正肝方影响肝癌前病变AFP依赖机制的研究
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摘要
目的：基于甲胎蛋白（AFP）对肝癌的发生发展及预后判断具有重要意义，以及前期正肝方具有较好防治肝癌前病变的临床及实验研究结果，本文应用RNAi技术构建AFP表达缺失的人肝癌HepG_2细胞系(AFPsiRNA-HepG_2细胞系)作为研究工具，观察AFP对肝癌细胞系HepG_2的影响，以及正肝方对表达AFP肝癌细胞系和AFPsiRNA-HepG_2细胞系的药效学差异，从而阐释AFP依赖机制是否是正肝方防治肝癌前病变的重要药理机制。
方法：本论文分为四个部分。
1.制备正肝方大鼠血清，应用Alamar blue方法筛选药物血清作用浓度及药物作用时间，干预人肝癌HepG_2细胞系，MTT法检测细胞增殖情况，试剂盒法测GGT水平，化学发光法和Western blot法检测AFP的表达，Hoechst33258染色观察细胞核形态，流式细胞术检测细胞的周期及凋亡，RT-PCR检测细胞端粒酶逆转录酶基因的表达。
2.通过http://rnaidesigner.invitrogen.com/rnaiexpress/网站软件设计基因干扰靶点序列，模板中的loop结构选用了TTCAAGAGA以避免形成终止信号。正义链模板的5’端添加T，与BbsI酶切后形成的粘端互补；反义链模板的5’端添加AGCT，与XhoI酶切后形成的粘端互补。正义链:5’-T-(GN_18)-(TTCAAGAGA)-(N_18C)-TTTTTTC-3’，反义链：3’-A(CN_18)-(AAGTTCTCT)-(N_18G)-AAAAAAGAGCT-5’。按此规则选择4个干扰靶点，分别记为AFP-639，AFP-1020，AFP-1432，AFP-1811。合成靶点序列，构建并鉴定质粒，包装、纯化病毒载体，转染，细胞计数计算感染效率，Western blot及RT-PCR检测各组AFP基因及蛋白的表达情况。选择干扰效率最高组，大量病毒包装构建AFPsiRNA-HepG_2细胞系，Westernblot及RT-PCR检测AFPsiRNA-HepG_2细胞及其传代后AFP基因及蛋白的表达。
3.不同浓度外源性AFP干预AFPsiRNA-HepG_2细胞系，MTT法检测细胞增殖情况，试剂盒法检测GGT水平，流式细胞术检测细胞的周期及凋亡，RT-PCR检测细胞端粒酶逆转录酶基因的表达。
4.将AFPsiRNA-HepG
2细胞分为8组，按5%正常大鼠血清、5%药物血清、10%正常大鼠血清、10%药物血清、AFP+5%正常大鼠血清、AFP+5%药物血清、AFP+10%正常大鼠血清、AFP+10%药物血清加入干预，24h后，MTT法检测细胞增殖情况，试剂盒法检测GGT水平，流式细胞术检测细胞的周期及凋亡。
结果：
1.Alamar blue结果提示，20%血清浓度组正常大鼠血清及中药大鼠血清有明显细胞毒性作用，所以选择5%、10%两个浓度组进行实验。药物干预时间为24h；与正常大鼠血清组比较，5%、10%药物血清组有显著阻滞细胞周期，抑制细胞增殖，诱导细胞凋亡，并下调细胞AFP基因及蛋白的表达，下调端粒酶基因的表达；5%、10%药物血清组显著改变细胞核形态结构；药物血清组对细胞GGT的表达无影响。
2.选择的四个靶序列，腺病毒少量包装后转染，计算感染效率，并测细胞AFP基因及蛋白的表达，结果提示，与野生型人肝癌HepG
2细胞比较，靶点“AFP-639”干扰效率最高(97%)。选择靶点“AFP-639”构建细胞模型(AFPsiRNA-HepG
2)，模型细胞传代后仍能稳定地沉默AFP基因及蛋白的表达，沉默效率达87%左右。
3.大剂量AFP(100ug/ml)抑制AFPsiRNA-HepG
2细胞增殖，阻滞细胞周期，下调端粒酶基因表达，并诱导其凋亡，小剂量AFP(≤10ug/ml)能促进细胞增殖，缩短细胞周期。不同剂量AFP对细胞GGT水平无影响。
4.无AFP共同干预时，与正常大鼠血清组比较，5%、10%药物血清组对细胞周期、细胞增殖、细胞凋亡及GGT水平均无影响。有AFP共同干预时，AFP+正常大鼠血清组比较，AFP+药物血清组将细胞周期阻滞于G_0/G_1期，显著抑制细胞增殖，延长细胞周期，诱导细胞凋亡；5%、10%药物血清组对细胞GGT水平无显著影响。
结论：
1.在体外实验中，正肝方药物血清显著下调肝癌细胞AFP的表达，抑制细胞生长并诱导其凋亡。
2.应用RNAi技术成功构建了AFP表达缺失的人肝癌模型细胞系（AFPsiRNA-HepG_2）。
3.低浓度AFP有促进AFPsiRNA-HepG_2细胞增殖作用。
4.正肝方依赖抑制AFP对肝癌细胞的促增殖作用防治肝癌前病变。
Objective:
RNAi technology was used to construct the model cell line(AFPsiRNA-HepG_2cell line)which is out of expression of the AFP of HepG_2cellline; To investigate The effects of exogenous alpha fetoprotein onproliferation and apoptosis in AFPsiRNA-HepG_2cells;and furtherexplore the specific mechanisms of Zheng Gan Fang formula oninhibiting precancerous lesion of liver.
Methods: This paper includes four parts.
1.The drug sera was collected from rats. Preliminary screening incytotoxicity and density of the drug sera was determined using thealamar blue method. HepG_2cells were cultured with drug sera, Cellproliferation was assayed by MTT method,GGT was detected by Kitassay,Chemiluminiscence method and Western blot assay were appliedto detect the expression of AFP. Nuclear morphometry wasinvestigated by IFA, Cell cycle and apoptosis was assayed by flowcytometry(FCM), Real Time PCR were applied to detect the expressionof Cell telomerase gene.
2.Gene sequence target AFP was designed from the website that ishttps://rnaidesigner.invitrogen.com/rnaiexpress/.The loopstructure in the template used TTCAAGAGA to avoid a terminationsigna.Positive Sense strand:5’-T-(GN18)-(TTCAAGAGA)-(N18C)-TTTTTTC-3’,Antisense strand:3’-A(CN18)-(AAGTTCTCT)-(N18G)-AAAAAAGAGCT-5’.Choose four targetspots,named AFP-639，AFP-1020，AFP-1432，AFP-1811. Synthesis oftarget sequence, Build and identifying plasmid, packaging andpurifing viral vector, Transfection, Cell count Computingefficiency of infection, Use Western blot and Real Time PCR methodto detect the gene and protein expression of AFP in all groups.Select interfere with the highest efficiency group, Large amountsof virus package build AFPsiRNA-HepG_2cell lines. And the expressionof. the gene and protein expression of AFP in AFPsiRNA-HepG_2celland after the wedding was detected by Western blot and Real TimePCR method.
3.AFPsiRNA-HepG_2cell lines were cultured with different levelsof exogenous AFP.Cell proliferation was assayed by MTT method, GGTwas detected by Kit assay; The nucleus morphological observationwas investigated by fluorescent assay, Cell cycle and apoptosis wasassayed by flow cytometry(FCM), Real Time PCR were applied to detectthe expression of cell telomerase.
4.AFPsiRNA-HepG_2cells was Divided into eight groups:5%of normallybig rat sera,5%of drug sera,10%of normally big rat sera,10%of drugsera,AFP+5%of normally big rat sera,AFP+5%of drug sera, AFP+10%ofnormally big rat sera,AFP+10%of drug sera, cultured24hours, Cellproliferation was assayed by MTT method,GGT was detected by Kitassay,Chemiluminiscence method and Western blot assay were applied to detect the expression of AFP.Nuclear morphometry wasinvestigated by IFA,cell cycle and apoptosis was assayed by flowcytometry(FCM),Real Time PCR were applied to detect the expressionof cell telomerase gene.
Results:
1.The results showed that concentrations of20%with normally bigrat sera and drug sera have obvious cytotoxic effect,so theconcentration of5%and10%were selected,and clture for24hours；Compared with the group of normally big rat sera，the concentrationof5%and10%groups have obvious effect in inhibiting cellproliferation, blocking the cell cycle, and inducing cell apoptosis,down-regulating the expression of AFP gene and protein,anddown-regulating the expression of telomerase gene;And have obviouseffect on changeing the shape and structure of the nucleus; Drugsera have no influence on the expression of GGT.
2.Four target sequences were choesed, Adenovirus transfectionafter a small amount of packaging,Computing efficiency ofinfection,detecting the gene and protein expression of AFP,theresults suggest that the target spot of “AFP-639”have the highestinterference efficiency(97%) compare withWild type humanhepatocellular carcinoma HepG_2cells.So the the target spot of“AFP-639” was selected to build cell model of AFPsiRNA-HepG_2，the model cells after the wedding can still steady silence theexpression of AFP gene and protein.
3.High dose AFP(≥100ug/ml) can inhibit AFPsiRNA-HepG_2cellproliferation, block the cell cycle, cut the telomerase geneexpression and induce cell death, low dose AFP(＜100ug/ml) canpromote cell proliferation, shorten the cell cycle.Different dose AFP have no influence on the expression of GGT.
4.Compared with the group of normally normal sera，the concentrationof5%and10%drug sera have obvious effect to inhibit cellproliferation, block the cell cycle in G_0/G_1, Speriod, prolong thecell cycle, significantly induced cell death, significantly cut thetelomerase gene expression, the concentration of5%and10%drugsera groups have no significantly influence on the expression ofGGT.
Conclusion:
1.Zheng Gan Fang Formula has obvious effect on reducing theexpression of AFP,inhibiting cell proliferation, blocking the cellcycle, and inducing cell apoptosis of HepG_2cells in vitro.
2.RNAi technology can be used to build HepG_2cell line out ofexpression of AFP.
3.The low concentration of AFP has effect on promoting cellproliferation of AFPsiRNA-HepG_2cells.
4.Zheng Gan Fang Formula prevent and treat precancerous lesion ofliver throug restraining the effect on promoting cell proliferitionof AFP.
方法：本论文分为四个部分。
1.制备正肝方大鼠血清，应用Alamar blue方法筛选药物血清作用浓度及药物作用时间，干预人肝癌HepG_2细胞系，MTT法检测细胞增殖情况，试剂盒法测GGT水平，化学发光法和Western blot法检测AFP的表达，Hoechst33258染色观察细胞核形态，流式细胞术检测细胞的周期及凋亡，RT-PCR检测细胞端粒酶逆转录酶基因的表达。
2.通过http://rnaidesigner.invitrogen.com/rnaiexpress/网站软件设计基因干扰靶点序列，模板中的loop结构选用了TTCAAGAGA以避免形成终止信号。正义链模板的5’端添加T，与BbsI酶切后形成的粘端互补；反义链模板的5’端添加AGCT，与XhoI酶切后形成的粘端互补。正义链:5’-T-(GN_18)-(TTCAAGAGA)-(N_18C)-TTTTTTC-3’，反义链：3’-A(CN_18)-(AAGTTCTCT)-(N_18G)-AAAAAAGAGCT-5’。按此规则选择4个干扰靶点，分别记为AFP-639，AFP-1020，AFP-1432，AFP-1811。合成靶点序列，构建并鉴定质粒，包装、纯化病毒载体，转染，细胞计数计算感染效率，Western blot及RT-PCR检测各组AFP基因及蛋白的表达情况。选择干扰效率最高组，大量病毒包装构建AFPsiRNA-HepG_2细胞系，Westernblot及RT-PCR检测AFPsiRNA-HepG_2细胞及其传代后AFP基因及蛋白的表达。
3.不同浓度外源性AFP干预AFPsiRNA-HepG_2细胞系，MTT法检测细胞增殖情况，试剂盒法检测GGT水平，流式细胞术检测细胞的周期及凋亡，RT-PCR检测细胞端粒酶逆转录酶基因的表达。
4.将AFPsiRNA-HepG
2细胞分为8组，按5%正常大鼠血清、5%药物血清、10%正常大鼠血清、10%药物血清、AFP+5%正常大鼠血清、AFP+5%药物血清、AFP+10%正常大鼠血清、AFP+10%药物血清加入干预，24h后，MTT法检测细胞增殖情况，试剂盒法检测GGT水平，流式细胞术检测细胞的周期及凋亡。
结果：
1.Alamar blue结果提示，20%血清浓度组正常大鼠血清及中药大鼠血清有明显细胞毒性作用，所以选择5%、10%两个浓度组进行实验。药物干预时间为24h；与正常大鼠血清组比较，5%、10%药物血清组有显著阻滞细胞周期，抑制细胞增殖，诱导细胞凋亡，并下调细胞AFP基因及蛋白的表达，下调端粒酶基因的表达；5%、10%药物血清组显著改变细胞核形态结构；药物血清组对细胞GGT的表达无影响。
2.选择的四个靶序列，腺病毒少量包装后转染，计算感染效率，并测细胞AFP基因及蛋白的表达，结果提示，与野生型人肝癌HepG
2细胞比较，靶点“AFP-639”干扰效率最高(97%)。选择靶点“AFP-639”构建细胞模型(AFPsiRNA-HepG
2)，模型细胞传代后仍能稳定地沉默AFP基因及蛋白的表达，沉默效率达87%左右。
3.大剂量AFP(100ug/ml)抑制AFPsiRNA-HepG
2细胞增殖，阻滞细胞周期，下调端粒酶基因表达，并诱导其凋亡，小剂量AFP(≤10ug/ml)能促进细胞增殖，缩短细胞周期。不同剂量AFP对细胞GGT水平无影响。
4.无AFP共同干预时，与正常大鼠血清组比较，5%、10%药物血清组对细胞周期、细胞增殖、细胞凋亡及GGT水平均无影响。有AFP共同干预时，AFP+正常大鼠血清组比较，AFP+药物血清组将细胞周期阻滞于G_0/G_1期，显著抑制细胞增殖，延长细胞周期，诱导细胞凋亡；5%、10%药物血清组对细胞GGT水平无显著影响。
结论：
1.在体外实验中，正肝方药物血清显著下调肝癌细胞AFP的表达，抑制细胞生长并诱导其凋亡。
2.应用RNAi技术成功构建了AFP表达缺失的人肝癌模型细胞系（AFPsiRNA-HepG_2）。
3.低浓度AFP有促进AFPsiRNA-HepG_2细胞增殖作用。
4.正肝方依赖抑制AFP对肝癌细胞的促增殖作用防治肝癌前病变。
Objective:
RNAi technology was used to construct the model cell line(AFPsiRNA-HepG_2cell line)which is out of expression of the AFP of HepG_2cellline; To investigate The effects of exogenous alpha fetoprotein onproliferation and apoptosis in AFPsiRNA-HepG_2cells;and furtherexplore the specific mechanisms of Zheng Gan Fang formula oninhibiting precancerous lesion of liver.
Methods: This paper includes four parts.
1.The drug sera was collected from rats. Preliminary screening incytotoxicity and density of the drug sera was determined using thealamar blue method. HepG_2cells were cultured with drug sera, Cellproliferation was assayed by MTT method,GGT was detected by Kitassay,Chemiluminiscence method and Western blot assay were appliedto detect the expression of AFP. Nuclear morphometry wasinvestigated by IFA, Cell cycle and apoptosis was assayed by flowcytometry(FCM), Real Time PCR were applied to detect the expressionof Cell telomerase gene.
2.Gene sequence target AFP was designed from the website that ishttps://rnaidesigner.invitrogen.com/rnaiexpress/.The loopstructure in the template used TTCAAGAGA to avoid a terminationsigna.Positive Sense strand:5’-T-(GN18)-(TTCAAGAGA)-(N18C)-TTTTTTC-3’,Antisense strand:3’-A(CN18)-(AAGTTCTCT)-(N18G)-AAAAAAGAGCT-5’.Choose four targetspots,named AFP-639，AFP-1020，AFP-1432，AFP-1811. Synthesis oftarget sequence, Build and identifying plasmid, packaging andpurifing viral vector, Transfection, Cell count Computingefficiency of infection, Use Western blot and Real Time PCR methodto detect the gene and protein expression of AFP in all groups.Select interfere with the highest efficiency group, Large amountsof virus package build AFPsiRNA-HepG_2cell lines. And the expressionof. the gene and protein expression of AFP in AFPsiRNA-HepG_2celland after the wedding was detected by Western blot and Real TimePCR method.
3.AFPsiRNA-HepG_2cell lines were cultured with different levelsof exogenous AFP.Cell proliferation was assayed by MTT method, GGTwas detected by Kit assay; The nucleus morphological observationwas investigated by fluorescent assay, Cell cycle and apoptosis wasassayed by flow cytometry(FCM), Real Time PCR were applied to detectthe expression of cell telomerase.
4.AFPsiRNA-HepG_2cells was Divided into eight groups:5%of normallybig rat sera,5%of drug sera,10%of normally big rat sera,10%of drugsera,AFP+5%of normally big rat sera,AFP+5%of drug sera, AFP+10%ofnormally big rat sera,AFP+10%of drug sera, cultured24hours, Cellproliferation was assayed by MTT method,GGT was detected by Kitassay,Chemiluminiscence method and Western blot assay were applied to detect the expression of AFP.Nuclear morphometry wasinvestigated by IFA,cell cycle and apoptosis was assayed by flowcytometry(FCM),Real Time PCR were applied to detect the expressionof cell telomerase gene.
Results:
1.The results showed that concentrations of20%with normally bigrat sera and drug sera have obvious cytotoxic effect,so theconcentration of5%and10%were selected,and clture for24hours；Compared with the group of normally big rat sera，the concentrationof5%and10%groups have obvious effect in inhibiting cellproliferation, blocking the cell cycle, and inducing cell apoptosis,down-regulating the expression of AFP gene and protein,anddown-regulating the expression of telomerase gene;And have obviouseffect on changeing the shape and structure of the nucleus; Drugsera have no influence on the expression of GGT.
2.Four target sequences were choesed, Adenovirus transfectionafter a small amount of packaging,Computing efficiency ofinfection,detecting the gene and protein expression of AFP,theresults suggest that the target spot of “AFP-639”have the highestinterference efficiency(97%) compare withWild type humanhepatocellular carcinoma HepG_2cells.So the the target spot of“AFP-639” was selected to build cell model of AFPsiRNA-HepG_2，the model cells after the wedding can still steady silence theexpression of AFP gene and protein.
3.High dose AFP(≥100ug/ml) can inhibit AFPsiRNA-HepG_2cellproliferation, block the cell cycle, cut the telomerase geneexpression and induce cell death, low dose AFP(＜100ug/ml) canpromote cell proliferation, shorten the cell cycle.Different dose AFP have no influence on the expression of GGT.
4.Compared with the group of normally normal sera，the concentrationof5%and10%drug sera have obvious effect to inhibit cellproliferation, block the cell cycle in G_0/G_1, Speriod, prolong thecell cycle, significantly induced cell death, significantly cut thetelomerase gene expression, the concentration of5%and10%drugsera groups have no significantly influence on the expression ofGGT.
Conclusion:
1.Zheng Gan Fang Formula has obvious effect on reducing theexpression of AFP,inhibiting cell proliferation, blocking the cellcycle, and inducing cell apoptosis of HepG_2cells in vitro.
2.RNAi technology can be used to build HepG_2cell line out ofexpression of AFP.
3.The low concentration of AFP has effect on promoting cellproliferation of AFPsiRNA-HepG_2cells.
4.Zheng Gan Fang Formula prevent and treat precancerous lesion ofliver throug restraining the effect on promoting cell proliferitionof AFP.
引文
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[49]姜雅堃，刘吉勇，韩翠萍等.反义人端粒酶RNA诱导肝癌细胞凋亡的分子生物学机制研究[J].山东大学学报(医学版)，2011，49(12):25-29.
[50]蔡俊义，孙超.张德忠治疗原发性肝癌经验[J].湖北中医杂志，2010，32（7）:30-31．
[51]钱祥夕．谢兆丰老中医治疗肝癌的经验之浅谈[J]．中医临床研究，2011，3(2):105．
[52]Peck A，Murgita RA，Wigzel H．Cellular and genetic restrictionsin the immunoregulatory activity of alpha fetoprotein. Sel--ective inhibition of anti-aassociated proliferativereactions[J]．J Exp Med，1978，147(11):667-672.
[53]Caballero OL，Chen YT．Cancer/testis(CT)antigens:potentialtargets for immunotherapy[J].Cancer Sci,2009,100(11):2014-2021.
[54]Yang XR，Xu Y，Yu B，et al.High expression levels of putativehe patic stem/progenitor cell biomarkers related to tumourangiogenesis and poor prognosis of hepatocellular carcinoma[J]．Gut,2010,59(7):953-962.
[55]卢彦琦，贺学礼．黄芪化学成分及药理作用综述[J]．保定师范专利学校学报，2004，17(4):40－42．
[56]孙婧,周信达,刘银坤.丹参对肝癌转移复发防治作用的研究[J].中国中西医结合杂志.1999,19(5):292-295.
[57]许惠.中药川芎应用与提取工艺的发展研究[J].重庆工商大学学报(自然科学版)，2005，22(3):233-236.
[58]曾永长，梁少瑜，罗佳波.白花蛇舌草水提部位体内外抗肿瘤实验研究[J].中药新药与临床药理2011,22(5):521-524.
[59]代志军,王西京,纪宗正.半枝莲提取物对DEN诱发大鼠肝癌的抑制作用[J].中药材，2009，32(4):568-571.
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[61]高福君.女贞子提取物抑制人肝癌细胞血管生长因子表达作用研究[J].中国实验方剂学杂志,2011,17(2):139-142.62.
[62]乔建卫,王慧铭.鳖甲的药用现状[J].浙江中西医结合杂志,2009,19(1):45-46.
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[3]Semenkova LN, Dudich EI, Dudich Ⅳ.Induction of apoptosis inhuman hepatona cells by alpha fetoprotein[J].Tumour Biol,1997,18(5):261-273.
[4]Mizejewski GJ.Alpha fetoprotein binding proteins:Implicationsfor transmembrane passage and subcellular localization[J].Life Sci,1995,56(1):1291.
[5]Villacampa MJ,Moro R, Naval J,etal.Alpha fetoprotein receptorsin a human breast cancer cell line[J]. Biochem Biophys ResCommun,1984,122(3):1322-1327.
[6]Uriel J,Failly CC,Villacampa MJ,etaL.In corporation ofalphafetoprotein by the MCF-7human breast cancer cell line[J].Tumour Biol1984,5(1):41-51.
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