| The research was carried out to study the localization and migration of skin stem cells, to construct suppression subtractive hybridization(SSH) libraries of secondary follicles in anagen and to search some candidate genes involved in cashmere development in Inner Mongolia cashmere goat with cDNA micrarray. The information generated in this study can reveal behavior characteristic and orientation distribution of follicle stem cells, especially biological characteristics of secondary follicle stem cells, also can privide theoretical basis and method on buliding mechanism model of dormancy, migration, proliferation, differentiation, decision processes of follicle stem cells.The healthy cashmere goats were divided into two groups: the test group labeled BrdU and the control group unlabeled BrdU.BrdU was injected intravenously for a total of two injections. According to skin stem cells characteristics of the slow cycle, skin samples were took from the back skin of the goats,fixed in 4% paraformaldehyde, embedded with paraffin and sectioned,detected by immunohistochemistry.BrdU could be detected only 10 weeks in Inner Mongolia cashmere goats and could be used for large animals in vivo short-term markers. Intraperitoneal injection 8 weeks, positive signals located mainly in hair bulb of primary and sencondary follicle,also in hair shaft. Intraperitoneal injection 8-10 weeks, positive signals obvirously decreased,only could be found in outer root sheath. After 10 weeks, almost no positive signals distribution. The back skin samples labeled BrdU for 8 and 10 weeks were incubated in 0.2% dispaseII at 4℃for 3h,isolated primary and secondary follicles under a dissecting microscope,then cultured label-retaining cells (LRC) of hair follicles(hair bulb and follicle midpiece) in vitro with segmentation culture method and collagen IV adhesion culture method respectively.The LRCs showed a large nuclear/cytoplasmic ratio and small size,and clonogenicity with the morphological characteristics of skin stem cells.To evaluate the BrdU LRCs, P63, a possible epidermal stem cell specific marker, was used. Immunohistochemical results idicated BrdU LRC were positive for P63. These results showed that BrdU LRCs were epidermal stem cells.The stem cells possiblly located in not only hair matrix and outer root sheath of primary,secondary follicle,but also the epidermal basal layer.To further clarify the startup–dormancy control genes of stem cells during secondary follicle reconstruction periodically,forward-SSH library was performed with cDNA from secondary follicle in anagen as the"tester"and cDNA from secondary follicle in telogen as the"driver". While reverse-SSH library was performed with cDNA from secondary follicle in telogen as the"tester"and cDNA from secondary follicle in anagen as the"driver".The gene fragments were sequenced, and analyzed by bioinformatics. Two SSH libraries had been constructed successfully. Twenty positive clones were amplificated by using nested PCR primer 1 and 2R, the size of inserts was 250-1000bp.309 genes and 344 genes were obtained respectively by DNA sequencing 750 clones picked randomly from two SSH libraries. Their average length were 693bp and 596bp.After nucleotide blast homological analysis,224 matched to known genes, seventy-six matched only to other ESTs in dbEST, and the remaining nine showed no match to any ESTs or known genes in forward-SSH library. While 300 matched to known genes, thirty-eight matched only to other ESTs in dbEST, and the remaining six showed no match to any ESTs or known genes in reverse-SSH library. 126 redundancy ESTs were registered in the GenBank,their accession numbers were 64970025~ 64970150. These were categorized into seven categories on the basis of gene function. They were divided into cell division(15,13), cell signaling/communication(44, 49),cell structure/motility(30,52),cell organism defense(13,21),metabolism(24,46),gene/protein expression(33,37) and unclassed (65,82).On this basis, the exchange of two fluorescent hybridizations of the cDNA chip were used to screen specific differentially expressed genes. A total of six differentially expressed genes were found in anagen including three up-regulated genes and three down-regulated genes.While ten differentially expressed genes were found in telogen including nine up-regulated genes and one down-regulated gene.In conclusion,it is the first time that stem cells are located in primary, secondary follicles and epidermis of cashmere goats by BrdU labeling method;it is the first time that differentially expressed genes in secondary follicles of anagen and telogen are isolated by SSH,combined with the exchange of fluorescent cDNA chip hybridization to screen differentially expressed genes.The results are useful for elucidating the possible molecular mechanism on keeping dormancy and starting differentiation by stem cells, but it is also necessary to elucidate biological functions of differentially expressed genes,especially genes with unknown functions.
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