本文選題：富血小板血漿　切入點(diǎn)：血小板　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究目的:通過對(duì)傳統(tǒng)的手工方法及已被認(rèn)可的系統(tǒng)制備方法的優(yōu)化,及對(duì)本實(shí)驗(yàn)制備方法的血小板和白細(xì)胞的濃度、血小板活性及生長因子濃度等方面的測(cè)定,探討分析該P(yáng)RP制備方法的穩(wěn)定性、經(jīng)濟(jì)性及實(shí)用性。研究方法:富血小板血漿的制備及血小板、白細(xì)胞計(jì)數(shù):取20例符合納入標(biāo)準(zhǔn)的健康成年男性志愿者全血各40 m L,與6ml抗凝劑搖勻后,取40 m L注入50ml無菌離心管中用于PRP制備;其兩次離心條件均為660g/min離心10min,最終PRP體積為(6±0.2)m L左右。利用血細(xì)胞分析儀對(duì)全血及PRP進(jìn)行血小板和白細(xì)胞的測(cè)定。血小板活性檢測(cè):利用流式細(xì)胞儀檢測(cè)全血及PRP中血小板表面的CD42b、CD63及CD62P(P-選擇素),分析制備前后血小板的活化程度。生長因子的檢測(cè):利用酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)激活后全血、激活后PRP及未激活PRP中的血小板源性生長因子-BB(PDGF-BB)、轉(zhuǎn)化生長因子-β(TGF-β)及血管內(nèi)皮細(xì)胞生長因子(VEGF)的濃度,分析生長因子的濃度與血小板數(shù)量的關(guān)系。研究結(jié)果:全血和PRP中血小板和白細(xì)胞濃度分析:全血和PRP中血小板濃度分別為(169.33±31.98)×109/L和(915.14±191.21)×109/L,比較差異有統(tǒng)計(jì)學(xué)意義(t=-20.384,P0.01);全血和PRP中白細(xì)胞濃度分別為(6.24±1.36)×109/L和(18.86±6.73)×109/L,比較差異有統(tǒng)計(jì)學(xué)意義(t=-10.141,P0.01)。PRP中血小板及白細(xì)胞的濃度與全血中血小板及白細(xì)胞的濃度呈正相關(guān)趨勢(shì),其相關(guān)系數(shù)分別為r=0.884(P0.01),r=0.877(P0.01)均呈顯著的正相關(guān)。血小板活性檢測(cè):用流式方法通過檢測(cè)血小板表面的CD42b、CD63、CD62p,計(jì)算出的血小板富集系數(shù)分別為5.26±0.68、6.03±1.09、5.22±0.66,與血常規(guī)檢測(cè)結(jié)果5.41±0.54比較接近,進(jìn)一步的驗(yàn)證了此方法對(duì)血小板的濃縮程度。另外,從流式圖中可看出全血與PRP中血小板表面標(biāo)志物(CD62p、CD63和CD42b)的表達(dá)率非常接近,說明在制備PRP的過程中并無血小板的活化,PRP中血小板保持著其原有活性。生長因子檢測(cè)結(jié)果分析:用ELISA試劑盒檢測(cè)的結(jié)果顯示PDGF-BB、TGF-β1、VEGF的濃度在激活后的PRP與激活后的全血中的的濃度存在顯著性的差異(P0.01);在未激活PRP中的PDGF-BB、TGF-β1、VEGF與激活PRP中的這三種生長因子之間的差異有顯著的統(tǒng)計(jì)學(xué)意義(P0.01);而統(tǒng)計(jì)結(jié)果顯示未激活PRP與激活全血中的這三種生長因子之間無統(tǒng)計(jì)學(xué)意義(P0.05)。研究結(jié)論:1.該P(yáng)RP制備方法所需的抗凝全血的量少,減少了患者不必要的血液浪費(fèi);2.該P(yáng)RP制備方法無需購買制備PRP的專門儀器及在常溫?zé)o菌條件下即可制備,減輕了患者的經(jīng)濟(jì)負(fù)擔(dān);3.該P(yáng)RP制備方法避免了離心過程中血小板的激活,保持了血小板原有的活性;4.此方法可以穩(wěn)定的制備出富含高濃度血小板的PRP,且PRP中生長因子的濃度較高,能夠很好的滿足臨床的需求。
[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 鹵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 鹵31.98 脳 10 9 / L and 915.14 鹵191.21 脳 10 9 / L, respectively. The white blood cell concentrations in PRP were 6.24 鹵1.36 脳 10 9 / L and 18.86 鹵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 鹵0.686.03 鹵1.095.22 鹵0.66, which was close to the result of routine blood test (5.41 鹵0.54), and the results of platelet activity were similar to that of routine blood test (5.41 鹵0.54), and the platelet enrichment coefficient was 5.26 鹵0.686.03 鹵1.095.22 鹵0.66, which was similar to the results of routine blood test (5.41 鹵0.54), and compared with that of routine blood test (5.41 鹵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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R457.1

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