Study Of Proliferation And Differentiation In Endometrium Side Population Stem/progenitor Cells

Background: Stem cells are characterized by the ability to renew themselves and differentiate into a range of specialized cell types. According to different period it can be divided into two types:embryonic stem cells and adult stem cells. Adult stem cells just as a type of undifferentiated cells be present in a variety of differentiated tissues.It is the basis for repairing and regeneration of tissues and organs .Adult stem cells are either unipotent or multipotent. They are small populations of quiescent cells with the potential to regenerate the entire tissue in which they reside when the tissue damaged. Adult stem cells have broad research and application prospects. Side-population cells(SP cells) depend on ATP-binding cassette superfamily G member 2(ABCG2) protein displaying a stem cell-like property that efflux fluorochrome Hoechst33342 . Recently, SP cells have been identified in many mature tissues, such as the intestine , colon, liver, lung and kidney.Endometrium is a tissue which have a highly proliferative activity and contains stem/progenitor cells . In previous study , we isolated SP cells from the endometrium of postpartum murine uterus. After identification, we found SP cells were a heterogeneous population of stem/progenitor cells. The ratio of SP cells becam increased first and then gradually decreased along with the time after parturition.Objective: In this study, we investigate the property of SP cells proliferation and differentiation in vivo and vitro, explore the regulation in SP cells proliferation and differentiation. By analyses of the serum estradiol level and the expression of ERαin the uterus of postpartum murine to approach the relationship of this two guys and the ratio of SP cells. Through using different concentrations of E₂ to affect on cultured SP cells in vitro to explore the effect of E₂ and ERαon the proliferation and differentiation of SP cells. We also take adventage of nude mouse to research the differentiation of SP cells in vivo under the effect of ectogenesis E₂. All these studies establish the foundation of investigating the effect of SP cells in repairing postpartum endometrium and in the pathomechanism of endometrium abnormal proliferation disease.Methods: (1)We separated SP cells according to former method of our laboratory. Using reverse transcription polymerase clain reaction(RT-PCR) and immunofluorescence to measure expression and location of ABCG2, CK-18,Vimentin andαSMA to characterize SP cells. (2)Detect the expression of ERαby RT-PCR and immunofluorescence.(3) Detect SP cells content in different postpartum days(d₁,d₃,d₇,d₁₄,d_17.5,d₂₁,d₂₈). Measure the level of serum E₂ in different postpartum time through radioimmunoassay(d₁,d₇,d₁₄,d₁₈,d₂₁,d₂₈). Realtime PCR,Westernblot and immunohistochemistry assay were employed to detect the expression and location of ERαin different time of postpartum uteri.(4) Through using different concentrations(10^-8M～10^-6M) of E₂ and E₂+ ICI182780(inhibiter of ERα) to affect on cultured SP cells in vitro 24h, then measure the proliferation of SP cells through MTT. After culture for 15 days,counted the colonies of SP cells and calculate the cloning efficiency(CE) and large CE. Detect the expression of CK-18 and Vimentin by immunofluorescence three weeks later.(5)SP cells were injected underneath the kidney capsules of nude mouse, ectogenesis E₂ was intramscular injected everyday from the first day of transplantation. The mouse was nephrectomized 8 weeks later. Serial slicing of the kidney undergo hematoxylin–eosin ( HE) staining and detect CK-18, Vimentin,αSMA by immunohistochemistry.Results:(1)? Phenotype analysis showed that SP cells were positive for ABCG2 though non-SP cells not. Most of SP cells positive for Vimentin, only a few expressed CK-18 andαSMA. (2)RT-PCR and immunofluorescence showed that SP cells expressed high percentage of ERα.(3) The level of serum E₂ and the expression of ERαin the uterus of postpartum murine were both show the trend that increasing first then decreasing gradually. SP cells content shows a trend of increasing by degrees then decreasing gradually after day 17.5(13.95%). Luminal epithelium expressed manipulus of ERαat d₁, at d₇ stroma and glandular epithelium to present some ERα, d₁₄～d₁₈ ERαexpressed Significantly increased in stroma and glandular epithelium and most of it located in stroma at d₁₄ though d₁₈ ERαmainly expressed in glandular epithelium, at d₂₁ the expression of ERαdecreased remarkable, only a little to present in stroma at d₂₈. In murine postpartum endometrium most BCRP1/ABCG2 were located in the stroma and vascular endothelium, a few reside in glandular epithelium and luminal epithelium expressed none. (4)Compared with the control, 10^-6M E₂ could promote the proliferation of SP cells notably after treatment for 24h(P<0.05).These effects could be inhibited by ICI182780(P<0.05).There were no significant differences of CE between each E₂ concentrations, but 10^-7M,10^-8M E₂ could impair the large CE of SP cells obviously(P<0.05).The effects could be inhibited by ICI182780, either(P<0.05). Compared with control, there were no significant differences of CK-18 expression among all experimental groups. In 10^-8M E₂ group there was an increasing tendency of Vimentin positive signal.(5) The nude mouse was nephrectomized 8 weeks after xenotransplantation. Through HE staining we found a mass of abnormal cells arranged tightly in the medullary substance of the kidney. Immunohistochemistry show that these cells expressed CK-18 and Vimentin notablely, a few cells expressed Desmin, negative ofαSMA.Conclusion: (1)SP cells of the endometrium of murine are a heterogeneous population of stem/progenitor and express ERαremarkably.(2) The proportion of postpartum mice uterus SP cells shows a trend of increasing by degrees then decreasing gradually. Along with the changing of postpartum days, the level of serum E₂ , expression of ERαin mouse uterus are both have the same variance tendency with SP cells ratio. Similar with ABCG2,The mainly location of ERαin postpartum murine uterus is stroma and glandular epithelium. These all indicate that SP cells participate in the repairing of postpartum endometrium under the effection of E₂ through ERα. (3)The influence of E₂ to SP cells maybe has two aspect:promote proliferation and promote differentiation. The effect of stimulate proliferation displays obviously while under high E₂ concentration.Though under closely to physiological E₂ concentration, perhaps the promote differentiation effect shows outstanding, especially to stimulate SP cells differentiate to stroma.(4)Under the effect of E₂ ,SP cells in vivo may have the potential to differentiate into stroma and epithelium-like structure, especially similar to stroma.(5) These studies settled the foundation of further approach in the effect of SP cells in repairing postpartum endometrium and in the pathomechanism of endometrium abnormal proliferation disease, at the same time, these researchs also accumulated research documents for experimental and clinical applications of endometrium SP cells.