| Objective:(1)The isolation and culture of placenta mesenchymal stem cells;(2)Using animal models establish atopic dermatitis;(3)To study the relationship ofPMSCs in AD-like model in mice;(4)To explore the influence of the PMSCs to Foxp3+Treg in AD-like model in mice;(5)To explore the influences and mechanisms of thePMSCs to IL-2, IL-4, IL-6, IL-10, IL-17A, TNF and IFN-γ inAD-like mice.Methods:(1)The PMSCs separation with tissue block method combined enzymedigestion method, PMSCs identification using flow cytometry instrument to detect cellsurface molecules, as well as in vitro induced PMSCs differentiate into fat cells,osteoblasts and chondrocytes;(2)1%5-fluorescein isothiocyanate (FITC) induced BALB/cmice to acute(once excitation)and chronic(once every3days excitation, a total of7times)AD;(3)Injecting the different doses of the PMSCs(0.5×106,1×106or2×106)in mousetail vein to treat the acute(one time),chronic(two times)AD. With macroscopic observationin mice ear and back skin changes, dynamic measuring the change of the thickness of earin mice, put to death before according to its weight, after executed, said its weight of earsand spleen, calculation about the difference of the ears weight and the spleenindex;(4)Using flow cytometry to detect the mice spleen and drainage of lymph node of earFoxp3+Treg cells;(5)Capture chip technology method with flow cytometry instrumentmicrospheres (CBA) detected IL-2, IL-4, IL-6, IL-10, IL-17A, TNF and IFN-γ content inmice back skin tissue homogenate;(6) Routine histopathologic sections and HE stainingcarried out in mice ear and back skin, observing the pathological changes of the skin,calculated the epidermis layer number and the number of inflammatory cells in the dermis.Results:1. We separated the placenta mesenchymal stem cells successfully. The cell surface of PMSCs expressed CD29,CD90,CD105,while CD34,CD45and HLA-DR wasnegative. In vitro, PMSCs could be divided into fat cells, osteoblasts and chondrocytes.2. Setting upAD-like model in mice.(1)Acute AD-like model in miceThe left ears thickness were (24.90±0.89)×10-3mm before excitation.At12h afterexcitation, the skin in auricle and back appeared light edematous erythema. After24h, thethickness of left ears reached (28.00±1.06)×10-3mm. And the thickness reached its peak(28.70±0.67)×10-3mm at48h. At this time, the weight of left ears were (13.88±0.61)mg,and the weight difference for ear to ear was (0.84±0.72)mg. The changes of ears and backskin tissue pathological was similar to acute dermatitis, such as, mild edema in skin andthickening, dermal shallow edema, expansion of small blood vessels, more inflammatorycells infiltrated (mainly single cells and less polymorphonuclear cells). Comparing with thesubstrate group, the number of infiltrating cells in ears and backs were increased (P<0.05),the thickness of back epidermis was increased (P<0.05).The Th1,Th2cytokines in themodel group lesions are elevated, IFN-γ/IL-4ratio lower than the matrix group,Th2in thedominant Thl/Th2balance disorders in model mice.(2) ChronicAD-like model in miceThe left ears thickness were (27.00±1.27)×10-3mm before the first excitation.At12hafter the first excitation, the ears were mildly edematous erythema, and under theincreasing of the excitation frequency, edematous erythema increased gradually, thenappeared scabby, scales and hair (mainly in the back). At28days before executing, thethickness ears were (39.30±4.15)×10-3mm, the weight of left ears were (19.60±3.86)mg,the weight difference for ear to ear was5.24±3.26mg. Under the light microscope, the skinchanged in subacute and chronic dermatitis, such as, ear skin visible in thickness skinstratum corneum, occasional serum scabs, thickening of stratum spinosum (4~5layers),leather with a large number of cell infiltration (mainly single cells and lesspolymorphonuclear cells);on the back, epidermal hyperkeratosis, parakeratosis, stratumspinosum markedly thickened (6~8layer), with offshoring cells, occasional serum scabs, a large number of inflammatory cells infiltrated in leather (mainly single nuclear cell),compared with the matrix group, infiltrating cells and spine layer upon layer wereincreased significantly (P<0.05~0.01).Model group mice were detected in IFN-γ/IL-4ratiois still lower than the matrix group and Th2disorder of the dominant Thl/Th2balanceremains.3. The influence of the PMSCs in AD-like model in mice.(1)Acute AD-like model in miceAt24h after excitation, in the PMSCs injection of high dose group, the difference ofbefore and after mice ear thickness was less than the model group (P<0.05). The middledose was also smaller than that of model group, but there was no significant difference(P>0.05). At48h, the thickness difference before and after the ear of the middle and highdose group was less than the model group (P<0.05), the correlation between dose andeffect was positive. In ear weight, high dose group was less than model group, but nostatistical significance (P>0.05). On histopathologic slice, the number of dermal cellsinfiltrating of ears and backs skin in the middle and high dose group and the thickness ofback epidermis were lower than the model group (P<0.05), suggesting that the PMSCs hastherapeutic effect on acute AD.(2) ChronicAD-like model in miceAt1day after excitation, compared with model group, before and after ear thicknessdifference in the high dose group was less than the model group(P<0.05). And after4days,before and after ear thickness difference in the high dose group was less than the modelgroup(P<0.05), as well as the curative effect of the high dose group was better than the lowdose group; The weight of ears with PMSCs in each dose group was lighter in the modelgroup, but there was no significant difference(P>0.05). In histopathological aspects, the earof the PMSCs high-dose group and back skin infiltrating cells in dermis and epidermisthickness of the back was less than the model group(P<0.05), the dose of ears and backskin of infiltrating cells was also lower than that of model group(P<0.05).In addition,compared with the number of infiltrating cells in the dermis and the thickness of back in high dose group was less than the middle dose group(P<0.05),showed that the PMSCs alsocould therapy chronic AD,and its curative effect was related to cell concentration.4. In the middle and high dose groups,PMSCs could raise the expression of Foxp3+Treg in acute stage drainage lymph nodes in AD mice,while in the spleen had no obviouseffect.5. The changes of IL-2,IL-4,IL-6,IL-10,IL-17a,TNF and IFN-γ were detected ingroups of mice.(1)Compared with the matrix control group, detecting in the lesions of acute modelgroup showed that the Th2type cytokines IL-4,IL-6,IL-10levels increased significantly,type Th1cytokines IL-2elevated, IFN-γ had no obvious change, and a significant rise ininflammatory factor TNF,Th17cells specificity mark IL-17A significantly higher(P<0.05),model group IFN-γ/IL-4(1.32±0.76)was lower than the matrix group(2.11±0.86),prompted that the model was immunizing response caused by the Th2.(2)Compared with the matrix control group,in the chronic model group,Th2typecytokines IL-4,IL-6,IL-10levels were increased, Th1cytokines IFN-γ moderately elevated,IL-2had no obvious change, and a significant rise in inflammatory factor TNF,Th17cellsspecificity mark IL-17had no significant changes.While in the chronic group, IFN-γ/IL-4(0.73±0.64) was less than the matrix group (1.34±0.41),suggested the chronic model groupalso was give priority to in order to type Th2immune response.(3) The mechanism of action of dexamethasone for the treatment of AD sample model inmice.Dexamethasone inhibited IL-2,IL-4in the lesions of acute AD sample significantly,slightly reduced TNF,IFN-γ and IL-10,and had no obvious effect with IL-6andIL-10,increased IFN-γ/IL-4(2.76±3.53),reduced the skin inflammatory factor TNF andIL-17A,showed that dexamethasone could through regulating Th1,Th2,Th17cellsimbalances to alleviate inflammation of the skin lesions.(4) The mechanism of action of PMSCs for the treatment ofAD sample model in mice.①Compared with acute model group,low and medium dose PMSCs in mice skin withacute AD sample model Th2cytokines IL-6,IL-10dropped apparently,IL-4dose group decreased,no significant changes in low dose group,and the IL-2,IL-4,IL-6,IL-10,IFN-γand IL-17A in the high dose group rised;PMSCs made IFN-γ/IL-4(0.86±0.28,0.97±0.22)reduced in the middle dose group as well as the high dose group was higher(2.12±1.55),and returned to normal. These results prompted that PMSCs influenced onTh1,Th2,Th17as concentration may have a two-way adjustment role.②Compared with chronic model group,PMSCs could make IL-10and TNF decrease inthe skin lesions,but other factor had no obvious change.Conclusions:(1)We separated the placenta mesenchymal stem cells successfully;(2)We made use of1%FITC induce BALB/c mice to set up AD-like model;(3)Bymaintaining the balance of Th1/Th2and Th17/Treg,PMSCs inhibited AD model on acuteand chronic skin lesions,and the curative effect was related to the dose. |
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