| Part1The effects of estrogen on proliferation and migration of human skin fibroblast and its regulatory mechanismObjective:To observe the effect of estrogen (17(3-estrogen,17β-E2) on the proliferation and migration of cultured normal human skin fibroblasts (Human skin fibroblast, HSFB), wheather mitogen-activated protein kinase signal transduction pathway (mitogen-activated protein kinases, MAPKs) involves in the regulation of estrogen on proliferation of human skin fibroblast, understanding the potential regulatory pathways and mechanisms.Methods:To culture normal human skin fibroblasts in vitro with enzyme digestion method, the3-6generation of cells were used for the experiments;1. To detect the proliferating activity of HSFB under the intervention of different concentrations of17β-E2,17β-E2+β receptor antagonist ICI-182780and blank at24,48,72,96h respectively by MTT;2. To establish the in vitro cell scratch (Scratch-Wound) model, the migrating situation of HSFB under the intervention of different concentrations of17β-E2,17β-E2+ICI-182780and blank were observed under phase contrast microscope at24,48,72h after preparation of the model;3. The expression changes of TGF-β1protein of HSFB under the intervention of17P-E2, 17β-E2+ICI-182780and blank were detected with immunocytochemical technique;4. The cell cycle distribution of HSFB under the intervention of17β-E2and ICI-182780was detected by flow cytometry;5. The cells were divided into6groups according to the different interference factors:control group (group A),17β-E2group (group B),17β-E2+ICI-182780group (group C),17β-E2+PD98059group (group D),17β-E2+SP600125group (group E) and17β-E2+SB203580group (group F), the cells under the intervention of the kinase inhibitor and (Or) estrogen were collected, after the extraction of total RNA and total protein, the expression of RNA and protein of proliferating cell nuclear antigen (Proliferation cell nuclear antigen, PCNA)were detected with fluorescence quantitative PCR and Western-Blot.Results:1(1) MTT,24h, there is no significant difference in cell proliferating effect between the group of estrogen stimulating concentration (10"8-10-12mol/L) and the control group (0.118±0.005,0.113±0.007,0.128±0.013,0.113±0.006,0.122±0.012vs0.115±0.007)(P>0.05);48,72,96h,10-10mol/l are the estrogen group of strongest proliferative effect (48h,0.208±0.015vs0.121±0.015,0.137±0.017,0.172±0.012,0.137±0.019);72,96h, the proliferating effect of this concentration estrogen was stronger than that of blank group (72h,0.245±0.028,0.303±0.022,0.378±0.041,0.276±0.036,0.232±0.022vs0.166±0.025;96h,0.344±0.036,0.406±0.033,0.485±0.031,0.386±0.032,0.368±0.034vs0.196±0.033)(P<0.01);(2)24h, ICI-182780+10’10mol/l estrogen group (group B) and the control group (group C),10"10mol/l estrogen group (group A) showed no significant difference of cell proliferation (0.120±0.015vs0.113±0.007.0.128±0.015)(P>0.05); and48,72,96h, the proliferation in group A was significantly stronger than that in the other two groups (48h,0.211±0.015vs0.119±0.016,0.136±0.015;72h,0.352±0.049vs0.168±0.030,0.232±0.025;0.486±0.028vs0.196±0.024,0.368±0.034)(P<0.01);48h, the cells proliferation in group B and group C had no difference (0.136±0.015vs0.119±0.016)(P>0.05), but that in group B was stronger than that in group C at72,96h(72h,0.232±0.025vs0.168±0.030;96h,0.368±0.034vs0.196±0.024)(P<0.01).2.(1)48h, compared to other groups, the cell migration in10-8mol/l estrogen group was the longest (568.4±11.305vs413.2±25.616,429.4±20.281,507.8±17.312,568.4±11.305,484.1±13.675,452.6±14.741)(P<0.01),10-7mol/l,10-9mol/l and10-10mol/l group also has a strong stimulating effect of transferation than blank group (507.8±17.312,484.1±13.675,452.6±14.741vs413.2±25.616)(P<0.01); but the10-6mol/l group had no obvious effect on cell migration (429.4±20.281vs413.2±25.616)(P>0.05);(2)24h,48h,72h, compared with ICl-182780+10-8mol/l estrogen group (group B) and control group (group C), the cell migrating distance in10" mol/1estrogen group (group A) has significant difference (24h,375±13.784vs225.6±10.761,243±14.747;72h,567±10.247vs407.6±11.718,416.6±16.134;96h,735.4±10.854vs603.8±18.887,623.2±15.057)(P<0.01), but there was no difference between group B and C(P>0.05);3.In cell climbing piece, the expression of TGF-β1protein in estrogen stimulating group was the highest (155.4±22.941vs103.2±10.281,87.4±9.343)(P<0.01), while no significant difference exsit in the other two groups (P>0.05);4.The percentage of cells during S phase in group A was more than in group B and C (58.20±1.387%vs13.02±1.052%,9.50±1.483%)(P<0.01), the percentage of cells during G0/G1phase in group A was significantly lower than that in group B and C (3.66±0.321%vs50.18±2.685%,62.36±2.474%)(P<0.01), the percentage of cells during G2/M phase in group A and group B has no difference (38.14±1.652%vs36.82±3.505%)(P>0.05), but that in group A was more than in group C (38.14±1.652%vs28.14±2.661%)(P<0.01); the percentage of cells in each phase in group B and C have significant differences (P<0.01);5. Compared with group A, the expression level of PCNA mRNA in group B was significantly higher(4.360±1.086vs1.000±0.000)(P<0.01), but compared with group B, that in group C and D was significantly inhibited (2.012±0.357,2.097±0.353vs4.360±1.086)(P<0.05), and that in group E and F showed no significant difference (4.313±0.962,4.319±0.975vs4.360±1.086)(P>0.05);6.The expressions of PCNA protein and mRNA in above groups were basical consistent.Conclusion:1. Estrogen has strong effects of promoting mitosis and proliferation, in the range of (10-8、10-9、10-10、10-11、10-12mol/L),the concentrations of10"10mol/l is the strongest;2.It has a strong chemotaxis, promoting the migration of HSFBs, in the range of (10-6、10-7、10-8、10-9、10-10mol/L),the concentrations of10-8mol/l is the strongest;3. ICI-182780can abate the above effect effectively.4. Estrogen can promote fibroblast secretion of TGFβ1protein;5. Estrogen β receptor and ERK/MAPK signal pathway should be involved in estrogen-Regulated proliferation of HSFB. Part2The experimental Study of the mechanism of estrogen delaying the aging of rats skinObjective:To observe the effect of estradiol benzoate on related factors of ovariectomized rat skin aging, explore the mechanism of estrogen affect on skin.Methods:40Wistar female rats were randomly divided into four groups: control group (group A), sham operation group (group B), model group (group C) and model+estradiol benzoate injecting group (group D), with10rats in each group.Through the operation of ovariectomization, rat aging model created (group C and group D), group A without any treatment, group B only took a few fat tissue around the ovary; continuous vaginal smear within one week after operation, to verify the success of modeling; one week after operation, the rats were injected with drugs (group A, B, C physiological saline, group D estradiol benzoate); administration of animal were killed after eight weeks, blood estradiol (E2)concentrations were determined in serum of rats, and clipping rats’back skin to make the tissue homogenate to detect superoxide dismutase (SOD) activity, hydroxyproline (HYP) content and malondialdehyde (MDA) content.Results:Compared with group A, the serum level of E2(pg/ml)(38.51±3.58vs49.31±3.52) and HYP content (mg/g wet weight)(3.26±0.259vs4.57±0.355) in group C were significantly decreased (P<0.01), further explained that ovariectomized rats’skin natural aging model was successfully established; compared with the group C, the serum level of E2(pg/ml)(50.69±3.61vs38.51±3.58)(P<0.01) and HYP content (mg/g wet weight)(4.06±0.231vs3.26±0.259)(P<0.01) in group D were significantly increased; compared with group A,the serum level of E2in group B (pg/ml)(46.43±4.86vs49.31±3.52) has no significant difference (P>0.05), but the content of HYP decreased significantly (mg/g wet weight)(4.20±0.172vs4.57±0.355)(P<0.01); compared with group A, the SOD activity in group C was significantly decreased (U/g protein)(100.68±7.92vs130.01±6.49)(P<0.01), the content of MDA increased significantly (P<0.01); compared with group C, the SOD activity in group D was significantly increased (U/g protein)(116.38±7.84vs100.68±7.92)(P<0.01), the content of MDA decreased (mmol/g protein)(2.55±0.29vs1.61±0.22)(P<0.01); compared with group A, the activity of SOD (U/g protein) and MDA content (mmol/g protein) in group B were not significantly different (125.57±8.66vs130.01±6.49,1.56±0.26vs1.61±0.22)(P>0.05), but the SOD activity in group D decreased significantly (U/g protein)(116.38±7.84vs130.01±6.49)(P<0.01), the MDA content in group D was significantly increased(2.06±0.24vs1.61±0.22)(P<0.01), so the estradiol benzoate can improve SOD activity, HYP content and serum E2concentration in the skin tissue of ovariectomized rats, but decreased the content of MDA.Conclusion:Estradiol benzoate can increase the expression of collagen fibers in rat skin tissue, while enhancing the antioxidant ability of ovariectomized rats to delay skin aging. |
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