豆状囊尾蚴体外培养方法的建立及其分泌产物的分离鉴定

Development of a system for long-term in vitro cultivation of Cysticercus pisiformis and isolation and identification of its secretory products

目的建立豆状囊尾蚴体外长期培养方法,并对其培养分泌产物进行鉴定。方法将豆状带绦虫虫卵经口感染新西兰白兔(2000枚/只),感染后3个月,从感染兔腹腔中收集豆状囊尾蚴。将虫体随机分为PBS组、RPMI 1640组、胆汁组(RPMI 1640+10%兔胆汁)和血清组(RPMI 1640+10%无外泌体胎牛血清),10个/组,37℃、5%CO_(2)培养后5 min、1 h、2 h、12 h、24 h、36 h、48 h及其后每天进行观察,记录豆状囊尾蚴头节翻出情况、虫体活力、体外存活时间及形态改变等,筛选适宜的培养条件。取50个幼虫在细胞瓶(25 cm^(2))中按筛选出的培养条件培养,每隔48小时收集培养液离心浓缩,取上清,蛋白质免疫印迹(Western blotting)检测豆状带绦虫烯醇化酶(Tpeno)和Tp14-3-3蛋白的表达情况。收集豆状囊尾蚴培养上清,提取外泌体,分别与阴性兔血清、CD63抗体、抗Tpeno单克隆抗体1D7和抗Tp14-3-3多克隆抗体孵育45 min,再加入胶体金标记的IgG,透射电镜观察外泌体形态,免疫电镜检测其标记蛋白CD63、Tpeno和Tp14-3-3表达情况。采用SPSS 20.0统计学软件进行数据分析,组间比较采用单因素方差分析。结果培养前的豆状囊尾蚴呈椭圆形,有内凹的点状乳白色头节;PBS组可存活7 d;RPMI 1640组可存活2个月;胆汁组存活时间最短,仅48 h;血清组幼虫存活时间最长,可达3个月以上,高于其他3个组(P<0.05)。存活期内,PBS组、RPMI 1640组、胆汁组和血清组幼虫囊泡长度分别为(1.38±0.39)、(1.30±0.12)、(1.18±0.59)和(1.83±0.10)cm;宽度分别为(0.45±0.10)、(0.68±0.05)、(0.25±0.06)和(0.85±0.05)cm;血清组虫体囊泡长度和宽度与其余3组相比差异均有统计学意义(F_(长度)=7.58、65.93、7.11,F_(宽度)=73.85、29.41、308.57,P<0.05或P<0.01)。培养12 h时,PBS组、RPMI 1640组、胆汁组和血清组幼虫头节翻出率分别为(33.3±5.8)%、(20.0±0.0)%、(100.0±0.0)%、(13.3±5.8)%,其中血清组与PBS和胆汁组头节翻出率差异有统计学意义(F=18.00、676.00,P<0.01)。对各组培养效果比较显示,血清组的培养液(RPMI 1640+10%无外泌体胎牛血清)为最适培养液。SDS-PAGE电泳分析结果显示,利用RPMI 1640+10%无外泌体胎牛血清培养豆状囊尾蚴,可成功提取到虫体的代谢-分泌产物和外泌体蛋白。Western blotting分析结果显示,用特异性抗体检测可见Tpeno和Tp14-3-3阳性条带,相对分子质量(Mr)分别约47000和28000,与预期相符。透射电镜下豆状囊尾蚴外泌体大小不一,直径为50~150 nm,为圆形或椭圆形的囊泡。免疫电镜分析显示,阴性兔血清不能识别外泌体蛋白,未见胶体金颗粒标记;外泌体标志蛋白CD63、虫体特异性蛋白Tpeno和Tp14-3-3均为阳性表达,胶体金颗粒标记明显。结论初步建立了豆状囊尾蚴体外培养体系,可连续培养豆状囊尾蚴至少3个月以上。采用该体外培养方法培养可在培养上清中成功提取豆状囊尾蚴的外泌体。

Objective To develop a long-term in vitro cultivation method for Cysticercus pisiformis and identify its secretory products in the culture.Methods New Zealand white rabbits were infected with Taenia pisiformis eggs by gavage at a dose of 2000 eggs per rabbit.Three months after infection,C.pisiformis were collected from the abdominal cavities and randomly assigned to four groups(10 larvae/group),including PBS group,RPMI 1640 group,bile group(RPMI 1640+10%rabbit bile)and serum group(RPMI 1640+10%exosome-free fetal bovine serum).The cysticerci were cultured at 37℃in a 5%CO_(2) incubator and were observed continuously at 5 min,1 h,2 h,12 h,24 h,36 h,48 h and daily for subsequent days to record the scolex evagination,activity,survival status and morphological changes of cysticerci in each group,for screening optimal culture condition.Under the optimized conditions,the cysticerci were cultured in cell culture bottle(25 cm^(2)),50 larvae each,and the culture medium was collected every 48 h for centrifugal concentration,from which the supernatants were used to detect the expression of enolase(Tpeno)and 14-3-3(Tp14-3-3)proteins by Western blotting.The exosomes were also extracted from the culture supernatants and incubated with negative rabbit serum,anti-CD63,monoclonal anti-Tpeno 1D7 and polyclonal anti-Tp14-3-3 antibodies for 45 min,followed by adding colloidal gold labelled IgG to observe the exosome morphology by transmission electron microscopy(TEM),and examine the expression of labeled proteins of CD63,Tpeno and Tp14-3-3 by immunoelectron microscopy(IEM).The data analysis was using SPSS 20.0 software,and one-way ANOVA was applied for comparison of group data.Results Before cultivation,the larvae were oval in shape,containing concaved dot-like white scolices.The larvae in the PBS group could survive for 7 days,while in RPMI 1640 group lasted for 2 months.However,the survival time of cysticerci in the bile group was the shortest of 48 h only,while that was longest in the serum group,which was over 3 months,and there was a significant difference between the groups(P<0.05).While the parasites were viable,the length of cysticerci vesicle in the PBS group,RPMI 1640 group,bile group and serum group was(1.38±0.39),(1.30±0.12),(1.18±0.59)and(1.83±0.10)cm,respectively.Meanwhile the vesicle width for cysticerci was(0.45±0.10),(0.68±0.05),(0.25±0.06)and(0.85±0.05)cm,respectively.Among these,the vesicle length and width of cysticerci in the serum group were significantly different from those in the other three groups(F_(length)=7.58,65.93,7.11;F_(width)=73.85,29.41,308.57,P<0.05 or P<0.01).In addition,at 12 h,the scolex evagination rates of cysticerci in PBS group,RPMI 1640 group,bile group and serum group were(33.3±5.8)%,(20.0±0.0)%,(100.0±0.0)%and(13.3±5.8)%,respectively,The differences among the serum group,the PBS group and the bile group were statistically significant(F=18.00,676.00;P<0.01).No statistically significant difference was found between the serum group and the RPMI 1640 group(F=4.00,P>0.05).Compared with the culture results of the other three groups,RPMI 1640+10%exosome-free fetal bovine serum was used as the optimal culture solution.SDS-PAGE analysis showed that the larvae-released products and exosome proteins could be successfully extracted from the culture supernatants with RPMI 1640+10%exosome-free fetal bovine serum.Using specific antibodies,two cysticerci-derived proteins,Tpeno and Tp14-3-3,were detected with the expected molecular weight(Mr)of 47000 and 28000,respectively.TEM showed cysticerci-derived exosomes are circular or elliptical,nano-sized vesicles(50-150 nm diameter)with disc-shaped morphology.IEM analysis demonstrated that the exosome proteins were not recognized by negative rabbit serum and did not show colloidal gold particle label.In contrast,the biomarker protein(CD63)and two parasite-specific proteins(Tpeno and Tp14-3-3)were positively expressed in exosomes with significant colloidal gold particle label.Conclusion This study established an in vitro culture system for C.pisiformis,being able to consecutively cultivate for at least 3 months.With this system,C.pisiformis-derived exosomes could be extracted from the culture supernatant.