Sca-1，CD24和Muc1在大鼠乳腺发育期分布的研究

发布时间：2018-04-29 07:56

本文选题：乳腺 + 干细胞　；参考：《第一军医大学》2007年硕士论文

【摘要】： 背景和目的哺乳动物的乳腺是一种复管泡状腺，具有分泌乳汁的功能；是动物出生后唯一可以多次重复再生的器官。根据乳腺发育的特征，啮齿类动物乳腺大致可以分为六个发育期：胚胎期、青春期、性成熟期、孕期、哺乳期和退化期。在乳腺细胞分化过程中，往往由于高度分化而完全失去再生能力，最终导致细胞的衰老死亡；乳腺在发育进化过程中为了补偿这一不足，保留了一部分未分化的原始细胞，称为乳腺干细胞。乳腺细胞的增殖和分化始终贯穿于乳腺发育及泌乳全过程，人们在细胞水平上进行了大量的乳腺上皮细胞增殖及分化的研究。这些研究揭示乳腺干细胞再生乳腺和分化异常导致乳腺癌变的机制，为乳腺肿瘤和癌症预防及治疗提供理论依据，为研究器官形成和细胞分化机理提供合适的模型。乳腺干细胞研究的标记蛋白主要有CK5，Sca-1，α6-integrin，MUC~-／ESA~+以及CK19。合适的乳腺干细胞特殊标记物的讨论仍在进行。在各报道中所确定的鉴别乳腺干细胞的标记物不同，这是值得重视和进一步研究的现象。这种不同可能有多方面的原因：1．材料的来源不同；2．体内和体外培养研究方法的不同；3．乳腺干细胞及其微环境随着个体的发育也处于发育变化过程中；4．乳腺干细胞在建立复杂的调控机制、信号转导系统和特殊功能过程中表达一系列相关的蛋白。我们选取青春期、性成熟期、孕期、哺乳期的大鼠乳腺组织进行形态学研究，运用免疫荧光和免疫酶标记以及Western免疫印迹法，观察Sca-1，CD24，Muc1在大鼠乳腺发育四个特殊时期的表达，研究这三种细胞标记物的分布，以探讨乳腺干细胞的增殖和分化。方法 1．乳腺组织石蜡切片的准备：6 w(体质量180～220g)、9 w(体质量210～230g)、孕15 d(体质量350～380g)、产后3 d(体质量330～350g)，每个时期1组，每组12只，乳腺组织块大小为1.0 cm×1.0 cm×0.5 cm，制成4μm厚切片。 2．HE染色：研究乳腺发育四个时期的形态和小亮细胞的形态分布。 3．免疫组化酶标记：研究Sca-1，CD24和Muc1在乳腺发育四个时期的抗原阳性情况。 4．免疫荧光单标记：研究Sca-1，CD24，Muc1在乳腺发育四个时期的抗原阳性情况。 5．Western免疫印迹法：取6 w、9 w、孕15 d、产后3 d的SPF级雌性大鼠，每个时期1组，每组3只，检测Sca-1，CD24，Muc1在乳腺发育四个时期的各自含量。 6．免疫荧光双标记：Sca-1分别和CD24以及Muc1标记，观察它们在四个时期的双阳性情况。结果 1．HE染色结果 6 w雌性大鼠乳腺末端主要是端芽结构；9 w雌性大鼠乳腺主要是腺小叶。在孕15 d乳腺组织中，乳腺的中、次级导管分支大量增加，腺泡迅速发育且数量增多。在产后3 d的乳腺组织中，乳腺小叶充满分泌腺泡，腺泡上皮的形态多样，细胞内出现较多空泡，在细胞核与细胞的游离面之间的胞质中空泡特别多，小叶内导管明显可见，结缔组织中的血管较多，小叶间隔很薄。在6 w乳腺组织中，小亮细胞大多是数个或成群地分布，常位于乳腺分支导管及末端芽的基底部；在9 w乳腺组织中，小亮细胞分布于导管及腺泡的基底膜上，也部分位于肌上皮与腺上皮细胞之间；在孕15 d乳腺组织中，小亮细胞分布于小叶导管中；在产后3 d乳腺组织中，小亮细胞大多分布在分支导管中。 2．免疫组化酶标记结果 Sca-1~+细胞分别位于6 w雌性大鼠乳腺的脂肪细胞之间和腺泡周围；9 w雌性大鼠乳腺脂肪细胞之间和腺泡周围；孕15 d雌性大鼠乳腺脂肪垫的脂肪细胞之间表达；以及产后3 d雌性大鼠乳腺的脂肪细胞之间，输乳管和乳腺脂肪垫中。 CD24~+细胞分别位于6 w雌性大鼠乳腺脂肪细胞之间；9 w雌性大鼠乳腺脂肪垫中；在孕15 d雌性大鼠乳腺腺泡周围；在产后3 d雌性大鼠乳腺的叶间导管。 Muc1~+细胞分别位于6w雌性大鼠乳腺的脂肪细胞之间，孕15 d雌性大鼠乳腺脂肪垫中；以及在产后3 d雌性大鼠乳腺叶间导管。 3．免疫荧光单标记结果 9 w雌性大鼠叶间导管分别有Sca-1~+；CD24~+和Muc1~+细胞；产后3 d雌性大鼠母鼠乳腺分支导管分别有Sca-1~+；CD24~+和Muc1~+细胞。 4．Western免疫印迹法结果 Sca-1在6 w和9 w乳腺中的含量没太大变化，产后3 d大鼠乳腺的Sca-1含量比孕15 d的少，活动期间的2个乳腺发育时期的Sca-1含量比静止期的含量少。CD24在大鼠乳腺发育前6w，9 w和孕15 d三个时期含量没太大变化，而在产后3 d大鼠乳腺CD24含量变少。Muc1在乳腺发育活动期的2个时期含量比静止期的2个时期高。 5．免疫免疫荧光双标记结果 6 w雌性大鼠乳腺脂肪垫；9 w雌性大鼠乳腺脂肪细胞间；孕15 d雌性大鼠乳腺脂肪细胞间；以及产后3 d雌性大鼠乳腺脂肪细胞间均发现有Sca-1和CD24双阳性细胞。 6 w雌性大鼠乳腺腺泡周围和分支导管；9 w雌性大鼠乳腺腺泡周围；孕15d雌性大鼠乳腺脂肪细胞之间以及产后3 d雌性大鼠乳腺分支导管存在Muc1和Sca-1双阳性细胞。结论 1．活动期的乳腺上皮细胞在乳导管基质中广泛增殖，乳腺中、次级导管分支增加；腺泡迅速发育，，数量增多且胞体变大。与静止期乳腺相比，这些明显变化应该是怀孕期间多种激素的作用结果；4个时期的生长增殖部位都出现小亮细胞。 2．部分Sca-1阳性细胞可能代表一类腔祖上皮细胞，而且在不同时期含量是有变化的。 3．关于乳腺干细胞的表面标志物在乳腺发育过程中可能有变化：(1)标志物性质的变化，即有不同标志物的保留或更替；(2)标志物表达量的变化。实验结果显示CD24在前三个时期含量没变化，在产后3 d发生变化可能是对称分裂发生的比较多，也可能是细胞横向分化，使得这个时期干细胞含量降低。 4．Muc1在分泌乳汁的腺泡周围和导管内侧以及脂肪等多个部位表达，Muc1在乳腺发育活动期的2个时期含量比静止期含量高，它可能是成熟腔上皮细胞的标志。 5．在乳腺发育时期，乳腺细胞(导管上皮细胞、腺泡上皮细胞、肌上皮细胞)大量扩增的主要来源有两种可能，可能Sca-1~+细胞具有双潜能，部分Sca-1~+细胞代表一类双潜能祖细胞。
[Abstract]:Background and Purpose

The mammary gland of mammal is a reconstituted alveolar gland with the function of secretion of milk ;
Animal mammary glands can be divided into six developmental stages : embryonic stage , adolescence , sexual maturity , pregnancy , lactation and degeneration .

In the process of mammary gland cell differentiation , the regeneration ability is often completely lost due to high differentiation , which leads to cell aging and death ;
In order to compensate for this deficiency in the course of development and evolution of the mammary gland , a portion of the undifferentiated primordial cells , referred to as mammary stem cells , are retained .

The proliferation and differentiation of mammary gland cells throughout the whole process of mammary gland development and lactation have been carried out in the whole process of mammary gland development and lactation .

There were CK5 , Sca - 1 , 伪6 - integrin , MUC ~ - / ESA ~ + and CK19 .

The discussion of a suitable marker for breast stem cells is still ongoing . This is a phenomenon that deserves attention and further research in the identification of markers for differentiation of breast stem cells identified in each report . This difference may have many reasons : 1 . the source of the material is different ;
2 . Different methods were developed in vivo and in vitro ;
3 . The mammary stem cells and their microenvironment are also in the process of development with the development of the individual ;
4 . Breast stem cells express a series of related proteins in the establishment of complex regulatory mechanisms , signal transduction systems and special functions .

In order to investigate the expression of Sca - 1 , CD24 and Muc1 during the four special periods of mammary gland development in rats , we observed the expression of Sca - 1 , CD24 and Muc1 in the rat mammary gland during the four special periods of mammary gland development , and studied the proliferation and differentiation of mammary stem cells .

method

1 . Preparation of paraffin section of breast tissue : 6 w ( body mass 180 ~ 220g ) , 9 w ( body mass 210 ~ 230g ) , pregnancy 15 days ( body mass 350 ~ 380g ) , postpartum 3 days ( body mass 330 ~ 350g ) , each time period 1 group , each group 12 , breast tissue mass size 1.0 cm 脳 1.0 cm 脳 0.5 cm , and made into 4 渭m thick section .

2 . HE staining : The morphology of four stages of mammary gland development and the morphological distribution of small bright cells were studied .

3 . Immunohistochemical staining was used to study the antigen - positive conditions of Sca - 1 , CD24 and Muc1 in four stages of mammary gland development .

4 . Immunofluorescent single labeling : study the antigen - positive condition of Sca - 1 , CD24 and Muc1 in four stages of mammary gland development .

5 . Western blot : SPF female rats with 6 w , 9 w , 15 d and 3 d postpartum were divided into 1 group , 3 in each group , and the contents of Sca - 1 , CD24 and Muc1 in four stages of mammary gland development were detected .

6 . Immunofluorescent double labeling : Sca - 1 and CD24 and Muc1 respectively were used to observe their double positive conditions in four periods .

Results

1 . HE Staining Results

The end - bud structure of the mammary gland of 6 w female rats was mainly the end bud structure .
The mammary gland of 9 w female rats is mainly glandular lobules . In the mammary gland tissue of 15 days after pregnancy , the branches of the secondary ducts increase and the number of acinar cells grow rapidly . In the mammary gland tissue of 3 days postpartum , there are many vacuoles in the mammary gland and the acinar cells , and there are more vacuoles in the cytoplasm of the nucleus and the free surface of the cells .

in 6 w breast tissue , most of that small bright cells are distribute in groups or groups , often located at the base of the breast branch catheter and the terminal bud ;
In 9 w breast tissue , the small bright cells were distributed on the basement membrane of catheter and acinar , and were also located between myoepithelial and glandular epithelial cells .
In the 15 d breast tissue , the small bright cells were distributed in the lobular duct .
In the postpartum 3 d breast tissue , the small bright cells are mostly distributed in the branch ducts .

2 . Immunohistochemical staining results

Sca - 1 ~ + cells were located between the fat cells and the acinar cells of the mammary gland of 6 w female rats .
9 w female rat mammary fat cells and around the acinar ;
The fat cells of mammary fat pad were expressed in 15 d female rats .
and postpartum 3 d female rat mammary gland fat cells , between the milk delivery tube and the breast fat pad .

CD24 ~ + cells were located between the mammary fat cells of 6 w female rats .
9 w female rat mammary fat pad ;
In the female rats of the 15th day of pregnancy , the mammary glands of the female rats were observed ;
Interleaf catheters in the mammary gland of female rat 3 d postpartum .

Muc1 ~ + cells were located in the fat cells of the mammary gland of 6w female rats , and in the mammary fat pad of 15 d female rats .
and in the postpartum 3 d female rat mammary gland duct .

3 . Immunofluorescence Single Sign Results

There were Sca - 1 ~ + in the leaves of 9 w female rats .
CD24 ~ + and Muc1 ~ + cells ;
Sca - 1 ~ + was found in the mammary gland branch ducts of female rats in 3 d postpartum .
CD24 ~ + and Muc1 ~ + cells .

4 . Western blot results

The content of Sca - 1 in the mammary glands of 6 w and 9 w was not significantly changed . The content of Sca - 1 in the mammary gland of 3 d rats was less than that of pregnant 15 d . The content of Sca - 1 in the mammary gland of rats was less than that in the rest period .

5 . Immune immunofluorescence double labeling results

6 w female rat mammary gland fat pad ;
9 w female rat mammary fat cells ;
The fat cells of mammary gland were observed in 15 d female rats .
Sca - 1 and CD24 - positive cells were found in fat cells of mammary gland in 3 d female rats .

6 w female rat mammary gland bubble around and branch catheter ;
9 w female rat mammary gland acinar ;
Muc1 and Sca - 1 double positive cells were present in the mammary gland branch ducts between 15 d female rat mammary fat cells and postpartum 3 d female rats .

Conclusion

1 . The mammary epithelial cells in the active period were proliferated in the matrix of the milk catheter , and the branches of the secondary ducts increased in the breast .
In contrast , these changes should be the result of multiple hormones during pregnancy .
Small bright cells appeared in the growth and multiplication sites in 4 periods .

2 . Some Sca - 1 - positive cells may represent a class of luminal epithelial cells and varied during different periods .

3 . There may be changes in the surface markers of breast stem cells in the mammary gland development : ( 1 ) changes in marker properties , i.e . retention or replacement of different markers ;
( 2 ) The results showed that CD24 was not changed during the first three stages , and the changes in 3 d after the postpartum period could be more or more likely to be a symmetric division , but also the lateral differentiation of the cells , so that the content of stem cells decreased during this period .

4 . Muc1 is expressed in the periphery of the gland of the secretion milk and the inner side of the catheter , as well as the fat and so on . Muc1 is higher than the rest period in 2 periods of the mammary gland development activity period , which may be the hallmark of mature luminal epithelial cells .

5 . There are two main sources of proliferation of mammary gland cells ( ductal epithelial cells , acinar epithelial cells , myoepithelial cells ) during the development of mammary glands . It is possible that Sca - 1 ~ + cells have dual potential , and some Sca - 1 ~ + cells represent a class of double - potential progenitor cells .

【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

【共引文献】