ObjectiveTo investigate the expression of keratinocyte growth factor (KGF) and cyclooxygen-ase-2 (COX-2) protein and microvessel density (MVD), and to explore their function and mechanism in the multistep process of gastric cancer. MethodsThe expressions of KGF and COX-2 protein in 64 samples of gastric cancer and 30 cases of normal gastric mucosa tissues were detected by immunohistochemistry. The MVD was detected by staining the endothelial cells in microvessles using anti-CD34 antibody. ResultsThe positive rate of KGF and COX-2 protein expression in gastric cancer were 65.6% (42/64) and 79.7% (51/64), respectively, which was significantly higher than that in normal gastric mucosa tissues 〔(23.3%, 7/30), P=0.046; (13.3%, 4/30), P=0.008〕. The MVD of gastric cancer was 31.8±8.0, which was significantly higher than that of normal gastric mucosa tissues (14.3±6.1), P=0.000. The MVD in gastric cancer with coexpressive KGF and COX-2 protein was 35.9±5.7, which was significant higher than that with non-coexpressive KGF and COX-2 protein (25.7±7.0), P=0.000. Both the expression of KGF and COX-2 protein were related to the invasion of serosa, lymph node metastasis and TNM staging (Plt;0.05, Plt;0.01). The MVD of gastric cancer tissues was related to lymph node metastasis and TNM staging (Plt;0.05), but unrelated to patient’s age, gender, and differentiation of tumor (Pgt;0.05). The co-expression of KGF and COX-2 protein was frequently found in patients with deeper invasion of serosa, lymph node metastasis, and higher TNM staging (Plt;0.05), but which was not associated withpatient’sage, gender, and differentiation of tumor (Pgt;0.05). The expression of KGF protein was positively correlated to the expression of COX-2 protein (r=0.610, P=0.000). There was positive correlation between MVD and the expression of KGF (r=0.675, P=0.000) and COX-2 protein (r=0.657, P=0.000) in gastric cancer, respectively. ConclusionKGF and COX-2 highly expressed by gastric cancer, which may be involved in the invasion and metastasis of gastric cancer by synergisticly promoting the angiogenesis.
Objective To investigate the outcome and histological changes of transplantation of acellular xeno-dermis combined with suspended keratinocytes.Methods Forty-two nude mice with full-thickness skin defect on the back were randomly divided into 2 groups, then acellular xeno-dermisand and suspended keratinocytes were adopted to cover the skin defect in the experimental group, pure suspended keratinocytes in the control group. The area of wound healing was calculated2, 3 and 5 weeks after transplantation, and the rates of wound contraction werealso calculated,and biopsy for histological examination was performed 3, 6and 12 weeks after transplantation. Results Compared with the experimental group,the control group showed delayed wound healing (P＜0.05), intensive wound contraction (P＜0.05), poor durability, elasticity, and cosmetic appearances as well asdisordered collagen fibers. In contrast, it was observed that the proliferationof collagen fibers was regularly organized, with no obvious acute immuno-rejection responses in the experimental group. Conclusion The composite transplantation of acellular xenodermis and suspended keratinocytes could promote the woundhealing with a satisfactory outcome.
OBJECTIVE: To fabricate artificial human skin with the tissue engineering methods. METHODS: The artificial epidermis and dermis were fabricated based on the successful achievements of culturing human keratinocytes(Kc) and fibroblasts (Fb) as well as fabrication of collagen lattice. It included: 1. Culture of epidermal keratinocytes and dermal fibroblasts: Kc isolated from adult foreskin by digestion of trypsin-dispase. Followed by comparison from aspects of proliferation, differentiation of the Kc, overgrowth of Fb and cost-benefits. 2. Fabrication of extracellular matrix sponge: collagen was extracted from skin by limited pepsin digestion, purified with primary and step salt fraction, and identified by SDS-PAGE. The matrix lattice was fabricated by freeze-dryer and cross-linked with glutaraldehyde, in which the collagen appeared white, fibrous, connected and formed pores with average dimension of 180 to 260 microns. 3. Fabrication artificial human skin: The artificial skin was fabricated by plating subcultured Kc and Fb separately into the lattice with certain cell density, cultured for one week or so under culture medium, then changed to air-liquid interface, and cultured for intervals. RESULTS: The artificial skin was composed of dermis and epidermis under light microscope. Epidermis of the skin consisted of Kc at various proliferation and differentiation stages, which proliferated and differentiated into basal cell layer, prickle cell layer, granular layer, and cornified layer. Conifilament not only increased in number, but also gathered into bundles. Keratohyalin granules at different development stages increased and became typical. The kinetic process of biochemistry of the skin was coincide with the changes on morphology. CONCLUSION: Tissue engineered skin equivalent has potential prospects in application of repairing skin defect with advantages of safe, effective and practical alternatives.
Objective To find new ways for wound healing and tissue expansion by reviewing of progress in recent years in functional molecules which are used for signaling channels of mechanical stress perception and mechanotransduction of keratinocyte. Methods The domestic and international articles were reviewed to summarize the functional molecules and signaling channels of mechanical stress perception and mechanotransduction of keratinocytes. Results The mechanism of mechanical stress perception includes mechano-sensitive channels, growth factor receptor-mediated mechanical stress perception, and mechanical stress perception by protein deformation. The mechanism of mechanotransduction includes cell adhesion-mediated signaling, mitogen-activated protein kinase signaling, the cytoskeleton and extracellular matrix, and so on. Conclusion Keratinocytes can response to the mechanical stress and transfer the effective information to undergo shaping, migration, proliferation, differentiation, and other biological behavior in order to adjust itself to adapt to the new environment.
OBJECTIVE: To investigate the selection and identification of human keratinocyte stem cells(KSC) in vitro. METHODS: According to the characteristics of KSC which can adhere to extracellular matrix very fast, we selected 3 groups of different time(5 minutes, 20 minutes and 60 minutes) and unselected as control group. And the cells were identified by monoclone antibody of beta 1-integrin and cytokeratin 19 (Ck19), then the image analysis was done. Furthermore we analyzed the cultured cells with flow cytometer(FCM) and observed the ultrastructure of the cell by transmission electron microscope(TEM). RESULTS: The cell clones formed in all groups after 10 to 14 days, while the cells of 5 minute group grew more slowly than those of the other groups, however, the clones of this group were bigger. The expression of beta 1-integrin and Ck19 were found in all groups. The positive rate of beta 1-integrin was significant difference between 5 minute group and the other groups (P lt; 0.05). And the expression of Ck19 was no significant difference between 5 minute group and 20 minute group(P gt; 0.05), and between 60 minute group and control group. But significant difference was observed between the former and the later groups(P lt; 0.05). The result of FCM showed that most cells of the 5 minute group lied in G1 period of cell cycle, which was different from those of the other groups. At the same time, the cells of 5 minute group were smaller and contained fewer organelles than those of the other groups. CONCLUSION: The above results demonstrate that the cells of 5 minute group have a slow cell cycle, characteristics of immaturity, and behaving like clonogenic cells in vitro. The cells have the general anticipated properties for KSC. So the KSC can be selected by rapid attachment to extracellular matrix and identified by monoclone antibody of beta 1-integrin and Ck19.
OBJECTIVE To search an ideal carrier of transferred keratinocytes for transplantation. METHODS The transferred keratinocytes were seeded on the surfaces of the artificial dermis and the silicone membrane and cultured in vitro for 2 weeks. The growth of the keratinocytes was observed by microscope and scanning electron microscope. RESULTS The keratinocytes implanted on the artificial dermis began to rupture and died after 2 to 3 days. While the keratinocytes adhered well on the surface of silicone membrane with pseudopodia formation after 1 week under scanning electron microscope, and the cells kept normal morphological and proliferative properties 2 weeks later. CONCLUSION The silicone membrane can be applied as an useful carrier for the keratinocytes transplantation.
Objective To investigate the different influence of the expression levels of the epidermal growth factor (EGF) and the keratinocyte growth factor (KGF) after the unilateral phrenectomy in piglets. Methods Thirty-six piglets were divided into 3 groups according to their ages during the operation (10 d,30 d,50 d). In each group, 6 piglets underwent the left cervical phrenectomy and 6 piglets were used as the shamoperation controls. The expression levels of EGF and KGF were determined by the real time quantitative RT-PCR at 2 weeks after operation.Results The melting curves of RTPCR showed that there was a single peark at the temperature of 80.0, 84.5 and 89.0℃ of EGF,KGF and GAPDH, respectively. In the experimental group, the expression levels of EGF were 3.53±0.36 and 1.73±0.29, and the expression levels of KGF were 4.71±0.42 and 2.77±0.29 in thepiglets undergiong the operation at their ages of 10 d and 30 d.Compared with the control group,the expression levels of EGF (4.60±0.41，2.18±0.24) and KGF(6.05±0.42，3.58±0.31) showed that there was a significant decrease postoperatively in the piglets undergoing the operation at their ages of 10 d and 30 d（P＜0.05）. However, there was no significant change in the piglets undergoing the operation at their ages of 50 d（P＞0.05）. The expression levels of EGF and KGF were significantly decreased with the lung development of the piglets（P＜0.05）. Conclusion The unilateral phrenectomy performed in the piglets younger than 30 d may cause abnormity of the EGF and KGF expression levels. The piglets older than 50 d may not cause a significant influence.
Objective Dermal papillae cells are widely applied to reconstruction of tissue engineered hair foll icle and skin. To investigate the difference of the biological characteristics of dermal papillae cells cultured with keratinocyte medium (KM)and normal medium (NM), and to determin whether it is feasible for the reconstruction of tissue engineered hair foll icle using dermal papillae cells cultured in KM. Methods Scalp samples were obtained in rhytidectomy procedure. Dermal papillaes were isolated by two steps digestive treatment, then cultured with KM and NM in two groups. The time of dermal papillae adherence and cell outgrowth was recorded and the rate of dermal papillae adherence was determined after 5 days. As well as, the difference of cell morphology was observed through inverted phase contrast microscope. The maximum generations were determined in two groups and the cell sheets were observed by HE staining. In third-generation cells, the number of aggregates in every dish and the prol iferation by MTT were compared between two groups. Meanwhile, the expression of α-smooth muscle actin (α-SMA) and ALP were detected by immunofluorescence and specific staining in two groups. Results Dermal papillaes of KM group had a higher rate of adherence and fast outgrowth. The rates of adherence were 54.17% and 36.78% in KM group and in NM group, respectively. In KM group, cells adhered after 24 hours and outgrew after 64 hours. While, cells adhered after 48 hours and outgrew after 80 hours in NM group. The cells were bigger in NM group than in KM group. In third-generation cells, 3.06 ± 1.12 and 9.25 ± 1.73 aggregates formed in NM group and KM group, respectively, the difference was significant (P lt; 0.05). In addition, cells could form cell sheets which were muti-layers in KM group. Mostly 7 and 15 generations could been subcultured in NMgroup and KM group, respectively. The result of MTT indicated that cells prol iferated more actively in KM group; absorbance value of KM group was significantly higher than that of NM group after 7 days (P lt; 0.05). The positive of α-SMA were detected in the third-generation cells of both groups. Ocassionally a l ittle few cells expressed ALP with (987 ± 146) m2 positive area in the sixth-generation cells of NM group. However, the cells still expressed ALP with (8 757 ± 558) μm2 positive area in the fourteenthgeneration cells of KM group and the difference was significant (P lt; 0.05). Conclusion Cells proliferate actively and aggregate obviously and could been subcultured more generations in KM. Therefore, culturing dermal papillae cells with KM is feasible for the reconstruction of tissue engineered hair foll icle.
To understand the reason of hyperpigmentation following full-thickness skin autograft, 54 guinea pigs were selected as skin autograft models. The changes of density of epidermal keratinocyte (KC) and the amount and distribution of melanin in the epidermal KC were observed by histological, histochemical and autoradiographic techniques. The results showed: (1) The histological changes of KC were in fact a wound-repair process; (2) The increased melanin in KC coordinated with "multi-overlapping screen" effect, a result of multiple layers of "supranuclear cap", was the direct cause of hyperpigmentation of the graft and (3) The hydrolytic disturbance of melanin in KC and the longer life span of the KC were probably the important reasons for the increase of melanin in the epidermis.
Objective To investigate the effects of heat injured keratinocytes (KC) supernatant on the expressions of collagen type I, collagen type III, and matrix metalloproteinase 1 (MMP-1) of dermal fibroblasts (Fb). Methods KC and Fb were isolated and cultured. Then the models of heat injured KC and Fb were reproduced in vitro, respectively. The heat injured and normal culture supernatant were collected respectively at 12 hours, and formulated as a 50% concentration of cell-conditioned medium. According to the culture medium, Fb at passage 3-5 was divided into 3 groups. Normal Fb was cultured with the conditioned medium containing 50% heat injured KC culture supernatant (group A), the conditioned medium containing 50% normal KC culture supernatant (group B), and DMEM (group C), respectively. The cells in 3 groups were collected at 24 hours. In addition， the cells in group A were collected at 0, 1, 2, 6, 12, 24, and 48 hours， respectively. Normal Fb was cultured with the conditioned medium containing 50% heat injured Fb culture supernatant. Then, the cells were collected at 0, 1, 2, 6, 12, 24, and 48 hours, respectively. The mRNA levels of the collagen type I, collagen type III, and MMP-1 of Fb were measured by real-time fluorescent quantitative PCR techniques. Results At 24 hours after cultured with supernatant of heat injured KC，mRNA relative expression levels of collagen type I, collagen type III, and MMP-1 in group A were significantly higher than those in groups B and C (P lt; 0.05). The mRNA relative expression levels of collagen type I, collagen type III, and MMP-1 in group A gradually increased with time going, showing significant differences between 0 hour and 2, 6, 12, 24, and 48 hours (P lt; 0.05); significant differences were found between different time points after 2 hours (P lt; 0.05). After Fb was treated with supernatant of heat injured Fb, the mRNA relative expression levels of MMP-1 gradually decreased with time going, showing significant differences between 0 hour and 1, 2, 6, 12, 24, and 24 hours (P lt; 0.05); after 2 hours of culture, significant differences were found among different time points (P lt; 0.05). Conclusion Heat injured KC supernatant may regulate the mRNA expressions of collagen type I, collagen type III, and MMP-1 of Fb.