富血小板血浆制备技术的优化及其组分的检测

发布时间：2018-03-07 06:11

本文选题：富血小板血浆　切入点：血小板　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的:通过对传统的手工方法及已被认可的系统制备方法的优化,及对本实验制备方法的血小板和白细胞的浓度、血小板活性及生长因子浓度等方面的测定,探讨分析该PRP制备方法的稳定性、经济性及实用性。研究方法:富血小板血浆的制备及血小板、白细胞计数:取20例符合纳入标准的健康成年男性志愿者全血各40 m L,与6ml抗凝剂摇匀后,取40 m L注入50ml无菌离心管中用于PRP制备;其两次离心条件均为660g/min离心10min,最终PRP体积为(6±0.2)m L左右。利用血细胞分析仪对全血及PRP进行血小板和白细胞的测定。血小板活性检测:利用流式细胞仪检测全血及PRP中血小板表面的CD42b、CD63及CD62P(P-选择素),分析制备前后血小板的活化程度。生长因子的检测:利用酶联免疫吸附试验(ELISA)检测激活后全血、激活后PRP及未激活PRP中的血小板源性生长因子-BB(PDGF-BB)、转化生长因子-β(TGF-β)及血管内皮细胞生长因子(VEGF)的浓度,分析生长因子的浓度与血小板数量的关系。研究结果:全血和PRP中血小板和白细胞浓度分析:全血和PRP中血小板浓度分别为(169.33±31.98)×109/L和(915.14±191.21)×109/L,比较差异有统计学意义(t=-20.384,P0.01);全血和PRP中白细胞浓度分别为(6.24±1.36)×109/L和(18.86±6.73)×109/L,比较差异有统计学意义(t=-10.141,P0.01)。PRP中血小板及白细胞的浓度与全血中血小板及白细胞的浓度呈正相关趋势,其相关系数分别为r=0.884(P0.01),r=0.877(P0.01)均呈显著的正相关。血小板活性检测:用流式方法通过检测血小板表面的CD42b、CD63、CD62p,计算出的血小板富集系数分别为5.26±0.68、6.03±1.09、5.22±0.66,与血常规检测结果5.41±0.54比较接近,进一步的验证了此方法对血小板的浓缩程度。另外,从流式图中可看出全血与PRP中血小板表面标志物(CD62p、CD63和CD42b)的表达率非常接近,说明在制备PRP的过程中并无血小板的活化,PRP中血小板保持着其原有活性。生长因子检测结果分析:用ELISA试剂盒检测的结果显示PDGF-BB、TGF-β1、VEGF的浓度在激活后的PRP与激活后的全血中的的浓度存在显著性的差异(P0.01);在未激活PRP中的PDGF-BB、TGF-β1、VEGF与激活PRP中的这三种生长因子之间的差异有显著的统计学意义(P0.01);而统计结果显示未激活PRP与激活全血中的这三种生长因子之间无统计学意义(P0.05)。研究结论:1.该PRP制备方法所需的抗凝全血的量少,减少了患者不必要的血液浪费;2.该PRP制备方法无需购买制备PRP的专门仪器及在常温无菌条件下即可制备,减轻了患者的经济负担;3.该PRP制备方法避免了离心过程中血小板的激活,保持了血小板原有的活性;4.此方法可以稳定的制备出富含高浓度血小板的PRP,且PRP中生长因子的浓度较高,能够很好的满足临床的需求。
[Abstract]:Objective: to optimize the traditional manual method and the approved preparation method, and to determine the concentration of platelet and white blood cell, platelet activity and growth factor, etc. To discuss and analyze the stability, economy and practicability of the preparation method of PRP. Methods: preparation of platelet-rich plasma and platelets, WBC count: the whole blood samples of 20 healthy adult male volunteers who met the inclusion criteria were collected. After shaking with 6ml anticoagulant, 40ml of anticoagulant was injected into 50ml aseptic centrifuge tube to prepare PRP. The two centrifugation conditions were 660g / min centrifugation for 10 min, and the final volume of PRP was about 6 卤0.2mL. Platelet and leukocyte were measured by blood cell analyzer. Platelet activity was detected by flow cytometry. The platelet surface CD42b CD63 and CD62PtP P- selectin in PRP were used to analyze the activation of platelets before and after preparation. The detection of growth factor: Elisa was used to detect the activated whole blood. The concentrations of platelet-derived growth factor (PDGF-BBF), transforming growth factor- 尾 (TGF- 尾) and vascular endothelial growth factor (VEGF) in activated PRP and unactivated PRP. Results: platelet and leukocyte concentrations in whole blood and PRP: platelet concentrations in whole blood and PRP were 169.33 卤31.98 脳 10 9 / L and 915.14 卤191.21 脳 10 9 / L, respectively. The white blood cell concentrations in PRP were 6.24 卤1.36 脳 10 9 / L and 18.86 卤6.73 脳 10 9 / L, respectively. The correlation coefficient was 0.884P0.01P0.01P0.01). The platelet activity was detected by flow cytometry. The platelet enrichment coefficient calculated by flow cytometry was 5.26 卤0.686.03 卤1.095.22 卤0.66, which was close to the result of routine blood test (5.41 卤0.54), and the results of platelet activity were similar to that of routine blood test (5.41 卤0.54), and the platelet enrichment coefficient was 5.26 卤0.686.03 卤1.095.22 卤0.66, which was similar to the results of routine blood test (5.41 卤0.54), and compared with that of routine blood test (5.41 卤0.54). In addition, from flow chart, we can see that the expression rate of platelet surface markers CD62pnCD63 and CD42b in whole blood is very close to that in PRP, and the expression rate of CD62pUCD63 and CD42b is similar to that of platelet surface markers (CD62ptCD63 and CD42b). The results of growth factor analysis showed that the concentration of PDGF-BBG TGF- 尾 _ 1 in the activated PRP and the activated PRP were determined by ELISA kit. There was a significant difference in blood concentration between PDGF-BBF- 尾 1 PRP and PRP, and there was a significant difference between the three growth factors in activated PRP, and the statistical results showed that there was a significant difference between the unactivated PRP and the activated PRP in the whole blood (P0.01a), and the difference was significant (P0.01a) between the unactivated PRP and the activated PRP (P0.01a), and the statistical results showed that there was a significant difference between the unactivated PRP and the activated PRP in the whole blood. There was no statistically significant difference between the three growth factors. Conclusion: 1. The anticoagulant whole blood volume needed for the preparation of PRP was low. The PRP preparation method does not require the purchase of special instruments for the preparation of PRP and can be prepared under aseptic conditions at room temperature. The PRP preparation method avoids platelet activation during centrifugation. This method can be used to produce high concentration platelets, and the concentration of growth factor in PRP is high, which can meet the needs of clinical.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R457.1

【参考文献】