本文選題：子宮內(nèi)膜　切入點：干細(xì)胞　出處：《暨南大學(xué)》2011年碩士論文

【摘要】：目的通過標(biāo)記滯留細(xì)胞技術(shù)檢測昆明小鼠子宮內(nèi)膜干細(xì)胞的存在及其分布情況；觀察Musashi-1和P63在不同周齡小鼠子宮內(nèi)膜的表達(dá),初步探討Musashi-1和P63在小鼠子宮內(nèi)膜的生長、發(fā)育過程中的作用,為子宮內(nèi)膜干細(xì)胞標(biāo)記物的篩選提供依據(jù)；通過免疫組化雙重標(biāo)記方法分別檢測Musashi-1和P63與BrdU的共表達(dá)情況,探求Musashi-1和P63作為子宮內(nèi)膜干細(xì)胞特異性標(biāo)記物的可能性。 方法取出生3天雌性乳鼠皮下注射BrdU。分別在1w、2w、3w、4w、6w、8w和10w處死小鼠取其子宮。采用免疫組化SP法分別檢測BrdU、Musashi-1和P63在各周齡小鼠子宮內(nèi)膜的表達(dá)情況：同時采用免疫組織化學(xué)雙重標(biāo)記(SP-SAP法)進(jìn)行P63/BrdU和Musashi-1/BrdU雙重染色,觀察Musashi-1和P63的表達(dá)與標(biāo)記滯留細(xì)胞重疊的情況。顯微鏡下觀察各指標(biāo)陽性細(xì)胞的分布情況并在高倍鏡下分別計算各指標(biāo)的上皮和基質(zhì)中的陽性細(xì)胞百分率。 結(jié)果1.BrdU在小鼠子宮內(nèi)膜的表達(dá)：注射BrdU后1w,子宮內(nèi)膜上皮和基質(zhì)中絕大部分細(xì)胞被BrdU標(biāo)記。第3w時BrdU陽性細(xì)胞主要分布在基質(zhì),表面上皮及腺上皮中也有較多BrdU陽性細(xì)胞。第6w時,BrdU陽性細(xì)胞已大幅度減少,陽性細(xì)胞仍以基質(zhì)居多,表面上皮中僅見零星BrdU陽性細(xì)胞。至第8w,上皮中幾乎未見BrdU陽性細(xì)胞,僅在基質(zhì)中存在極少量的BrdU陽性細(xì)胞。第10w的子宮內(nèi)膜上皮及基質(zhì)中均未發(fā)現(xiàn)BrdU陽性細(xì)胞。統(tǒng)計學(xué)分析表明,上皮和基質(zhì)的BrdU的陽性細(xì)胞百分率具有顯著性差異(P0.05)。 2.P63在小鼠子宮內(nèi)膜的表達(dá)：出生后1w,P63陽性細(xì)胞廣泛分布于小鼠子宮內(nèi)膜的表面上皮和基質(zhì)。隨著小鼠周齡的增加,腺體逐漸形成、發(fā)育,P63開始在腺體細(xì)胞上表達(dá)。3w時在腺體及其周圍可見較多陽性細(xì)胞,表面上皮陽性細(xì)胞較少。之后,陽性細(xì)胞數(shù)量隨小鼠子宮內(nèi)膜的發(fā)育不斷下降。第4w時陽性細(xì)胞明顯減少,主要表達(dá)于基質(zhì),腺體周圍較多,上皮表達(dá)較少。第6w時陽性細(xì)胞進(jìn)一步減少,但集中分布在腺上皮周圍的趨勢明顯。第8w時可見少量陽性細(xì)胞分布于基質(zhì),大多位于腺體周圍,上皮中未見陽性細(xì)胞。至第10w時僅在基質(zhì)中看到零星P63陽性細(xì)胞,上皮中未見。 3.Musashi-1在小鼠子宮內(nèi)膜的表達(dá)：出生后1w的子宮內(nèi)膜組織可見Musashi-1陽性細(xì)胞在上皮和基質(zhì)中廣泛分布。隨著小鼠周齡的增加,Musashi-1在子宮內(nèi)膜的表達(dá)逐漸減少。在第4w時,子宮內(nèi)膜基質(zhì)中仍有較多的Musashi-1陽性細(xì)胞,主要集中分布于基質(zhì)靠近肌層處,上皮也有少量陽性細(xì)胞表達(dá)。第6w時Musashi-1陽性細(xì)胞的分布以基質(zhì)中為多,常呈片帶狀集中分布。腺上皮中亦有少量陽性細(xì)胞分布,且主要表達(dá)于腺上皮基底部。第8w時Musashi-1表達(dá)進(jìn)一步減少,基質(zhì)中可見散在的陽性細(xì)胞,以基質(zhì)與肌層交接處居多,腺上皮中也可見少量陽性細(xì)胞。第10w時,陽性細(xì)胞表達(dá)降低,僅在基質(zhì)發(fā)現(xiàn)少量陽性細(xì)胞,且表達(dá)以基質(zhì)與肌層交接處居多。 4.免疫組織化學(xué)雙重染色結(jié)果：第8w的小鼠子宮內(nèi)膜中,檢測到零星存在的P63/BrdU雙陽性細(xì)胞,分布于基質(zhì),以內(nèi)膜與肌層連接處居多；表面上皮及腺體中均未發(fā)現(xiàn)P63/BrdU雙陽性細(xì)胞。Musashi-1/BrdU雙陽性細(xì)胞呈零星分布,主要分布于基質(zhì)血管周圍；上皮未見Musashi-1/BrdU雙陽性細(xì)胞。 結(jié)論 1.小鼠子宮內(nèi)膜標(biāo)記滯留細(xì)胞(LRCs)主要位于基質(zhì)血管周圍及內(nèi)膜與肌層交界處,這些細(xì)胞的分布與推測的子宮內(nèi)膜干細(xì)胞的分布部位相符,這些細(xì)胞(或其中一部分)為子宮內(nèi)膜干細(xì)胞。 2.早期小鼠子宮內(nèi)膜組織中P63.Musashi-1陽性細(xì)胞數(shù)量較多,隨著子宮內(nèi)膜的逐漸發(fā)育成熟,P63.Musashi-1的表達(dá)逐漸減少,提示二者參與子宮內(nèi)膜的增殖及分化過程。 3.P63/BrdU和Musashi-1/BrdU免疫組化雙重標(biāo)記顯示P63及Musashi-1表達(dá)與BrdU有部分重疊,進(jìn)一步提示P63和Musashi-1作為干細(xì)胞的標(biāo)記物的可能性。
[Abstract]:Objective to detect label retaining cells of Kunming mice endometrial stem cell and its distribution; to observe the expression of Musashi-1 and P63 in the endometrium of mouse uterus at different ages, a preliminary study of Musashi-1 and P63 in mouse endometrial growth and development process, provide the basis for the screening of stem cell marker for endometrial; through immunohistochemical double labeling method were used to detect Musashi-1 and P63 and BrdU co expression of Musashi-1 and P63, to explore the possibilities for endometrial stem cell specific markers.
Methods 3 days of birth female rat subcutaneous injection of BrdU. respectively in 1W, 2W, 3W, 4W, 6W, 8W and 10W mice were killed from the uterus. SP immunohistochemical method was used to detect BrdU, Musashi-1 and P63 expression in the mice endometrium: immunohistochemical double labeling (SP-SAP) P63/BrdU and Musashi-1/BrdU double staining to observe the expression of Musashi-1 markers and P63 and residual cell overlap. Microscope was used to observe the distribution of each index of positive cells and calculated the percentage of positive cells of each index epithelium and stroma in high magnification.
The expression of 1.BrdU in mouse endometrium: after injection of BrdU 1W, endometrial epithelial and stromal cells in most labeled by BrdU. The 3W BrdU positive cells were mainly distributed in the matrix, there are also many BrdU positive cells in the surface epithelium and glandular epithelium. The 6W, BrdU positive cells have been greatly reduced. The positive cells in the matrix are only sporadic BrdU positive cells in the surface epithelium. The epithelium to 8W, almost no BrdU positive cell, there are only very small amounts of BrdU positive cells in the stroma. Endometrial epithelial and stromal 10W were not detected in BrdU positive cells. The statistical analysis showed that the percentage of positive cells of epithelium and the matrix BrdU has significant difference (P0.05).
The expression of 2.P63 in mouse endometrium: 1W after birth, the surface epithelium and stroma of P63 positive cells are widely distributed in the mouse endometrium. With the increase in age, the gland gradually formed, development, P63 expression of.3w in glandular cells in the gland on and around the more visible positive cells, positive cells less surface epithelium the number of positive cells. After that, with the development of mouse endometrium decreased. Positive cells significantly reduced the 4W, mainly expressed in the stroma, glandular epithelial expression around more, less. To further reduce the 6W positive cells, but concentrated in the glandular epithelium around the trend is obvious. The 8W positive cells distributed in the the matrix, mostly located in the periphery of the gland, no positive epithelial cells. See only sporadic P63 positive cells in the matrix to the 10W, was found in the epithelium.
The expression of 3.Musashi-1 in mouse endometrium: positive cells showed Musashi-1 endometrial tissue 1W is widely distributed in the epithelium and stroma after birth. With the increase of mice age, the expression of Musashi-1 in endometrium decreased gradually. In 4W, there are still more Musashi-1 positive cells in endometrial stroma, mainly distributed in the matrix near the muscle layer, there are a few positive expression of epithelial cells. The 6W distribution of Musashi-1 positive cells in the matrix as much, often a banded concentration distribution in glandular epithelium. There is little positive cell distribution, and the main expression in gland on the bottom. During the first 8W Musashi-1 expression decreased further, positive cells were scattered in the matrix, the matrix and the muscle layer at the junction of the majority, the glandular epithelium also showed a few positive cells. 10W, decreased expression of positive cells, found only a small amount of positive cells in the stroma, It is expressed mainly at the junction of the stroma and the myometrium.
4. double immunohistochemical staining results: 8W mouse endometrium, detect the sporadic presence of P63/BrdU double positive cells distributed in the matrix, in connection with the muscular layer of endometrium are; surface epithelium and glands were not found in the double positive cells of P63/BrdU.Musashi-1/BrdU double positive cells were scattered, mainly distributed in the vascular matrix around; no epithelial Musashi-1/BrdU double positive cells.
conclusion
1. mouse endometrial marker retention cells (LRCs) were mainly located around the stromal vessels and the interface between the intima and the muscular layer. The distribution of these cells was consistent with the location of the presumed endometrial stem cells. These cells (or part of them) were endometrial stem cells.
2., the number of P63.Musashi-1 positive cells in the early mouse endometrial tissue was large. With the gradual maturation of the endometrium, the expression of P63.Musashi-1 gradually decreased, suggesting that the two participate in the process of endometrial proliferation and differentiation.
Double labeling of 3.P63/BrdU and Musashi-1/BrdU immunohistochemistry showed that P63 and Musashi-1 expression partly overlapped with BrdU, further suggesting the possibility of P63 and Musashi-1 as markers of stem cells.

【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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