No abstract available.
Culture Media, Conditioned* ; Thymocytes*

During bone remodeling, there is requirement of differentiation of osteoblastic cells. Previously, we identified proteins differentially expressed in soft tissue during bone healing. Of these proteins, we focused the effect of LTF on differentiation of osteoblast. In order to analyze the osteogenic ability of LTF, we treated conditioned media collected from human LTF-stably transfected HEK293T cells into osteoblastic MC3T3-E1. The results showed that the activity and expression of alkaline phosphatase were increased in MC3T3-E1 cells treated with conditioned media containing LTF in dose- and time-dependent manner. At the same time, we observed the significant increase of the expression of osteoblastic genes, such as ALP, BSP, COL1A1, and OCN, and along with matrix mineralization genes, such as DMP1 and DMP2, in LTF conditioned media-treated groups. Moreover, the result of treating recombinant human LTF directly into osteoblastic MC3T3-E1 showed the same pattern of treating conditioned media containing LTF. Our study demonstrated that LTF constitutively enhances osteoblastic differentiation via induction of osteoblastic genes and activation of matrix mineralization in MC3T3-E1 cells.
Alkaline Phosphatase ; Bone Remodeling ; Culture Media, Conditioned ; Humans ; Lactoferrin* ; Osteoblasts*

Dental pulp is a highly vascularized tissue with high regenerative potential. Revascularization of severed vasculature in the tooth is required for pulp healing during avulsed tooth treatment. In this study, the relative expression of angiogenesis-related proteins was determined in human dental pulp cells using a human angiogenesis proteome profiler array. The proteome profiler array detected differentially expressed angiogenesis-related factors under conditions of hypoxia, which enhances the angiogenic potential of dental pulp cells. We confirmed that hypoxia regulates the mRNA expression of angiogenesis-related factors, including CXCL16 in dental pulp cells. Furthermore, conditioned media of hypoxic pulp cells induced tube-like structures of vascular endothelial cells, which were reduced by the neutralization of CXCL16 function. In conclusion, our data show that angiogenesis-related factors are differentially expressed by hypoxia in dental pulp cells and suggest that CXCL16 may involve in the revascularization of hypoxic dental pulp.
Anoxia* ; Culture Media, Conditioned ; Dental Pulp* ; Endothelial Cells ; Humans* ; Proteome ; RNA, Messenger ; Tooth ; Tooth Avulsion

To evaluate the effects of kerationocytes on the growth of melanocytes, keratinocyte conditioned media (K-CM) with different molecular weight obtained by dialysis were added to melanocyte growth medium (M-GM). In addition, K-CM only, and K-CM mixed with each component of M-GM, such as TPA(12-tetradecanoyl- phorbol-13-acetate), IBMX(isobutylmethylxanthine) and CT(cholera toxin), were used for the culture of melanocytes. 1) The proliferation of melanocytes was incresased to 2.86 x 10(5)+/-0.87 x 10(5) cells/ well and 2.87 x 10(5)+/-0.71 x 10(5) cells/well in 25% K-CM with a cut-off molecular weight of 2,000 and 25% K-CM with a cut,-off molecular weight between 6000 8000 respectively, as compared to 1.88 x 10(5)+/-0.45 x 10(5) cells/well in the control group (p < 0.05). 2) The amount of melanin was increased to 0.2987+/-0.0830ng/mlin 25% un- dialyzed K-CM, as compared to 0.2264+/-0.0643ng/ml in the control group, but this differnce was not statistically significant. 3) Maximum proliferation of melanocytes was observed in 35% concentration of K-CM with a cut-off molecular weight of 6000 8000. 4) Maximum of melanin production was observed in 35% concentration of undialyzed K-CM 5) As compared to 7.86 x 10+/-1.74 x 10(5) cells/well in M-GM,proliferation of melanocytes in 35% K-CM with a cut-off molecular weight of 6000 8000 was de- creased to 1.38 X 10(5)+/-0.97 X 10(5) cells/well. 6) There was no difference in melanocyte proliferation between 6.81 x 10(5)+/-2.19 x 10(5) cells/well in 35% 6,000 8,000 M.W. cut-off dialyzed K-CM, with IBMX only, and 7.86 x 10(5)+/-1.74 x 10(5) cells/well in M-GM. 7) Compared to 0.2303+/-0.0700ng/well cell in M-GM, the amount of melanin was increased to 0.3227+/-0.0900ng/cell, 0.3624+/-0.0900ng/cell and 0.2928+/-0.0500ng/cell, respectively, when TPA, IBMX, CT was added to 35% undialyzed K-CM. It also increased to 0.3176+/-0.1100 in 35% undialyzed K-CM(p<0.05). In summary, the results proved that cellular activating substances released from keratinocytes affect the proliferation of melanocyte and the synthesis of melain. It is also expected that methods used in this study can be clinically utilized because melanocyte culture is possible on K-CM without adding tumor promotors.
1-Methyl-3-isobutylxanthine ; Culture Media, Conditioned ; Dialysis ; Keratinocytes* ; Melanins ; Melanocytes* ; Molecular Weight

BACKGROUND: The regulatory rnechanisrns of melanocytes underlying ultraviolet(UV) melanogenesis have been an interest tu many investigators. They have shown that several materials produced and secreted frorn norm il human keratinocytes play roles as mitogens of human melanocytes, and demonstrated that UVB exposure stirnulated highly the paracrine linkage of endothelin(ET) be tween keratinocytes and melanocytes. It suggest that ET is one of keratinocytes-derived intrinsic mitogens in UVB induced hyperpigmentation. OBJECTIVE: To evaluate whether ET secreted from keratinocytes under the UV irradiation works as paracrine effects such as melanocyte pro)iferation and function, the present, study was under taken. METHODS: Primary kevatinocytes and rnelanocytes cultures from neonatal foreskins were grown in complete MCDB 154 medium. Cultured human keratinocytes were irradiated with UVB 50mJ/ cm2. Twenty fours hour later, conditioned medium of keratinocytes was added into the growth medium of melanocytes of concentration in 10%, 20%, and 30%, respectively. Four days later, in order to detect of melanocytes proliferat.ion and function, number of melanocytes, melanin granules and tyrosinase activity v ere measured. RESULTS: J. The number of me anocytes were higher increase in groups of incubation with conditioned medium of irradiated keratinocytes than that of incubation with conditioned mediurn of irradiated keratinocytes and treatment of anti-ET-1(5ug/ml)(p>0.05). 2. The melanin contents were significantly higher increase in groups of incubation with conditioned medium(20%, 30%) of irradiated keratinocytes than that of incubation with conditioned medium of irradiated keiatinocytes and treatment of anti-ET-1(5ug/ml)(p<0.05). 3. The tyrosinase activity of melanocyte incubated with 30% COllcentration of conditioned medium from cultured keratinocyte irradiated with UVB was significantly higher increase than that of melanocyte incubated with 30% concentration of conditioned mediurn from cultured keratinocytes irradiated with UVB and treated with anti-ET-1(p<0.05). CONCLUSION: This stud provided an important confirmation of the proposal that ET-1 is intrin sic factor for proliferation and differentiation of human melanocytes. These findings suggest that keratinocyte derived ET-1 make a considerable effect on human melanocyte proliferation and function in UV melanog nesis.
Culture Media, Conditioned ; Endothelin-1* ; Foreskin ; Humans* ; Hyperpigmentation ; Keratinocytes* ; Melanins ; Melanocytes* ; Mitogens ; Monophenol Monooxygenase ; Research Personnel

No abstract available.
Animals ; Antibody Formation* ; Bone Marrow Cells* ; Bone Marrow* ; Culture Media, Conditioned* ; Mice* ; Thymocytes*

OBJECTIVETo observe the effects of conditioned medium from keloid fibroblasts under hypoxia on angiogenesis, and to investigate the role of hypoxic microenvironment in invasive growth of keloid.

METHODSPrimary keloid fibroblasts and human umbilical endothelial cells (HUVEC) were cultured as conventional method. Keloid fibroblasts were cultured either in a hypoxic incubator (2% O2) for 48 h or in a normoxic incubator (20% O2) as control. Then those cell culture mediums were collected and mixed with endothelial cell medium by the proportion of 1:1 as conditioned medium. The mRNA and secreted protein of pro-angiogenic factors such as vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and periostin of keloid fibroblasts under hypoxia were detected by real time PCR and ELISA. The proliferation, migration and invasion, tube formation of HUVEC cultured with conditioned medium were evaluated by CCK-8 assay, Transwell assay and matrigel tube formation assay, respectively.

RESULTSHypoxia increased the expression of VEGF, Ang-1 and periostin in both mRNA (increased by 75%, 43% and 118% respectively, P < 0.05) and secreted protein (increased by 30.2%, 14.2% and 19.5% respectively, P < 0.05) levels; the proliferations of HUVEC in hypoxic conditioned medium in 1, 2 and 3 d were 0.67 +/- 0.07, 0.84 +/- 0.09 and 1.08 +/- 0.10 respectively, which were higher compared to those in control group (0.52 +/- 0.08, 0.72 +/- 0.10 and 0.91 + 0.14, P < 0.05); the numbers of migration, invasion and tube formation of HUVEC were (73.2 +/- 8.9), (56.3 +/- 12.5), (9.66 +/- 1.96) cells/HP, which were higher compared to those in control group [(59.0 +/- 8.0), 35.5 +/- 8.5), (6.5 +/- 1.87) cells/HP, P < 0.05].

CONCLUSIONSHypoxia increases the expression of pro-angiogenic factors of keloid fibroblasts, and its conditioned medium under hypoxia could promote angiogenesis. The results suggest hypoxic microenvironment may play a significant role in the invasive growth of keloid by inducing angiogenesis.

OBJECTIVETo investigate the influence of the materials commonly used in the assisted reproduction procedure on human germ cells and embryo development.

METHODSWe used human sperm survival assay to detect the influence of two brands of culture dishes, two brands of injection needles, washed and unwashed ovum aspiration needles, embryo transfer catheters and surgical gloves on sperm motility. All the data obtained went through variance analysis with SPSS 13.0.

RESULTSSperm motility differed significantly between Nunclons and Falcon's culture dishes (52.68 +/-16.21 vs 45.36 +/- 15.25, P < 0.01) but not between the BD and the Jie-rui injection needles (P > 0.05), nor among the washed and unwashed ovum aspiration needles, embryo transfer catheters and surgical gloves (P > 0.05).

CONCLUSIONDifferent brands of assisted reproduction materials of similar use had different or similar influences on germ cells and embryos, while no difference existed in the influences of the washed and unwashed ovum aspiration needles, embryo transfer catheters and surgical gloves.

As a nonhistone DNA-binding protein, high mobility group box 1 (HMGB1) is released in large amounts into the extracellular space immediately after ischemic insult and plays a role in the release of proinflammatory cytokines. Here, we the examined cytokine-like or signaling molecule-like function of extracellular HMGB1 in primary cortical cultures. We found that a large amount of HMGB1 was released following zinc-induced neuronal cell death in primary cortical cultures and that this extracellular HMGB1 might aggravate neuronal damage. The conditioned media collected from zinc-treated primary cortical cultures decreased neuronal cell survival to 69.6+/-1.4% of control values when added to fresh primary cortical cultures. In contrast, treatment with HMGB1-depleted conditioned media produced by cultures treated with an HMGB1 siRNA-expression vector suppressed the induction of neuronal death. A mutant HMGB1 siRNA-expression vector did not suppress the induction of neuronal death, demonstrating a role of HMGB1 in neuronal death. Moreover, HMGB1-depletion in media conditioned by cotreatment with anti-HMGB1 antibody or with anti-RAGE antibody, a potential receptor for HMGB1, recovered neuronal cell survival to 81.0+/-4.0% and 79.0+/-4.0%, respectively, when added to fresh primary cortical cultures. These results indicate that extracellular HMGB1 released after zinc treatment induces neuronal death, which might aggravate zinc toxicity.
Cell Death ; Cell Survival ; Culture Media, Conditioned ; Cytokines ; Extracellular Space ; HMGB1 Protein ; Neurons ; Zinc

OBJECTIVE: To establish a primary culture method of human omental preadipocytes. METHODS: Using enzyme-digesting method, fibroblast-like cells from the human omental adipose tissues were cultured, and then differentiated by conditional medium, and identified by oil red O staining. RESULTS: The cultured cells were highly homogeneous, and highly proliferative in 4-5th generation. During the process of induction by conditional medium, the cells became round-like and larger, and more adipose droplets were aggregated. By oil red O staining, we confirmed the differentiated cells were mature adipocytes. CONCLUSION In human omental adipose tissues, there are some preadipocytes, which can differentiate into mature adipocytes with appropriate stimulus.
Adipocytes ; cytology ; Adult ; Cell Differentiation ; Cells, Cultured ; Culture Media, Conditioned ; Humans ; Male ; Omentum ; cytology