摘要
第一部分自体富血小板凝胶在糖尿病难治性皮肤溃疡中的应用—预试验研究
目的：评价自体富血小板凝胶(Autologous Platelet-Rich Gel，APG)治疗糖尿病难治性皮肤溃疡的可行性、有效性和安全性，探索治疗糖尿病难治性皮肤溃疡的新方法。
对象与方法：自2005年7月至2007年4月，我科共收治糖尿病皮肤溃疡患者117例。将入院后经严格控制血糖、血压、抗凝、抗感染及溃疡局部标准(清创、引流、减压、交换敷料等)治疗，2周后溃疡无好转迹象或恶化的患者定义为难治性皮肤溃疡。13例难治性皮肤溃疡入选APG治疗，其中12例有窦道，1例有骨髓炎；背部深部溃疡2例，褥疮3例(Ⅳ级)，小腿溃疡1例，足部溃疡7例(Wagner分级Ⅲ-Ⅳ级)。取患者自身外周静脉血，经离心、分离、浓缩制得的富含血小板血浆(platelet-rich plasma，PRP)按一定比例与凝血酶—钙剂混合凝固形成APG，然后用双通注射器将APG均匀喷洒至溃疡表面或注射至深部窦道内。治疗前和治疗后每周测量溃疡面积直至溃疡愈合或第12周末，有窦道的患者测量窦道体积，同时记录患者的体温和不良反应。
结果：治疗12周后溃疡愈合率为69.2%(9/13)，显效率7.7%(1/13)，有效率7.7%(1/13)，无效率15.4%(2/13)，无效者1例足底窦道有骨髓炎，1例为左髋部褥疮；治疗前3周溃疡面积愈合速度最快(P＜0.05)；APG对窦道治愈率为83.3%，能在短时间内使窦道愈合；治疗过程中无不良反应发生。
结论：APG用于治疗糖尿病难治性皮肤溃疡是安全有效的，特别适用于溃疡面积较大和有窦道的患者，局部血供较好的神经性糖尿病足溃疡患者，但不适用于缺血性溃疡及存在骨髓炎且感染未得到控制的患者。
第二部分自体富血小板凝胶的制备及其所含生长因子分析
目的：探讨手工操作二次差速离心制备APG的方法，通过改变离心速度比较不同的离心力对血小板富集的影响，以期找到较好的离心方法，提高血小板的富集率；分析全血和APG中转化生长因子(TGF-β1)、血管内皮生长因子(VEGF)、血小板源性生长因子(PDGF-BB)、表皮生长因子(EGF)和胰岛素样生长因子(IGF-1)浓度。
方法：(1)富血小板血浆(platelet-rich plasma)的制备：抽取静脉血，注入无菌离心管，离心后汲取上层血浆至红细胞层；将汲取的血浆再次离心，弃去上层血浆，下层即为PRP。共选择3种离心速度：A：先以2600rpm离心4分钟，再以3800rpm离心6分钟；B：先以2000rpm离心4分钟，再以4000rpm离心6分钟；C：先以1500rpm离心5分钟，再以4000rpm离心5分钟，离心前后进行血细胞计数。(2)APG的制备：将PRP与凝血酶-钙剂以10：1混合凝固后为APG。(3)酶联免疫吸附法测定全血和APG中PDGF-BB、VEGF、IGF-1、EGF和TGF-β1等5种生长因子浓度。统计采用配对t检验和Pearson相关分析。
结果：①3种离心速度的结果比较：B法PRP中血小板数量最高为1363.80x10~9/L，比治疗前提高5.91倍，血小板回收率为75.2%，是3种方法中最好的离心方法；②离心后血小板数量平均为957.63×10~9/L，提高4.35倍；红细胞和血红蛋白明显减少，白细胞数量增多；③全血和APG中生长因子浓度比较，APG中含有的PDGF-BB、EGF、IGF-1、TGF-β1浓度高于全血(P＜0.05)，VEGF浓度升高，但两者间没有显著差异；④血小板数量与PDGF-BB和TGF-β1浓度呈正相关。结论：二次差速离心法中B法(以313×g力离心4分钟，再以1252×g力离心6分钟)为最佳离心方法；APG中生长因子浓度高于全血中生长因子浓度；血小板数量与PDGF-BB、TGF-β1浓度呈正相关。
第三部分自体富血小板凝胶治疗糖尿病难治性皮肤溃疡可能的作用机制
目的：通过对APG治疗前后不同治疗时间段溃疡肉芽组织生长因子水平的测定，观察组织中生长因子浓度变化与溃疡愈合之间的关系，探讨APG治疗糖尿病难治性皮肤溃疡可能的作用机制。
材料与方法：分别于治疗前和治疗后3、6、9、15天取溃疡局部肉芽组织，-80℃冰箱保存；使用酶联免疫吸附法测定组织中转化生长因子(TGF-β1)、血管内皮生长因子(VEGF)、血小板源性生长因子(PDGF-BB)、表皮生长因子(EGF)和胰岛素样生长因子(IGF-1)浓度；观察相应时间溃疡缩小面积。统计采用配对t检验和Wilcoxon秩和检验。
结果：组织中生长因子浓度均在治疗后第3天明显升高，PDGF-BB在治疗后第3天达到最高，而VEGF、IGF-1、EGF和TGF-β1在治疗后第9天达到最高；其中VEGF较治疗前升高2.10倍，IGF-1较治疗前升高1.95倍，EGF较治疗前升高1.75倍，PDGF-BB较治疗前升高1.89倍，TGF-β1较治疗前升高1.67倍。溃疡面积在APG治疗后3天及9-15天明显缩小。
结论：APG中血小板激活后逐步释放多种生长因子导致溃疡组织局部生长因子水平升高可能是APG治疗糖尿病难治性溃疡的主要作用机制之一。
Part one:
The use of autologous platelet gel to treat refractory diabetic dermal ulcer—A preliminary study
Objective: To evaluate the feasibility, efficacy and security of autologous platelet gel (APG) in the treatment of refractory diabetic dermal ulcer.
Methods: APG was produced by platelet-rich plasma (PRP) with thrombin and calcium gluconate. 13 cases with refractory diabetic dermal ulcers were enrolled in treatment with APG from July 2005 to April 2007. APG treatment consisted of wound dressing with APG, followed by topical washing and cleaning. The APG was then covered with Vaseline gauze and left for 72 hours, after which the ulcers were treated by standard care. The standard care consisted of daily topical washing, cleaning, debridement and dressing changing of the wounds. Patients underwent antibiotic therapy were required. The clinical endpoints of the study were the healing rate, and/or the time required to bring about adequate tissue regeneration in order to undergo reconstructive plastic surgery.
Results: 69.2% ulcers were cured, 15.4% of patients were no response; the ulcers area were reduced significantly in the first 3 weeks. No adverse reactions were observed.
Conclusion: Topical therapy with APG may be considered as an effective adjuvant method in treating refractory diabetic dermal ulcer.
Part two:
The preparation of autologous platelet gel and the platelet quantification in PRP and the growth factors analysis from APG
Objective:①To compare the platelet enrich ratio in the PRP prepared by the different centrifgual methods, search for the best effective centrifgual method.②To analyse the growth factors released from APG.
Methods:①Preparation of PRP: The patient's venous blood samples were drawn into steriled centrifuge tube. The superstratum plasma was sucted after the first centrifugalization, and the superstratum plasma was centfifugated again, the plasma of underlayer was defined of PRP. 3 centrifgual methods were selected: A: 2600rpm for 4 minutes and 3800rpm for 6 minutes. B: 2000rpm for 4minutes and 4000rpm for 6 minutes. C: 1500rpm for 5 minutes and 4000rpm for 5 minutes, counting the blood cell before and after centrifugalization.②Preparation of APG: The PRP mixed with Thrombin-calcium for APG.③To measure the PDGF-BB, VEGF, IGF-1, EGF and TGF-β1 in the APG and the whole blood using the Enzyme-linked immunoadsordent assay method.
Results:①The B method was the best centfifgual method, the average platelet concentration was 1363.80×10~9/L and the platelet recovery rate was 75.2%.②The average platelet concentration in PRP was 957.63×10~9/L, an average 4.35-fold increase in platelet concentration was found in the PRP compared with that of whole blood.③The concentration of growth factors all increased with increasing platelet number. However, growth factor concentration varied from patient to patient. On average for the whole blood as compared with PRP, the IGF—1 concentration increased from 14.54±35.34 ng/ml to 110.56±84.36 ng/ml, the EGF concentration increased from 160.73±71.10pg/ml to 265.95±138.43pg/ml, the PDGF-BB concentration increased from 145.94±133.24pg/ml to 503.81±197.86pg/ml, the TGF—β1 concentration increased from 3.31±2.27ng/ml to 5.67±4.80 ng/ml. No increase was found for VEGF.④There were positive correlation between the platelet concentration and PDGF-BB, TGF—β1.
Conclusion: The B method is the best centrifgual method. A variety of therapeutic growth factors were detected and released from the platelets in significant levels in APG. There were positive correlation between the platelet concentration and PDGF-BB, TGF—β1.
Part three:
The probable mechanism of autologous platelet gel to treat the refractory diabetic dermal ulcer
Objective: By measuring the growth factors concentration in the granulation tissue pre-treatment and post-treatment of APG, to investigate the probable mechanism of APG to treat refractory diabetic dermal ulcer.
Methods: The PDGF-BB, VEGF, IGF-1, EGF and TGF-β1 in the granulation tissues were detected by ELISA of pre-treatment and 3, 6, 9, 15 days after APG treatment.
Results: The concentration of 5 growth factors all began to increase in the granulation tissues after 3 days treatment, on average for pre-treatment as compared with post-treatment, the VEGF concentration increased 2.1-fold, the IGF-1 concentration increased 1.95-fold, the EGF concentration increased 1.75-fold, the PDGF-BB concentration increased 1.89-fold and the TGF-β1 concentration increased 1.67-fold. The ulcers areas were reduced obviously in 3 days and in 9-15 days after APG treatment.
Conclusion: The one of the main mechanism of APG to treat the refractory diabetic dermal ulcer may be that the growth factors of granulation tissue increase after APG treatment.
引文
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