ABCC4与低位直肠癌放疗耐受的预后探讨与机制研究
发布时间：2018-07-26 07:22
【摘要】：尽管实验研究人员在不断的了解癌症的病因学，并且一直在尝试开发新的诊断和治疗方法，但是，恶性肿瘤的发病率和死亡率的仍然没有得到有效的控制。造成这种情况的其中一个主要原因是患者化疗后，仍然没有行之有效的办法来抑制部分耐药肿瘤的复发。对化疗的耐药性可能是先天存在的，即从治疗开始之前便存在的，也可能是在治疗的过程中逐渐产生的，这是一种变化的情况。举例来说，几乎与化疗治疗的所有非小细胞肺癌患者最终都会发展为抗癌药物耐受。化疗药物的生物学机制是十分复杂的，通常包括以下一种或多种机制：抑制细胞凋亡，诱导并且抑制的DNA修复机制，变更药物靶标的结构，修改细胞膜的成分或者结构（从而降低了药物的吸收)，以及药物的外排。关于后者，一个主要的问题是交叉耐药性，其中涉及广谱药物因为癌细胞膜中存在的转运蛋白产生的耐药性，这不仅是针对单一活性药物或化学相关药物，患者体内耐药蛋白的表达会增加，而且对于全身性症状的化疗剂，甚至尚未施加过任何化疗药物的患者，都可能产生耐药的作用。这一现象被称为多药耐药（MDR)，其中参与了这一过程的蛋白质称为MDR蛋白。人们对于多药耐药蛋白的研究始于1974年，当时Victor Ling和Larry Thompson阐述了他们的实验结果，选取稳定的CHO细胞系，成功复制增殖了对秋水仙素耐药的细胞，并且发现耐药细胞并没有让秋水仙碱进入细胞质。研究人员还发现这种细胞也能够抗地美可辛，放线菌素D和长春碱。有人进一步指出，对秋水仙碱摄取敏感的细胞，参与到细胞分子转运过程是被动的，耐药细胞则是经历一个主动的过程，原因是耐药细胞可能被氰化物，叠氮化钠和二硝基苯酚等多种化合物抑制。据进一步实验结果证明，未成熟细胞和抗性细胞之间的主要区别是一个170kDa的质膜糖蛋白是否表达，这个蛋白被称为P-糖蛋白。P-糖蛋白也被称为P-gp、 ABCB1或MDR1。它能够在很短的时间内大量的产生，P糖蛋白与其他的膜转运蛋白有所不同，它参与多药耐药机制。举例来说，在1990年，有人描述了MCF-7/AdrVp耐药细胞株中含有蒽环类95kDa的膜蛋白，这个蛋白后来被称为BCRP (乳腺癌耐药蛋白,又名ABCG2)。在1992年，Cole和其他合作研究人员发现并克隆另一个磷酸糖蛋白，这种酶在阿霉素耐药细胞株H69AR中过度表达，人们将这种蛋白命名为MRP (也称为多药耐药相关蛋白）。在之后的不久，研究人员明确指出，所有这些蛋白质均含有一些相同的基因序列和发挥同源性的功能，并且均被定义为ATP结合蛋白超家族(ATP-binding cassette superfamily,ABCs)的成员。ATP结合蛋白超家族是最大的跨膜蛋白家族，根据序列和同源性，分为A到G七个亚型。 ABCC家族成员主要由大约200个氨基酸构成，这种独特的氨基酸跨膜结构把ABCC家族成员与其他的ABC成员区分开来。ABCC4是ABC转运C亚家族的成员。ABC转运蛋白主要是跨膜蛋白，能够介导ATP-依赖的细胞通路运转其他分子物质穿过细胞膜。ABCC4基因位于人染色体13q32.1位点，在ABCC4基因编码后表达的蛋白质是ABCC/MRP家族中长度最短的成员，其作用是介导各种外源性和内源性分子的流出。 在对ABCC4基因敲除小鼠的实验研究显示，ABCC4具有重要的生理作用，特别是作用在肝脏，脑组织，和肾脏。在胆汁淤积性损伤的病例中，胆汁通过肝脏时受阻，在大鼠和小鼠胆管结扎后，会出现ABCC4上调，并且验证在患者中也存在进行性的家族性肝内胆汁淤积。ABCC4介导发生的谷胱甘肽-依赖性胆汁酸外排，在特定条件下的这种诱导是一种保护性反应，作用是防止肝坏死。除了清除代谢产物，ABCC4能够通过外排活性药物，以抵抗各种外来物质可能对重要组织造成的损伤。ABCC4缺陷的小鼠对多种药物（抗癌药拓扑替康，抗病毒药物阿德福韦和替诺福韦）表现出肾脏排泄功能降低。相应的，在血浆和某些组织中检测到托泊替康的水平升高。ABCC4缺乏也使得拓扑替康能够穿过血脑屏障，进而渗透到中枢神经系统和脑。此外， ABCC4基因敲除小鼠与野生型同窝小鼠相比，基因敲除小鼠在骨髓，胸腺，脾脏和肠中都增强了抗病毒药物的吸收。另外已经有实验研究表明，ABCC4在髓系祖细胞中高度表达，通过主动外排机制，以保护这些细胞免受硫嘌呤诱导产生的造血毒性。 对于直肠癌的预后评判，目前公认的判断方法是TNM分期，细胞分化程度，区域淋巴结及血清肿瘤标志物的表达，这些指标均与直肠癌的长期预后相关。.然而，对LARC患者的术前放疗短期效果验证和长期预后效果检测，到目前为止被证明是金标准的生物活性标记物还很少。目前比较新型的辅助治疗模型是，对局部晚期直肠癌患者的治疗是结合了5-FU为基础的化学疗法和其他的放射疗法。因此，在使用ABCC4作为一种新型的生物标记物在预测LARC患者抗辐射能力的最大优点在于，这个比较理想的分子标记同时与照射和细胞毒性药物都具有相关联意义。ABCC4的耐药相关性在一些研究中已经报道了，其中涉及到多种癌细胞类型（包含直肠癌）。总之，这些研究突出表明ABCC4作为转运体能够发挥多个功能，保护生命器官，防止生物代谢物的积累，防止化疗药物的损伤。 本实验首先采用细胞增殖实验，在五株直肠癌细胞系中挑选出放疗敏感株LOVO和抗性株RKO，然后用免疫组化的方法，观察了敏感株LOVO细胞和抗性株RKO细胞中ABCC4表达的情况，结果显示，仅能在细胞膜和细胞质中观察到染色颗粒，在细胞核中看不到ABCC4的棕黄色染色颗粒。这个实验结果与国外研究人员在其他肿瘤细胞系中观察到的结果是一致的（Zaman以及他的同事在肺癌细胞中做过相应的验证），更进一步的实验结果是，敏感株LOVO细胞，其ABCC4的高表达率为17%，而放射抵抗株RKO细胞的高表达率为71%，敏感株细胞明显低于抵抗株细胞，差异具有统计学意义。这个结果说明，ABCC4的高表达很可能参与到放射抵抗的生物学分子通路中。Havie等人在前期的研究文献中已经指明，ABCC4在T淋巴细胞白血病细胞中也具有高表达的情况，同时这种细胞也会产生放疗抗性。作者还指出，在接受放疗失败后，即使使用化学疗法的手段，耐药性依然没有消失。随后，展开更深入的研究，对总计93例患者接受经腹前切除术，28例患者接受腹会阴联合切除术的治疗手段。根据术后病理报告判断环周切缘均为阴性。术后中位随访时间34.5个月(SD=8.6)。与预后相关的临床病理多因素进行分析。Cox风险分析显示ABCC4高表达(P=.036)、p53突变型(P=.047)、病理反应差(P=.044)的患者预后不良，3年总生存率低；ABCC4高表达(P=.027)、p53突变型(P=.019)、最终病理分期差(P=.032)的患者预后不良，3年无病生存率相对较低。慢病毒转染获得稳定下调ABCC4蛋白的细胞株。Real-time PCR以及Western Blot结果均证实，其在RKO-KD、HT29-KD细胞中持续有效抑制abcc4基因表达，为后续细胞功能实验提供一定的理论基础，可以继续分子机制研究方面的探索。Real-time PCR结果显示abcc4mRNA在HT29-KD组的表达量(0.20±0.02)明显少于HT29-NC组(0.99±0.02)和HT29-CON组(0.98±0.03)(P 0.05)；Western Blot实验表明ABCC4蛋白表达水平在HT29-KD组(0.25±0.02)显著低于HT29-NC组(0.98±0.02)和HT29-CON组(0.98±0.03)(P 0.05)。而在阴性对照组HT29-NC和空白对照组HT29-CON之间比较，两者之间均无显著差异(P0.05)。通过测量肿瘤直径计算的方法，我们发现，与对照组相比，放疗后RKO-KD裸鼠移植瘤模型肿瘤生长状态呈现时间依赖性显著抑制(P .05)，观察终点测量平均肿瘤重量，RKO-KD组瘤重2.01克(±0.18)明显轻于RKO-NC组3.97克(±0.21)以及RKO-CON组4.01克(±0.19)(P .05)。在三组的空白对照组当中，裸鼠的肿瘤体积大小与重量均无统计学显著差异(P.05)。4Gy剂量射线照射后，细胞内cAMP含量迅速上升后，呈现时间依赖性减少。。平均cAMP浓度在RKO-KD组0.66(±0.25)%比RKO-NC组0.45(±0.15)%和RKO-CON组0.46(±0.16)%显著升高(P .05)。同样处理后的细胞检测细胞周期分布，RKO-KD组细胞43.54(±3.07)%处于G0/G1期的比例明显少于RKO-NC组细胞65.73(±3.78)%以及RKO-CON组细胞66.52(±3.64)%(P .05)，表明在RKO-KD组细胞丧失G1-S监测点DNA修复能力，进入相对放疗敏感的G2期从而易于遭受辐射损伤。在未经照射的三组空白对照中，实验结果显示，分析细胞周期分布情况，无统计学意义上的显著性差异(P.05)。 这里值得强调的是，耐药的分子机制与辐射抗性之间有很大的不同。细胞毒性药物是发挥一种持续的，缓慢的和系统性的生物化学作用，因此对于在细胞凋亡信号通路的关键蛋白，这些蛋白具有足够的时间做出改变，，对药物进行应答，这可能很好地解释化疗耐药的潜在机制，然而，由于照射的方式，是给机体直接提供了一个瞬时高能量并且直达癌细胞内部，通过产生的活性氧诱导DNA的大规模损伤。细胞凋亡信号通路可能不是最主要的决定因素。在此，我们确定了下调ABCC4表达，在射线辐照后，会提高细胞内cAMP浓度积累，细胞周期G1-S期检查点明显不足，这可能解释了其中的机制，帮助阐明抑制ABCC4的shRNA表达后，会诱导促进大肠癌细胞凋亡。 总之，目前的实验研究表明，ABCC4是体内实验和体外实验中，都表现出对射线辐照的调节作用，是极其重要的敏感分子，并与局部侵袭性直肠癌同时接收术前放疗患者的不良预后有显著相关。这些分析数据支持以下理论，ABCC4可以看成是一个有用的预测指标或者靶点，对于局部侵袭性直肠癌患者是否应该接受术前放疗，和预后的情况评估有一定的指导意义。提示未来可以更加深入的研究，明确ABCC4在治疗前筛查的临床意义，为提高预后，改善患者生活质量提供了新的思路。
[Abstract]:Although the researchers are constantly learning the etiology of cancer and have been trying to develop new methods of diagnosis and treatment, the incidence and mortality of malignant tumors are still not effectively controlled. One of the main reasons for this is that the patients are still not effective after chemotherapy. The relapse of some drug-resistant tumors. The resistance to chemotherapy may be innate, that is, before the treatment begins and may also be produced in the process of treatment. This is a change. For example, almost all patients with non small cell lung cancer treated with chemotherapy will eventually develop to anticancer drug tolerance. The biological mechanism of chemotherapeutic drugs is very complex, usually including one or more mechanisms: inhibiting apoptosis, inducing and inhibiting the DNA repair mechanism, changing the structure of the drug target, modifying the composition or structure of the cell membrane (and reducing the absorption of drugs), as well as the efflux of the drug. A major question about the latter The problem is cross resistance, which involves the resistance of broad-spectrum drugs to transporters in the membrane of cancer cells, not only for single active drugs or chemical related drugs, but also in the expression of drug resistant proteins in the patient's body, and for patients with systemic symptoms, or even those who have not had any chemotherapy drugs. This phenomenon is known as multidrug resistance (MDR), and the protein involved in this process is called MDR protein. The study of multidrug resistant proteins began in 1974, when Victor Ling and Larry Thompson elaborated their experimental results, selected a stable CHO cell line, and successfully replicated and proliferated to autumn. Narcissuin resistant cells and found that drug-resistant cells did not allow colchicine to enter cytoplasm. The researchers also found that the cells were also able to resist actinomycin, actinomycin D and Changchun base. One active process is that resistant cells may be inhibited by a variety of compounds such as cyanide, sodium azide, and two nitrophenol. According to further experimental results, the main difference between the immature and resistant cells is whether a 170kDa plasma membrane glycoprotein is expressed, and this protein is called the P- glycoprotein.P- glycoprotein. P-gp, ABCB1, or MDR1. can be produced in a very short period of time, and P glycoproteins are different from other membrane transporters. It is involved in multidrug resistance mechanisms. For example, in 1990, a membrane protein containing anthracycline 95kDa was described in MCF-7/AdrVp resistant cell lines, which was later called BCRP (breast cancer resistance). Protein, also known as ABCG2). In 1992, Cole and other cooperative researchers found and cloned another phosphate glycoprotein, which was overexpressed in the doxorubicin resistant cell strain H69AR. People named the protein MRP (also called multidrug resistance associated protein). The ATP binding protein superfamily (ATP-binding cassette superfamily, ABCs), a member of the.ATP binding protein superfamily, is the largest family of transmembrane proteins, which are divided into seven subtypes from A to G according to sequence and homology.
The members of the ABCC family are mainly composed of about 200 amino acids. This unique amino acid transmembrane structure distinguishes ABCC family members from other ABC members..ABCC4 is a member of the ABC transshipment C subfamily,.ABC transporters are mainly transmembrane proteins, and can mediate ATP- dependent cells through other molecular substances through the cell membrane.ABCC4 The gene is located at the 13q32.1 site of the human chromosome. The protein expressed by the ABCC4 gene is the shortest member of the ABCC/MRP family, and its role is to mediate the outflow of a variety of exogenous and endogenous molecules.
The experimental study of ABCC4 gene knockout mice showed that ABCC4 had important physiological functions, especially in the liver, brain tissue, and kidney. In cases of cholestasis, bile was blocked through the liver. After ligation of the bile ducts in rats and mice, the ABCC4 up-regulated and a progressive home was also found in the patients. Human intrahepatic cholestasis.ABCC4 mediated glutathione dependent bile acid efflux, which is a protective reaction under specific conditions, is a protective reaction to prevent liver necrosis. In addition to removing metabolites, ABCC4 can pass out active drugs to resist the possible damage to important tissues by various external substances in.ABCC4 deficiency. The trapped mice showed a decrease in renal excretion by a variety of drugs (antitumor drugs topotecan, antiviral drugs A Duff Vee and Nuo Fuwei). Correspondingly, the elevated levels of.ABCC4 deficiency in plasma and some tissues showed that topotecan was able to penetrate the blood brain barrier and then infiltrate into the central nervous system and the brain. In addition, ABCC4 gene knockout mice have enhanced the absorption of antiviral drugs in bone marrow, thymus, spleen and intestines compared with wild type mice. In addition, experimental studies have shown that ABCC4 is highly expressed in medullary progenitor cells and protects these cells from sulfur purine induced by the active platoon system. Hematopoiesis.
For the prognosis of rectal cancer, TNM staging, cell differentiation, regional lymph nodes and the expression of tumor markers in the region are associated with the long-term prognosis of rectal cancer. However, the short-term effect of preoperative radiotherapy and the long-term prognosis of LARC patients have been proved so far. There are few biomarkers for the gold standard. A new model of adjuvant therapy is the combination of 5-FU based chemotherapy and other radiation therapy for patients with locally advanced rectal cancer. Therefore, the greatest advantage of using ABCC4 as a new biomarker in predicting the radiation resistance of LARC patients is In some studies, this relatively ideal molecular marker associated with both exposure and cytotoxic drugs has been reported in some studies, involving a variety of cancer cell types (including rectal cancer). In conclusion, these studies suggest that ABCC4 can play multiple functions and protect life as a transporter. Organs prevent the accumulation of biological metabolites and prevent the damage of chemotherapeutic drugs.
In this experiment, the cell proliferation test was first used to select the radiotherapy sensitive strain LOVO and the resistant strain RKO in five rectal cancer cell lines. Then the expression of ABCC4 in the sensitive LOVO cells and the resistant strain RKO cells was observed by immunohistochemical method. The results showed that the staining particles could be observed only in the cell membrane and cytoplasm, and the nuclei were in the nucleus. The results were consistent with the results observed by foreign researchers in other tumor cell lines (Zaman and his colleagues in lung cancer cells), and further experimental results were that the sensitive strain of LOVO cells, the high expression of ABCC4, was 17%, and radiation resistance was 17%. The high expression rate of RKO cells was 71%, and the sensitive strain cells were significantly lower than those of the resistant strain cells. The results showed that the high expression of ABCC4 was likely to be involved in the biological pathway of radiation resistance, and.Havie et al. In the previous research literature has indicated that ABCC4 is also found in T lymphocyte leukemia cells. The authors also pointed out that, after the failure of the radiotherapy, the drug resistance was still not disappearing even with the means of chemotherapy. Then, a more in-depth study was carried out for the total of 93 patients undergoing pre abdominal excision, and 28 patients received a combination of abdominal perineal resection. The circumferential margin of the circumferential margin was negative according to the postoperative pathological report. The median follow-up time was 34.5 months (SD=8.6). The.Cox risk analysis showed that the high expression of ABCC4 (P=.036), the p53 mutant (P=.047), the pathological response poor (P=.044) patients had poor prognosis, the 3 year total survival rate was low, and the ABCC4 high expression (P=). .027), p53 mutant (P=.019), the final pathological staging (P=.032) patients have poor prognosis, and the 3 year disease free survival rate is relatively low. The lentivirus transfection obtained the stable and down-regulation of the ABCC4 protein cell line.Real-time PCR and Western Blot results all confirmed that the gene expression in RKO-KD and HT29-KD cells continued to effectively inhibit the expression of the ABCC4 gene, for subsequent cell work. .Real-time PCR results showed that the expression of abcc4mRNA in HT29-KD group (0.20 + 0.02) was significantly less than that of HT29-NC group (0.99 + 0.02) and HT29-CON group (0.98 + 0.03) (P 0.05), and Western Blot experiment showed that the expression level of ABCC4 protein was in HT29-KD group (0.25 + 0.02). Significantly lower than group HT29-NC (0.98 + 0.02) and group HT29-CON (0.98 + 0.03) (P 0.05), but there was no significant difference between the negative control group HT29-NC and the blank control group HT29-CON (P0.05). By measuring the tumor diameter calculation method, we found that the tumor growth pattern of the RKO-KD nude mice after radiotherapy was compared with those in the radiotherapy group. The state showed a significant time dependence inhibition (P.05), the average tumor weight was measured at the end point, the tumor weight of the RKO-KD group was 2.01 grams (+ 0.18) significantly lighter than that of the RKO-NC group (3.97 g) and 4.01 grams (0.19) (P.05) in the RKO-CON group. In the blank control group, there was no significant difference between the size and weight of the tumor in the nude mice (P.05).4Gy dose. After the radiation, the intracellular cAMP content increased rapidly and showed a time dependent decrease.. the average cAMP concentration was 0.66 (+ 0.25)% in the RKO-KD group (+ 0.15)% and 0.46 (0.16)% in the RKO-CON group (P.05). The cell cycle distribution of the treated cells and the percentage of 43.54 (+ 3.07)% in RKO-KD group cells were obviously in the G0/G1 stage. Less than 65.73 (+ 3.78)% of cells in group RKO-NC and 66.52 (+ 3.64)% of cells in group RKO-CON (P.05), indicating that the cells lost the ability to repair DNA at the G1-S monitoring point in RKO-KD group, and were easily exposed to radiation damage. In the three blank controls of unirradiated groups, the experimental results showed that the distribution of cell cycle was analyzed and no system was found. Significant difference in the meaning of the study (P.05).
It is worth emphasizing that there is a great difference between the molecular mechanism of drug resistance and the radiation resistance. Cytotoxic drugs play a continuous, slow and systematic biochemical role, so for the key proteins in the apoptotic signaling pathway, these proteins have enough time to make changes and respond to drugs. It may well explain the potential mechanism of chemoresistance, however, as a result of exposure, the body directly provides an instantaneous high energy and directly to the inside of the cancer cells, and induces a massive damage to DNA through the production of reactive oxygen. The apoptotic signaling pathway may not be the most important determinant. Here, we have determined the downregulation of ABCC 4 expression, after radiation, can increase the accumulation of intracellular cAMP concentration, and the cell cycle G1-S phase checkpoint is obviously inadequate, which may explain the mechanism, helping to clarify the inhibition of shRNA expression of ABCC4 and induce the apoptosis of colorectal cancer cells.
In conclusion, the current experimental study shows that ABCC4 is a very important sensitive molecule in both in vivo and in vitro experiments, which is a very important sensitive molecule, and has a significant correlation with the poor prognosis of patients receiving preoperative radiotherapy for local invasive rectal cancer. These data support the following theory that ABCC4 can be considered as a one. A useful predictor or target is of guiding significance for the evaluation of preoperative radiotherapy for local invasive rectal cancer and the evaluation of the prognosis. It is suggested that a more in-depth study should be made in the future to clarify the ABCC4 screening before treatment.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R735.37;R730.55
[Abstract]:Although the researchers are constantly learning the etiology of cancer and have been trying to develop new methods of diagnosis and treatment, the incidence and mortality of malignant tumors are still not effectively controlled. One of the main reasons for this is that the patients are still not effective after chemotherapy. The relapse of some drug-resistant tumors. The resistance to chemotherapy may be innate, that is, before the treatment begins and may also be produced in the process of treatment. This is a change. For example, almost all patients with non small cell lung cancer treated with chemotherapy will eventually develop to anticancer drug tolerance. The biological mechanism of chemotherapeutic drugs is very complex, usually including one or more mechanisms: inhibiting apoptosis, inducing and inhibiting the DNA repair mechanism, changing the structure of the drug target, modifying the composition or structure of the cell membrane (and reducing the absorption of drugs), as well as the efflux of the drug. A major question about the latter The problem is cross resistance, which involves the resistance of broad-spectrum drugs to transporters in the membrane of cancer cells, not only for single active drugs or chemical related drugs, but also in the expression of drug resistant proteins in the patient's body, and for patients with systemic symptoms, or even those who have not had any chemotherapy drugs. This phenomenon is known as multidrug resistance (MDR), and the protein involved in this process is called MDR protein. The study of multidrug resistant proteins began in 1974, when Victor Ling and Larry Thompson elaborated their experimental results, selected a stable CHO cell line, and successfully replicated and proliferated to autumn. Narcissuin resistant cells and found that drug-resistant cells did not allow colchicine to enter cytoplasm. The researchers also found that the cells were also able to resist actinomycin, actinomycin D and Changchun base. One active process is that resistant cells may be inhibited by a variety of compounds such as cyanide, sodium azide, and two nitrophenol. According to further experimental results, the main difference between the immature and resistant cells is whether a 170kDa plasma membrane glycoprotein is expressed, and this protein is called the P- glycoprotein.P- glycoprotein. P-gp, ABCB1, or MDR1. can be produced in a very short period of time, and P glycoproteins are different from other membrane transporters. It is involved in multidrug resistance mechanisms. For example, in 1990, a membrane protein containing anthracycline 95kDa was described in MCF-7/AdrVp resistant cell lines, which was later called BCRP (breast cancer resistance). Protein, also known as ABCG2). In 1992, Cole and other cooperative researchers found and cloned another phosphate glycoprotein, which was overexpressed in the doxorubicin resistant cell strain H69AR. People named the protein MRP (also called multidrug resistance associated protein). The ATP binding protein superfamily (ATP-binding cassette superfamily, ABCs), a member of the.ATP binding protein superfamily, is the largest family of transmembrane proteins, which are divided into seven subtypes from A to G according to sequence and homology.
The members of the ABCC family are mainly composed of about 200 amino acids. This unique amino acid transmembrane structure distinguishes ABCC family members from other ABC members..ABCC4 is a member of the ABC transshipment C subfamily,.ABC transporters are mainly transmembrane proteins, and can mediate ATP- dependent cells through other molecular substances through the cell membrane.ABCC4 The gene is located at the 13q32.1 site of the human chromosome. The protein expressed by the ABCC4 gene is the shortest member of the ABCC/MRP family, and its role is to mediate the outflow of a variety of exogenous and endogenous molecules.
The experimental study of ABCC4 gene knockout mice showed that ABCC4 had important physiological functions, especially in the liver, brain tissue, and kidney. In cases of cholestasis, bile was blocked through the liver. After ligation of the bile ducts in rats and mice, the ABCC4 up-regulated and a progressive home was also found in the patients. Human intrahepatic cholestasis.ABCC4 mediated glutathione dependent bile acid efflux, which is a protective reaction under specific conditions, is a protective reaction to prevent liver necrosis. In addition to removing metabolites, ABCC4 can pass out active drugs to resist the possible damage to important tissues by various external substances in.ABCC4 deficiency. The trapped mice showed a decrease in renal excretion by a variety of drugs (antitumor drugs topotecan, antiviral drugs A Duff Vee and Nuo Fuwei). Correspondingly, the elevated levels of.ABCC4 deficiency in plasma and some tissues showed that topotecan was able to penetrate the blood brain barrier and then infiltrate into the central nervous system and the brain. In addition, ABCC4 gene knockout mice have enhanced the absorption of antiviral drugs in bone marrow, thymus, spleen and intestines compared with wild type mice. In addition, experimental studies have shown that ABCC4 is highly expressed in medullary progenitor cells and protects these cells from sulfur purine induced by the active platoon system. Hematopoiesis.
For the prognosis of rectal cancer, TNM staging, cell differentiation, regional lymph nodes and the expression of tumor markers in the region are associated with the long-term prognosis of rectal cancer. However, the short-term effect of preoperative radiotherapy and the long-term prognosis of LARC patients have been proved so far. There are few biomarkers for the gold standard. A new model of adjuvant therapy is the combination of 5-FU based chemotherapy and other radiation therapy for patients with locally advanced rectal cancer. Therefore, the greatest advantage of using ABCC4 as a new biomarker in predicting the radiation resistance of LARC patients is In some studies, this relatively ideal molecular marker associated with both exposure and cytotoxic drugs has been reported in some studies, involving a variety of cancer cell types (including rectal cancer). In conclusion, these studies suggest that ABCC4 can play multiple functions and protect life as a transporter. Organs prevent the accumulation of biological metabolites and prevent the damage of chemotherapeutic drugs.
In this experiment, the cell proliferation test was first used to select the radiotherapy sensitive strain LOVO and the resistant strain RKO in five rectal cancer cell lines. Then the expression of ABCC4 in the sensitive LOVO cells and the resistant strain RKO cells was observed by immunohistochemical method. The results showed that the staining particles could be observed only in the cell membrane and cytoplasm, and the nuclei were in the nucleus. The results were consistent with the results observed by foreign researchers in other tumor cell lines (Zaman and his colleagues in lung cancer cells), and further experimental results were that the sensitive strain of LOVO cells, the high expression of ABCC4, was 17%, and radiation resistance was 17%. The high expression rate of RKO cells was 71%, and the sensitive strain cells were significantly lower than those of the resistant strain cells. The results showed that the high expression of ABCC4 was likely to be involved in the biological pathway of radiation resistance, and.Havie et al. In the previous research literature has indicated that ABCC4 is also found in T lymphocyte leukemia cells. The authors also pointed out that, after the failure of the radiotherapy, the drug resistance was still not disappearing even with the means of chemotherapy. Then, a more in-depth study was carried out for the total of 93 patients undergoing pre abdominal excision, and 28 patients received a combination of abdominal perineal resection. The circumferential margin of the circumferential margin was negative according to the postoperative pathological report. The median follow-up time was 34.5 months (SD=8.6). The.Cox risk analysis showed that the high expression of ABCC4 (P=.036), the p53 mutant (P=.047), the pathological response poor (P=.044) patients had poor prognosis, the 3 year total survival rate was low, and the ABCC4 high expression (P=). .027), p53 mutant (P=.019), the final pathological staging (P=.032) patients have poor prognosis, and the 3 year disease free survival rate is relatively low. The lentivirus transfection obtained the stable and down-regulation of the ABCC4 protein cell line.Real-time PCR and Western Blot results all confirmed that the gene expression in RKO-KD and HT29-KD cells continued to effectively inhibit the expression of the ABCC4 gene, for subsequent cell work. .Real-time PCR results showed that the expression of abcc4mRNA in HT29-KD group (0.20 + 0.02) was significantly less than that of HT29-NC group (0.99 + 0.02) and HT29-CON group (0.98 + 0.03) (P 0.05), and Western Blot experiment showed that the expression level of ABCC4 protein was in HT29-KD group (0.25 + 0.02). Significantly lower than group HT29-NC (0.98 + 0.02) and group HT29-CON (0.98 + 0.03) (P 0.05), but there was no significant difference between the negative control group HT29-NC and the blank control group HT29-CON (P0.05). By measuring the tumor diameter calculation method, we found that the tumor growth pattern of the RKO-KD nude mice after radiotherapy was compared with those in the radiotherapy group. The state showed a significant time dependence inhibition (P.05), the average tumor weight was measured at the end point, the tumor weight of the RKO-KD group was 2.01 grams (+ 0.18) significantly lighter than that of the RKO-NC group (3.97 g) and 4.01 grams (0.19) (P.05) in the RKO-CON group. In the blank control group, there was no significant difference between the size and weight of the tumor in the nude mice (P.05).4Gy dose. After the radiation, the intracellular cAMP content increased rapidly and showed a time dependent decrease.. the average cAMP concentration was 0.66 (+ 0.25)% in the RKO-KD group (+ 0.15)% and 0.46 (0.16)% in the RKO-CON group (P.05). The cell cycle distribution of the treated cells and the percentage of 43.54 (+ 3.07)% in RKO-KD group cells were obviously in the G0/G1 stage. Less than 65.73 (+ 3.78)% of cells in group RKO-NC and 66.52 (+ 3.64)% of cells in group RKO-CON (P.05), indicating that the cells lost the ability to repair DNA at the G1-S monitoring point in RKO-KD group, and were easily exposed to radiation damage. In the three blank controls of unirradiated groups, the experimental results showed that the distribution of cell cycle was analyzed and no system was found. Significant difference in the meaning of the study (P.05).
It is worth emphasizing that there is a great difference between the molecular mechanism of drug resistance and the radiation resistance. Cytotoxic drugs play a continuous, slow and systematic biochemical role, so for the key proteins in the apoptotic signaling pathway, these proteins have enough time to make changes and respond to drugs. It may well explain the potential mechanism of chemoresistance, however, as a result of exposure, the body directly provides an instantaneous high energy and directly to the inside of the cancer cells, and induces a massive damage to DNA through the production of reactive oxygen. The apoptotic signaling pathway may not be the most important determinant. Here, we have determined the downregulation of ABCC 4 expression, after radiation, can increase the accumulation of intracellular cAMP concentration, and the cell cycle G1-S phase checkpoint is obviously inadequate, which may explain the mechanism, helping to clarify the inhibition of shRNA expression of ABCC4 and induce the apoptosis of colorectal cancer cells.
In conclusion, the current experimental study shows that ABCC4 is a very important sensitive molecule in both in vivo and in vitro experiments, which is a very important sensitive molecule, and has a significant correlation with the poor prognosis of patients receiving preoperative radiotherapy for local invasive rectal cancer. These data support the following theory that ABCC4 can be considered as a one. A useful predictor or target is of guiding significance for the evaluation of preoperative radiotherapy for local invasive rectal cancer and the evaluation of the prognosis. It is suggested that a more in-depth study should be made in the future to clarify the ABCC4 screening before treatment.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R735.37;R730.55
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