| BackgroundHair follicles were appendix organs of skin which controls the hair growth and experiences the stage of anagen, catagen and telogen repeatedly during the whole life of mammals. By periodic growth, hair follicles revealed adequate self-constructing ability. What’s more, we found that different species have different follicle growth cycle and rhythm. For example, the growth cycle rhythm of hair follicles of different part of people and guinea pig were not the same. However, the growth rhythm of most vertebrates was synchronized. Then where was the biological clock which controls the growth cycle rhythm and what were the regulator factors. These problems had to be elucidated.The research focus of hair follicle growth cycle had been about all kinds of growth factors or cytokines all the time. After studying the three different cycles comparatively, such as environmental stimulation, difference of cytokine expression and cell apoptosis, the possible regulatory effect was put forward by scholars and all these theoretical researches need to be further confirmed. At present, we had identified the soluble factors which participate in the regulation of hair follicle growth cycle, such as, insulin-like growth factor, an important factor which was expressed during the anagen, transforming growth factor which participates in the shift of anagen to the catagen and bone morphogenetic proteins which participate in the shift of tologen to anagen. The mechanism of these factors was not that clear. On the other hand, relevant scholars had done a lot of research on the transformation mechanism of CuiMao to bristles, part of hair follicle exiting the growth cycle, the roles of macrophage and mastocyte in the growth cycle, the regulation of central nervous system on hair follicle growth cycle and the influence of hair follicle on skin biology. Certain progress had been achieved.The growth of hair follicle was a very complicated process in which multiple factors regulating and controlling each other. The development and maturation of hair follicle needed the interaction between cells which was promoted by a series of signal transmission produced by the interstitial cells and epithelial matrix components. In the process of hair follicle cycle, some cytokines, active protein, enzymes, hormones which exist in the extracellular matrix secreted by the dermal papilla promoted or inhibited the growth of hair follicle. Depending on the extracellular matrix, all kinds of cells constructed the basic structure. So a variety of components of ECM played a key role in cell growth and proliferation.Matrix metallproteinases was a kind of polygenic and endogenous Zn dependent enzyme family which was constituted by a lot of neutral protease with similar structure and function. It could degrade all components of extracellular matrix except polysaccharide and was produced by a variety of cells, such as mononuclear cells, vascular smooth muscle cells and endothelial cells. The degradation of MMPs lead to the reshaping of the extracellular matrix, especially separating the cortex of the hair follicle (such as outer root sheath) and the basement membrane of interstitial layer (such as dermal sheath and dermal papilla), which was necessary for the growth and development of the hair follicle. The basement membrane remolding was indispensable to the signal transfer between epithelium and stromal element during the hair follicle growth cycle and the extension and invasion of subcutaneous tissue during the last period of anagen.Tissue inbitors of metalloproteinases was the endogenous specific inhibitors of MMPs and there were four members in its family, TIMP-1, TIMP-2, TIMP-3 and TIMP-4. The degradation and accurate positioning of ECM were involved in normal physiological process, such as growth and development, tissue repair, etc and this was the remodeling of ECM. It may be result in some disease in the process, for example cancer, arthritis and cardiovascular disease. Although it was a kind of repair behavior, it was often the specific and destructive expression. Matrix metalloproteinases family was the main enzymes in various types of known proteolytic enzymes at present. Research showed that MMPs played an important role in regulating the bioactive molecules on the surface of cells and the environment of cells and ultimately affected the cell behavior. The enzyme with specific function had different functional performance in normal or pathological condition. Inhibitors were the main research direction in clearing the role of MMP.A further exploration was made in the rules of dynamic change and mechanism of MMp-2, MMP-9, TIMP-1 and TIMP-2 in the hair cycle. The hair growth cycle of C57BL/6 female mice which are 3 to 12 weeks were taken as the standard in the study. Through systematic test combined with animal experiments, we analyzed the functional rules and influence of Matrix metalliproteinases and inhibitors on hair cycle and explored the mechanism preliminarily, which provided a new research strategy for understanding the dispelling reason of the extracellular matrix which was excreted by dermal papilla and its effect on hair follicle cycle. The results of the study had great significance in clarifying the regulation mechanism of hair follicle growth cycle, studying hair follicle biology, hair formation and reconstruction and developing effective method for the treatment of hair loss eventually.Objective1、To explore the dynamic change regularity of MMp-2, MMP-9, TIMP-1 and TIMP-2 during the hair cycle.2、To explore the influence of Tissue inbitors metalloproteinases, SB-3CT on hair growth.3、To explore the possible mechanism of inhibitors on the process of hair growth.Methods1、The dynamic analysis of MMp-2, MMP-9, TIMP-1 and TIMP-2 during the hair cycle.1.1 Gelatinases Spectrum50mg skin tissue of each cycle from the C57BL/6 female mice which were 3 to 12 weeks was taken. Then 500ul lysis solution was added for grinding organization and later 10% homogenate solution was prepared. After centrifuging it for 10 minutes (14000rpm,4℃), took the supernatant. Mixed the 100ul solution with the 25ul 5× loading buffer and separating gel and stacking gel were made up. The samples were 5ul in each hole (the amount was determined according to the expression intensity and the protein concentration). Later SDS-PAGE electrophoresis of 100v were taken for 1.5 hours at 4℃ (put on ice around when electrophoresis was going on, which is beneficial to make the stripe running straight). After electrophoresis, gel was put in the eluent for oscillation elution twice and 40 minutes every time. Then it was rinsed by wash water twice and 20 minutes each time. Later, we put the gel in incubation at 37℃ for 42 hours. After incubation, they were put in dyeing liquid (0.05% Coomassic brilliant blue,30% methanol,10% acetic acid) for 3 hours and then in the destaining liquid for 3 hours. It demonstrated that MMP-2 (72KD) and MMP-9(92KD) were located in the bright belt on blue background.1.2 Immunohistochemical1.2.1 Conventional HE:Tissue specimens of each cycle were fixed with formalin fixed fluid for 6 hours and then they were dehydrated and embedded. Later, routine biopsy and roast were carried out and finally they were dyed and sealed by neutral gum.1.2.2 Immunohistochemical:After the paraffin section was roasted and dewaxed to water, we washed it with distilled water for 3 minutes and then high-pressure repairment was carried out by citrate buffer for 3 minutes. Cooling to room temperature naturally, it was incubated with 3% H2O2 inhibition of endogenous peroxidase for 10 minutes at room temperature. Washed it with PBS and we diluted the Anti-TIMP1、Anti-TIMP2、Anti-MMP9 and Anti-MMP2 respectively with antibody diluents at the proportion of 1:200. Then added the antibody on the tissue section, incubated it at 37℃ for 30 minutes with Goat anti rabbit IgG antibody-HRP polymer. Later, DAB chromogenic drops were added, it was restained by hematoxylin, differentized by 0.1% HCL, rinsed by tap water, blued, dehydrated by gradient alcohol, make it transparent by xylene, sealed by neutral gum and observation was carried out after drying.1.2.3 ELISA:All the standards and samples were detected by ELISA to single hole plate after the kit and samples were balanced at room temperature (18-25℃). Added 100uL prepared standards and samples in the ELISA plate which has been coated by antibody, and then sealed the slats with microplate sealers. The plates of transformations were incubated overnight at 4℃.Added the prepared 1x lotion to the washer,clean the slats 4 times, added 300 μL wash and 100 μL well formulated detection antibody (biotinylated antibody) in each hole and incubated at room temperature for 1h. Cleaned the slats and added 100 uL prepared HRP-streptavidin-biotin to the holes and incubated at room temperature for 45 min.100 μL TMB Color-substrate solution was added after the plates were cleaned again, incubated at room temperature away from light for 30 min.Finally, added 50 μL of Stop Solution to each hole and read the microplate reader 450 nm at once.1.2.4 qRT-PCR:Total RNA purity and integrity were detected after the tissue was grounded to powders by liquid nitrogen and the total RNA was extracted and the genome was knocked out. Detected reverse transcription when the total RNA was extracted proved relatively intact. Primer test was on the first before quantitative PCR detection. When orienting computer contents of the samples were determined, arranged the samples regularly on board, and then make a formal experiment. The total system was loaded to calculate according to the specific configuration of the experimental system to the eight-link-tubes. At last, use the quantitative PCR instrument, each sample was repeated three times to get the final result.2. Animal experimentsFirst, hairs on the back of 8 weeks old C57BL/6 female rats was cut short, in which to unhair an area about 8 cm2 by depilatory creams with cotton ball. And the unhairing parts on the back of rats were respectively coated with different concentrations of SB-3CT matrix metalloproteinases inhibitor, which sticked to the schedule 2 times a day and five days a week for a total of 30 days. During the 30 days’ experimental period, photos of rat’s back were taken once a day. Hair regeneration of rats was evaluated by observing images to calculate hair regeneration rate. After euthanasia regularly carried out to each rat, the back skin of rats was excised for HE staining, simultaneously went through the immunohistochemistry and ELISA tests. Image analysis was done by Image J software to calculate the number and length of hair follicles at different periods. Finally, all data and image information was needed to be processed.3. The role of VEGF-IGF-1-TGF-β1 signaling pathway in the hair regeneration and its relationship with MMP3.1 Fluorescent quantitative PCR3.1.1 Total RNA extraction of skin tissueLiquid nitrogen was added in the mortar in which organization was cut into small pieces and then to be ground into powder.50 to 100mg powder was taken out by the spoon precooled with liquid nitrogen, and then which was put into the EP tube containing 1ml Trizol fluid (the total volume of organization and powder cannot be more than 10% of the volume of Trizol) to be mixed evenly. After placed at room temperature for 5 minutes, the EP tube was then to be added 200ul chloroform and covered tightly to do violent oscillation for 15 seconds. Went through 12000RPM centrifuge for 10 minutes, the upper layer water within the EP tube was taken (Forbidden taking liquid mixed with precipitation in the middle and lower layer, otherwise to do the centrifugal separation again) and added in 500ul isopropyl alcohol and to be turned upside down moderately. The new EP tube was placed at room temperature for 10 minutes and then do another 12000RPM centrifuge for 10 minutes. Carefully to discard supernatant liquor, about lml 75% ethanol was add into the new EP tube with vortex blending and then to accept 12000RPM centrifuge under 4℃ for 5 minutes, which should be repeat again. Discarded the supernatant liquor (Remove residual liquid as much as possible), the new EP tube was placed at room temperature or vacuum drying for 5-10 minutes (Don’t dry too much, which will reduce the solubility of RNA). RNA was dissolved with water processed by 30ul DEPC, water bath between 55℃～60℃ for 10 minutes can be done when necessary. Subsequently, RNA can be proceed with the mRNA isolation or kept in 70% ethanol under-70℃.3.1.2 Reverse transcription and PCR reaction of RNA sampleslug total RNA was taken as template for reverse transcription, under 37℃ for 60 minutes and at 98℃ for 10 minutes, which in order to compose cDNA first chain. Subsequently, cDNA chain as a template was to proceed with PCR experiments.3.2 ELISA3.2.1 Extraction of total protein40mg solid tissue was taken to be cut into pieces and then added 0.5ml lysate solution (Add PMSF, cocktail and phosphatase inhibitor, etc, in proportion before), which was put into a glass homogenizer to and shaken up and down to reach the homogenate. After all samples are ground as much as possible, the protein solution was transferred into 1.5ml EP tube, which was placed on the ice for full 30 minutes’ pyrolysis and then undergone 14000 RPM centrifuge for 10 minutes under 4℃ to get the supernatant liquor that to be called the total protein solution.3.2.2 ELISA detectionThe standard hole and sample hole were set up, respectively. Every hole was added 100ul standard panel or samples under test, followed with shaking gently to be blending and overlying sheet for 1.5 hours under temperature of 37℃. After discarded liquid and washed for five times, every hole was added with 100ul biotinylated antibody working liquid and then covered with a new sheet for 1 hour incubation under 37℃. After discarded liquid inside the hole, washed for five times and spin-dry, each hole was added with 100ul enzyme combination working liquid and then covered a new sheet for 30 minutes incubation under 37℃. Still after discarded liquid inside the hole, washed for five times and spin-dry, each hole was added with 100ul chromogenic substrate solution (TMB) developing for 15 minutes under 37℃ avoiding light. At last, each hole was added with 100ul termination solution to terminate the reaction. Within 5 minutes after the termination of reaction, ELIASA was used to measure the optical density (OD value) of each hole in the 450nm wavelength in order.4. Statistical analysisThe data were analyzed with SPSS 17.0 statistical software (SPSS, Chicago, IL, USA). These measurement data were expressed as mean±SD. Multiple sets of measurement data were analyzed with analysis of variance. Homogeneity test of variances was analyzed with One-way ANOVA and LSD test was used for comparison between two groups. Heterogeneity of variance was analyzed with Welch’s test and Dunnett’s T3 test was used for comparison between two groups. The comparison between two groups at the same time point was used independent samples t test. A P<0.05 level was used to determine a significant difference.Results1. In three weeks to 12 weeks of the back hair cycle in the C57BL/6 female mice, the enzyme activity of MMP-2 and MMP-9 in each cycle exist. While, the enzyme activity of MMP-2 and MMP-9 in the hair cycle remain in a state of fluctuation. In the period of the first hair growth period, the enzyme activity of MMP-2 has experienced the tendency of decreasing firstly and then increasing with reaching the lowest value on the activity of enzyme; And in the period of the second a longer rest period, the enzyme activity present the trend of rise first then fall with reaching the highest value on the enzyme activity. What is more, during the first growing stage to the regression stage, the enzyme activity of MMP-2 and MMP-9 has increased gradually; during the regression stage to the resting stage, the enzyme activity of MMP-2 and MMP-9 has decreased gradually; during the resting stage to the growing stage, the enzyme activity has declined rapidly. Comparison with the curve of the MMP-9 enzyme activity, we reached the similar conclusion. The typical organization image by HE staining has also be obtained.The fluctuations of expression in the enzyme activity of MMP 2 and MMP-9 are similar with the level of the fluctuations in the mRNA and protein measured by qRT PCR and ELIS A. It can prove that the expression of both mRNA and protein in the matrix metalloproteinase MMP-2 and MMP-9 are similar and detectable in the hair cycle. Their level of value is at a minimum in the 5th and 7th week while at a maximum in the 10th week of the hair cycle.In the next step, we detect whether the expression of TIMP 1 and TIMP-2 is different from their corresponding activator as MMP-9 and MMP-2. Finally we found that the fluctuations level on the mRNA and protein of TIMPs at a maximum in the corresponding 5th and 7th week but at a minimum in 10th week. The change of the fluctuation trend in the hair cycle is opposite with the trend of MMPs. All of these have showed that the MMPs and TIMPs have the relationship of exciting agent and the inhibitor each other.In order to determine the expression of the location in the MMP-2, MMP-9, TIMP-1, and TIMP-2 at each stage in the hair cycle, we display them through the image of immunohistochemical. The image expresses negative or weakly positive for MMP-2, MMP-9 and TIMP-1 in the hair follicles while TIMP-2 has been expressed as strong positive in the hair follicle. They are all visible for the expression of MMP-2, MMP-9 and TIMP-1, TIMP-2 in the skin, sebaceous glands and subcutaneous tissue layers.2. During the animal experiments, SB-3ct as the effectively selective gelatin inhibitor has been acted on mouse back skin after shaving which is the experimental group. Compared with the blank control group, the hair growth of the mice in the experimental group has an overall slower speed and the hair length and quantity per unit area are low in comparison with control groups as a whole. The typical image of HE staining has also proved this point. Through the immunohistochemical staining, it showed that the expression of MMP-2, MMP-9 in hair follicles were negative or weakly positive which has proved the front experimental results once again.3. Through the tissues sampling and trimming for the mouse back skin after shaving, SB-3 c group and blank group have been tested respectively for the level of mRNA and protein in VEGF, IGF 1, TGF-beta. They found that at various stages of the hair cycle as the growing period, the regression phase and the regression stage, the value of VEGF, IGF 1 in the experimental group are decreased obviously compared with the blank control group while the values of TGF-beta in the experimental group are increased compared with the blank control group.Conclusions1. This study has researched the dynamic change rule of hair cycle in mice with the MMP-2, MMP-9, TIMP-1 and TIMP-2, which stated that, during the growing period, because a lot of matrix metalloproteinase are needed to digest extracellular matrix for releasing one of the key cytokines or hormones, so the resting stage need a long time to accumulate enough amount of matrix metalloproteinase with a significant decline in the early growth period. For this reason, the matrix metalloproteinase may play a key role on the cyclical growth of hair follicle organs.We can be suggested that MMP-2, MMP-9, TIMP-1 and TIMP-2 has participated in modeling of the extracellular matrixin dermal papillae, while the reshaping has been closely related to the hair follicle growth cycle. The results of immunohistochemical show that the matrix metalloproteinase has existed in hair follicles organs and accessory organs, not only expressed in the single tissues and organs.2. The matrix metalloproteinase inhibitor has inhibitory effect on the growth of hair. On the other hand, the cyclical hair growth has also been confirmed referring to MMP-2 and MMP-9 which may be affected by VEGF, IGF 1 and TGF-β access to affect hair growth. |
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