自体富血小板凝胶的制备及其生长因子分析

目的 探讨不同离心方法 制备自体富血小板凝胶(autologous platelet-rich gel,APG)的方法 ,通过改变离心速度比较不同的离心力对PLT富集的影响分析全血和APG中5种生长因子浓度.方法 对13例糖尿病难治性皮肤溃疡患者行APG治疗.取11例自身外周静脉血,分别用3种离心速度制备富血小板血浆(platelet-rich plasma,PRP).A组(n=6)先以529×g离心4 min,再以854×g离心6 minB组(n=5)先以313×g离心4 min,再以1252×g离心6 min;C组(n=5)先以176×g离心5 min,再以1 252×g离心5 min.将离心后制得的PRP与凝血酶.钙剂以101混合凝固后制备APG,用于患者皮肤溃疡的治疗.采用全自动血细胞分析仪计数各组全血和PRP中PLT数量.采用酶联免疫吸附法测定全血和APG中PDGF.BB、VEGF、IGF.1、EGF和TGF.a1 5种生长因子浓度.结果 A组PRP中PLT数量为(779.67±352.39)×109/L,较全血的(263.50±76.63)×109/L提高(2.98±1.42)倍,差异有统计学意义(P＜0.05);PLT回收率为51.5%±22.2%.B组PRP中PLT数量最高,为(1363.80±919.74)X 109/L,较全血的(232.80±127.99)×109/L提高(5.91±2.04)倍,差异有统计学意义(P＜0.05);PLT回收率为75.2%±21.0%,明显高于A组(P＜0.05).全血和APG中PDGF-BB、EGF、IGF-1以及TGF-a.浓度分别为(145.94±133.24)、(503.81±197.86)pg/mL,(160.73±71.10)、(265.95±138.43)pg/mL,(14.54±35.34)、(110.56±84.36)ng/mL,(3.31±2.27)、(5.67±4.80)ng/mL,差异有统计学意义(P＜0.05);VEGF浓度升高,两者间差异无统计学意义(P＞0.05).对数转换后的PLT数量与PDGF-BB、TGF-a1浓度成正相关,相关系数r分别为0.627和0.437(P＜0.05).13例患者共行18次APG治疗,其中9例治疗12周溃疡愈合,愈合率为69.2%;10例窦道愈合,愈合率为83.3%.结论 以313×g离心4 min,再以1252×g离心6 min是制备PRP的最佳方法 ;APG中生长因子浓度高于全血;PLT数量与PDGF-BB、TGF-a1浓度成正相关.

PREPARATION OF AUTOLOGOUS PLATELET-RICH GEL FOR DIABETIC REFRACTORY DERMAL ULCER AND GROWTH FACTORS ANALYSIS FROM IT

OBJECTIVE: To compare the platelet enrichment ratio of platelet-rich plasma (PRP) prepared by different centrifuge methods and to compare the concentration of growth factors released from autologous platelet-rich gel (APG) with the whole blood. METHODS: Thirteen diabetic patients with refractory skin lesions were enrolled in APG treatment. (1) Three kinds of centrifuge methods were selected for PRP by 11 diabetic patients: A (n = 6): 529 x g for 4 minutes in the first centrifuge and 854 x g for 6 minutes in the second centrifuge; B (n = 5): 313 x g for 4 minutes in the first centrifuge and 1,252 x g for 6 minutes in the second centrifuge; C (n = 5): 176 x g for 5 minutes in the first centrifuge and 1,252 x g for 5 minutes in the second centrifuge. Platelet counted on the whole blood and PRP was determined. The APG, produced by combining the PRP with thrombin and calcium gluconate (10:1) was used by patients. (2) PDGF-BB, TGF-beta1, VEGF, EGF, and IGF-1 were measured in the APG and the whole blood using the enzyme-linked immunosorbent assay method. RESULTS: (1) The average platelet concentration was higher in group B (1,363.80 +/- 919.74) x 10(9)/L] than in groups A (779.67 +/- 352.39) x 10(9)/L)] and C (765.00 +/- 278.78) x 10(9)/L] and the platelet recovery rate was 75.2% +/- 21.0% in group B. (2) The concentration of growth factors all increased with the increasing platelet number. On average, for the whole blood as compared with APG, the PDGF-BB concentration increased from (145.94 +/- 133.24) pg/mL to (503.81 +/- 197.86) pg/mL (P < 0.05); TGF-beta1 concentration increased from (3.31 +/- 2.27) ng/mL to (5.67 +/- 4.80) ng/mL (P < 0.05); IGF-1 concentration increased from (14.54 +/- 35.34) ng/mL to (110.56 +/- 84.36) ng/mL (P < 0.05); and EGF concentration increased from (160.73 +/- 71.10) pg/mL to (265.95 +/- 138.43) pg/mL (P < 0.05). No increase was found for VEGF (P > 0.05). (3) There was positive correlation between the platelet concentration and PDGF-BB and TGF-beta1 (r = 0.627, r = 0.437, P < 0.05). (4) Thirteen diabetic repractory dermal ulcers received APG treatment for 18 times, 9 ulcers (69.2%) and 10 sinuses (88.3%) were cured at the end of 12-week treatment. CONCLUSION: The method of group B is the best centrifuge method. A variety of growth factors are detected and released from the platelets at significant levels in APG. There is positive correlation between the platelet concentration and PDGF-BB and TGF-beta1.