ART1对2型糖尿病高NE状态大肠癌生长影响及机制研究
发布时间：2018-07-26 12:23
【摘要】：研究背景及目的:大肠癌是最常见的恶性肿瘤之一,近20年来,在我国发病率呈迅速增长的趋势,其死亡率已上升至恶性肿瘤的第三位。2型糖尿病的发病率,在世界范围内也在不断增加,预计2030年将上升至4.4%,约4.39亿人次。流行病学和临床研究均显示2型糖尿病和大肠癌之间有着共同的危险因素,且相互促进。2型糖尿病人群中,大肠癌的发病及死亡风险增加,且预后差。现有研究显示,2型糖尿病时高胰岛素、活性氧增加等与大肠癌发生发展有关,同时,2型糖尿病时去甲肾上腺素水平增高也是大肠癌发生风险增高的重要诱因。精氨酸特异性单ADP核糖基转移酶1(ART1)是一种重要的单ADP核糖基转移酶,被认为与多种细胞生物学行为有着密切的关系。课题组前期研究显示,ART1的表达变化,可以影响大肠癌细胞的增殖、侵袭转移、分化、微血管形成、自噬及凋亡,参与大肠癌的发生及发展;ART1与胰岛素信号通路肝糖原调节的负性调节子磷酸化GSK-3β蛋白表达有关,提示ART1可能在糖代谢性疾病中发挥作用。但ART1对2型糖尿病高NE状态下大肠癌生长影响及机制研究,尚未见相关报道。本课题旨在前期研究的基础上,从三方面入手,首先探讨2型糖尿病合并大肠癌患者癌组织ART1的表达变化,其次建立balb/C2型糖尿病小鼠模型,采用课题组前期制备的不同ART1表达水平的CT26细胞,建立小鼠移植瘤模型,此外,以去甲肾上腺素诱导不同ART1表达水平的CT26细胞,从在体及离体两方面,探讨ART1对2型糖尿病高NE状态大肠癌生长的影响及其可能的分子机制。为ART1作为治疗2型糖尿病合并大肠癌的候选靶点提供实验依据。方法:本研究分为三部分进行。1.2型糖尿病合并大肠癌癌组织ART1表达变化采用免疫组化方法,检测大肠癌组织ART1表达。比较大肠癌合并2型糖尿病(CRCD)组(n=37例)与大肠癌未合并糖尿病(CRCO)组(n=23例)ART1表达强度的变化,并分析ART1表达强度与糖尿病的关系。2.ART1对2型糖尿病高NE状态小鼠大肠癌生长的影响(1)离体实验选用四组CT26细胞:ART1高表达组(GFP-ART1组),空载体组(GFP-Vector组),ART1沉默组(GFP-Sh ART1组),未转染组(Un-transfection组)。1)CCK8法,评估ART1对不同NE浓度诱导CT26细胞增殖活性的影响及ART1对NE不同时间诱导CT26细胞增殖活性影响。2)流式细胞术评估ART1对NE诱导各组CT26细胞周期的影响。(2)在体实验1)2型糖尿病小鼠模型的建立采用高脂饲料喂养及腹腔注射1%链脲菌素(STZ)的方法建立2型糖尿病Balb/c小鼠模型。对照组Balb/c小鼠腹腔注射等体积生理盐水。尾静脉采血检测小鼠血糖,以确定模型的建立,同时检测去甲肾上腺素及胰岛素水平。2)不同ART1水平2型糖尿病大肠癌移植瘤体积及重量和小鼠荷瘤生存时间检测以糖尿病Balb/c小鼠为实验组,非糖尿病Balb/c小鼠为对照组,小鼠右侧腋窝皮下分别接种四组CT26细胞,即ART1高表达CT26细胞(GFP-ART1组)、ART1沉默CT26细胞(GFP-sh ART1组)、未转染CT26细胞(Un-transfection组)和空载体转染CT26细胞(GFP-Vector组),建立Balb/c小鼠大肠癌移植瘤模型。观测各组小鼠体重变化、移植瘤体积及重量;观测各组小鼠荷瘤生存时间。3)ART1高表达对2型糖尿病小鼠不同NE水平大肠癌移植瘤生长的影响采用左肾交感神经离断术,建立糖尿病血循环低NE水平Balb/c小鼠模型。尾静脉采血检测小鼠去甲肾上腺素(NE)以确定模型的建立,同时检测血糖及胰岛素水平。在小鼠右侧腋窝皮下接种ART1高表达CT26细胞。以手术组(LRD组)为实验组,未手术组(GFP-ART1组)和假手术组(LSO组)为对照组,观测各组小鼠移植瘤体积及重量。3、ART1对2型糖尿病高NE状态小鼠大肠癌生长影响的机制(1)离体实验ART1对高NE诱导的小鼠大肠癌CT26细胞p-AKT、AKT、m TOR、STAT3、Cyclin D1及c-myc蛋白表达影响选用四组CT26细胞:ART1高表达组(GFP-ART1组),空载体组(GFP-Vector组),ART1沉默组(GFP-Sh ART1组),未转染组(Un-transfection组)。以终浓度为1μmmol/l NE诱导各组CT26细胞。采用western blot检测各组细胞ART1、p-AKT、m TOR、STAT3、Cyclin D1、c-myc蛋白表达变化。(2)在体实验1)2型糖尿病高NE状态小鼠大肠癌移植瘤ART1、P-AKT、m TOR及STAT3表达变化采用Western blot方法,以非糖尿病小鼠移植瘤为对照组,检测糖尿病小鼠移植瘤组织ART1、p-AKT、AKT、m TOR、STAT3的蛋白表达变2)ART1高表达对2型糖尿病不同NE水平小鼠大肠癌移植瘤组织p-AKT、AKT、m TOR、STAT3蛋白表达影响采用western blot方法,以未手术组(GFP-ART1组)和假手术组(LSO组)为对照组,检测手术组(LRD组)移植瘤组织ART1、p-AKT、AKT、m TOR、STAT3蛋白表达变化。结果:1、2型糖尿病合并大肠癌癌组织ART1表达变化(1)免疫组化检测人大肠癌组织中ART1的表达ART1着色部位主要位于肿瘤细胞胞质及胞膜。CRCD组ART1的表达强度明显高于CRCO组,且有统计学差异(p0.05)。即使对年龄、性别、肿瘤部位、浸润深度、等因素进行较正后,2型糖尿病仍是ART1表达强度的独立相关因素。2、ART1对2型糖尿病高NE状态小鼠大肠癌生长的影响(1)离体实验1)ART1对不同NE浓度诱导CT26细胞增殖活性影响CCK8法检测各组细胞的增殖活性。结果显示,随着NE浓度的增加,GFP-ART1、Un-transfection、GFP-Vector组细胞的增殖活性增加,但GFP-Sh ART1组细胞增殖活性无明显变化。GFP-ART1组,相较其余三组,细胞增殖活性最高,GFP-Sh ART1组相较其余三组增殖活性最低(p0.05),Un-transfection、GFP-Vector组细胞间则无统计学差异(P0.05)。2)ART1对NE不同时间诱导CT26细胞增殖活性影响CCK8结果显示,以终浓度为1μmmol/l NE分别诱导四组CT26细胞,在各诱导处理时间,GFP-ART1组,相较其余三组,细胞增殖活性最高,GFP-Sh ART1组相较其余三组增殖活性最低(p0.05)。且随诱导时间的延长,GFP-ART1,Un-transfection和GFP-Vector组细胞增殖活性增加(p0.05),但GFP-Sh ART1组细胞增殖活性似乎没有明显变化(P0.05)。Un-transfection、GFP-Vector组细胞间则无统计学差异(P0.05)。3)ART1对NE诱导CT26细胞周期影响NE诱导CT26细胞后,未经NE诱导的各对照组细胞:(1)ART1高表达组,未转染组及空载体组可见G1期比例明显减少,S期比例明显增加,细胞增殖指数PI明显增加(p0.05);(2)GFP-sh ART1组,即ART1沉默组,各时期细胞分布及PI无明显改变(P0.05)。无论各组CT26细胞有无NE诱导,GFP-ART1组,G1期比例最少,S期比例最多,PI增加(p0.05)。Un-transfection、GFP-Vector组细胞间则无统计学差异(P0.05)。(2)在体实验1)2型糖尿病小鼠模型的建立高脂饮食饲养及腹腔注射1%STZ后,小鼠体型肥胖,体重明显重于对照组(P0.05);小鼠血糖、胰岛素水平明显高于对照组(P0.01),小鼠NE也明显高于对照组(P0.01),表明糖尿病动物模型建立成功。2)不同ART1水平2型糖尿病大肠癌移植瘤体积及重量和小鼠荷瘤生存时间变化相同ART1表达水平CT26细胞接种后,与CRCO组比较,CRCD组小鼠移植瘤体积更大,重量更重,荷瘤生存时间更短,差异均具有统计学意义(p0.05)。CRCD组和CRCO组中,接种GFP-ART1 CT26细胞组移植瘤体积均为最大,重量最重,荷瘤生存时间最短,差异均具有统计学意义(p0.05);接种GFP-sh ART1 CT26细胞,移植瘤体积均最小,重量最轻,荷瘤生存时间最长,差异均具有统计学意义(P0.05);未转染组与空载组则均无明显差异(P0.05)。3)ART1高表达对2型糖尿病小鼠不同NE水平大肠癌移植瘤生长的影响与未手术组(GFP-ART1组)和假手术组(LSO组)比较,LRD组小鼠,NE水平,胰岛素水平及血糖水平明显下降(P0.01);移植瘤重量最轻,体积最小,差异具有统计学意义(P0.01)。GFP-ART1组和LSO组间则无统计学意义差异(P0.05)。3、ART1对2型糖尿病高NE状态小鼠大肠癌生长影响的机制(1)离体实验ART1对高NE诱导的小鼠大肠癌CT26细胞p-AKT、m TOR、STAT3、Cyclin D1及c-myc蛋白表达影响1)GFP-ART1组与un-transfection组相比较,ART1、p-AKT、m TOR、STAT3、Cyclin D1、c-myc蛋白表达明显增加(p0.05)。2)GFP-sh ART1组与GFP-Vector组相比较,ART1、p-AKT、m TOR、STAT3、Cyclin D1、c-myc蛋白表达明显减少(p0.05)。3)un-transfection组与GFP-Vector组,各蛋白表达无明显差异(P0.05)。(2)在体实验1)2型糖尿病高NE状态小鼠移植瘤ART1、P-AKT、m TOR及STAT3表达变化Western blot显示:与CRCO组比较,CRCD移植瘤组织,ART1、m TOR、STAT3、P-AKT蛋白表达明显增加,(p0.01),AKT的表达,两组间无明显差异(P0.05)。2)ART1高表达对2型糖尿病不同NE水平小鼠大肠癌移植瘤组织p-AKT、AKT、m TOR、STAT3蛋白表达影响Western blot显示:未手术组(GFP-ART1组)、假手术组(LSO组)、LRD组,瘤体组织,ART1、AKT蛋白表达三组间无明显差异(P0.05),GFP-ART1组及LSO组P-AKT,m TOR、STAT3蛋白表达明显高于LSD组及GFP-ART1组(p0.05)。结论:1、大肠癌合并2型糖尿病时,癌组织ART1表达明显增强,且伴淋巴转移组表达强度高于未转移组。ART1表达强度与性别、分化程度及肿瘤部位无关;但对年龄、性别、肿瘤部位、侵润深度、等因素进行较正后,糖尿病仍是ART1表达强度的独立相关因素。提示ART1可能在2型糖尿病合并大肠癌时,对肿瘤发生发展发挥了一定作用。2、2型糖尿病小鼠大肠癌移植瘤生长更快,体积更大,重量更重,生存时间更短,且在ART1高表达时,上述现象更明显;沉默ART1对NE诱导的大肠癌CT26细胞增殖活性具有抑制作用,细胞主要阻滞在G1期。提示ART1对2型糖尿病小鼠大肠癌移植瘤生长具有促进作用。3、糖尿病高NE状态下,ART1可通过对AKT调节,进而影响AKT/m TOR/STAT3信号通路,干预其下游基因cyclin D1、c-myc表达,影响大肠癌细胞的增殖。ART1有望成为糖尿病伴高NE水平时合并大肠癌治疗候选靶点。
[Abstract]:Background and purpose: colorectal cancer is one of the most common malignant tumors. In the past 20 years, the incidence of the disease is increasing rapidly in China. The incidence of the third.2 type diabetes which has risen to malignant tumor is increasing in the world. It is expected to rise to 4.4%, about 439 million people in 2030. The bed studies have shown that there is a common risk factor between type 2 diabetes and colorectal cancer and that among people with type.2 diabetes, the risk of colorectal cancer and death is increased and the prognosis is poor. The present study shows that high insulin and active oxygen increase in type 2 diabetes are associated with the development of colorectal cancer, and the normethylene kidney in type 2 diabetes mellitus The increase of the level of the gland is also an important cause of the increased risk of colorectal cancer. Arginine specific single ADP ribonucleotransferase 1 (ART1) is an important single ADP ribonucleotransferase, which is considered to be closely related to a variety of cellular biological behavior. Proliferation, invasion, metastasis, differentiation, microvascular formation, autophagy and apoptosis are involved in the development and development of colorectal cancer; ART1 is associated with the expression of GSK-3 beta protein expression of the negative regulator of liver glycogen regulated by insulin signaling pathway, suggesting that ART1 may play a role in glycometabolic diseases. But ART1 is the growth of colorectal cancer in the high NE state of type 2 diabetes. The study of influence and mechanism has not yet been reported. On the basis of early study, this topic begins with three aspects, first to discuss the changes in the expression of ART1 in the cancer tissue of patients with type 2 diabetes and colorectal cancer, secondly to establish a model of balb/C2 type diabetes in mice, and to establish a small CT26 cell with different ART1 expression levels prepared by the project group. In addition, CT26 cells with different ART1 expression levels were induced by norepinephrine, and the effect of ART1 on the growth of high NE colorectal cancer in type 2 diabetes mellitus and its possible molecular mechanism were investigated from two aspects in vivo and in vitro. This provides an experimental basis for the treatment of ART1 as a candidate target for the treatment of type 2 diabetes with colorectal cancer. The study was divided into three parts, which were divided into three parts. The expression of ART1 expression in the tissues of type.1.2 diabetes and colorectal cancer by immunohistochemical method was used to detect the expression of ART1 in colorectal cancer tissue. The changes in the expression of ART1 in the group of large intestine cancer with type 2 diabetes mellitus (CRCD) and colorectal cancer (CRCO) group (n=23 cases) were compared, and the expression intensity and sugar of ART1 were analyzed. The effect of.2.ART1 on the growth of colorectal cancer in type 2 diabetic mice with high NE status (1) in vitro, four groups of CT26 cells were selected: ART1 high expression group (GFP-ART1 group), GFP-Vector group (group GFP-Vector), ART1 silence group (GFP-Sh ART1 group), non transfection group (Un-transfection group).1) CCK8 method Effect of activity and effect of ART1 on the proliferation of CT26 cells induced by NE at different time.2) flow cytometry was used to evaluate the effect of ART1 on CT26 cell cycle induced by NE. (2) in body experiment 1) the establishment of a model of type 2 diabetic mice by feeding high fat feed and intraperitoneal injection of 1% streptozotocin (STZ) to establish a model of type 2 diabetic Balb/c mice The Balb/c mice in the control group were intraperitoneally injected with equal volume of normal saline. The blood glucose was detected in the tail vein to determine the model, at the same time, the volume and weight of the transplanted tumor and the tumor survival time of the mice with different ART1 levels of type 2 diabetic colorectal cancer were detected at the same level of norepinephrine and insulin level.2). The experimental group of diabetic Balb/c mice was tested. Non diabetic Balb/c mice as control group, four groups of CT26 cells were inoculated subcutaneously in the right armpit of mice, namely, ART1 high expression CT26 cells (group GFP-ART1), ART1 silent CT26 cells (GFP-sh ART1 group), non transfected CT26 cells (Un-transfection group) and empty carrier transfected CT26 cells (Group). The mice model of colorectal carcinoma transplantation tumor was established. The body weight change, the volume and weight of the transplanted tumor, the tumor bearing time of the mice in each group.3) ART1 high expression on the growth of different NE levels of colorectal cancer in type 2 diabetic mice, the left renal sympathetic disconnection was used to establish a Balb/c mouse model with low blood circulation NE level in diabetic mice. Adenin (NE) was used to determine the establishment of the model, and at the same time the blood glucose and insulin levels were detected. ART1 high expression CT26 cells were subcutaneously inoculated in the right armpit of mice. The operation group (group LRD) was the experimental group, the non operation group (group GFP-ART1) and the sham operation group (group LSO) were used as the control group. The volume and weight of the transplanted tumor in each group were observed and.3, and ART1 was highly NE state of type 2 diabetes. The mechanism of the effect of the growth of colorectal cancer in mice (1) in vitro experiment, the effects of ART1 on the expression of p-AKT, AKT, m TOR, STAT3, Cyclin D1 and c-myc protein in CT26 cells induced by high NE in mice were selected as the expression of CT26 cells: the high expression group, the empty body group, the silent group and the untransfected group. CT26 cells were induced with final concentration of 1 mmol/l NE. Western blot was used to detect ART1, p-AKT, m TOR, STAT3, Cyclin D1, and protein expression changes. (2) in body experiment 1) colorectal cancer transplantation tumor of type 2 diabetic mice For the control group, the expression of ART1, p-AKT, AKT, m TOR, STAT3 was 2) in the transplanted tumor tissues of diabetic mice. The high expression of ART1 on the colorectal cancer transplanted tumor tissues of the mice with type 2 diabetes, p-AKT, AKT, m TOR, was used as the control group, and the control group and the sham operation group as the control group. The changes of the expression of ART1, p-AKT, AKT, m TOR, STAT3 protein in the transplanted tumor tissue of the operation group (group LRD). Results: the expression of ART1 expression in the tissues of 1,2 type diabetes combined with colorectal cancer (1) the expression of ART1 stained part of ART1 in human colorectal cancer tissue was mainly located in the cytoplasm and the cell membrane.CRCD group. And there was a statistical difference (P0.05). Even for age, sex, tumor site, depth of infiltration, and other factors, type 2 diabetes was still an independent factor in ART1 expression.2, ART1 had an effect on the growth of colorectal cancer in the high NE state of type 2 diabetes in mice (1) 1 in vitro.) ART1 had an effect on the proliferation of CT26 cells induced by different NE concentrations. The results showed that the proliferation activity of GFP-ART1, Un-transfection, GFP-Vector group increased with the increase of NE concentration, but the proliferation activity of GFP-Sh ART1 group had no obvious changes in.GFP-ART1 group, compared with the other three groups, the cell proliferation activity was the highest, GFP-Sh ART1 group compared with the other three groups, the proliferation activity was the lowest (P). 0.05), there was no statistical difference between Un-transfection and GFP-Vector groups (P0.05).2) ART1 on the proliferation activity of CT26 cells induced by NE at different times, and CCK8 results showed that four groups of CT26 cells were induced at the final concentration of 1 u mmol/l NE respectively. The cell proliferation activity was the highest in the induction time, GFP-ART1 group and the other three groups. The proliferation activity of the other three groups was the lowest (P0.05). And with the prolongation of the induction time, the proliferation activity of GFP-ART1, Un-transfection and GFP-Vector increased (P0.05), but the proliferation activity of GFP-Sh ART1 group did not seem to be significantly changed (P0.05).Un-transfection, and there was no statistical difference between the GFP-Vector groups (P0.05).3) Cell cycle influence NE induced CT26 cells, without NE induced cells: (1) ART1 high expression group, untransfected group and empty body group can obviously decrease the proportion of G1 stage, the proportion of S phase obviously increase, cell proliferation index PI obviously increase (P0.05); (2) GFP-sh ART1 group, that is, ART1 silencing group, cell distribution and PI have no obvious changes at all times. No matter whether the CT26 cells were induced by NE, the proportion of GFP-ART1, the G1 stage was the least, the S stage was the most, PI increased (P0.05).Un-transfection, and there was no statistical difference between the GFP-Vector group (P0.05). (2) in the body experiment 1) the establishment of high fat diet and celiac injection 1%STZ in the model of type 2 diabetes mice was obese and weight was significantly heavier than that in the model of type 2 diabetic mice. Control group (P0.05), mice blood glucose, insulin level was significantly higher than the control group (P0.01), mice NE was also significantly higher than the control group (P0.01), indicating the establishment of diabetic animal model successful.2) different ART1 Level 2 diabetes large intestine cancer transplantation tumor volume and weight and mice bearing tumor survival time changes the same ART1 expression level CT26 cell inoculation, and CRC Compared with group O, the tumor size of the mice in group CRCD was larger, weight was heavier, and the survival time of the tumor was shorter. The difference was statistically significant (P0.05) in group.CRCD and CRCO, the size of the transplanted tumor in the GFP-ART1 CT26 cell group was the largest, the weight was the heaviest, the survival time of the tumor bearing was the shortest, the difference was statistically significant (P0.05); GFP-sh ART1 CT26 was inoculated. Cells, the size of the transplanted tumor was the smallest, the weight was the lightest, the life time of the tumor bearing was the longest, and the difference was statistically significant (P0.05), and there was no significant difference between the untransfected group and the empty group (P0.05).3). The effect of high expression of ART1 on the growth of colorectal cancer in different NE levels of type 2 diabetic mice was compared with that of the unoperated group (group GFP-ART1) and the sham operation group (group LSO). Compared with group LRD, NE level, insulin level and blood glucose level decreased significantly (P0.01), the weight of the transplanted tumor was the lightest, the volume was the smallest, the difference was statistically significant (P0.01), there was no statistical difference between.GFP-ART1 and LSO groups (P0.05).3, ART1 on the growth of colorectal cancer in mice with high NE status of type 2 diabetes mellitus (1) ART1 on high N E induced mouse colorectal cancer CT26 cells p-AKT, m TOR, STAT3, Cyclin D1 and c-myc protein expression influence 1) GFP-ART1 group compared with un-transfection group. There was no significant difference in the expression of protein in group un-transfection and GFP-Vector (P0.05). (2) (2) in body experiment 1) the expression of ART1, P-AKT, m TOR and STAT3 expressed in the high NE state of type 2 diabetes mellitus mice. There was no significant difference between the two groups (P0.05).2) ART1 high expression of p-AKT, AKT, m TOR, and STAT3 protein expression in different NE mice of type 2 diabetes mellitus with the expression of Western blot: the non operative group (GFP-ART1 group), the sham operation group, the tumor body tissue, the tumor tissue, the protein expression of three groups had no significant difference. The expression of P-AKT, m TOR and STAT3 protein in group RT1 and LSO group was significantly higher than that in group LSD and GFP-ART1 group (P0.05). Conclusion: 1, the expression of ART1 in colorectal carcinoma with type 2 diabetes is obviously enhanced, and the expression intensity of the lymph node group is higher than that in the non metastatic group, and the intensity of.ART1 expression is not related to the sex, the degree of differentiation and the tumor site, but the age, sex, and tumor part are not related. Diabetes is still an independent factor in the expression intensity of ART1. It suggests that ART1 may play a role in the development of cancer in type 2 diabetes with colorectal cancer. It may play a role in the development of cancer in type 2 diabetic mice. The growth of the tumor is faster, the volume is greater, the weight is heavier, the survival time is shorter, and the ART1 is higher. At the time of expression, the above phenomenon is more obvious. Silence ART1 can inhibit the proliferation activity of NE induced colorectal CT26 cells, and the cells are mainly blocked at G1 stage. It suggests that ART1 has a promoting effect on the growth of colorectal carcinoma in type 2 diabetic mice, and ART1 can be regulated by AKT under the high NE state of diabetes, and then affects AKT/m TOR/STAT3 signaling pathway. Interfering with the downstream gene cyclin D1, c-myc expression and affecting the proliferation of colorectal cancer cells is expected to be a candidate target for the treatment of colorectal cancer with high NE levels in diabetes.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.34;R587.1
本文编号：2146000
[Abstract]:Background and purpose: colorectal cancer is one of the most common malignant tumors. In the past 20 years, the incidence of the disease is increasing rapidly in China. The incidence of the third.2 type diabetes which has risen to malignant tumor is increasing in the world. It is expected to rise to 4.4%, about 439 million people in 2030. The bed studies have shown that there is a common risk factor between type 2 diabetes and colorectal cancer and that among people with type.2 diabetes, the risk of colorectal cancer and death is increased and the prognosis is poor. The present study shows that high insulin and active oxygen increase in type 2 diabetes are associated with the development of colorectal cancer, and the normethylene kidney in type 2 diabetes mellitus The increase of the level of the gland is also an important cause of the increased risk of colorectal cancer. Arginine specific single ADP ribonucleotransferase 1 (ART1) is an important single ADP ribonucleotransferase, which is considered to be closely related to a variety of cellular biological behavior. Proliferation, invasion, metastasis, differentiation, microvascular formation, autophagy and apoptosis are involved in the development and development of colorectal cancer; ART1 is associated with the expression of GSK-3 beta protein expression of the negative regulator of liver glycogen regulated by insulin signaling pathway, suggesting that ART1 may play a role in glycometabolic diseases. But ART1 is the growth of colorectal cancer in the high NE state of type 2 diabetes. The study of influence and mechanism has not yet been reported. On the basis of early study, this topic begins with three aspects, first to discuss the changes in the expression of ART1 in the cancer tissue of patients with type 2 diabetes and colorectal cancer, secondly to establish a model of balb/C2 type diabetes in mice, and to establish a small CT26 cell with different ART1 expression levels prepared by the project group. In addition, CT26 cells with different ART1 expression levels were induced by norepinephrine, and the effect of ART1 on the growth of high NE colorectal cancer in type 2 diabetes mellitus and its possible molecular mechanism were investigated from two aspects in vivo and in vitro. This provides an experimental basis for the treatment of ART1 as a candidate target for the treatment of type 2 diabetes with colorectal cancer. The study was divided into three parts, which were divided into three parts. The expression of ART1 expression in the tissues of type.1.2 diabetes and colorectal cancer by immunohistochemical method was used to detect the expression of ART1 in colorectal cancer tissue. The changes in the expression of ART1 in the group of large intestine cancer with type 2 diabetes mellitus (CRCD) and colorectal cancer (CRCO) group (n=23 cases) were compared, and the expression intensity and sugar of ART1 were analyzed. The effect of.2.ART1 on the growth of colorectal cancer in type 2 diabetic mice with high NE status (1) in vitro, four groups of CT26 cells were selected: ART1 high expression group (GFP-ART1 group), GFP-Vector group (group GFP-Vector), ART1 silence group (GFP-Sh ART1 group), non transfection group (Un-transfection group).1) CCK8 method Effect of activity and effect of ART1 on the proliferation of CT26 cells induced by NE at different time.2) flow cytometry was used to evaluate the effect of ART1 on CT26 cell cycle induced by NE. (2) in body experiment 1) the establishment of a model of type 2 diabetic mice by feeding high fat feed and intraperitoneal injection of 1% streptozotocin (STZ) to establish a model of type 2 diabetic Balb/c mice The Balb/c mice in the control group were intraperitoneally injected with equal volume of normal saline. The blood glucose was detected in the tail vein to determine the model, at the same time, the volume and weight of the transplanted tumor and the tumor survival time of the mice with different ART1 levels of type 2 diabetic colorectal cancer were detected at the same level of norepinephrine and insulin level.2). The experimental group of diabetic Balb/c mice was tested. Non diabetic Balb/c mice as control group, four groups of CT26 cells were inoculated subcutaneously in the right armpit of mice, namely, ART1 high expression CT26 cells (group GFP-ART1), ART1 silent CT26 cells (GFP-sh ART1 group), non transfected CT26 cells (Un-transfection group) and empty carrier transfected CT26 cells (Group). The mice model of colorectal carcinoma transplantation tumor was established. The body weight change, the volume and weight of the transplanted tumor, the tumor bearing time of the mice in each group.3) ART1 high expression on the growth of different NE levels of colorectal cancer in type 2 diabetic mice, the left renal sympathetic disconnection was used to establish a Balb/c mouse model with low blood circulation NE level in diabetic mice. Adenin (NE) was used to determine the establishment of the model, and at the same time the blood glucose and insulin levels were detected. ART1 high expression CT26 cells were subcutaneously inoculated in the right armpit of mice. The operation group (group LRD) was the experimental group, the non operation group (group GFP-ART1) and the sham operation group (group LSO) were used as the control group. The volume and weight of the transplanted tumor in each group were observed and.3, and ART1 was highly NE state of type 2 diabetes. The mechanism of the effect of the growth of colorectal cancer in mice (1) in vitro experiment, the effects of ART1 on the expression of p-AKT, AKT, m TOR, STAT3, Cyclin D1 and c-myc protein in CT26 cells induced by high NE in mice were selected as the expression of CT26 cells: the high expression group, the empty body group, the silent group and the untransfected group. CT26 cells were induced with final concentration of 1 mmol/l NE. Western blot was used to detect ART1, p-AKT, m TOR, STAT3, Cyclin D1, and protein expression changes. (2) in body experiment 1) colorectal cancer transplantation tumor of type 2 diabetic mice For the control group, the expression of ART1, p-AKT, AKT, m TOR, STAT3 was 2) in the transplanted tumor tissues of diabetic mice. The high expression of ART1 on the colorectal cancer transplanted tumor tissues of the mice with type 2 diabetes, p-AKT, AKT, m TOR, was used as the control group, and the control group and the sham operation group as the control group. The changes of the expression of ART1, p-AKT, AKT, m TOR, STAT3 protein in the transplanted tumor tissue of the operation group (group LRD). Results: the expression of ART1 expression in the tissues of 1,2 type diabetes combined with colorectal cancer (1) the expression of ART1 stained part of ART1 in human colorectal cancer tissue was mainly located in the cytoplasm and the cell membrane.CRCD group. And there was a statistical difference (P0.05). Even for age, sex, tumor site, depth of infiltration, and other factors, type 2 diabetes was still an independent factor in ART1 expression.2, ART1 had an effect on the growth of colorectal cancer in the high NE state of type 2 diabetes in mice (1) 1 in vitro.) ART1 had an effect on the proliferation of CT26 cells induced by different NE concentrations. The results showed that the proliferation activity of GFP-ART1, Un-transfection, GFP-Vector group increased with the increase of NE concentration, but the proliferation activity of GFP-Sh ART1 group had no obvious changes in.GFP-ART1 group, compared with the other three groups, the cell proliferation activity was the highest, GFP-Sh ART1 group compared with the other three groups, the proliferation activity was the lowest (P). 0.05), there was no statistical difference between Un-transfection and GFP-Vector groups (P0.05).2) ART1 on the proliferation activity of CT26 cells induced by NE at different times, and CCK8 results showed that four groups of CT26 cells were induced at the final concentration of 1 u mmol/l NE respectively. The cell proliferation activity was the highest in the induction time, GFP-ART1 group and the other three groups. The proliferation activity of the other three groups was the lowest (P0.05). And with the prolongation of the induction time, the proliferation activity of GFP-ART1, Un-transfection and GFP-Vector increased (P0.05), but the proliferation activity of GFP-Sh ART1 group did not seem to be significantly changed (P0.05).Un-transfection, and there was no statistical difference between the GFP-Vector groups (P0.05).3) Cell cycle influence NE induced CT26 cells, without NE induced cells: (1) ART1 high expression group, untransfected group and empty body group can obviously decrease the proportion of G1 stage, the proportion of S phase obviously increase, cell proliferation index PI obviously increase (P0.05); (2) GFP-sh ART1 group, that is, ART1 silencing group, cell distribution and PI have no obvious changes at all times. No matter whether the CT26 cells were induced by NE, the proportion of GFP-ART1, the G1 stage was the least, the S stage was the most, PI increased (P0.05).Un-transfection, and there was no statistical difference between the GFP-Vector group (P0.05). (2) in the body experiment 1) the establishment of high fat diet and celiac injection 1%STZ in the model of type 2 diabetes mice was obese and weight was significantly heavier than that in the model of type 2 diabetic mice. Control group (P0.05), mice blood glucose, insulin level was significantly higher than the control group (P0.01), mice NE was also significantly higher than the control group (P0.01), indicating the establishment of diabetic animal model successful.2) different ART1 Level 2 diabetes large intestine cancer transplantation tumor volume and weight and mice bearing tumor survival time changes the same ART1 expression level CT26 cell inoculation, and CRC Compared with group O, the tumor size of the mice in group CRCD was larger, weight was heavier, and the survival time of the tumor was shorter. The difference was statistically significant (P0.05) in group.CRCD and CRCO, the size of the transplanted tumor in the GFP-ART1 CT26 cell group was the largest, the weight was the heaviest, the survival time of the tumor bearing was the shortest, the difference was statistically significant (P0.05); GFP-sh ART1 CT26 was inoculated. Cells, the size of the transplanted tumor was the smallest, the weight was the lightest, the life time of the tumor bearing was the longest, and the difference was statistically significant (P0.05), and there was no significant difference between the untransfected group and the empty group (P0.05).3). The effect of high expression of ART1 on the growth of colorectal cancer in different NE levels of type 2 diabetic mice was compared with that of the unoperated group (group GFP-ART1) and the sham operation group (group LSO). Compared with group LRD, NE level, insulin level and blood glucose level decreased significantly (P0.01), the weight of the transplanted tumor was the lightest, the volume was the smallest, the difference was statistically significant (P0.01), there was no statistical difference between.GFP-ART1 and LSO groups (P0.05).3, ART1 on the growth of colorectal cancer in mice with high NE status of type 2 diabetes mellitus (1) ART1 on high N E induced mouse colorectal cancer CT26 cells p-AKT, m TOR, STAT3, Cyclin D1 and c-myc protein expression influence 1) GFP-ART1 group compared with un-transfection group. There was no significant difference in the expression of protein in group un-transfection and GFP-Vector (P0.05). (2) (2) in body experiment 1) the expression of ART1, P-AKT, m TOR and STAT3 expressed in the high NE state of type 2 diabetes mellitus mice. There was no significant difference between the two groups (P0.05).2) ART1 high expression of p-AKT, AKT, m TOR, and STAT3 protein expression in different NE mice of type 2 diabetes mellitus with the expression of Western blot: the non operative group (GFP-ART1 group), the sham operation group, the tumor body tissue, the tumor tissue, the protein expression of three groups had no significant difference. The expression of P-AKT, m TOR and STAT3 protein in group RT1 and LSO group was significantly higher than that in group LSD and GFP-ART1 group (P0.05). Conclusion: 1, the expression of ART1 in colorectal carcinoma with type 2 diabetes is obviously enhanced, and the expression intensity of the lymph node group is higher than that in the non metastatic group, and the intensity of.ART1 expression is not related to the sex, the degree of differentiation and the tumor site, but the age, sex, and tumor part are not related. Diabetes is still an independent factor in the expression intensity of ART1. It suggests that ART1 may play a role in the development of cancer in type 2 diabetes with colorectal cancer. It may play a role in the development of cancer in type 2 diabetic mice. The growth of the tumor is faster, the volume is greater, the weight is heavier, the survival time is shorter, and the ART1 is higher. At the time of expression, the above phenomenon is more obvious. Silence ART1 can inhibit the proliferation activity of NE induced colorectal CT26 cells, and the cells are mainly blocked at G1 stage. It suggests that ART1 has a promoting effect on the growth of colorectal carcinoma in type 2 diabetic mice, and ART1 can be regulated by AKT under the high NE state of diabetes, and then affects AKT/m TOR/STAT3 signaling pathway. Interfering with the downstream gene cyclin D1, c-myc expression and affecting the proliferation of colorectal cancer cells is expected to be a candidate target for the treatment of colorectal cancer with high NE levels in diabetes.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.34;R587.1
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