| Skin aging is a complex biological process, influences all layers of the skin, but mainly in the dermis. elastic fibres net is formed from elastic fiber in the dermis, it is one of the important structure to maintain the skin elasticity and plays an important role in skin aging, the main components of the elastic fiber elastic protein was synthesized from skin fibroblasts.Objective: This topic focuses on the main of the skin cells skin fibroblasts, Study the role of aldosterone、IGF- 1 in fibroblasts and adjusting skin elastin. After cultivate skin fibroblasts, intervention different concentrations of aldosterone 、 IGF 1, then observe their effects on fibroblast; Through the influence to elastin at different concentrations of IGF- 1、aldosterone, explore the possible mechanism; Observation the promotion of the synthesis elastin of fibroblasts under aldosterone receptor antagonist to aldosterone, aldosterone and the effects of combination of IGF 1 in fibroblasts on elastic protein, influence of aldosterone after impact on IGF 1 signaling pathways and targets, and discuss the possible mechanism.Methods:1. Determined by MTT method to detect different times and different concentration of IGF 1 influence on skin fibroblasts proliferation, fibroblasts are divided into six groups: zero group(no cell culture), the control group without intervention(cells only), 50 ng/ml IGF- 1, 100 ng/ml IGF- 1, 1, 200 ng/ml IGF- 500 ng/ml IGF- 1continuous observation 12 h, 24 h, 48 h, 72 h, 96 h.2. Determined by MTT method to detect different times and different concentrations of aldosterone skin fibroblasts proliferation, there are 5 groups: zero group(no cell culture), the control group without intervention(cells only), 10 nmol/L aldosterone,50 nmol/L aldosterone, 1μ mol/L aldosterone continuous observation 12 h, 24 h, 48 h,72 h, 96 h.3. Immunofluorescence test the elastin’s regulation by different concentration of IGF-I: according to the 50 ng/ml IGF-I, 100 ng/ml IGF-I, 200 ng/ml IGF-I, 500ng/ml IGF-I concentration gradient dosing treat fibroblasts, and set a negative control.4. Immunofluorescence method to detect the elastin’s regulation of different concentrations of aldosterone: according to the 10 nmol/L aldosterone, 50 nmol/L, 1μmol/L aldosterone concentration gradient dosing treat fibroblasts, and set a negative control.5. Immunofluorescence test of elastin’s regulating mechanism of aldosterone:according to the result of the experiment selected the best concentration of aldosterone, cultured with the best concentration of IGF 1, the biggest concentration of IGF- 1 and aldosterone receptor antagonist spironolactone respectively, and set a negative control.6. Tyrosinase inhibitor cultured with the optimal concentration of aldosterone and IGF-Irespectively, phosphatide acyl alcohol inhibitors cultured with the optimal concentration of aldosterone and IGF-I respectively, set a negative control, then do cellular immune confocal fluorescence and observe the changes of elastin.7. Western blot semi-quantitative detection of elastin aldosterone adjustment mechanism:Results:1. MTT method to detect the skin fibroblast’s proliferation in different times and concentration of IGF-I: Compared with the control group, 50 ng/ml IGF-I, 100 ng/ml IGF-I and 200 ng/ml IGF-I group have obvious difference, P<0.05; Compared with50 ng/ml IGF-I, 100 ng/ml IGF-I to 200 ng/ml IGF-I group have obvious difference,is greater than the group, P < 0.05; Compared with the control group, 500 ng/ml IGF-I group has obvious difference, less than the control group, P < 0.05.2. MTT method to detect the skin fibroblasts proliferation in different times and concentrations: Compared with the control group, 50 nmol/L and 10 nmol/L aldosterone have obvious difference, P < 0.05. 1μmol/L aldosterone group obvious difference, less than the control group, P < 0.05.3. Immunofluorescence method to detect the regulation to elastin in different concentration of IGF-I: compared with the blank control, 50 ng/ml IGF-I, 100 ng/ml IGF-I, 200 ng/ml IGF-I group, elastin in fibroblasts(red fluorescent substances) is increase gradually. in 500 ng/ml IGF-I group, the elastin is drop.4. Immunofluorescence method to detect the elastin’s regulation in different concentrations of aldosterone: compared with the blank control, 10 nmol/L aldosterone and 50nmol/L aldosterone, elastin in fibroblasts(red fluorescent substances) is increase gradually. 1μmol/L has no obvious change.5. Immunofluorescence method and Western Blot method to detect the mechanism of aldosterone to adjust elastin: Compared with 50nmol/L aldosterone, 50nmol/L +spironolactone aldosterone 2 um/ml, 50 nmol/L aldosterone+100 ng/ml IGF-I, the elastin in fibroblasts is increase(red fluorescence material), 50nmol/L aldosterone+1000ng/ml IGF-I is reduced. Compared with blank control, 100ng/ml IGF-I+10umol/L tyrosine kinase inhibitors, the elastin is increase(red fluorescence material);in 50nmol/L aldosterone+tyrosine kinase inhibitor, 50nmol/L aldosterone+phosphatide acyl alcohols inhibitor, 100ng/ml IGF-I+phosphatide acyl alcohols inhibitor group, there is no obvious change in the elastin.6. Western Blot method to detect the mechanism of aldosterone to adjust elastin:Screen out the best concentration :aldosterone 50 nmol/L, IGF- 100 ng/ml. Cultured in spironolactone2um/ml+ aldosterone50nmol/L, aldosterone50nmol/L+100 ng/ml IGF-I, 100ng/ml IGF-I+PP210umol/L, the elastin increased; 50nmol/L aldosterone +1000ng/ ml IGF-I, aldosterone 50 nmol/L + 1 umol/L tyrosine kinase inhibitors,aldosterone 50 nmol/L + phosphatide acyl alcohols inhibitor 1 umol/L, 100ng/ml IGF-I+ phosphatide acyl alcohols inhibitor 1 umol/L, there is no obvious change in the elastin. Results consistent with immunohistochemistry.Conclusion:1.From 50ng/ml IGF-I-200ng/ml, skin fibroblast proliferation of IGF-I is dose-depend,in100ng/ml-200 ng/ml concentration the promoting proliferation effect is remarkable.High concentration of IGF- 1(500 ng/ml) has inhibitory effect on cell proliferation.2. In 50nmol/L aldosterone concentration, it has the most significant effect to promoting cell proliferation, high concentration of aldosterone has inhibitory effect on cell proliferation.3. From 50ng/ml IGF-I-200ng/ml, elastin modulate of IGF-Iis dose-depend,in100ng/ml-200 ng/ml concentration the up-modulation effect is remarkable. High concentration of IGF-I can inhibit the action of the fibroblasts to produce elastin.4. Aldosterone increasing elastic protein synthesis is dose dependent and aldosterone receptor inhibitor spironolactone can’t eliminate the effect, its function is through the aldosterone receptor independent way.5.Aldosterone can obviously increase the IGF-I receptor tyrosine phosphorylation,and PP2 can suppress the elastin synthesis induced by aldosterone. Thus infer that to promote the synthesis of elastin aldosterone is in a aldosterone receptor independent pathways but IGF-I receptor signaling pathways.6.Wortmain can eliminate the influence of aldosterone and IGF-I, aldosterone’s effection is not depend aldosterone receptor, but IGF-I receptor, influence the downstream PI3 kinase/AKT signaling system.Due to the steroid hormone is easily penetrate the skin, when topical steroids penetrate into skin, it can stimulate elastin synthesis to postpone skin aging, this provides ua a new way. |
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