【作者】 李晓艳；

【导师】 王泽华；

【作者基本信息】 华中科技大学 ， 妇产科学， 2006， 博士

【摘要】 目的探讨低频超声终止早期妊娠的可能性。利用低频聚焦超声(353 kHZ)直接照射兔早孕胚胎，观察其抗孕有效性，检测有效抗孕声功率；同时观察低频聚焦超声有效抗孕声功率直接辐射兔胚胎后不同时间胎盘及子宫光镜和电镜下的形态学改变、胎盘组织细胞凋亡的变化和凋亡调控基因Fas、FasL、p53、Bax和Bcl-2表达的改变，以期为临床研究提供重要的实验依据。方法1．将妊娠第10 d摸胎确诊孕兔随机分成6组，孕兔麻醉后开腹，置双侧子宫于腹壁上，选取左侧子宫的全部着床胚胎为实验组，右侧为自身对照，分别用353 kHZ低频聚焦超声声功率30 W／cm~2×60 s、35 W／cm~2×60 s、40 W／cm~2×60 s、45 W／cm~2×60 s、50 W／cm~2×60 s和55 W／cm~2×60 s直接辐照胚胎着床点，照射后关腹继续饲养至妊娠第20 d(照后第10 d)，再次开腹，肉眼观察子宫内胚胎的发育情况，计算出对照组和实验组胚胎的死亡率，即照射后第10 d子宫内已死亡(包括已流产和死胎)的胚胎数与该侧照射前的胚胎数相比。2．将妊娠第10 d摸胎确诊孕兔随机分成6组，孕兔麻醉后开腹，置双侧子宫于腹壁上，将对照组妊娠第10 d、20 d各3只动物和353 kHZ低频聚焦超声6种声功率辐照组照后即刻(妊娠第10 d)、照后10 d(妊娠第20 d)各10只动物的部分胚胎取出，选取胎盘组织，生理盐水冲净血液后，一部分组织置4％多聚甲醛溶液中固定24 h，常规石蜡包埋、切片、HE染色，光镜下观察胎盘(子宫底蜕膜和绒毛)及子宫的组织学变化情况；另一部分组织置2.5％戊二醛溶液中前固定2 h以上，常规电镜包埋、超薄切片，H-600型透射电镜观察胎盘及子宫的超微结构的改变。3．妊娠第10 d摸胎确诊孕兔对照组胚胎(右侧子宫胚胎为自身对照)不照射，实验组(左侧子宫胚胎)选用已证实为完全终止孕兔早孕声功率(353 kHZ低频聚焦超声40 W／cm~2×60 s)辐照胚胎，留取正常妊娠第10 d和照射后24、48、72、96、120、168、192 h的胎盘组织，生理盐水洗净血液后置4％多聚甲醛中固定，常规石蜡包埋、切片，末端脱氧核糖核苷酸转移酶标记技术(TUNEL reaction)技术观察胎盘组织细胞的凋亡情况。4．免疫组织化学(immunohistochemistry)技术检测353 kHZ低频聚焦超声40 W／cm~2×60 s辐照胚胎后24、48、72、96、120、168、192 h胎盘组织细胞Fas、FasL、Bax和Bcl-2蛋白的表达改变。5．原位杂交(In situ hybridization)技术检测353 kHZ低频聚焦超声40W／cm~2×60 s辐照胚胎后24、48、72、96、120、168、192 h胎盘组织细胞p53 mRNA、Bax mRNA、Bcl-2 mRNA的表达改变。结果1．对照组胚胎总死亡率为12.65％(32／253)。照射组中声功率30 W／cm~2、35 W／cm~2及≥40 W／cm~2组的胚胎死亡率分别为25.00％(12／48)、72.88％(43／59)和100％(64／64、43／43、40／40、35／35)；从35 W／cm~2组开始胚胎死亡率剧增，与对照组相比有非常显著的统计学差异(P＜0.005)。2．353 kHZ低频聚焦超声辐照后的即刻改变：光镜下主要为胎盘(绒毛及子宫蜕膜)出血、坏死；电镜下胎盘细胞正常结构消失，子宫平滑肌、内膜上皮细胞中线粒体肿胀。变化随剂量的增加而明显。照射后10 d观察：光镜下见绒毛、蜕膜细胞仍有变性或坏死；电镜下胎盘组织细胞坏死呈致密团块状，子宫内膜上皮、平滑肌及血管内皮细胞均未见异常。3．353 kHZ低频聚焦超声40 W／cm~2×60 s照射后24、48、72、96、120、168 h胎盘组织凋亡细胞数量与对照组相比均有显著差异(P＜0.05)；照射后24h凋亡细胞开始增加，照射后96 h凋亡细胞增加最多，与其他各组相比差异均有显著性意义(P＜0.05)；照后192 h凋亡细胞数量接近对照组水平，两者相比差别无显著性(P＞0.05)。4．353 kHZ低频聚焦超声40 W／cm~2×60 s照射胚胎后48、72、96、120、168 h胎盘组织细胞Fas蛋白、FasL蛋白、p53 mRNA、Bax蛋白和mRNA表达阳性率与对照组相比均有显著升高，照射后96h增加至高峰，照射后192h接近对照组水平；照射后24、48、72、96、120、168 h胎盘组织细胞表达Bcl-2蛋白和mRNA的水平与对照组相比均有显著降低，照射后96h下降至最低点，照后192h接近对照组水平。结论1．低频超声抗早孕具有潜在的可行性，值得进一步研究。2．胎盘较子宫对低频超声更敏感，更易受损，子宫照射后变化轻微且可恢复。3．低频聚焦超声(353 kHZ)辐照兔胚胎可诱导胎盘组织细胞凋亡增加，但有时间依赖性，即可复性。4．低频聚焦超声(353 kHZ)辐照兔胚胎可诱导胎盘组织细胞Fas、FasL、p53和Bax表达增加，Bcl-2表达下降，从而诱导胎盘组织细胞凋亡增加，但具有时间依赖性，即可复性。本研究的创新性1．首次使用353 kHz低频聚焦超声进行终止早期妊娠实验研究。2．首次检测低频聚焦超声辐照胚胎后不同时间胎盘组织细胞的凋亡变化情况。3．首次检测低频聚焦超声辐照胚胎后不同时间胎盘组织细胞凋亡调控基因的表达改变。更多还原

【Abstract】 OBJECTIVE To investigate the availability of early pregnancy termination with low frequency ultrasound . The early embryos were irradiated directly with low frequency focused ultrasound (353 kHz ) and embryonic mortality and morphology changes of placenta and uterus were observed . At the same time , the apoptosis and expression of regulatory genes of apoptosis of placenta were tested with methods of TUNEL , immunohistochemistry and in situ hybridization respectively at 24、 48、 72、 96、 120、168、 192 hours after irradiating . The studies mightprovide important experimental foundation for clinical studies .METHODS1. The implantation sites of left uterus of 10th day rabbit embryos were exposured with an intensity of 30 W/cm~2、 35 W/cm~2、 40 W/cm~2、 45 W/cm~2、 50 W/cm~2 and 55 W/cm~2 for 60 seconds . At same time , the embryos of right uterus belonged to the control group . At 20 th day of gestation , the animals were sacrificed . Embryonic mortality was observed .2. Choosing three rabbits on 10 th day and three on 20 th day of pregnancy of control group , three at once and three 10 days after irradiating of different experimental groups , opening the abdominal cavity of pregnant rabbits , choosing part of the placenta and putting them into 4 % paraformaldehyde for 24 hours and 2.5 % glutaraldehyde for more than 2 hours respectively . Paraffin embedding , slices and dye with HE as routine procedure . Morphology changes of placenta and uterus were observed under optical microscope . Embedding using for electron microscope as routine procedure and ultrathin slices . The ultrastructure changes of placenta and uterus were observed under electron microscope .3. The embryos of right uterus , as control group , were not irradiated . The embryos of left uterus of 10th day rabbit were irradiated with 40 W/cm~2 for 60 seconds which had been proved asan totally pregnancy terminating intensity . The apoptosis of placenta cells were tested with TUNEL at 24、 48、 72、 96、 120、 168、192 hours after irradiating . Choosing part of the placenta of 10 th day of control group and 24、48、72、 96、 120、 168、 192 hours after irradiating , dipping them in 4 % paraformaldehyde for 24 hours , paraffin embedding , slices and dye with HE as routine procedure . The apoptosis of placenta cells was detected with TUNEL reaction .4. The expression of Fas、 FasL、 Bax and Bcl-2 proteins of placenta cells was detected with immunohistochemistry at 24、 48、 72、 96、120、 168、 192 hours after irradiating with 40 W/cm~2 for 60 seconds .5. The expression of p53 mRNA、 Bax mRNA、 Bcl-2 mRNA of placenta cells was detected with In situ hybridization at 24、 48、 72、96、 120、 168、 192 hours after irradiating with 40 W/cm~2 for 60 seconds.RESULTS1. It was found that total embryonic mortality of control group was 12.65 % ( 32/253 ) and those of experimental group were 25.00 % (12/48 )、 72.88 % (43/59 ) and 100 % ( 64/64, 43/43 , 40/40 , 35/35 ) respectively with an intensity of 30 W/cm~2、 35W/cm~2 and equal or more than 40 W/cm~2. There was a rough increase of embryonic mortality from the group of 35 W/cm~2 and ther after . Ther was a very significant difference compared with control group .2. Under optical microscope , necrosis and hemorrhage were observed in placenta (villosity ang decidua) at once after irradiating . Under electron microscope , the cells of placenta were found to have lost normal structures and there was mitochondrial swelling in endometrial and myometrial cells at once after irradiating . On 10 th day after irradiating , degeneration and necrosis were also observed in villosity , decidua cells and there was no obvious change in endometrial, myometrial and vascula endothelial cells under optical microscope.3. There was a significant apoptosis increase at 24、48、72、 96、 120、 168、 192 hours after irradiating compared with control group . The incidence of placenta cell apoptosis was increased at 24 hours after irradiating and peaked at 96 hours , but then decreased gradually , and almost recovered to the level of controls by 192 hours after irradiating .4. There was a significant increase of expression of Fas protein, FasL protein、p53 mRNA、Bax protein and mRNA at 24、48、72、96、120、168、192 hours after irradiating comparingto control group . The rate of these genes expression of placenta cells was increased at 24 hours after irradiating and peaked at 96 hours, but then decreased gradually , and almost recovered to control levels by 192 hours . The incidence of Bcl-2 protein and mRNA was decreased at 24、48、72、96、120、168 hours after irradiating compared with those of control group and least at 96 hours after irradiating. But then recovered gradually, and to almost same levels as control by 192 hours .CONCLUSIONS1. It is feasible for early pregnancy termination of rabbit with low frequency ultrasound . But the further studies were needed in the future.2. The placenta was more sensitive and injured to low frequency ultrasonic irradiation than uterus . There was a slight and reparable changes in uterus after irradiating .3. The low frequency ultrasound may result in more placenta cells apoptosis in rabbits and the number of apoptosis of placenta cell can be recovered gradually.4. The low frequency ultrasoundmay result in more Fas, FasL, p53 expressions of placenta cell and less Bcl-2 expression . That might result in more apoptosis in placenta cells, but the changes were reparable.In summary , we concluded the points of creation as follows :1. For the first time , we applied low frequency ultrasound for terminating animal pregnancy.2. For the first time , we detected the apoptosis changes in placenta cells after irradiating rabbit embryos with low frequency ultrasoundmay.3. For the first time , we detected the expression changes of regulatory genes of apoptosis in placenta cells after irradiating rabbit embryos with low frequency ultrasound .更多还原