利用人子宫内膜异体移植模型对月经出血和蜕膜化发生的初步研究

发布时间：2018-08-29 18:26

【摘要】：月经是自然界中人和部分灵长类动物以及少数其它动物(如蝙蝠、大象)所独有的一种生理现象,是子宫内膜生长、血管增生、腺体生长分泌以及子宫内膜崩解脱落并伴随出血的生理性周期变化。这种周期性变化正是女性生殖系统的生理特点之一,自上世纪80年代以来,包括我们实验室在内的国内外的研究者们通过人工给予激素或借助假孕动物模型的方式建立了小鼠月经模型,但小鼠并非行经动物,因此通过人工外在刺激模拟产生的小鼠月经模型并不能够真正反应人类月经发生和发展的过程,该模型的运用具有一定的局限性。因此,建立一个人源化的月经发生模型来研究月经发生的细胞和分子生物学基础具有十分重要的意义。在此月经模型基础之上,探讨月经出血以及环氧合酶在月经蜕膜化中的作用,本研究的主要内容分为如下三个部分。我们首先建立人子宫内膜组织异体移植模型。将人子宫内膜组织异种移植到SCID小鼠皮下后,人工给予雌、孕激素。观察移植后不同时间点,移植组织的形态和结构变化,激素水平,波形蛋白和角蛋白标志物表达情况,并与在体周期性变化的子宫内膜组织比较。研究表明,组织移植后同样发生与在体人子宫内膜相似增殖、分化和崩解的组织形态学变化。对基质金属蛋白酶1、2和9的表达,蜕膜化标志物催乳素以及环氧合酶1和2的表达进行了研究,结果显示,上述过程与在体人子宫内膜组织和灵长类月经研究所揭示的特征相符合。另外,在白细胞浸润结果发现,不同类型白细胞参与月经周期不同阶段,发挥不同的生物学作用。移植子宫内膜组织与小鼠组织形成血管嵌合体。在前期结果的启发下,我们进一步深入探索了环氧合酶在子宫内膜蜕膜化过程中的作用。在人子宫内膜组织异体移植模型基础上,一方面在代谢水平层面,给予宿主小鼠环氧合酶抑制剂吲哚美辛和塞来昔布,观察蜕膜化发生程度。结果显示,COX2对于蜕膜化的启动是必须的,而对于蜕膜化的维持不是必须的,环氧合酶抑制剂抑制蜕膜化发生具有时间特异性。吲哚美辛同时抑制PGE2和PGF2α的合成,由于COX1代偿作用,塞来昔布对PGE2和PGF2。的合成未见影响。另一方面,我们进行了分子水平的前期研究工作,主要是建立人子宫内膜组织块体外慢病毒转染体系,通过对组织培养条件、转染条件、病毒数量等方面进行优化,达到了50%的转染阳性率,为下一步进行RNA干扰下调环氧合酶基因、以及子宫月经发生的分子机制研究奠定实验基础和提供技术保障。在改良子宫内膜细胞原代分离培养方法的基础之上,我们将人端粒酶基因通过脂质体转染的方法导入体外培养的人子宫内膜基质细胞,以建立永生化人子宫内膜基质细胞系。结果表明,原代分离得到的子宫内膜基质和上皮细胞分别达到97%和90%的纯度。RT-PCR检测显示,通过脂质体传染的方式,成功地将人端粒酶基因导入细胞,并稳定表达。经过G418筛选,得到2个单克隆,并扩大传代培养,目前传至第10代和15代。综上所述,我们成功的建立了人子宫内膜异种移植月经模型,并初步表明在这个过程中,环氧合酶在月经蜕膜化启动过程中发挥着重要的作用,为进一步的完善分子生物学研究模型,我们进行了建立人子宫内膜基质细胞永生化细胞系和体外组织块慢病毒转染体系研究,初步取得了较好的结果,为后续的研究打下了坚实的基础。
[Abstract]:Menstruation is a unique physiological phenomenon in humans, some primates and a few other animals, such as bats and elephants. It is a physiological cycle of endometrial growth, vascular proliferation, glandular growth and secretion, and endometrial disintegration accompanied by bleeding. One of the characteristics is that since the 1980s, researchers at home and abroad, including our laboratory, have established mouse menstrual models by artificial hormones or by means of pseudo-pregnant animal models, but the mice are not menstruating animals, so the mouse menstrual models produced by artificial external stimulation can not really respond. Therefore, it is of great significance to establish a humanized model of menstruation to study the cellular and molecular biological basis of menstruation. The main contents of this study are divided into three parts.
We first established a human endometrial allograft model. After xenograft of human endometrial tissue into SCID mice, estrogen and progesterone were given artificially. The morphological and structural changes, hormone levels, the expression of vimentin and keratin markers were observed at different time points after transplantation. Tissue comparison of endometrium showed that the same histomorphological changes of proliferation, differentiation and disintegration occurred after tissue transplantation as in vivo human endometrium. The expression of matrix metalloproteinase 1, 2 and 9, prolactin and cyclooxygenase 1 and 2, markers of decidualization, were studied in vivo. In addition, leukocyte infiltration showed that different types of leukocytes participated in different stages of the menstrual cycle and played different biological roles.
Inspired by the earlier results, we further explored the role of cyclooxygenase in endometrial decidualization. On the basis of human endometrial allograft model, on the one hand, at the metabolic level, cyclooxygenase inhibitors indomethacin and celecoxib were given to the host mice to observe the degree of decidualization. Indomethacin also inhibited the synthesis of PGE2 and PGF2alpha. Celecoxib had no effect on the synthesis of PGE2 and PGF2. Previous studies at the molecular level mainly focused on the establishment of lentiviral transfection system in vitro for human endometrial tissue blocks. Through optimization of tissue culture conditions, transfection conditions and the number of viruses, 50% of the positive transfection rate was achieved, and cyclooxygenase gene was down-regulated by RNA interference for the next step, as well as the molecular mechanism of uterine menstruation. System research lays the foundation for experiments and provides technical support.
On the basis of improving the method of primary isolation and culture of endometrial cells, human telomerase gene was transfected into human endometrial stromal cells by liposome to establish immortalized human endometrial stromal cell lines. RT-PCR assay showed that the human telomerase gene was successfully transfected into the cells by liposome infection and expressed stably. Two monoclones were screened by G418 and extended to the 10th and 15th generations.
In conclusion, we have successfully established a menstrual model of human endometrial xenograft and preliminarily demonstrated that cyclooxygenase plays an important role in the initiation of menstrual decidualization. To further improve the molecular biological model, we have established immortalized human endometrial stromal cell lines and established immortalized human endometrial stromal cells. Good results have been obtained in the study of lentivirus transfection system in vitro, which lays a solid foundation for the follow-up study.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R-332

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