摘要
第一部分TRAIL、阿霉素、顺铂、IFN-γ分别对MG-63细胞增殖及凋亡的调节作用
背景与目的肿瘤坏死因子相关凋亡诱导配体(TRAIL)能够诱导多种肿瘤细胞发生凋亡，但并不是所有的肿瘤细胞都对其敏感。以往研究表明一些化疗药物能够增强TRAIL所诱导的肿瘤细胞凋亡。本部分实验旨在研究TRAIL、阿霉素、顺铂、IFN-γ分别作用于人骨肉瘤MG-63细胞时，对其增殖的抑制作用及诱导凋亡作用。
方法采用MTT法检测TRAIL、阿霉素、顺铂、IFN-γ在不同浓度及不同时间下，分别作用于人骨肉瘤MG-63细胞时，对其增殖的抑制作用。用流式细胞仪检测TRAIL、阿霉素、顺铂、IFN-γ分别作用于人骨肉瘤MG-63细胞时，诱导其凋亡的作用。DNA凝胶电泳检测TRAIL作用于人骨肉瘤MG-63后人骨肉瘤MG-63的凋亡率。
结果(1)不同浓度TRAIL对MG-63细胞均具有杀伤作用。当TRAIL浓度较低(小于1000ng／ml)时，杀伤作用的增加不明显，相同时间不同浓度组比较，差异无显著性(P＞0．05)；而当其浓度增高时，相同时间不同浓度组比较，差异有显著性(P＜0．05)。(2)阿霉素可杀伤MG-63细胞，药物对细胞的杀伤率随着药物浓度的增高而增加，相同时间不同浓度组间比较差异具有显著性(P＜0．05)。(3)顺铂可杀伤人骨肉瘤MG-63细胞，药物对细胞的杀伤率随着药物浓度的增高而增加，相同时间不同浓度组间比较差异具有显著性(P＜0．05)。(4)IFN-γ可杀伤骨肉瘤细胞，这种杀伤作用在24小时内较弱，24小时后显著增大(P＜0．05)。
结论(1)TRAIL浓度在高于1000 ng／ml时，对人骨肉瘤MG-63细胞的杀伤效果增加明显。(2)阿霉素浓度在高于4．3μl／ml时，对人骨肉瘤MG-63细胞的杀伤效果增加明显。(3)顺铂浓度在高于1μmol／L时，对人骨肉瘤MG-63细胞的杀伤效果增加明显。(4)IFN-γ杀伤作用在24小时内对人骨肉瘤MG-63较弱，24小时后显著增大。
第二部分TRAIL、阿霉素、顺铂、IFN-γ联合作用对MG-63细胞增殖及凋亡的调节作用
背景与目的化疗是治疗骨肉瘤的一种常用手段，但化疗药物对机体的毒副作用大，因而限制了其临床上的应用。因此，如何调整化疗药物剂量、减少其副作用是当前非手术治疗骨肉瘤的重点。本实验旨在研究TRAIL、阿霉素、顺铂、IFN-γ联合对人骨肉瘤MG-63细胞凋亡的作用。
方法采用MTT法检测当由TRAIL、阿霉素、顺铂、IFN-γ组成的不同组合，分别作用于人骨肉瘤MG-63时，对其增殖的抑制作用。用流式细胞仪检测由TRAIL、阿霉素、顺铂、IFN-γ组成的不同组合，分别作用于人骨肉瘤MG-63时，诱导其凋亡的作用。
结果(1)TRAIL联合阿霉素及INF-γ组对人骨肉瘤MG-63细胞生长的影响相比其他联合应用组明显增加，差异有显著性(P＜0．05)。(2)TRAIL联合顺铂及INF-γ组对人骨肉瘤MG-63细胞生长的影响相比其他联合应用组增加明显，差异有显著性(P＜0．05)。
结论(1)TRAIL联合阿霉素及INF-γ组对人骨肉瘤细胞MG-63的杀伤效果增加明显。(2)TRAIL联合顺铂及INF-γ组对人骨肉瘤细胞MG-63的杀伤效果增加明显。
第三部分TRAIL、阿霉素、IFN-γ联合作用于人骨肉瘤MG-63，TRAIL相关受体表达研究
背景与目的目前认为细胞表面死亡受体的表达状况，可以从一定程度上反映靶细胞对TRAIL诱导凋亡的敏感度。如何通过上调死亡受体的表达水平而增强肿瘤细胞对TRAIL的敏感性，使TRAIL选择性杀伤癌细胞成为近年来的研究热点本实验旨在研究TRAIL、阿霉素、IFN-γ联合作用于骨肉瘤MG-63细胞引起凋亡作用的机制。
方法采用免疫组化法检测收集的病人骨肉瘤石蜡切片中DR5及YY1基因表达情况。用RT-PCR法检测TRAIL、阿霉素、IFN-γ组成的不同组合，分别作用于人骨肉瘤MG-63后人骨肉瘤MG-63中DR5及YY1的表达情况。用western blot法检测TRAIL、阿霉素、IFN-γ组成的不同组合，分别作用骨肉瘤MG-63后人骨肉瘤MG-63中DR5及YY1的表达情况。
结果(1)收集的病人骨肉瘤细胞中DR5与YY1基因表达情况未发现有显著相关性(P＞0．05)。(2)TRAIL联合阿霉素及INF-γ对人骨肉瘤MG-63细胞中DR5及YY1的表达有较明显影响，差异有显著性(P＜0．05)。
结论(1)收集的病人骨肉瘤石蜡切片中DR5与YY1基因表达情况未发现有显著相关性。(2)TRAIL联合阿霉素及INF-γ组对人骨肉瘤MG-63细胞的凋亡作用强于其他组合(TRAIL组、阿霉素组和TRAIL+可霉素组)，可能是因为他们联合作用时，通过增强TRAIL相关受体DR5基因的表达及降低YY1基因表达引起的。
PARTⅠExperimental Study on Apoptosis-inducing Effect ofTRAIL,Adriamycin,Cisplatin and IFN-γrespectively onMG-63 cells
OBJECTIVE To investigate the synergistic induction of cytoxicity and apoptosis bytumor necrosis factor related apoptosis inducing ligand(TRAIL),adriamycin(ADM),cisplatin(DDP) and IFN-γrespectively on osteosarcoma cell line MG-63.
METHODS Methyl thiazolyl tetrazolium(MTT) assays were used to evaluate thecytotoxic effect induced by TRAIL,Adriamycin,Cisplatin and IFN-γrespectively onosteosarcoma cell line MG-63,Flow cytometric analyses were used for the assessment ofapoptosis rates after different TRAIL,Adriamycin,Cisplatin and IFN-γrespectivelytreating osteosarcoma cell line MG-63.DNA gel electrophoresis was used for theassessment of apoptosis rates after TRAIL treating osteosarcoma cell line MG-63.
RESULTS a) ALL concentrations of TRAIL had different degree effect onosteosarcoma cell line MG-63.When the concentration was under 1000ng/ml,TRAIL did not show statistically significant effect on osteosarcoma cell line MG-63,with theconcentration increasing,the correlation among groups which have different concentrationunder the same stimulating periods had statistically significant(P＜0.05);b) adriamycin(ADM) could kill osteosarcoma cell line MG-63,which show a dose-dependent cytotoxiceffect relationship between drug and cells.When the concentration was under 4.3μl/ml,adriamycin did not show statistically significant effect on osteosarcoma cell lineMG-63,with the concentration increasing,the correlation among groups which havedifferent concentration under the same stimulating periods had statistically significant(P＜0.05);c) cisplatin(DDP) could kill osteosarcoma cell line MG-63,which show adose-dependent cytotoxic effect relationship between drug and cells.When theconcentration was under 1μl/ml,cisplatin did not show statistically significant effect onosteosarcoma cell line MG-63,with the concentration increasing,the correlation amonggroups which have different concentration under the same stimulating periods hadstatistically significant(P＜0.05);d) IFN-γshow statistically significant effect after treatingosteosarcoma cells MG-63 24 hours.
CONCLUSION a) TRAIL show statistically significant effect on osteosarcoma cellsMG-63,when the concentration was higher than 1000ng/ml;b) adriamycin showstatistically significant effect on osteosarcoma cells MG-63,when the concentration washigher than 4.3μl/ml;c) cisplatin show statistically significant effect on osteosarcoma cellsMG-63,when the concentration was higher than 1μmol/L;d) IFN-γshow statisticallysignificant effect after treating osteosarcoma cells MG-63 24 hours.
PARTⅡExperimental Study on Apoptosis-inducing Effect ofdifferent combinations of TRAIL,Adriamycin,Cisplatin andIFN-γon MG-63 cells
OBJECTIVE To investigate the synergistic induction of cytoxicity and apoptosis bythe different combinations of tumor necrosis factor related apoptosis inducing ligand (TRAIL),adriamycin(ADM),cisplatin(DDP) and IFN-γin osteosarcoma cell lineMG-63
METHODS Methyl thiazolyl tetrazolium(MTT) assays were used to evaluate thecytotoxic effect by the different combinations of tumor necrosis factor related apoptosisinducing ligand(TRAIL),adriamycin(ADM),cisplatin(DDP) and IFN-γonosteosarcoma cell line MG-63,Flow cytometric analyses were used for the assessment ofapoptosis rates after different combinations of tumor necrosis factor related apoptosisinducing ligand(TRAIL),adriamycin(ADM),cisplatin(DDP) and IFN-γtreatingosteosarcoma cell line MG-63.
RESULTS a)The combination of tumor necrosis factor related apoptosis inducingligand(TRAIL),adriamycin(ADM),and IFN-γinduced significantly higher apoptosisand cytotoxicity than exposure to TRAIL,ADM or IFN-γalone(P＜0.05),but thecombination of two of the three agents did not show significantly synergistic effect onosteosarcoma cell line MG-63.b)The combination of tumor necrosis factor relatedapoptosis inducing ligand(TRAIL),cisplatin(DDP),and IFN-γinduced significantlyhigher apoptosis and cytotoxicity than exposure to TRAIL,DDP or IFN-γalone(P＜0.05),but the combination of two of the three agents did not show significantly synergistic effecton osteosarcoma cell line MG-63.
CONCLUSION a)The combination of tumor necrosis factor related apoptosisinducing ligand(TRAIL),adriamycin(ADM),and IFN-γinduced significantly higherapoptosis and cytotoxicity than exposure to TRAIL,ADM or IFN-γalone;b)Thecombination of tumor necrosis factor related apoptosis inducing ligand(TRAIL),cisplatin(DDP),and IFN-γinduced significantly higher apoptosis and cytotoxicity thanexposure to TRAIL,DDP or IFN-γalone.
PARTⅢThe expression of TRAIL and its receptors inosteosarcoma cells and the apoptosis effect of a combination of
TRAIL,adriamycin and IFN-γon osteosarcoma cell lineMG-63
OBJECTIVE Tumor necrosis factor related apoptosis-inducing ligand(TRAIL) is adeath receptor ligand that can induce apoptosis in a variety of cancer cell lines,but not allcancer cells.Past studies suggested that some chemotherapeutic drugs intensified thesensitivity of TRAIL induced apoptosis.We investigated the effect and potentialmechanism of combined TRAIL,adriamycin and IFN-γon apoptosis of humanosteosarcoma cell line MG-63 in vitro.
Methods Paraffin embedded surgical specimens from 39 cases of human osteosarcomaare used to detect DR5 and YY1 proteins by immunohistochemical staining method.Expression of DR5 and Yin Yangl(YY1)mRNA after different combinations of TRAIL,Adriamycin and IFN-γwas examined by RT-PCR.Expression of DR5 and YinYangl(YY1)mRNA after different combinations of TRAIL,Adriamycin and IFN-γwasexamined by western blot.
RESULT a) DR5 protein was detected in 53.8 %(21/39) cases in human osteosarcoma,YY1 protein was detected in 38.5%(15/39) cases in human osteosarcoma:nosignificant correlate expression of DR5 proteins and YY1 protein was found in humanosteosarcoma.No obvious correlations were found between the expression of DR5 andYY1 and the tumor staging,grading,major vascular involvement and lung metastasis inhuman osteosarcoma.B) The combination of the three treatments induced significantlyhigher apoptosis and cytotoxicity than exposure to TRAIL,ADM or IFN-γalone,but thecombination of two of the three agents did not show synergistic effect.
Conclusions a) DR5 and YY1 down regulation seem to be an important event in tumorgenesis and progression in human osteosarcoma.In human osteosarcoma,none of thesetwo tumor inhibiting genes seems to play important role.b) There might be a synergisticalinteraction between paclitaxel in terms of cytotoxicity and its mechanisms might beinvolved in the up-regulation of DR5 gene and the down-regulation of YY1.
引文
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