| BACKGROUND AND DESTINATIONMammary gland of Mammal, which is a compound tubuloacinar gland with thefunction of lactating, and it is the only organ that can repetitional regeneration inpostnatal animal. On the basis of mammogenesis' characteristic, rodents animal'smammary gland roughly has six developmental phase: embryonic period, puberty,Sexual maturity, gestation, lactation and degeneration.Mammary gland cell lose it's regeneration ability for the high degreedifferentiation during the differentiated progress, which finally cause the cells death.In order to compensate this insufficiency, Mammary gland retain a part ofundefferentiated archaeocyte during growth and evolution, which is called mammarystem cells.Proliferation and differentiation of mammary gland cell occur during the wholeprocess of mammogenesis and lactation. Many researches about the proliferation anddifferentiated of mammary gland epithelium in cell level have been carried out. Theseresearch data revealed the mechanism of mammary gland stem cell regeneration andprosoplasia that result in mammary gland tumor, preparing theory for breast tumor,cancer's precaution and healing servation, which providing suitable standard forstudying organofaction and cellular differentiation. CK5, Sca-1,α6-integrin, MUC-/ESA+-and CK19 were used as the symbolproteinum in the formerly experiments.The discussion about suitable mammary gland stem cell marks still continued,and there were many different symbol proteinum have been used in each researches.This phenomenon is worth reconstruction and further research. The difference mayexist in Versatile cause:1. The source of materials were different; 2. The methods ofinternal and culture in vitro were different; 3. The mammary gland stem cell and it'smicroenvironment develop along with individual development, 4. The mammarygland stem cell establish complicate control mechanism, signal transduction systemand special function.We research morphology of female rat mammary gland, which contains pubertas,Sexual maturity, gestation, lactation. We apply methods of immunofluorescence,enzyme labeling and Western blotting to observating Sca-1, CD24 and Muc1'sexpression during the four special period of rat mammary gland. We researchdistribution of the three kinds of cell marker for approaching the proliferation anddifferentiation of mammary gland stem cell.EMPIRICAL METHOD1. The preparation of mammary gland's tissue paraffin section: 6-week-old(180-220g), 9-week-old (210-230 g), 15 day of gestation (350-380 g), 3 day ofpostnatal (330-350 g), every stage represent a group, the amount of a group is 12, Thepiece of mammary gland's tissue is 1.0 cm×1.0 cm×0.5 cm, which is sliced in 4μmthick section.2. HE dyeing: We researched the four period mammogenesis's morphous anddistribution of the SLC. 3. Immunohistochemical enzyme labeling:We observe Sca-1+, CD24+ and Mucl+cell in the four mammogenesis period.4. Immunofluorescence single labeling: We observe Sca-1+, CD24+ and Mucl+cell in the four mammogenesis period.5. Western blotting: We use 6-week-old, 9-week-old, 15 day of gestation, 3 dayof postnatal SPF female rat, every stage represent a group, the amount of a group is 3,which is used for detecting the content of Sca-1, CD24 and Muc1 in the fourmammogenesis period.6. Immunofluorescence double labeling: double labeling with Sca-1 and CD24,double labeling with Sca-1 and Muc1, We observe the information of co-staining inthe the four period.CONSEQUENCE1. The result of HE dyeing6-week-old rat's mammary gland, which contains much distal budding structure;9-week-old rat's mammary gland, which contains much glandular lobule; 15 day ofgestation rat's mammary gland, in which middle and secondary ductus multiplyincreasing, gland alveolus develop fast and the number is increasing; 3 day ofpostnatal female elder's mammary gland: glandular lobule is full of secretory glandalveolus, the morphous of gland alveolus are diversify, intra-cellular emerge morevacuole, The blood vessel was much in connective tissue, interlobular septum is verythinness.6-week-old rat's mammary gland: SLC distribute in branching ductus and basilarpart of terminal shoot; 9-week-old rat's mammary gland: SLC distribute in the ductusof lobule; 15 day of gestation rat's mammary gland: SLC distribute in the ductus of lobule; 3 day of postnatal female elder's mammary gland: SLC multiply distribute inthe ductus of lobule.2. The result of Immunohistochemical enzyme labelingSca-1+ cells distribute around the 6-week-old rat's mammary gland alveolus andfat cell; Sca-1+ cells distribute around the 9-week-old rat's mammary glandalveolus;Sca-1+ cells distribute in 15 day of gestation rat's mammary gland fatpad;Sca-1+ cells distribute in the 3 day of postnatal rat's mammary gland fat pad.CD24+ cells distribute around the 6-week-old mammary gland fat cell; CD24+cells distribute in the 9-week-old mammary gland fat pad; CD24+ cells distributearound the 15 day of gestation rat's mammary gland alveolus; CD24+ cells distributein the 3 day of postnatal rat's mammary gland ducts.Muc1+ cells distribute around the 6-week-old mammary gland alveolus; Muc1+cells distribute in the 15 day of gestation rat's mammary gland fat pad; Muc1+ cellsdistribute around the 3 day of postnatal rat's mammary gland duct.3. The result of immunofluorescence single labelingSca-1+, CD24+ and Muc1+ cell respectively distribute in duct of the 9-week-oldSD female rat's mammary gland.Sca-1+, CD24+ and Muc1+ cell respectively distribute in branching duct of 3 dayof postnatal rat's mammary gland4. The Result of Western blottingThe content of Sca-1 in 6-week-old mammary gland and in 9-week-old's areunchanging, The content of Sca-1 in the 3 day of postnatal mammary gland is lessthan 15 day of gestation's, The content of Sca-1 in two developmental period ofactive phase mammary gland is less than in quiescence's; The content of CD24 is unchanging in 6-week-old, 9-week-old and the day of 15 gestation, however, in 3 dayof postnatal, which is decrease; The content of Muc1 in two developmental periods ofactive phase mammary gland is more than in quiescence's.5. The result of immunofluorescence double labelingSca-1+ and CD24+ cell distribute in the fat pad of 6-week-old rat's mammarygland; Around mammary gland fat cell of 9-week-old rat; Around the mammary glandfat cell of 15 day of gestation rat; or Around the mammary gland fat cell of 3 day ofpostnatal rat.Muc1+ and Sca-1+ cell distribute Around mammary gland alveolus of6-week-old rat; Around mammary gland alveolus of 9-week-old rat;Aroundmammary gland fat cell of 15 day of gestation rat; or In mammary gland branchingduct of 3 day of postnatal rat.CONCLUSION1. In active stage, mammary gland epithelium proliferate extensivly in the basematerial of mammary duct, center and secondary duct branch was increase, glandalveolus develop fast, the quantity is increasing and cell body greaten, comparingwith the quiescent period's mammary gland, these marked changing should be theresult of multiple endocrine effect during the pregnancy;SLC appear in growing andproliferating part of the four stage.2. Part of Sca-1+ cell possible represent a kind of cavity ancestor epithelium, ofwhich content was vicissitudinous.3. Surface marker of mammary gland stem cell possible changing duringmammary gland development, first, The surface marker's qualitative was changed, itmeans different surface marker retention or alternation, the second, Expressing quantitative of the surface marker was variance. Experimental result showed thatCD24 was not changing during the forward three period, 3 day of postnatal elderfemale's change may be the result of symmetry mitogenesis or cell transversaldifferentiation, The content of stem cell in the period was step down.4. Mucl distributed surrounding Lactating gland alveolus, intraductal and fat,Mucl mammogenesis active stage's content were more than quiescent period's, whichpossible represent a mature cavity epithelial identification marker.5. In mammogenesis period, mammary gland cell (duct epithelium, glandalveolus epithelium, myoepithelium cell) generous amplification has two main source,Sca-1+ cell has bipotentiality, part of Sca-1+ cell represent a species of bipotentialityancestral cell.
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