【摘要】：月經(jīng)是自然界中人和部分靈長類動物以及少數(shù)其它動物(如蝙蝠、大象)所獨有的一種生理現(xiàn)象,是子宮內(nèi)膜生長、血管增生、腺體生長分泌以及子宮內(nèi)膜崩解脫落并伴隨出血的生理性周期變化。這種周期性變化正是女性生殖系統(tǒng)的生理特點之一,自上世紀(jì)80年代以來,包括我們實驗室在內(nèi)的國內(nèi)外的研究者們通過人工給予激素或借助假孕動物模型的方式建立了小鼠月經(jīng)模型,但小鼠并非行經(jīng)動物,因此通過人工外在刺激模擬產(chǎn)生的小鼠月經(jīng)模型并不能夠真正反應(yīng)人類月經(jīng)發(fā)生和發(fā)展的過程,該模型的運用具有一定的局限性。因此,建立一個人源化的月經(jīng)發(fā)生模型來研究月經(jīng)發(fā)生的細(xì)胞和分子生物學(xué)基礎(chǔ)具有十分重要的意義。在此月經(jīng)模型基礎(chǔ)之上,探討月經(jīng)出血以及環(huán)氧合酶在月經(jīng)蛻膜化中的作用,本研究的主要內(nèi)容分為如下三個部分。 我們首先建立人子宮內(nèi)膜組織異體移植模型。將人子宮內(nèi)膜組織異種移植到SCID小鼠皮下后,人工給予雌、孕激素。觀察移植后不同時間點,移植組織的形態(tài)和結(jié)構(gòu)變化,激素水平,波形蛋白和角蛋白標(biāo)志物表達(dá)情況,并與在體周期性變化的子宮內(nèi)膜組織比較。研究表明,組織移植后同樣發(fā)生與在體人子宮內(nèi)膜相似增殖、分化和崩解的組織形態(tài)學(xué)變化。對基質(zhì)金屬蛋白酶1、2和9的表達(dá),蛻膜化標(biāo)志物催乳素以及環(huán)氧合酶1和2的表達(dá)進(jìn)行了研究,結(jié)果顯示,上述過程與在體人子宮內(nèi)膜組織和靈長類月經(jīng)研究所揭示的特征相符合。另外,在白細(xì)胞浸潤結(jié)果發(fā)現(xiàn),不同類型白細(xì)胞參與月經(jīng)周期不同階段,發(fā)揮不同的生物學(xué)作用。移植子宮內(nèi)膜組織與小鼠組織形成血管嵌合體。 在前期結(jié)果的啟發(fā)下,我們進(jìn)一步深入探索了環(huán)氧合酶在子宮內(nèi)膜蛻膜化過程中的作用。在人子宮內(nèi)膜組織異體移植模型基礎(chǔ)上,一方面在代謝水平層面,給予宿主小鼠環(huán)氧合酶抑制劑吲哚美辛和塞來昔布,觀察蛻膜化發(fā)生程度。結(jié)果顯示,COX2對于蛻膜化的啟動是必須的,而對于蛻膜化的維持不是必須的,環(huán)氧合酶抑制劑抑制蛻膜化發(fā)生具有時間特異性。吲哚美辛同時抑制PGE2和PGF2α的合成,由于COX1代償作用,塞來昔布對PGE2和PGF2。的合成未見影響。另一方面,我們進(jìn)行了分子水平的前期研究工作,主要是建立人子宮內(nèi)膜組織塊體外慢病毒轉(zhuǎn)染體系,通過對組織培養(yǎng)條件、轉(zhuǎn)染條件、病毒數(shù)量等方面進(jìn)行優(yōu)化,達(dá)到了50%的轉(zhuǎn)染陽性率,為下一步進(jìn)行RNA干擾下調(diào)環(huán)氧合酶基因、以及子宮月經(jīng)發(fā)生的分子機(jī)制研究奠定實驗基礎(chǔ)和提供技術(shù)保障。 在改良子宮內(nèi)膜細(xì)胞原代分離培養(yǎng)方法的基礎(chǔ)之上,我們將人端粒酶基因通過脂質(zhì)體轉(zhuǎn)染的方法導(dǎo)入體外培養(yǎng)的人子宮內(nèi)膜基質(zhì)細(xì)胞,以建立永生化人子宮內(nèi)膜基質(zhì)細(xì)胞系。結(jié)果表明,原代分離得到的子宮內(nèi)膜基質(zhì)和上皮細(xì)胞分別達(dá)到97%和90%的純度。RT-PCR檢測顯示,通過脂質(zhì)體傳染的方式,成功地將人端粒酶基因?qū)爰?xì)胞,并穩(wěn)定表達(dá)。經(jīng)過G418篩選,得到2個單克隆,并擴(kuò)大傳代培養(yǎng),目前傳至第10代和15代。 綜上所述,我們成功的建立了人子宮內(nèi)膜異種移植月經(jīng)模型,并初步表明在這個過程中,環(huán)氧合酶在月經(jīng)蛻膜化啟動過程中發(fā)揮著重要的作用,為進(jìn)一步的完善分子生物學(xué)研究模型,我們進(jìn)行了建立人子宮內(nèi)膜基質(zhì)細(xì)胞永生化細(xì)胞系和體外組織塊慢病毒轉(zhuǎn)染體系研究,初步取得了較好的結(jié)果,為后續(xù)的研究打下了堅實的基礎(chǔ)。
[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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R-332

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本文編號：2212037