apoptotic cells upwards as mifepristone concentration increased.The ratios of cell apoptosis in different mifepristone group were higher than that in control group,and in 10μmol/L RU486 group the apoptotic rate was the highest(14.96%vs 4.69%).6.Ultrastructure of the villous tissues in mifepristone and control groupUltrastructure features of villous tissues in the normal early pregnancy:the free surface of syncytotrophoblast was rich in microvilli.The cytoplasm contained numerous mitochondria,rough endoplasmic reticulum(rER) and smooth endoplasmic reticulum(sER),scattered ribosomes and lipid droplets of various sizes.The cytoplasm of cytotrophoblast was rich in ribosomes,mitochondria which were often surrounded by numerous RER.The capillary endothelial cells were rich in cytoplasmic projections,and there were more cell-to-cell communication with the pericytes and stromal cells.Ultrastructural features of villous tissue in mifepristone group:the syncytophoblast shed off partly.The microvilli in the surface of the remained syncytotrophoblast became short and thin,even absent.The RER dilated, mitochondria shrinked and some even became vacuolization.The nucleus of syncytotrophoblast showed some apoptotic features including peripheral nuclear chromatin condensation.In the cytoplasm of cytotrophoblast,plenty ofα-glycogen accumulated,some mitochondria became vacuolizaed.The nucleus of endothelial cells revealed an apoptotic-like feature including peripheral nuclear chromatin condensation.The cytoplasm contained less cytoplasmic projections,and there were less cell-to-cell communications with the pericytes and stromal cells.Conclusions:1.Under the effects of mifepristone,villous angiogenesis and consequently might be disturbed,consequently it is harmful to the embryo’ s survival.2.The secretory functions of trophoblastic cells are destroyed by mifepristone,the dynamic balance of angiogenic factors may be broken,and the angiogenesis will be destroyed.3.The balance of trophoblastic proliferation and apoptosis is important for the placental function.Mifepristone can blockade the trophoblastic proliferation and up-regulate the expression of caspases family proteins which can induce the cellular apoptosis.4.Mifepristone can induce the apoptosis of BeWo and JEG-3 cells in vitro, indicating that it can induce the apoptosis of human trophoblastic cells directly.5.In comparison to the control group,the trophoblastica cells of mifepristone group show morphological changes.Furthermore,these changes reflect the functional changes of the trophoblastic cells in secretion,proliferation and apoptosis.更多还原