重組L-門冬酰胺酶前體脂質(zhì)體對急性淋巴白血病小鼠的治療效果

1、    重組L-門冬酰胺酶前體脂質(zhì)體 對急性淋巴白血病小鼠的治療效果        摘要考察L-門冬酰胺酶前體脂質(zhì)體(L-ASNasePL)與L-ASNase對移植性小鼠急性淋巴細(xì)胞白血病的作用。采用DBA/2小鼠兩種L1210、P388腹水瘤模型,脂質(zhì)體組和L-ASNase組小鼠每天ip一次（按L-門冬酰胺酶計(jì)7400,3700,1850KU/kg）, 連續(xù)10d。設(shè)生理鹽水組（0.9%NaCl,0.4ml/20g）及阿霉素注射劑（2mg/kg）組為陰性和陽性對照

2、組。結(jié)果L-ASNase PL及L-門冬酰胺酶（L-ASNase）皆能顯著延長移植性淋巴細(xì)胞白血病的小鼠存活天數(shù)（P<0.01）, L-ASNase PL對 L1210、P388移植小鼠，生命延長率分別最高達(dá)184.7%和183.3%。L-ASNase組生命延長率最高分別為132.9%和132.1%。L-ASNasePL與相同劑量的L-ASNase相比,其高、中劑量組（7400,3700kU/kg）皆顯著延長L1210、P388小鼠的存活天數(shù)（P<0.05）。關(guān)鍵詞L-門冬酰胺酶前體脂質(zhì)體（L-ASNasePL）；L-門冬酰胺酶（L-ASNase）；急性淋巴白血病 Effect o

3、f Recombinant -L-Asparaginase Proliposome on the Acute Lymphatic Leukaemia in MiceWang Hong（College of Biopharmaceutics, China Pharmaceutical University, Nanjing 210009）Wu Wutong（College of Biopharmaceutics, China Pharmaceutical University, Nanjing 210009）Guo Qinglong（College of Biopharmaceutics, Ch

4、ina Pharmaceutical University, Nanjing 210009）Gu Xueqiu（Pharmaceutical Research Laboratory, Shenyang Pharmaceutical University, Shenyang 110015）AbstractOBJECTIVE：To examine the effect of L-Asparaginase proliposomes(L-ASNasePL)and L-Asparaginase(L-ASNase)on acute lymphatic leukaemia in mice.METHODS:

5、The dose of L-ASNasePL and L-ASNase group mice(7400,3700,1850kU/kg)was given ip one time every day for 10 days adopting L1210 and P388 acute tumor model. Saline and Doxorubicin used as negative and positive control. RESULTS: L-ASNasePL and L-ASNase could extend acute lymphatic leukaemia mice surviva

6、l time significantly（P<0.01）. The life lengthen ratio of L-ASNasePL was the highest to 184.7% in L1210 group and 183.3% in P388 group respectively, and that of L-ASNase was the highest to 132.9% in L1210 group and 132.1% in P388 group respectively. CONCLUSION: Compared with the same dose of L-ASN

7、ase ,the high and middle dose （7400,3700KU/kg）L-ASNasePL extend survival time of L1210 and P388 mice（P<0.05）. Key WordsL-asparaginase proliposomes, L-asparaginase , Acute lymphatic leukaemia脂質(zhì)體作為藥物載體已進(jìn)入深入研究1,也可作為肽類和蛋白類藥物的載體,以提高重組蛋白的使用性,使之適宜于藥物的研制和開發(fā)。L-門冬酰胺酶（L-ASNase）是治療人急性淋巴細(xì)胞白血病的有效抗癌制劑2，3，是目前聯(lián)合治

8、療轉(zhuǎn)移癌和急性淋巴細(xì)胞白血病的較好藥物，由于靜脈注射L-ASNase可能引起某些不良反應(yīng)，如發(fā)燒、皮疹和過敏性休克、血栓出血癥嚴(yán)重者死亡，使臨床應(yīng)用受到限制4，5。為此將L-ASNase研制成脂質(zhì)體制劑，使酶的抗原決定簇被包封物掩蓋，以達(dá)到降低免疫原性、減少過敏反應(yīng)目的，利于白血病人的治療。1材料瘤譜：腹水型小鼠淋巴白血病細(xì)胞（L1210），腹水型小鼠淋巴白血病細(xì)胞（P388），均購自上海細(xì)胞所細(xì)胞庫。動(dòng)物：DBA/2小鼠，由中科院上海實(shí)驗(yàn)動(dòng)物中心提供，合格證：中國動(dòng)管會(huì)005號，滬動(dòng)管會(huì)90（10）號文。藥品與試劑：前體脂質(zhì)體自制(批號96072701,96072702,96072703)，

9、通過空白前體脂質(zhì)體加入L-門冬酰胺酶液進(jìn)行水合包封,配成所需濃度的L-門冬酰胺酶脂質(zhì)體混懸液；L-ASNase由中國藥科大學(xué)生物制藥學(xué)院提供。阿霉素（DR）浙江海門制藥廠。2方法與結(jié)果21L-門冬酰胺酶前體脂質(zhì)體對移植性小鼠淋巴細(xì)胞白血病L1210生命延長作用取DBA/2小鼠18g22g,雌雄各半,80只,按移植性腫瘤研究法6接種L1210腹水瘤,按種后隨機(jī)分為8組,每組10只,雌雄各半,分別按以下三個(gè)劑量給藥:L-門冬酰胺酶前體脂質(zhì)體（按L-門冬酰胺酶計(jì)7400,3700,1850kU/kg）;L-門冬酰胺酶注射液（7400,3700,1850kU/kg）;另設(shè)生理鹽水空白對照組（0.9%N

10、aCl,0.4ml/20g）及阿霉素注射劑（2mg/kg）組,于接種后24h腹腔注射,每天一次,共給藥10d,給藥后觀察荷瘤DBA/2小鼠存活天數(shù)（存活60d以上小鼠按60d計(jì)）,所得實(shí)驗(yàn)結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)（t檢驗(yàn)）處理.試驗(yàn)重復(fù)三次, 生命延長率計(jì)算如下式:生命延長率(T-C)/C×100%式中C為對照組平均生存天數(shù);T為給藥組平均生存天數(shù)；實(shí)驗(yàn)結(jié)果見表1。Tab 1The mean survival time of L-asparaginase preparations on L1210 by ip route , ±s, n=10ExperimentLot No.Prep

11、arationAnimalsNumberDosekU/kgThe mean survivaltime(day)(%)Prolong ratio(%)Control108.5±1.57L-ASNasePL10740024.2±4.16*184.710379918.7±3.38*120.010185012.5±3.81*47.096072701L-ASNase10740019.8±4.73*132.910370015.1±3.84*77.610185011.8±3.26*38.8DR102mg31.1±7.87*265

12、.9Control108.6±1.43L-ASNasePL10740024.3±4.24*182.610370018.6±3.43*116.310185012.3±3.65*43.096072702L-ASNase10740019.5±4.43*126.710370015.2±3.49*76.710185011.5±2.80*33.7DR102mg29.6±6.01*244.2Control108.6±1.52L-ASNasePL10740023.9±4.12*177.910370018.9&#

13、177;3.63*119.810185010.6±3.1423.396072703L-ASNase10740019.3±4.14*124.410370014.7±3.93*70.91018509.7±3.0512.8DR102mg30.4±8.10*253.5?*P<0.01, Compared with control ; P<0.05 Compared with L-ASNase 實(shí)驗(yàn)結(jié)果表明：與生理鹽水對照組相比L-門冬酰胺酶脂質(zhì)體（L-ASNase liposomes）、L-門冬酰胺酶（L-ASNase）及阿霉素（DR）皆能

14、顯著延長移植性小鼠淋巴細(xì)胞白血病的存活天數(shù)（P<0.01）,對L1210的生命延長作用DR作用最強(qiáng),其生命延長率為265.9%;其次為L-ASNase LP,其生命延長率最高達(dá)184.7%。L-ASNase作用最弱,其生命延長率最高為132.9%。L-ASNase LP與相同劑量的L-ASNase相比,其高、中劑量組（7400,3700kU/kg）皆有顯著延長L1210小鼠的存活天數(shù)作用（P<0.05）,試驗(yàn)重復(fù)三次,結(jié)果相近。2.2L-門冬酰胺酶前體脂質(zhì)體對移植性小鼠淋巴細(xì)胞白血病P388的生命延長作用取DBA/2小鼠1822g,雌雄各半,80只,按移植性腫瘤研究法接種P388腹

15、水瘤,接種后,隨機(jī)分為8組,每組10只,雌雄各半,分別按以下劑量給藥:L-ASNase LP（按L-ASNase計(jì)7400,3700,1850kU/kg）;L-ASNase注射劑(7400,3700,1850kU/kg）;另設(shè)生理鹽水空白對照組（0.9%NaC1,0.4ml/20g）及DR注射劑（20mg/kg）組,于接種后24h腹腔注射,每天一次,連續(xù)10d,給藥后記錄荷瘤DBA/2小鼠平均生存時(shí)間（存活60d以上小鼠按60d計(jì)）,計(jì)算給藥組生命延長率,并分別進(jìn)行統(tǒng)計(jì)學(xué)（t檢驗(yàn)）處理。試驗(yàn)重復(fù)三次,結(jié)果見表2。Tab 2The mean survival time of L-asparagi

16、nase preparations on P388 by ip route , ±s, n=10ExperimentLot No.PreparationAnimalsNumberDosekU/kgThe mean survivaltime(day)(%)Prolong ratio(%)Control108.9±1.10L-ASNasePL10740024.5±4.97*175.310379918.8±3.55*111.210185012.7±3.59*42.796072701L-ASNase10740019.9±4.75*123.61

17、0370015.3±3.83*71.910185011.9±3.01*33.7DR102mg30.9±7.53*247.1Control108.4±1.35L-ASNasePL10740023.8±3.60*183.310370018.5±3.44*120.210185012.2±2.94*45.296072702L-ASNase10740019.5±4.50*126.710370015.3±3.37*76.710185011.7±2.95*39.3DR102mg29.6±6.26*2

18、52.4Control108.6±1.43L-ASNasePL10740023.4±3.94*172.110370018.9±3.51*114.010185010.8±3.0125.696072703L-ASNase10740019.5±4.09*126.710370015.0±3.64*74.410185010.4±2.8720.9DR102mg30.4±8.10*253.5*P<0.01 , Compared with control ; P<0.05 , Compared with L-ASNase 實(shí)驗(yàn)結(jié)果表明:與生理鹽水空白對照組相比,L-ASNase LP、L-ASNase及DR皆均能顯著地延長移植小鼠淋巴細(xì)胞白血病P388的存活天數(shù)（P<0.01）。對P388的生命延長作用DR作用最強(qiáng),生命延長率為253.4%,其次為L-ASNase LP其生命延長率最高達(dá)183.3%,L-ASNase作用最弱,其生命延長率最高為132.1%。與相同劑量的L-ASNase注射劑相比,L-ASNase LP高、中劑量組（7400、3700kU/kg）皆有顯著延長P388小鼠的存活天數(shù)（P<0.05）。實(shí)驗(yàn)重復(fù)三次,結(jié)果相近