弓形虫profilin抗体体外抗速殖子增殖及保护作用动态观察

Dynamic observation of inhibition of tachyzoite proliferation and protective action of anti-Toxoplasma profilin antibody in vitro

目的探讨弓形虫profilin(TgPRF)抗体对弓形虫体外增殖的影响以及细胞生长和凋亡的动态变化。方法1)将rTgPRF蛋白背部皮下多点注射免疫新西兰兔4次,末次免疫后10 d心脏采血,ELISA检测抗体效价。2)检测抗体对速殖子的直接杀伤作用,用10^8个速殖子(RH-GFP)感染小鼠胚胎成纤维细胞20 h后,加入1∶40抗血清以及YOYO-1死亡探针孵育观察5 h。3)评估TgPRF抗体对速殖子增殖的影响:将3×10^3细胞/孔接种96孔板并加入6×10^3个速殖子(RH-GFP),同时分别加入1∶40、1∶80、1∶160稀释的抗血清作为干预组,设空白组(仅培养基)和阴性血清组(1∶40稀释免疫前兔血清),培养72 h并间隔12 h拍照,通过Image pro 6.0软件分析虫体增殖情况。4)采用IncuCyte ZOOMTM系统对TgPRF抗体干预后的细胞生长和凋亡进行动态监测:设未感染孔为正常对照组和抗血清对照组(加入1∶40抗TgPRF血清)。感染孔加入6×10^3个速殖子(RH),分别在感染时或感染12 h后加入1∶40、1∶80、1∶160抗TgPRF血清作同步干预或延迟干预,空白组及阴性血清组设置如前。各孔均加入caspase3/7凋亡探针继续观察72 h,记录细胞凋亡情况。结果rTgPRF诱导特异性抗体产生,ELISA检测抗体效价>1∶10^5。镜下未见TgPRF抗体对速殖子的直接杀伤作用。体外干预试验中,空白组和阴性血清组的相对虫体量在实验结束时分别为24.27±7.78和25.72±4.04,差异无统计学意义(P>0.05),并伴有大量细胞缺失;TgPRF抗体抑制虫体增殖,实验结束时1∶40组、1∶80组和1:160组相对虫体量分别为6.32±1.10、9.35±1.32和17.88±1.47,抑制率分别为73.5%、61.5%和26.3%,呈剂量-效应关系。同步干预时各干预组均显示通过抑制虫体增殖不同程度地保护细胞,其中1∶40组细胞生长恢复正常,细胞融合度显著高于空白组和阴性血清组(P<0.01);弓形虫诱导细胞凋亡增加,但各感染组间差异无统计学意义(P>0.05)。TgPRF抗体延迟干预的保护效果有所降低,但诱导细胞凋亡增强,其中1∶40组细胞凋亡率显著高于空白组和阴性血清组(P<0.05)。镜下观察TgPRF抗体干预使凋亡多集中在细胞缺失区域或速殖子增殖区域,而空白组和阴性血清组的凋亡则散布视野。结论弓形虫TgPRF抗体镜下未见直接杀伤弓形虫,但在体外能抑制弓形虫速殖子增殖并促进感染细胞凋亡。

Objective To investigate the effectiveness with which anti-Toxoplasma profilin(TgPRF)antibody inhibited the proliferation of tachyzoites in vitro and dynamic changes of cell growth and apoptosis.Methods New Zealand rabbits were immunized with rTgPRF via multiple subcutaneous injections on the back.Blood was collected from the rabbit heart 10 d after the last immunization,and the specific antibody was titrated using ELISA.First,the direct killing effect of anti-TgPRF antibody on tachyzoites was detected.Mouse embryonic fibroblasts were challenged with 10^8 tachyzoites(RH-GFP)for 20 h,and then 1:40 antiserum and a YOYO-1 cell death probe were added.The mixture was incubated for 5 h and observed.Second,to evaluate the effect of anti-TgPRF antibody on tachyzoite proliferation,3×10^3 cells/well were inoculated onto 96-well plates and challenged with 6×10^3 tachyzoites(RH-GFP).Anti-TgPRF serum with a final concentration of 1:40,1:80,or 1:160 was added as an intervention;1:40 pre-immune rabbit serum served as a negative control,and medium alone served as a blank control.Each sample was cultured for 72 h and photographed at 12-h intervals,and the proliferation of tachyzoites was analyzed with the software Image pro 6.0.The IncuCyte ZOOM system was used to further monitor the growth and apoptosis of cells after intervention with the anti-TgPRF antibody.Uninfected wells were divided into the normal control and antiserum control supplemented with 1:40 anti-TgPRF serum.The infected wells were challenged with 6×10^3 tachyzoites(RH),and 1:40,1:80,or 1:160 antiserum was added during infection as a synchronized intervention or 12 hours after infection as a delayed intervention.The blank control and the negative serum control were prepared as described.A caspase 3/7 apoptosis probe was added to wells,and wells were monitored for 72 h at 1-h intervals.Results rTgPRF effectively induced the production of specific antibodies,and the antibody titer exceeded 1:105.Anti-TgPRF antibody did not directly kill tachyzoites according to observations under a microscope.In vitro intervention experiments indicated that there was no significant difference in the relative number of parasites between the blank control and the negative serum control(P>0.05).Parasites numbered 24.27±7.78 in the former and 25.72±4.04 in the latter at the end of the experiment.A decrease in the number of cells was noted.Anti-TgPRF antibody effectively inhibited the proliferation of tachyzoites,and the relative number of parasites was 6.32±1.10 in the 1:40 intervention group,9.35±1.32 in the 1:80 intervention group,and 17.88±1.47 in the 1:160 intervention group at the end of the experiment.The rate of inhibition was 73.5%,61.5%,and 26.3%,respectively.Real-time dynamic monitoring during the synchronized intervention indicated that anti-TgPRF serum restored cell growth to a varying degree.The cell confluence in the normal control and antiserum control plateaued(>98%)at 64 h,while that in 1:40 antiserum plateaued(>98%)at 71 h and that in 1:80 antiserum plateaued(about 91%)at 69 h.The cell confluence in 1:160 antiserum peaked(84.7%)at 74 h and then decreased to 84.9%,while that in the blank control and negative serum control peaked(about 88.0%)at 66 h and then decreased to 70.5%and 66.1%,respectively.A delayed intervention with the TgPRF antibody resulted in less protective action.The cell confluence in 1:40 antiserum peaked(93.3%)at 75 h and then decreased to 89.9%,while that in 1:80 antiserum peaked(90.0%)at 70 h and then decreased to 85.0%and that in 1:160 antiserum peaked(85.9%)at 61 h and then decreased to 79.6%.Infection with tachyzoites induced apoptosis.With simultaneous intervention,the rate of apoptosis(%)was 5.74±0.97 for 1:40 antiserum,5.97±1.67 for 1:80 antiserum,and 5.11±2.02 for 1:160 antiserum at the end of the experiment.The rate did not differ significantly from that in the negative serum control(5.40±1.72)or the blank control(5.12±1.80)(P>0.05).Delayed intervention induced more pronounced apoptosis.The rate of apoptosis(%)was 7.97±0.78 for 1:40 antiserum,7.16±1.23 for 1:80 antiserum,and 6.63±0.71 for 1:160 antiserum at the end of the experiment.The rate of apoptosis in 1:40 antiserum was significantly higher than that in the blank control or negative serum control(P<0.05).Microscopically,a large number of cells were absent in the negative serum control,and there were more apoptotic cells in the visual field.In each intervention group,cell growth recovered to different degrees,and apoptosis was mainly concentrated in areas of tachyzoites proliferation or cell loss,and this was especially true for the 1:40 antiserum.Conclusion Anti-TgPRF antibody does not directly kill Toxoplasma,but it effectively inhibits the proliferation of tachyzoites and it promotes the apoptosis of infected cells in vitro.