EFFECTS OF HEAT INJURED KERATINOCYTES SUPERNATANT ON EXPRESSIONS OF COLLAGEN TYPE I, COLLAGEN TYPE III, AND MATRIX METALLOPROTEINASE 1 OF DERMAL FIBROBLASTS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YANG Xuekang , BAI Xiaozhi , ZHANG Yue , LI Xiaoqiang , SHI Jihong , TANG Chaowu , HU Dahai.

Burn Center of Chinese PLA, Xijing Hospital, the Fourth Military Medical University, Xi’an Shaanxi, 710032, P.R.China. Corresponding author: HU Dahai, E-mail: burns@fmmu.edu.cn;

Corresponding author：

Keywords：

Keratinocytes; Dermal fibroblasts; Heat injury; Collagen type I; Collagen type III; Matrix metalloproteinase 1

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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.

Citation： YANG Xuekang,BAI Xiaozhi,ZHANG Yue,LI Xiaoqiang,SHI Jihong,TANG Chaowu,HU Dahai.. EFFECTS OF HEAT INJURED KERATINOCYTES SUPERNATANT ON EXPRESSIONS OF COLLAGEN TYPE I, COLLAGEN TYPE III, AND MATRIX METALLOPROTEINASE 1 OF DERMAL FIBROBLASTS. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(12): 1497-1500. doi: Copy

1.	沈丹蓓, 夏隆庆. 角质形成细胞与皮肤创面愈合瘢痕形成. 中华整形外科杂志, 2005, 21(1): 69-71.
2.	白晓智, 吕根法, 谢松涛, 等. 离体角质形成细胞热损伤模型建立及细胞凋亡观察. 中华烧伤杂志, 2009, 25(3): 189-192.
3.	Xia W, Phan TT, Lim IJ, et al. Complex epithelial-mesenchymal interactions modulate transforming growth factor-beta expression in keloid-derived cells. Wound Repair Regen, 2004, 12(5): 546-556.
4.	El Ghalbzouri A, Hensbergen P, Gibbs S, et al. Fibroblasts facilitate re-epithelialization in wounded human skin equivalents. Lab Invest, 2004, 84(1): 102-112.
5.	Werner S, Krieg T, Smola H. Keratinocyte-fibroblast interactions in wound healing. J Invest Dermatol, 2007, 127(5): 998-1008.
6.	van der Veer WM, Bloemen MC, Ulrich MM, et al. Potential cellular and molecular causes of hypertrophic scar formation. Burns, 2009, 35(1): 15-29.
7.	Wang X, Liu Y, Deng Z, et al. Inhibition of dermal fibrosis in self-assembled skin equivalents by undifferentiated keratinocytes. J Dermatol Sci, 2009, 53(2): 103-111.
8.	Bellemare J, Roberge CJ, Bergeron D, et al. Epidermis promotes dermal fibrosis: role in the pathogenesis of hypertrophic scars. J Pathol, 2005, 206(1): 1-8.
9.	Harrison CA, Gossiel F, Bullock AJ, et al. Investigation of keratinocyte regulation of collagen I synthesis by dermal fibroblasts in a simple in vitro model. Br J Dermatol, 2006, 154(3): 401-410.
10.	Tandara AA, Kloeters O, Mogford JE, et al. Hydrated keratinocytes reduce collagen synthesis by fibroblasts via paracrine mechanisms. Wound Repair Regen, 2007, 15(4): 497-504.
11.	马忠锋, 柴家科. 表皮细胞的培养移植的现状与展望. 中华烧伤杂志, 2005, 21(2): 158-160.
12.	Vashishta A, Saraswat Ohri S, Vetvickova J, et al. Procathepsin D secreted by HaCaT keratinocyte cells—A novel regulator of keratinocyte growth. Eur J Cell Biol, 2007, 86(6): 303-313.
13.	白晓智, 胡大海, 石继红, 等. 热损伤角质形成细胞培养上清对真皮成纤维细胞增殖与胶原mRNA表达的影响. 中华实验外科杂志, 2011, 28(3): 380-382.
14.	白晓智, 胡大海, 张万福, 等. 热损伤角质形成细胞培养上清液对成纤维细胞生物学行为的影响. 中华烧伤杂志, 2010, 26(2): 133-137.
15.	Moulin V, Larochelle S, Langlois C, et al. Normal skin wound and hypertrophic scar myofibroblasts have differential responses to apoptotic inductors. J Cell Physiol, 2004, 198(3): 350-358.
16.	陈苏丽, 刘流. 上皮细胞-干细胞在创伤愈合中的作用. 中国组织工程研究与临床康复, 2010, 14(6): 1092-1096.
17.	Kisseleva T, Brenner DA. Mechanisms of fibrogenesis. Exp Biol Med, 2008, 233(2): 109-122.
18.	Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology, 2008, 134(6): 1655-1669．.
19.	Risinger GM Jr, Hunt TS, Updike DL, et al. Matrix metalloproteinase-2 expression by vascular smooth muscle cells is mediated by both stimulatory and inhibitory signals in response to growth factors. J Biol Chem, 2006, 281(36): 25915-25925.
20.	Ravanti L, Kähäri VM. Matrix metalloproteinases in wound repair (review). Int J Mol Med, 2000, 6(4): 391-407.
21.	Longaker MT, Whitby DJ, Adzick NS, et al. Studies in fetal wound healing, VI. Second and early third trimester fetal wounds demonstrate rapid collagen deposition without scar formation. J Pediatr Surg, 1990, 25(1): 63-69.

1. 沈丹蓓, 夏隆庆. 角质形成细胞与皮肤创面愈合瘢痕形成. 中华整形外科杂志, 2005, 21(1): 69-71.
2. 白晓智, 吕根法, 谢松涛, 等. 离体角质形成细胞热损伤模型建立及细胞凋亡观察. 中华烧伤杂志, 2009, 25(3): 189-192.
3. Xia W, Phan TT, Lim IJ, et al. Complex epithelial-mesenchymal interactions modulate transforming growth factor-beta expression in keloid-derived cells. Wound Repair Regen, 2004, 12(5): 546-556.
4. El Ghalbzouri A, Hensbergen P, Gibbs S, et al. Fibroblasts facilitate re-epithelialization in wounded human skin equivalents. Lab Invest, 2004, 84(1): 102-112.
5. Werner S, Krieg T, Smola H. Keratinocyte-fibroblast interactions in wound healing. J Invest Dermatol, 2007, 127(5): 998-1008.
6. van der Veer WM, Bloemen MC, Ulrich MM, et al. Potential cellular and molecular causes of hypertrophic scar formation. Burns, 2009, 35(1): 15-29.
7. Wang X, Liu Y, Deng Z, et al. Inhibition of dermal fibrosis in self-assembled skin equivalents by undifferentiated keratinocytes. J Dermatol Sci, 2009, 53(2): 103-111.
8. Bellemare J, Roberge CJ, Bergeron D, et al. Epidermis promotes dermal fibrosis: role in the pathogenesis of hypertrophic scars. J Pathol, 2005, 206(1): 1-8.
9. Harrison CA, Gossiel F, Bullock AJ, et al. Investigation of keratinocyte regulation of collagen I synthesis by dermal fibroblasts in a simple in vitro model. Br J Dermatol, 2006, 154(3): 401-410.
10. Tandara AA, Kloeters O, Mogford JE, et al. Hydrated keratinocytes reduce collagen synthesis by fibroblasts via paracrine mechanisms. Wound Repair Regen, 2007, 15(4): 497-504.
11. 马忠锋, 柴家科. 表皮细胞的培养移植的现状与展望. 中华烧伤杂志, 2005, 21(2): 158-160.
12. Vashishta A, Saraswat Ohri S, Vetvickova J, et al. Procathepsin D secreted by HaCaT keratinocyte cells—A novel regulator of keratinocyte growth. Eur J Cell Biol, 2007, 86(6): 303-313.
13. 白晓智, 胡大海, 石继红, 等. 热损伤角质形成细胞培养上清对真皮成纤维细胞增殖与胶原mRNA表达的影响. 中华实验外科杂志, 2011, 28(3): 380-382.
14. 白晓智, 胡大海, 张万福, 等. 热损伤角质形成细胞培养上清液对成纤维细胞生物学行为的影响. 中华烧伤杂志, 2010, 26(2): 133-137.
15. Moulin V, Larochelle S, Langlois C, et al. Normal skin wound and hypertrophic scar myofibroblasts have differential responses to apoptotic inductors. J Cell Physiol, 2004, 198(3): 350-358.
16. 陈苏丽, 刘流. 上皮细胞-干细胞在创伤愈合中的作用. 中国组织工程研究与临床康复, 2010, 14(6): 1092-1096.
17. Kisseleva T, Brenner DA. Mechanisms of fibrogenesis. Exp Biol Med, 2008, 233(2): 109-122.
18. Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology, 2008, 134(6): 1655-1669．.
19. Risinger GM Jr, Hunt TS, Updike DL, et al. Matrix metalloproteinase-2 expression by vascular smooth muscle cells is mediated by both stimulatory and inhibitory signals in response to growth factors. J Biol Chem, 2006, 281(36): 25915-25925.
20. Ravanti L, Kähäri VM. Matrix metalloproteinases in wound repair (review). Int J Mol Med, 2000, 6(4): 391-407.
21. Longaker MT, Whitby DJ, Adzick NS, et al. Studies in fetal wound healing, VI. Second and early third trimester fetal wounds demonstrate rapid collagen deposition without scar formation. J Pediatr Surg, 1990, 25(1): 63-69.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECTS OF HEAT INJURED KERATINOCYTES SUPERNATANT ON EXPRESSIONS OF COLLAGEN TYPE I, COLLAGEN TYPE III, AND MATRIX METALLOPROTEINASE 1 OF DERMAL FIBROBLASTS

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