乳腺癌循环肿瘤细胞的分离鉴定及槐耳清膏抑制其产生的作用和机制

发布时间：2018-06-07 19:09

本文选题：乳腺癌 + 循环肿瘤细胞　；参考：《第三军医大学》2016年博士论文

【摘要】：侵袭转移是恶性肿瘤重要的生物学特征,是导致癌症复发和患者死亡的重要原因。乳腺癌是威胁全球女性健康的恶性肿瘤之一,早期即可发生远处转移,循环肿瘤细胞被认为是血行转移的关键步骤,它对指导临床制定治疗方案、监测治疗效果、提示乳腺癌的复发或转移、以及评估患者预后情况都有重要意义。循环肿瘤细胞的研究正在深入,愈来愈多的目光集中到临床应用方面,而对于各种治疗能否降低循环肿瘤细胞仍有争议。因此,我们开展一项meta分析,整理了已经发表的在治疗前后检测循环肿瘤细胞的研究论文,并评估现有的抗肿瘤治疗对循环肿瘤细胞的影响。结论是肯定的,但要将其应用于临床实践还需更多更深入的研究。至今,化疗仍然是乳腺癌治疗的基本手段之一,一线药物紫杉类、蒽环类和抗代谢类等化疗药物的使用,尽管可以在很大程度上改善患者预后,但其毒副反应也不容忽视。随着传统中药开发新药治疗癌症成为新的研究方向。越来越多的报道证实槐耳清膏具有抗肿瘤作用,是有效的乳腺癌临床辅助治疗药物。然而,槐耳清膏的抗肿瘤作用机制尚不完全明了,尤其对循环肿瘤细胞的形成的影响尚不清楚。有研究表明上皮细胞间质化(EMT)与循环肿瘤细胞间存在密切的联系,而且循环肿瘤细胞本身也会表达EMT的相关标志物。因此我们提出假设:槐耳清膏可能调节EMT影响循环肿瘤细胞的形成,从而降低乳腺癌的转移风险。由于商品化的仪器,比如Cellsearch?更侧重于临床,主要用于预后监测等方面,不能够分离活细胞进行后续实验。基于此,我们建立适合本科室检测乳腺癌循环肿瘤细胞的方法对32例临床样本进行检测应用,然后在小鼠乳腺癌原位移植瘤模型上探讨槐耳清膏抑制乳腺癌循环肿瘤细胞产生的作用与机制。其主要结果和结论如下:1.循环肿瘤细胞反映抗肿瘤治疗效果的meta分析。(1)通过随机效应模型分析治疗前后循环肿瘤细胞的阳性率,总体分析显示:与治疗前相比,治疗后循环肿瘤细胞的阳性率显著下降(RR=0.68,95%CI:0.61 to 0.76,P0.00001;I2=73%,P0.00001)。(2)亚组分析显示不同治疗手段对循环肿瘤细胞的阳性率的影响不尽相同。新辅助化疗、化疗和联合治疗后循环肿瘤细胞阳性率显著下降:新辅助化疗(RR=0.65,95%CI:0.52 to 0.81,P=0.0001;I2=32%,P=0.16),化疗(RR=0.81,95%CI:0.69 to 0.95,P=0.009;I2=72%,P0.0001)和联合治疗(RR=0.64,95%CI:0.54 to 0.75,P0.0001;I2=67%,P0.0001),而手术亚组治疗后循环肿瘤细胞阳性率未显著降低(RR=1.40,95%CI:0.56 to 3.50,P=0.47;I2=45%,P=0.16)。(3)亚组分析显示乳腺癌不同分子分型对循环肿瘤细胞阳性率有影响:与治疗前相比,HER2阳性患者和HER2阴性患者的循环肿瘤细胞阳性率显著下降:HER2阳性患者(RR=0.68,95%CI:0.57 to 0.82,P0.0001;I2=0%,P=0.59)和HER2阴性患者(RR=0.52,95%CI:0.31 to 0.86,P=0.01;I2=66%,P=0.01),但是三阴性患者的循环肿瘤细胞阳性率没有变化(RR=0.38,95%CI:0.06 to 2.33,P=0.29;I2=72%,P=0.06)。(4)循环肿瘤细胞状态与患者预后间关系的亚组分析显示:与治疗后循环肿瘤细胞上升或者持平的患者相比,循环肿瘤细胞下降的患者有更长的总生存期(均值差=11.61月,95%CI:8.63 to 14.59,P0.00001;I2=69%,P=0.07);其疾病进展概率更低(OR=0.54,95%CI:0.33 to 0.89,P=0.01;I2=45%,P=0.05),无进展生存期更长(均值差=5.07月,95%CI:2.70 to 7.44,P0.0001;I2=96%,P0.00001)。2.乳腺癌循环肿瘤细胞检测方法的建立及在临床样本中的初步应用(1)通过组合密度梯度离心、免疫磁珠分离和流式细胞仪检测技术,建立起乳腺癌外周血循环肿瘤细胞的分离方法。免疫荧光用于鉴定分离的循环肿瘤细胞。结果表明所建立的分离方法能有效地从乳腺癌患者外周血分离循环肿瘤细胞,鉴定结果显示,乳腺癌循环肿瘤细胞是Epcam阳性、CK18阳性、CD45阴性的有核细胞,其细胞直径较大,核浆比例较高,符合目前对循环肿瘤细胞的定义。虽然该测定方法以人工操作为主,但较商品化的仪器Cellsearch?等能够提供更多信息,有利于对循环肿瘤细胞进行更深入的基础和临床研究。(2)对32例乳腺癌患者外周血进行了循环肿瘤细胞检测,27例能检出循环肿瘤细胞(≥1 CTC/7.5m L),占比为84.4%,而作为对照组的纤维腺瘤患者、健康自愿者中的血液均为阴性。在32例患者中,10例转移患者的循环肿瘤细胞阳性且≥5 CTCs/7.5mL检出率为80%,而无转移患者的阳性且≥5 CTCs/7.5mL检出率仅为22.7%,P=0.002。Ki67阳性患者的循环肿瘤细胞阳性且≥5 CTCs/7.5m L检出率50%,高于Ki67阴性患者的循环肿瘤细胞阳性且≥5 CTCs/7.5mL检出率(12.5%),P=0.061。上述结果显示,我们建立的循环肿瘤细胞检测方法稳定性和可靠性好,能够较好的反映乳腺癌的进展。3.槐耳清膏抑制乳腺癌移植瘤的EMT表型,继而抑制其循环肿瘤细胞产生的作用和机制(1)构建带绿色荧光的GFP-4T1细胞系,然后建立BALB/c小鼠乳腺癌原位移植瘤模型,再通过流式细胞仪检测外周血中绿色荧光细胞的数量而反映乳腺癌循环肿瘤细胞状态。通常在建模第12天即可从外周血中检测到绿色荧光细胞,表明模型建立成功。(2)建模第二周末开始药物干预,通过流式细胞仪动态监测血液中GFP-4T1细胞变化,每周1次,共4次,以反映乳腺癌荷瘤小鼠外周血循环肿瘤细胞的状态。第一周各组间循环肿瘤细胞未见显著差异。而在第2、3和4周,槐耳清膏给药组(50mg和100mg)检出的循环肿瘤细胞数量显著少于对照组(P值均小于0.01),而多西他赛组则无显著差异。这些结果表明槐耳清膏较多西他赛能更有效地抑制循环肿瘤细胞的形成。(3)槐耳清膏能减少转移灶的形成,在对照组中有22处转移灶,而槐耳清膏100mg组只有13处,P0.05;而多西他赛组与对照组无显著差异,P0.05。通过调控移植瘤EMT表型,继而减少循环肿瘤细胞的产生,P0.05。(4)槐耳清膏处理后,显著增高E-cadherin表达而降低N-cadherin表达,与降低循环肿瘤细胞数量及减少转移灶形成相一致,提示槐耳清膏能通过调控移植瘤的EMT表型降低循环肿瘤细胞的产生进而减少转移的发生。(5)相较于对照组,槐耳清膏和多西他赛处理均能有效抑制肿瘤细胞的生长(P值均小于0.05)。槐耳清膏能够提高小鼠的生存率且对体重与白细胞无明显影响,而多西他赛则相反。综上所述,循环肿瘤细胞可以作为监测乳腺癌治疗效果、乳腺癌患者预后的指标,同时也能够辅助临床医生为病人定制个体化诊疗的决策。槐耳清膏通过调控乳腺癌的EMT表型从而抑制循环肿瘤细胞的生成,最终减少乳腺癌的转移几率。
[Abstract]:Invasion and metastasis is an important biological characteristic of malignant tumor, which is an important cause of cancer recurrence and patient death. Breast cancer is one of the malignant tumors that threaten the health of women in the world. Distant metastasis can occur in the early stage. Circulating tumor cells are considered to be the key steps of blood transfer. The therapeutic effects, which suggest the recurrence or metastasis of breast cancer, and the assessment of the prognosis of the patients are of great significance. The research on circulating tumor cells is going deep and more and more attention is focused on the clinical application, and it is still controversial whether various treatments can reduce circulating tumor cells. Therefore, we have carried out a meta analysis. Published research papers on circulating tumor cells before and after treatment, and assess the impact of existing antitumor therapies on circulating tumor cells. Conclusion is affirmative, but more in-depth studies are needed to apply it to clinical practice. To date, chemotherapy is still one of the basic methods for the treatment of breast cancer, first line medicine yew, anthracene ring The use of chemotherapeutic drugs such as class and antimetabolic drugs, although it can improve the prognosis of patients to a great extent, can not be ignored. With the development of new drugs in the development of new drugs with traditional Chinese medicine, it has become a new research direction. More and more reports have confirmed that the ointment has anti tumor use and is an effective clinical adjuvant therapy for breast cancer. However, the anti-tumor mechanism of the cream is still not fully understood, especially the effect of the formation of circulating tumor cells. Studies have shown that there is a close relationship between EMT and circulating tumor cells, and the circulating tumor cells themselves also express EMT related markers. Therefore we propose that: It may regulate the formation of circulating tumor cells and reduce the risk of metastasis of breast cancer. As commercial instruments, such as Cellsearch, are more focused on clinical and mainly for prognosis monitoring, it is not possible to separate living cells for follow-up experiments. Based on this, we set up a suitable laboratory for the detection of breast cancer in the undergraduate room. The tumor cell method was used to detect 32 cases of clinical samples, and then the effect and mechanism of the inhibition of the circulating tumor cells in breast cancer were investigated in the mouse model of breast cancer in situ transplantation. The main results and conclusions were as follows: the meta analysis of the 1. circulating tumor cells reflecting the therapeutic effect of anti-tumor treatment. (1) through random Xiao Yingmo The positive rate of circulating tumor cells before and after treatment was analyzed. The overall analysis showed that the positive rate of circulating tumor cells decreased significantly after treatment (RR=0.68,95%CI:0.61 to 0.76, P0.00001; I2=73%, P0.00001). (2) subgroup analysis showed that the effect of different treatment methods on the positive rate of circulating tumor cells was different. The positive rate of circulating tumor cells decreased significantly after chemotherapy, chemotherapy and combined therapy: neoadjuvant chemotherapy (RR=0.65,95%CI:0.52 to 0.81, P=0.0001; I2=32%, P=0.16), chemotherapy (RR=0.81,95%CI:0.69 to 0.95, P=0.009; I2=72%, P0.0001), and combined therapy (RR=0.64,95%CI: 0.54 to 0.75), and surgery subgroup after treatment of circulating tumor The positive rate of cell was not significantly decreased (RR=1.40,95%CI:0.56 to 3.50, P=0.47; I2=45%, P=0.16). (3) the subgroup analysis showed that different molecular types of breast cancer had an influence on the positive rate of circulating tumor cells: compared with before treatment, the positive rate of circulating tumor cells in HER2 positive patients and HER2 negative patients decreased significantly: HER2 positive patients (RR=0.68,95%CI:0.5) 7 to 0.82, P0.0001; I2=0%, P=0.59) and HER2 negative patients (RR=0.52,95%CI:0.31 to 0.86, P=0.01; I2=66%, P=0.01), but there was no change in the positive rate of circulating tumor cells in three negative patients (RR=0.38,95%CI:0.06 to 2.33). (4) a subgroup analysis of the relationship between the state of the circulating tumor cells and the prognosis of the patients showed that after treatment Patients with circulatory tumor cells rising or flat had longer total survival (=11.61 months, 95%CI:8.63 to 14.59, P0.00001; I2=69%, P=0.07) in patients with decreased circulating tumor cells; the progression of the disease was lower (OR=0.54,95%CI:0.33 to 0.89, P=0.01; I2=45%, P=0.05), and the progression - free survival period was longer (mean mean =5.07 months) 2.70 to 7.44, P0.0001, I2=96%, P0.00001) the establishment of a method for detecting circulating tumor cells in.2. breast cancer and its preliminary application in clinical samples (1) a separation method was established by combining density gradient centrifugation, immunomagnetic beads separation and flow cytometry, and the immunofluorescence was used to identify and isolate the tumor cells in the peripheral blood of breast cancer. The results show that the circulating tumor cells can be isolated from the peripheral blood of the breast cancer patients effectively. The results show that the circulating tumor cells of breast cancer are Epcam positive, CK18 positive and CD45 negative nucleated cells. The cell diameter is larger and the proportion of the nuclear plasma is high, which conforms to the current circulating tumor cells. Definition. Although this method is based on manual operation, but more information can be provided than commercial instrument Cellsearch? It is beneficial to further basic and clinical study of circulating tumor cells. (2) circulating tumor cell detection in peripheral blood of 32 patients with breast cancer and 27 cases of circulating tumor cells (> 1 CTC/7.5m) can be detected. L) was 84.4%, and the blood of the healthy volunteers was negative in the fibroadenoma patients of the control group. In the 32 patients, 10 cases of metastatic tumor cells were positive and the detection rate of 5 CTCs/7.5mL was 80%, while no metastases were positive and the rate of 5 CTCs/7.5mL was only 22.7%, and the circulating tumor in P=0.002.Ki67 positive patients The positive rate of cell positive and 5 CTCs/7.5m L was 50%, higher than the positive rate of circulating tumor cells in Ki67 negative patients and the positive rate of 5 CTCs/7.5mL (12.5%). The above results of P=0.061. showed that the method of circulating tumor cell detection established by us was stable and reliable, and the good progress in anti screening of breast cancer was to inhibit the migration of breast cancer. The EMT phenotype of the tumor, then inhibits the role and mechanism of its circulating tumor cells (1) to construct a GFP-4T1 cell line with green fluorescence, and then establish an in situ xenograft model of breast cancer in BALB/c mice, and then detect the number of green fluorescent cells in the peripheral blood by flow cytometry. Green fluorescent cells could be detected from the peripheral blood for Twelfth days, indicating that the model was established successfully. (2) the drug intervention was started at the end of the second week of modeling, and the changes of GFP-4T1 cells in the blood were monitored dynamically by flow cytometry. 1 times a week, a total of 4 times, in order to reflect the state of peripheral blood circulating tumor cells in the breast cancer bearing mice. There was no significant difference in tumor cells, but in the 2,3 and 4 weeks, the number of circulating tumor cells in the group (50mg and 100mg) was significantly less than that of the control group (P value was less than 0.01), but there was no significant difference in the docetaxel group. These results showed that Huaxi ointment was more effective than docetaxel in inhibiting the formation of circulating tumor cells. (3) Sophora japonica Ointment can reduce the formation of metastases, and there are 22 metastatic foci in the control group, while group 100mg only 13, P0.05, but there is no significant difference between the docetaxel group and the control group. P0.05. can reduce the production of circulating tumor cells by regulating the EMT phenotype of the transplanted tumor, and after the treatment of P0.05. (4), the E-cadherin expression is significantly increased. Low N-cadherin expression was consistent with reducing the number of circulating tumor cells and reducing the formation of metastatic foci. It was suggested that Hua ear ointment could reduce the occurrence of circulating tumor cells by regulating the EMT phenotype of the transplanted tumor and then reduce the occurrence of metastasis. (5) compared to the control group, the growth of the tumor cells could be effectively suppressed by the control group (P The value is less than 0.05). It can improve the survival rate of mice and have no obvious effect on the body weight and the white blood cells, while the docetaxel is the opposite. In summary, the circulating tumor cells can be used to monitor the therapeutic effect of breast cancer and the prognosis of the breast cancer patients, and can also assist the clinicians to make the decision making and individualized diagnosis and treatment for the patients. Conclusion: the black ear extract can inhibit the formation of circulating tumor cells by regulating the EMT phenotype of breast cancer, and ultimately reduce the metastatic probability of breast cancer.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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