小鼠子宫内膜BrdU、Musashi-1和P63的表达及其意义

发布时间：2018-04-04 10:17

  本文选题：子宫内膜　切入点：干细胞　出处：《暨南大学》2011年硕士论文

【摘要】：目的通过标记滞留细胞技术检测昆明小鼠子宫内膜干细胞的存在及其分布情况；观察Musashi-1和P63在不同周龄小鼠子宫内膜的表达,初步探讨Musashi-1和P63在小鼠子宫内膜的生长、发育过程中的作用,为子宫内膜干细胞标记物的筛选提供依据；通过免疫组化双重标记方法分别检测Musashi-1和P63与BrdU的共表达情况,探求Musashi-1和P63作为子宫内膜干细胞特异性标记物的可能性。 方法取出生3天雌性乳鼠皮下注射BrdU。分别在1w、2w、3w、4w、6w、8w和10w处死小鼠取其子宫。采用免疫组化SP法分别检测BrdU、Musashi-1和P63在各周龄小鼠子宫内膜的表达情况：同时采用免疫组织化学双重标记(SP-SAP法)进行P63/BrdU和Musashi-1/BrdU双重染色,观察Musashi-1和P63的表达与标记滞留细胞重叠的情况。显微镜下观察各指标阳性细胞的分布情况并在高倍镜下分别计算各指标的上皮和基质中的阳性细胞百分率。 结果1.BrdU在小鼠子宫内膜的表达：注射BrdU后1w,子宫内膜上皮和基质中绝大部分细胞被BrdU标记。第3w时BrdU阳性细胞主要分布在基质,表面上皮及腺上皮中也有较多BrdU阳性细胞。第6w时,BrdU阳性细胞已大幅度减少,阳性细胞仍以基质居多,表面上皮中仅见零星BrdU阳性细胞。至第8w,上皮中几乎未见BrdU阳性细胞,仅在基质中存在极少量的BrdU阳性细胞。第10w的子宫内膜上皮及基质中均未发现BrdU阳性细胞。统计学分析表明,上皮和基质的BrdU的阳性细胞百分率具有显著性差异(P0.05)。 2.P63在小鼠子宫内膜的表达：出生后1w,P63阳性细胞广泛分布于小鼠子宫内膜的表面上皮和基质。随着小鼠周龄的增加,腺体逐渐形成、发育,P63开始在腺体细胞上表达。3w时在腺体及其周围可见较多阳性细胞,表面上皮阳性细胞较少。之后,阳性细胞数量随小鼠子宫内膜的发育不断下降。第4w时阳性细胞明显减少,主要表达于基质,腺体周围较多,上皮表达较少。第6w时阳性细胞进一步减少,但集中分布在腺上皮周围的趋势明显。第8w时可见少量阳性细胞分布于基质,大多位于腺体周围,上皮中未见阳性细胞。至第10w时仅在基质中看到零星P63阳性细胞,上皮中未见。 3.Musashi-1在小鼠子宫内膜的表达：出生后1w的子宫内膜组织可见Musashi-1阳性细胞在上皮和基质中广泛分布。随着小鼠周龄的增加,Musashi-1在子宫内膜的表达逐渐减少。在第4w时,子宫内膜基质中仍有较多的Musashi-1阳性细胞,主要集中分布于基质靠近肌层处,上皮也有少量阳性细胞表达。第6w时Musashi-1阳性细胞的分布以基质中为多,常呈片带状集中分布。腺上皮中亦有少量阳性细胞分布,且主要表达于腺上皮基底部。第8w时Musashi-1表达进一步减少,基质中可见散在的阳性细胞,以基质与肌层交接处居多,腺上皮中也可见少量阳性细胞。第10w时,阳性细胞表达降低,仅在基质发现少量阳性细胞,且表达以基质与肌层交接处居多。 4.免疫组织化学双重染色结果：第8w的小鼠子宫内膜中,检测到零星存在的P63/BrdU双阳性细胞,分布于基质,以内膜与肌层连接处居多；表面上皮及腺体中均未发现P63/BrdU双阳性细胞。Musashi-1/BrdU双阳性细胞呈零星分布,主要分布于基质血管周围；上皮未见Musashi-1/BrdU双阳性细胞。 结论 1.小鼠子宫内膜标记滞留细胞(LRCs)主要位于基质血管周围及内膜与肌层交界处,这些细胞的分布与推测的子宫内膜干细胞的分布部位相符,这些细胞(或其中一部分)为子宫内膜干细胞。 2.早期小鼠子宫内膜组织中P63.Musashi-1阳性细胞数量较多,随着子宫内膜的逐渐发育成熟,P63.Musashi-1的表达逐渐减少,提示二者参与子宫内膜的增殖及分化过程。 3.P63/BrdU和Musashi-1/BrdU免疫组化双重标记显示P63及Musashi-1表达与BrdU有部分重叠,进一步提示P63和Musashi-1作为干细胞的标记物的可能性。
[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.

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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