重楼抗肿瘤活性及中药对氟尿嘧啶类药物疗效相关分子调节作用的初步研究

发布时间：2018-06-23 02:53

本文选题：重楼 + 胃癌　；参考：《南京中医药大学》2017年博士论文

【摘要】：[目的]研究重楼乙醇提取物(Parispolyphlla ethanol extract,PPE)对ICR小鼠的急性毒性剂量,探讨重楼在肿瘤综合治疗中的应用,并考察其体外的抗肿瘤作用及机制;探讨胃癌患者的术前血清肿瘤标记物水平和手术后脉管内癌栓之间的关系,初步探讨胃癌临床应用中药联合氟尿嘧啶类化疗的疗效及其对疗效相关分子调节作用。[方法]1、用ICR小鼠进行急性毒性实验,以生存状态、血液学指标变化、腹腔脏器变化为观察指标,并计算PPE的半数致死量(median leath death,LD50)剂量。2、用MTT法及细胞克隆形成方法检测PPE,以及PPE联合放疗对人胃癌细胞MKN-45的生长抑制;用PI染色法检测PPE,以及PPE联合放疗对人胃癌细胞株MKN-45细胞周期的影响;Western-blot检测PPE对凋亡相关蛋白水平的影响;运用RT-PCR法检测PPE对氟尿嘧啶类药物敏感基因胸苷酸合成酶(thymidylatesynthase,TS)的mRNA水平的变化。3、通过对我院普通外科进行胃癌根治性手术的1005例患者,检测手术前一周内的外周血肿瘤标志物CEA、CA19-9、CA242、CA72-4、CA125的水平,并对术后标本进行包括脉管癌栓在内的多项病理学检查。入组患者基线水平数据按照各外周血肿瘤标志物的状况被分组,分析每个亚组标志物水平对脉管癌栓状态的影响。评价各亚组肿瘤标志物水平预测脉管癌栓的敏感性、特异性、阳性预测价值、阴性预测价值;追踪胃癌根治术Ⅲ期的辅助化疗患者共270名,化疗前后患者均进行外周血TS mRNA检测。将入组患者分成中药结合化疗和单纯化疗组。入组患者每月检测外周血肿瘤标志物CEA、CA19-9、CA242、CA72-4、CA125的水平,每三月复查影像学评价病情进展。比较两组患者的外周血肿瘤学标志物变化情况,1年内无复发生存率,以及化疗前后外周血TS mRNA水平变化。[结果]1、单次给药后获得PPE对ICR小鼠的LD50值约为:12mg/ml,给药剂量越接近LD50值的小鼠,其白细胞较正常小鼠组明显下降,红细胞及血小板无明显变化,药物对小鼠脏器未见明显毒性作用。2、PPE对人胃癌细胞MKN-45的抑制率呈时间和剂量依赖性;重楼联合放疗可以降低肿瘤细胞的生存率;重楼醇提物单独作用将MKN-45细胞阻滞在S期,而与放疗联合细胞周期阻滞在G2/M期;凋亡分析显示PPE可以诱导肿瘤细胞凋亡,同时PPE与放疗以协同作用方式诱导肿瘤细胞凋亡。PPE可下调Bcl-2蛋白的表达及上调Bax蛋白的表达。PPE可下调TS mRNA水平,在MKN-45细胞系中下调至0.59 ±0.07倍。3、入组的1005名患者中共有540名(53.73%)患者发现术后存在血管内癌栓。对于至少有一项外周血肿瘤标志物升高的患者来说,其脉管癌栓发生危险性是没有肿瘤标志物升高患者的2.9倍。对于至少有四项外周血肿瘤标志物升高的患者,对比没有肿瘤标志物升高的患者,其脉管癌栓发生风险是其16.4倍;辅助化疗联合中药治疗组与单纯辅助化疗组比较,6周期辅助化疗后外周血肿瘤标志物较化疗前上升超过20%者分别有27名(19.3%)及34名(26.2%,P=0.178);1年内病情复发的患者分别有31名(22.1%)及43名(33.1%,P=0.044);两组间化疗前外周血TS mRNA表达水平无显著差异(联合组:1.31,化疗组:1.28,P=0.93),6周期化疗后单纯化疗组患者外周血TS mRNA表达平均水平较化疗前上升(1.28,1.42,P=0.43),化疗联合中药治疗组患者外周血TS mRNA表达平均水平较化疗前下降(1.31,0.98,P=0.12),但均未达到统计学显著性差异。[结论]1、PPE具有毒性,可能是通过血液白细胞的变化所致,对于腹腔脏器没有明显影响,临床应用需关注血液学检测指标。2、PPE具有显著的体外抗肿瘤作用,其抗肿瘤作用的机理可能与影响细胞周期,下调Bcl-2蛋白的表达及上调Bax蛋白的表达来诱导肿瘤细胞凋亡,下调TS基因表达水平提高化疗药物敏感性等作用有关;PPE与放疗结合可以阻滞细胞周期,诱导肿瘤细胞凋亡,其机制可能与释放活性氧相关。重楼是一味在肿瘤综合治疗中非常有前景的中药。3、术前外周血肿瘤标记物可以用于预测术后静脉癌栓状态,对于术前血浆肿瘤标志物升高的患者我们需要考虑更加积极的术前治疗。4、胃癌Ⅲ期根治术后辅助化疗患者中,联合中药治疗较单纯化疗能显著减少肿瘤复发风险,其机制可能与中药能够降低患者TS mRNA表达从而提高氟尿嘧啶类化疗药物疗效相关。
[Abstract]:[Objective] to study the acute toxic dose of Parispolyphlla ethanol extract (PPE) on ICR mice, to explore the application of heavy building in the comprehensive treatment of tumor, and to investigate the anti-tumor effect and mechanism in vitro, and to explore the relationship between the level of serum tumor markers before operation and the relationship between the tumor thrombus in the pulse tube after operation. The effect of the combination of traditional Chinese medicine and fluorouracil in the clinical application of gastric cancer and the regulating effect on the therapeutic effect of fluorouracil. [methods]1, ICR mice were used for acute toxicity test, the survival state, the change of Hematology Index, the change of abdominal organ as the observation index, and the dosage of median leath death, LD50), and the dosage of.2, and MTT. The method and cell clone formation method were used to detect the growth inhibition of PPE and PPE combined with radiotherapy for human gastric cancer cell MKN-45; the effect of PPE and PPE combined with radiotherapy on the MKN-45 cell cycle of human gastric cancer cell line were detected by PI; Western-blot was used to detect the effect of PPE on the level of apoptosis related proteins; PPE on fluorouracil was detected by RT-PCR method. Changes in the mRNA level of the thymidylatesynthase (TS) of the substance sensitive gene.3. The level of peripheral blood tumor markers, CEA, CA19-9, CA242, CA72-4, and CA125, were detected in 1005 patients undergoing radical gastrectomy for gastric cancer in general surgery in our hospital, and many of the postoperative specimens were included in the postoperative specimens including the vascular tumor thrombus. The baseline level data of the patients were grouped according to the status of the peripheral blood tumor markers, and the effects of each subgroup marker level on the vascular tumor thrombus status were analyzed. The sensitivity, specificity, positive predictive value and negative predictive value of the tumor markers in each subgroup were evaluated. 270 patients were treated with adjuvant chemotherapy in stage III, and peripheral blood TS mRNA detection was performed before and after chemotherapy. The patients were divided into groups of Chinese medicine combined with chemotherapy and chemotherapy alone. The patients enrolled in the group were detected the level of peripheral blood tumor markers CEA, CA19-9, CA242, CA72-4, CA125 every month, and evaluated the progress of the disease every March. Compare the two groups. There was no recurrence survival rate within 1 years, and the change of TS mRNA in peripheral blood before and after chemotherapy. [results]1, the LD50 value of PPE to ICR mice after a single dose of drug was about 12mg/ml, the more the dosage was closer to the LD50 value, the leukocytes were significantly lower than those of the normal mice, and the red blood cells and platelets had no obvious changes. The drug has no obvious toxic effect on mouse organs.2, and the inhibitory rate of PPE on human gastric cancer cell MKN-45 is time and dose dependent; heavy building combined with radiotherapy can reduce the survival rate of tumor cells; heavy building alcohol extracts separate MKN-45 cells in S phase alone, and the cell cycle block in G2/M phase with radiotherapy; apoptosis analysis shows PPE. The apoptosis of tumor cells could be induced, and the apoptosis of tumor cells was induced by PPE and radiotherapy in a synergistic manner..PPE could down regulate the expression of Bcl-2 protein and up regulate the expression of Bax protein,.PPE can down regulate the level of TS mRNA, down to 0.59 + 0.07 times.3 in MKN-45 cell line, and 540 (53.73%) patients in the 1005 patients of the group were found to exist after the operation. Intravascular thrombus. For patients with at least one increase in peripheral blood tumor markers, the risk of the vascular tumor thrombus was 2.9 times as high as those without a tumor marker. For patients with at least four peripheral blood tumor markers, the risk of the vascular tumor thrombus was 16. compared to those who had no higher tumor markers. 4 times, compared with the adjuvant chemotherapy group and the simple adjuvant chemotherapy group, there were 27 (19.3%) and 34 (26.2%, P=0.178) of peripheral blood tumor markers in the 6 cycle adjuvant chemotherapy than before chemotherapy, and 31 (22.1%) and 43 (33.1%, P=0.044) in 1 years, respectively, and TS mRNA of peripheral blood before chemotherapy before chemotherapy, and TS mRNA before chemotherapy. There was no significant difference in expression level (1.31, 1.31, chemotherapy group: 1.28, P=0.93). The average level of TS mRNA expression in peripheral blood of patients with chemotherapy after 6 cycles was higher than that before chemotherapy (1.28,1.42, P=0.43). The level of TS mRNA expression in peripheral blood of patients with chemotherapy combined with Chinese medicine treatment group was lower than that before chemotherapy (1.31,0.98, P=0.12), but no statistics were reached. [conclusion]1, PPE is toxic and may be caused by changes in blood leucocyte, it has no obvious influence on abdominal organs. Clinical application should pay attention to hematology detection index.2, PPE has significant antitumor effect in vitro, its anti-tumor mechanism can affect cell cycle, down regulation of Bcl-2 protein expression and The expression of Bax protein can induce apoptosis of tumor cells and reduce the level of TS gene expression to improve the chemosensitivity. The combination of PPE and radiotherapy can block cell cycle and induce apoptosis of tumor cells. The mechanism may be related to the release of reactive oxygen species. The anterior peripheral blood tumor markers can be used to predict the postoperative venous tumor thrombus status. For patients with elevated plasma tumor markers, we need to consider more active preoperative treatment of.4. In patients with adjuvant chemotherapy after stage III radical gastrectomy, the combination of traditional Chinese medicine and chemotherapy can significantly reduce the risk of tumor recurrence. The mechanism may be related to the possible mechanism. Chinese medicine can reduce the expression of TS mRNA in patients, thereby enhancing the efficacy of fluorouracil chemotherapy drugs.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2

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