• 1 北京華信醫(yī)院整形外科（北京，100084）;;
  • 2天津市中心婦產(chǎn)科醫(yī)院乳腺科;

【摘 要】 目的 尋找制備富血小板血漿 （platelet-rich plasma，PRP）的最佳離心方案，并制備PRP凝膠用于皮下注射促進(jìn)大鼠皮瓣成活，探討其有效性及作用機(jī)制。 方法 取12周齡健康Wistar大鼠72只，體重250～300 g。于24只大鼠心臟取動脈血8～10 mL/只，分別采用3種離心方法制備PRP。A組：以200 × g離心15 min、500 × g離心10 min；B組：以312 × g離心10 min、1 248 × g離心10 min；C組：以200 × g離心15 min，200 × g離心10 min。在PRP制作過程中取各組全血、PRP及貧血小板血漿（platelet-poor plasma，PPP）行血小板計(jì)數(shù)，根據(jù)血小板計(jì)數(shù)結(jié)果選擇最佳離心方案，并取對應(yīng)的PRP、PPP及第1次離心后血清，采用ELISA法測定PDGF-BB和TGF-β1濃度；并制備PRP及PPP凝膠。于48只大鼠背部制備大小為11 cm × 3 cm的皮瓣，隨機(jī)分為3組（n=16）：PRP組每只大鼠皮瓣下注射100 mL PRP凝膠，PPP組同法注射100 mL PPP凝膠，對照組不作處理。術(shù)后大體觀察皮瓣成活情況，7 d后取材計(jì)算成活率；組織學(xué)觀察計(jì)數(shù)炎性細(xì)胞，免疫組織化學(xué)染色計(jì)數(shù)微血管；于術(shù)后8、24 h，3、7 d行實(shí)時熒光定量PCR檢測VEGF、EGF、PDGF-AA和PDGF-BB mRNA表達(dá)。 結(jié)果 血小板計(jì)數(shù)結(jié)果示，A組血小板濃縮倍數(shù)最高，為最佳制備方法。A組PRP中TGF-β1、PDGF-BB濃度明顯高于血清及PPP （P  lt; 0.05）。與對照組相比，術(shù)后PRP組傷口無膿性分泌物；PRP組皮瓣成活率為61.2% ± 9.1%，與PPP組35.8% ± 11.3%及對照組28.0% ± 5.4%比較，差異有統(tǒng)計(jì)學(xué)意義（P  lt; 0.05）。PRP組炎性細(xì)胞計(jì)數(shù)較PPP組及對照組明顯減少， 微血管計(jì)數(shù)明顯高于其余兩組，差異均有統(tǒng)計(jì)學(xué)意義（P  lt; 0.05）。實(shí)時熒光定量PCR檢測示與PPP組及對照組比較，PRP組VEGF及PDGF-BB mRNA術(shù)后均高表達(dá)，而EGF mRNA僅在術(shù)后24 h內(nèi)呈高表達(dá)，PDGF-AA mRNA在3 d后開始高表達(dá)。術(shù)后3 d及7 d PRP組PDGF-AA、8 h PDGF-BB、24 h及3 d VEGF、24 h EGF的mRNA相對表達(dá)量與其他兩組比較，差異有統(tǒng)計(jì)學(xué)意義（P  lt; 0.05）。 結(jié)論 以200 × g離心15 min、500 × g離心10 min是制備PRP的最佳離心方案。PRP凝膠可通過調(diào)節(jié)血管生發(fā)相關(guān)基因促進(jìn)大鼠皮瓣成活。

引用本文： 李薇薇 ,馮銳. 富血小板血漿凝膠制備及對大鼠皮瓣成活的作用分析. 中國修復(fù)重建外科雜志, 2012, 26(1): 64-69. doi: 復(fù)制

1. platelet-rich plasma: characterization in vitro and in the ischemic hind limb of the mouse. J Vasc Surg, 2009, 50(4): 870-879.
2. bone in dogs. J Oral Maxillofac Surg, 2009, 67(9): 1850-1858.
3. of flap tensile strength. J Periodontol, 2009, 80(6): 985-992.
4. dermal fibroblasts. Plast Reconstr Surg, 2008, 122(5): 1352-1360.
5. using thrombin receptor agonist peptide. J Oral Maxillofac Surg, 2005, 63(4): 529-535.
6. J Oral Maxillofac Surg, 2000, 58(3): 297-300.
7. chronic wounds. Transfus Apher Sci, 2004, 30(2): 145-151．.
8. Surg, 2004, 6(4): 489-496.
9. differentiation and regeneration during equine wound healing. Exp Mol Pathol, 2003, 74(3): 244-255.
10. Plast Reconstr Surg, 2004, 114(6): 1502-1508．.
11. of cultured smooth muscle cells from spontaneously hypertensive rats by platelet-derivled growth factor A-chain homodimer. Cell Regul, 1990, 1(11): 821-831.
12. IUBMB Life, 2001, 52(1-2): 61-66.
13. Saldalamacchia G, Lapice E, Cuomo V, et al. A controlled study of the use of autologous platelet gel for the treatment of diabetic foot ulcers. Nutr Metab Cardiovase Dis, 2004, 14(6): 395-396.
14. Bir SC, Esaki J, Marui A, et al. Angiogenic properties of sustained release.
15. Tajima N, Sotome S, Marukawa E. A three-dimensional cell-loading system using autologous plasma loaded into a porous β-tricalcium-phosphate block promotes bone formation at extraskeletal sites in rats. Materials Science and Engineering: C, 2007, 27(4): 625-632.
16. Choi BH, Zhu SJ, Kim BY, et al. Effect of platelet-rich plasma (PRP) concentration on the viability and proliferation of alveolar bone cells: an in vitro study. Int J Oral Maxillofac Surg, 2005, 34(4): 420-424.
17. Li NY, Yuan RT, Chen T. Effect of platelet-rich plasma and latissimus dorsi muscle flap on osteogenesis and vascularization of tissue-engineered.
18. Powell CA, Bannister SR, Mackey SA, et al. Periodontal wound healing with and without platelet-rich plasma: histologic observations and assessment.
19. Kakudo N, Minakata T, Mitsui T, et al. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human.
20. Landesberg R, Burke A, Pinsky D, et al. Activation of platelet-rich plasma.
21. Landesberg R, Roy M, Glickman RS. Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation.
22. 宋揚(yáng), 宮蘋, 侯睿, 等. 傳統(tǒng)二次離心法和改良法制備富血小板血漿的比較研究. 實(shí)用口腔醫(yī)學(xué)雜志, 2005, 21(5): 629-632.
23. Sclafani AP. Applications of platelet-rich fibrin matrix in facial plastic surgery. Facial Plast Surg, 2009, 25(4): 270-276.
24. Crovetti G, Martinelli G, Issi M, et al. Platelet gel for healing cutaneous.
25. 馮揚(yáng), 唐休發(fā). 富血小板血漿在頜面部非血管化骨移植中的應(yīng)用. 中國修復(fù)重建外科雜志, 2006, 20(9): 944-946．.
26. Mazor Z, Peleg M, Garg AK, et al. Platelet-rich plasma for bone graft enhancement in sinus floor augmentation with simultaneous implant placement: patient series study. Implant Dent, 2004, 13(1): 65-72．.
27. Marx RE. Platelet-rich plasma: evidence to support its use. J Oral Maxillofac.
28. Carter CA, Jolly DG, Worden CE Sr, et al. Platelet-rich plasma gel promotes.
29. Epplev BL, Woodell JE, Higgins J. Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing.
30. Griffin XL, Smith CM, Costa ML. The clinical use of platelet-rich plasma in the promotion of bone heating: a systematic review. Injury, 2009, 40(2): 158-162．.
31. Werner S, Grose R. Regulation of wound healing by Growth factors and cytokines. Physiol Rev, 2003, 83(3): 835-870．.
32. Resink TJ, Scott-Burden T, Hahh AW, et al. Specific growth stimulation.
33. Kliche S, Waltenberg J. VEGF receptor signaling and endothelial function.
34. Adelmann-Grill BC, Wach F, Cully Z, et al. Chemotactic migration of normal dermal fibroblasts towards epidermal growth factor and its modulation by platelet-derived growth factor and transforming growth factor-beta. Eur J Cell Biol, 1990, 51(2): 322-326.
35. Eppley BL, Pietrzak WS, Blanton M. Platelet-rich plasma: a review of biology and applications in plastic surgery. Plast Reconstr Surg, 2006, 118(6): 147e-159e.
36. Akeda K, An HS, Pichika R, et al. Platelet-rieh plasma (PRP) stimulates the extracellular matrix metabolism of porcine nucleus pulposus and anulus fibrosus cells cultured in alginate beads. Spine (Phila Pa 1976), 2006, 31(9): 959-966．.
  1. 1. platelet-rich plasma: characterization in vitro and in the ischemic hind limb of the mouse. J Vasc Surg, 2009, 50(4): 870-879.
  2. 2. bone in dogs. J Oral Maxillofac Surg, 2009, 67(9): 1850-1858.
  3. 3. of flap tensile strength. J Periodontol, 2009, 80(6): 985-992.
  4. 4. dermal fibroblasts. Plast Reconstr Surg, 2008, 122(5): 1352-1360.
  5. 5. using thrombin receptor agonist peptide. J Oral Maxillofac Surg, 2005, 63(4): 529-535.
  6. 6. J Oral Maxillofac Surg, 2000, 58(3): 297-300.
  7. 7. chronic wounds. Transfus Apher Sci, 2004, 30(2): 145-151．.
  8. 8. Surg, 2004, 6(4): 489-496.
  9. 9. differentiation and regeneration during equine wound healing. Exp Mol Pathol, 2003, 74(3): 244-255.
  10. 10. Plast Reconstr Surg, 2004, 114(6): 1502-1508．.
  11. 11. of cultured smooth muscle cells from spontaneously hypertensive rats by platelet-derivled growth factor A-chain homodimer. Cell Regul, 1990, 1(11): 821-831.
  12. 12. IUBMB Life, 2001, 52(1-2): 61-66.
  13. 13. Saldalamacchia G, Lapice E, Cuomo V, et al. A controlled study of the use of autologous platelet gel for the treatment of diabetic foot ulcers. Nutr Metab Cardiovase Dis, 2004, 14(6): 395-396.
  14. 14. Bir SC, Esaki J, Marui A, et al. Angiogenic properties of sustained release.
  15. 15. Tajima N, Sotome S, Marukawa E. A three-dimensional cell-loading system using autologous plasma loaded into a porous β-tricalcium-phosphate block promotes bone formation at extraskeletal sites in rats. Materials Science and Engineering: C, 2007, 27(4): 625-632.
  16. 16. Choi BH, Zhu SJ, Kim BY, et al. Effect of platelet-rich plasma (PRP) concentration on the viability and proliferation of alveolar bone cells: an in vitro study. Int J Oral Maxillofac Surg, 2005, 34(4): 420-424.
  17. 17. Li NY, Yuan RT, Chen T. Effect of platelet-rich plasma and latissimus dorsi muscle flap on osteogenesis and vascularization of tissue-engineered.
  18. 18. Powell CA, Bannister SR, Mackey SA, et al. Periodontal wound healing with and without platelet-rich plasma: histologic observations and assessment.
  19. 19. Kakudo N, Minakata T, Mitsui T, et al. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human.
  20. 20. Landesberg R, Burke A, Pinsky D, et al. Activation of platelet-rich plasma.
  21. 21. Landesberg R, Roy M, Glickman RS. Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation.
  22. 22. 宋揚(yáng), 宮蘋, 侯睿, 等. 傳統(tǒng)二次離心法和改良法制備富血小板血漿的比較研究. 實(shí)用口腔醫(yī)學(xué)雜志, 2005, 21(5): 629-632.
  23. 23. Sclafani AP. Applications of platelet-rich fibrin matrix in facial plastic surgery. Facial Plast Surg, 2009, 25(4): 270-276.
  24. 24. Crovetti G, Martinelli G, Issi M, et al. Platelet gel for healing cutaneous.
  25. 25. 馮揚(yáng), 唐休發(fā). 富血小板血漿在頜面部非血管化骨移植中的應(yīng)用. 中國修復(fù)重建外科雜志, 2006, 20(9): 944-946．.
  26. 26. Mazor Z, Peleg M, Garg AK, et al. Platelet-rich plasma for bone graft enhancement in sinus floor augmentation with simultaneous implant placement: patient series study. Implant Dent, 2004, 13(1): 65-72．.
  27. 27. Marx RE. Platelet-rich plasma: evidence to support its use. J Oral Maxillofac.
  28. 28. Carter CA, Jolly DG, Worden CE Sr, et al. Platelet-rich plasma gel promotes.
  29. 29. Epplev BL, Woodell JE, Higgins J. Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing.
  30. 30. Griffin XL, Smith CM, Costa ML. The clinical use of platelet-rich plasma in the promotion of bone heating: a systematic review. Injury, 2009, 40(2): 158-162．.
  31. 31. Werner S, Grose R. Regulation of wound healing by Growth factors and cytokines. Physiol Rev, 2003, 83(3): 835-870．.
  32. 32. Resink TJ, Scott-Burden T, Hahh AW, et al. Specific growth stimulation.
  33. 33. Kliche S, Waltenberg J. VEGF receptor signaling and endothelial function.
  34. 34. Adelmann-Grill BC, Wach F, Cully Z, et al. Chemotactic migration of normal dermal fibroblasts towards epidermal growth factor and its modulation by platelet-derived growth factor and transforming growth factor-beta. Eur J Cell Biol, 1990, 51(2): 322-326.
  35. 35. Eppley BL, Pietrzak WS, Blanton M. Platelet-rich plasma: a review of biology and applications in plastic surgery. Plast Reconstr Surg, 2006, 118(6): 147e-159e.
  36. 36. Akeda K, An HS, Pichika R, et al. Platelet-rieh plasma (PRP) stimulates the extracellular matrix metabolism of porcine nucleus pulposus and anulus fibrosus cells cultured in alginate beads. Spine (Phila Pa 1976), 2006, 31(9): 959-966．.