中药清毒栓对HPV相关宫颈癌防治的实验研究
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摘要
目的
通过研究中药清毒栓含药血清对人宫颈癌SiHa细胞体外增殖、细胞周期及凋亡的影响以及其对MDM2基因和p53基因表达的影响,从细胞及分子生物学水平探讨清毒栓防治宫颈癌的作用机制。方法
1.含药血清的提取:参照李仪奎法提取含药血清,56℃水浴灭活后分装,-70℃冰箱保存备用;
2.体外生长抑制实验:以不同浓度含药血清培养液分别作用于SiHa细胞24、48、72、96h,并设立空白血清及含中、西药阳性对照药血清为对照组,通过MTT法检测该药对细胞生长及增殖的影响,计算抑制率并选出最佳量效时点以进行下一步实验;
3.细胞周期及细胞凋亡实验:用流式细胞分析技术进行细胞周期及细胞调亡检测,根据MTT的最佳量效时点条件的药血清培养液处理SiHa细胞后,用RNAase消化半小时,PI避光染色1小时,上机检测,Modi Fit软件进行细胞周期分析并绘制DNA分布图,同时设立空白血清及含中、西药阳性对照药血清为对照组;
4.用PT-PCR法半定量检测对原癌基因MDM2及抑癌基因p53mRNA表达的影响:根据MTT的最佳量效时点条件的含药血清培养液处理SiHa细胞后,提取总RNA,进行RT-PCR反应后,电泳、凝胶图像分析仪采集图像,分析灰度值,同时设立空白血清及含中、西药阳性对照药血清为对照组。
结果
1.清毒栓含药血清对SiHa细胞体外增殖有明显的抑制作用,并随着血清浓度的增加其抑制作用更加明显,在一定浓度范围内呈剂量依赖性。口服组清毒栓含药血清与其它对照组相比72h时对SiHa细胞抑制作用最强,96h抑制作用明显减弱,阴道给药组各含药血清组与空白血清对照组比较对细胞也有明显的抑制作用。
2.细胞周期及细胞凋亡的研究:
2.1细胞周期各时相中,口服给药各含药血清组G0-G1期所占比例较空白血清对照组增加,其差异有显著性(P<0.01)。而口服各组含药血清组S期所占比例较空白血清对照组减少,其差异有显著性(P<0.05),尤以干扰素组明显(P<0.01),清毒栓组次之。而阴道给药各含药血清组与空白血清对照组比较在G0-G1期所占比例有增加的趋势;
2.2口服给药组清毒栓组及干扰素组含药血清对细胞凋亡有促进作用,与空白血清对照组相比有统计学差异(P<0.05);保妇康组含药血清未见明显差异,阴道给药各含药血清组与空白血清对照组比较在细胞凋亡方面未见明显差异。
3. MDM2-p53基因反馈环表达:
3.1对MDM2基因表达的影响:经RT-PCR并测定电泳条带吸光度值,计算MDM2与β2m基因扩增片段的吸光度比值,可见各组含药血清与空白对照组相比,MDM2 mRNA的表达量降低(P<0.01),以干扰素组最佳,清毒栓次之,清毒栓与干扰素组相比较无显著性差异。
3.2对p53基因表达的影响:经RT-PCR并测定电泳条带吸光度值,计算p53与β2m基因扩增片段的吸光度比值,可见各组含药血清与空白对照组相比,p53 mRNA的表达量增加(P<0.01),以干扰素组最佳,清毒栓次之。阴道给药清毒栓组及干扰素组与空白血清对照组比较p53 mRNA的表达量增加(P<0.01),其余各组含药血清与空白血清组比较未见明显差异。
结论清毒栓含药血清不仅可直接抑制宫颈癌细胞的生长、调节细胞周期、促进细胞凋亡,并可能通过调控MDM2-p53反馈环的分子机制,达到抑制肿瘤的目的。
Objact :To investigate the effect of the Serum pharmacological effect Qingdu Suppository on the SiHa cell growth in vitro.To approach the mechanism of action on Cell biology and Molecular biology level which Qingdu Suppository inhibiting the SiHa cell growth.
Method:
1. the Serum Containing Qingdu Suppository preparation :In accordance with the methode of Liyikui’s to extract the Serum Containing Qingdu Suppository and Blank Serum , Baofukang Suppository , Inf-αcontrolling group . Inactivate the Serum by waterbath at 56℃for half an hour. Store in -70℃Refrigerator. The Serologic pharmacological method.
2. the SiHa cell growth in vitro:After different density and different Serum treating to SiHa cells 72h , we evaluated photoabsorption value ,calculating inhibition ration and getting quantity and effect data .After treating SiHa cells by Serum 24,48,72,96h, we evaluated photoabsorption value ,calculating inhibition ration and getting quantity and effect data .
3. To detect the effect of cell cycle and apoptosis to SiHa cells treated with Serums by Flow cytometry After 8% density Serum treating SiHa cells 72h ,fixing cells ,digeting half an hour by RNAase ,dyeing 1h by PI away from light ,we detected it , anayzeed cell cycle by Modi Fit Sofeware and draw a DNA profile . 4. To detect the effect of regulating to p53-mRNA and MDM2-mRNA expression of SiHa cells te reated with Serums by RT-PCR Semiquantitative detection methode:After 8% density Serums treating SiHa cells , we extracted RNA , did RT-PCR reaction ,collected image by electrophoresis and gel image analytical apparatus and anayaed gray scale.
Results:
1. The Suppress rate of cells treated with Qingdu Suppository Serum were Significantly higher in 8% -72h group than those in the control group treated with Rat Serum,and those treated with IFN Serum were also significantly higher than those in the control group treated with Rat Serum .
2. Cell cycle and apoptosis :
2.1 Cell ratio had Significantly higher at G0-G1 phase by treating with 8% -72hr OR-group Qingdu Suppository Serum ,on the other hand that Cell ratio had Significantly lower at S phase by treating with 8% -72hr group Qingdu Suppository Serum. But there was no obviously change at each phase by treating with 8% -72h V-group Qingdu Suppository Serum.
2.2 The 8% -72h OR-group Qingdu Suppository Serum accelerated SiHa cell apoptosis,and so did 8% -72hr OR-group IFN Serum . But there no apoptosis peak appear in V-group Serums.
3. p53 and MDM2 mRNA expression:
3.1 The suppress rate of MDM2 gene expression of cells treated with were Significantly lower in than those in the control group treated with Rat Serum,and those treated with IFN Serum were also Significantly lower than those in the control group treated with Blank Rat Serum .
3.2 The Suppress rate of p53 gene expression of cells treated with Qingdu Suppository Serum were significantly higher in 8% -72h group than those in the control group treated with Rat Serum ,and those treated with IFN Serum were also significantly lower than those in the control group treated with Rat Serum .
ConcluSion: The Qingdu Suppository Serum suppress tumor by direct repression the cervical cancer cell proliferation , regulate cell cycle, accelerated SiHa cell apoptosis and also regulate the MDM2 - p53 feedback loop molecule mechanism.
通过研究中药清毒栓含药血清对人宫颈癌SiHa细胞体外增殖、细胞周期及凋亡的影响以及其对MDM2基因和p53基因表达的影响,从细胞及分子生物学水平探讨清毒栓防治宫颈癌的作用机制。方法
1.含药血清的提取:参照李仪奎法提取含药血清,56℃水浴灭活后分装,-70℃冰箱保存备用;
2.体外生长抑制实验:以不同浓度含药血清培养液分别作用于SiHa细胞24、48、72、96h,并设立空白血清及含中、西药阳性对照药血清为对照组,通过MTT法检测该药对细胞生长及增殖的影响,计算抑制率并选出最佳量效时点以进行下一步实验;
3.细胞周期及细胞凋亡实验:用流式细胞分析技术进行细胞周期及细胞调亡检测,根据MTT的最佳量效时点条件的药血清培养液处理SiHa细胞后,用RNAase消化半小时,PI避光染色1小时,上机检测,Modi Fit软件进行细胞周期分析并绘制DNA分布图,同时设立空白血清及含中、西药阳性对照药血清为对照组;
4.用PT-PCR法半定量检测对原癌基因MDM2及抑癌基因p53mRNA表达的影响:根据MTT的最佳量效时点条件的含药血清培养液处理SiHa细胞后,提取总RNA,进行RT-PCR反应后,电泳、凝胶图像分析仪采集图像,分析灰度值,同时设立空白血清及含中、西药阳性对照药血清为对照组。
结果
1.清毒栓含药血清对SiHa细胞体外增殖有明显的抑制作用,并随着血清浓度的增加其抑制作用更加明显,在一定浓度范围内呈剂量依赖性。口服组清毒栓含药血清与其它对照组相比72h时对SiHa细胞抑制作用最强,96h抑制作用明显减弱,阴道给药组各含药血清组与空白血清对照组比较对细胞也有明显的抑制作用。
2.细胞周期及细胞凋亡的研究:
2.1细胞周期各时相中,口服给药各含药血清组G0-G1期所占比例较空白血清对照组增加,其差异有显著性(P<0.01)。而口服各组含药血清组S期所占比例较空白血清对照组减少,其差异有显著性(P<0.05),尤以干扰素组明显(P<0.01),清毒栓组次之。而阴道给药各含药血清组与空白血清对照组比较在G0-G1期所占比例有增加的趋势;
2.2口服给药组清毒栓组及干扰素组含药血清对细胞凋亡有促进作用,与空白血清对照组相比有统计学差异(P<0.05);保妇康组含药血清未见明显差异,阴道给药各含药血清组与空白血清对照组比较在细胞凋亡方面未见明显差异。
3. MDM2-p53基因反馈环表达:
3.1对MDM2基因表达的影响:经RT-PCR并测定电泳条带吸光度值,计算MDM2与β2m基因扩增片段的吸光度比值,可见各组含药血清与空白对照组相比,MDM2 mRNA的表达量降低(P<0.01),以干扰素组最佳,清毒栓次之,清毒栓与干扰素组相比较无显著性差异。
3.2对p53基因表达的影响:经RT-PCR并测定电泳条带吸光度值,计算p53与β2m基因扩增片段的吸光度比值,可见各组含药血清与空白对照组相比,p53 mRNA的表达量增加(P<0.01),以干扰素组最佳,清毒栓次之。阴道给药清毒栓组及干扰素组与空白血清对照组比较p53 mRNA的表达量增加(P<0.01),其余各组含药血清与空白血清组比较未见明显差异。
结论清毒栓含药血清不仅可直接抑制宫颈癌细胞的生长、调节细胞周期、促进细胞凋亡,并可能通过调控MDM2-p53反馈环的分子机制,达到抑制肿瘤的目的。
Objact :To investigate the effect of the Serum pharmacological effect Qingdu Suppository on the SiHa cell growth in vitro.To approach the mechanism of action on Cell biology and Molecular biology level which Qingdu Suppository inhibiting the SiHa cell growth.
Method:
1. the Serum Containing Qingdu Suppository preparation :In accordance with the methode of Liyikui’s to extract the Serum Containing Qingdu Suppository and Blank Serum , Baofukang Suppository , Inf-αcontrolling group . Inactivate the Serum by waterbath at 56℃for half an hour. Store in -70℃Refrigerator. The Serologic pharmacological method.
2. the SiHa cell growth in vitro:After different density and different Serum treating to SiHa cells 72h , we evaluated photoabsorption value ,calculating inhibition ration and getting quantity and effect data .After treating SiHa cells by Serum 24,48,72,96h, we evaluated photoabsorption value ,calculating inhibition ration and getting quantity and effect data .
3. To detect the effect of cell cycle and apoptosis to SiHa cells treated with Serums by Flow cytometry After 8% density Serum treating SiHa cells 72h ,fixing cells ,digeting half an hour by RNAase ,dyeing 1h by PI away from light ,we detected it , anayzeed cell cycle by Modi Fit Sofeware and draw a DNA profile . 4. To detect the effect of regulating to p53-mRNA and MDM2-mRNA expression of SiHa cells te reated with Serums by RT-PCR Semiquantitative detection methode:After 8% density Serums treating SiHa cells , we extracted RNA , did RT-PCR reaction ,collected image by electrophoresis and gel image analytical apparatus and anayaed gray scale.
Results:
1. The Suppress rate of cells treated with Qingdu Suppository Serum were Significantly higher in 8% -72h group than those in the control group treated with Rat Serum,and those treated with IFN Serum were also significantly higher than those in the control group treated with Rat Serum .
2. Cell cycle and apoptosis :
2.1 Cell ratio had Significantly higher at G0-G1 phase by treating with 8% -72hr OR-group Qingdu Suppository Serum ,on the other hand that Cell ratio had Significantly lower at S phase by treating with 8% -72hr group Qingdu Suppository Serum. But there was no obviously change at each phase by treating with 8% -72h V-group Qingdu Suppository Serum.
2.2 The 8% -72h OR-group Qingdu Suppository Serum accelerated SiHa cell apoptosis,and so did 8% -72hr OR-group IFN Serum . But there no apoptosis peak appear in V-group Serums.
3. p53 and MDM2 mRNA expression:
3.1 The suppress rate of MDM2 gene expression of cells treated with were Significantly lower in than those in the control group treated with Rat Serum,and those treated with IFN Serum were also Significantly lower than those in the control group treated with Blank Rat Serum .
3.2 The Suppress rate of p53 gene expression of cells treated with Qingdu Suppository Serum were significantly higher in 8% -72h group than those in the control group treated with Rat Serum ,and those treated with IFN Serum were also significantly lower than those in the control group treated with Rat Serum .
ConcluSion: The Qingdu Suppository Serum suppress tumor by direct repression the cervical cancer cell proliferation , regulate cell cycle, accelerated SiHa cell apoptosis and also regulate the MDM2 - p53 feedback loop molecule mechanism.
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