P38 MAPK在顺铂和热疗致肝癌细胞凋亡过程中的作用
摘要
目的:探讨P38 MAPK在顺铂化疗和热疗诱导肝癌细胞凋亡的机制,以及对正常肝细胞的影响,为临床应用提供实验依据。
方法:通过体外培养正常肝细胞HL-7702、癌旁肝硬化肝细胞QSG-7701和肝癌细胞QGY-7703,应用免疫组织细胞化学、MTT、激光扫描共聚焦显微镜、Western Blot、流式细胞仪和TUNEL法等方法观察顺铂和热诱导对其凋亡的影响,然后加入P38 MAPK的特异性抑制剂SB203580,再观察顺铂和热诱导对细胞凋亡情况的变化。
结果:P38 MAPK表达从正常肝细胞、癌旁肝硬化肝细胞到肝癌细胞逐渐增高。P38 MAPK的特异性抑制剂SB203580可以使细胞的凋亡率降低。3株培养细胞加顺铂后凋亡率均明显增加,存活率明显下降,加用P38 MAPK的特异性抑制剂SB203580后,可使细胞凋亡率下降。3株培养细胞对于顺铂的反应性不同,肝癌细胞最敏感,凋亡率和坏死率最高。热诱导可以使3株培养细胞发生凋亡,其中肝癌细胞最敏感,引起凋亡和坏死的细胞最多。
结论1顺铂诱导细胞凋亡有P38 MAPK通路的参与。
2热诱导可以引起细胞凋亡,这过程中有P38 MAPK通路的参与。
3癌旁肝硬化肝细胞的生物学特性与正常肝细胞和肝癌细胞都不同,属于正处在转化过程中的癌前期细胞,值得进一步研究。
Objective:To investigate the effects of P38 MAP kinase in apoptosis of the hcc cell line and study the mechanism of carcinogenesis of HCC,Based on the DDP and heating caused in HCC apoptosis by means of P38 MAPK,as well as investigate the effects of the normal liver cell line, and provided the scientific basis for the clinical application.
Methods:The present study was designed to cultured cell lines(the normal liver cell line HL-7702,the paratumor cirrhosis cell line QSG-7701 and the HCC cell line QGY-7703)in vitro,p38MAPK were detected by immunohistochemical,confocal microscope,Western Blot.MTT,Flow cytometer and TUNEL method were used to detect apoptosis to observe heating and DDP affect cell lines,together with P38 MAPK specificity inhibitor SB203580.observe the influence of apoptosis regulation at the before and the after block by SB203580 in disparity the cell lines.Investigating the mechanisms of inducement apoptosis by means of heat treat and DDP chemo-treatment.It can be used to provide theoretical basis for clinical prevention and cure tumor.
Results:The express of P38 MAPK gradually increase from normal liver cell line,the paratumor cirrhosis cell line to the HCC cell line,The P38 MAPK specificity inhibitor SB203580 may to reduce cell apoptosis.The rate of apoptosis were markedly increased after to together with DDP.when plusing the P38 MAPK specificity inhibitor SB203580,the rate of apoptosis was decreased.The reaction of the 3 cultured cell lines to DDP were different,HCC cell line were the most sensitive,the rate.of apoptosis and necrosis were the obvious,Heating may induce a change to apoptosis in the 3 cultured cell lines.the reactions of the 3 cultured cell lines to heating inducement were different,HCC cell line were the most sensitive,the cell number of apoptosis and necrosis were the most.
Conclusions:1 DDP induced apoptosis by the P38 MAPK pathway.
2 Heating inducement may lead to cell apoptosis,which involved the P38 MAPK pathway.
3 The paratumor cirrhosis cells had an individual biology characteristic,and the growth curve of them was similar to that of HCC cells,which demonstrated paratumor cirrhosis cells belonged to precancerous cells transforming.
方法:通过体外培养正常肝细胞HL-7702、癌旁肝硬化肝细胞QSG-7701和肝癌细胞QGY-7703,应用免疫组织细胞化学、MTT、激光扫描共聚焦显微镜、Western Blot、流式细胞仪和TUNEL法等方法观察顺铂和热诱导对其凋亡的影响,然后加入P38 MAPK的特异性抑制剂SB203580,再观察顺铂和热诱导对细胞凋亡情况的变化。
结果:P38 MAPK表达从正常肝细胞、癌旁肝硬化肝细胞到肝癌细胞逐渐增高。P38 MAPK的特异性抑制剂SB203580可以使细胞的凋亡率降低。3株培养细胞加顺铂后凋亡率均明显增加,存活率明显下降,加用P38 MAPK的特异性抑制剂SB203580后,可使细胞凋亡率下降。3株培养细胞对于顺铂的反应性不同,肝癌细胞最敏感,凋亡率和坏死率最高。热诱导可以使3株培养细胞发生凋亡,其中肝癌细胞最敏感,引起凋亡和坏死的细胞最多。
结论1顺铂诱导细胞凋亡有P38 MAPK通路的参与。
2热诱导可以引起细胞凋亡,这过程中有P38 MAPK通路的参与。
3癌旁肝硬化肝细胞的生物学特性与正常肝细胞和肝癌细胞都不同,属于正处在转化过程中的癌前期细胞,值得进一步研究。
Objective:To investigate the effects of P38 MAP kinase in apoptosis of the hcc cell line and study the mechanism of carcinogenesis of HCC,Based on the DDP and heating caused in HCC apoptosis by means of P38 MAPK,as well as investigate the effects of the normal liver cell line, and provided the scientific basis for the clinical application.
Methods:The present study was designed to cultured cell lines(the normal liver cell line HL-7702,the paratumor cirrhosis cell line QSG-7701 and the HCC cell line QGY-7703)in vitro,p38MAPK were detected by immunohistochemical,confocal microscope,Western Blot.MTT,Flow cytometer and TUNEL method were used to detect apoptosis to observe heating and DDP affect cell lines,together with P38 MAPK specificity inhibitor SB203580.observe the influence of apoptosis regulation at the before and the after block by SB203580 in disparity the cell lines.Investigating the mechanisms of inducement apoptosis by means of heat treat and DDP chemo-treatment.It can be used to provide theoretical basis for clinical prevention and cure tumor.
Results:The express of P38 MAPK gradually increase from normal liver cell line,the paratumor cirrhosis cell line to the HCC cell line,The P38 MAPK specificity inhibitor SB203580 may to reduce cell apoptosis.The rate of apoptosis were markedly increased after to together with DDP.when plusing the P38 MAPK specificity inhibitor SB203580,the rate of apoptosis was decreased.The reaction of the 3 cultured cell lines to DDP were different,HCC cell line were the most sensitive,the rate.of apoptosis and necrosis were the obvious,Heating may induce a change to apoptosis in the 3 cultured cell lines.the reactions of the 3 cultured cell lines to heating inducement were different,HCC cell line were the most sensitive,the cell number of apoptosis and necrosis were the most.
Conclusions:1 DDP induced apoptosis by the P38 MAPK pathway.
2 Heating inducement may lead to cell apoptosis,which involved the P38 MAPK pathway.
3 The paratumor cirrhosis cells had an individual biology characteristic,and the growth curve of them was similar to that of HCC cells,which demonstrated paratumor cirrhosis cells belonged to precancerous cells transforming.
引文
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[2]Livingstone C,Patel G,Jones N.ATF-2 contains a phosphorylation-dependent transcriptional activation domain.EMBO J,1995,14:1785-1797
[3]Nakata S,Ito K,Fujimori M,et al.Involvement of vascular endothelial growth factor and urokinase-type plasminogen activator receptor in microvessel invasion in human colorectal cancers.Int J Cancer,1998,17:179-186
[4]Schumann RR,Pfeil D,Lamping N,et al.Lipopolysaccharide induces the rapid tyrosine phosphorylation of the Mitogen-activated protein kinases erk-1 and p38 in cultured human vascular endothelial cells requiring the presence of soluble CD14.Blood,1996,87:2805-2814
[5]Simon C,Goepfert H,Boyd D.Inhibition of the p38 Mitogen-activated protein -kinase by SB 203580 blocks PMA-induced Mr 92000 type Ⅳ collagenase secretion and in vitro invasion.Cancer Res,1998,58:1135-1139
[6]Martin-Blanco E.P38 MAPK signaling cascades:ancient roles and new function.Bioessays,2000,22:637-645.
[7]Ellinger-Ziegelbauer H,Kelly K,Siebenlist U.Cell cycle arrest and reversion of Ras-induced transformation bya conditionally activated form of mitogen-activated protein kinase kinase kinase3.Mol Cell Biol,1999,19:3857-3868.
[8]Sivash insk JiM S,Salganik R I.Heterogenous reaction of cancercells to cisp latin.Vopr Onkol,2001,47(1):66-68
[9]Zhang X,Yamamo to N,So ramo to S,et al.Cisp latin induced germ cell apoptosis in mouse testes.Arch And rol,2001,46(1):43-49.
[10]EvansDL,D ire C.Effects of cisp latin on the induction of apoptosis in p ro liferating hepatoma cells and nonp ro liferating immature thymocytes.Cancer Res,1993,53(9):2133-2139
[11]郑壬鸿,窦科峰,梁克明等.体外顺铂诱导肝癌SMMC-7721细胞凋亡时细胞信号转 导因子活性变化 第四军医大学学报,2002,23(9):831-834
[12]毛华,袁爱力,赵敏芳等.P38 MAPK信号通路影响血管内皮细胞生长因子诱导肝癌细胞超微结构变化.世界华人消化杂志,2000,8(5):536-538
[13]Abe JI,Eusuhara M,Ulevitch RJ,et al.Big mitogen-activated protein kinase 1(BMK1)is a redox-sensitive kinase.J Biol Chem,1996,271(28):16586
[14]MU Jin-ye,CHEN Klao-Guang..The progress of the study on the mitogen-activated protein kinase pathways.Chin Bull Life Sci,2002,14(4):208-211
[15]Han J,Lee JD,Bibbs L,et al.A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells,science,1994,265(5173):808-811
[16]Wang XS,Diener K,Manthey CL,et al.Molecula cloning and characterization of a novel p38 mitogen-activated protein kinases.J Biol Chemii,1997,2/2:23668-23674
[17]Raingeaud J,Gupta S,Rogers J,et al.Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine.J Biol Chem,1995,270:7420-7426
[18]Raingeaud J,Whitmarsh AJ,Barrett T,et al.MKK3 and MKK6 regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transducion pathway.Mol Cell Biol,1996,16(3):1247
[19]Martin-Blanco E.P38 MAPK signaling cascades:ancient roles and new function.Bioessays,2000,22:637-645
[19]Ellinger-Ziegelbauer H,Kelly K,Siebenlist U.Cell cycle arrest and reersion of Ras-induced transformation by a conditionally activated formm of mitogen-activated protein kinase kinase kinase3.Mol Cell Biol,1999,19:3857-3868
[20]Bulavin Dr,Kovalsky O,Hollander MC,et al.Loss of oncogenic H-ras-induced cell arrest and p38 mitogen-activated protein kinase activatin by disruption of Gadd45a.Mol Cell Biol,2003,23:3859-3871
[21]De Souze cp,Ellemka,Gabrielli bc.Centrosomal and cytoplasmic Cdc2/cyclinB1activation precedes nuclear mitotic events.Exp Cell Res,2000,257:11-21
[22]Lin SB,Li CH,Lee SS,Kan LS.Triterpene-enriched extracts from Ganoderma lucidum inhibit growth of hepatoma cells via suppressing protein kinase C.Activating mitogen-activated protein kinases and G2-phase cell cycle arrest.2003,72(21):2381-90
[23]Xuhui Tong,Shigang Lin.Makoto Fujii et al,Molecular mechanisms of echinocystic acid-induced apoptosis in HepG2 cells.Biochom Biophys Res Commun,2004,321:539-546
[24]Wen J,Zhang Y,Chen X,Shen L,et al.Enhancement of diallyl disulfide-induced apoptosis by inhibitors of MAPKs in human nepG2 hepatoma cells.Biochem Pharmacol,2004,68(2):323-331
[25]A Zauberman,D Zipori,M Krupsky et al.Stress activated protein kinase p38is involved in IL-6 induced transcriptional activation of STAT3.Oncogene,1999,18:3886-3893
[26]肖恩华,胡国栋,刘鹏程等.化疗栓塞后肝细胞癌细胞凋亡的研究.中华放射学杂志,1999,33(3):153-156
[27]Ketam S,John F,Brion N,etal.Inhibition of p38 mitogen-activated protein kinase increases LPS induced inhibition of apoptosis in neutrophils by activating extracellular signal regulated kinase.Surgery,2001,130(2):242-247
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