亚精胺对日本血吸虫培养细胞影响的研究
摘要
本文以琥珀酸脱氢酶(succinate dehydrogenase，SDH)、核仁组织区相关嗜银蛋白(nucleolarorganizerregions，Ag-NORs)、碱性磷酸酶(alkaline phosphatase，AKP)和酸性磷酸酶(acidic phosphatase，ACP)为指标，观察亚精胺(Spermidine)对日本血吸虫培养细胞的影响。旨在探寻一种能促进日本血吸虫培养细胞分裂增殖的外源物质，为最终建立一个无限增殖的日本血吸虫传代细胞系奠定良好基础。
1．Spd对日本血吸虫成虫培养细胞SDH的影响
实验一：将联合法接种培养至第4d的日本血吸虫成虫培养细胞，用含Spd终浓度分别为0(对照)、25、50、75、80、90、100、150、200μmol／L的无血清培养基处理24h，然后换用常规培养基继续培养，至第8d时，对处理过的培养细胞进行SDH细胞化学染色，Olympus-BH_2显微镜下观察，并将结果输入HPIAS-2000图像分析仪进行图像分析，结果表明：处理日本血吸虫培养细胞的最佳Spd浓度为75μmol／L。实验二：日本血吸虫细胞接种培养第4d时，用终浓度为75μmol／L的Spd分别处理0(对照)、12、24、36、48、60、72h，继续培养至第8d，对培养细胞进行SDH细胞化学染色，并作图像分析。结果显示，血吸虫培养细胞以终浓度为75μmol／L的Spd处理48h，实验效果最佳。
2．Spd对日本血吸虫成虫培养细胞Ag-NORs的影响
将日本血吸虫成虫培养细胞接种于小盖玻片上，培养于常规培养基中。接种后第3d，随机分为实验组和对照组。将实验组细胞用无血清培养基配制的终浓度为75μmol／L的Spd处理48h，对照组细胞则用不含Spd的无血清培养基处理相同时间；然后换用常规培养基继续培养。从第7d起每周定时对培养细胞进行Ag-NORs染色，直至第5w。将染色结果输入HPIAS-2000图像分析仪，结果显示，Spd处理组细胞的AgNORs颗粒数目、颗粒面积与核面积百分比、平均光密度值均较对照组细胞高(P＜0.01)。结果表明Spd具有一定促进细胞增殖的能力。
3．Spd对日本血吸虫成虫培养细胞AKP和ACP的影响
实验随机分为Spd组、肝基质组、Spd+肝基质组3组。Spd+肝基质组和肝基质
组在日本血吸虫成虫细胞接种前预先在盖玻片上涂抹肝基质。细胞接种后第3d，sPd
组和spd十肝基质组细胞用含75 p mol几sPd的无血清培养基处理48h;肝基质组细胞
用不含Spd的无血清培养基处理相同时间。每周定时对各组培养细胞进行A对、AcP
细胞化学染色。图像分析显示，培养前两周Spd组培养细胞的AKP和ACP活性强于
肝基质组细胞(P<0.01):当Spd与肝基质联合应用时，培养细胞的AKp和AcP活性
低于肝基质组细胞(P<0.05);21天后，肝基质组细胞AKP和ACP活性较spd组细
胞强(P<0.05)，联合组细胞两种酶活性最弱。spd组细胞的酶活性下降较快，而肝基
质组细胞的酶活性下降较平缓，说明肝基质对日本血吸虫培养细胞的作用较稳定。:而
肝基质和spd的联合作用对日本血吸虫培养细胞的物质代谢无协同增加作用。总之，
sPd既能增强日本血吸虫成虫培养细胞的有氧代谢和物质代谢功能，还具一定促进培
养细胞增殖的作用。
The effects of Spermidine(Spd)on the growth and proliferation of cultured cells were reaseached by observing the influence on succinate dehydrogenase(SDH),the distribution and dynamics changes of nucleolarorganizerregions (NORs) of the cultured cells from Schistosoma japoncum(Ag-NORs)and alkaline/acidic phosphatase (AKP/ACP) in the cultured cells. It was expected to lay a good foundation for the establishment of cell line of 5. japonicum. 1.Effects of SPD on SDH of the cultured cells from S. japonicum
The first experiment: To study the effect of Spd on the growth and proliferation of cultured cells from adult Schstosoma japonicum,cells collected were cultured in medium RPMI-1640 containing 20% calf serum and a moderate amount of antibiotics for 4 days.The cells were directly treated by Spd with concentrations of 0, 25,50,75, 80,90,150,200μm/L for 24 hours in medium RPMI-1640 with free serum respectively.Afterwards,they were moved to original medium.Then,the cultured cells were stained for SDH in 8cultured day.The contents of SDH in the cells of each group were measured by the photometer-image analytical instrument(HPIA-2000).The results showed the contents of SDH were strongest in 75μm/L Spd. The second experiment:the cultured cells were treated with 75μm/L Spd for 0,12,24,6,48,60,72h after cultured for 4 days in routine medium.Samely,stained for SDH on 8 day.The result showed that the cultured cells grow well after treated with 75μm/L Spd for 48h. 2 .Effects of Spd on Ag-NORs of the cultured cells from S
. japonicum
In the experiment the cells from S. japonicum were inoculated on the small glass coverslipS. The cells were divided into test and control groups after cultured for 3 days in routine medium. Cells in the test group were treated with with Spd of the concentration of 75|μm/L for 48 hours in the medium without serum.Afterwars,the cells were cultured in routine medium.The culture condition for the cells in the control group was the same as the
test group except for no addition of Spd in the media.The cells were stained according to the routine cytochemical methods regularly from Iweek to 5 week. The three parameters of AgNORs: number of granules, percentage of AgNORs area/Nucleus area, average optical density were measured. Compared with control group, contents of AgNORs were rising by using Spd in the culture. The three parameters of AgNORs were all statisticaly different between control group and test group (P<0.01). It was suggested that Spd have some ability to increase the potential of transformation and proliferation in cultured cells. 3. Effects of Spd on AKP and ACP of the cultured cells from S. japonicum
In this experiment,3 groups were made:Spd group,liver matix group,Spd+liver matix group. The contents of AKP and ACP in the cultured cells of each group were measured by using HPIA-2000. The results showed the contents of AKP and ACP in cells of Spd group were strongest in three groups in 14 cultured days(P<0.01). AKP and ACP in Spd+liver matix group were the weakest in three groups(P<0.01);21 days later, AKP and ACP in cells of liver matix group were stronger than those of Spd group and the concentrations of the two enzymes in the cells of Spd+liver matix group were lower than 2 weeks before.The activities of AKP and ACP in Spd group were descended more sharply than those in liver matix group. It was suggested that liver matix was more stable and steady to advance the cultured cells than Spd.group.There was adverse-operation between Spd and liver matix.lt was proved that Spd could increase the metabolism in cultured cells and give an impetus to cell growth and proliferation.
1．Spd对日本血吸虫成虫培养细胞SDH的影响
实验一：将联合法接种培养至第4d的日本血吸虫成虫培养细胞，用含Spd终浓度分别为0(对照)、25、50、75、80、90、100、150、200μmol／L的无血清培养基处理24h，然后换用常规培养基继续培养，至第8d时，对处理过的培养细胞进行SDH细胞化学染色，Olympus-BH_2显微镜下观察，并将结果输入HPIAS-2000图像分析仪进行图像分析，结果表明：处理日本血吸虫培养细胞的最佳Spd浓度为75μmol／L。实验二：日本血吸虫细胞接种培养第4d时，用终浓度为75μmol／L的Spd分别处理0(对照)、12、24、36、48、60、72h，继续培养至第8d，对培养细胞进行SDH细胞化学染色，并作图像分析。结果显示，血吸虫培养细胞以终浓度为75μmol／L的Spd处理48h，实验效果最佳。
2．Spd对日本血吸虫成虫培养细胞Ag-NORs的影响
将日本血吸虫成虫培养细胞接种于小盖玻片上，培养于常规培养基中。接种后第3d，随机分为实验组和对照组。将实验组细胞用无血清培养基配制的终浓度为75μmol／L的Spd处理48h，对照组细胞则用不含Spd的无血清培养基处理相同时间；然后换用常规培养基继续培养。从第7d起每周定时对培养细胞进行Ag-NORs染色，直至第5w。将染色结果输入HPIAS-2000图像分析仪，结果显示，Spd处理组细胞的AgNORs颗粒数目、颗粒面积与核面积百分比、平均光密度值均较对照组细胞高(P＜0.01)。结果表明Spd具有一定促进细胞增殖的能力。
3．Spd对日本血吸虫成虫培养细胞AKP和ACP的影响
实验随机分为Spd组、肝基质组、Spd+肝基质组3组。Spd+肝基质组和肝基质
组在日本血吸虫成虫细胞接种前预先在盖玻片上涂抹肝基质。细胞接种后第3d，sPd
组和spd十肝基质组细胞用含75 p mol几sPd的无血清培养基处理48h;肝基质组细胞
用不含Spd的无血清培养基处理相同时间。每周定时对各组培养细胞进行A对、AcP
细胞化学染色。图像分析显示，培养前两周Spd组培养细胞的AKP和ACP活性强于
肝基质组细胞(P<0.01):当Spd与肝基质联合应用时，培养细胞的AKp和AcP活性
低于肝基质组细胞(P<0.05);21天后，肝基质组细胞AKP和ACP活性较spd组细
胞强(P<0.05)，联合组细胞两种酶活性最弱。spd组细胞的酶活性下降较快，而肝基
质组细胞的酶活性下降较平缓，说明肝基质对日本血吸虫培养细胞的作用较稳定。:而
肝基质和spd的联合作用对日本血吸虫培养细胞的物质代谢无协同增加作用。总之，
sPd既能增强日本血吸虫成虫培养细胞的有氧代谢和物质代谢功能，还具一定促进培
养细胞增殖的作用。
The effects of Spermidine(Spd)on the growth and proliferation of cultured cells were reaseached by observing the influence on succinate dehydrogenase(SDH),the distribution and dynamics changes of nucleolarorganizerregions (NORs) of the cultured cells from Schistosoma japoncum(Ag-NORs)and alkaline/acidic phosphatase (AKP/ACP) in the cultured cells. It was expected to lay a good foundation for the establishment of cell line of 5. japonicum. 1.Effects of SPD on SDH of the cultured cells from S. japonicum
The first experiment: To study the effect of Spd on the growth and proliferation of cultured cells from adult Schstosoma japonicum,cells collected were cultured in medium RPMI-1640 containing 20% calf serum and a moderate amount of antibiotics for 4 days.The cells were directly treated by Spd with concentrations of 0, 25,50,75, 80,90,150,200μm/L for 24 hours in medium RPMI-1640 with free serum respectively.Afterwards,they were moved to original medium.Then,the cultured cells were stained for SDH in 8cultured day.The contents of SDH in the cells of each group were measured by the photometer-image analytical instrument(HPIA-2000).The results showed the contents of SDH were strongest in 75μm/L Spd. The second experiment:the cultured cells were treated with 75μm/L Spd for 0,12,24,6,48,60,72h after cultured for 4 days in routine medium.Samely,stained for SDH on 8 day.The result showed that the cultured cells grow well after treated with 75μm/L Spd for 48h. 2 .Effects of Spd on Ag-NORs of the cultured cells from S
. japonicum
In the experiment the cells from S. japonicum were inoculated on the small glass coverslipS. The cells were divided into test and control groups after cultured for 3 days in routine medium. Cells in the test group were treated with with Spd of the concentration of 75|μm/L for 48 hours in the medium without serum.Afterwars,the cells were cultured in routine medium.The culture condition for the cells in the control group was the same as the
test group except for no addition of Spd in the media.The cells were stained according to the routine cytochemical methods regularly from Iweek to 5 week. The three parameters of AgNORs: number of granules, percentage of AgNORs area/Nucleus area, average optical density were measured. Compared with control group, contents of AgNORs were rising by using Spd in the culture. The three parameters of AgNORs were all statisticaly different between control group and test group (P<0.01). It was suggested that Spd have some ability to increase the potential of transformation and proliferation in cultured cells. 3. Effects of Spd on AKP and ACP of the cultured cells from S. japonicum
In this experiment,3 groups were made:Spd group,liver matix group,Spd+liver matix group. The contents of AKP and ACP in the cultured cells of each group were measured by using HPIA-2000. The results showed the contents of AKP and ACP in cells of Spd group were strongest in three groups in 14 cultured days(P<0.01). AKP and ACP in Spd+liver matix group were the weakest in three groups(P<0.01);21 days later, AKP and ACP in cells of liver matix group were stronger than those of Spd group and the concentrations of the two enzymes in the cells of Spd+liver matix group were lower than 2 weeks before.The activities of AKP and ACP in Spd group were descended more sharply than those in liver matix group. It was suggested that liver matix was more stable and steady to advance the cultured cells than Spd.group.There was adverse-operation between Spd and liver matix.lt was proved that Spd could increase the metabolism in cultured cells and give an impetus to cell growth and proliferation.
引文
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