瘦素信号通路对T淋巴瘤细胞葡萄糖代谢的影响及其临床意义

发布时间：2018-06-03 12:53

本文选题：T细胞淋巴瘤 + 瘦素　；参考：《山东大学》2016年博士论文

【摘要】：淋巴瘤是来源于淋巴组织的恶性肿瘤,根据肿瘤细胞来源主要分为B细胞淋巴瘤和T细胞淋巴瘤(T cell lymphoma, TCL)。近年来,其发病率和死亡率逐年增加。随着分子生物学研究的进展,特别是酪氨酸激酶抑制剂、CD20单抗等靶向治疗药物广泛应用于临床,B细胞来源的白血病、淋巴瘤的预后得到了明显的改善。而T细胞白血病／淋巴瘤的生物学行为、临床特点具有高度异质性,其发病机制尚未完全清楚,以CHOP、ESHAP为基础的常规化疗方案治疗反应不佳,缺少有效的分子生物学靶向治疗药物,难治、复发病例多见,预后极差。因此需要对T细胞淋巴瘤／白血病的发病的分子机制、增殖、代谢的特点进行深入研究,以期为T细胞恶性肿瘤的靶向治疗寻找新的方向。葡萄糖是维持机体内环境稳定的主要能源物质。肿瘤细胞的增殖速度和能量消耗均明显高于正常细胞。干扰葡萄糖摄取将会影响肿瘤细胞的增殖、代谢、侵袭能力,从而影响疾病进展,改善疾病预后。葡萄糖转运蛋白(glucose transporters,Gluts)是介导哺乳动物细胞糖转运的主要载体。主要包括三类：第一类：Glut1-lut4,主要转运葡萄糖；第二类：Glut5, Glut7, Glut9,lut11,主要转运果糖；第三类：Glut6, Glut8, Glut10, Glut 12和HMIT等,功能尚不明确。在胰腺癌、胃癌、卵巢癌、宫颈癌、肺癌、鼻咽癌等肿瘤中Glut1的表达均出现异常增多。在原发胃的淋巴瘤中(病理类型包括弥漫大B细胞淋巴瘤和粘膜相关淋巴组织淋巴瘤),肿瘤组织表达Glut1水平与PET/CT中肿瘤摄取18FDG的SUV值明显相关。瘦素(leptin)是一种由肥胖基因(OB)编码的多肽类激素,主要由脂肪组织分泌,由146个氨基酸构成,其结构与人粒细胞刺激因子以及白介素6相似。瘦素受体(ObR)属于I类受体家族,在人体内存在广泛的生物学效应。瘦素与其受体(ObR)结合,通过JAK/STAT、PI3K/Akt和MAPK信号通路参与摄食和能量消耗、细胞增殖及凋亡以及炎症反应等多种病理生理过程。近年来已有大量的研究证实瘦素在多种恶性肿瘤的发生、发展过程中起重要作用。在弥漫大B细胞淋巴瘤(diffuse large B cell lymophoma, DLBCL)中,ObR的表达明显高于反应性增生的淋巴结组织；并且瘦素通过激活P13K/AKT信号通路促进DLBCL细胞增殖、抑制凋亡。瘦素对机体葡萄糖代谢的影响主要是通过作用于中枢神经系统和周围组织两个水平实现的。在中枢神经系统,瘦素作用于下丘脑神经元,(如：葡萄糖兴奋性神经元和葡萄糖抑制性神经元)激活细胞内的P13K信号通路,进而调节葡萄糖代谢。在周围组织,瘦素主要作用于代谢相关细胞,如胰岛细胞、脂肪细胞、肌肉细胞等。此外,近年来瘦素对免疫细胞,特别是活化T细胞葡萄糖代谢的影响也逐渐引起了研究者的重视。但瘦素对恶性肿瘤细胞糖代谢的作用目前仅见报道于乳腺癌细胞：高糖能促进乳腺癌细胞表达ObR,激活瘦素信号通路,促进其对葡萄糖的生物利用。在T细胞淋巴瘤中,瘦素对TCL细胞葡萄糖代谢和细胞增殖的作用尚未有相关研究报道,而瘦素信号通路对TCL预后的意义还不清楚。为此,我们进行了以下两部分实验：1、研究不同浓度的瘦素干预下,T细胞淋巴瘤／白血病细胞Molt-3增殖能力和葡萄糖摄取能力的变化。经过瘦素干预后,通过提取细胞膜蛋白和Western blot法测定葡萄糖转运体Glut1向细胞膜转运情况的变化；通过RT-PCR和Western blot法检测Glut1在mRNA和蛋白水平表达情况的变化。2、收集T细胞淋巴瘤病例的临床资料,采用免疫组织化学法检测ObR和Glut1在T细胞淋巴瘤组织和反应性增生的淋巴组织中的表达情况。并对上述两个指标与临床特点、疾病预后的相关性进行分析。第一部分瘦素及其受体对T淋巴瘤细胞Molt-3葡萄糖摄取的影响及其机制的探讨目的：葡萄糖是维持细胞生长的主要能源物质。与正常细胞相比,肿瘤细胞具有增殖活跃,生长迅速,能量需求大的特点。葡萄糖代谢不仅参与肿瘤细胞增殖和凋亡的过程,还影响肿瘤预后。在急性淋巴细胞白血病中,葡萄糖代谢的抑制可以使肿瘤细胞对糖皮质激素的杀伤作用更加敏感。瘦素(leptin)是一种蛋白质类激素,由OB基因编码,其结构与生长激素、粒细胞刺激因子等相似。主要由脂肪细胞分泌,一直以来被认为是调节机体葡萄糖代谢的重要物质。在中枢神经系统,它通过与下丘脑部位相应受体的结合,参与机体对食物摄入、能量消耗、脂肪代谢等的调节作用,维持葡萄糖稳态。在周围组织中,瘦素-瘦素受体-葡萄糖转运蛋白通路能直接作用于组织细胞,为细胞摄取利用葡萄糖的重要通路。但目前人们对瘦素在肿瘤细胞糖代谢中的作用还知之甚少。本研究选择了人T细胞淋巴瘤细胞株Molt-3,研究瘦素及其受体对其葡萄糖代谢的影响,并探讨其作用方式。材料和方法：1.TCL细胞株Molt-3与重组人瘦素共培养2.CCK-8测定细胞增殖3. 葡萄糖检测试剂盒检测细胞内葡萄糖浓度4.提取RNA,进行实时定量PCR5.膜蛋白、总蛋白提取以及蛋白印迹分析6.ObR特异性siRNA质粒转染Molt-3细胞,测定转染后细胞内葡萄糖浓度的变化,Western blot法测定转染后ObR、Gluts蛋白水平的变化7.统计学分析结果：1.CCK-8结果显示：随着瘦素作用时间的延长和瘦素浓度的增加,Molt-3细胞活性逐渐增加。其差异均有统计学意义。2. 对细胞内葡萄糖浓度测定结果显示：在瘦素作用30mmin后,Molt-3细胞内葡萄糖浓度以浓度依赖的方式增加；在瘦素作用48h后,Molt-3细胞内葡萄糖浓度增加,其差异均具有统计学意义。3. Western blot结果显示：与对照组比较,100ng/ml瘦素与Molt-3细胞共孵育30min后细胞膜蛋白中Glut1的表达水平最高,与细胞内葡萄糖浓度的变化一致(P0.05)。4. RT-PCR与Western blot结果分别证实在瘦素作用48h后,Molt-3细胞中Glut1的mRNA和蛋白表达水平均上调,而ObR和Glut4表达无明显变化。5.ObR特异性siRNA质粒阻断瘦素信号通路后,ObR、Glut1蛋白表达水平下调,细胞内葡萄糖水平明显下降。结论：1.瘦素促进TCL细胞Molt-3细胞增殖。2.瘦素促进Molt-3细胞摄取葡萄糖。3.瘦素对Molt-3细胞糖代谢的影响可能是通过上调Glut1的表达以及促进Glut1募集固定于细胞膜而实现的。第二部分T细胞淋巴瘤中ObR和Glut1的表达情况及临床意义目的：T细胞淋巴瘤／白血病是一组来源于T淋巴细胞的恶性肿瘤,在非霍奇金淋巴瘤(non-Hodgkin lymphoma, NHL)中占10-15%,在亚洲国家的发病率高于西方国家。与B细胞来源的肿瘤相比,T细胞淋巴瘤／白血病病情进展快,对常规化疗的反应率不高,缺少靶向治疗药物,预后差,因此,研究T细胞恶性肿瘤的生物学特点,以期找寻新的治疗策略非常必要。瘦素参与肿瘤细胞的增殖和代谢过程,而其在T细胞淋巴瘤中的临床意义尚不清楚。本研究收集T细胞淋巴瘤患者的临床资料、病理标本,研究瘦素受体ObR和葡萄糖转运体Glut1在T细胞淋巴瘤组织中的表达情况及其临床意义。材料和方法：1.收集T细胞淋巴瘤患者临床资料。2.免疫组化的方法检测手术切除的T细胞淋巴瘤和反应性增生的淋巴组织中CbR、Glut1、Glut4的表达情况。3. Fisher确切概率法分析ObR与其他临床、病理指标的相关性。4.Cox回归法分析ObR与T细胞淋巴瘤的预后相关性。结果：1.共收集T细胞淋巴瘤病例36例。其中：外周T细胞淋巴瘤(非特指型)12例,NK/T细胞淋巴瘤8例,血管免疫母T细胞淋巴瘤3例,T免疫母细胞淋巴瘤3例,T淋巴母细胞淋巴瘤4例,间变性大细胞T细胞淋巴瘤6例。中位年龄62岁、中位生存期16.5个月,3年生存率14.0%。2.T细胞淋巴瘤组织中ObR和Glut1的表达率高于反应性增生的淋巴结组织(58.3% vs 22.2%, P=0.012),而Glut4在二者中的表达情况无统计学差异。3.ObR的表达情况与与Glut1表达存在相关性(P=0.007),与Glut4表达不相关(P=0.292),并且与患者的年龄、性别、分期、LDH水平、B症状、合并糖尿病等亦不相关。4.单因素分析显示：年龄大于60岁以及共同表达ObR、Glut1者预后不良,多因素分析提示：共同表达ObR、Glut1为影响T细胞淋巴瘤预后的独立危险因素(HR=3.420,95%CI 1.293-9.049, P=0.013)结论：1.ObR和Glut1在T细胞淋巴瘤组织中的表达高于反应性增生的淋巴结组织。2.在细胞淋巴瘤组织中ObR和Glut1的表达存在相关性。3.T细胞淋巴瘤预后差,在T细胞淋巴瘤中,共同表达ObR和Glut1为独立的不良预后因素。
[Abstract]:Lymphoma is a malignant tumor derived from lymphoid tissue, which is mainly divided into B cell lymphoma and T cell lymphoma (T cell lymphoma, TCL) based on the source of tumor cells. In recent years, its incidence and mortality have increased year by year. With the progress of molecular biology research, especially tyrosine kinase inhibitors, CD20 McAbs, and other targeted therapies. The prognosis of B cell leukemia and lymphoma has been significantly improved in clinical use. The biological behavior and clinical characteristics of T cell leukemia / lymphoma are highly heterogeneous, its pathogenesis is not completely clear, and the conventional chemotherapy based on CHOP and ESHAP is not effective and lacks effective molecular biological targets. It is necessary to study the molecular mechanism, proliferation and metabolism of T cell lymphoma / leukemia, so as to find a new direction for the targeting therapy of T cell malignant tumor. The proliferation rate and energy consumption are significantly higher than that of normal cells. Interference with glucose uptake will affect the proliferation, metabolism and invasiveness of tumor cells, which affect the progression of the disease and improve the prognosis of the disease. Glucose transporter (glucose transporters, Gluts) is the main carrier of sugar transport in mammalian cells. It mainly includes three types: First class: Glut1-lut4, mainly transporting glucose; second types: Glut5, Glut7, Glut9, lut11, mainly transporting fructose; the third types: Glut6, Glut8, Glut10, Glut 12 and HMIT, etc. the function is not yet clear. The Glut1 expression in pancreatic, gastric, ovarian, cervical, cervical, nasopharyngeal cancer and other tumors. The pathological types include diffuse large B cell lymphoma and mucosa associated lymphoid tissue lymphoma. The expression of Glut1 in tumor tissue is significantly related to the SUV value of 18FDG in PET/CT. Leptin (leptin) is a polypeptide hormone encoded by the obesity gene (OB), which is mainly secreted by adipose tissue and consists of 146 amino acids. It is similar to human granulocyte stimulating factor and interleukin 6. Leptin receptor (ObR) belongs to the I receptor family, and has extensive biological effects in human body. Leptin and its receptor (ObR) are combined with JAK/STAT, PI3K/Akt and MAPK signaling pathways to participate in feeding and energy consumption, cell proliferation and apoptosis, and inflammatory reactions. In recent years, a large number of studies have shown that leptin plays an important role in the development of a variety of malignant tumors. In the diffuse large B cell lymphoma (diffuse large B cell lymophoma, DLBCL), the expression of ObR is significantly higher than the reactive proliferation of lymph nodes; and leptin promotes DLBCL by activating the P13K/AKT signaling pathway. The effect of leptin on the metabolism of glucose is realized mainly by two levels of the central nervous system and the surrounding tissue. In the central nervous system, leptin acts on the hypothalamic neurons, such as glucose excitatory neurons and glucose suppressor neurons, to activate the P13K signaling pathway in the cells. In the surrounding tissue, leptin mainly acts on metabolic related cells, such as islet cells, adipocytes, and muscle cells. In addition, the effect of leptin on the metabolism of glucose in immune cells, especially in activated T cells, has gradually aroused the attention of researchers in recent years. It is only reported in breast cancer cells: high glucose can promote the expression of ObR in breast cancer cells, activate leptin signaling pathway and promote the biological utilization of glucose. In T cell lymphoma, the effect of leptin on glucose metabolism and cell proliferation in TCL cells has not been reported, but the significance of leptin signaling pathway to the prognosis of TCL is not yet significant. To this end, we conducted the following two experiments: 1, to study the changes in the proliferation and glucose uptake of T cell lymphoma / leukemia cells under the intervention of different concentrations of leptin. After leptin intervention, the transport of glucose transporter Glut1 into the cell membrane was determined by the extraction of cell membrane protein and Western blot method. The changes in the expression of Glut1 in mRNA and protein levels were detected by RT-PCR and Western blot. The clinical data of the cases of T cell lymphoma were collected. The expression of ObR and Glut1 in T cell lymphoma and reactive proliferation of lymphoid tissues was detected by immunohistochemistry. The above two indexes and clinical manifestations were observed. Characteristics, analysis of the correlation between the prognosis of the disease. Part 1 the effect of leptin and its receptor on the uptake of Molt-3 glucose in T lymphoma cells and its mechanism: glucose is the main source of energy for the maintenance of cell growth. Compared with normal cells, the tumor cells have the characteristics of active proliferation, rapid growth and large energy demand. Glucose metabolism not only participates in the process of tumor cell proliferation and apoptosis, but also affects the prognosis of the tumor. In acute lymphoblastic leukemia, the inhibition of glucose metabolism can make the tumor cells more sensitive to the killing of glucocorticoids. Leptin is a kind of protein irritable, encoded by the OB gene, and its structure and growth hormone are fine. It is believed to be an important substance that regulates the metabolism of glucose in the body. In the central nervous system, it participates in the regulation of food intake, energy consumption, fat metabolism and so on by combining with the corresponding receptors in the hypothalamus to maintain the glucose homeostasis. In the fabric, leptin leptin receptor glucosglucose transporter pathway can directly act on tissue cells, and it is an important pathway for the uptake of glucose in cells. However, little is known about the role of leptin in the glucose metabolism of tumor cells. This study selected human T cell lymphoma cell line Molt-3 and studied leptin and its receptor to its glucose. The effects of metabolism and methods of action. Materials and methods: 1.TCL cell line Molt-3 and recombinant human leptin co culture 2.CCK-8 determination of cell proliferation 3. glucose detection kit to detect intracellular glucose concentration 4. extract RNA, real-time quantitative PCR5. membrane protein, total protein extraction and Western blot analysis of 6.ObR specific siRNA plasmid Molt-3 cells were transfected to determine the changes of glucose concentration in the cells after transfection. Western blot assay was used to determine ObR after transfection. The changes of Gluts protein level were 7. statistical analysis results: 1.CCK-8 results showed that the activity of Molt-3 cells increased gradually with the prolongation of the action time of leptin and the increase of leptin concentration. The difference was statistically significant.2. The results of intracellular glucose concentration measurement showed that after leptin action 30mmin, the glucose concentration in Molt-3 cells increased in a concentration dependent manner, and the glucose concentration in Molt-3 cells increased after leptin action 48h, and the difference was statistically significant.3. Western blot results showed that 100ng/ml leptin and Molt were compared with the control group. The expression level of Glut1 in the membrane protein of -3 cells was the highest, which was the same as that of the intracellular glucose concentration (P0.05). (P0.05).4. RT-PCR and Western blot results showed that the Glut1 mRNA and protein expression level in Molt-3 cells increased after the action of leptin, respectively. After the plasmid blocked the leptin signaling pathway, the expression level of ObR, Glut1 protein was down, and the glucose level in cells decreased significantly. Conclusion: 1. leptin promotes the proliferation of.2. leptin in Molt-3 cells of TCL cells and promotes the uptake of glucose.3. leptin by Molt-3 cells to the glucose metabolism of Molt-3 cells by up regulating the expression of Glut1 and promoting Glut1 recruitment. The expression of ObR and Glut1 in second part of T cell lymphoma and its clinical significance: T cell lymphoma / leukemia is a group of malignant tumors derived from T lymphocyte, which accounts for 10-15% in non Hodgkin lymphoma (non-Hodgkin lymphoma, NHL), and the incidence in Asian countries is higher than that in western countries. Compared with the tumor derived from B cells, T cell lymphoma / leukemia progress rapidly, the response rate of conventional chemotherapy is not high, the target therapy drug is short, the prognosis is poor. Therefore, it is necessary to study the biological characteristics of T cell malignant tumor in order to find new treatment strategies. Leptin is involved in the proliferation and metabolic process of tumor cells, and it is in T. The clinical significance of cell lymphoma is still unclear. This study collects the clinical data of T cell lymphoma patients, pathological specimens, and studies the expression and clinical significance of leptin receptor ObR and glucose transporter Glut1 in T cell lymphoma tissue. Materials and methods: 1. the immunohistochemical staining of the clinical data of T cell lymphoma patients was collected by.2. Methods to detect the expression of CbR, Glut1, Glut4 in surgical excised T cell lymphoma and reactive proliferative lymphoid tissue,.3. Fisher accurate probability analysis of ObR and other clinical and pathological indexes, correlation.4.Cox regression analysis of the prognostic correlation between ObR and T cell lymphoma. 36 cases of T cell lymphoma were collected in 1. cases. 12 cases of peripheral T cell lymphoma (non special type), 8 cases of NK/T cell lymphoma, 3 cases of angioimmuno T cell lymphoma, 3 cases of T immunblastial lymphoma, 4 cases of T lymphoblastic lymphoma and 6 cases of T cell lymphoma of anaplastic large cells. The median age is 62, the median survival time is 16.5 months, and the 3 year survival rate of 14.0%.2.T cell lymphoma tissue is ObR. The expression rate of and Glut1 was higher than that of reactive hyperplasia (58.3% vs 22.2%, P=0.012), and there was no statistical difference in the expression of Glut4 in the two. The expression of.3.ObR was correlated with the expression of Glut1 (P=0.007), not associated with the expression of Glut4 (P=0.292), and was associated with the patient's age, sex, stage, LDH level, B symptoms. Diabetes and other unrelated.4. analysis showed that age is more than 60 years old and co expression of ObR and Glut1 has poor prognosis. Multivariate analysis suggests that common expression of ObR, Glut1 as an independent risk factor for the prognosis of T cell lymphoma (HR=3.420,95%CI 1.293-9.049, P=0.013): 1.ObR and Glut1 in T cell lymphoma tissue The expression of ObR and Glut1 in lymphoid tissue, which is higher than reactive hyperplasia, has a poor prognosis for the expression of.3.T cell lymphoma. In T cell lymphoma, the common expression of ObR and Glut1 is an independent prognostic factor in T cell lymphoma.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.1

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