几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.     

几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.     

几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.     

几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.     

几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.     

几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.     

几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.     

几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.     

几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.     

人角质形成细胞复合异种脱细胞真皮基质三维培养的形态学观察

Objective To establish the tridimensional culture method for tissue-engineered skin to observe the histomorphological change in human immortal KC strain (HacaT)cocultured with xenogenic acel-lular dermal matrix (ADM). Methods The ADM was prepared from SD rats by a modified method. HaCaTs were cultured in defined KC-serum free medium. HaCaTs in log growth phase were inoculated on ADM at the cell density of 2 × 105/cm2. They were submergedly cultured for 5 days and then changed to air-liquid phase culture for another 5 days. ADM and growth of HaCaTs on day 1 and 5 after cocultured with ADM were observed with scanning electron microscope. The histological change in ADM and HaCaTs on day 1, 5, and 10 after cocultured with ADM were examined by HE staining. Results The gross appearance of ADM was white with smooth and soft texture, and intact collagen bundles without cellular residue. HaCaTs adhered and stretched out pseudopodia on the surface of the ADM on day 1 after combined culture, and a monolayer of cells was formed on day 5, growing into 3 - 6 layers of cells on day 10 with a tendency to grow into ADM. Conclusions SD rats ADM is benefit for the adhesion of HaCaTs and the permeation of nutri-ent solution, from which an engineered multiple-layered human skin can be obtained within 10 days.