| Part 1 The preparation of acne model in SD ratObjective: To prepare a ance inflammation in SD rat by hypodermic injection with P.acne suspension.Methods: The 21 healthy female rats(6-8weeks,250±30kg)were assigned randomly to blank control group(group BC),acne group(group AC,n=7)and saline control group(group NS,n=7).Group BC does not any treatment,other grous respectively by hypodermic injection with 50?L P.ance bacterial suspention and 50?L saline on day 1.The changes of auricula's appearance of three groups were observed after 24 hours,and the swelling of auricula's tissue in group AC were selected and inoculated on petri dish,than cultured 48 hours in anaerobic jar,which to prove the infection of P.acne.Rats were sacrificed afther day 7 and histopathological observation were performed in local skin.,then auricula tissure TNF-? ? MMP-2 m RNA expression levels were detected by real-time fluprescen-ce quantitative PCR(RT-PCR).Results: Appearance: group BC of rat has no change.Red and swelling of bilateral auricula were seen in group AC after 24 hours of post-injection and it not dispear after one week,red papules has appear at the same time.Swelling of bilateral auricula were seen in group NS on 24 hours after post-injection and dispear complately after one week.Microbiological texting:The growth of P.acne can be seen under microscope in group AC,which proves infection of P.acnes.Histopathology: The BC group showed epidermis,dermis and subcutaneous tissue,and the structure was clear.No obvious inflammatory cell infiltration was observed.In the AC group,the subcutaneous tissue structure disappeared,the inflammatory cells were diffuse,patially formed into abscess,thefollicular orifice has expanded and the sebaceous glands has disappeared.The structure of the skin of NS group was clear,and no obvious inflammatory cell infiltration was observed Gene.dection:TNF-?and mmp-2 were detected in auricle's tissue of three groups.There was no signification difference between the group NS and the group BC on expression level of TNF-?and mmp-2(P>0.05).Compared with group NS,the expression level of TNF-?was statistically higher in group AC(P<0.01),the expression level of mmp-2 was higher on mmp-2 in group AC(P<0.05).Conclusion:The way of hypodermic injection in SD rat with P.acne suspension can build the inflammation model of acne successfully.Part 2 Effects of culture supernate from human amniotic mesenchymal stem cell(h AMSCs)on expression of TNF-?,MMP-2 gene in a rat model of acneObjective:To clarify the existence of anti-inflammatory cytokines IL-10 in the supernate of human amniotic mesenchymal stem cells(h AMSCs).To observe the effect of the supernatant on the gene expression level of TNF-? and MMP-2 in rat inflammation model and preliminary disscuss the possible anti-inflammatory action of h AMSCs.Methods:The ABC-Elisa method was used to determine the concentration of anti-inflammatory cytokines IL-10 in h AMSCs culture.The 49(6-8weeks,250±30kg)healthy female SD rats by the method of random Numbers are divided into blank group and group(BC),normal saline group(NS group),acne group(AC),untreatment group of acne model(P group)and the treatment group by injected of h AMSCs culture supernatant(H1),the treatment group by smeared of h AMSCs culture supernatant smear(H2),the treatment group by smeared of vitamin A acid erythromycin gel(YW),a total of 7 groups,each group of seven.The BC group was not treated,and the AC and NS groups processed same as the part I.P group: hypodermic injection with 50?L P.ance bacterial suspention on bilateral central auricle on day 1 and after one week acne inflammation model in rats were build successfully,later will not be any processing,observed after 4 weeks to be put to death.H1 group: After one week of modeling,h AMSCs suspention of 25 ?L were local subcutaneous injeced to rats and 2 times one week;H2group: h AMSCs suspention were local smeared to rats after one week of construction of model and 2 times one week,YW group was treated with erythromycin hydrochloride gel.H1,H2 and YW group were all killed after 4 weeks of treatment.The expression levels of TNF-? and mmp-2 in the auricle were determined by RT-pcr.Results:The differences in each indicator were statistically significant(P< 0.01).The levels of TNF-? and mmp-2 in the P group were higher than those in the AC group,and the differences in the indexes were not statistically significant(P>0.05).The expression level of TNF-? and mmp-2 in H2 group was lower than that in H2 group,and there was no statistically significant difference in each indicator(P > 0.05).The expression levels of TNF-? and mmp-2 were lower in the YW group than in the H1 group,and the differences in the indexes were not statistically significant(P>0.05).Conclusion:1.With the help of h AMSCs,it can reduce the inflammation of the auricle of acne rats.The mechanism may be to inhibit the expression of TNF-? and mmp-2 by IL-10 and TIMPs respectively,thereby improving the inflammatory skin lesions of acne.2.There was no significant difference in the inflammatory efficacy between local injection and smear on h AMSCs. |
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