PI3K-AKT Pathway Protects Cardiomyocytes Against Hypoxia-Induced Apoptosis by MitoKATP-Mediated Mitochondrial Translocation of pAKT.

BACKGROUND/AIMS: The phosphatidylinositol-3-kinase -AKT (PI3K-AKT) is an important intracellular signal pathway in regulating cell proliferation, differentiation and apoptosis. In previous studies, we've demonstrated that PI3K-AKT pathway protects cardiomyocytes from ischemic and hypoxic apoptosis through mitochondrial function. However, the molecular mechanisms underlying hypoxia-induced cardiomyocyte apoptosis via PI3K-AKT pathway remain ill-defined. Here, we addressed this question. METHODS: Cardiomyocytes were exposed to hypoxia, with/without different inhibitors and then protein levels were assessed by Western blotting. RESULTS: We found that the PI3K-AKT pathway was activated in cardiomyocytes that were exposed to hypoxia. Moreover, the phospho-AKT (pAKT) translocated from cytosol to mitochondria via mitochondrial adenosine triphosphate-dependent potassium (mitoKATP), leading to an increase in cytochrome c oxidase (CcO) activity to suppress apoptosis. On the other hand, the mitoKATP specific blocker, 5-hydroxydecanote (5-HD), or suppression of CcO using siRNA, inhibited the pAKT mitochondrial translocation to maintain the CcO activity, resulting in mitochondrial dysfunction and cellular apoptosis induced by hypoxia. CONCLUSION: These findings suggest that the anti-apoptotic effect of the PI3K-AKT pathway through pAKT translocation to mitochondrial via mitoKATP may be conducted through modification of CcO activity.

The phosphatidylinositol 3-kinase-Akt pathway protects cardiomyocytes from ischaemic and hypoxic apoptosis via mitochondrial function.

1. After a severe burn, a marked decrease in myocardial blood flow results in ischaemic and hypoxic injury, which subsequently leads to apoptosis or necrosis. Phosphatidylinositol 3-kinase (PI3-K)/Akt is an important intracellular signal transduction molecule that regulates cell proliferation, differentiation, glucose metabolism and migration. However, the function and mechanisms of the PI3-K-Akt pathway in cardiomyocyte apoptosis after a burn remain unclear. 2. In the present study, an in vivo rat model of burn injury and an in vitro hypoxic model using rat cardiomyocytes were established. In burned rats, the expression of PI3-K and phosphorylated (p-) Akt expression increased, as did myocardial apoptosis. Inhibition of the PI3-K-Akt pathway with 1.4 mg/kg LY294002 caused a significant increase in the myocardial apoptotic index compared with hypoxia alone in the in vivo model. 3. Cardiomyocytes cultured under hypoxic conditions exhibited increased apoptosis, decreased cell viability, enhanced caspase 3 activity, a decreased mitochondrial membrane potential, increased cytoplasmic calcium transients and increased p53 and Bax mRNA expression. Pretreatment with 50 mumol/L LY294002 significantly enhanced all these negative indicators compared with hypoxia alone. In contrast, pretreatment of cells with 200 ng/mL insulin-like growth factor-1, an activator of PI3-K-Akt, significantly ameliorated the effects of hypoxia, although control levels were not reached. 4. These findings indicate that activation of the PI3-K-Akt pathway induced by ischaemia and hypoxia after a severe burn can protect cardiomyocytes from apoptosis. This anti-apoptotic effect is most likely mediated via the mitochondria and changes in p53 and Bax gene expression, intracellular [Ca(2+)] and caspase 3 activity.

Astragaloside IV attenuates hypoxia-induced cardiomyocyte damage in rats by upregulating superoxide dismutase-1 levels.

1. Astragaloside IV (AST-IV) is purified from a natural plant product. Previous studies have shown that AST-IV has anti-oxidant activity. In the present study, we investigated the effect and mechanism of action AST-IV on rat cardiomyocytes subjected to hypoxic conditions (up to 12 h). 2. Cardiomyocytes were prepared from neonatal rats and cultured under normoxic or hypoxic conditions in the absence or presence of AST-IV (12.5, 25 or 50 microg/mL). Cell viability, malondialdehyde (MDA) levels, activity and expression of superoxide dismutase (SOD)-1 (mRNA and protein levels determined by reverse transcription-polymerase chain reaction and western blotting, respectively) and reactive oxygen species (ROS; determined by 2',7'-dichlorodihydrofluorescein diacetate) were investigated under these culture conditions. Intracellular localization of AST-IV was tested using fluorescein isothiocyanate-labelled AST-IV. 3. Hypoxic culture reduced the viability of cardiomyocytes, which was improved following treatment with 25 or 50 microg/mL AST-IV. Under hypoxic conditions, MDA levels were double those under control conditions. Astragaloside IV (25 and 50 microg/mL) dose-dependently reduced the increase in MDA seen in hypoxic cardiomyocytes. 4. Fluorescein isothiocyanate-labelled AST-IV entered cardiomyocytes and was localized mainly within the cytoplasm. 5. Under hypoxic conditions, SOD-1 activity was decreased, but mRNA and protein expression increased, compared with normoxia. Following treatment with 25 microg/mL AST-IV, SOD-1 activity and expression were increased under both normoxic and hypoxic conditions. The ROS scavenging effect of AST-IV was abolished in the presence of the SOD inhibitor sodium diethyl dithiocarbamate (25 micromol/L). 6. These in vitro results show that AST-IV protects cardiomyocytes from oxidative stress-mediated injury under hypoxic conditions. A major part of this action is achieved by upregulation of SOD-1 content and activity within the cell cytoplasm.

Clinical features and treatment of 140 cases of Marjolin's ulcer at a major burn center in southwest China.

Marjolin's ulcer is a type of malignant tumor that occurs in scar tissue. The present study aimed to summarize and analyze the aetiology, clinical characteristics, treatment methods, metastasis and prognosis of this disease. A total of 140 cases of Marjolin's ulcer encountered at the Institute of Burn Research, Southwest Hospital (Chongqing, China) between January 2013 and December 2017 were retrospectively analyzed. Demographic data, clinical characteristics, occurrence of bone invasion and lymph node metastasis, as well as treatment and prognosis were statistically analyzed. Among the 140 patients, the initial injury or primary disease occurred at 1-75 years of age, while Marjolin's ulcer occurred at 15-85 years of age (mean, 53.3±1.2 years). The mean latency period was 28.8±1.7 years. The most common initial injury of the patients was flame burns, followed by skin masses, trauma, skin ulcerations caused by repeated scratching/friction, and scalding. The age at onset of initial injury or disease in patients had a significantly negative correlation with the latency period (P<0.01). The most common lesion locations were the lower limbs (42.1%), followed by the head, face and neck (34.5%). Of the 140 patients, 46 cases (32.9%) had bone invasion, 33 cases (23.6%) had lymph node enlargement and only 5 cases (3.6%) had lymph node metastasis. The skull was the bone that was most susceptible to Marjolin's ulcer invasion. The prevalence of bone invasion in patients with head, face and neck lesions was significantly higher than that in patients with lesions in other locations (P<0.01). The surgical methods applied were skin grafting, local flap repair, amputation and island flap repair. In the 65 cases who underwent follow-up, recurrence mainly occurred within 1 year after surgery. In conclusion, Marjolin's ulcer mainly occurred in males and was a scar carcinoma after a flame burn in most cases. Autologous skin grafting and local skin flap repair were the major repair methods. The peak period of recurrence was within one year after surgery and patients should receive regular follow-ups.

Tumor necrosis factor receptor-associated protein 1 regulates hypoxia-induced apoptosis through a mitochondria-dependent pathway mediated by cytochrome c oxidase subunit II.

BACKGROUND: Tumor necrosis factor receptor-associated protein 1 (TRAP1) plays a protective effect in hypoxic cardiomyocytes, but the precise mechanisms are not well clarified. The study is aimed to identify the mechanism of TRAP1 on hypoxic damage in cardiomyocytes. METHODS: In this study, the effects of TRAP1 and cytochrome c oxidase subunit II (COXII) on apoptosis in hypoxia-induced cardiomyocytes were explored using overexpression and knockdown methods separately. RESULTS: Hypoxia induced cardiomyocyte apoptosis, and TRAP1 overexpression notably inhibited apoptosis induced by hypoxia. Conversely, TRAP1 silencing promoted apoptosis in hypoxic cardiomyocytes. Further investigation revealed that the proapoptotic effects caused by the silencing of TRAP1 were prevented by COXII overexpression, whereas COXII knockdown reduced the antiapoptotic function induced by TRAP1 overexpression. Additionally, changes in the release of cytochrome c from mitochondria into the cytosol and the caspase-3 activity in the cytoplasm, as well as reactive oxygen species production, were found to be correlated with the changes in apoptosis. CONCLUSIONS: The current study uncovered that TRAP1 regulates hypoxia-induced cardiomyocyte apoptosis through a mitochondria-dependent apoptotic pathway mediated by COXII, in which reactive oxygen species presents as an important component.

p38 MAP kinase mediates burn serum-induced endothelial barrier dysfunction: involvement of F-actin rearrangement and L-caldesmon phosphorylation.

The aim of this study was to test the hypothesis that circulating factors released after a severe burn cause endothelial barrier dysfunction by triggering endothelial cell (EC) contraction through a p38 mitogen-activated protein (MAP) kinase-dependent mechanism. Human umbilical vein ECs (ECV304 cell line) were cultured to create a monolayer of cells that were then cultured with 20% human normal or burn serum. Monolayer permeability was measured by the influx of labeled albumin across the cells. Endothelial cells contraction was determined by alterations of cell surface area and formation of intracellular gaps. P38 MAP kinase activation, F-actin arrangement, and L-caldesmon phosphorylation were assessed by Western blots or immunofluorescence staining. These studies showed that exposure to burn serum resulted in a significant increase in endothelial permeability in a time-dependent manner, which was paralleled by a rapid and persistent activation of p38 MAP kinases. Morphologically, increased intercellular gaps, reduced cell surface area, and a unique rearrangement of F-actin cytoskeleton were observed in burn serum-treated ECs. Inhibition of p38 MAP kinase suppressed the rearrangement of F-actin cytoskeleton, reduced the occurrence of burn serum-induced formation of intercellular gaps, and ameliorated endothelial hyperpermeability. Further study showed that phosphorylation of L-caldesmon was enhanced in burn serum-treated cells via p38 MAP kinase; overexpression of L-caldesmon by adenovirus transfection, however, attenuated the increase in endothelial permeability by burn serum challenge. Collectively, these results have demonstrated for the first time that p38 MAP kinase is an important participant in mediating burn serum-induced endothelial barrier dysfunction through rearrangement of the F-actin cytoskeleton and phosphorylation of L-caldesmon. Inhibition of p38 MAP kinase in vivo, thus, would be a promising therapeutic strategy in ameliorating burn shock development.

[Influence of microtubule depolymerization of myocardial cells on mitochondria distribution and energy metabolism in adult rats].

OBJECTIVE: To investigate the influence of microtubule depolymerization of myocardial cells on distribution and activity of mitochondria, and energy metabolism of cells in adult rats. METHODS: Myocardial cells of SD adult rats and SD suckling rats were isolated and cultured. They were divided into adult and suckling rats control groups (AC and SC, normally cultured without any stimulating factor), adult and suckling rats microtubule depolymerization agent groups (AMDA and SMDA, cultured with 8 micromol/L colchicine containing nutrient solution for 30 minutes) according to the random number table. (1) The expression of polymerized beta tubulin in myocardial cells of adult and suckling rats was detected with Western blot. (2) Myocardial cells of rats in AC and AMDA groups were collected. The expression of cytochrome c was detected with Western blot. Distribution of voltage-dependent anion channels (VDAC) and polymerized beta tubulin in myocardial cells were observed with immunofluorescent staining. Mitochondrial inner membrane potential was determined with immunocytochemical method. Activity of myocardial cells was detected with MTT method. Contents of ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) and energy charge of cells were determined with high performance liquid chromatography. RESULTS: (1) The expression of polymerized beta tubulin:in AMDA group it was 0.52 + or - 0.07, which was obviously lower than that (1.25 + or - 0.12) in AC group (F = 31.002, P = 0.000); in SMDA group it was 0.76 + or - 0.12, which was significantly lower than that (1.11 + or - 0.24) in SC group (F = 31.002, P = 0.000), but was obviously higher than that in AMDA group (F = 31.002, P = 0.009). (2) The expression of cytochrome c in AC group was 0.26 + or - 0.03, which was obviously lower than that (1.55 + or - 0.13) in AMDA group (t = -24.056, P = 0.000). (3) Immunofluorescent staining result: in AC group, microtubules of myocardial cells were in linear tubiform, distributed in parallel with myocardial fiber; VDAC staining result showed that mitochondria were in granular form, distributed in the same direction as microtubules. In AMDA group, the normal distribution regularity of microtubules was destroyed, with weakened immune fluorescence intensity, microtubules structure indistinct, continuity lost, rough in appearance, and the distribution of mitochondria became disrupted. (4) Mitochondrial inner membrane potential in AC group fluorescent intensity was 1288 + or - 84, which was obviously higher than that (331 + or - 27) in AMDA group (t = 26.508, P = 0.000). (5) Cellular activity: in AC group absorbance value was 1.75 + or - 0.11, which was obviously lower than that (0.81 + or - 0.07) in AMDA group (t = 17.348, P = 0.000). (6) Energy metabolism: compared with those in AC group, content of ATP decreased, contents of ADP and AMP increased, and ATP/ADP value and energy charge decreased in AMDA group. CONCLUSIONS: Microtubules and mitochondria distribute in the same direction in normal myocardial cells in adult rats. After microtubule depolymerization, mitochondria are arranged in disorder fashion; cytochrome c leaks from mitochondria; mitochondrial membrane potential, energy supply, and cellular activity decrease in the myocardial cells.

[Effects of glycine on apoptosis in murine cardiomyocyte suffering from ischemia and hypoxia].

OBJECTIVE: To investigate the effects of glycine on apoptosis in murine cardiomyocyte suffering from ischemia and hypoxia. METHODS: The primary passage of cultured cardiomyocytes from neonatal rats were subjected to ischemia and hypoxia, and the cells were divided into IH (without other treatment), and G (with treatment of 5 mmol/L glycine) groups. Normal murine cardiomyocytes served as control (C group). Cardiomyocytes were cultured for 6 hours in vitro. Apoptosis, mitochondrial membrane potential and its distribution, the condition of mitochondria permeability transition pore (mPTP) were observed with expression of fluorescence intensity. The activity of caspase-3 was observed by Laser Scanning staining. RESULTS: (1) Apoptosis: the fluorescence intensity in IH group was obviously higher than that in G and C groups (P < 0.01). (2) Mitochondrial membrane potential: the fluorescence intensity in IH group was 32 +/- 7, which was obviously lower than that in G and C groups (52 +/- 4, 73 +/- 4, respectively, P < 0.01). (3) The condition of mPTP: the intensity in IH group was 27 +/- 4, which was obviously lower than that in G and C groups (62 +/- 8, 90 +/- 7, respectively, P < 0.01). (4) The activity of caspase-3: the activity of caspase-3 in IH group was obviously higher than that in G and C groups (P < 0.01). CONCLUSION: Glycine can inhibit apoptosis in cardiomyocytes subjected to ischemia and hypoxia,and the effect may be attributable to changes in mitochondrial membrane potential, lessening opening of mPTP, alleviation of calcium overload , and decrease in activity of caspase-3.

[Protective effects of enalaprilat on the myocardial kinetics in rats at early stage of severe scald].

OBJECTIVE: To investigate the therapeutic effects of Enalaprilat on the myocardial kinetics in rats at early stage of severe scald. METHODS: Eighty-four SD rats were inflicted with 30% TBSA full-thickness scald, and randomly divided into scald (S, with intraperitoneal injection of isotonic saline according to Parkland formula, n=30), L (n=30), M (n=12) and H (n=12) groups. The rats in L,M,H groups were intraperitoneally injected with 1,2,4 mg/kg Enalaprilat. Other 6 healthy rats were enrolled into study as control (C group). The myocardial kinetic parameters including left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), +/- dp/dt max and the levels of A II in myocardium were observed at 1,3,6,12 and 24 post scald hour (PBH) in L and S groups,and at 6,12 PBH in M and H groups. The above indices in C group were also examined. RESULTS: The levels of LVSP, LVEDP, +/- dp/dt max in C group were higher than those in other groups during 3-24 PBH (P < 0.05 or P < 0.01), while those in L,M,H groups were obviously higher than those in S group (P < 0.05 or P < 0.01). The level of +/- dp/dt max in H group at 6,12 PBH were obviously lower than those in L and M groups. The level of A II in S group at 1 PBH was (53.0 +/- 2.6) pg/200 mg, which was significantly higher than thatin C group [(14.8 +/- 0.7) pg/200 mg, P < 0.05 or P < 0.01]; it peaked at6 PBH and lowered afterwards, and they were significantly higher than that in C group at 24 PBH (P < 0.01). The levels of A II in L group during 3-24 PBH were obviously higher than those in C group (P < 0.01), which were also lower than those in S group. The level of A II in S group was significantly higher than in L,M,H groups at 6 PBH [(145.2 +/- 14.5) pg/200 mg. vs. (65.1 +/- 0.9) pg/200 mg, (53.6 +/- 1.1) pg/200 mg, (34.2 +/- 0.9) pg/200 mg, respectively, P < 0.01]. CONCLUSION: Myocardium can be obviously damaged at early stage after severe scald,cardiac function is impaired. Enalaprilat injection (especially at low dose) can significantly ameliorate the myocardial kinetics indices, and it seems to exert a protective effect on cardiac function.

[The influence of insulin growth factor-I on the apoptosis of cardiomyocytes subjected to ischemia and hypoxia].

OBJECTIVE: To investigate the influence of insulin growth factor-I (IGF-I) on apoptosis of cardiomyocytes subjected to ischemia and hypoxia and its possible mechanism. METHODS: Cardiomyocytes were cultured in vitro, and randomized into hypoxia group, treatment group (T, the cells were treated with IGF-1 before subjected to hypoxia and ischemia) and control group (C, normal cardiomyocytes as controls). Changes in the OD value of cell apoptosis, mitochondrial membrane potential and relative amount of phospho-Akt protein were observed at different time-points by ELISA, laser scanning with TMRE staining and Western blot, respectively. RESULTS: The OD value of cell apoptosis in control group was 0.18 +/- 0.03, while that in hypoxia group was gradually increased to 0.33 +/- 0.05, 0.61 +/- 0.06, 1.17 +/- 0.08, 2.25 +/- 0.11, respectively at 1, 3, 6, 12 post-hypoxia hours (PHH), showing an increasing tendency (P < 0.01). The OD values of cell apoptosis in T group were 0.26 +/- 0.04, 0.49 +/- 0.05, 0.84 +/- 0.06, 1.63 +/- 0.09, respectively, which were obviously lower than those in hypoxia group (P < 0.05 or P < 0.01). The mitochondrial membrane potential (Dymt) values in hypoxia group at 6 and 12 PHH were 18.7 +/- 5.1 and 6.3 +/- 1.9, respectively, which were obviously lower than that in control group (40.2 +/- 10.1, P < 0.01). The DYmt in T group at 6 and 12 PHH were 28.8 +/- 6.2, 12.5 +/- 3.1, respectively, which were obviously higher compared with those in hypoxia group (P < 0.05). The amount of phospho-Akt protein was increased by IGF-I administration. CONCLUSION: IGF-I exhibits an anti-apoptotic effect on cardiomyocytes subjected to ischemia and hypoxia, and this may be related to the activation of PI3K/Akt signal pathway and stabilization of mitochondrial membrane potential.

[Protective effects of Astragaloside and Quercetin on rat myocardial cells after hypoxia].

OBJECTIVE: To investigate and compare the protective effects of Astragaloside IV (AST) and Quercetin (QUE) on rat myocardial cells after their exposure to hypoxia, and to determine their dose-effect relationship. METHODS: Myocardial cells from fetal SD rat were cultured in vitro and divided into 7 groups: i.e. A (hypoxia), B (hypoxia and 100 mg/L of QUE), C (hypoxia and 50 mg/L of QUE), D (hypoxia and 25 mg/L of QUE), E (hypoxia and 50.0 mg/L of AST), F (hypoxia and 25.0 mg/L of AST), G (hypoxia and 12.5 mg/L AST) H(hypoxia and 10 mg/L of VitE) groups. Different doses of AST and QUE were added into the culture media cells in each group before the myocardial cells receiving hypoxia for 12 hrs. The number of viable cells (CCK-8) and the content of lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), active oxygen (ROS, with detection only in A, C, F and H groups) were determined after hypoxia. RESULTS: The amount of LDH, MDA, ROS (C, F groups) in group B - G decreased significantly compared with those of group A, while the number of viable cells and the SOD content increased significantly. The protective effects were better in group B - G than that of the group H. With the same dosage, levels of LDH, CCK-8 in AST-treated groups were significantly lower than those in QUE-treated group (the number of viable cells in group C, F was 0.454 +/- 0.018, 0.471 +/- 0.017, and the content of lactate dehydrogenase was 2800 +/- 9,2312 +/- 52). There were no significant differences in MDA, SOD and ROS levels between AST and QUE treated groups (ROS in C and F groups were 16.0 +/- 5.3 vs 22.4 +/- 8.7, P > 0.05). CONCLUSION: AST and QUE might be beneficial in the protection of myocardial cells against hypoxia because of attenuation of oxidative damage. The protective effects of both AST and QUE are better than that of VitE, and that of AST is better than QUE as shown by a decrease in the amount of LDH and increase in the number of viable cells with the same dosage, but no obvious difference is shown between them in attenuating oxidative damage.

[Study on the influence of hypoxia induced microtubule damage on the opening of mitochondrial permeable transition pore of cardiac myocytes in rat].

OBJECTIVE: To investigate the influence of hypoxia induced microtubule damage on the opening of mitochondrial permeable transition pore (MPTP)of cardiac myocytes and on the decrease of respiratory function in rat. METHODS: Primary cultured myocardial cells from 30 neonatal rats were randomized as normoxic group (A), hypoxia group (B), normoxia with microtubule destabilizing agent group (C, with treatment of 8 micromol/L colchicines for 30 minutes before normoxia), and hypoxia with microtubule stabilizing agent group (D, with treatment of 10 micromol/L taxol for 30 minutes before hypoxia). beta-tubulin immunofluorescence ,the opening of mitochondria permeability transition pore, and the mitochondrial inner membrane potential were detected at 0.5, 1, 3, 6 and 12 post-treatment hours (PTH), and the mitochondrial respiratory function was determined by MTT method. The changes in these indices were also determined in A group at the corresponding time-points. RESULTS: Obvious damage of polymerized microtubule, opening of MPTP, mitochondrial inner membrane potential loss and decrease of myocardial respiratory activity were observed in both group B and C at 0.5 PTH, and they became more and more serious afterwards. However, the changes in the above indices in D group were much better than those in B group (P < 0.05 or 0.01), and no difference was found between D (92.8 +/- 4.0)% and C [(100.0 +/- 0.0) %, P > 0.05] groups. CONCLUSION: Hypoxia played a role in the myocardial microtubule damage as well as in the opening of MPTP. Moreover, hypoxia could also impair the mitochondrial respiratory function. Microtubule destabilizing agent could reproduce well the process of hypoxia induced microtubule damage, while the stabilizing agent exerted protective effect by improving the transition of mitochondrial permeability and the mitochondria respiratory function.

[Biological characteristics of human keratinocytes cultured in serum-free medium without feeder layer].

OBJECTIVE: To establish an optimal method for serum-free and feeder layer-free culture of human keratinocytes and to investigate their biological characteristics. METHODS: The keratinocytes were harvested from human foreskin of 5 children (aged 5-10 yr) and 5 adults (aged 20-30 yr). The samples were isolated by two-step digestion and the quantities of primary harvested HKCs were determined. The HKCs were then cultured in KCS serum-free culture medium. The morphology of HKCs were observed under light microscope. The HKCs and their growing speed were observed and identified under fluorescent microscope. The growth curve of HKCs was detected with MTT method, and the cell cycle was determined with flow cytometry. RESULTS: The number of harvested HKCs from children [(1.780 +/- 0.010) x 10(6)/cm(2)] was obviously higher than that from adults [(1.490 +/- 0.120) x 10(6)/cm(2)], (P < 0.01). Freshly isolated primary HKCs were round and transparent, and 94% of them were trypan blue resistant. The adherent speed and rate and lucent degree of multiply passaged HKCs increased followed by each passage. Under the fluorescent microscope, the cells exhibited strong Kelly fluorescence in the cytoplasm and with no staining in the nucleolus, thus the cells were identified as HKCs. The HKCs from children for skin could be passaged for more times [(11.0 +/- 1.2) times] than that from adults [(9.2 +/- 0.8) times], (P < 0.05). There was no clear sign of incubation period in the growth curve of HKCs, and both cellular proliferating speed and rate of proliferation were high. The percentage of cells in G1, G2 and S phase and the proliferation index was 36.15%, 25.17%, 38.68% and 63.85%, respectively. CONCLUSION: Serum-free and feeder layer-free culture seems to be an ideal method for the cultivation of HKCs.