不同浓度的葡萄糖和胰岛素对Colon26肿瘤细胞分泌免疫抑制物质的影响及其分子机制的研究
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摘要
目的:肿瘤和糖尿病已成为当今严重危害人类健康和生命的重大疾病,它们的发病率和死亡率逐年升高。近年来,越来越多的临床和流行病学资料显示,糖尿病人易患结直肠癌。糖尿病患者与非糖尿病患者比较,患结直肠癌的风险明显增加,已有人提出糖尿病是结直肠癌患病的独立危险因素。糖尿病患者结直肠癌的发病率明显高于非糖尿病患者,糖尿病人群中结直肠癌的复发率和死亡率亦明显增加。糖尿病与结直肠癌的关系已经成为备受人们关注的热点;研究和揭示两者之间的病理生理关系及其分子机制,已成为该领域中迫切需要进行探索的重要课题。
肿瘤免疫学的研究已经揭示,肿瘤的发生、发展,不仅与机体的免疫功能密切相关,在某种意义上更与肿瘤细胞的免疫抑制作用密切相关。肿瘤细胞分泌免疫抑制物质抑制机体免疫功能,即肿瘤细胞的免疫抑制作用,是肿瘤细胞逃避机体免疫监视,使肿瘤得以发生和发展的重要机制,在促进机体肿瘤的发生、发展中具有极其重要的作用。肿瘤细胞通过其分泌的免疫抑制物质,不但有可能在远离肿瘤部位的机体其他部位发挥免疫抑制作用,更可在肿瘤局部形成深度免疫抑制区,即所谓“黑洞”区,以至于不仅使原本就在此区之中的原位免疫细胞功能严重受抑,而且还能使由此区之外进入其中的功能正常甚至已经活化的免疫细胞成为功能受抑的“沉默”细胞,从而使之失去有效清除肿瘤细胞的能力,显然这是在即使免疫功能完全正常的机体中肿瘤也能逃避机体免疫监视而得以发生和发展的重要机制。
糖尿病重要的病理生理特征之一,就是患者血中葡萄糖和胰岛素水平的异常改变。现代医学也正是依据患者血中葡萄糖和胰岛素水平的异常改变,将糖尿病的自然病程分为四个阶段。糖尿病自然病程不同阶段血中的葡萄糖和胰岛素,各有与其所处病程阶段相应的不同浓度。糖尿病患者易患结直肠癌,是否与其自然病程不同阶段血中不同浓度的葡萄糖和胰岛素能影响结直肠癌肿瘤细胞分泌免疫抑制物质有关,目前国内外尚未见报道,十分需要对之进行研究和探讨。迄今,已见于报道能由肿瘤细胞分泌的免疫抑制物质多达20余种。其中,转化生长因子-β1(TGF-β1)、血管内皮生长因子(VEGF)和白细胞介素-6(IL-6),是见诸报道较多、免疫抑制作用较强,并且也恰是可由结直肠癌肿瘤细胞株Colon26肿瘤细胞分泌的三种重要的免疫抑制物质。因此,本研究选用小鼠结直肠癌肿瘤细胞株Colon26肿瘤细胞作为研究对象,以模拟糖尿病自然病程血中不同浓度的葡萄糖和胰岛素对之进行作用,测定是否对Colon26肿瘤细胞分泌这三种免疫抑制物质有影响和有何影响,并初步探索是通过何种分子机制导致了这种影响。从影响结直肠癌肿瘤细胞分泌免疫抑制物质的角度,为研究糖尿病与结直肠癌之间的病理生理关系,揭示糖尿病患者为何易患结直肠癌,提供新的研究思路和实验依据。
方法:体外常规培养Colon26肿瘤细胞。根据培养液(RPMI1640完全培养基, CM)中外加葡萄糖和胰岛素的终浓度不同,分为12个组:
A组(对照组, Control),CM;B组(常糖组,nG),5mmol /L葡萄糖;C组(中糖组,mG),10mmol /L葡萄糖;D组(高糖组,hG),25mmol /L葡萄糖;E组(常胰岛素组,nI),5μIU/mL胰岛素;F组(中胰岛素组,mI),25μIU/mL胰岛素;G组(高胰岛素组,hI),125μIU/mL胰岛素;H组(常糖常胰岛素组,nGnI),5mmol/L葡萄糖和5μIU/mL胰岛素;K组(常糖高胰岛素组,nGhI),5mmol/L葡萄糖和125μIU/mL胰岛素;L组(高糖高胰岛素组,hGhI),25mmol/L葡萄糖和125μIU/mL胰岛素;M组(高糖常胰岛素组,hGnI),25mmol/L葡萄糖和5μIU/mL胰岛素;N组(高糖低胰岛素组,hGloI),25mmol/L葡萄糖和1.25μIU/mL胰岛素。每组均设3个复孔,置37℃5 %CO2饱和湿度的培养箱培养48小时。培养结束后,分别收集细胞培养上清和细胞。ELISA法分别检测细胞培养上清中VEGF、TGF-β1和IL-6的含量(pg/mL)。将所收集细胞抽提总RNA,采用RT-PCR法扩增目的片段VEGF、TGF-β1、IL-6和内参照β-actin,扩增产物行琼脂糖凝胶电泳,在凝胶成像系统上扫描分析,分别计算VEGF、TGF-β1和IL-6 mRNA相对表达量,表达强度以相对表达系数RC表示。全部实验重复三次,结果数据以三次重复实验的x±s表示。统计学分析采用t检验和单因素方差分析,以P<0.05为有统计学差异。
结果:
1不同浓度的葡萄糖对Colon26肿瘤细胞VEGF分泌的影响
B(nG)组、C(mG)组和D(hG)组细胞培养上清中VEGF的浓度(分别为9.4167±0.4646,9.4667±0.1528和9.6167±0.2887),与A(Control)组细胞培养上清中VEGF的浓度(9.5583±0.2691)相比较,均无显著性差异(均为P>0.05)。显示所试三个浓度([5、10和25)mmol/L]的葡萄糖,对Colon26肿瘤细胞分泌VEGF均无影响。
2不同浓度的胰岛素对Colon26肿瘤细胞VEGF分泌的影响
G(hI)组细胞培养上清中VEGF的浓度(27.7667±0.2517),明显高于A(Control)组、E(nI)组和F(mI)组细胞培养上清中的VEGF浓度(分别为9.5583±0.2691,9.5833±0.0764和9.6167±0.1258),差异均有显著性(均为P<0.01);而A(Control)组、E组和F组三组之间,无显著性差异(P>0.05)。显示只有高浓度(125μIU/mL)胰岛素能显著增加Colon26肿瘤细胞VEGF的分泌。
3不同浓度的葡萄糖和胰岛素联合对Colon26肿瘤细胞VEGF分泌的影响
K(nGhI)组和L(hGhI)组细胞培养上清中VEGF的浓度(分别为27.8333±0.3512和27.6000±0.6557),均明显高于A(Control)组细胞培养上清中的VEGF浓度(9.5583±0.2691),差异均有显著性(均为P<0.01);而K组与L组细胞培养上清中的VEGF浓度无差异(P>0.05)。H(nGnI)组、M(hGnI)组和N(hGloI)组细胞培养上清中VEGF的浓度(分别为9.7167±0.00289,9.8667±0.0577和9.9000±0.1732)与A组相比较均无显著性差异(均为P>0.05)。显示,不同浓度的葡萄糖和胰岛素联合未明显改变各自单独对Colon26肿瘤细胞分泌VEGF的影响模式,仍显示为仅是加有高浓度(125μIU/mL)胰岛素者(K组和L组)能显著增加Colon26肿瘤细胞VEGF的分泌;两者联合,既无协同效应也无拮抗效应。
4不同浓度的葡萄糖和胰岛素对Colon26肿瘤细胞VEGF基因转录的影响
培养液中加有高浓度(125μIU/mL)胰岛素的G(hI)组、K(nGhI)组和L(hGhI)组:它们的VEGF mRNA扩增产物的电泳条带,与A(Control)组相比明显增粗变亮;它们的VEGF mRNA的表达(分别为0.8802±0.1900、0.8605±0.1800和0.8569±0.2200),与A组(0.4653±0.0200)相比均有显著性差异(均为P<0.01)。而其他组与A组相比,电泳条带在宽度和亮度上没有差异;且VEGF mRNA的表达与A组相比亦无显著性差异(均为P>0.05)。显示高浓度(125μIU/mL)的胰岛素显著增加Colon26肿瘤细胞VEGF的基因转录;提示上述高浓度的胰岛素显著增加Colon26肿瘤细胞分泌VEGF的分子机制,应是在基因水平提高VEGF mRNA转录所致。
5不同浓度的葡萄糖对Colon26肿瘤细胞TGF-β1分泌的影响
D(hG)组细胞培养上清中TGF-β1的浓度(863.12±20.26)明显高于A(Control)组(625.14±12.02),差异有显著性(P<0.01);而B(nG)组和C(mG)组细胞培养上清中TGF-β1的浓度(分别为635.51±16.97和632.52±17.88)与A组相比均无显著性差异(均为P >0.05 )。显示高浓度的葡萄糖显著增加Colon26肿瘤细胞分泌TGF-β1。
6不同浓度的胰岛素对Colon26肿瘤细胞TGF-β1分泌的影响
E(nI)组、F(mI)组和G(hI)组细胞培养上清中TGF-β1的浓度(分别为622.18±10.53、614.85±14.92和617.12±16.12)与A(Control)组(625.14±12.02)相比均无显著性差异(均为P>0.05)。显示所试三个浓度[(5、25和125)μIU/mL ]的胰岛素均不影响Colon26肿瘤细胞TGF-β1的分泌。
7不同浓度的葡萄糖和胰岛素联合对Colon26肿瘤细胞TGF-β1分泌的影响
培养液中均加有高浓度(25mmol/L)葡萄糖的L(hGhI)组、M(hGnI)组和N(hGloI)组细胞培养上清中TGF-β1的浓度(分别为982.78±19.02、882.54±17.08和871.46±16.21)均明显高于A(Control)组(625.14±12.02),差异均有显著性(均为P<0.01);而培养液中均未加有高浓度葡萄糖的H(nGnI)组和K(nGhI)组细胞培养上清中TGF-β1的浓度(分别为626.18±15.98和630.18±18.95)与A组相比均无显著性差异(均为P>0.05)。这些结果显示,葡萄糖与胰岛素联合未改变各自单独作用模式,仍显示为仅高浓度葡萄糖显著增加Colon26肿瘤细胞TGF-β1的分泌;两者联合既无协效应同也无拮抗效应。
8不同浓度的葡萄糖和胰岛素对Colon26肿瘤细胞TGF-β1基因转录的影响
TGF-β1 mRNA的表达水平,在培养液加有高浓度(25mmol/L)葡萄糖的D (hG)组(0.768±0.017)、L组(0.889±0.028)、M (hGnI)组(0.764±0.027)和N (hGloI)组(0.752±0.035)均显著高于A组(0.540±0.012) (均P <0.01),而在培养液没加高浓度葡萄糖的B (nG)组(0.545士0.033)、C (mG)组(0.558±0.035)、E (nI)组(0.566±0.039)、F (mI)组(0.549±0.030)、G (hI)组(0.531±0.022)、H (nGnI)组(0.550±0.037)和K (nGhI)组(0.537±0.025)与A组相当(均为P>0.05)。显示只有高浓度(25mmol/L)的葡萄糖能增加Colon26肿瘤细胞的TGF-β1 mRNA转录;提示上述高浓度的葡萄糖能增加Colon26肿瘤细胞分泌TGF-β1的分子机制应是由在基因水平增加TGF-β1 mRNA转录所致。
9不同浓度的葡萄糖对Colon26肿瘤细胞IL-6分泌的影响
D(hG)组细胞培养上清中IL-6的浓度(27.70±1.53)明显高于A(Control)组(18.33±2.52),差异有显著性(P <0.01);而B(nG)组和C(mG)组细胞培养上清中IL-6的浓度(分别为19.33±2.31和22.67±0.58),与A组相比均无显著性差异(均为P >0. 05)。显示高浓度(25mmol/L)的葡萄糖显著提高Colon26肿瘤细胞IL-6分泌。
10不同浓度的胰岛素对Colon26肿瘤细胞IL-6分泌的影响
E(nI)组、F(mI)组和G(hI)组细胞培养上清中IL-6的浓度(分别为19.67±1.53、21.33±4.04和21.67±2.08)与A组(18.33±2.52)相比均无显著性差异(均为P>0. 05)。显示所试三个浓度([5, 25和125)μIU/mL]胰岛素均不影响Colon26肿瘤细胞分泌IL-6。
11不同浓度的葡萄糖和胰岛素联合对Colon26肿瘤细胞IL-6分泌的影响
培养液中均加有高浓度(25 mmo1/L)葡萄糖的L(hGhI)组、M(hGnI)组和N(hGloI)组细胞培养上清中IL-6的浓度(分别为27.67±2.52、27.33±2.08和27.33±1.53)均明显高于A(Control)组(18.33±2.52),差异均有显著性(均为P<0.01),而培养液中均未加高浓度葡萄糖的H(nGnI)组和K(nGhI)组细胞培养上清中IL-6的浓度(分别为20.33±1.53和22.33±2.08)与A组相比均无显著性差异(均为P >0. 05)。显示,不同浓度的葡萄糖和胰岛素联合未改变各自单独作用模式,仍是显示为高浓度的葡萄糖显著增加Colon26肿瘤细胞分泌IL-6;两者联合对Colon26肿瘤细胞分泌IL-6,既无协同也无拮抗效应。
12不同浓度的葡萄糖和胰岛素对Colon26肿瘤细胞IL-6基因转录的影响
D(hG)组IL-6 mRNA的表达(0.560±0.011)高于A(Control)组(0.406±0.012),差异有显著性(P<0.05);而B(nG)组和C(mG)组IL-6 mRNA的表达(分别为0.413±0.031和0.426±0.001)与A组相比均无显著性差异(均为P >0. 05)。E(nI)组、F(mI)组和G(hI)组IL-6 mRNA的表达(分别为0.416±0.002、0.422±0.013和0.426±0.016)与A组相比均无显著性差异(均为P>0.05)。培养液中均加有高浓度(25 mmo1/L)葡萄糖的L组、M组和N组的IL-6 mRNA表达(分别为0.560±0.011、0.560±0.011和0.563±0.002)均高于A组,差异均有显著性(均为P <0.05);而培养液中未加高浓度葡萄糖的H组和K组的IL-6 mRNA表达(分别为0.420±0.001和0.426±0.002)与A组相比均无显著性差异(均为P>0.05)。这些结果显示,高浓度的葡萄糖(25mmol/L)显著增加Colon26肿瘤细胞IL-6 mRNA转录;提示上述高浓度葡萄糖增加Colon26肿瘤细胞分泌IL-6的分子机制应是在基因水平增加Colon26肿瘤细胞IL-6 mRNA转录所致。
结论:
1高浓度(25mmol/L)的葡萄糖可显著增加Colon26肿瘤细胞分泌TGF-β1和IL-6,其分子机制应是由促进Colon26肿瘤细胞TGF-β1和IL-6基因转录所致。
2不同浓度[(5—25)mmol/L]的葡萄糖对Colon26肿瘤细胞VEGF的分泌和基因转录无影响。
3高浓度(125μIU/mL)的胰岛素可显著增加Colon26肿瘤细胞分泌VEGF,其分子机制应是由促进Colon26肿瘤细胞VEGF基因转录所致。
4不同浓度[(1.25-125)μIU/mL]的胰岛素对Colon26肿瘤细胞TGF-β1和IL-6的分泌及基因转录无影响。
5不同浓度的葡萄糖和胰岛素联合,不改变各自单独对Colon26肿瘤细胞VEGF、TGF-β1和IL-6的分泌及基因转录的作用模式。
6不同浓度的葡萄糖和胰岛素联合,对Colon26肿瘤细胞VEGF、TGF-β1和IL-6的分泌及基因转录,既无协同效应也无拮抗效应。
7糖尿病自然病程中不同浓度的葡萄糖和胰岛素可显著增加结直肠癌肿瘤细胞某些免疫抑制物质分泌,应是糖尿病患者易患结直肠癌的内在分子机制之一。
Objective: Tumor and diabetes are the two kinds of severe diseases harming the health and life of human at present and the morbidity and mortality of them are increasing year by year. Of late years, more and more clinical and epidemiological data discovered that the patients with diabetes easily suffer from colorectal cancer. The risk suffering from colorectal cancer by the patients with diabetes is significantly higher than the non-diabetes patients, and it was suggested that diabetes is the independent risk factor for colorectal cancer. The morbidity, relapse rate and mortality of colorectal cancer in the diabetes patients are notably higher than that in the non-diabetes patients. The relationship between diabetes and colorectal cancer has become a highly interested heat point. Studying and discovering the pathological physiology relationship between diabetes and colorectal cancer and its molecular mechanisms has become an important question for exploration needed very in this scientific territory.
The tumor immunological studies have discovered that the tumor development closely related not only to the immunofunction of body, in a sense, but also even more to the immunosuppression mediated by tumor cells. That the tumor cells secrete immunosuppressive substances to suppress the immunofunction of body, i.e. the immunosuppression of tumor cells, is the important mechanism with which the tumor cells escape the immunosurveillance of body to develop. The immunosuppressive substances secreted by tumor cells not only possibly could play a immunosuppression in the sites apart from the tumor in the body, but also even more can form the deeply immunosuppressive area, i.e. called“collapsar”, severely suppressing not only the immunofunction of the immunocytes in situ within the area, but also that of the function-normal and even activated immunocytes entered into the area to form“silence”cells, making all immunocytes in the area to loss the ability of effectively killing the tumor cells, and thus this apparently should be the important mechanism that in the body with completely normal immunofunctions the tumor yet could escape the immunosurveillance to develop.
One of the important pathological physiology characteristics of diabetes is the abnormal changes of the concentrations of glucose and insulin in blood of the patients. Just according to the abnormal changes of the concentrations of glucose and insulin in blood of the patients,the modern medicine divided the natural course of diabetes into four phases, and there are the different concentrations of glucose and insulin in blood corresponding to the different phases. It was not reported at present that the patients with diabetes easily suffer from colorectal cancer would be related either to the different concentrations of glucose and insulin in blood in the different phases of diabetes or not , very needing to study on it. Up to now, it has been reported that there are about twenty kinds of immunosuppressive substances which could be secreted by tumor cells. Among these, those reported more frequently, having more intensive immunosuppression and just to be secreted by the colorectal cancer cell line Colon26 tumor cells are the vascular endothelial growth factor(VEGF), the transforming growth factorβ1(TGF-β1)and interleukin-6(IL-6). Thus, in this study using the different concentrations of glucose and insulin mimicing the different phases of diabetes to affect Colon26 tumor cells, the effect on the secretion of VEGF, TGF-β1 and IL-6 from Colon26 tumor cells was detected and the molecular mechanism leading to the effect was explored primarily, so that from the aspect of affecting the secretion of immunosuppressive substances from colorectal cancer cells, for the investigating the relationship between diabetes and colorectal cancer and discovering why the diabetes patients easily suffer from colorectal cancer, to supply new studying way and experimental evidences.
Methods: Colon26 tumor cells were cultured conventionally in vitro. According to the final concentrations of glucose and insulin added into the cell medium (RPMI1640 complete medium,CM), the Colon26 tumor cells cultured were divided into 12 groups: A group(control), CM; B group (normal glucose, nG), 5mmol/L glucose; C group(middle glucose, mG), 10mmol/L glucose ; D group(high glucose, hG), 25mmol/L glucose; E group (normal insulin, nI), 5μIU/mL insulin; F group(middle insulin, mI), 25μIU/mL insulin; G group(high insulin, hI), 125μIU/mL insulin; H group (nGnI), 5mmol/L glucose and 5μIU/mL insulin; K group (nGhI), 5mmol/L glucose and 125μIU/mL insulin; L group(hGhI), 25mmol/L glucose and 125μIU/mL insulin; M group(hGnI), 25mmol/L glucose and 5μIU/mL insulin; N group(high glucose and low insulin, hGloI), 25mmol/L glucose and 1.25μIU/mL insulin. All groups were triple holes, placed in 37℃5 % CO2 saturated humidity to be cultured for 48h. After culture completion the cells and the supernatants were collected respectively. The concentrations (pg/mL) of TGF-β1, VEGF and IL-6 in the supernatant were detected by ELISA respectively. The total RNA of the cells collected was extracted. The cDNA of VEGF, TGF-β1, IL-6 and interior referenceβ-actin were amplificated by RT-PCR respectively. The agarose gel electrophoresis of amplificated products was performed and analyzed by scaning in the gel imaging system, and the mRNA relative expressive values of TGF-β1, VEGF and IL-6 were calculated respectively and the expressive intensity was shown as the relative expressive coefficient RC. The all experiments were triplicated and the results were shown as x±s. The statistical analysis was performed using the t test and the sigle factor analysis of variance, and the significant size of test was P<0.05.
Results:
1 The effect of glucose at different concentrations on the secretion of VEGF from Colon26 tumor cells
The concentrations of VEGF in the supernatants of B (nG) group, C (mG) group and D (hG) group (9.4167±0.4646,9.4667±0.1528 and 9.6167±0.2887 respectively) had not significant difference compared with that of A (control) group (9.6167±0.2887) (all P>0.05), indicating that the three concentrations [(5, 10 and 25) mmol/L] of glucose tested did not affect the secretion of VEGF from Colon26 tumor cells.
2 The effect of insulin at different concentrations on the secretion of VEGF from Colon26 tumor cells
The concentration of VEGF in the supernatant of G (hI) group (27.7667±0.2517) was significantly higher than that of A (control) group (9.5583±0.2691), E (nI) group (9.5833±0.0764) and F (mI) group (9.6167±0.1258) ( all P<0.01), but there was no significant difference among the three (i.e. A, E and F) groups (P>0.05), indicating only the high concentration (125μIU/mL) of insulin enabaling to increase the secretion of VEGF from Colon26 tumor cells.
3 The effect of the combination of glucose and insulin at different concentrations on the secretion of VEGF from Colon26 tumor cells
The concentrations of VEGF in the supernatants of K(nGhI) group and L(hGhI) group(27.8333±0.3512 and 27.6000±0.6557respectively)were significantly higher than that of A group(9.5583±0.2691)(all P<0.01) and there was no difference between K group and L group(P>0.05), and the concentrations of VEGF in the supernatants of H(nGnI) group, M(hGnI) group and N(hGloI) group(9.7167±0.00289,9.8667±0.0577 and 9.90±0.1732, respectively) had no differences compared with A group(all P>0.05), indicating that the combination of glucose and insulin at different concentrations could not affect the pattern of the effect by oneself on the secretion of VEGF from Colon26 tumor cells, yet showing that only that adding high concentration(125μIU/mL)of insulin(K group and L group) could increase the secretion of VEGF from Colon26 tumor cells, and that the combination of both had neither synergistic effect nor antagonistic effect.
4 The effect of glucose and insulin at different concentrations on the VEGF genetic transcription of Colon26 tumor cells
In G, K and L groups in which in the cell medium the final concentration of insulin all was high(125μIU/mL), their electrophoresis strip of the amplificated product of VEGF mRNA had become thickening and bright compared with A group, and their VEGF mRNA expression level (0.8802±0.1900,0.8605±0.1800 and 0.8569±0.2200 respectively) compared with A group(0.4653±0.0200) had significant differences(all P<0.01). In other groups compared with A group either the electrophoresisi strips or the VEGF mRNA expression levels had not significant differences(all P>0.05). These findings indicated that the high concentration(125μIU/mL)of insulin could increase the VEGF genetic transcription and suggested that this should be the molecular mechanism that the high concentration of insulin could increase the secretion of VEGF from Colon26 tumor cells as above-mentioned.
5 The effect of glucose at different concentrations on the secretion of TGF-β1 from Colon26 tumor cells
The concentration of TGF-β1 in the supernatant of D(hG) group(863. 12±20.26) was significantly higher than that of A group (625.14±12.02) (P<0.01), and that of B (nG) group and C (mG) group (635.51±16.97 and 632.52±17.88 respectively) compared with A group had not significant differences(all P>0.05). These findings indicated that the high concentration (25mmol/L) of glucose could increase significantly the secretion of TGF-β1 from Colon26 tumor cells.
6 The effect of insulin at different concentrations on the secretion of TGF-β1 from Colon26 tumor cells
The concentrations of TGF-β1 in the supernatants of E(nI), F(mI) and G (hI) groups(622.18±10.53, 614.85±14.92 and 617.12±16.12, respectively) compared with A group(625.14±12.02) had not significant differences(all P>0.05), indicating that the three concentrations [(5, 25 and 125)μIU/mL ] of insulin tested had not affect the secretion of TGF-β1 from Colon26 tumor cells.
7 The effect of the combination of glucose and insulin at different concentrations on the secretion of TGF-β1 from Colon26 tumor cells
The concentrations of TGF-β1 in the supernatants of L(hGhI),M(hGnI)and N(hGloI)groups in which the high concentration(25mmol/L)of glucose had be added into the cell medium were 982.78±19.02, 882.54±17.08 and 871.46±16.21 and significantly higher than that of A group (625.14±12.02) (all P<0.01). The concentrations of TGF-β1 in the supernatants of H(nGnI)group and K(nGhI)group in which the high concentration of glucose had not be added into the cell medium were 626.18±15.98 and 630.18±18.95 respectively and compared with A group had not significant differences (all P>0.05). These findings indicated that the combination of glucose and insulin did not change the effect patterns by oneself, yet showing that only the high concentration of glucose could increase significantly the secretion of TGF-β1 from Colon26 tumor cells, and that the combination of both had neither synergistic effect nor antagonistic effect.
8 The effect of glucose and insulin at different concentrations on the TGF-β1 genetic transcription of Colon26 tumor cells
The expression level of TGF-β1 mRNA in D(hG) group(0.768±0.017), L(hGhI) group(0.889±0.028), M(hGnI) group(0.764±0.027) and N (hGloI) group (0.752±0.035) in which the high concentration (25mmol/L) of glucose was added into the cell medium were significantly higher than that of A group(0.540±0.012()all P<0.01) and that in B(nG) group(0.545±0.033),C(mG) group(0.558±0.035),E(nI) group(0.566±0.039), F(mI) group(0.549±0.030), G(hI) group(0.531±0.022), H(nGnI) group(0.550±0.037) and K(nGhI) group(0.537±0.025) in which the high concentration of glucose did not be added into the cell medium were equivalent to that in A group(all P>0.05), indicating that only the high concentration(25mmol/L) of glucose could increase the TGF-β1 mRNA transcription of Colon26 tumor cells and suggesting that the molecular mechanism with which the high concentration of glucose could increase the secretion of TGF-β1 from Colon26 tumor cells as above-mentioned should be due to the increasing the TGF-β1 mRNA transcription at the genetic level.
9 The effect of glucose at different concentration on the secretion of IL-6 from Colon26 tumor cells
The concentration of IL-6 in the supernatant of D(hG) group(27.70±1.53) was significantly higher than that of A group(18.33±2.52)(P <0.01), but that of B(nG) group(19.33±2.31) and C(mG) group(22.6±0.58) compared with A group had not significant differences(all P>0.05), indicating that the high concentration(25mmol/L)of glucose could increase significantly the secretion of IL-6 from Colon26 tumor cells.
10 The effect of insulin at different concentration on the secretion of IL-6 from Colon26 tumor cells
The concentrations of IL-6 in the supernatants of E(nI) group, F(mI) group and G(hI) group(19.67±1.53, 21.33±4.04 and 21.67±2.08, respectively) compared with that of A group(18.33±2.52) had not significant differences (all P>0.05), indicating that the three kinds of concentrations [(5, 25 and 125 )μIU/mL] of insulin tested had not the effect on the secretion of IL-6 from Colon26 tumor cells.
11 The effect of the combination of glucose and insulin at different concentration on the secretion of IL-6 from Colon26 tumor cells
The concentrations of IL-6 in the supernatants of L group , M group and N group (27.67±2.52, 27.33±2.08 and 27.33±1.53, respectively) in which the high concentration (25mmol/L) of glucose was added into the cell medium were significantly higher than that of A group (18.33±2.52)( all P <0. 01) and that of H group and K group (20.33±1.53 and 22.33±2.08, respectively) in which the high concentration of glucose did not be added into the cell medium compared with that of A group had not significant differences (all P>0.05). These findings indicated that the combinations of glucose and insulin at different concentrations did not change the pattern of effect by oneself, yet showing that the high concentration of glucose could increase the secretion of IL-6 from Colon26 tumor cells, and that the combination of both had neither synergistic effect nor antagonistic effect.
12 The effect of glucose and insulin at different concentrations on the IL-6 genetic transcription of Colon26 tumor cells
The IL-6 mRNA expression in D(hG) group(0.560±0.011) was significantly higher than that in A group(0.406±0.012 )(P <0.05), that in B (nG)group and C(mG) group(0.413±0.031 and 0.426±0.001 respectively) compared with A group had not significant differences(all P >0. 05), that in E(nI)group, F(mI)group and G(hI)group(0.416±0.002, 0.422±0.013 and 0.426±0.016 respectively) compared with A group had not significant differences(all P >0. 05), that in the L , M and N groups in which the high concentration(25mmo1/L)of glucose was added into the cell medium ( 0.560±0.011, 0.560±0.011 and 0.563±0.002, respectively) were significantly higher than that in A group(all P <0.05), and that in the H and K groups in which the high concentration of glucose was not added into the cell medium (0.420±0.001 and 0.426±0.002 respectively) compared with A group had not significant differences(all P >0.05). These findings indicated that the high concentration(25 mmo1/L)of glucose can significantly increase the IL-6 mRNA transcription of Colon26 tumor cells and suggested that the molecular mechanism with which the high concentration of glucose could increase the secretion of IL-6 from Colon26 tumor cells as above-mentioned should be due to the increasing the IL-6 mRNA transcription at the genetic level.
Conclusion:
1 The high concentration(25mmol/L of glucose can increase significantly the secretion of TGF-β1 and IL-6 from Colon26 tumor cells, the molecular mechanism of which should be due to that the genetic transcription of TGF-β1 and IL-6 within Colon26 tumor cell had be promoted.
2 The different concentrations ([5-25)mmol/L] of glucose had not the effect on the secretion and genetic transcription of VEGF of Colon26 tumor cells.
3 The high concentration(125μIU/mL)of insulin can increase significantly the secretion of VEGF from Colon26 tumor cells, the molecular mechanism of which should be due to that the genetic transcription of VEGF within Colon26 tumor cell had be promoted.
4 The different concentrations [(1.25-125)μIU/mL] of insulin had not the effect on the secretion and genetic transcription of the TGF-β1 and IL-6 of Colon26 tumor cells.
5 The combination of glucose and insulin at different concentrations did not change the pattern of effect by oneself on the secretion and genetic transcription of VEGF, TGF-β1 and IL-6 of Colon26 tumor cells.
6 The effect of the combination of glucose and insulin at different concentrations on the secretion and genetic transcription of VEGF, TGF-β1 and IL-6 of Colon26 tumor cells was neither synergistic nor antagonistic.
7 That the different concentrations of glucose and insulin in the natural course of diabetes could increase significantly the secretion of some immunosuppressive substances from the colorectal cancer cells should be one of the internal molecular mechanism with which the diabetes patients easily suffer from the colorectal cancer.
肿瘤免疫学的研究已经揭示,肿瘤的发生、发展,不仅与机体的免疫功能密切相关,在某种意义上更与肿瘤细胞的免疫抑制作用密切相关。肿瘤细胞分泌免疫抑制物质抑制机体免疫功能,即肿瘤细胞的免疫抑制作用,是肿瘤细胞逃避机体免疫监视,使肿瘤得以发生和发展的重要机制,在促进机体肿瘤的发生、发展中具有极其重要的作用。肿瘤细胞通过其分泌的免疫抑制物质,不但有可能在远离肿瘤部位的机体其他部位发挥免疫抑制作用,更可在肿瘤局部形成深度免疫抑制区,即所谓“黑洞”区,以至于不仅使原本就在此区之中的原位免疫细胞功能严重受抑,而且还能使由此区之外进入其中的功能正常甚至已经活化的免疫细胞成为功能受抑的“沉默”细胞,从而使之失去有效清除肿瘤细胞的能力,显然这是在即使免疫功能完全正常的机体中肿瘤也能逃避机体免疫监视而得以发生和发展的重要机制。
糖尿病重要的病理生理特征之一,就是患者血中葡萄糖和胰岛素水平的异常改变。现代医学也正是依据患者血中葡萄糖和胰岛素水平的异常改变,将糖尿病的自然病程分为四个阶段。糖尿病自然病程不同阶段血中的葡萄糖和胰岛素,各有与其所处病程阶段相应的不同浓度。糖尿病患者易患结直肠癌,是否与其自然病程不同阶段血中不同浓度的葡萄糖和胰岛素能影响结直肠癌肿瘤细胞分泌免疫抑制物质有关,目前国内外尚未见报道,十分需要对之进行研究和探讨。迄今,已见于报道能由肿瘤细胞分泌的免疫抑制物质多达20余种。其中,转化生长因子-β1(TGF-β1)、血管内皮生长因子(VEGF)和白细胞介素-6(IL-6),是见诸报道较多、免疫抑制作用较强,并且也恰是可由结直肠癌肿瘤细胞株Colon26肿瘤细胞分泌的三种重要的免疫抑制物质。因此,本研究选用小鼠结直肠癌肿瘤细胞株Colon26肿瘤细胞作为研究对象,以模拟糖尿病自然病程血中不同浓度的葡萄糖和胰岛素对之进行作用,测定是否对Colon26肿瘤细胞分泌这三种免疫抑制物质有影响和有何影响,并初步探索是通过何种分子机制导致了这种影响。从影响结直肠癌肿瘤细胞分泌免疫抑制物质的角度,为研究糖尿病与结直肠癌之间的病理生理关系,揭示糖尿病患者为何易患结直肠癌,提供新的研究思路和实验依据。
方法:体外常规培养Colon26肿瘤细胞。根据培养液(RPMI1640完全培养基, CM)中外加葡萄糖和胰岛素的终浓度不同,分为12个组:
A组(对照组, Control),CM;B组(常糖组,nG),5mmol /L葡萄糖;C组(中糖组,mG),10mmol /L葡萄糖;D组(高糖组,hG),25mmol /L葡萄糖;E组(常胰岛素组,nI),5μIU/mL胰岛素;F组(中胰岛素组,mI),25μIU/mL胰岛素;G组(高胰岛素组,hI),125μIU/mL胰岛素;H组(常糖常胰岛素组,nGnI),5mmol/L葡萄糖和5μIU/mL胰岛素;K组(常糖高胰岛素组,nGhI),5mmol/L葡萄糖和125μIU/mL胰岛素;L组(高糖高胰岛素组,hGhI),25mmol/L葡萄糖和125μIU/mL胰岛素;M组(高糖常胰岛素组,hGnI),25mmol/L葡萄糖和5μIU/mL胰岛素;N组(高糖低胰岛素组,hGloI),25mmol/L葡萄糖和1.25μIU/mL胰岛素。每组均设3个复孔,置37℃5 %CO2饱和湿度的培养箱培养48小时。培养结束后,分别收集细胞培养上清和细胞。ELISA法分别检测细胞培养上清中VEGF、TGF-β1和IL-6的含量(pg/mL)。将所收集细胞抽提总RNA,采用RT-PCR法扩增目的片段VEGF、TGF-β1、IL-6和内参照β-actin,扩增产物行琼脂糖凝胶电泳,在凝胶成像系统上扫描分析,分别计算VEGF、TGF-β1和IL-6 mRNA相对表达量,表达强度以相对表达系数RC表示。全部实验重复三次,结果数据以三次重复实验的x±s表示。统计学分析采用t检验和单因素方差分析,以P<0.05为有统计学差异。
结果:
1不同浓度的葡萄糖对Colon26肿瘤细胞VEGF分泌的影响
B(nG)组、C(mG)组和D(hG)组细胞培养上清中VEGF的浓度(分别为9.4167±0.4646,9.4667±0.1528和9.6167±0.2887),与A(Control)组细胞培养上清中VEGF的浓度(9.5583±0.2691)相比较,均无显著性差异(均为P>0.05)。显示所试三个浓度([5、10和25)mmol/L]的葡萄糖,对Colon26肿瘤细胞分泌VEGF均无影响。
2不同浓度的胰岛素对Colon26肿瘤细胞VEGF分泌的影响
G(hI)组细胞培养上清中VEGF的浓度(27.7667±0.2517),明显高于A(Control)组、E(nI)组和F(mI)组细胞培养上清中的VEGF浓度(分别为9.5583±0.2691,9.5833±0.0764和9.6167±0.1258),差异均有显著性(均为P<0.01);而A(Control)组、E组和F组三组之间,无显著性差异(P>0.05)。显示只有高浓度(125μIU/mL)胰岛素能显著增加Colon26肿瘤细胞VEGF的分泌。
3不同浓度的葡萄糖和胰岛素联合对Colon26肿瘤细胞VEGF分泌的影响
K(nGhI)组和L(hGhI)组细胞培养上清中VEGF的浓度(分别为27.8333±0.3512和27.6000±0.6557),均明显高于A(Control)组细胞培养上清中的VEGF浓度(9.5583±0.2691),差异均有显著性(均为P<0.01);而K组与L组细胞培养上清中的VEGF浓度无差异(P>0.05)。H(nGnI)组、M(hGnI)组和N(hGloI)组细胞培养上清中VEGF的浓度(分别为9.7167±0.00289,9.8667±0.0577和9.9000±0.1732)与A组相比较均无显著性差异(均为P>0.05)。显示,不同浓度的葡萄糖和胰岛素联合未明显改变各自单独对Colon26肿瘤细胞分泌VEGF的影响模式,仍显示为仅是加有高浓度(125μIU/mL)胰岛素者(K组和L组)能显著增加Colon26肿瘤细胞VEGF的分泌;两者联合,既无协同效应也无拮抗效应。
4不同浓度的葡萄糖和胰岛素对Colon26肿瘤细胞VEGF基因转录的影响
培养液中加有高浓度(125μIU/mL)胰岛素的G(hI)组、K(nGhI)组和L(hGhI)组:它们的VEGF mRNA扩增产物的电泳条带,与A(Control)组相比明显增粗变亮;它们的VEGF mRNA的表达(分别为0.8802±0.1900、0.8605±0.1800和0.8569±0.2200),与A组(0.4653±0.0200)相比均有显著性差异(均为P<0.01)。而其他组与A组相比,电泳条带在宽度和亮度上没有差异;且VEGF mRNA的表达与A组相比亦无显著性差异(均为P>0.05)。显示高浓度(125μIU/mL)的胰岛素显著增加Colon26肿瘤细胞VEGF的基因转录;提示上述高浓度的胰岛素显著增加Colon26肿瘤细胞分泌VEGF的分子机制,应是在基因水平提高VEGF mRNA转录所致。
5不同浓度的葡萄糖对Colon26肿瘤细胞TGF-β1分泌的影响
D(hG)组细胞培养上清中TGF-β1的浓度(863.12±20.26)明显高于A(Control)组(625.14±12.02),差异有显著性(P<0.01);而B(nG)组和C(mG)组细胞培养上清中TGF-β1的浓度(分别为635.51±16.97和632.52±17.88)与A组相比均无显著性差异(均为P >0.05 )。显示高浓度的葡萄糖显著增加Colon26肿瘤细胞分泌TGF-β1。
6不同浓度的胰岛素对Colon26肿瘤细胞TGF-β1分泌的影响
E(nI)组、F(mI)组和G(hI)组细胞培养上清中TGF-β1的浓度(分别为622.18±10.53、614.85±14.92和617.12±16.12)与A(Control)组(625.14±12.02)相比均无显著性差异(均为P>0.05)。显示所试三个浓度[(5、25和125)μIU/mL ]的胰岛素均不影响Colon26肿瘤细胞TGF-β1的分泌。
7不同浓度的葡萄糖和胰岛素联合对Colon26肿瘤细胞TGF-β1分泌的影响
培养液中均加有高浓度(25mmol/L)葡萄糖的L(hGhI)组、M(hGnI)组和N(hGloI)组细胞培养上清中TGF-β1的浓度(分别为982.78±19.02、882.54±17.08和871.46±16.21)均明显高于A(Control)组(625.14±12.02),差异均有显著性(均为P<0.01);而培养液中均未加有高浓度葡萄糖的H(nGnI)组和K(nGhI)组细胞培养上清中TGF-β1的浓度(分别为626.18±15.98和630.18±18.95)与A组相比均无显著性差异(均为P>0.05)。这些结果显示,葡萄糖与胰岛素联合未改变各自单独作用模式,仍显示为仅高浓度葡萄糖显著增加Colon26肿瘤细胞TGF-β1的分泌;两者联合既无协效应同也无拮抗效应。
8不同浓度的葡萄糖和胰岛素对Colon26肿瘤细胞TGF-β1基因转录的影响
TGF-β1 mRNA的表达水平,在培养液加有高浓度(25mmol/L)葡萄糖的D (hG)组(0.768±0.017)、L组(0.889±0.028)、M (hGnI)组(0.764±0.027)和N (hGloI)组(0.752±0.035)均显著高于A组(0.540±0.012) (均P <0.01),而在培养液没加高浓度葡萄糖的B (nG)组(0.545士0.033)、C (mG)组(0.558±0.035)、E (nI)组(0.566±0.039)、F (mI)组(0.549±0.030)、G (hI)组(0.531±0.022)、H (nGnI)组(0.550±0.037)和K (nGhI)组(0.537±0.025)与A组相当(均为P>0.05)。显示只有高浓度(25mmol/L)的葡萄糖能增加Colon26肿瘤细胞的TGF-β1 mRNA转录;提示上述高浓度的葡萄糖能增加Colon26肿瘤细胞分泌TGF-β1的分子机制应是由在基因水平增加TGF-β1 mRNA转录所致。
9不同浓度的葡萄糖对Colon26肿瘤细胞IL-6分泌的影响
D(hG)组细胞培养上清中IL-6的浓度(27.70±1.53)明显高于A(Control)组(18.33±2.52),差异有显著性(P <0.01);而B(nG)组和C(mG)组细胞培养上清中IL-6的浓度(分别为19.33±2.31和22.67±0.58),与A组相比均无显著性差异(均为P >0. 05)。显示高浓度(25mmol/L)的葡萄糖显著提高Colon26肿瘤细胞IL-6分泌。
10不同浓度的胰岛素对Colon26肿瘤细胞IL-6分泌的影响
E(nI)组、F(mI)组和G(hI)组细胞培养上清中IL-6的浓度(分别为19.67±1.53、21.33±4.04和21.67±2.08)与A组(18.33±2.52)相比均无显著性差异(均为P>0. 05)。显示所试三个浓度([5, 25和125)μIU/mL]胰岛素均不影响Colon26肿瘤细胞分泌IL-6。
11不同浓度的葡萄糖和胰岛素联合对Colon26肿瘤细胞IL-6分泌的影响
培养液中均加有高浓度(25 mmo1/L)葡萄糖的L(hGhI)组、M(hGnI)组和N(hGloI)组细胞培养上清中IL-6的浓度(分别为27.67±2.52、27.33±2.08和27.33±1.53)均明显高于A(Control)组(18.33±2.52),差异均有显著性(均为P<0.01),而培养液中均未加高浓度葡萄糖的H(nGnI)组和K(nGhI)组细胞培养上清中IL-6的浓度(分别为20.33±1.53和22.33±2.08)与A组相比均无显著性差异(均为P >0. 05)。显示,不同浓度的葡萄糖和胰岛素联合未改变各自单独作用模式,仍是显示为高浓度的葡萄糖显著增加Colon26肿瘤细胞分泌IL-6;两者联合对Colon26肿瘤细胞分泌IL-6,既无协同也无拮抗效应。
12不同浓度的葡萄糖和胰岛素对Colon26肿瘤细胞IL-6基因转录的影响
D(hG)组IL-6 mRNA的表达(0.560±0.011)高于A(Control)组(0.406±0.012),差异有显著性(P<0.05);而B(nG)组和C(mG)组IL-6 mRNA的表达(分别为0.413±0.031和0.426±0.001)与A组相比均无显著性差异(均为P >0. 05)。E(nI)组、F(mI)组和G(hI)组IL-6 mRNA的表达(分别为0.416±0.002、0.422±0.013和0.426±0.016)与A组相比均无显著性差异(均为P>0.05)。培养液中均加有高浓度(25 mmo1/L)葡萄糖的L组、M组和N组的IL-6 mRNA表达(分别为0.560±0.011、0.560±0.011和0.563±0.002)均高于A组,差异均有显著性(均为P <0.05);而培养液中未加高浓度葡萄糖的H组和K组的IL-6 mRNA表达(分别为0.420±0.001和0.426±0.002)与A组相比均无显著性差异(均为P>0.05)。这些结果显示,高浓度的葡萄糖(25mmol/L)显著增加Colon26肿瘤细胞IL-6 mRNA转录;提示上述高浓度葡萄糖增加Colon26肿瘤细胞分泌IL-6的分子机制应是在基因水平增加Colon26肿瘤细胞IL-6 mRNA转录所致。
结论:
1高浓度(25mmol/L)的葡萄糖可显著增加Colon26肿瘤细胞分泌TGF-β1和IL-6,其分子机制应是由促进Colon26肿瘤细胞TGF-β1和IL-6基因转录所致。
2不同浓度[(5—25)mmol/L]的葡萄糖对Colon26肿瘤细胞VEGF的分泌和基因转录无影响。
3高浓度(125μIU/mL)的胰岛素可显著增加Colon26肿瘤细胞分泌VEGF,其分子机制应是由促进Colon26肿瘤细胞VEGF基因转录所致。
4不同浓度[(1.25-125)μIU/mL]的胰岛素对Colon26肿瘤细胞TGF-β1和IL-6的分泌及基因转录无影响。
5不同浓度的葡萄糖和胰岛素联合,不改变各自单独对Colon26肿瘤细胞VEGF、TGF-β1和IL-6的分泌及基因转录的作用模式。
6不同浓度的葡萄糖和胰岛素联合,对Colon26肿瘤细胞VEGF、TGF-β1和IL-6的分泌及基因转录,既无协同效应也无拮抗效应。
7糖尿病自然病程中不同浓度的葡萄糖和胰岛素可显著增加结直肠癌肿瘤细胞某些免疫抑制物质分泌,应是糖尿病患者易患结直肠癌的内在分子机制之一。
Objective: Tumor and diabetes are the two kinds of severe diseases harming the health and life of human at present and the morbidity and mortality of them are increasing year by year. Of late years, more and more clinical and epidemiological data discovered that the patients with diabetes easily suffer from colorectal cancer. The risk suffering from colorectal cancer by the patients with diabetes is significantly higher than the non-diabetes patients, and it was suggested that diabetes is the independent risk factor for colorectal cancer. The morbidity, relapse rate and mortality of colorectal cancer in the diabetes patients are notably higher than that in the non-diabetes patients. The relationship between diabetes and colorectal cancer has become a highly interested heat point. Studying and discovering the pathological physiology relationship between diabetes and colorectal cancer and its molecular mechanisms has become an important question for exploration needed very in this scientific territory.
The tumor immunological studies have discovered that the tumor development closely related not only to the immunofunction of body, in a sense, but also even more to the immunosuppression mediated by tumor cells. That the tumor cells secrete immunosuppressive substances to suppress the immunofunction of body, i.e. the immunosuppression of tumor cells, is the important mechanism with which the tumor cells escape the immunosurveillance of body to develop. The immunosuppressive substances secreted by tumor cells not only possibly could play a immunosuppression in the sites apart from the tumor in the body, but also even more can form the deeply immunosuppressive area, i.e. called“collapsar”, severely suppressing not only the immunofunction of the immunocytes in situ within the area, but also that of the function-normal and even activated immunocytes entered into the area to form“silence”cells, making all immunocytes in the area to loss the ability of effectively killing the tumor cells, and thus this apparently should be the important mechanism that in the body with completely normal immunofunctions the tumor yet could escape the immunosurveillance to develop.
One of the important pathological physiology characteristics of diabetes is the abnormal changes of the concentrations of glucose and insulin in blood of the patients. Just according to the abnormal changes of the concentrations of glucose and insulin in blood of the patients,the modern medicine divided the natural course of diabetes into four phases, and there are the different concentrations of glucose and insulin in blood corresponding to the different phases. It was not reported at present that the patients with diabetes easily suffer from colorectal cancer would be related either to the different concentrations of glucose and insulin in blood in the different phases of diabetes or not , very needing to study on it. Up to now, it has been reported that there are about twenty kinds of immunosuppressive substances which could be secreted by tumor cells. Among these, those reported more frequently, having more intensive immunosuppression and just to be secreted by the colorectal cancer cell line Colon26 tumor cells are the vascular endothelial growth factor(VEGF), the transforming growth factorβ1(TGF-β1)and interleukin-6(IL-6). Thus, in this study using the different concentrations of glucose and insulin mimicing the different phases of diabetes to affect Colon26 tumor cells, the effect on the secretion of VEGF, TGF-β1 and IL-6 from Colon26 tumor cells was detected and the molecular mechanism leading to the effect was explored primarily, so that from the aspect of affecting the secretion of immunosuppressive substances from colorectal cancer cells, for the investigating the relationship between diabetes and colorectal cancer and discovering why the diabetes patients easily suffer from colorectal cancer, to supply new studying way and experimental evidences.
Methods: Colon26 tumor cells were cultured conventionally in vitro. According to the final concentrations of glucose and insulin added into the cell medium (RPMI1640 complete medium,CM), the Colon26 tumor cells cultured were divided into 12 groups: A group(control), CM; B group (normal glucose, nG), 5mmol/L glucose; C group(middle glucose, mG), 10mmol/L glucose ; D group(high glucose, hG), 25mmol/L glucose; E group (normal insulin, nI), 5μIU/mL insulin; F group(middle insulin, mI), 25μIU/mL insulin; G group(high insulin, hI), 125μIU/mL insulin; H group (nGnI), 5mmol/L glucose and 5μIU/mL insulin; K group (nGhI), 5mmol/L glucose and 125μIU/mL insulin; L group(hGhI), 25mmol/L glucose and 125μIU/mL insulin; M group(hGnI), 25mmol/L glucose and 5μIU/mL insulin; N group(high glucose and low insulin, hGloI), 25mmol/L glucose and 1.25μIU/mL insulin. All groups were triple holes, placed in 37℃5 % CO2 saturated humidity to be cultured for 48h. After culture completion the cells and the supernatants were collected respectively. The concentrations (pg/mL) of TGF-β1, VEGF and IL-6 in the supernatant were detected by ELISA respectively. The total RNA of the cells collected was extracted. The cDNA of VEGF, TGF-β1, IL-6 and interior referenceβ-actin were amplificated by RT-PCR respectively. The agarose gel electrophoresis of amplificated products was performed and analyzed by scaning in the gel imaging system, and the mRNA relative expressive values of TGF-β1, VEGF and IL-6 were calculated respectively and the expressive intensity was shown as the relative expressive coefficient RC. The all experiments were triplicated and the results were shown as x±s. The statistical analysis was performed using the t test and the sigle factor analysis of variance, and the significant size of test was P<0.05.
Results:
1 The effect of glucose at different concentrations on the secretion of VEGF from Colon26 tumor cells
The concentrations of VEGF in the supernatants of B (nG) group, C (mG) group and D (hG) group (9.4167±0.4646,9.4667±0.1528 and 9.6167±0.2887 respectively) had not significant difference compared with that of A (control) group (9.6167±0.2887) (all P>0.05), indicating that the three concentrations [(5, 10 and 25) mmol/L] of glucose tested did not affect the secretion of VEGF from Colon26 tumor cells.
2 The effect of insulin at different concentrations on the secretion of VEGF from Colon26 tumor cells
The concentration of VEGF in the supernatant of G (hI) group (27.7667±0.2517) was significantly higher than that of A (control) group (9.5583±0.2691), E (nI) group (9.5833±0.0764) and F (mI) group (9.6167±0.1258) ( all P<0.01), but there was no significant difference among the three (i.e. A, E and F) groups (P>0.05), indicating only the high concentration (125μIU/mL) of insulin enabaling to increase the secretion of VEGF from Colon26 tumor cells.
3 The effect of the combination of glucose and insulin at different concentrations on the secretion of VEGF from Colon26 tumor cells
The concentrations of VEGF in the supernatants of K(nGhI) group and L(hGhI) group(27.8333±0.3512 and 27.6000±0.6557respectively)were significantly higher than that of A group(9.5583±0.2691)(all P<0.01) and there was no difference between K group and L group(P>0.05), and the concentrations of VEGF in the supernatants of H(nGnI) group, M(hGnI) group and N(hGloI) group(9.7167±0.00289,9.8667±0.0577 and 9.90±0.1732, respectively) had no differences compared with A group(all P>0.05), indicating that the combination of glucose and insulin at different concentrations could not affect the pattern of the effect by oneself on the secretion of VEGF from Colon26 tumor cells, yet showing that only that adding high concentration(125μIU/mL)of insulin(K group and L group) could increase the secretion of VEGF from Colon26 tumor cells, and that the combination of both had neither synergistic effect nor antagonistic effect.
4 The effect of glucose and insulin at different concentrations on the VEGF genetic transcription of Colon26 tumor cells
In G, K and L groups in which in the cell medium the final concentration of insulin all was high(125μIU/mL), their electrophoresis strip of the amplificated product of VEGF mRNA had become thickening and bright compared with A group, and their VEGF mRNA expression level (0.8802±0.1900,0.8605±0.1800 and 0.8569±0.2200 respectively) compared with A group(0.4653±0.0200) had significant differences(all P<0.01). In other groups compared with A group either the electrophoresisi strips or the VEGF mRNA expression levels had not significant differences(all P>0.05). These findings indicated that the high concentration(125μIU/mL)of insulin could increase the VEGF genetic transcription and suggested that this should be the molecular mechanism that the high concentration of insulin could increase the secretion of VEGF from Colon26 tumor cells as above-mentioned.
5 The effect of glucose at different concentrations on the secretion of TGF-β1 from Colon26 tumor cells
The concentration of TGF-β1 in the supernatant of D(hG) group(863. 12±20.26) was significantly higher than that of A group (625.14±12.02) (P<0.01), and that of B (nG) group and C (mG) group (635.51±16.97 and 632.52±17.88 respectively) compared with A group had not significant differences(all P>0.05). These findings indicated that the high concentration (25mmol/L) of glucose could increase significantly the secretion of TGF-β1 from Colon26 tumor cells.
6 The effect of insulin at different concentrations on the secretion of TGF-β1 from Colon26 tumor cells
The concentrations of TGF-β1 in the supernatants of E(nI), F(mI) and G (hI) groups(622.18±10.53, 614.85±14.92 and 617.12±16.12, respectively) compared with A group(625.14±12.02) had not significant differences(all P>0.05), indicating that the three concentrations [(5, 25 and 125)μIU/mL ] of insulin tested had not affect the secretion of TGF-β1 from Colon26 tumor cells.
7 The effect of the combination of glucose and insulin at different concentrations on the secretion of TGF-β1 from Colon26 tumor cells
The concentrations of TGF-β1 in the supernatants of L(hGhI),M(hGnI)and N(hGloI)groups in which the high concentration(25mmol/L)of glucose had be added into the cell medium were 982.78±19.02, 882.54±17.08 and 871.46±16.21 and significantly higher than that of A group (625.14±12.02) (all P<0.01). The concentrations of TGF-β1 in the supernatants of H(nGnI)group and K(nGhI)group in which the high concentration of glucose had not be added into the cell medium were 626.18±15.98 and 630.18±18.95 respectively and compared with A group had not significant differences (all P>0.05). These findings indicated that the combination of glucose and insulin did not change the effect patterns by oneself, yet showing that only the high concentration of glucose could increase significantly the secretion of TGF-β1 from Colon26 tumor cells, and that the combination of both had neither synergistic effect nor antagonistic effect.
8 The effect of glucose and insulin at different concentrations on the TGF-β1 genetic transcription of Colon26 tumor cells
The expression level of TGF-β1 mRNA in D(hG) group(0.768±0.017), L(hGhI) group(0.889±0.028), M(hGnI) group(0.764±0.027) and N (hGloI) group (0.752±0.035) in which the high concentration (25mmol/L) of glucose was added into the cell medium were significantly higher than that of A group(0.540±0.012()all P<0.01) and that in B(nG) group(0.545±0.033),C(mG) group(0.558±0.035),E(nI) group(0.566±0.039), F(mI) group(0.549±0.030), G(hI) group(0.531±0.022), H(nGnI) group(0.550±0.037) and K(nGhI) group(0.537±0.025) in which the high concentration of glucose did not be added into the cell medium were equivalent to that in A group(all P>0.05), indicating that only the high concentration(25mmol/L) of glucose could increase the TGF-β1 mRNA transcription of Colon26 tumor cells and suggesting that the molecular mechanism with which the high concentration of glucose could increase the secretion of TGF-β1 from Colon26 tumor cells as above-mentioned should be due to the increasing the TGF-β1 mRNA transcription at the genetic level.
9 The effect of glucose at different concentration on the secretion of IL-6 from Colon26 tumor cells
The concentration of IL-6 in the supernatant of D(hG) group(27.70±1.53) was significantly higher than that of A group(18.33±2.52)(P <0.01), but that of B(nG) group(19.33±2.31) and C(mG) group(22.6±0.58) compared with A group had not significant differences(all P>0.05), indicating that the high concentration(25mmol/L)of glucose could increase significantly the secretion of IL-6 from Colon26 tumor cells.
10 The effect of insulin at different concentration on the secretion of IL-6 from Colon26 tumor cells
The concentrations of IL-6 in the supernatants of E(nI) group, F(mI) group and G(hI) group(19.67±1.53, 21.33±4.04 and 21.67±2.08, respectively) compared with that of A group(18.33±2.52) had not significant differences (all P>0.05), indicating that the three kinds of concentrations [(5, 25 and 125 )μIU/mL] of insulin tested had not the effect on the secretion of IL-6 from Colon26 tumor cells.
11 The effect of the combination of glucose and insulin at different concentration on the secretion of IL-6 from Colon26 tumor cells
The concentrations of IL-6 in the supernatants of L group , M group and N group (27.67±2.52, 27.33±2.08 and 27.33±1.53, respectively) in which the high concentration (25mmol/L) of glucose was added into the cell medium were significantly higher than that of A group (18.33±2.52)( all P <0. 01) and that of H group and K group (20.33±1.53 and 22.33±2.08, respectively) in which the high concentration of glucose did not be added into the cell medium compared with that of A group had not significant differences (all P>0.05). These findings indicated that the combinations of glucose and insulin at different concentrations did not change the pattern of effect by oneself, yet showing that the high concentration of glucose could increase the secretion of IL-6 from Colon26 tumor cells, and that the combination of both had neither synergistic effect nor antagonistic effect.
12 The effect of glucose and insulin at different concentrations on the IL-6 genetic transcription of Colon26 tumor cells
The IL-6 mRNA expression in D(hG) group(0.560±0.011) was significantly higher than that in A group(0.406±0.012 )(P <0.05), that in B (nG)group and C(mG) group(0.413±0.031 and 0.426±0.001 respectively) compared with A group had not significant differences(all P >0. 05), that in E(nI)group, F(mI)group and G(hI)group(0.416±0.002, 0.422±0.013 and 0.426±0.016 respectively) compared with A group had not significant differences(all P >0. 05), that in the L , M and N groups in which the high concentration(25mmo1/L)of glucose was added into the cell medium ( 0.560±0.011, 0.560±0.011 and 0.563±0.002, respectively) were significantly higher than that in A group(all P <0.05), and that in the H and K groups in which the high concentration of glucose was not added into the cell medium (0.420±0.001 and 0.426±0.002 respectively) compared with A group had not significant differences(all P >0.05). These findings indicated that the high concentration(25 mmo1/L)of glucose can significantly increase the IL-6 mRNA transcription of Colon26 tumor cells and suggested that the molecular mechanism with which the high concentration of glucose could increase the secretion of IL-6 from Colon26 tumor cells as above-mentioned should be due to the increasing the IL-6 mRNA transcription at the genetic level.
Conclusion:
1 The high concentration(25mmol/L of glucose can increase significantly the secretion of TGF-β1 and IL-6 from Colon26 tumor cells, the molecular mechanism of which should be due to that the genetic transcription of TGF-β1 and IL-6 within Colon26 tumor cell had be promoted.
2 The different concentrations ([5-25)mmol/L] of glucose had not the effect on the secretion and genetic transcription of VEGF of Colon26 tumor cells.
3 The high concentration(125μIU/mL)of insulin can increase significantly the secretion of VEGF from Colon26 tumor cells, the molecular mechanism of which should be due to that the genetic transcription of VEGF within Colon26 tumor cell had be promoted.
4 The different concentrations [(1.25-125)μIU/mL] of insulin had not the effect on the secretion and genetic transcription of the TGF-β1 and IL-6 of Colon26 tumor cells.
5 The combination of glucose and insulin at different concentrations did not change the pattern of effect by oneself on the secretion and genetic transcription of VEGF, TGF-β1 and IL-6 of Colon26 tumor cells.
6 The effect of the combination of glucose and insulin at different concentrations on the secretion and genetic transcription of VEGF, TGF-β1 and IL-6 of Colon26 tumor cells was neither synergistic nor antagonistic.
7 That the different concentrations of glucose and insulin in the natural course of diabetes could increase significantly the secretion of some immunosuppressive substances from the colorectal cancer cells should be one of the internal molecular mechanism with which the diabetes patients easily suffer from the colorectal cancer.
引文
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2 Kim ES, Kim MS, Moon A. Transforming growth factor (TGF)-beta in conjunction with H-ras activation promotes malignant progression ofMCF10A breast epithelial cells. Cytokine,2005,29(2): 84-91
3 Pockaj BA, Basu GD, Pathangey LB, et al. Reduced T-cell and dendritic cell function is related to cyclooxygenase-2 over-Expression and prostaglandin E2 secretion in patients with breast cancer. Ann Surg Oncol, 2004, 11(3): 328-339
4 Sredni B, Weil M, Khomenok G, et al. Ammonium trichloro (dioxoethyene- o, o’) tellurate (AS101) sensitizes tumors to chemotherapy by inhibiting the tumor interleukin 10 autocrine loop. Cancer Res, 2004, 64(5): 1843-1852
5 Wei-Qing Xu , Xu-Cheng Jiang , Lin Zheng , et al. Expression of TGF-β1, TβRII and Smad4 in colorectal carcinoma. Experimental and Molecular Pathology, 2007,82:284-291
6 Bairey O, Boycov O, Kaganovsky E, et al. All three receptors for vascular endothelial growth factor (VEGF) are expressed on B-chronic lymphocytic leukemia(CLL) cells. Leuk Res, 2004, 28(3)221-222
7 Weber F, Byrne SN, Le S, et al. Transforming growth factor-beta1 immobilises dendritic cells within skin tumours and facilitates tumour escape from the immune system. Cancer Immunol Immunother, 2005, 54(9): 898-906
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9 Hajos S, Waldner C. IL-2, IL-10, IL-15 and TNF are key regulators of murine T-cell lymphoma growth. Int J Mol Med, 2003, 12(4): 627-632
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