| Objective:Preeclampsia(PE)is a common pregnancy complication based on ischemia and hypoxia caused by placental hypoperfusion.In this study,the inflammatory factors(IL-2/4/6/10/17,TNF-α,INF-γ)levels in PE and normal maternal placenta tissues and the biological characteristics of umbilical cord mesenchymal stem cells were compared and observed,so as to explore the effect of inflammatory microenvironment in preeclampsia on the biological characteristics of offspring umbilical cord mesenchymal stem cells.To provide reference for clinical improvement of maternal and fetal health of preeclampsia.Methods:1.Grouping and tissue pretreatment:10 pregnant women who gave birth from October 2021 to October 2022 in Shanxi Maternal and Child Health Hospital were collected and divided into PE group(5 cases)and normal control group(5 cases).The umbilical cord and placenta tissues were collected immediately after caesarean section.The umbilical cord was extracted and cultured with UC-MSCs,and the placenta tissues were frozen.2.Isolation and culture of UC-MSCs:The umbilical cord tissues of the two groups were transported to the laboratory at low temperature within a short time after collection.After removing the capsule and blood vessels of the umbilical cord under the ultra-clean work,the Wharton’s jelly was separated and cut to less than 1mm~3.The primary culture was performed by tissue adhesion method.3.Morphology of UC-MSCs:The morphological characteristics of the two groups of UC-MSCs were observed with an inverted microscope and photographed.4.Proliferation capacity of UC-MSCs:Cell counting method was used to compare the proliferation capacity of UC-MSCs in the two groups.Cell proliferation culture was carried out with 24-well plates.From the next day after 48 hours of planting plates,three holes were taken every day for cell count and average value,and the growth curve was recorded and drawn for 7 consecutive days.5.Migration ability of UC-MSCs:The cell scribing method was used to compare the migration ability of the two groups of UC-MSCs,and photographs were taken at different time points(0,6,12,24 h),and Image J software was used for analysis and comparison.6.Differentiation ability of UC-MSCs:Bone and lipid formation were induced in the two groups.After 21 days,alizarin red staining was used to determine bone formation and oil red O staining was used to determine lipid formation.The Integrated optical density(IOD)was analyzed by IPP software.7.Comparison of placental water content:The water content of placental tissue was determined by dry weight method,which was used to measure the edema of placental tissue in the two groups.8.Expression of inflammatory factors in placenta:The expression of inflammatory factors in placenta tissue was detected by magnetic microsphere immunofluorescence assay using double antibody sandwich method.The magnetic beads were coated with IL-2/4/6/10/17,TNF-αand INF-γtrapping antibodies,which were specifically bound to the corresponding factors in the samples and then bound to phycoglobin labeled detection antibodies.The fluorescence intensity of each phycoglobin channel was correlated with the corresponding antigen concentration in the samples.The fluorescence intensity was measured by flow cytometry,and the concentration of corresponding factors in samples was calculated according to the standard curve.Results:1.Morphological differences of umbilical cord that observed by naked eye and UC-MSCs under microscope between the two groups:Naked eye observation:the umbilical cord tissue in the normal group was cord-like in shape,smooth and transparent in surface,and edema,vasodilation and intravascular congestion of Watton’s gel tissue were rare in section.The shape of umbilical cord tissue in PE group was more like twisted rope,the section showed obvious edema of umbilical cord tissue,Watton’s glue was transparent and wire-drawn,blood vessels were obviously dilated,and large blood clots were common.Microscopic observation of UC-MSCs:UC-MSCs in the normal group crawled out successively after 5-7 days of tissue adherence,and the cells showed slender spindle-shaped,parallel to each other or in a whirlpool arrangement.When the cell density increased,the cell shape became slender.After passage,the cells were dominated by uniform long spindle-shaped cells in a whirlpool arrangement.UC-MSCs in PE group crawled out slowly after 8-10 days of tissue adherence,and most of the cells separated from each other and showed scattered distribution.The cell shape was polygonal,with large cell bodies and slow proliferation.After passage,the cell bodies became large and flat,the arrangement was disorganized,and the cell density increased slowly.2.Differences in the proliferation capacity of UC-MSCs between the two groups:for 7 consecutive days,the proliferation status of UC-MSCs between the two groups was detected by cell counting method.The results showed that the growth curve trend of UC-MSCs in normal group and PE group was roughly the same,both of which went through the incubation period,exponential growth period and stagnation period.However,the UC-MSCs in PE group entered the plateau in advance and cell proliferation was slow compared with that in normal group.3.Differences in the migration ability of UC-MSCs between the two groups:An inverted microscope was taken and the scratched area of cells was measured by Image J software.It was found that the migration ability of UC-MSCs in PE group was significantly weaker than that in normal group.4.Differences in differentiation ability of UC-MSCs between the two groups:After osteogenic induction of UC-MSCs in both groups,mineralization nodules and lipid drome formation could be observed,indicating that UC-MSCs in both groups had osteogenic lipid differentiation ability.IOD values showed that the differentiation degree of UC-MSCs was different between the two groups.The differentiation degree of PE component bone(2224.54±62.29)was lower than that of normal component bone(5087.52±95.00),and the differentiation degree of PE component fat(8631.60±102.58)was lower than that of normal component fat(13030.58±727.60).The differentiation degree of osteogenesis lipids was P<0.001.5.Comparison of water content of placenta between the two groups:The water content of placental tissue in PE group(0.8540±0.019)was significantly higher than that in normal group(0.8424±0.006)and P<0.001,indicating that there was edema in placental tissue in PE group.6.Differences in expression of inflammatory cytokines in placental tissue between the two groups:Flow cytometry showed that the content of IL-2 was 1.65±0.13 in PE group and1.53±0.14 in normal group.The content of IL-4 was 2.52±0.19 in PE group and2.49±0.51 in normal group.The content of IL-6 was 28.07±15.27 in PE group and15.00±3.64 in normal group.The content of IL-10 was 0.62±0.37 in PE group and0.34±0.22 in normal group.The content of IL-17A was 7.25±1.56 in PE group and6.59±1.57 in normal group.The content of TNF-αwas 1.88±0.55 in PE group and1.70±0.32 in normal group.INF-γcontent was 2.04±0.36 in PE group and 1.72±0.29 in normal group.The content of inflammatory factors in PE group was higher than that in normal group,indicating that placental tissue of PE group was infiltrated in inflammatory microenvironment.Conclusions:1.The water content of placental tissue in PE group was significantly higher than that in normal group,and there was edema in placental tissue.2.The content level of inflammatory factors in placental tissue of PE group was significantly higher than that of normal group,and placental tissue was in an inflammatory state.3.The growth,proliferation,migration and differentiation of UC-MSCs in PE group were inhibited. |
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