川芎嗪联合顺铂对小细胞肺癌细胞的影响

【作者】 刘志良；

【导师】 刘学政；

【作者基本信息】 辽宁中医药大学 ， 中西医结合基础， 2009， 博士

【摘要】 目的:小细胞肺癌约占肺癌的20%,其恶性程度高、发展迅速、早期可有远处广泛的转移,预后较差。虽然小细胞肺癌对化疗敏感,近期疗效好,但远期容易出现复发和耐药。尽管新一代化疗药物不断面世,抗癌作用进一步增强,但其毒副作用仍不可避免。并且未能完全解决化疗药物的抗药问题,从而限制其临床应用,致使部分患者的存活率未能提高。川芎嗪具有抑制某些实体肿瘤生长、转移的作用,其机制与促进机体免疫功能及诱导细胞调亡有关。顺铂为当前治疗小细胞肺癌最常用的药物之一,具有抗癌谱广、作用强、与多种抗肿瘤药有协同作用、且无交叉耐药等特点。近年来的一些研究表明,某些化疗药物与川芎嗪联合应用,可以提高肿瘤的治疗效果。本试验通过应用川芎嗪与顺铂对接种人小细胞肺癌细胞株NCI-H446裸鼠进行药物作用后,通过测量裸鼠肿瘤体积及肿瘤重量的变化,观察以上药物治疗对瘤体生长情况的影响。同时通过应用川芎嗪与顺铂对人小细胞肺癌细胞株NCI-H446进行一定的药物影响,观察以上药物作用后对小细胞肺癌细胞的增殖、凋亡的影响情况。材料与方法:动物实验分为四组:对照组,川芎嗪治疗组,顺铂治疗组,川芎嗪联合顺铂治疗组。各组裸鼠右臀部皮下分别一次性注射约1×10⁷个NCI-H446细胞,液体总量为0.2ml。对照组接种小细胞肺癌细胞后常规饲养动物。各用药组接种后一周开始给药。川芎嗪治疗组给予川芎嗪注射液100 mg/kg腹腔内注射,每天1次,连续21天。顺铂治疗组给予顺铂注射液2mg/kg腹腔内注射,每3天注射一次,连续7次。联合治疗组依前述方法将川芎嗪组和顺铂联合应用。各组裸鼠在治疗结束后5天脱颈法处死,75%酒精中浸泡2～3分钟。取出瘤体,将瘤体移入培养皿中,清除外周血块及结缔组织,用含4万IU/ml青霉素+4万IU/ml链霉素的生理盐水冲洗。用精密天平测量瘤体的重量,并用游标卡尺测量瘤体的长与宽,计算体积。切取各组新鲜瘤块组织,置于4%多聚甲醛固定液中固定72小时后,常规制作石蜡切片,HE染色,光镜下观察,拍照。切取各组新鲜瘤块组织1mm³大小数块,迅速放入4℃预冷的戊二醛和多聚甲醛混合固定液中固定,1%锇酸后固定,环氧树脂包埋,超薄切片用醋酸铀和柠檬酸铅染色,透射电镜观察瘤组织超微结构。人小细胞肺癌细胞株NCI-H446,在含10%胎牛血清的1640完全培养液中37℃和5%CO₂条件下培养,每3天换液一次,并以0.25%的胰蛋白酶消化传代。细胞学实验分成四组:空白组、川芎嗪治疗组、顺铂治疗组及川芎嗪联合顺铂治疗组,分别通过MTT试验、集落形成试验、流式细胞仪检测、RT-PCR和Western blot检测各组NCI-H446细胞增殖、凋亡以及增殖相关基因cyclin D1、P16和凋亡相关基因Bcl-2、p53的改变。结果:1治疗方案结束时,通过精神状态、活动状况、对刺激的反应、体重下降幅度、食欲5项指标的观察,评价裸鼠生存质量和对药物的毒副反应。结果显示:川芎嗪治疗组裸鼠无明显的药物毒副反应,生存质量好;对照组裸鼠也无明显的药物毒副反应,生存质量一般;顺铂治疗组和川芎嗪联合顺铂治疗组药物毒副反应均较重,联合治疗组的毒副反应比顺铂治疗组的毒副反应轻,表明川芎嗪联合顺铂治疗可降低单独应用顺铂治疗的毒副反应。2治疗后观察裸鼠肿瘤的体积:川芎嗪治疗组（14.36±2.54）、顺铂治疗组（12.12±1.57）以及联合治疗组（8.22±3.05）肿瘤的体积小于对照组（15.13±6.38）,差异有显著性（P＜0.05）。裸鼠肿瘤的重量:川芎嗪治疗组（0.0948±0.0328）、顺铂治疗组（0.0496±0.0299）以及联合治疗组（0.0264±0.0257）肿瘤的重量低于对照组（0.1255±0.0412）,差异有显著性（P＜0.05）。联合治疗组裸鼠肿瘤体积减小、重量降低的更加明显（与顺铂治疗组相比P＜0.05）,差异有显著性。3裸鼠瘤块光镜、电镜观察检测瘤体符合小细胞肺癌绌胞特点。光镜结果显示川芎嗪治疗组瘤组织变性坏死,瘤细胞内可见较多脂滴。顺铂治疗组瘤细胞排列疏松,部分细胞体积变小。联合治疗组瘤细胞稀少,体积变小,核固缩,瘤细胞间有结缔组织增生,纤维化明显,可见较多成纤维细胞。透射电镜检查结果显示川芎嗪治疗组瘤细胞脂肪变性多见,细胞浆内脂滴增多,可见较多坏死细胞及其碎片,细胞胞膜不完整,细胞器减少,并且结构不清,呈空泡状。顺铂治疗组瘤细胞分布稀少,且体积明显变小,并可见较多细胞碎片,部分细胞胞浆内线粒体肿胀,细胞核内异染色质增多,肿瘤细胞凋亡现象常见。联合治疗组瘤细胞分布稀少,瘤细胞体积小,组织内细胞碎片多,细胞胞浆内线粒体肿胀,细胞核内异染色质增多,肿瘤细胞凋亡现象更加常见,可见较多凋亡小体形成。4经川芎嗪、顺铂及联合治疗后NCI-H446细胞存活的细胞减少,细胞变圆,坏死。MTT实验显示在72小时及96小时的吸光度值川芎嗪治疗组（1.031±0.034）（1.129±0.049）,顺铂治疗组（0.986±0.028）（1.018±0.038）以及联合治疗组（0.854±0.036）（0.923±0.041）与空白组相比（1.336±0.067）（1.437±0.045）,明显降低（P＜0.05）。提示用药后各组的细胞增殖性降低,且联合治疗组降低的程度最大（与顺铂治疗组相比P＜0.05）,差异有显著性。集落形成实验显示NCI-H446细胞集落形成率川芎嗪治疗组（17.9±3.7）,顺铂治疗组（15.8±3.5）以及联合治疗组（11.2±2.9）与空白组相比（23.8±4.2）,明显减少（P＜0.05）。结果同样显示用药后各组的NCI-H446细胞增殖性下降,且联合治疗组下降的程度最大（与顺铂治疗组相比P＜0.05）,差异有显著性。RT-PCR及Western blot实验结果显示川芎嗪、顺铂以及联合用药后,NCI-H446细胞的增殖性下降的同时P16基因的表达水平升高,cyclin D1的表达水平下降,差异有显著性（P＜0.05）。5经吖啶橙（AO）和嗅乙啶（EB）染色检测细胞凋亡显示川芎嗪治疗组（11.36±2.18）、顺铂治疗组（15.73±3.25）及联合治疗组（21.32±3.27）与空白组比较（5.21±1.94）细胞凋亡率增加,差异有显著性（P＜0.05）,且联合治疗组凋亡率增加最明显（与顺铂治疗组相比P＜0.05）,差异有显著性。流式细胞仪检测细胞凋亡显示川芎嗪治疗组（16.37±1.23）、顺铂治疗组（19.2±1.51）及联合治疗组（21.2±1.79）与空白组比较（5.23±1.01）细胞凋亡率增加,差异有显著性（P＜0.05）,且联合治疗组捌亡率增加最明显（与顺铂治疗组相比P＜0.05）,差异有显著性。DNA电泳显示,除对照组外,其余各组NCI-H446细胞均可引起DNA断裂,两种药物联合应用与单独应用相比DNA损伤程度增加。RT-PCR及western blot实验结果显示川芎嗪、顺铂及联合治疗作用后各组NCI-H446细胞Bcl-2基因的表达水平降低,p53基因的表达水平升高。结论:1川芎嗪、顺铂及联合治疗均可抑制成瘤裸鼠的肿瘤生长。二者联合作用对成瘤裸鼠的肿瘤生长抑制作用强于单独应用顺铂。2川芎嗪单独应用可一定程度提高裸鼠的生活质量,川芎嗪联合顺铂治疗可以降低顺铂的毒副反应。3川芎嗪、顺铂以及二者联合应用均可诱导小细胞肺癌成瘤裸鼠肿瘤细胞凋亡。4川芎嗪、顺铂及联合治疗均可降低小细胞肺癌细胞株NCI-H446的增殖能力。二者联合作用对小细胞肺癌细胞株NCI-H446的增殖抑制作用强于单独应用顺铂。各组小细胞肺癌细胞株NCI-H446增殖能力降低的同时细胞cyclin D1的表达水平降低,P16的表达水平升高。5川芎嗪、顺铂及二者联合应用均可诱导小细胞肺癌细胞株NCI-H446细胞凋亡,二者联合作用对小细胞肺癌细胞株NCI-H446细胞凋亡作用强于单独应用顺铂。诱导NCI-H446细胞凋亡同时其Bcl-2表达水平降低,p53表达水平升高。更多还原

【Abstract】 Purpose:Small cell lung cancer accounts for about 20 percent of lung cancer.It has high degree of malignancy,rapid development,wide range of distant metastasis and poor prognosis. Although small cell lung cancer is sensitive to chemotherapy in short-term effect,it is prone to relapse and drug resistance in long-term.Despite the continued launch of a new generation of chemotherapeutic agents further enhance its role in cancer,its side effects are inevitable. Chemotherapy can not completely solve the problem of drug resistance,which limits its clinical application.The survival rate of some patients with Small cell lung cancer can not be improved.Tetramethylpyrazine（TMP） can inhibit the growth and the metastasis of certain solid tumors.Its mechanism is related to promoting immune function and inducing apoptosis of the cell.Cisplatin（DDP） is one of the most effective single-drug in the current treatment of small cell lung cancer.It has such characteristics as a broad spectrum of anti-cancer effect,a variety of antineoplastic synergistic effect and no cross-resistance.In recent years a number of studies have shown that the combination of certain chemotherapeutic drugs and TMP can enhance the effectiveness of tumor treatment.In this study,we apply TMP and DDP on nude mice vaccinating with small cell lung cancer cell line NCI-H446 and observe the impact on nude mice tumor growth after the drug effect by measuring the tumor size and tumor weight changes of the nude mice inoculated small-cell lung cancer cells.Cytology test is to observe the proliferation and apoptosis changes of small cell lung cancer cell line NCI-H446 through the application of TMP and DDP on small cell lung cancer cell line NCI-H446 to a certain extent.Material and method:Animal experiments were divided into four groups:control group,TMP treatment group,DDP treatment group,combined TMP and DDP therapy group. We respectively injected about 1×10⁷ NCI-H446 cells in the right hip of nude mice in each group.The total liquid was 0.2ml,Control group of nude mice were conventional fed after vaccinating small cell lung cancer cells.The drugs were given one week after the nude mice were vaccinated with small cell lung cancer cell on the remaining treatment groups.The TMP treatment group:TMP 100mg/kg intraperitoneal injection,1 times per day,for 21 days.DDP treatment group were given DDP 2mg/kg intraperitoneal injection,one injection every three days,for 7 times.Combined TMP and DDP therapy group:the combination of TMP group and DDP group In accordance with the above-mentioned method.The nude mice in each group were killed through neck dislocation 5 days after finishing the treatment,dipping for 2-3 minutes in 75%alcohol.The tumors were removed and put into the dish.The peripheral blocks and connective tissue were cleared.The tumors were washed with saline containing strong antibiotics by 40,000 IU/ml penicillin and 40,000 IU/ml streptomycin.The weights of tumors were measured with precision scales.Vernier caliper measurement of the tumor length and width were taken with vernier caliper,and calculated the volume of the tumers.Cut fresh tumor of each group after treatment,put it under 4%neutral formaldehyde fixative,fixed for 72 hours.Paraffin sections were conventional produced,HE staining,observed under light microscope.We cut several pieces of 1mm3 blocks of fresh tumor after treatment and quickly added them into 4℃precooling 2.5%glutaraldehyde and 1%paraformaldehyde mixed fixative,1%osmium tetroxide post-fixation.Epoxy resin-embedded,staining with uranyl acetate and lead citrate to ultrathin sections,observed the ultrastructure of tumor tissue under electron microscopy（TMP） and photographed the record.Small cell lung cancer cell line NCI-H446 were cultured in the 1640 complete culture medium with 10%fetal bovine serum,under the conditions of 37℃and 5%CO₂ training.The solution were changed for every three days.The celles digested and subcultured with 0.25% trypsin.Cytology tests were divided into four groups:control group,TMP treatment group, DDP treatment group and combined TMP and DDP therapy group.MTT experiment, colony-forming experiment,flow cytometry detection,RT-PCR and Western blot detection were respectively cayyied out.Observed the cell proliferation and apoptosis in each group and the changes of proliferation-related genes cyclin D1,P16 and apoptosis-related gene Bcl-2, p53.Results:1 Through observing the following five indicators:the mental state,activities,response to the stimulation,weight loss and appetite,We evaluated the quality of life and toxicity in the nude.The results showed that nude TMP group had no significant toxicity of the drug,and has the best quality of life,Followed by co-treatment group,again as the control group,while the toxicity in DDP group had the worst quality of life.The nude’s toxicity in Combined TMP and DDP therapy group was significantly lower than in DDP group,the quality of life was superior to DDP group,indicating that combination treatment could reduce DDP toxicity.2 Observed the size of nude mice tumor after treatment:TMP treatment group（14.36±2.54）, DDP treatment group（12.12±1.57） and combined TMP and DDP therapy group（8.22±3.05）. The size.of tumors in the groups above were all smaller than the size in control group（15.13±6.38）.The tumor weight of nude mice:TMP treatment group（0.0948±0.0328）,DDP treatment group（0.0496±0.0299） and combined TMP and DDP therapy group（0.0264±0.0257）.The weight of tumors in the groups above were all lower than those in control group （0.1255±0.0412）.The reduction of tumor size and weight in combined TMP and DDP therapy group were more significant,showing that combined TMP and DDP therapy group can more enhance the therapeutic effect than DDP treatment group.3 By light microscopy and electron microscopy detection,the nude mice tumors were consistent with the characteristics of small cell lung cancer cells.Through light microscopy detection,degeneration and necrosis of the tumor tissue could be seen in TMP treatment group.More lipid droplets appeared in the tumor cells.The tumor cells lined along with osteoporosis in DDP treatment group.Part of the tumor cells became smaller in size.The tumor cells in-combined TMP and DDP therapy group rared.The tumor cells became smaller in size.The cell nuclear became pyknosis.Connective tissue growthed between the tumor cells.Fibrosis was obvious.More fibroblasts could be seen in the tumor tissue.Through electron microscopy examination,in TMP treatment group we found more fatty degeneration in the tumor cells.Lipid droplets inside the cytoplasm increased.Necrotic cells and its debris could be seen more.The cell membrane was not complete.Reduction of cellular organ, structural unclear and vacuolization was like.In DDP treatment group,the tumor cells rared. The tumor cells became significantly smaller in size.More cell debris could be seen.In part of the tumor cells,mitochondria in cytoplasm swelled.The heterochromatin increased in the nucleus.Tumor cell apoptosis was common.In combined TMP and DDP therapy group,the tumor cells were scarce,and the patterns were similar to in the DDP treatment group.4 By TMP,DDP and joint treatment on small cell lung cancer cell line NCI-H446,the survival of the cells decreased.The cells became round and necrosis.MTT test showed the absorbance value in 72h and 96h:TMP treatment group（1.031±0.034）（1.129±0.049）,DDP treatment group（0.986±0.028）（1.018±0.038 ）,combined TMP and DDP therapy group（0.854±0.036） （0.923±0.041）,all decreased compared with the control group（1.336±0.067）（1.437±0.045 ）, （P＜0.05）.Those indicated that the cell proliferation decreased in each treatment group and the absorbance value reduced to the extent of the largest in combined TMP and DDP therapy group.（compared with DDP treatment group P＜0.05）.The colony-forming experiments showed the rate of colony-forming cells:TMP treatment group（17.9±3.7）,DDP treatment group（15.8±3.5）,combined TMP and DDP therapy group（11.2±2.9）.Decreased significantly compared with the control group（23.8±4.2）.The results also showed that the cell proliferation in each treatment group is reduced after administration of the drugs,and the combined TMP and DDP therapy group decreased to the largest extent in the number of the colony formation（compared with DDP treatment group P＜0.05）.The results of RT-PCR and Western blot showed that the expression level of cyclin D1 gene in the small-cell lung cancer cell line NCI-H446 decreased,and increased the expression level of P16 gene,after treated by TMP,DDP and the combination of both.There was a significant difference（P＜0.05）.5 The result of detecting apoptosis by acridine orange（AO） and olfactory bromide（EB） staining showed:TMP treatment group（11.36±2.18）,DDP treatment group（15.73±3.25）, combined TMP and DDP therapy group（21.32±3.27）.The apoptosis rate of the cells increased compared with the control group（5.21±1.94）.There was a significant difference （P＜0.05）.The result of flow cytometry detecting showed:TMP treatment group （16.37±1.23）、DDP treatment group（19.2±1.51）,combined TMP and DDP therapy group （21.2±1.79）.The apoptosis rate of the cells increased compared with the control group （5.23±1.01）.There was a significant difference（P＜0.05）.The apoptosis rate increased to the largest extent（compared with DDP treatment group P＜0.05）.DNA electrophoresis revealed that each group could result in DNA fragmentation in addition to the control group. The level of DNA damage increased after combined application of the two drugs compared to separate application.The results of RT-PCR and Western blot showed TMP,DDP and combination therapy could reduce the level of Bcl-2 gene expression,and increase p53 gene expression level.Conclusion:1 TMP,DDP and combination therapy can inhibit tumor growth in nude mice tumors.The role of TMP is weak,a stronger role in DDR the role of joint treatment of the two is stronger than DDP alone.2 Application TMP after DDP in the treatment of nude mice can significantly improve the quality of life in nude mice.3 TMP DDP and the combination of two can induce apoptosis of small-cell lung cancer cell in nude mice tumor.4 TMP,DDP and combination therapy can significantly reduce the small-cell lung cancer cell line NCI-H446 proliferation capacity.The role of TMP is weak,a stronger role in DDP,the role of joint treatment of the two is stronger than DDP alone.The expression level of cyclin D1 gene in the small-cell lung cancer cell line NCI-H446 decreased,and increased the expression level of P16 gene.5 TMP,DDP and combination of both can induce small cell lung cancer cell line NCI-H446 cell apoptosis.The role of TMP is weak,a stronger role in DDP,the role of joint treatment of the two is stronger than DDP alone.Meanwhile the expression level of Bcl-2 gene decreased, and increased the expression level of p53 gene.更多还原