654●中國實(shí)驗(yàn)血液學(xué)雜志Journal of Experimetal Hemaology 2003; 11(6):654 - 658文章編號(Aricle ID):109 . 2137(2003)06 - 0654- os.●論著.甲氧基聚乙二醇修飾改造RhD( + )血型的初步研究李立立，王捷熙,檀英霞,章?lián)P培軍事醫(yī)學(xué)科學(xué)院輸血研究所,北京100850摘要Rh血型與ABO血型-樣是重要的血型 系統(tǒng), Rh血型不符會(huì)引起嚴(yán)重的輸血反應(yīng)及新生兒溶血病。中國.人群中RhD陰性血型的比例僅為0.2% - 0.5%,將RhD陽性紅細(xì)胞改造成RhD朗性紅細(xì)胞在臨床輸血中有重要的意義。本研究用4種不同端基的甲氧基聚乙二醇(mPEG),修飾A型.B型.AB型和o型的RhD(+ )紅細(xì)胞,比較4種mPEG衍生物對RhD抗原的修飾效果。同時(shí)觀察mPEG修飾對紅細(xì)胞形態(tài)結(jié)構(gòu)和功能的影響。結(jié)果表明，mPEG BTC(苯并三唑雞基PEC)較其他3種PEG的修飾效果好,在1 mmol/L時(shí)可以有效地遮藏紅細(xì)胞表面RhD抗原,而對紅細(xì)胞形態(tài)滲透脆性、自身溶血、乙酰膽堿酯酶、膽因醇、ATP.2, 3-DPG;含量以及變形性無影響。結(jié)論:初步實(shí)現(xiàn)了將RhD( + )紅細(xì)胞修飾改造成RhD( - )紅細(xì)胞的目的。關(guān)鍵詞甲氧基聚乙二醇; RhD血型;血型改造中團(tuán)分類號R457.11文獻(xiàn)標(biāo)識碼Preliminary Study on Conversion of RhD Positive Red Blood Cells to RhDNegative by Modification with Methoxy Polyethylene Glycol1.I LiLi, WANG Jie-Xi, TAN Ying Xia, ZHANG Yang-PeiInstinte of Blood Transfusion, Academy of Military Medical Sciences, Bejing 100850, ChinaAhstractRh is a very important blood group like ABO blood system in transusion mdicine. It causes severetransfusion reaction and hemolytic disease of the newbom (HDN) if RhD blood group does not match between the donorand the reipient. The population of RhD negative is only about 0.2%一0. 5% in Chinese. Conversion of RhD pesitiveRBCs to RhD negative is very important in clinical transfusion. This study was to try to modify RhD antigen located onthe surface of A, B,0 and AB red blood cells in order to convert RhD positive to RhD negative by the modification of fourkinds of methoxypolyethylene glycol ( mPEG) derivatives and to observe the effect of mPEG modification on cellmorphology，structure and function. The result demonstrated that modification fficiency of mPEG-BTC ( mPEG-benzotriazole carbonate) was better than other three kinds of mPEG derivatives. It could camouflage RhD antigenefficiently when the concentration reached to 1 mmol/L. The result also showed that there were no harmnful efs ofmPEG modification on cell morphology, asmotic fragilty, hemolysis, AchE, cholesterol, ATP, 2, 3-DPG anddeformability. It is suggested that succes in convering RhD positive RBCs to RhD negative was preliminarily achieved.Key words nethoxy polyethylene glyool; RhD blood group; blood group conversionJ Exp Hemaol 2003; 11(6):654 - 658Rh血型系統(tǒng)自1939年被發(fā)現(xiàn)以來,因?qū)е滦律鷥?薩克族、錫伯族、烏孜別克族和柯爾克孜族中，Rh陰溶血病(HDN)、溶血性輸血反應(yīng)而成為紅細(xì)胞中僅性約占5%,高于國內(nèi)其他民族;而漢族人Rh陰性次于ABO血型的重要血型系統(tǒng)。Rh血型系統(tǒng)主要僅占0.2% -0.5% 2]由此可以看出,我國漢族人抗原有RhD, RhCe, RhEe, 其中RhD抗原性最強(qiáng)，Rh陰性血的比例遠(yuǎn)小于白種人和黑人。Rh陰性血50% - 75% Rh陰性個(gè)體通過輸血和妊娠,可受D的供應(yīng)長期存在不足,在出現(xiàn)大量傷員(如災(zāi)害、戰(zhàn)抗原紅細(xì)胞免疫而產(chǎn)生抗D。給RhD陰性人輸用爭)及一些大型的國際交流活動(dòng)(如奧運(yùn)會(huì).世博會(huì))D陽性血200ml,85%產(chǎn)生抗D"。如果將RhD陽時(shí),對Rh陰性血的需求會(huì)大大增加。因此,將RhD性血輸給已產(chǎn)生抗D的人,可能產(chǎn)生危及生命的溶(+ )血改造成RhD(- )血有重要的政治、軍事意血性輸血反應(yīng)。義。Rh血型中的D抗原具有較大的臨床意義,因此在常規(guī)檢查中一般只鑒定RhD陰性和陽性。在基金項(xiàng)目:國家973課題，編號202CB713804白種人中, Rh陰性約占15%;在黑人中約占4%;在通訊作者;章?lián)P培,教授,博土生導(dǎo)師.電話: (010)66931545. Enail: zhaneyp@nic.' bmi. a. cn蒙古人種中不到1%。居住在新疆的維吾爾族、哈2003中國煤化工YHCNMHG甲氧基聚乙二醇修飾改造RhD(+ )血型的初步研究在本課題中研究了4種攜帶不同端基的甲氧基mPEG修飾對紅細(xì)胞結(jié)構(gòu)功能的影響聚乙二醇(mPEG)對RhD( + )血型抗原的修飾效果取mPEG修飾前后的紅細(xì)胞, 37保溫48小時(shí),檢及其對紅細(xì)胞形態(tài)、結(jié)構(gòu)、功能的影響,以期將RhD測上清液在540nm處的光吸收值,計(jì)算紅細(xì)胞的(+)改造成RhD(-),從根本上解決臨床輸血對自身溶血率;紅細(xì)胞懸液10 μl加入1.25 m!的RhD( - )血型紅細(xì)胞的需求。0.32%-0.64%的NaCl梯度溶液,判斷紅細(xì)胞的滲透脆性是否發(fā)生變化;乙酰膽堿酯酶(AchE)活性材料與方法用微量羥胺比色法檢測;異丙醇氯仿抽提紅細(xì)胞膜脂質(zhì)，膽固醇與FeCl,可生成紫紅色膽固醇~鐵復(fù)合血液標(biāo)本采A型RhD(+),B型RhD(+),AB型RhD(+),0.物，在560nm處比色,確定紅細(xì)胞膜膽固醇含量;型RhD( + )志愿獻(xiàn)血者外周靜脈血,肝素抗凝,生紅細(xì)胞ATP用試劑盒(Stock no. fl-asc blolumines-理鹽水洗旅,獲取紅細(xì)胞, CPDA-1保存液4C保存。cent somatic cell assay kit, Sigma) 測定;紅細(xì)胞2, 3-DPG含量用試劑盒(Diagnostics, 2, 3-dpg, proce-不同端基PEG衍生物dure no. 665, Sigma) 測定;紅細(xì)胞懸浮于4%聚乙烯mPEG BTC( mPEG benzotriazole carbonate)(苯并三吡咯烷溶液中,用紅細(xì)胞變形儀(techiconekta.唑端基PEG)(20 kD); mPEG ALD (mPEG propi-cytometer)測定紅細(xì)胞變形性。onaldehyde)(丙醛端基PEG)(20 kD); mPEG2-NHS統(tǒng)計(jì)學(xué)方法(mPEG2-N-hydroxysuccinimide) (N羥基琥珀酰胺應(yīng)用SAS6.0統(tǒng)計(jì)軟件進(jìn)行方差分析,數(shù)值以平均酯端基PEG)(20 kD); mPEG-MAL( PEG maleim-值土標(biāo)準(zhǔn)差(元士s)表示。ide)(馬來酰亞胺端基PEG)(20 kD)。上述衍生物均購自Shearwater公司(USA)。結(jié)果PEG修飾紅細(xì)胞生理鹽水洗滌紅細(xì)胞3次,用PBS等滲液(pH 8.0)mPEG修飾紅細(xì)胞RhD抗原性調(diào)整壓積到12%。加入不同濃度mPEG,其中人RhD(+)的A型B型、AB型和0型紅細(xì)胞與抗mPEG BTC(用pH 8.0的PBS溶解)終濃度分別為_D單抗反應(yīng)發(fā)生凝集。mPEG BTC修飾后的紅細(xì)0,0.2,0.5, 1.0 mmol/L, 25C反應(yīng)1小時(shí); mPEG-胞隨著mPEG濃度增加,凝集反應(yīng)逐漸臧弱,依度ALD(用pH 8.0的PBS溶解)終濃度分別為0,0.5,達(dá)到0.5 mmol/L時(shí)與抗_D單抗反應(yīng)消失。mPEG-1.0, 3.0 mmol/L,并加入占反應(yīng)體積1:10的硼氰化ALD, mPEG2-NHS, mPEG-MAL雖然隨著濃度增鈉(NaCNBH3), 25個(gè)分別反應(yīng)1, 6, 12小時(shí);加，凝集反應(yīng)也逐漸減弱,但直到4.0, 6.0 mmol/LmPEG2-NHS(分別用pH8.0的PBS和pH8.0的.時(shí),凝集反應(yīng)仍未完全消失(表1)(顯微鏡觀察xHEPES溶解')終濃度分別為0,2,4, 6 mmol/L,10),提示mPEG BTC對RhD( + )的修飾效果最好,25C和4C分別反應(yīng)1, 6, 12小時(shí);mPEG-MAL(pH可以有效遮蔽RhD(+)抗原，將RhD(+)血型改造8.0的PBS溶解)終濃度分別為0, 2, 4, 6 mmol/L,成RhD(- )血型。25C和4C分別反應(yīng)1,6,12小時(shí)。反應(yīng)完后,用生抗_D單抗( Immucor, USA)為IgM類抗體,而理鹽水洗滌3次,并調(diào)整壓積到45%,4C放置。天然的RhD抗體為IgG類抗體,用IgG類抗ID多mPEG修飾對紅細(xì)胞RbD抗原性的影響克隆抗體( Immucor, USA)及羊抗人lgG( Immucor,mPEG修飾紅細(xì)胞后對紅細(xì)胞RhD血型抗原的遮USA)與上述0.5, 1.0 mmol/L mPEG-BTC修飾的蔽效果通過凹形血型鑒定卡、間接抗人球蛋白實(shí)驗(yàn)紅細(xì)胞及對照組紅細(xì)胞進(jìn)行間接抗人球蛋白(IAT)(IAT)來檢測(1。實(shí)驗(yàn),以檢測修飾后的紅細(xì)胞表面是否還存在弱滴度的RhD抗原。顯微鏡觀察(X 40)說明, 0.5mPEG修飾對紅細(xì)胞形態(tài)的影響在顯微鏡下觀察mPEG修飾紅細(xì)胞。用戊二醛固mmol/L時(shí)仍有凝集發(fā)生, 1. 0 mmol/L時(shí)無凝集。.定紅細(xì)胞,原子力顯微鏡( SPM-9500J3, SHIMAD-這表明經(jīng)1.0 mmol/L mPEG-BTC修飾的紅細(xì)胞表面已檢測不出RhD抗原(圖1)。zU, JAPAN)觀察紅細(xì)胞形態(tài)S。中國煤化工MYHCNMHG656中國實(shí)驗(yàn)血液學(xué)雜志J Exp Hemutol 2003; 11(6)Table 1 Comparison of the mditication efets of four kindsmPEG on RhD antigenmPEG對紅細(xì)胞結(jié)構(gòu)功能的影響mPEGreaction conditionconcentration對mPEGBTC修飾后的紅細(xì)胞進(jìn)行結(jié)構(gòu)功能檢測(mmoVL)Anti-D表明:修飾前后,紅細(xì)胞的滲透脆性、自身溶血、乙酰膽堿酯酶活性、膽固醇含量、ATP、2, 3-DPG以及變mPEG-BIC25t 1h4+pH 8.0 PBS dsolutio0.2+形性均無顯著改變,說明mPE(- BTC修飾對紅細(xì)胞的結(jié)構(gòu)及攜氧功能、變形力沒有影響(表2)。0.51.00討論mPEG-ALD 25T 1- 12h"0.PEG的基本結(jié)構(gòu)是HO (CH2(H2O)。CH、CH2OH,plI 8.0 PBS disolution是一種無毒、低免疫原性的化合物，可用于體內(nèi),NacCNBH3PEG修飾的白蛋白己經(jīng)被美國FDA批準(zhǔn)用于臨mPEG2-NIS 25t: 1- 12h'床[6]。Jeongh'" 及Hortinl 等在1996年首次報(bào)道pH 8.0 PBS dssolution了將PEG用于修飾紅細(xì)胞以遮蔽紅細(xì)胞表面血型σr4C 1-12h"抗原。隨后,不少作者進(jìn)行了PEG修飾紅細(xì)胞的實(shí)pH8. 0 IEPES dssdus驗(yàn)[9 14)。 大部分實(shí)驗(yàn)結(jié)果表明PEG修飾紅細(xì)胞以mPEG2-MAL 25t 1- 12h'后,可以遮敞紅細(xì)胞的血型抗原。Hortin["4I 和Hu-pH 8.0 PBS dssalutioangl5]的實(shí)驗(yàn)表明，紅細(xì)胞經(jīng)過修飾以后在小鼠體or4C 1- 12h*內(nèi)的存活時(shí)間減短,紅細(xì)胞離心容易破碎及溶血。pH8.0 PIS dsuionnFisherl31的實(shí)驗(yàn)又顯示PEG修飾后并沒有完全遮蔽紅細(xì)胞的血型抗原。作者的實(shí)驗(yàn)室曾用氰脲酰氯* ; 12 hours incubation resulus. 4 + : complete agglutinabiliy. 2 +50% aglulinabiliy. 1 + ": less than 5% aglutinobility. 0: no agglu-活化的PEG(C-mPEG)(5 kD)修飾AB型RhD( + )tinabilits紅細(xì)胞,當(dāng)CmPEG濃度達(dá)到12. 0 mg/ml時(shí)，紅細(xì)胞抗原性大大減弱,形態(tài)、結(jié)構(gòu).功能不受影響,但濃mPEG修飾對紅細(xì)胞形態(tài)的影響度較高會(huì)使小鼠紅細(xì)胞壽命明顯縮短"61。1.0 mmol/L mPEG-BTC修飾紅細(xì)胞形態(tài)正常,進(jìn)一為了降低修飾紅細(xì)胞所需的濃度,尋找更適合步用原子力電鏡觀測mPEG-BTC修飾的紅細(xì)胞呈用于人體的PEG,我們選擇了mPEG-BTC, mPEG-典型的雙凹圓盤狀(圖2)。0 mmol/L0.5 mmoLL1.0 mmol/L.Figure 1 IAT test of mPEG-BTC-modifhed RhD(+ ) RBCs中國煤化工MHCNMHG甲氧基聚乙二醇修飾改造RhD( + )血型的初步研究657●的1010201.00001博00.001.期017開D網(wǎng)INm 5相明0.000 mmol/L1.0 mmol/LFigure 2 Morphology of RBCs before and after mPEG-BIC moditication obsered by Atomic Force microscopeTable2 Eftetes of mPEG-BTC modincation on cell sructur, function and deorombillyParameter0 mmol/I.0.5 mol/L1.0 mmol/I.Osnotic fraglit( %)0.460.44Hemolysis( %)2.45士0. 377#1. 91 +0.0361. 52+0.071AchE( umol)0.754土0.0220.789土0.0570.782士0.146Cholesterol° (%)100110.33+ 18.23103.73土15.736ATP( x 10-8 mo/50 μl RBC)2.55+0.211.72+0. 19.2.04土0.252,3-0PG*(%)110.72土13.9599.43+5.89 .DhMux0. 6413 t 0.0170.6852土0.0070.6607 t 0.025#: Nornal range +SD. * : Relative contentAI.D, mPEG2- NHS, mPEG MAL,其中mPEG-BTC驗(yàn)結(jié)果說明mPEGBTC修飾對紅細(xì)胞結(jié)構(gòu)和功能與紅細(xì)胞膜上的氨基反應(yīng),形成氨基甲酸酯的穩(wěn)定均無影響,我們初步實(shí)現(xiàn)了將RhD(+)血改造成連接;mPEGALD是醛基的mPEG術(shù)生物,與胺類RhD( - )血型的目的。反應(yīng); mPEG2-NHS 是分枝的mPEG衍生物;mPEGMAL與紅細(xì)胞膜上的巰基反應(yīng)。4種參考文獻(xiàn)mPEG衍生物分子量均為20 kD。實(shí)驗(yàn)表明, 4種不.同端基的mPEG以mPEGBTC遮蔽紅細(xì)胞表面. 李勇,楊貴珍. RH血型系統(tǒng).見李勇,楊貴珍主編.人類紅RhD抗原的效果最好，可將RhD( + )紅細(xì)胞改造成細(xì)胞血型學(xué)實(shí)用理論與實(shí)驗(yàn)技術(shù).北京:中國科學(xué)枝術(shù)出版社，1999:73-92RhD(- )。我們還對mPEG BTC修飾的紅細(xì)胞進(jìn)行了形2趙桐茂RH血型.見趙桐茂主編.人類血型遺傳學(xué)。北京;中國科學(xué)技術(shù)出版社, 1987:91- 109態(tài)及結(jié)構(gòu)功能的檢測。滲透脆性、自身溶血和膽固醇指標(biāo)反映了紅細(xì)胞膜完整性。其中滲透脆性是觀3 Hortin GL, Iok HT, Huang sT. Progres towerd preparation ofuniversal donor red cells. Arif Cells Blod Substit Immcbi Biotcch.察紅細(xì)胞在不同濃度鹽水中的溶血程度,判斷其對nol, 1997; 25: 487 - 491低滲鹽水的抵抗力，與紅細(xì)胞表面積和體積比值有4童軍,楊明.紅細(xì)胞血型鑒定.見李勇,楊貴珍主編.人類紅關(guān);自身溶血說明紅細(xì)胞膜是否有缺陷,如果膜缺陷細(xì)胞血型學(xué)實(shí)用理論與實(shí)驗(yàn)技術(shù).北京:中國科學(xué)技術(shù)出版社,會(huì)表現(xiàn)為溶血程度增加;膽固醇含量是膜的流動(dòng)性1999:279 - 288的重要指標(biāo),膽固醇含量增加則膜流動(dòng)性降低;ATPs Ouerghi O, Toohani A, Othnane A. et al. Investigting specilice和2,3 DPG是紅細(xì)胞代謝中間產(chǎn)物,也是反映RBCantigen/antibody binding with the atormic force microscope. Biomol攜氧能力最重要的指標(biāo);DImx反映紅細(xì)胞的最大變Eng, 2002; 19:183- 188形指數(shù),說明紅細(xì)胞變形通過毛細(xì)血管的能力。實(shí)5 Se中國煤化工er b sbsiurte amYHCNMHG658●中國實(shí)驗(yàn)血液學(xué)雜志J Exp Hemaol 2003; 11(6)tigenically inert erythroeytes. In: Winslow RM，Vandegriff KD,tional consequences of antigenic modulation of red cells with me-Intagletta M (eds). Advances in Blood Substitutes: Industrial Opthoxypoly (ethylene giycol). Blood, 1999; 93:2121 - 2127portunities and Medical Challengcs. Boston: MA Birkhauser, 1997:133-15012 Scott MD, Murad KL, Koumpours F, et al. Chemicel eamoulgeof antigenic detemninants: tealth erythrocytes. Proc Natl Acad Sxi7 Jeong ST, Byun SM. The derase of egutinablity of buman ABUSA, 1997; 94:7566 - 7571type red blood cell by attechment of methoxy-polyethylene glyool.Art Cells Blood Substit Immobiol Biotechnol, 1996; 24: 358 (ab-13 Fisher TC, Arnstong JK, Miselman HU, et al. 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