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作者:博醫(yī)康來源:北京博醫(yī)康實(shí)驗(yàn)儀器有限公司 發(fā)表日期:2015年3月22日 11:45
【摘要】 本研究探討冷凍干燥保存血小板的方法,以獲得可長期凍干保存的血小板制品,使之能在常溫條件下保存、占用空間小、重量輕、便于長距離運(yùn)輸,能夠滿足突發(fā)事件和戰(zhàn)傷救治的需要。在冷凍干燥保存過程中添加血小板可逆性激活抑制劑、DMSO和海藻糖等低溫保護(hù)劑,進(jìn)行預(yù)處理、冷凍、一級(jí)干燥、二級(jí)干燥,再水化,并同時(shí)測(cè)定血小板回收率,凝血酶聚集反應(yīng),促凝血功能,CD62p表達(dá)率和PAC1表達(dá)率等。結(jié)果表明:血小板回收率為56.29%,其對(duì)凝血酶的聚集反應(yīng)與對(duì)照組無明顯差異,對(duì)ADP和丙基沒食子酸誘導(dǎo)的聚集反應(yīng)較對(duì)照組分別降低49.34%和26.25%,促凝血功能與對(duì)照組比較也無統(tǒng)計(jì)學(xué)差異;凍干血小板CD62p表達(dá)率為42.36%,PAC1表達(dá)率為2.12%,凝血酶激活后CD62p再表達(dá)率為50.88%,PAC1再表達(dá)率為54.55%。結(jié)論:添加血小板可逆性激活抑制劑,海藻糖和DMSO后的凍干血小板,其聚集活性和促凝血功能與新鮮血小板無明顯差異,血小板可逆性激活抑制劑降低了凍干血小板的CD62p表達(dá),增強(qiáng)了凍干血小板的生存能力,因而延長了其生存時(shí)間,因此可以該凍干方法為基礎(chǔ)進(jìn)一步提高凍干保存血小板的效率。
【關(guān)鍵詞】 可逆性激活抑制劑
Experimental Study on Lyophilization of Plaets
Abstract The aim of this study was to search a procedure of plaet lyophilization and find a way of longterm sto rage of human plaets at normal temperature with smaller size and lighter weight, to be convenient to transport at long distance thus to meet the demands in accidents and war time. Human plaets were pretreated by freezing, the first and the second desiccation, and were added with reversible activationinhibitors of plaets, DMSO and trehalose, then were rehydrated. At the same time, the recovery rate of plaets, plaet maximal aggregation induced by thrombin, coagulation of plaets, CD62p expression and PAC1 expression were assayed. The results indicated that the recovery rate of the plalelets was 56.29%. The plaet maximal aggregation induced by thrombin had no significant difference between lyophilized plaets and the fresh plaetrich plasma (FPRP), but the aggregation of plaets induced by ADP or propyl gallate was decreased by 49.34% and 26.25%. Coagulation of the lyophilized plaets was not significantly different from FPRP. CD62p expression of the lyophilized plaets (42.36%) was higher than that in FPRP while PAC1 expression was 2.12%. CD62p reexpression rate induced by thrombin was 50.88% and PAC1 reexpression was 54.55%. It is concluded that the ability of recovered lyophilized plaets added with reversible activationinhibitors, DMSO and trehalose to aggregate and coagulate has showed no significant difference as compared with FPRP. The reversible activationinhibitors can decrease CD62p expression of lyophilized plaets, and may enhance their survival ability and prolongate survival time. Therefore the efficiency of lyophilizing plaets can be improved based on this freezedrying procedure.
Key words plaet; lyophilization; activationinhibitor; trehalose; DMSO
由于凍干制品能在常溫下保存、性能穩(wěn)定、便于運(yùn)輸,因而冷凍干燥技術(shù)已廣泛應(yīng)用于各種生物材料的保存。目前的血小板保存方法使其應(yīng)用和運(yùn)輸受到很大的限止,凍干是血小板的保存方法,凍干的血小板能在常溫條件下保存、占用空間小、重量輕、便于運(yùn)輸?shù)?,尤其能?yīng)付突發(fā)事件以及戰(zhàn)場(chǎng)救治的需要。因此,凍干保存血小板的研究幾十年來一直吸引著各國學(xué)者的關(guān)注。本研究利用海藻糖、可逆性血小板功能抑制劑和二甲亞砜等低溫保護(hù)劑,冷凍干燥保存血小板,現(xiàn)將研究結(jié)果報(bào)告如下。
材料和方法
血小板來源
血小板取自自愿供血者。自愿供血者獻(xiàn)血前1天內(nèi)禁飲含酒精類飲料,2周內(nèi)未服用阿司匹林等抗血小板及抗凝血的藥物。應(yīng)用COBE Spectra血液分離機(jī)采集濃縮血小板(PCs)。
主要試劑及溶液
試劑 植酸鈉(Phylocid sodium, Sigma公司產(chǎn)品),百維利肽(Bivalirudin, Medicine公司產(chǎn)品)左旋精氨酸(Larginine, Sigma公司產(chǎn)品),PGE1(商品名保達(dá)新)、海藻糖(trehalose)購自南寧中諾生物工程公司)、二甲亞砜(DMSO,天津醫(yī)藥公司產(chǎn)品)。熒光素標(biāo)記的特異性血小板膜表面抗體及相關(guān)多肽:PAC1-FITC, CD62P-PE,CD61-PerCP,MIgGI-PE等購自Becton Dickinson公司;RGDS肽和GPRP (GlyProArgPro乙酸鹽)購自Sigma公司產(chǎn)品。血小板聚集誘導(dǎo)劑包括:凝血酶(thrombin, Sigma公司產(chǎn)品),二磷酸腺苷(ADP, Sigma公司產(chǎn)品),丙基沒食子酸(Propyl gallate, Analytical ControlSystems公司產(chǎn)品)。
MEK6108K型全自動(dòng)血細(xì)胞分析儀(Nihon Kohden公司產(chǎn)品),APACT2聚集儀(德國Lanbo公司產(chǎn)品),F(xiàn)ACSCalibur流式細(xì)胞儀(Becton Dickinson公司產(chǎn)品)和CellQuest分析軟件, Centrifuge 5415D離心機(jī)(Eppendorf),Biofuge15R(Heraeus公司產(chǎn)品),臺(tái)式冷凍干燥機(jī)(TFFD1)。
實(shí)驗(yàn)分組
實(shí)驗(yàn)為配伍設(shè)計(jì),5份PCs,各準(zhǔn)備15 ml。設(shè)對(duì)照組和2個(gè)實(shí)驗(yàn)組,對(duì)照組為新鮮富含血小板血漿(FPRP),不進(jìn)行任何處理。每份PCs分3個(gè)試管,2ml/tube,分至各組,剩余的離心(2 287×g,15分鐘),留取少量血小板血漿(PPP)備用。
前處理(海藻糖負(fù)載)
血小板中加入預(yù)處理液,實(shí)驗(yàn)組1含海藻糖 45 mmol/L和DMSO 3%,實(shí)驗(yàn)組2含海藻糖 45 mmol/L,DMSO 3%,PGE1 1 μmol/L,LArg 5 mmol/L,Phy 0.5 mmol/L和Bivaridin 0.5 μmol/L。將各組樣品混勻后,37℃水浴4小時(shí),每20分鐘振搖1次。
血小板冷凍干燥、預(yù)水化和再水化
血小板懸液1 111×g離心8分鐘,用冷凍干燥液1 ml(HEPES 9.5 mmol/L,NaCl 142.5 mmol/L,KCl 4.8 mmol/L,MgCl2 1.0 mmol/L, trehalose 150.0 mmol/L,PGE1 1 μmol/L, LArg 5 mmol/L,Phy 0.5 mmol/L, Bivaridin 0.5 μmol/L,DMSO 3%,人血清白蛋白 5%)在硅化后的玻璃瓶中重懸血小板,血小板PCT為40%-50%,干燥厚度<1 cm,進(jìn)行冷凍干燥。
冷凍 ①以5℃/分鐘的速度從22℃降至-5℃; ②以2℃/分鐘從-5℃降至-60℃; ③ -60℃以下維持1小時(shí)以上。
干燥 一級(jí)干燥,-30℃,1小時(shí),50 mTorr;二級(jí)干燥,架上溫度以0.2℃/分鐘的速度從-30℃升至20℃,50 mTorr。一級(jí)干燥和二級(jí)干燥之間采用快速直接升溫,避免梯度升溫。真空干燥完畢后,樣品室溫下在凍干機(jī)中至少放置24小時(shí),然后用于試驗(yàn)或封裝保存室溫或4℃放置。
預(yù)水化 凍干的血小板在37℃濕度飽和的密閉環(huán)境中水浴4小時(shí),使水含量達(dá)25%左右。
再水化 預(yù)水化處理后,按1∶4進(jìn)入等滲的復(fù)水液/H2O(3/1, V/V)中再水化。復(fù)水液含NaCl 116.0 mmol/L,枸櫞酸鈉 13.6 mmol/L,Na2HPO4 8.6 mmol/L, KH2PO4 1.6 mmol/L, EDTA 0.9 mmol/L,葡萄糖 11.1 mmol/L,PGE1 1μmol/L,LArg 5 mmol/L,Phy 0.5 mmol/L,Bivaridin 0.5 μmol/L,DMSO 3%,pH=6.8。
血小板回收率
血小板回收率=(復(fù)水后血小板數(shù)目/凍干前血小板數(shù)目)×100%
血小板聚集試驗(yàn)
各誘導(dǎo)劑作用濃度為thrombin 1 U/ml,ADP 20 μmol/L, propyl gallate 50 μl。PPP聚集率為0%,PRP聚集率為100%。原血漿調(diào)整PRP血小板濃度至(200-300)×106/ml,反應(yīng)體積250 μl。
血小板膜表面CD62p、PAC1的表達(dá)率
采用流式細(xì)胞術(shù)(FCM)測(cè)定血小板膜表面CD62p、PAC1d陽性表達(dá)率,操作參照常規(guī)檢查步驟。以CD61表達(dá)和SSC作為選定血小板的條件R1,獲取10 000個(gè)血小板(速度300個(gè)/秒)進(jìn)行FL1(PAC1-FITC)和FL2(CD62p-PE)雙參數(shù)分析。以陰性對(duì)照管,調(diào)節(jié)陽性區(qū)的假陽性率<1%,用單純血小板和各陽性單染(抗CD61單染,抗CD62p單染和抗PAC1單染對(duì)照)加陰性對(duì)照校正熒光FL1,F(xiàn)L2。建立獲取模式后檢測(cè)測(cè)定管。
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