The Research On The Development Related Genes Of Sweat Glands And Combined Effect Of Them And BM-MSCs On The Repair And Regeneration Of Sweat Glands

Objective: (1) To investigate the character of the formation and the antigens expression of the fetal skin in different developmental stage, as well as the dynamic changes of gene expression in skin during the development of sweat glands, then find out the principal of sweat glands development and the related genes which play key roles in it. (2) To investigate the effect of sweat glands development-related genes and bone marrow stem cells on the damaged sweat glands, and discuss the feasibility of the co-application of them to repair and regenerate sweat glands.Methods: (1) The skin histological specimens of the aborted fetus (fetal age: 10-36 weeks) were observed by H&E stain and immunohistochemical method. The sweat glands development-related genes and its expression products were detected by RT-PCR and western blotting method. (2) The DNA fragments of human sweat glands development-related gene were cloned from the human fetal fibroblasts, and then the eukaryotic plasmids containing those genes were constructed. The wounds of the scald nude mice were treated by the local injection of naked DNA or transplantation of BM-MSCs transfected with sweat glands development-related genes in vitro, and the gene expression and product level of target genes in the transfected cells was detected by RT-PCR and western blotting, H&E stain and immunohistochemical method were used to observe the repair and regeneration of sweat glands to evaluate the curative effect, and the iodostarch sweat test was performed to evaluate the sweat glands function.Results: (1) The skin structures of fetus at different development stage have organized succession, and there are three stages during sweat glands development: fetal age from 12 to 16 weeks is the first stage, the second stage is fetal age 17-24 weeks, and the third stage is 25w to birth. (2)Immunohistochemistry test: beta1integrin, CK7/8/18 are negative in the sweat glands primodium while CEA and CK14/19 are positive. At the second stage, CK7 is positive and shift into deep portion. CEA and CK19 are positve in sweat glands stablely while CK14 is positive only in induct portion.CK8/18 appear in secretory portion at the end of development period. (3)The result of PCR and western blotting show that the transcript and expression of EDA and FGF10 are up-regulated to peak during the first stage then sustaining in the second stage then down-regulated at last stage. FGF7 look like has this kind of organized change, but there is no significant deviation between three stage after statistical analysis. (4)The DNA fragments encoding FGF10 and EDA-A1 protein were obtained from fetal dermal fibroblasts, and inserted into eukaryotic vector. Identitified with the method of restriction map analysis and sequencing showed that the DNA sequence of insert fragments are accord with the data supplied by Gene Bank. (4) The results of PCR, western blotting and ICC all showed that the transfected BM-MSCs transcript and expression the target gene, and sustain at a high level to the 9d at least. Pathological findings show that the damaged sweat glands in the paw of scald nude mice had been repaired perfectly 2w after naked eukaryotic plasmids DNA injection or transfected BM-MSCs transplantation. A number of BrdU and CK8 amphophilic cells was found in the repaired sweat glands. The sweat test result showed distinguished positive reaction.Conclusions: (1) The development of sweat glands in fetal period has a distinguished character of three stages, more over, the transcription and expression of FGF10 and EDA genes has the same organized alternation in accord with sweat glands development periods, there is correlation between them. (2) Local injection of Naked EDA-A1 and FGF10 DNA local multipoint injected can promot the repair and regeneration of damaged sweat glands.Gene modified BM-MSCs could have the same curative effect, more over that, they can transdifferentiate into the cells that show the same phenotype with sweat gland cells. The combination of gene therapy and stem cell therapy bring us new hope in the skin functional reconstruction of burns.