| Axillary Osmidrosis (AO) is a common inherited disease characterized by its symptomatic unpleasant odor from axilla. Although itself cause no direct damage to the body health, but its unpleasant emissions may bring inferiority and psychological burden, cause trouble to the interpersonal communication and daily life. As a result, AO patients often continue to seek treatment, and mostly require the complete elimination of odor. The current treatment of body odor can be classified into conservative methods and surgical treatments. The conservative approach is temporary effective and generally difficult to achieve any comprehensive cure. Surgery is still the most radical way in dealing with axillary osmidrosis. However, the pain it caused is often unacceptable besides the relative complications. Therefore, constant research is still needed in order to seek a long-lasting effective method with less pain and fewer complications, which will be well-recognized.There are bottlenecks of bromhidrosis in clinical diagnosis and treatment that should be noticed:Firstly, the exact pathogenesis of bromhidrosis syndrome is still unclear. It is generally considered that axillary osmidrosis is related to the abnormal secretions of the apocrine sweat gland. Its secretion was carried to the skin surface and decomposited by bacteria, generating smelly unsaturated fatty acids. But the specific molecular mechanism is still unclear and the typical autosomal dominant hereditary is unexplainable. Therefore, further study is required to explore the genetic basis of bromhidrosis, which may delight new approaches for bromhidrosis treatment.Secondly, the current clinical diagnosis of bromhidrosis is primarily based on the patient’s subjective feeling. Sometimes the presence of odor is difficult to determine especially for patients with less severe odor. And no uniform evaluation for treatments made the scientific comparison study with different methods invalid. Therefore, further study of the pathogenesis of bromhidrosis is still needed, which would helpful to find reliable indicators and related therapeutic drug.Objectives:1. Correlation Analysis between single nucleotide polymorphism of ABCC11gene and the pathogenesis of AO. As well as the relationship between the different genotypes and the severity of bromhidrosis;2. Study the axillary apocrine sweat glands protein expression level of ABCC11between AO patients and normal people; Explore the role of ABCC11single nucleotide polymorphisms in the pathogenesis of bromhidrosis mechanisms.3. Apocrine sweat gland cells culture and vitro models establishment.The growth characteristics of different genotypes were observed and the proliferation was analyzed.4. Analysis of the protein levels of ApoD and ABCC11/MRP8in human apocrine sweat glands between the different genotypes. Correlation analysis of the two proteins. Provide for a fuller understanding of the mechanism, and seek new objective indicators for clinical diagnosis and treatment evaluation of bromhidrosis.5. Correlation analysis between the different genotypes and the severity of bromhidrosis as well as its relationship with efficacy and prognosis. The new surgical therapeutic method was applicated combines Xiaozhiling perfusion with vacuum stripping. Follow-up and protein was tested to evaluate the efficiency, providing a theoretical basis for clinical diagnosis and evaluation of surgical treatment improvement.Methods:1. Take peripheral blood from both the normal adult and AO patients to extract DNA. Polymerase chain reaction-high resolution melting curve (PCR-HRM) technology, combined with sequencing was used to detect the genotype of ABCC11rs17822931. Analysis the distribution of different alleles in AO patients and normal population. Discuss its correlation with the bromhidrosis onset in Chinese Han population 2. Cultured the apocrine sweat glands in vitro. Observe and analysis the growth characteristics, made the growth curve and analysis the difference of proliferation between different genotypes.3. Randomly collected armpit full-thickness skin samples from the AO Subjects with different genotype. Observed the morphology of apocrine sweat glands in tissue sections. Immunohistochemistry, real-time PCR and western blot assay were performed to measure the mRNA and protein expression of ABCC11/MRP8and ApoD protein. SPSS13.00software and rank sum test was used for statistical analysis, the difference was considered statistically significant when P <0.05.Analysis of the correlation between the two protein and ABCC11SNP, further clarify the mechanism of the occurrence of the bromhidrosis.4. The general information and relevant clinical data of AO patients were collected in cosmetic surgery outpatient of Xiangya Hospital. The vacuum stripping combine with Xiaozhiling perfusion treatment was applied and followed-up. Records were done including the efficiency and complications, the results were compared with other reported methods; ApoD and ABCC11/MRP8protein expression was analyzed by real-time-PCR and western-blot to help evaluate the efficacy, which may help seek objective indicators. The advantages and disadvantages of the technique were discussed, as well as the relation of SNP and efficacy. Provide a theoretical basis for seeking further improve of surgical technique and evaluation methods.Results:1. There are three genotypes of ABCC11gene among AO patients: G/G, A/G and A/A and the frequencies were9.62%,88.46%and1.92%seperately, while all of the normal people got the AA genotype. The G allele accounted for98.08%of the AO patients, indicating the important role of G allele in the bromhidrosis occurrence. There are statistically differences of severity between different genotypes.2.58full-thickness skin samples from axillary were collected from8normal people and50AO patients (5GG and45GA). Immunohistochemical staining results showed that the ABCC11/MRP8 and ApoD are both specifically expressed in human axillary apocrine sweat glands and their expression levels are different according to both severity and genotypes. The rank test analysis shows ABCC11/MRP8positive expression rates was significantly higher in AO patients with GG or GA genotypes than the normal control group with the AA genotype; But no statistic difference was found between the GG and GA group.3. Skin tissue sections observed by light microscope showed:the apocrine sweat glands in AO patients were significantly richer than the control group, as well as the proportion of active sweat glands. No statistically significant difference was found between the GG and GA type.4. Apocrine sweat gland cell culture observation suggests that cells with different genotypes share the common characteristics:The incubation period is1-2days; cell proliferation activate into the logarithmic growth phase in the3rd day; cells were ’cobblestone’-like, larger with better transparency.The logarithmic growth phase lasts about10-12days, peak on the7th day and proliferation slowed down after about12days, when the apocrine sweat gland cell subject to different degrees of contact inhibition. By comparing and analyzing growth curve, we found the human apocrine sweat gland cells with GG and GA genotype was statistically significant in number than the AA type on the6th,9thand12th days.5. Immunohistochemistry, Western blotting and PCR results showed that there was significant difference in the expression of ABCC11and ApoD between AO patients and healthy controls.There is a correlation between genotype and its mRNA and protein levels. ABCC11/MRP8protein expression levels were positively correlated with the ApoD protein.6. From March2009to March2013,171patients in cosmetic surgery outpatient of Xiangya Hospital were collected and followed-up (among them severe in63, moderate in81and mild in27cases). The patients were treated with vacuum stripping combined with Xiaozhiling perfusion to eliminate the bromhidrosis. The patients were followed up for6months to26months and the result showed that the treatment was effective in26side(7.6%) and316sides were healed (92.4%);The total efficiency is100%;The satisfaction survey showed that157patients were very satisfied (91.8%),13patients expressed general satisfaction (7.6%), the total satisfaction percent is up to99.4%. Comparison with the existing surgical treatments shows the new technique is relatively high in satisfaction rate with fewer incidences of complications.7. Western blotting and PCR showed the expression of ApoD and ABCC11/MRP8in axillary skin is significantly reduced after surgery. The rank test analysis showed that the cure rate in mild to moderate patients is relatively higher than the severe group;Conclusion1.ABCC11gene single nucleotide polymorphism loci rs17822931are important genetic factors of bromhidrosis, the presence of G allele is the genetic basis of bromhidrosis occurrence; It is specifically expressed in axillary apocrine secretary cells, and the expression levels is closely related to different genotypes, which seems also affect the severity of body odor. As the result, the detection of SNP genotype could be helpful for clinical diagnosis and judging odor extent.2.The MRP8protein expression and mRNA levels in apocrine glands differ in different genotypes. We speculate that the gene may regulate secretion activity by the transcription of protein which is the transporter of the precursors of variety lipid.3.Expression of ABCC11/MRP8and ApoD was closely related to differential severity of odors, and both proteins decreased after the elimination surgery. Although the expression levels of both proteins were weakly positive correlated, they may serve as indicator of the AO diagnosis and treatment evaluation.4.The SNP of ABCC11may play a regulatory role in the morphology and metabolism of apocrine sweat glands and the further study is needed to explore the exact mechanism.Figure25, Table23,... |
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