EGFR inhibitor AG1478 blocks the formation of 3D structures mainly through ERK signaling pathway in Matrigel-induced 3D reconstruction of eccrine sweat gland-like structures.

EGFR signaling plays important roles in the development of eccrine sweat glands. We previously demonstrate that Matrigel induces eccrine sweat gland cells to reconstruct the three-dimensional (3D) structures of eccrine sweat glands, but the mechanisms are still unknown. In the study, eccrine sweat gland cells were cultured within a 3D Matrigel, and EGFR inhibitor AG1478, or MEK1/2 inhibitor U0126, were added to the medium respectively. The morphology of the 3D-reconstructed eccrine sweat gland-like structures was observed, the localization of phospho-EGFR was detected, and protein levels of EGFR, phospho-EGFR, phospho-JAK, phospho-AKT and phospho-ERK were examined. The results showed that cells treatment with AG1478 from Day 0 of 3D cultures blocked formation of spheroid-like structures. AG1478 administration caused reduced phospho-EGFR, concomitant with downregulation of phospho-ERK1/2, but not phospho-JAK or phospho-AKT. Phospho-EGFR and phospho-ERK were reduced, and only a small number of 3D-structures were formed following treatment with U0126. We conclude that EGFR plays important roles in Matrigel-induced 3D structures of eccrine sweat gland-like structures, and ERK1/2 signaling is responsible, at least in part, for the effect of EGFR.

K31 as a novel marker for clear secretory cells in human eccrine sweat glands.

K31 was previously considered as one of the hair keratins. During a study on differential markers between hair follicles and eccrine sweat glands, we observed that K31 was expressed in eccrine sweat gland cells in a scattered pattern, similar to the distribution of dark or clear secretory cells. To investigate the precise cell localization of K31 in human eccrine sweat glands and find new marker for eccrine sweat gland cells, human skin samples were fixed, paraffined and sectioned. The serial sections were stained for K31, dark secretory cell marker gross cystic disease fluid protein 15 (GCDFP15) and clear secretory cell marker carbonic anhydrase II (CAII). The exact cell localization of K31 was detected by double immunofluorescence staining of K31 and a serial of cell-specific markers, and further by dual stain using a combination of periodic acid-Schiff (PAS) and immunofluorescence for K31 and GCDFP15. The expression pattern of K31-positive cells was similar to that of CAII-positive cells but was different from that of GCDFP15-positive staining in serial sections. Double immunofluorescent staining showed that K31-positive cells co-expressed K7 and CAII, but not S100P, α-SMA or GCDFP15. Dual stain by combined PAS and immunofluorescence showed that K31-positive cells are negative for PAS staining. We conclude that K31 is a previously unreported eccrine clear cell marker that allows for distinction between clear and dark secretory cells, as well as between secretory coils and ducts of eccrine sweat glands in human eccrine sweat glands.

Differential antigen expression between human eccrine sweat glands and hair follicles/pilosebaceous units.

Eccrine sweat glands and hair follicles are two primary skin appendages that serve different functions. Although the two appendages exhibit unique morphological patterns in adults, it is difficult to distinguish them morphologically in the early stages of development and regeneration. To research and compare the development, differentiation and regeneration between eccrine sweat glands and hair follicles/pilosebaceous units, specific antigen markers must be found first. Human skin samples were fixed, paraffin-embedded, and cut. The expression of K5, K7, K8, K14, K27, K31, K73, AE13, α-smooth muscle actin (α-SMA), epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA), Na+/K+-ATPase α and Na+-K+-2Cl cotransporter 1 (NKCC1) in eccrine sweat glands, hair follicles and sebaceous glands was detected by immunofluorescence staining. The results showed that eccrine sweat glands expressed K5, K7, K8, K14, K31, α-SMA, CEA, EMA, Na+/K+-ATPase α and NKCC1, but did not express K27, K73 or K31. Hair follicles expressed K5, K8, K14, K27, K31, K73, α-SMA and AE13, but did not express K7, CEA, Na+/K+-ATPase α or NKCC1. Sebaceous glands expressed K5, K14, K73, and EMA, but did not express K7, K8, K31, α-SMA, CEA, EMA, Na+/K+-ATPase α or NKCC1. We concluded that K7, CEA, Na+/K+-ATPase and NKCC1 can be used as specific markers for eccrine sweat glands, K27 and AE13 can be used as specific markers for hair follicles, and K73 can be used as a specific marker for pilosebaceous unit. These specific markers may contribute to differentiate between eccrine sweat glands and hair follicle/pilosebaceous units.

Expression and localization of Forkhead transcription factor A1 in the three-dimensional reconstructed eccrine sweat glands.

Previously studies showed that Forkhead transcription factor A1 (FoxA1) was associated with sweat secretion. To investigate the expression and localization of FoxA1 in the three-dimensional (3D) reconstructed eccrine sweat glands, eccrine sweat gland cells were transplanted subcutaneously into nude mice with Matrigel, and at 2, 3, 4, 5, 6, 8, 10 and 12 weeks post-transplantation, the reconstructed eccrine sweat glands were removed and immunostained for FoxA1 and co-immunostained for FoxA1 and eccrine sweat markers, K7, carbonic anhydrase II (CA Ⅱ), gross cystic disease fluid protein-15 (GCDFP-15) and α-smooth muscle actin (α-SMA), and FoxA1 and sweat secretion-related proteins, Na+-K+-ATPase α and Na+-K+-2Cl- cotransporter 1 (NKCC1). The results showed that FoxA1-positive cells weren't detected until 3 weeks post-implantation, a time point of the differntiation of secretory coil-like structures. From the fourth week on, the number of FoxA1-positive cells increased and thereafter maintained at a high number. Double immunofluorescence staining showed that FoxA1-positive cells co-expressed dark cell marker GCDFP-15 and myoepithelial cell marker α-SMA, as well as secretion-related proteins, Na+-K+-ATPase α and NKCC1 in both the native and reconstructed eccrine sweat glands. In conclusion, FoxA1 might be related to the development and differentiation of secretory coil-like structures, as well as the secretory function of the 3D reconstructed eccrine sweat glands.

Serum is an indispensable factor in the maintenance of the biological characteristics of sweat gland cells.

The tolerance of sweat gland cells for in vitro amplification and subcultivation is low as they are somatic cells. The present study aimed to formulate an optimal medium for the culture of human eccrine sweat gland cells (HESGCs) and to establish a method for induction of HESGCs proliferation, whilst maintaining the characteristics of sweat gland cells. HESGCs cultured in sweat gland (SG):keratinocyte growth medium­2 (KGM­2) (1:1) medium had a higher proliferation rate and a stable morphology compared with cells cultured in SG and KGM­2 medium only. Reverse transcription­quantitative polymerase chain reaction indicated that cells cultured in the SG:KGM­2 (1:1) medium exhibited higher expression levels of α­smooth muscle actin, keratin (K)77, carcinoembryonic antigen, K8, K18, ectodysplasin A receptor, c­Myc, Kruppel­like factor 4 and octamer­binding transcription factor 4 compared with cells cultured in SG only or KGM­2 only medium. Three­dimensional culture analysis revealed that HESGCs cultured in SG:KGM­2 1:1 medium differentiated into sweat gland­like structures, whereas cells cultured in KGM­2 only medium underwent cornification. The present study also determined that the maintenance of the biological characteristics of HESGCs occurred due to the presence of fetal bovine serum (FBS). Cells cultured in medium without FBS differentiated into keratinocytes. Therefore, the SG:KGM­2 (1:1) medium may be a suitable culture medium for HESGCs. In conclusion, this mixed medium is a valuable compound and should be considered to be a potential supplemental medium for HESGCs.

Cells in 3D-reconstitutued eccrine sweat gland cell spheroids differentiate into gross cystic disease fluid protein 15-expressing dark secretory cells and carbonic anhydrase II-expressing clear secretory cells.

Secretory coils of eccrine sweat glands are composed of myoepithelial cells, dark secretory cells and clear secretory cells. The two types of cells play important roles in sweat secretion. In our previous study, we demonstrated that the 3D-reconstituted eccrine sweat gland cell spheroids differentiate into secretory coil-like structures. However, whether the secretory coil-like structures further differentiate into dark secretory cells and clear secretory cells were is still unknown. In this study, we detected the differentiation of clear and dark secretory cells in the 3D-reconstituted eccrine sweat gland cell spheroids using the dark secretory cell-specific marker, GCDFP-15, and clear secretory cell-specific marker, CAII by immunofluorescence staining. Results showed that there were both GCDFP-15- and CAII-expressing cells in 12-week-old 3D spheroids, similar to native eccrine sweat glands, indicating that the spheroids possess a cellular structure capable of sweat secretion. We conclude that the 12-week 3D spheroids may have secretory capability.

Identification of a new sweat gland progenitor population in mice and the role of their niche in tissue development.

Sweat gland cells are responsible for the regulation of body temperature and are critical for wound repair. Furthermore, they have the regenerative potential in response to injury, and show a substantial turnover during both wound healing and homeostasis. However, as a usual research model of sweat gland, mice have not too much glandular cells for experiments. In this study, we identify previously unreported sweat gland progenitor population in mice and characterize them. The progenitor characteristics of sweat gland were confirmed using cellular immunofluorescence assay and quantitative real-time PCR assay. K8 and K18 expression was barely detected in the early stage of skin development (Embryo 17.5d) and increased to a high level at P5d (postnatal 5d), then showed reduction at adult stage (P28d). Further investigation of K8 and K18 positive cells using tissue immunofluorescence revealed the presence of sweat gland progenitors in back epidermis of mice at early stage of sweat gland development and continuous reduction during the developmental process. In vivo transplantation assay with animal models elucidated that sweat gland specific niche in paw pads was critical for the development of sweat gland cells. Although the relationship between new sweat gland progenitors and their niche still needs to be further investigated, the presence of these cells implicates that there is more source ascribed to sweat glands in addition to serving as progenitors in mice.

[Optimization of primary human eccrine sweet glands' isolation in vitro].

OBJECTIVE: To optimize the methods of isolating human eccrine sweat gland cells in vitro so as to get efficiently primary human sweat glands. METHODS: The fresh and normal skin tissue was cut into pieces of microskin about 1mm3 and the following 3 group digestion buffer was applied to isolated gland cells. The digestion buffer of group A was the equivoluminal mixture of Trypsin-Ethylene Diamine Tetraacetic Acid (EDTA) and collagenase-II (2 mg/ml). The digestion buffer of group B was collagenase-II (2 mg/ml) traditionally and group C was Trypsin-EDTA. These three groups were placed into an incubator simultaneously and the emerging time of dissociated sweat glands was calculated. Sweat glands were sorted out and then placed in culture dish. The adherence and the growth of cells were observed. The proliferation index was detected by flow cytometry. The identification of cultured cells was performed by immunocytochemical staining. RESULTS: After digesting 30 min in group A and C, a very few of dissociated sweat glands were emerging. But after digesting for 2 h, there were lots of dissociated sweat glands emerging in group A rather than in group C. The emergence of dissociated sweat glands in group B would require at least 6 hours. After seeded in culture dishes, the sweat glands in group C couldn't adhere to the wall of dish, but the sweat glands in group A and B adhered very well and even grew like paving stones after 9 days. In addition, the proliferation index were (18 ± 4) % and (17 ± 6) % respectively, there was no statistical difference. The results of immunocytochemical staining showed that the cells expressed carcino-embryonic antigen (CEA) and cytokeratin 7(CK7) in group A and B. CONCLUSION: Trypsin-EDTA combined with collagenase-II can shorten the time of isolating sweat gland cells and have no effect on cell activity and proliferation.

Three-dimensional co-culture of BM-MSCs and eccrine sweat gland cells in Matrigel promotes transdifferentiation of BM-MSCs.

Victims with extensive and deep burns are unable to regenerate eccrine sweat glands. Combining of stem cells and biomimetic ECM to generate cell-based 3D tissues is showing promise for tissue repair and regeneration. We co-cultured BrdU-labeled bone marrow-derived mesenchymal stem cells (BM-MSCs) and eccrine sweat gland cells in Matrigel for 2 weeks in vitro and then evaluated for BM-MSCs differentiation into functional eccrine sweat gland cells by morphological assessment and immunohistochemical double staining for BrdU/pancytokeratin, BrdU/ZO-2, BrdU/E-cadherin, BrdU/desmoglein-2, BrdU/Na(+)-K(+)-ATPase α, BrdU/NHE1 and BrdU/CFTR. Cells formed spheroid-like structures in Matrigel, and BrdU-labeled BM-MSCs were involved in the 3D reconstitution of eccrine sweat gland tissues, and the incorporated BM-MSCs expressed an epithelial cell marker (pancytokeratin), epithelial cell junction proteins (ZO-2, E-cadherin and desmoglein-2) and functional proteins of eccrine sweat glands (Na(+)-K(+)-ATPase α, NHE1 and CFTR). In conclusion, three-dimensional co-culture of BM-MSCs and eccrine sweat gland cells in Matrigel promotes the transdifferentiation of BM-MSCs into potentially functional eccrine sweat gland cells.

Site-specific migration of human fetal melanocytes in volar skin.

BACKGROUND: Melanocytes originate from the neural crest and migrate ventrally from the dorsal neural tube during embryogenesis. How human melanocytes locate at their suitable positions during embryogenesis, however, is unclear. Although a growing body of evidence indicates that melanocytes, melanoblasts, and melanocyte stem cells are closely related to hair follicles, little is known about volar skin. OBJECTIVE: The aim of this study was to observe skin development during human fetal period and clarify the site-specific migration process of human fetal sole melanocytes. METHODS: We obtained 4-mm punch biopsies from the scalp, back, abdomen, and right sole of 36 aborted fetuses (gestational age 12-21 weeks). We compared the migration process between hairly areas and volar areas by immunohistochemical staining. RESULTS: Immunohistochemical examination revealed that gp100 (HMB-45) sensitively detects human melanocytes in embryogenesis. Melanocytes were present at the epidermal base, where hair placodes/buds form at 12-15 weeks gestation. Fetal melanocytes in hair follicles are supplied from the epidermis. In volar skin, melanocytes originally localize only in the acrosyringium, where they migrate deeper into with gland development at 16-18 weeks gestation. Palmoplantar melanocyte migration and maturation processes differ considerably from those of the other hairy skin sites. CONCLUSION: Eccrine sweat glands seem to have a central role in the palmoplantar melanocyte migration process, similar to the role of hair follicles in hairy sites.

Matrigel basement membrane matrix induces eccrine sweat gland cells to reconstitute sweat gland-like structures in nude mice.

BACKGROUND: Severe burn results in irreversible damage to eccrine sweat glands, for which no effective treatment is available. Interaction between the extracellular matrix and epithelial cells is critical for proper three-dimensional organization and function of the epithelium. METHODS: Matrigel-embedded eccrine sweat gland cells were subcutaneously implanted into the inguinal regions of nude mice. Two weeks later, the Matrigel plugs were removed and evaluated for series of detection items. RESULTS: Sweat gland cells developed into sweat gland-like structures in the Matrigel plugs based on: (1) de novo formation of tubular-like structures with one or more hollow lumens, (2) expression of epithelial and sweat gland markers (pancytokeratin, CK5/7/14/19, α-SMA and CEA), (3) basement membrane formation, (4) myoepithelial cells presenting in and encompassing the tubular-like structures, (5) cellular polarization, evident by the expression of tight junction proteins (claudin-1 and ZO-2), anchoring junctions (desmoglein-1 and -2 and E-cadherin) and CEA in the luminal membrane, (6) expression of proteins related to sweat secretion and absorption (Na(+)-K(+)-ATPase α/ß, Na(+)-K(+)-2Cl-cotranspoter 1, Na(+)/H(+) exchanger 1, aquaporin-5, epithelial sodium channel, cystic fibrosis transmembrane conductance regulator, potassium channel and vacuolar-type H+-ATPase), and (7) about 20% of the tubular-like structures are de novo coils and 80% are de novo ducts. CONCLUSIONS: This study provides not only an excellent model to study eccrine sweat gland development, cytodifferentiation and reconstitution, but also an in vivo model for regeneration of eccrine sweat glands.

[Three-dimensional culture and morphological observation of human eccrine sweat gland cells].

OBJECTIVE: To investigate the three-dimensional (3D) culture and morphology of human eccrine sweat gland cells. METHODS: The human eccrine sweat gland cells were isolated from normal abdominal full thickness skin by digestion of type II collagenase, and cultured in defined-keratinocyte serum free medium supplemented with 5 ng/mL recombinant human epidermal growth factor, 25 mg/mL bovine pituitary extract, 100 U/mL penicillin, and 100 microg/mL streptomycin at 37 degrees C in a humidified atmosphere of 5%CO2/95% air incubator. When the cell fusion reached above 80%, the cells were harvested and the concentration was adjusted to 1 x 10(5) cells/mL. The mixture of 0.3 mL cell suspension and 0.3 mL Matrigel basement-membrane matrix was cultured in 12-well plate. The cell growth was observed under an inverted phase contrast microscope. At 14 days after culture, frozen sections were prepared and were stained with HE to observe the cells morphology, and immunohistochemical analysis was used to detect the antigen expressions of cytokeratin 7 (CK7) and CK19. RESULTS: Inverted phase contrast microscope observation showed that many free eccrine sweat gland tissues were seen after digestion of type II collagenase; eccrine sweat gland cells grew adhering to the wall at 3-5 days and continued division for 2-3 weeks to form single ring around the block sweat glands; cellular senescence were observed after 3-4 weeks. During the process of 3D culture, the single eccrine sweat gland cell divided into 2-4 cells after 2-3 days, and these cells subsequently formed small cell clusters, tubular-like structures and finally spheric-like shapes. After cultured for about 2 weeks, there was crack in part of the gelled mixture or liquefaction occurred. HE staining of frozen sections of the 3D cultures showed some of the tubular-like structures composed of 1-2 layers of epithelial cells, which were similar to the secretion part and the duct part of the eccrine sweat gland. Immunohistochemical analysis showed that CK7 and CK19 antigens expressed positively in the cells. CONCLUSION: Human eccrine sweat gland cells cultured in Matrigel can form the 3D structures which simulate the morphology of eccrine sweat glands in vivo.

The secretory clear cell of the eccrine sweat gland as the probable source of excess sweat production in hyperhidrosis.

Primary hyperhidrosis is characterized by excessive sweating in palmar, plantar and axillary body regions. Gland hypertrophy and the existence of a third type of sweat gland, the apoeccrine gland, with high fluid transporting capabilities have been suggested as possible causes. This study investigated whether sweat glands were hypertrophied in axillary hyperhidrotic patients and if mechanisms associated with fluid transport were found in all types of axillary sweat glands. The occurrence of apoeccrine sweat glands was also investigated. Axillary skin biopsies from control and hyperhidrosis patients were examined using immunohistochemistry, image analysis and immunofluorescence microscopy. Results showed that glands were not hypertrophied and that only the clear cells in the eccrine glands expressed proteins associated with fluid transport. There was no evidence of the presence of apoeccrine glands in the tissues investigated. Preliminary findings suggest the eccrine gland secretory clear cell as the main source of fluid transport in hyperhidrosis.

Matrigel-induced tubular morphogenesis of human eccrine sweat gland epithelial cells.

Human eccrine sweat glands are tubule-structured glands of the skin that are vital in thermoregulation, secretion, and excretion of water and electrolytes. A study of tubular morphogenesis in vitro would facilitate the development of a tissue engineering model for eccrine sweat glands and other tubule-structured glands. Matrigel, a basement membrane matrix, has been shown to promote differentiation and morphogenesis of many different cell types, including tubular cells. This study investigated the growth, differentiation, and tubular morphogenesis of human eccrine sweat gland epithelial cells cultured in Matrigel. Human eccrine gland epithelial cells were isolated and cultured in vitro. The cell growth in Matrigel was evidenced by the formation of cell clusters, which were observed under an inverted microscope. The internal structure of the cell clusters was further investigated by hematoxylin-eosin (HE) staining and confocal laser scanning microscopy (CLSM) of propidium iodide-stained nuclei. The results demonstrated that although on a plastic surface or in a collagen gel the cells could not form tubular structures, they formed tubular structures when cultured in Matrigel. Consequently, we conclude that Matrigel can promote tubular morphogenesis of human eccrine sweat gland epithelial cells.

Development and structure of the glandopreputial sulcus of the human clitoris with a special reference to glandopreputial glands.

The structure and development of the sulcus between the glans and prepuce of the human clitoris have hardly been investigated. Interest in its structure was raised when in the female, in contrast to the male, glands were found to develop from the solid lamella-like precursor of the glandopreputial sulcus. It prompted a further histological analysis of the sulcus in female fetuses and newborn and an extension of that study to clitorises of adult women. The investigation showed that in the clitoris, in contrast to the penis, the transformation of the glandopreputial lamella into the open sulcus was mostly incomplete and apparently remained so throughout life. As a most striking and probably exclusively female feature, two to eight eccrine glands developed from the base of the lamella in fetuses older than 14.5 weeks gestation. These glands formed secretory coils near and occasionally inside the adjacent distal corpora cavernosa. Some glands showed atresia, cystic dilatation, and squamous metaplasia. A remarkably similar picture was observed in the adult clitorises, in which the secretory coils were often found between the large blood vessels and nerves to the glans and were connected to the sulcus by long excretory ducts. All glands revealed unmistakably eccrine features. It is suggested that their secretion moistens the female glandopreputial sulcus, which is not lubricated by urethral secretion as in the male. The findings may explain the rare clitoral phimosis, cysts, and some pilonidal sinuses.

Estradiol rapidly induces the translocation and activation of the intermediate conductance calcium activated potassium channel in human eccrine sweat gland cells.

BACKGROUND AND AIMS: Steroid hormones target K+ channels as a means of regulating electrolyte and fluid transport. In this study, ion transporter targets of Estradiol (E2) were investigated in the human eccrine sweat gland cell line NCL-SG3. RESULTS: Whole cell patch-clamp studies revealed E2 (10 nM) rapidly activates a whole cell K+ conductance, which is abolished by clotrimazole (30 microM), an inhibitor of the intermediate conductance calcium activated K+ channel (IKCa). The estrogen receptor (ER) antagonist ICI 182, 780 had no effect on this E2 activated K+ conductance, suggesting an estrogen receptor independent mechanism of activation. Confocal microscopy studies revealed under basal conditions that the IKCa channel is located within the cell cytoplasm and in the presence of E2, rapidly translocates to both the apical and basolateral membrane. In the presence of E2, tyrosine phosphorylation of calmodulin, which is known to regulate trafficking of the IKCa channel, is increased, and treatment of cells with the calmodulin inhibitor trifluoperazine (TFP) prevents the E2-induced translocation. CONCLUSIONS: Estradiol rapidly regulates a K+ conductance through the IKCa channel in an estrogen receptor independent manner. E2 stimulates the translocation of IKCa to the cell membrane in a calmodulin dependent manner, representing a novel paradigm of estrogen action in sweat gland epithelial cells.

Antigen expression of human eccrine sweat glands.

BACKGROUND: The proliferating abilities of sweat glands are very limited, so researches on the repair and regeneration of sweat glands are important. First of all, we must find out reliable and specific antigen markers of sweat glands. OBJECTIVE: To investigate the antigen expression of human eccrine sweat glands. METHODS: The development of eccrine sweat glands was investigated by hematoxylin and eosin staining, and the antigen expression was detected by immunohistochemical techniques. RESULTS: Human eccrine sweat glands expressed cytokeratin (CK) 7, CK8, CK14, CK18, CK19 and epithelial membrane antigen (EMA). Carcinoembryonic antigen (CEA) was only expressed in sweat glands in the adult skin. Developing and developed sweat glands all had some cells expressing Ki67 and p63 antigens. Epidermal growth factor (EGF) was mainly localized in the secretory cells and ductal cells. Some myoepithelial cells were also labeled with anti-EGF antibody. In the older fetus, positive staining for EGF was seen in the lumen of the secretory portion. EGF receptor (EGFR) was expressed in the ducts. CONCLUSIONS: Human eccrine sweat glands express CK7, CK8, CK14, CK18, CK19, CEA, EMA, Ki67, p63, EGF and EGFR. In skin, CEA can be used as a specific immunological marker of sweat glands.

[Effect of hepatocyte growth factor on proliferation of cultured human eccrine sweat gland epithelial cells].

OBJECTIVE: To investigate the effect of hepatocyte growth factor (HGF) on proliferation of cultured human eccrine sweat gland epithelial cells (hESGc) and the involvement of phosphorylation of ERK1/2. METHODS: hESGc were cultured in keratinocyte serum free medium (KSFM) and the first generation of hESGc was harvested. The expression of C-met was detected by immunocytochemistry. MTT assay was used to detect the effect of HGF on the proliferation of hESGc. The cells were divided into blank group, control group and experimental group. The culture density was 2 x 10(3) cells/hole in control group and experimental group. Two hundred microL KSFM with HGF in different levels was added to every hole. hESGc were cultured in KSFM with HGF at different levels (2, 20, 40 and 80 ng/mL) in experimental group, in KSFM without HGF in control group, and in KSFM without HGF and no hESGc in blank group. The cell proliferation was observed in experimental group 2 and 4 days later. Western blot was used to detect the expression of phosphorylated ERK1/2 at 40 ng/mL HGF after 0, 5, 30, 90 and 120 minutes. RESULTS: The results were positive for anti-C-met staining in the cytoplasm. HGF (40 ng/mL and 80 ng/mL) significantly improved the proliferation of hESGc (P < 0.05). When cultured in the KSFM with 40 ng/mL HGF, the cell proliferation rate and the absorbance were 74.2%, 0.2393 +/- 0.0709 at 2 days and 74.8%, 0.2878 +/- 0.0743 at 4 days; showing significant differences when compared with control group (P < 0.05). When cultured in KSFM with 80 ng/mL HGF, the cell proliferation rate and the absorbance were 54.5%, 0.2123 +/- 0.0592 at 2 days and 40.3%, 0.2310 +/- 0.0567 at 4 days; showing significant differences when compared with control group (P < 0.05). The expression of p-ERK1/2 reached to the maximum after stimulation of 40 ng/mL HGF for 5 minutes, and relative integral absorbance (RIA) was 0.5932 +/- 0.1922, increased 8.1 times compared with instant stimulation (P < 0.01). CONCLUSION: HGF could induce the proliferation of hESGc and activate the phosphorylation of ERK 1/2 protein.

Comparison of proliferating cells between human adult and fetal eccrine sweat glands.

Studies of sweat glands had demonstrated that there were degenerating cells and proliferating cells in the eccrine sweat glands. To compare the differences in the proliferating cells between human adult and fetal eccrine sweat glands, immunostaining of proliferating-associated proliferating cell nuclear antigen (PCNA) and Ki67 nuclear antigen (Ki67) was performed, and the location and the percentage of the positive staining cells were analyzed. The results showed that a few cells of the secretory and ductal portion in both the adult and fetal eccrine sweat glands stained positive with Ki67 and PCNA. The labeling index of PCNA in adult eccrine sweat glands was 34.71 +/- 8.37%, while that in the fetal was 62.72 +/- 6.54%. The labeling index of PCNA in fetal eccrine sweat glands was higher than that in adult. Myoepithelial cells were negative staining with anti-PCNA antibody in adult eccrine sweat glands, while in the fetal a few myoepithelial cells were positive staining. Labeling index of Ki67 in adult eccrine sweat glands was similar to that in the fetal, ranging from 0.5 to 4.3%. Myoepithelial cells of the adult and fetal eccrine sweat glands both were negative staining with anti-Ki67 antibody. We concluded that the myoepithelial cells had proliferating ability only in fetal eccrine sweat glands, and that the proliferating ability of fetal eccrine sweat glands was stronger than that of the adult.