Efficient Cultivation Conditions for Human Limbal Epithelial Cells

To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding casette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.

Effects of Damaged Human Corneal Epithelial Cells on Differentiation of Human Mesenchymal Stem Cell

PURPOSE: To identify the effects of microenvironmental changes caused by human corneal epithelial damages to characteristics or differentiation of human mesenchymal stem cells (hMSCs). METHODS: Artificial corneal damage was induced onto a cultured monolayer of human corneal epithelial cells. hMSCs were then co-cultured with damaged human corneal epithelial cells (dIHCE). Morphological changes in the co-cultured hMSCs were observed. To elucidate the differentiation of hMSCs into corneal keratocytes or epithelial cells, the expressions of alpha-smooth muscle actin, keratin-3/-12, and E-cadherin were confirmed by immunofluorescence. RESULTS: hMSCs co-cultured with dIHCE showed enhanced adherence in the neighborhood of dIHCE and morphological change into dendritic shapes at 6 days post-seeding. Although the expression of alpha-smooth muscle actin, known as hMSCs marker, significantly decreased at the dIHCE-contacted site of hMSCs; there were no expressional changes on keratin-3/-12 and E-cadherin, the markers of corneal epithelial cells. Interestingly, positive expression of corneal epithelial marker keratin-3/-12 was observed in dIHCE co-cultured hMSCs. hMSCs co-cultured with normal human corneal epithelial cells (nIHCE) were unable to attach, and showed no change in the expression of alpha-smooth muscle actin. CONCLUSIONS: It is proposed that dIHCE causes a morphological change in hMSCs, and decreased expression of alpha-smooth muscle actin. These results suggest that dIHCE can affect a change in the characteristics and differentiation of hMSCs.

Isolation of Putative Corneal Epithelial Stem Cells from Cultured Limbal Tissue

PURPOSE: To investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue. METHODS: Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus. Limbal tissues were extracted by incubation at 37 degrees C or 4 degrees C for 1 or 16 hours, respectively, with 1.2U/ml dispase/trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid (EDTA) at 37 degrees C in single procedure. Collected cells were cultured on NIH/3T3-seeded plates, and colony forming efficiency (CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed with a Coulter EPICS 753 after incubation with Hoechst 33342 and propidium iodide (PI). Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 nm BP filter. Gated cells of each fraction were re-cultured to assess the capability of colony formation. RESULTS: The mean numbers of viable cells obtained from treatment with dispase and trypsin was 3x10(4) cell/ml and 8.06x10(5) cell/ml at 37 degrees C and 4 degrees C incubations; the number increased to 1.21x10(6) cell/ml with a trypsin/EDTA treatment (p<0.05). CFE was 9.67+/-2.13% and 6.63+/-2.35% in rabbit and human cells, respectively. Likewise, the Hoechst negative fraction was 3.61+/-0.42% and 5.21+/-4.91% in rabbit and human cells, respectively. The sorted Hoechst negative cells were cultured through four passages, forming small round colonies. In rabbit cells, the CFEs of Hoechst negative and positive fractions after FACS, were 12.67+/-2.24% and 1.17+/-6.13%, respectively (p<0.05). CONCLUSIONS: Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS. This technique may be very useful in tissue engineered stem cell therapy.

Isolation of Putative Limbal Epithelial Stem Cells by Fluorescein Activated Cell Sorting

PURPOSE: To analyze the isolating pattern of slow cycling cells as putative limbal epithelial stem cells (PLESCs) using Hoechst exclusive cell sorting. METHODS: Rabbits were injected with 5-bromo-2-deoxyuridine (Brd U) 1 month prior to be sacrificed. After obtaining limbal tissues, fluorescence-activated cells were sorted on a Coulter EPICS 753 after they had been incubated with Hoechst 33342 and propidium iodide. Two different methods were applied to sort PLESCs. Side-population(Sp) cells were obtained using gates with dichroic mirror to detect low Hoechst blue and red after verapamil was treated. Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 BP filter. Brd U-retaining cells were counted and their sizes were evaluated in each gated sample to compare isolating pattern of PLESCs in each method. RESULTS: The percentages of Sp cells and of the Hoechst negative fraction were 0.96 +/- 0.79% and 16.01 +/- 13.60%, respectively(p=0.021). Homogeneity and density of the small cells were higher in Hoechst negative fraction than in Sp cells. The percentage of Brd U-retaining cells was 47.36 +/- 10.34% and 47.14 +/- 14.94% in Sp cells and Hoechst negative fraction, respectively(p>0.05), and they were 10 times higher than in non-Sp and Hoechst positive fraction(p=0.000). CONCLUSIONS: Hoechst negative exclusion without verapamil more efficiently isolated PLESCs than Sp did.

Three Dimensional Reconstitution of Oral Mucosal Keratinocytes and Its Biologic Characteristics

The purposes of this study were to develop an in vitro co-culture model of epithelial tissue with dermal equivalent, cultured at an air-liquid interface, and to evaluate the effects of extracellular matrix and concentration of calcium and fetal bovine serum in medium to find optimized culture condition. Oral keratinizing epithelial cells in monolayer culture were grown in Mitomycin-treated 3T3 feeder. Primary cultured oral epithelial cells were reconstituted onto the dermal equivalents consisting of 3T3 fibroblast and type I collagen, and co-culture was grown at the air-liquid interface. The histomorphological development of reconstituted oral epithelium in vitro for 21 days revealed 10~12 layered statified epithelium, closely similar to the parakeratinized gingival epithelium. Neither laminin nor type IV collagen was able to induce keratinocyte differentiation. But a mixture of laminin and type IV collagen induced well-polarized keratinizing tissue with anchoring structure of basal cells. When the reconstituted oral epithelium was incubated in 1.0% and 0.5% serum-containing medium, the granular cell layers with orthokeratinization developed. The reconstituted epidermis generated in serum-free keratinocyte growth medium (KGM)-containing pituitary extract showed features of incomplete differentiation. The present study shows that the dermal equivalents containing fibroblasts will support epidermal morphogenesis and differentiation. And these results suggest that extracellular matrix and calcium concentration are important factors during the reconstitution of keratinizing epithelium in vitro.

cDNA Cloning and Expression of Angiostatin, an Angiogenesis Inhibitor , from Human Liver Tissue mRNA / 대한암학회지

PURPOSE: Angiostatin, a 38 kDa internal fragment of plasminogen, is a potent inhibitor of angiogenesis. It blocks neovascularization and growth of primary and metastatic tumors in mice. To produce recombinant angiostatin protem comprising kringle 1-4 of plasminogen, we cloned the angiostatin cDNA from human liver tissue mRNA and expressed it in E. coli. MATERIALS AND METHODS: We cloned angiostatin cDNA from human liver tissue mRNA using reverse transcriptase polymerase chain reaction (RT-PCR) method. Cloned cDNA was ligated to pET22b (+) expression vector, transformed into E. coli stram BL21 (DE3) and expressed by IPTG induction. Recombinant human angiostatin protein was purified from the inclusion bodies of lysated bacterial pellet with 8 M urea solubilization, refolding, single step Lysine-Sepharose 4B affinity chromatography and 0.2 M E-aminocarproic acid elution. The anti-angiogenic activity of purified recombinant angiostatin was assayed with endothelial cell proliferation assay and chorioallantoic membrane assay (CAM). RESULTS: The identification of cloned angiostatin cDNA was confirmed by Southern hybridization and Pst I restriction enzyme digestion pattern. Angiostatin cDNA was expressed in E. coli, refolded in vitro and purified by Lysine Sepharose 4B affinity chromatography. The molecular weight of purified recombinant angiostatin was about 55 kDa on the SDS-PAGE. It inhibited the proliferation of bovine capillary endothelial (BCE) cells in vitro with a half-maximal inhibition concentration (ED50) of approximately 500 ng/mL. It also suppressed neovasculrization on the CAM assay. CONCLUSION: These results demonstrated that recombinant human angiostatin has similar function and biological activity compared with human angiostatin which is purified from porcine elastase digested human plasminogen fragment.

Expression of urokinase: type plasminogen activator, its receptor, and its inhibitor in gastric adenocarcinoma tissues

The plasminogen and plasmin system, which is mainly regulated by urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1), is generally believed to play a role in cancer invasion and metastasis. This study was conducted to investigate the role of uPA, uPAR and PAI-1 in the invasion and metastasis of gastric adenocarcinoma. The expression of mRNAs for uPA and PAI-1 was determined by Northern blot analysis in nine primary gastric cancer tissues, nine paired metastatic lymph nodes and normal gastric mucosa. The mRNA of uPA was not or faintly detected in normal mucosa, while the expression was increased in both primary gastric cancer tissues and metastatic lymph nodes to a similar degree. The mRNA expression for PAI-1 in the gastric cancer tissues was not different from that in the paired metastatic lymph nodes and normal mucosae. uPAR was determined by immunohistochemical staining, demonstrating that five (56%) and six (67%) out of nine primary gastric cancer tissues and nine paired metastatic lymph nodes were positive, respectively and the intensity was stronger in metastatic lymph nodes. The results support the concept that most gastric cancer cells may have an innately moderate level of uPA and uPAR, and that increase of uPAR expression can be considered to be closely associated with cancer invasion and metastasis.

Effects of Calcipotriol ( MC 903 ) on Proliferation and Differentiation of Cultured Human Epidermal Keratinocytes / 대한피부과학회지

BACKGROUND: Calcipotriol(MC903), a new vitamin D(3) analogue, has been reported to be effective in the treatment of patients with psoriasis. OBJECTIVE: The purpose of this study is to examine the effects of calcipotriol on proliferation and differentiation of the keratinocytes in monolayer cultures and three-dimensional cultures. METHODS: Using moriolayer cultures, we examined morphological changes of keratinocytes and performed [(3)H]thymidine incorporation after calcipotriol was added into the medium. Using three dimensional cultures, we performed two experiments: one with cultures treated with calcipotriol immediately after the keratinocytes had been exposed to the air and another set of cultures treated with calcipotriol after three dimensional morphogenesis of the keratinocytes. We examined morphological changes of keraitinocytes and performed a immunohistochemical study for proliferation differentiation markers RESULTS: In monolayer cultures, at calcipotriol concentrations of 10(-9)M-10(-6)M, keratinocytes became larger, more irregular, and flattened in a dose-dependent manner. At 10(-9)M-10(-6)M, [3Hl thymidine incorporatiorn was decreased dose-dependently as compared to the control culture. In the first experiment using three-dimensional cultures, at 10(-9)M-10(-6)M, total epidermal layers were thinned. This was associated with thinnings of nucleated and horny layers in a dose dependent manner. In the seconcd experiment using three-dimensional cultures, at 10(-8)M-10(-6)M, nucleated layers were thinned in a dose dependent manner, but the horny layer was slightly thickened, as compared to the control culture. Immunohistochemical studies showed a reduction of differentiation markers such as keratin 1, involucrin, filaggrin, loricrin consistent with a thinning of nucleated layers in the epidermal architecture in both experiments. In the basal layer, at 10(-9)M-10(-6), PCNA-positive cells were and BrdU-positive cells were decreased dose-dependently as compared to the control culture. CONCLUSIONS: In this study, we demonstrated that at 10(-9)M-10(-6) calcipotriol inhibited keratinocytes proliferation and stimulated keratinocytes differentiation in a dose-dependent manner.

Overexpression of tissue inhibitors of metalloproteinase-1 and -2 in the stroma of gastric cancer

The fundamental event of cancer invasion and metastasis is the complicated interaction of cancer cells with host cells, in which event, a number of proteases and their inhibitors are involved. Matrix metalloproteinases are the potent proteases in degrading the basement membrane and extra cellular matrix and are inhibited by specific endogeneous inhibitors, tissue inhibitors of metalloproteinases-1(TIMP-1) and TIMP-2. The expression of mRNA for TIMP-1 and -2 was investigated by Northern blot analysis in specimens taken from 27 patients with primary gastric adenocarcinoma; 25 samples from the primary site, six from the metastatic lymph nodes and two from the peritoneal fluids. The expression for TIMP-1 and -2 was compared in primary gastric cancer tissues, metastatic lymph nodes and normal gastric mucosae. TIMP-1 mRNA was overexpressed in 24 (96%) out of 25 primary cancer tissues compared with the paired normal mucosae, while TIMP-2 was in 10 (40%). In six specimens of metastatic lymph nodes, TIMP-1 and -2 were overexpressed in 6 (100%) and 4 (67%) specimens, respectively. Of two specimens prepared from the peritoneal fluids, all specimens overexpressed TIMP-1 compared with the those of primary cancer tissues, while one (50%) specimen overexpressed TIMP-2. Immunohistochemical staining was done to investigate the localization of TIMP-1 and -2, demonstrating that the immunoreactivity for TIMP-1 and -2 was clearly detected in the cytoplasm of the stromal cells. These results suggest that both TIMP-1 and -2 are overexpressed by stromal cells in most of primary and some metastatic gastric cancer tissues and that TIMP-1 and TIMP-2, produced by stromal cells, may play an important role in inhibiting the proteolytic activity of matrix metalloproteinases originated from cancer cells, in gastric cancer.

Overexpression of tissue inhibitors of metalloproteinase-1 and -2 in the stroma of gastric cancer

The fundamental event of cancer invasion and metastasis is the complicated interaction of cancer cells with host cells, in which event, a number of proteases and their inhibitors are involved. Matrix metalloproteinases are the potent proteases in degrading the basement membrane and extra cellular matrix and are inhibited by specific endogeneous inhibitors, tissue inhibitors of metalloproteinases-1(TIMP-1) and TIMP-2. The expression of mRNA for TIMP-1 and -2 was investigated by Northern blot analysis in specimens taken from 27 patients with primary gastric adenocarcinoma; 25 samples from the primary site, six from the metastatic lymph nodes and two from the peritoneal fluids. The expression for TIMP-1 and -2 was compared in primary gastric cancer tissues, metastatic lymph nodes and normal gastric mucosae. TIMP-1 mRNA was overexpressed in 24 (96%) out of 25 primary cancer tissues compared with the paired normal mucosae, while TIMP-2 was in 10 (40%). In six specimens of metastatic lymph nodes, TIMP-1 and -2 were overexpressed in 6 (100%) and 4 (67%) specimens, respectively. Of two specimens prepared from the peritoneal fluids, all specimens overexpressed TIMP-1 compared with the those of primary cancer tissues, while one (50%) specimen overexpressed TIMP-2. Immunohistochemical staining was done to investigate the localization of TIMP-1 and -2, demonstrating that the immunoreactivity for TIMP-1 and -2 was clearly detected in the cytoplasm of the stromal cells. These results suggest that both TIMP-1 and -2 are overexpressed by stromal cells in most of primary and some metastatic gastric cancer tissues and that TIMP-1 and TIMP-2, produced by stromal cells, may play an important role in inhibiting the proteolytic activity of matrix metalloproteinases originated from cancer cells, in gastric cancer.

Peripheral blood lymphocyte subsets in patients with stomach cancer

The present study was conducted in order to investigate the immunologic alterations alongside the numerical changes in peripheral blood lymphocytes(PBL) and their subsets in stomach cancer patients. Lymphocyte surface markers were determined in 85 stomach cancer patients and 49 controls by indirect immunofluorescence technique using monoclonal antibodies. Monoclonal antibodies used were Leu 2a(CD8, suppressor/cytotoxic T cells), Leu 3a(CD4, inducer/helper T cells), Leu 4(CD3, pan T reagent), Leu 11(CD16, natural killer cells) and Leu 12(CD19, B cells). The numbers of PBL, CD3+, CD4+, CD8+, CD16+ and CD19+ cells significantly decreased and the CD4: CD8 value increased in 85 patients with stomach cancer compared to those in controls(p < 0.01). In stage I(n = 17), neither PBL, their subsets nor the CD4: CD8 value were significantly different from those of the controls. In stage II(n = 17), the numbers of PBL, CD3+, CD4+ and CD8+ cells decreased(p < 0.01). In stage III(n = 24) and IV(n = 27), PBL and all subsets measured decreased(p < 0.01). The CD4: CD8 value showed significant increases in stages III and IV(p < 0.01), because the CD8+ cells decreased to a greater extent than did the CD4+ cells. The results demonstrating that the lymphocyte subsets are depressed differentially with the stage suggest that host immunity is impaired with the progression of stomach cancer.

Effects of combination timing of recombinant human interferon-?and adriamycin on cytotoxicity to human stomach cancer cells / 대한면역학회지

No abstract available.

Mutations of p53 tumor suppressor gene in human lung cancer cell lines / 결핵

No abstract available.