宫颈癌差异蛋白的表达及化疗药物对宫颈癌Hela细胞的作用
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摘要
研究背景
子宫颈癌是全世界妇女中最常见的恶性肿瘤之一,在我国一直高居妇科恶性肿瘤的首位。各国妇女宫颈癌的发病率均随年龄增长而上升,40岁以后发病率显著增加。近30年以来世界范围内宫颈癌的发病率和死亡率均有明显下降趋势,但在许多不发达国家,宫颈癌仍是女性癌症死亡的主要原因。宫颈癌早期诊断是改善预后的最有效方法。尽管已作为已婚女性防癌普查的重要内容,宫颈细胞学涂片等检查手段仍缺乏足够的敏感性。近年来也有血清肿瘤标志物鳞状细胞癌抗原(SCCA)、癌胚抗原(CEA)曾用于宫颈癌前瞻性监测,发现早期患者无明显变化,但肿瘤复发或进展时,SCCA显著上升。总之,目前仍然没有理想的肿瘤标志物用来早期发现宫颈癌或作为病情随访中的监测指标,因此寻找早期诊断宫颈癌敏感而有效的方法就显得尤为迫切。
蛋白质芯片技术在临床方面有着广泛的应用,尤其是能够同时检测出生物样品中与某种疾病或环境因素损伤可能相关的全部蛋白质的含量变化情况,对疾病的诊断或筛查、病情的进展及预后、疗效判断等均有重要意义。蛋白质组学为宫颈癌研究提供了新平台,可从整体上全面、动态的定量分析比较正常及病变标本中蛋白质种类和数量的改变,有助于阐明蛋白质间的调控网络,从而有希望发现控制宫颈癌进程的关键分子,为宫颈癌的诊断、药物研究带来新的思路和途径。
宫颈癌治疗强调个体化,应根据患者的临床分期、年龄、一般情况、肿瘤相关因素等决定治疗方案。宫颈癌早期(Ⅰ～ⅡA期)主要采用手术治疗,宫颈鳞癌是放射敏感肿瘤,对早期不宜采用手术治疗的患者,放疗可以取得与手术相同的治疗效果。局部晚期宫颈癌(ⅡB～ⅢB期)目前采用放化疗同步治疗。晚期(Ⅳ期)转移、治疗后肿瘤未能控制或复发多采用化疗加放疗。近年来,由于含顺铂的联合化疗的进展,宫颈癌的化疗也从对晚期复发癌的姑息治疗变成宫颈癌综合治疗的重要组成部分。治疗宫颈癌的传统有效药物包括顺铂(cisplatin,DDP)、卡铂(carboplatin,CBP)、氟尿嘧啶(fluorouracil,5-Fu)、环磷酰胺(cyclophosphamide,CTX)、异环磷酰胺(ifosfamide,IFO)、阿霉素(adriamycin,ADM)、氨甲喋呤(amethopterin,MTX)、博莱霉素(bleomycin,BLM)、丝裂霉素(mitomycin,MMC)等。近年来出现的新药如紫杉醇(paclitaxel,Taxol)、吉西他滨(gemcitabine,GEM)、伊立替康(irinotecan,CPT-11),长春瑞滨(vinorelbine,NVB)、拓扑替康(topotecan,TPT)等也是治疗宫颈癌的有效药物。顺铂在是目前治疗复发及转移性宫颈癌中最有活性的单药细胞毒类化疗药物,联合化疗能明显提高化疗效率,效果较好的有BIP、PF、TP、CPT-11+DDP及TPT+DDP等方案。随着紫杉醇在实体肿瘤中的广泛应用,紫杉醇联合顺铂(TP)的方案应用于晚期及复发性宫颈癌,其有效率为40%～51%。紫杉醇脂质体是与紫杉醇相似的药物,主要用于非小细胞肺癌、乳腺癌,卵巢癌的治疗,临床显示了良好的疗效。在最新版的美国NCCN指南中,顺铂单药、顺铂联合紫杉醇及顺铂联合拓扑替康等方案均为复发性宫颈癌的一线化疗方案。但是,目前并未证明联合化疗较单药化疗能明显改善患者的生存时间及生存质量,仍需进一步探讨新的药物、用药方案和给药途径,以期提高化疗的有效性和延长患者生存时间。
研究目的
1.寻找宫颈鳞癌早期和中晚期的差异蛋白质,建立早期和中晚期宫颈癌的诊断模型。
2.了解化疗药物顺铂、紫杉醇、紫杉醇脂质体单药或联合对宫颈癌Hela细胞的生长抑制作用和凋亡诱导作用。
研究方法
1.检测宫颈癌早期和中晚期差异蛋白质的表达
(1)样品收集:宫颈癌患者共44例,临床分期为早期26例,中晚期18例。采集患者静脉血,4℃离心后取上清,-70℃冰箱保存。
(2)用SELDI-TOF-MS、弱阳离子结合芯片,检测血清蛋白质峰,在CiphergenProteinChip3.1.1软件中设定读片程序,读取芯片数据并绘制出蛋白质质谱图。Biomarker Pattern软件和Biomarker Wizard软件分析蛋白质质谱峰值,建立诊断树模型。
(3)ELISA方法检测血清SCCA浓度(正常参考值＜1.5ng/ml),以SCCA正常为对照组,SSCA高出正常值3倍以上为实验组,应用Biomarker Wizard软件分析,检测对照组与实验组的蛋白质质谱峰值。
2.化疗药物对宫颈癌Hela细胞体外生长的抑制作用
(1)宫颈癌Hela细胞体外传代培养,细胞培养至对数生长期后实验细胞分为6组:分别为空白对照组、DDP单药组、Taxol单药组、Liposome单药组、DDP+Taxol联合组、DDP+Liposome联合组。检测细胞增殖抑制率时DDP药物剂量分别为2.5、5、10、20μg/ml;Taxol药物剂量分别为4.5、9、18、36μg/ml;Liposome药物剂量分别为4.5、9、18、36μg/ml;DDP+Taxol联合组药物剂量分别为(2.5+4.5)、(5+9)、(10+18)μg/ml;DDP+Liposome联合组药物剂量分别为(2.5+4.5)、(5+9)、(10+18)μg/ml。检测细胞凋亡时,各组药物剂量分别为;单药组DDP 10μg/ml、Taxol18μg/ml、Liposome 18μg/ml,联合组DDP+Taxol(10+18)μg/ml、DDP+Liposome(10+18)μg/ml。
(2)以MTT法检测24、48、72小时后药物抑制人宫颈癌Hela细胞增殖的作用,根据OD值计算细胞生长抑制率。采用协同作用q值判断DDP和Taxol联用、DDP和Liposome联用性质:q＞1.15为协同作用,0.85≤q≤1.15为相加作用,q＜0.85为拮抗作用。
(3)Annexin V-FITC/PI双染法、流式细胞仪于药物作用后24小时检测细胞凋亡,并计算凋亡率。
(4)所有数据采用SPSS 13.0统计软件进行统计处理,实验数据用均数±标准差(x~-±s)表示,各组间细胞抑制率比较采用析因方差分析;其余均采用单因素方差分析(One-Way ANOVA),不同作用时间点各组间比较采用多重比较,方差齐性时采用LSD法,方差不齐采用Dunnett's T3方法。同一时间同一浓度两组间比较采用两独立样本,检验。各组间的药物IC_(50)值比较采用析因分析,不同作用时间点各组间的IC_(50)值采用多重比较。细胞凋亡率的比较采用One-Way ANOVA,多重比较在方差齐性时采用LSD检验,方差不齐时采用Dunnett's T3方法。P＜0.05(双侧)表示差异有显著性。
研究结果
1.宫颈癌早期和中晚期差异蛋白质的表达
(1)宫颈癌早期与中晚期血清中共测得79个蛋白质质谱峰值,相对分子质量在1002～18369范围内,其中43个质谱峰值有显著性差异(P＜0.05),其中质荷比(M/Z)为11523.83、11679.13、5841.076、7971.04、16109.71、15932.17、11480.07、5902.02、15861.78、18368.15、8141.23的差异蛋白质在宫颈癌早期患者血清中含量明显低于中晚期组,P＜0.001。
(2)建立宫颈癌早期与中晚期的诊断树模型。模型一以单个差异蛋白质11679.13(M/Z)作为分类变量,其分类正确率为93.18%(41/44)。模型二以单个差异蛋白质5902.02(M/Z)作为分类变量,其分类正确率为81.81%(36/44)。
(3)将44例血清标本根据SCCA值进行分组,其中对照组为SCCA＜1.5ng/ml,共22例;实验组为SCCA≥4.5ng/ml,共16例。应用Biomarker Wizard软件分析,结果显示在相对分子质量1002～18369范围内,对照组与实验组共测得78个蛋白质质谱峰值,其中43个质谱峰值有显著性差异(P＜0.05),其中M/Z为11523.83、11679.13、5841.076、7971.04、16109.71、15932.17、11480.07、5902.02、15861.78、18368.15、8141.23的差异蛋白质在对照组中的含量明显低于实验组,P＜0.001。比对蛋白质质谱峰值,发现这些差异蛋白质即26例宫颈癌早期组与18例中晚期组血清之间的差异蛋白质。
2.化疗药物对宫颈癌Hela细胞体外生长的抑制作用
(1)MTT检测结果显示,不同浓度DDP、Taxol、Liposome单独作用于Hela细胞,增殖抑制程度随着药物浓度和作用时间的增加呈现增强的趋势,表现为时间剂量依赖效应(P＜0.001)。随着浓度的增加和作用时间的延长,DDP对Hela细胞的抑制率不断增加,DDP 20μg/ml作用72小时仍未出现明显的平台期。当Taxol药物浓度达到36μg/ml时,对细胞的生长抑制作用不再随着作用时间的延长而出现变化。Liposome对细胞的抑制作用也随着浓度的增加更明显,但作用时间较短时,浓度的增加并不能同步增加对细胞生长的抑制。
(2)联合组药物对细胞增殖的抑制作用也表现为时间剂量依赖效应(P＜0.001)。DDP+Taxol联合低剂量组(DDP 2.5μg/ml+Taxol 4.5μg/ml)分别作用48小时与72小时对细胞的抑制率有明显差异。并不是药物浓度越高,作用时间越长,对细胞的抑制作用就越强,联合中剂量组(DDP5μg/ml+Taxol 9μg/ml)作用48小时的抑制作用最强,此时的药物作用已进入平台期。DDP+Liposome联合低剂量组(DDP 2.5μg/ml+Liposome4.5μg/ml)随时间延长抑制作用不断增强,并且有显著性差异;但中剂量组(DDP 5μg/ml+Liposome 9μg/ml)在48小时与72小时时的抑制作用已无显著性差异,高剂量组(DDP 10μg/ml+Liposome 18μg/ml)出现相同结果。从作用时间来看,48小时后,中剂量组(DDP 5μg/ml+Liposome9μg/ml)和高剂量组(DDP 10μg/ml+Liposome 18μg/ml)的抑制作用已无差异,72小时的结果同48小时。总的来说,DDP+Liposome联合在作用到达一定时间(48小时)及药物浓度(中等剂量)后抑制率不再增加。
(3)计算各组药物的IC_(50)值并进行分析,结果表明药物作用时间越长、各组化疗药的IC_(50)值越低。作用24小时,DDP+Taxol组与DDP+Liposome组无显著性差异;作用48小时,DDP与联合组DDP+Taxol、DDP+Liposome无显著性差异;作用72小时,各组之间均有显著性差异。
(4)协同作用q值判断。DDP+Taxol的联合,显示了DDP、Taxol的协同和相加作用,随着作用时间的延长,药物浓度的增加,联合组q值逐渐变小,最大的q值为2.19,出现在联合组(DDP 2.5μg/ml+Taxol 4.5μg/ml)作用48小时。DDP+Liposome的联合与DDP+Taxol的联合结论一致,最大q值1.90出现在(DDP 2.5μg/ml+Liposome 4.5μg/ml)作用48小时。
(5)流式细胞术检测细胞凋亡显示,单药与联合用药组均能促进细胞的凋亡,与对照组比较,P＜0.001;而且联合组与单药组之间也有显著差别,联合组间DDP+Taxol组的细胞凋亡率也显著高于DDP+Liposome组。
结论
1.宫颈癌早期组与中晚期组血清中存在差异蛋白质,以单个差异蛋白质作为分类变量建立宫颈癌早期与中晚期的诊断树模型,可以作为早期诊断及临床分期的参考。血清SCCA升高,尤其升高至正常值3倍以上(SCCA≥4.5ng/ml)与临床分期为中晚期有关。SCCA正常组与SCCA升高3倍组也可检测到差异蛋白质,与早期、中晚期组中存在的差异蛋白质相同。
2.DDP、Taxol、Liposome单独作用,对Hela细胞均有抑制增殖作用,并表现为一定的时间剂量依赖效应。DDP+Taxol联合、DDP+Liposome联合有协同作用。单药及联合用药均可明显促进Hela细胞的凋亡。
Background:
Cervical cancer is one of the most common cancers of women in the world and on the top of gynecology cancers in our country.The morbidity of cervical cancer is increased with the age,which increased significantly after 40 years old.In the past 30 years,the morbidity and mortality of cervical cancer were decreased all over the world.But cervical cancer is still the main cause of women died of cancer.Early diagnosis in cervical cancer is the most efficient method in improving prognostic. Examination method,such as cervix smear,is still lack of sufficient sensitivity. SCCA and CEA,serum tumor markers,are used in prospective detection of cervical cancer.SCCA increase significantly when tumor recur or progress,while has no marked change in earlier period.In a word,there are no ideal tumor markers to discovery cervical cancer early or to be used as monitoring index in follow-up visit. It is very urgent to find a sensitiveness and effective method in early diagnosis of cervical cancer.
Protein array technique is used widely in clinic.It is very important to diagnose and screen disease,judge the progression,prognosis and therapeutic effect.Proteome provided a new platform for cervical cancer research,which analyze quantitatively and compare change of proteins category and quantity in normal and process sample. It is helpful to illuminate the regulation network among proteins and hoped to find the key molecule controlling the progress of cervical cancer.It brings new idea and pathway to diagnosis and medicine research of cervical cancer.
Therapeutic regimen of cervical cancer is decided with clinical stage,age, general state of health and tumor correlation factor.The earlier stage(Ⅰ～ⅡA stage) is treated by operation.Patients who are not suitable for operation can be treated with radiotherapy,which has the same therapeutic efficiency with operation.Part advanced stage(ⅡB～ⅢB stage)is treated by radiotherapy and chemotherapy together. Advanced stage(Ⅳstage),tumor metastasis and recurrence,is treated by chemotherapy plus radiotherapy.For few years,chemotherapy of cervical cancer is changed from the alleviative treatment to an important ingredient of cervical cancer combined therapy because of the progress of combination chemotherapy based on DDP.Traditional active drug in treatment of cervical cancer include cisplatin(DDP)、carboplatin(CBP)、fluorouracil(5-Fu)、cyclophosphamide(CTX)、ifosfamide (IFO)、adriamycin(ADM)、amethopterin(MTX)、bleomycin(BLM)、mitomycin (MMC)and so on.New drugs such as paclitaxel(Taxol)、gemcitabine(GEM)、irinotecan(CPT-11),vinorelbine(NVB)、topotecan(TPT)are the active drug in treatment of cervical cancer.DDP is the most competent cytotoxic drug in the treatment of recurrence and metastatic cervical cancer.Combination chemotherapy increases the therapeutic effect.Including BIP、PF、TP、CPT-11+DDP and TPT +DDP.In the American NCCN guideline of latest edition,DDP、combination of DDP+ Taxol and combination of DDP+CPT-11 are the first-line chemotherapy of recurrence cervical cancer.Paclitaxel liposome,the similar drug with paclitaxel,is used in treatment of nonsmall-cell lung cancer、breast cancer and ovarian cancer. Paclitaxel liposome has good therapeutic effect in clinic.But it is not proved that combination chemotherapy can improve obviously the survival time and quality of patients compared with simple chemotherapy.It needs to approach new drug and administration route to elevate the chemotherapy efficacy and prolong survival time of patients.
Objectives:
1.To search the differentially expressed proteins and set up diagnostic model in earlier period and advanced stage of cervical cancer.
2.To investigate the inhibitory proliferation effect and apoptosis induction of Taxo1、Liposome single or combined with DDP on Hela human cervical carcinoma cells.
Methods:
1.Identify the differentially expressed proteins between earlier period and advanced stage in the serum of patients with cervical cancer.
(1)Sample collection:44 cervical carcinoma patients were divided into two groups,26 patients in earlier period and 18 patients in advanced stage. Collect the venous blood of all patients.Centrifuge blood at 4℃and take supernatant,which was preserved in frigidaire at-70℃.
(2)Serum protein mass peak were detected by SELDI-TOF-MS and weak canonic chips.Fetch array data and draw protein mass spectra figure.The protein fingerprints were obtained,two diagnostic models was set up,and bioinformatic analysis was performed to identify the differentially expressed proteins in the serum of the patients.
(3)The density of SCCA in serum was detected by ELISA method.Control group included the normal value of SCCA;treatment group included the level of SCCA which surpass 3 times of normal value.The peak value of protein mass spectra in control group and treatment group were detected.
2.Inhibitory effect of chemotherapeutics on Hela human cervical carcinoma cells in vitro
(1)Hela human cervical carcinoma cells were cultured in RPMI 1640 containing 10%fetal calf serum.Exponentially growing cells were chosen for experiment. Hela cells were divided into 6 groups:control group,DDP-treated group, Taxol-treated group,Liposome-treated group,DDP+Taxol combination treatment group and DDP+Liposome combination treatment group.MTT assay included cells treated with DDP(2.5、5、10、20μg/ml),Taxol(4.5、9、18、36μg/ml),Liposome(4.5、9、18、36μg/ml)alone and combination DDP+Taxol(2.5+4.5、5+9、10+18)(μg/ml)and combination DDP+Liposome (2.5+4.5、5+9、10+18)(μg/ml)for 24,48 and 72 hours respectively.Apoptosis analysis included cells treated with DDP(10μg/ml),Taxol(18μg/ml)and Liposome(10μg/ml)alone and combination DDP+Taxol(10+18)μg/ml and combination DDP+Liposome(10+18)μg/ml.
(2)Group as the above mentioned,the inhibitory of Hela cells were detected by MTT assay after treatment.Calculate the inhibitory rates of cells according OD value.Interaction between DDP and Taxol,DDP and Liposome were assessed using the q value,where q＞1.15,0.85≤q≤1.15,q＜0.85 indicated synergistic,additive,and antagonistic effects respectively.
(3)Group as the above mentioned,collected cells and analyzed apoptosis by Annexin V-FITC/PI and flow cytometry.Calculate the inhibitory rates of apoptosis.
(4)All datas were analysised by SPSS 13.0 statistical softwire.Experimental data were expressed as mean±S.D.The inhibitory rates were analyzed by univariate analysis of variance.The groups with same dose and same time were analyzed by independent-samples t test.The others data were analyzed by One-way ANOVA.The method of comparision within different time and groups was LSD multiple comparison test if homogeneity of variance,and Dunnett's T3 test if heterogeneity of variance..P-Values were considered to be significant at＜0.05(2-sided)
Results:
1.Differentially expressed proteins in earlier stage and advanced stage of cervical cancer
(1)In the range of 1002～18369 in relative molecular mass,79 differentially expressed proteins were detected in the serum of cervical cancer patients in two groups and 43 mass spectra peak value had significant difference (P＜0.05),among which 11 proteins with mass/charge ratio of 11523.83、11679.13、5841.076、7971.04、16109.71、15932.17、11480.07、5902.02、15861.78、18368.15 and 8141.23,respectively,showed lower expression in the serum of cervical cancer patients in earlier period,P＜0.001.
(2)Two diagnostic models for cervical cancer were generated to discriminate earlier period and advanced stage,including 11679.13(M/Z)protein with the correct classification of 93.18%(41/44)and 5902.02(M/Z)protein with the correct classification of 81.81%(36/44).
(3)Divide the 44 serum sample into two groups according to the value of SCCA. Control group included 22 patients with normal value of SCCA.Treatment group included 16 patients with the level of SCCA which surpass 3 times of normal value.The peak value of protein mass spectra in control group and treatment group were detected.The result indicated:in the range of 1002～ 18369 in relative molecular mass,78 differentially expressed proteins were detected in two groups and 43 mass spectra peak value had significant difference(P＜0.05),among which 11 proteins with mass/charge ratio of 11523.83、11679.13、5841.076、7971.04、16109.71、15932.17、11480.07、5902.02、15861.78、18368.15 and 8141.23,respectively,showed lower expression in control group.These differentially expressed proteins were the same with which in earlier period and advanced stage.
2.The inhibitory effect of chemotherapeutics on Hela cells in vitro
(1)The MTT result indicated:DDP、Taxol and Liposome alone inhibited cell proliferation time-and dose-dependently(P＜0.00l).With the increase of dose and the lasting of the time,inhibition ratio of DDP increased accordingly。The platform stage didn't appear when DDP(20μg/ml)had affected cells for 72 hours.The inhibition of Taxol didn't increase accordingly with the change of time when the dose of Taxol achieved 36μg/ml.In a short time,the inhibition of Liposome didn't increase accordingly with the increasing of dose.
(2)Combination groups inhibited cell proliferation time-and dose-dependently (P＜0.001).Combination group(DDP 2.5μg/ml+Taxol 4.5μg/ml)had a different inhibition in 24h and 72h.Combination group(DDP 5μg/ml+ Taxol 9μg/ml)had the best inhibition in 48h.Combination group(DDP 5μg/ml+ Liposome 9μg/ml)had the same inhibition in 48h and 72h.So did combination group(DDP 10μg/ml+Liposome 18μg/ml).All in all,the inhibition of combination between DDP and Liposome didn't increase after 48h and medium dosage.
(3)Calculate and analysis the value of IC_(50)every group.The result indicated: the value of IC_(50)diminished with the drugs worker longer,there was no significant difference in combination between DDP and Taxol and combination between DDP and Liposome in 24h.There was no significant difference in DDP、combination between DDP and Taxol、combination between DDP and Liposome in 48h.There was a significant difference in every group at 72h..
(4)q Value of DDP combined with Taxol showed:Coadministration of DDP with Taxol showed synergism and additive effect of all concentration levels at 24, 48,and 72h(0.85≤q≤1.15).Combination group(DDP 2.5μg/ml+ Taxol 4.5μg/ml)had the max q value(2.19)at 48h.Combination group(DDP 2.5μg/ml+ Liposome 4.5μg/ml)had the max q value(1.90)at 48h.
(5)Single and combination group induced Hela cells apoptosis.Cells apoptosis was detected after treated with single and combination group(P＜0.001). There was a significant difference in single and combination group.The rate of apoptosis in combination between DDP and Taxol was higher than in combination between DDP and Liposome.
Conclusion:
1.Differential protein expression existed in earlier period and advanced stage in cervical carcinoma.Two diagnostic models for cervical cancer were generated using software,which was used to reference of early diagnosis and stage in clinic.Serum SCCA at the level of 3 times of normal value was considered has a relationship with advanced stage.Differential protein expression existed in both group which SCCA at normal value and group which SCCA at the level of 3 times of normal value.These differential protein were the same with which in earlier period and advanced stage.
2.DDP、Taxol and Liposome alone inhibited cell proliferation time-and dose-dependently.DDP has synergism effect with Taxol and Liposome.The single and combination could promote Hela cells apoptosis.
子宫颈癌是全世界妇女中最常见的恶性肿瘤之一,在我国一直高居妇科恶性肿瘤的首位。各国妇女宫颈癌的发病率均随年龄增长而上升,40岁以后发病率显著增加。近30年以来世界范围内宫颈癌的发病率和死亡率均有明显下降趋势,但在许多不发达国家,宫颈癌仍是女性癌症死亡的主要原因。宫颈癌早期诊断是改善预后的最有效方法。尽管已作为已婚女性防癌普查的重要内容,宫颈细胞学涂片等检查手段仍缺乏足够的敏感性。近年来也有血清肿瘤标志物鳞状细胞癌抗原(SCCA)、癌胚抗原(CEA)曾用于宫颈癌前瞻性监测,发现早期患者无明显变化,但肿瘤复发或进展时,SCCA显著上升。总之,目前仍然没有理想的肿瘤标志物用来早期发现宫颈癌或作为病情随访中的监测指标,因此寻找早期诊断宫颈癌敏感而有效的方法就显得尤为迫切。
蛋白质芯片技术在临床方面有着广泛的应用,尤其是能够同时检测出生物样品中与某种疾病或环境因素损伤可能相关的全部蛋白质的含量变化情况,对疾病的诊断或筛查、病情的进展及预后、疗效判断等均有重要意义。蛋白质组学为宫颈癌研究提供了新平台,可从整体上全面、动态的定量分析比较正常及病变标本中蛋白质种类和数量的改变,有助于阐明蛋白质间的调控网络,从而有希望发现控制宫颈癌进程的关键分子,为宫颈癌的诊断、药物研究带来新的思路和途径。
宫颈癌治疗强调个体化,应根据患者的临床分期、年龄、一般情况、肿瘤相关因素等决定治疗方案。宫颈癌早期(Ⅰ～ⅡA期)主要采用手术治疗,宫颈鳞癌是放射敏感肿瘤,对早期不宜采用手术治疗的患者,放疗可以取得与手术相同的治疗效果。局部晚期宫颈癌(ⅡB～ⅢB期)目前采用放化疗同步治疗。晚期(Ⅳ期)转移、治疗后肿瘤未能控制或复发多采用化疗加放疗。近年来,由于含顺铂的联合化疗的进展,宫颈癌的化疗也从对晚期复发癌的姑息治疗变成宫颈癌综合治疗的重要组成部分。治疗宫颈癌的传统有效药物包括顺铂(cisplatin,DDP)、卡铂(carboplatin,CBP)、氟尿嘧啶(fluorouracil,5-Fu)、环磷酰胺(cyclophosphamide,CTX)、异环磷酰胺(ifosfamide,IFO)、阿霉素(adriamycin,ADM)、氨甲喋呤(amethopterin,MTX)、博莱霉素(bleomycin,BLM)、丝裂霉素(mitomycin,MMC)等。近年来出现的新药如紫杉醇(paclitaxel,Taxol)、吉西他滨(gemcitabine,GEM)、伊立替康(irinotecan,CPT-11),长春瑞滨(vinorelbine,NVB)、拓扑替康(topotecan,TPT)等也是治疗宫颈癌的有效药物。顺铂在是目前治疗复发及转移性宫颈癌中最有活性的单药细胞毒类化疗药物,联合化疗能明显提高化疗效率,效果较好的有BIP、PF、TP、CPT-11+DDP及TPT+DDP等方案。随着紫杉醇在实体肿瘤中的广泛应用,紫杉醇联合顺铂(TP)的方案应用于晚期及复发性宫颈癌,其有效率为40%～51%。紫杉醇脂质体是与紫杉醇相似的药物,主要用于非小细胞肺癌、乳腺癌,卵巢癌的治疗,临床显示了良好的疗效。在最新版的美国NCCN指南中,顺铂单药、顺铂联合紫杉醇及顺铂联合拓扑替康等方案均为复发性宫颈癌的一线化疗方案。但是,目前并未证明联合化疗较单药化疗能明显改善患者的生存时间及生存质量,仍需进一步探讨新的药物、用药方案和给药途径,以期提高化疗的有效性和延长患者生存时间。
研究目的
1.寻找宫颈鳞癌早期和中晚期的差异蛋白质,建立早期和中晚期宫颈癌的诊断模型。
2.了解化疗药物顺铂、紫杉醇、紫杉醇脂质体单药或联合对宫颈癌Hela细胞的生长抑制作用和凋亡诱导作用。
研究方法
1.检测宫颈癌早期和中晚期差异蛋白质的表达
(1)样品收集:宫颈癌患者共44例,临床分期为早期26例,中晚期18例。采集患者静脉血,4℃离心后取上清,-70℃冰箱保存。
(2)用SELDI-TOF-MS、弱阳离子结合芯片,检测血清蛋白质峰,在CiphergenProteinChip3.1.1软件中设定读片程序,读取芯片数据并绘制出蛋白质质谱图。Biomarker Pattern软件和Biomarker Wizard软件分析蛋白质质谱峰值,建立诊断树模型。
(3)ELISA方法检测血清SCCA浓度(正常参考值＜1.5ng/ml),以SCCA正常为对照组,SSCA高出正常值3倍以上为实验组,应用Biomarker Wizard软件分析,检测对照组与实验组的蛋白质质谱峰值。
2.化疗药物对宫颈癌Hela细胞体外生长的抑制作用
(1)宫颈癌Hela细胞体外传代培养,细胞培养至对数生长期后实验细胞分为6组:分别为空白对照组、DDP单药组、Taxol单药组、Liposome单药组、DDP+Taxol联合组、DDP+Liposome联合组。检测细胞增殖抑制率时DDP药物剂量分别为2.5、5、10、20μg/ml;Taxol药物剂量分别为4.5、9、18、36μg/ml;Liposome药物剂量分别为4.5、9、18、36μg/ml;DDP+Taxol联合组药物剂量分别为(2.5+4.5)、(5+9)、(10+18)μg/ml;DDP+Liposome联合组药物剂量分别为(2.5+4.5)、(5+9)、(10+18)μg/ml。检测细胞凋亡时,各组药物剂量分别为;单药组DDP 10μg/ml、Taxol18μg/ml、Liposome 18μg/ml,联合组DDP+Taxol(10+18)μg/ml、DDP+Liposome(10+18)μg/ml。
(2)以MTT法检测24、48、72小时后药物抑制人宫颈癌Hela细胞增殖的作用,根据OD值计算细胞生长抑制率。采用协同作用q值判断DDP和Taxol联用、DDP和Liposome联用性质:q＞1.15为协同作用,0.85≤q≤1.15为相加作用,q＜0.85为拮抗作用。
(3)Annexin V-FITC/PI双染法、流式细胞仪于药物作用后24小时检测细胞凋亡,并计算凋亡率。
(4)所有数据采用SPSS 13.0统计软件进行统计处理,实验数据用均数±标准差(x~-±s)表示,各组间细胞抑制率比较采用析因方差分析;其余均采用单因素方差分析(One-Way ANOVA),不同作用时间点各组间比较采用多重比较,方差齐性时采用LSD法,方差不齐采用Dunnett's T3方法。同一时间同一浓度两组间比较采用两独立样本,检验。各组间的药物IC_(50)值比较采用析因分析,不同作用时间点各组间的IC_(50)值采用多重比较。细胞凋亡率的比较采用One-Way ANOVA,多重比较在方差齐性时采用LSD检验,方差不齐时采用Dunnett's T3方法。P＜0.05(双侧)表示差异有显著性。
研究结果
1.宫颈癌早期和中晚期差异蛋白质的表达
(1)宫颈癌早期与中晚期血清中共测得79个蛋白质质谱峰值,相对分子质量在1002～18369范围内,其中43个质谱峰值有显著性差异(P＜0.05),其中质荷比(M/Z)为11523.83、11679.13、5841.076、7971.04、16109.71、15932.17、11480.07、5902.02、15861.78、18368.15、8141.23的差异蛋白质在宫颈癌早期患者血清中含量明显低于中晚期组,P＜0.001。
(2)建立宫颈癌早期与中晚期的诊断树模型。模型一以单个差异蛋白质11679.13(M/Z)作为分类变量,其分类正确率为93.18%(41/44)。模型二以单个差异蛋白质5902.02(M/Z)作为分类变量,其分类正确率为81.81%(36/44)。
(3)将44例血清标本根据SCCA值进行分组,其中对照组为SCCA＜1.5ng/ml,共22例;实验组为SCCA≥4.5ng/ml,共16例。应用Biomarker Wizard软件分析,结果显示在相对分子质量1002～18369范围内,对照组与实验组共测得78个蛋白质质谱峰值,其中43个质谱峰值有显著性差异(P＜0.05),其中M/Z为11523.83、11679.13、5841.076、7971.04、16109.71、15932.17、11480.07、5902.02、15861.78、18368.15、8141.23的差异蛋白质在对照组中的含量明显低于实验组,P＜0.001。比对蛋白质质谱峰值,发现这些差异蛋白质即26例宫颈癌早期组与18例中晚期组血清之间的差异蛋白质。
2.化疗药物对宫颈癌Hela细胞体外生长的抑制作用
(1)MTT检测结果显示,不同浓度DDP、Taxol、Liposome单独作用于Hela细胞,增殖抑制程度随着药物浓度和作用时间的增加呈现增强的趋势,表现为时间剂量依赖效应(P＜0.001)。随着浓度的增加和作用时间的延长,DDP对Hela细胞的抑制率不断增加,DDP 20μg/ml作用72小时仍未出现明显的平台期。当Taxol药物浓度达到36μg/ml时,对细胞的生长抑制作用不再随着作用时间的延长而出现变化。Liposome对细胞的抑制作用也随着浓度的增加更明显,但作用时间较短时,浓度的增加并不能同步增加对细胞生长的抑制。
(2)联合组药物对细胞增殖的抑制作用也表现为时间剂量依赖效应(P＜0.001)。DDP+Taxol联合低剂量组(DDP 2.5μg/ml+Taxol 4.5μg/ml)分别作用48小时与72小时对细胞的抑制率有明显差异。并不是药物浓度越高,作用时间越长,对细胞的抑制作用就越强,联合中剂量组(DDP5μg/ml+Taxol 9μg/ml)作用48小时的抑制作用最强,此时的药物作用已进入平台期。DDP+Liposome联合低剂量组(DDP 2.5μg/ml+Liposome4.5μg/ml)随时间延长抑制作用不断增强,并且有显著性差异;但中剂量组(DDP 5μg/ml+Liposome 9μg/ml)在48小时与72小时时的抑制作用已无显著性差异,高剂量组(DDP 10μg/ml+Liposome 18μg/ml)出现相同结果。从作用时间来看,48小时后,中剂量组(DDP 5μg/ml+Liposome9μg/ml)和高剂量组(DDP 10μg/ml+Liposome 18μg/ml)的抑制作用已无差异,72小时的结果同48小时。总的来说,DDP+Liposome联合在作用到达一定时间(48小时)及药物浓度(中等剂量)后抑制率不再增加。
(3)计算各组药物的IC_(50)值并进行分析,结果表明药物作用时间越长、各组化疗药的IC_(50)值越低。作用24小时,DDP+Taxol组与DDP+Liposome组无显著性差异;作用48小时,DDP与联合组DDP+Taxol、DDP+Liposome无显著性差异;作用72小时,各组之间均有显著性差异。
(4)协同作用q值判断。DDP+Taxol的联合,显示了DDP、Taxol的协同和相加作用,随着作用时间的延长,药物浓度的增加,联合组q值逐渐变小,最大的q值为2.19,出现在联合组(DDP 2.5μg/ml+Taxol 4.5μg/ml)作用48小时。DDP+Liposome的联合与DDP+Taxol的联合结论一致,最大q值1.90出现在(DDP 2.5μg/ml+Liposome 4.5μg/ml)作用48小时。
(5)流式细胞术检测细胞凋亡显示,单药与联合用药组均能促进细胞的凋亡,与对照组比较,P＜0.001;而且联合组与单药组之间也有显著差别,联合组间DDP+Taxol组的细胞凋亡率也显著高于DDP+Liposome组。
结论
1.宫颈癌早期组与中晚期组血清中存在差异蛋白质,以单个差异蛋白质作为分类变量建立宫颈癌早期与中晚期的诊断树模型,可以作为早期诊断及临床分期的参考。血清SCCA升高,尤其升高至正常值3倍以上(SCCA≥4.5ng/ml)与临床分期为中晚期有关。SCCA正常组与SCCA升高3倍组也可检测到差异蛋白质,与早期、中晚期组中存在的差异蛋白质相同。
2.DDP、Taxol、Liposome单独作用,对Hela细胞均有抑制增殖作用,并表现为一定的时间剂量依赖效应。DDP+Taxol联合、DDP+Liposome联合有协同作用。单药及联合用药均可明显促进Hela细胞的凋亡。
Background:
Cervical cancer is one of the most common cancers of women in the world and on the top of gynecology cancers in our country.The morbidity of cervical cancer is increased with the age,which increased significantly after 40 years old.In the past 30 years,the morbidity and mortality of cervical cancer were decreased all over the world.But cervical cancer is still the main cause of women died of cancer.Early diagnosis in cervical cancer is the most efficient method in improving prognostic. Examination method,such as cervix smear,is still lack of sufficient sensitivity. SCCA and CEA,serum tumor markers,are used in prospective detection of cervical cancer.SCCA increase significantly when tumor recur or progress,while has no marked change in earlier period.In a word,there are no ideal tumor markers to discovery cervical cancer early or to be used as monitoring index in follow-up visit. It is very urgent to find a sensitiveness and effective method in early diagnosis of cervical cancer.
Protein array technique is used widely in clinic.It is very important to diagnose and screen disease,judge the progression,prognosis and therapeutic effect.Proteome provided a new platform for cervical cancer research,which analyze quantitatively and compare change of proteins category and quantity in normal and process sample. It is helpful to illuminate the regulation network among proteins and hoped to find the key molecule controlling the progress of cervical cancer.It brings new idea and pathway to diagnosis and medicine research of cervical cancer.
Therapeutic regimen of cervical cancer is decided with clinical stage,age, general state of health and tumor correlation factor.The earlier stage(Ⅰ～ⅡA stage) is treated by operation.Patients who are not suitable for operation can be treated with radiotherapy,which has the same therapeutic efficiency with operation.Part advanced stage(ⅡB～ⅢB stage)is treated by radiotherapy and chemotherapy together. Advanced stage(Ⅳstage),tumor metastasis and recurrence,is treated by chemotherapy plus radiotherapy.For few years,chemotherapy of cervical cancer is changed from the alleviative treatment to an important ingredient of cervical cancer combined therapy because of the progress of combination chemotherapy based on DDP.Traditional active drug in treatment of cervical cancer include cisplatin(DDP)、carboplatin(CBP)、fluorouracil(5-Fu)、cyclophosphamide(CTX)、ifosfamide (IFO)、adriamycin(ADM)、amethopterin(MTX)、bleomycin(BLM)、mitomycin (MMC)and so on.New drugs such as paclitaxel(Taxol)、gemcitabine(GEM)、irinotecan(CPT-11),vinorelbine(NVB)、topotecan(TPT)are the active drug in treatment of cervical cancer.DDP is the most competent cytotoxic drug in the treatment of recurrence and metastatic cervical cancer.Combination chemotherapy increases the therapeutic effect.Including BIP、PF、TP、CPT-11+DDP and TPT +DDP.In the American NCCN guideline of latest edition,DDP、combination of DDP+ Taxol and combination of DDP+CPT-11 are the first-line chemotherapy of recurrence cervical cancer.Paclitaxel liposome,the similar drug with paclitaxel,is used in treatment of nonsmall-cell lung cancer、breast cancer and ovarian cancer. Paclitaxel liposome has good therapeutic effect in clinic.But it is not proved that combination chemotherapy can improve obviously the survival time and quality of patients compared with simple chemotherapy.It needs to approach new drug and administration route to elevate the chemotherapy efficacy and prolong survival time of patients.
Objectives:
1.To search the differentially expressed proteins and set up diagnostic model in earlier period and advanced stage of cervical cancer.
2.To investigate the inhibitory proliferation effect and apoptosis induction of Taxo1、Liposome single or combined with DDP on Hela human cervical carcinoma cells.
Methods:
1.Identify the differentially expressed proteins between earlier period and advanced stage in the serum of patients with cervical cancer.
(1)Sample collection:44 cervical carcinoma patients were divided into two groups,26 patients in earlier period and 18 patients in advanced stage. Collect the venous blood of all patients.Centrifuge blood at 4℃and take supernatant,which was preserved in frigidaire at-70℃.
(2)Serum protein mass peak were detected by SELDI-TOF-MS and weak canonic chips.Fetch array data and draw protein mass spectra figure.The protein fingerprints were obtained,two diagnostic models was set up,and bioinformatic analysis was performed to identify the differentially expressed proteins in the serum of the patients.
(3)The density of SCCA in serum was detected by ELISA method.Control group included the normal value of SCCA;treatment group included the level of SCCA which surpass 3 times of normal value.The peak value of protein mass spectra in control group and treatment group were detected.
2.Inhibitory effect of chemotherapeutics on Hela human cervical carcinoma cells in vitro
(1)Hela human cervical carcinoma cells were cultured in RPMI 1640 containing 10%fetal calf serum.Exponentially growing cells were chosen for experiment. Hela cells were divided into 6 groups:control group,DDP-treated group, Taxol-treated group,Liposome-treated group,DDP+Taxol combination treatment group and DDP+Liposome combination treatment group.MTT assay included cells treated with DDP(2.5、5、10、20μg/ml),Taxol(4.5、9、18、36μg/ml),Liposome(4.5、9、18、36μg/ml)alone and combination DDP+Taxol(2.5+4.5、5+9、10+18)(μg/ml)and combination DDP+Liposome (2.5+4.5、5+9、10+18)(μg/ml)for 24,48 and 72 hours respectively.Apoptosis analysis included cells treated with DDP(10μg/ml),Taxol(18μg/ml)and Liposome(10μg/ml)alone and combination DDP+Taxol(10+18)μg/ml and combination DDP+Liposome(10+18)μg/ml.
(2)Group as the above mentioned,the inhibitory of Hela cells were detected by MTT assay after treatment.Calculate the inhibitory rates of cells according OD value.Interaction between DDP and Taxol,DDP and Liposome were assessed using the q value,where q＞1.15,0.85≤q≤1.15,q＜0.85 indicated synergistic,additive,and antagonistic effects respectively.
(3)Group as the above mentioned,collected cells and analyzed apoptosis by Annexin V-FITC/PI and flow cytometry.Calculate the inhibitory rates of apoptosis.
(4)All datas were analysised by SPSS 13.0 statistical softwire.Experimental data were expressed as mean±S.D.The inhibitory rates were analyzed by univariate analysis of variance.The groups with same dose and same time were analyzed by independent-samples t test.The others data were analyzed by One-way ANOVA.The method of comparision within different time and groups was LSD multiple comparison test if homogeneity of variance,and Dunnett's T3 test if heterogeneity of variance..P-Values were considered to be significant at＜0.05(2-sided)
Results:
1.Differentially expressed proteins in earlier stage and advanced stage of cervical cancer
(1)In the range of 1002～18369 in relative molecular mass,79 differentially expressed proteins were detected in the serum of cervical cancer patients in two groups and 43 mass spectra peak value had significant difference (P＜0.05),among which 11 proteins with mass/charge ratio of 11523.83、11679.13、5841.076、7971.04、16109.71、15932.17、11480.07、5902.02、15861.78、18368.15 and 8141.23,respectively,showed lower expression in the serum of cervical cancer patients in earlier period,P＜0.001.
(2)Two diagnostic models for cervical cancer were generated to discriminate earlier period and advanced stage,including 11679.13(M/Z)protein with the correct classification of 93.18%(41/44)and 5902.02(M/Z)protein with the correct classification of 81.81%(36/44).
(3)Divide the 44 serum sample into two groups according to the value of SCCA. Control group included 22 patients with normal value of SCCA.Treatment group included 16 patients with the level of SCCA which surpass 3 times of normal value.The peak value of protein mass spectra in control group and treatment group were detected.The result indicated:in the range of 1002～ 18369 in relative molecular mass,78 differentially expressed proteins were detected in two groups and 43 mass spectra peak value had significant difference(P＜0.05),among which 11 proteins with mass/charge ratio of 11523.83、11679.13、5841.076、7971.04、16109.71、15932.17、11480.07、5902.02、15861.78、18368.15 and 8141.23,respectively,showed lower expression in control group.These differentially expressed proteins were the same with which in earlier period and advanced stage.
2.The inhibitory effect of chemotherapeutics on Hela cells in vitro
(1)The MTT result indicated:DDP、Taxol and Liposome alone inhibited cell proliferation time-and dose-dependently(P＜0.00l).With the increase of dose and the lasting of the time,inhibition ratio of DDP increased accordingly。The platform stage didn't appear when DDP(20μg/ml)had affected cells for 72 hours.The inhibition of Taxol didn't increase accordingly with the change of time when the dose of Taxol achieved 36μg/ml.In a short time,the inhibition of Liposome didn't increase accordingly with the increasing of dose.
(2)Combination groups inhibited cell proliferation time-and dose-dependently (P＜0.001).Combination group(DDP 2.5μg/ml+Taxol 4.5μg/ml)had a different inhibition in 24h and 72h.Combination group(DDP 5μg/ml+ Taxol 9μg/ml)had the best inhibition in 48h.Combination group(DDP 5μg/ml+ Liposome 9μg/ml)had the same inhibition in 48h and 72h.So did combination group(DDP 10μg/ml+Liposome 18μg/ml).All in all,the inhibition of combination between DDP and Liposome didn't increase after 48h and medium dosage.
(3)Calculate and analysis the value of IC_(50)every group.The result indicated: the value of IC_(50)diminished with the drugs worker longer,there was no significant difference in combination between DDP and Taxol and combination between DDP and Liposome in 24h.There was no significant difference in DDP、combination between DDP and Taxol、combination between DDP and Liposome in 48h.There was a significant difference in every group at 72h..
(4)q Value of DDP combined with Taxol showed:Coadministration of DDP with Taxol showed synergism and additive effect of all concentration levels at 24, 48,and 72h(0.85≤q≤1.15).Combination group(DDP 2.5μg/ml+ Taxol 4.5μg/ml)had the max q value(2.19)at 48h.Combination group(DDP 2.5μg/ml+ Liposome 4.5μg/ml)had the max q value(1.90)at 48h.
(5)Single and combination group induced Hela cells apoptosis.Cells apoptosis was detected after treated with single and combination group(P＜0.001). There was a significant difference in single and combination group.The rate of apoptosis in combination between DDP and Taxol was higher than in combination between DDP and Liposome.
Conclusion:
1.Differential protein expression existed in earlier period and advanced stage in cervical carcinoma.Two diagnostic models for cervical cancer were generated using software,which was used to reference of early diagnosis and stage in clinic.Serum SCCA at the level of 3 times of normal value was considered has a relationship with advanced stage.Differential protein expression existed in both group which SCCA at normal value and group which SCCA at the level of 3 times of normal value.These differential protein were the same with which in earlier period and advanced stage.
2.DDP、Taxol and Liposome alone inhibited cell proliferation time-and dose-dependently.DDP has synergism effect with Taxol and Liposome.The single and combination could promote Hela cells apoptosis.
引文
1.HaieMeder C,de Crevoisier R,Bruna A,et al.Concomitant chemoradiation in patients with cervix cancer[J].Bull Cancer,2005,92(12):1032-1038.
2.Schoell WM,Janicek MF,Mirhasphemi R.Epidemiology and biology of cervical cancer[J].Semin Surg Oncol,1999,16(3):203-211.
3.Lonky NM.Risk factors related to the development and mortality from invasive cervical cancer:clinical utility and impact on prevention[J].Obstet Gynecol Clin North Am,2002,29(4):817-842.
4.TangN,Tornatore P,Weinbexger S.Current developments in SELDI affinity technology[J].Mass Spectrom Rev,2004,23:34-44.
5.Kas K.On the technicalities of discovering and applying protein biomarkers for cancer prevention[J].Eur J Cancer revention,2004,(13):437-446.
6.Lin SC,Tseng FG,Huang HM,et al.Microsized 2D protein arrays in mobilized by micro-stamps and micro-wells for disease diagnosis and drug screening [J].Fresenius J Anal Chem,2001,371(2):202-208.
7.Wonga YF,Cheunga TH,Loa KW,et al.Protein profiling of cervical cancer by protein-biochips:proteomic scoring to discriminate cervical cancer from normal cervix[J].Cancer Letters,2004,211(2):227-234.
8.郭社珂,乔玉环,赵先兰.宫颈癌组织蛋白质组图谱分析[J].郑州大学学报(医学版),2007,42(2):305-307.
9.Lin YW,Lai HC,Lin CY,at al.Plasma proteomic profiling for detecting and differentiating in situ and invasive carcinomas of the uterine cervix[J].Int J Gynecol Cancer,2006,16(3):1216-1224.
10.夏婷,郑智国,高赟,等.宫颈癌患者血清蛋白指纹图谱的检测及其意义[J].癌症,2008,27(3):279-282.
11.郭社珂,刘红彦,乔玉环.六种蛋白质质谱在宫颈癌发生发展中的变化[J].肿瘤基础与临床,2007,20(5):375-377.
12.Yim EK,Lee KH,Namkoong SE,et al.Proteomic analysis of ursolic acid-induced apoptosis in cervical carcinoma cells[J].Cancer Letters,2006,235(2):209-220.
13.Benedetti Panici P,Greggi S,Colombo A,et al.Neoadjuvant chemotherapy and radical surgery versus exclusive radiotherapy in locally advanced squamous cell cervical cancer:results from the Italian multicenter randomized study[J].J Clin Oncol,2002,20(1):179-188.
14.Tinker AV,Bhagat K,Swenerton KD,et al.Carboplatin and paclitaxel for advanced and recurrent cervical carcinoma:the British Columbia Cancer Agency experience[J].Gynecol Oncol,2005,98:542-581
15.Park EK,Lee SB,Lee YM.Preparation and characterization of methoxy poly (ethylene glycol)/poly(epsilon-caprolactone)amphiphilic block copolymeric nanospheres for tumor-specific folate-mediated targeting of anticancer drugs[J].Biomaterials,2005,26(9):1053-1061.
16.Stevens PJ,Sekido M,Lee RJ.A folate receptor-targeted lipid nanoparticle formulation for a lipophilic paclitaxel prodrug[J].Pharm Res,2004,21(12):2153-2157.
17.Wu J,Liu Q,Lee RJ.A folate receptor-targeted liposomal formulation for paclitaxel[J].Int J Pharm,2006,316(1-2):148-153.
1.Conrads TP,Hood BL,Issaq HJ,et al.Proteomic patterns as a diagnostic tool for early-stage cancer:a review of it sprogress to a clinically relevant tool[J].Mol Diagn,2004,8(2):77-85.
2.Issaq HJ.The role of separation science in proteomics research[J].Electrophoresis,2001,22(17):3629-38.
3.Tang N,Tornatore P,Weinbexger S.Current developments in SELDI affinity technology[J].Mass Spectrom Rev,2004,23:34-44.
4.Wu SP,Lin YW,Lai HC,et al.SELDI-TOF MS profiling of plasma proteins in ovarian cancer[J].Taiwanese J Obstet Gynecol,2006,45(1):26-32.
5.Pang YL,Zhang B,Wang JX,et al.Application of protein finger print pattern models in liver cancer diagnosis[J].J Zhengzhou University,2006,5:69-76.
6.Zinkin NT,Grall F,Bhaskar K,et al.Serum protemics and biomarkers in hepatocellular carcinomal and chronic liver disease[J].Clini Cancer Rea,2008,15,14(2):470-7.
7.Leong S,Christopherson RI,Baxter RC.Profiling of apoptotic changes in human breast cancer cells using SELDI-TOF mass spectrometry[J].Cell Physiol Biochem,2007,20(5):579-90.
8.Scarlett CJ,Samra JS,Xue A,et al.Classification of pancreatic cystic lesions using SELDI-TOF mass spectrometry[J].ANZ J Surg,2007,77(8):648-53.
9.Wong YF,Cheunga TH,Loa KW,et al.Protein profiling of cervical cancer by protein-biochips:proteomic scoring to discriminate cervical cancer from normal cervix[J].Cancer Letters,2004,211(2):227-34.
10.夏婷,郑智国,高赟,等.宫颈癌患者血清蛋白指纹图谱的检测及其意义.癌症[J],2008,27(3):279-282.
11.Kato H,Torigoe T.Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma[J].Cancer,1977,40(4):1621-1628.
12.MaimanM,Feuev G.Value of squamous cell carcinoma antigen levels in invasive cervical carcinoma.Gynecol Oncol,1989,34:312-316.
13.Rose PG,Baker S.Serum squamous cell carcinoma antigen levels in invasive cervical cancer:predictive of response and recurrence.Am J Obstet Gynecol,1993,168(3):942-946.
14.Duk JM,Groenier KH,de Bruljn HW,et al.Pretreatment serum squamous cell carcinoma antigen:a newly identified prognostic factor in early-stage cervical carcinoma.J Clin Oncol,1996,14(1):111-118.
15.Bonfrer JM,Gaarenstroom KN,Korse CM,et al.Cyfra21-1 in monitoring cervical cancer:a comparison with tissue polypep tide antigen and squamous cell carcinoma antigen.Anticancer Res,1997,17(30):2329-2334.
16.柯晓慧,胡芝,孙杭临.血清SCCAg在宫颈癌诊疗中的意义.临床医学[J],2006,26(5):65-67.
1.金正均.合并用药中的相加[J].中国药理学报,1980,1(2):70-76.
2.Van Engeland M,Nieland LJ,Ramaekers FC,et al.Annein V-affinity assay:a review on an apop tosis detection system based on phosphatidylserine exposure[J].Cytometry,1998,31(1):1-9.
3.Kumar L,Bhargava VL.Chemotherapy in recurrent and advanced cervical cancer[J].Gynecol Oncol,1991,40(2):1072-1111
4.Kigawa J,Kanamori Y,Ishihara H,et al.Response rate and cellcycle changes due to intra-arterial infusion chemotherapy with cisplatin and bleomycin for locally recurrent uterine cervical cancer[J].Am J Clin Oncol,1992,15(6):4742-4791
5.Aoki Y,Tomita M,Sato T,et al.Neoadiuvant chemotherapy for patients younger than 50 years with high risk squamous cell carcinoma of the cervix[J].Gynecol Oncol,2001,83(2):263.
6.Zanetta G,Fei F,Parma G,et al.Paclitaxel,ifosfamide and cisplatin(TIP)chemotherapy for recurrent or persistent squamous cell cervical cancer[J].Ann Oncol,1999,10:1171.
7.Christos A,Papadimitriou,Kyrillos S,et al.Phase Ⅱ trial of paclitaxel and cisplatin in metastatic and recurrent carcinoma of the uterine cervix[J].J Clin Oncol,1999,17(3):7612-7661
8.Tinker AV,Bhagat K,Swenerton KD,et al.Carboplatin and paclitaxel for advanced and recurrent cervical carcinoma:the British Columbia Cancer Agency experience[J].Gynecol Oncol,2005,98:542-581
9.Toru S,Michiaki Y,Kiichiro N,et al.Phase Ⅱ study of irinotecan and cisplatin as first2line chemotherapy in advanced or recurrent cervical cancer[J].Oncology,2000,58:312371
10.Imjai C,Anun T,Vimol S,et al.Phase Ⅱ clinical study of irinotecan and cisplatin as first-line chemotherapy in metastatic or recurrent cervical cancer[J].Gynecol Oncol,2003,89:4022-4071
11.Kastritis E,BamiasA,Efstathiou E,et al.The outcome of advanced or recurrent nonsquamous carcinoma of the uterine cervix after platinum-based combination chemotherapy[J].Gynecol Oncol,2005,99(2):3762-3821.
12.Tinker AV,Bhagat K,Swenerton KD,et al.Carbop latin and paclitaxel for advanced and recurrent cervical carcinoma:the British Columbia Cancer Agency experience[J].Gynecol Oncol,2005,98:542-581
13.Yang T,Cui FD,ChoiMK,et al.Liposome formulation of paclitaxel with enhanced solubility and stability[J].DrugDeliv,2007,14(5):301-308.
14.Straubinger RM,Sharma A,MurrayM,et al.Novel taxol formulations:taxol-containing liposomes[J].J Natl Cancer Inst Monogr,1993,(15):69-78.
15.贾晋伟,吴亚梅,郭述良,等.紫杉醇脂质体与传统紫杉醇联合顺铂治疗老年非小细胞肺癌对比研究[J].第四军医大学学报,2008,29(17):1604-1606
2.Schoell WM,Janicek MF,Mirhasphemi R.Epidemiology and biology of cervical cancer[J].Semin Surg Oncol,1999,16(3):203-211.
3.Lonky NM.Risk factors related to the development and mortality from invasive cervical cancer:clinical utility and impact on prevention[J].Obstet Gynecol Clin North Am,2002,29(4):817-842.
4.TangN,Tornatore P,Weinbexger S.Current developments in SELDI affinity technology[J].Mass Spectrom Rev,2004,23:34-44.
5.Kas K.On the technicalities of discovering and applying protein biomarkers for cancer prevention[J].Eur J Cancer revention,2004,(13):437-446.
6.Lin SC,Tseng FG,Huang HM,et al.Microsized 2D protein arrays in mobilized by micro-stamps and micro-wells for disease diagnosis and drug screening [J].Fresenius J Anal Chem,2001,371(2):202-208.
7.Wonga YF,Cheunga TH,Loa KW,et al.Protein profiling of cervical cancer by protein-biochips:proteomic scoring to discriminate cervical cancer from normal cervix[J].Cancer Letters,2004,211(2):227-234.
8.郭社珂,乔玉环,赵先兰.宫颈癌组织蛋白质组图谱分析[J].郑州大学学报(医学版),2007,42(2):305-307.
9.Lin YW,Lai HC,Lin CY,at al.Plasma proteomic profiling for detecting and differentiating in situ and invasive carcinomas of the uterine cervix[J].Int J Gynecol Cancer,2006,16(3):1216-1224.
10.夏婷,郑智国,高赟,等.宫颈癌患者血清蛋白指纹图谱的检测及其意义[J].癌症,2008,27(3):279-282.
11.郭社珂,刘红彦,乔玉环.六种蛋白质质谱在宫颈癌发生发展中的变化[J].肿瘤基础与临床,2007,20(5):375-377.
12.Yim EK,Lee KH,Namkoong SE,et al.Proteomic analysis of ursolic acid-induced apoptosis in cervical carcinoma cells[J].Cancer Letters,2006,235(2):209-220.
13.Benedetti Panici P,Greggi S,Colombo A,et al.Neoadjuvant chemotherapy and radical surgery versus exclusive radiotherapy in locally advanced squamous cell cervical cancer:results from the Italian multicenter randomized study[J].J Clin Oncol,2002,20(1):179-188.
14.Tinker AV,Bhagat K,Swenerton KD,et al.Carboplatin and paclitaxel for advanced and recurrent cervical carcinoma:the British Columbia Cancer Agency experience[J].Gynecol Oncol,2005,98:542-581
15.Park EK,Lee SB,Lee YM.Preparation and characterization of methoxy poly (ethylene glycol)/poly(epsilon-caprolactone)amphiphilic block copolymeric nanospheres for tumor-specific folate-mediated targeting of anticancer drugs[J].Biomaterials,2005,26(9):1053-1061.
16.Stevens PJ,Sekido M,Lee RJ.A folate receptor-targeted lipid nanoparticle formulation for a lipophilic paclitaxel prodrug[J].Pharm Res,2004,21(12):2153-2157.
17.Wu J,Liu Q,Lee RJ.A folate receptor-targeted liposomal formulation for paclitaxel[J].Int J Pharm,2006,316(1-2):148-153.
1.Conrads TP,Hood BL,Issaq HJ,et al.Proteomic patterns as a diagnostic tool for early-stage cancer:a review of it sprogress to a clinically relevant tool[J].Mol Diagn,2004,8(2):77-85.
2.Issaq HJ.The role of separation science in proteomics research[J].Electrophoresis,2001,22(17):3629-38.
3.Tang N,Tornatore P,Weinbexger S.Current developments in SELDI affinity technology[J].Mass Spectrom Rev,2004,23:34-44.
4.Wu SP,Lin YW,Lai HC,et al.SELDI-TOF MS profiling of plasma proteins in ovarian cancer[J].Taiwanese J Obstet Gynecol,2006,45(1):26-32.
5.Pang YL,Zhang B,Wang JX,et al.Application of protein finger print pattern models in liver cancer diagnosis[J].J Zhengzhou University,2006,5:69-76.
6.Zinkin NT,Grall F,Bhaskar K,et al.Serum protemics and biomarkers in hepatocellular carcinomal and chronic liver disease[J].Clini Cancer Rea,2008,15,14(2):470-7.
7.Leong S,Christopherson RI,Baxter RC.Profiling of apoptotic changes in human breast cancer cells using SELDI-TOF mass spectrometry[J].Cell Physiol Biochem,2007,20(5):579-90.
8.Scarlett CJ,Samra JS,Xue A,et al.Classification of pancreatic cystic lesions using SELDI-TOF mass spectrometry[J].ANZ J Surg,2007,77(8):648-53.
9.Wong YF,Cheunga TH,Loa KW,et al.Protein profiling of cervical cancer by protein-biochips:proteomic scoring to discriminate cervical cancer from normal cervix[J].Cancer Letters,2004,211(2):227-34.
10.夏婷,郑智国,高赟,等.宫颈癌患者血清蛋白指纹图谱的检测及其意义.癌症[J],2008,27(3):279-282.
11.Kato H,Torigoe T.Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma[J].Cancer,1977,40(4):1621-1628.
12.MaimanM,Feuev G.Value of squamous cell carcinoma antigen levels in invasive cervical carcinoma.Gynecol Oncol,1989,34:312-316.
13.Rose PG,Baker S.Serum squamous cell carcinoma antigen levels in invasive cervical cancer:predictive of response and recurrence.Am J Obstet Gynecol,1993,168(3):942-946.
14.Duk JM,Groenier KH,de Bruljn HW,et al.Pretreatment serum squamous cell carcinoma antigen:a newly identified prognostic factor in early-stage cervical carcinoma.J Clin Oncol,1996,14(1):111-118.
15.Bonfrer JM,Gaarenstroom KN,Korse CM,et al.Cyfra21-1 in monitoring cervical cancer:a comparison with tissue polypep tide antigen and squamous cell carcinoma antigen.Anticancer Res,1997,17(30):2329-2334.
16.柯晓慧,胡芝,孙杭临.血清SCCAg在宫颈癌诊疗中的意义.临床医学[J],2006,26(5):65-67.
1.金正均.合并用药中的相加[J].中国药理学报,1980,1(2):70-76.
2.Van Engeland M,Nieland LJ,Ramaekers FC,et al.Annein V-affinity assay:a review on an apop tosis detection system based on phosphatidylserine exposure[J].Cytometry,1998,31(1):1-9.
3.Kumar L,Bhargava VL.Chemotherapy in recurrent and advanced cervical cancer[J].Gynecol Oncol,1991,40(2):1072-1111
4.Kigawa J,Kanamori Y,Ishihara H,et al.Response rate and cellcycle changes due to intra-arterial infusion chemotherapy with cisplatin and bleomycin for locally recurrent uterine cervical cancer[J].Am J Clin Oncol,1992,15(6):4742-4791
5.Aoki Y,Tomita M,Sato T,et al.Neoadiuvant chemotherapy for patients younger than 50 years with high risk squamous cell carcinoma of the cervix[J].Gynecol Oncol,2001,83(2):263.
6.Zanetta G,Fei F,Parma G,et al.Paclitaxel,ifosfamide and cisplatin(TIP)chemotherapy for recurrent or persistent squamous cell cervical cancer[J].Ann Oncol,1999,10:1171.
7.Christos A,Papadimitriou,Kyrillos S,et al.Phase Ⅱ trial of paclitaxel and cisplatin in metastatic and recurrent carcinoma of the uterine cervix[J].J Clin Oncol,1999,17(3):7612-7661
8.Tinker AV,Bhagat K,Swenerton KD,et al.Carboplatin and paclitaxel for advanced and recurrent cervical carcinoma:the British Columbia Cancer Agency experience[J].Gynecol Oncol,2005,98:542-581
9.Toru S,Michiaki Y,Kiichiro N,et al.Phase Ⅱ study of irinotecan and cisplatin as first2line chemotherapy in advanced or recurrent cervical cancer[J].Oncology,2000,58:312371
10.Imjai C,Anun T,Vimol S,et al.Phase Ⅱ clinical study of irinotecan and cisplatin as first-line chemotherapy in metastatic or recurrent cervical cancer[J].Gynecol Oncol,2003,89:4022-4071
11.Kastritis E,BamiasA,Efstathiou E,et al.The outcome of advanced or recurrent nonsquamous carcinoma of the uterine cervix after platinum-based combination chemotherapy[J].Gynecol Oncol,2005,99(2):3762-3821.
12.Tinker AV,Bhagat K,Swenerton KD,et al.Carbop latin and paclitaxel for advanced and recurrent cervical carcinoma:the British Columbia Cancer Agency experience[J].Gynecol Oncol,2005,98:542-581
13.Yang T,Cui FD,ChoiMK,et al.Liposome formulation of paclitaxel with enhanced solubility and stability[J].DrugDeliv,2007,14(5):301-308.
14.Straubinger RM,Sharma A,MurrayM,et al.Novel taxol formulations:taxol-containing liposomes[J].J Natl Cancer Inst Monogr,1993,(15):69-78.
15.贾晋伟,吴亚梅,郭述良,等.紫杉醇脂质体与传统紫杉醇联合顺铂治疗老年非小细胞肺癌对比研究[J].第四军医大学学报,2008,29(17):1604-1606
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