AGEs对人结肠癌细胞SW-480增殖的影响及罗格列酮干预作用的研究
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摘要
[背景]
随着社会经济的发展和人民生活水平的提高,糖尿病的患病率日渐增高。糖尿病所引起的一系列急、慢性并发症极大地降低了糖尿病患者的生活质量和生存率。目前,全球糖尿病患者人数已达到2.5亿,而这个数字在未来的20年内将上升至3.8亿。因此,糖尿病已成为一种严重威胁全球人类健康的疾病。
近年来,大量流行病学研究发现,糖尿病患者结直肠癌的发病率显著高于非糖尿病人群。糖尿病与结直肠癌的发生可能存在着密切联系。有学者认为晚期糖基化终产物(advanced glycation end products, AGEs)与其受体RAGE的相互作用可能是糖尿病患者包括结直肠癌在内的各种恶性肿瘤发病率增加的重要机制之一。
AGEs为还原糖如葡萄糖等的羰基与蛋白质、脂质的游离氨基端,通过非酶糖基化作用(Mailard反应)形成可逆的Schiff碱,并经一系列的分子重排形成酮氨类化合物(Amadori products),再进一步脱水和凝聚,形成不可逆的终产物。糖尿病的高血糖状态会促使糖基化进程加快,导致体内AGEs蓄积。在机体的组织和细胞中均存在AGEs受体,其中RAGE是研究最为深入的AGEs受体。以往研究认为,AGEs与RAGE结合后可启动一系列受体后信号转导通路,导致多种细胞因子与生长因子的合成与释放,引起血管内皮损伤、血流动力学和血液流变学改变、细胞基质异常增生等病理变化,从而参与了糖尿病慢性并发症的发生和发展。
目前研究认为,AGEs-RAGE途径还与肿瘤形成相关,其可能机制是AGEs通过与RAGE相互作用促进氧化应激的产生,导致DNA损伤,而氧化应激反过来又促进AGEs的形成并上调RAGE的表达；另外,AGEs-RAGE介导的ROS水平增多可以激活多条与细胞增殖和凋亡相关的信号通路,最终促进了癌细胞的生长。目前,关于AGEs-RAGE途径与结直肠癌之间的研究较少,因此有必要进行更多、更深入的研究。
近年来,尽管糖尿病的治疗和癌症的关系引发了一些争议,甚至有流行病学研究提示胰岛素和磺脲类药物可能增加患癌症风险。但是,另外一些研究却发现：罗格列酮作为一种胰岛素增敏剂除了可以降血糖外,还具备抑制肿瘤细胞增殖的作用。
罗格列酮是PPAR-γ的一种合成配体,作为PPAR-γ激动剂,它除了可以增加胰岛素敏感性、调节血脂、抗炎等作用外,它和其他PPAR-γ配体在肿瘤发生、发展中的作用受到了广泛的关注。在多种恶性肿瘤细胞(肺癌、胃癌、结肠癌、乳腺癌、肝癌、胰腺癌等)实验研究中,发现PPAR-γ配体可以通过促进细胞分化、细胞周期阻滞,诱导细胞凋亡,抑制肿瘤血管形成和下调基质金属蛋白酶以抑制肿瘤细胞的侵袭性等机制发挥抗癌作用。目前,关于PPAR-γ与结直肠癌的关系尚存分歧。尽管大多数研究肯定了PPAR-γ在结直肠肿瘤的抑制作用,但也有部分实验得出了与之相反的结果。因此,需要更多、更细致的研究来证实PPAR-γ在结直肠肿瘤中的作用。
既往研究提示,PPAR-γ激动剂可以抑制.AGEs诱导的促动脉粥样硬化和促炎作用。在血管平滑肌细胞、脐静脉内皮细胞的研究中,有学者发现PPAR-γ激动剂可以通过下调RAGE的表达来抑制AGEs-RAGE途径,从而减轻AGEs引起的促动脉粥样硬化作用；在人单核细胞源树突状细胞(DC)中,罗格列酮可以减轻AGEs的促T淋巴细胞增殖作用,抑制了DC细胞因子IL-10、IL-12、INF-γ的分泌,从而发挥了抗炎作用。那么,在结肠癌细胞中,罗格列酮是不是也可以抑制.AGEs诱导的促增殖作用呢?目前鲜见报导。
因此,本研究拟采用人结肠癌细胞SW-480,观察罗格列酮和AGEs对其增殖的影响,并探讨其可能机制。为糖尿病患者结肠癌的防治提供理论依据。
本研究包括以下三部分：
第一章AGEs对人结肠癌细胞SW-480增殖的影响及机制研究
[目的]
观察不同浓度AGEs对人结肠癌细胞SW-480细胞增殖的影响,并探讨其可能机制。
[研究对象和方法]
1、以人结肠癌细胞株SW-480细胞为实验对象；SW-480细胞株培养于含37℃、5%CO2、10%胎牛血清、1%青链霉素的RPMI 1640完全培养基中,隔天传代1次。
2、体外制备AGEs修饰牛血清白蛋白(AGE-BSA)和BSA：将BSA与葡萄糖混合于37℃孵育60天,4℃透析24h以去除未结合的葡萄糖。体外制备的AGE-BSA经荧光分光光度计鉴定,AGEs含量为102.67U／mg蛋白,BSA对照为12.84U／mg。
3、MTT测细胞活力情况：取生长对数期的细胞消化成单细胞,以细胞数3000个／孔接种至96孔培养板,每孔100ul。细胞贴壁后以无血清培养基继续培养24h,然后进行分组干预,分为正常对照组、AGEs组(100ug/ml、200ug/ml、500ug/ml)组、BSA组(500ug/ml)组,并设置空白对照组(培养液中无细胞),每组7复孔,孵育24h后,每孔加MTT (4mg/ml) 20ul,继续孵育4h,弃上清,加150ulDMSO,振荡10分钟后用酶标仪上测各孔490nm处吸光值(OD值)。
4、流式细胞术测细胞周期：取生长对数期的细胞消化,接种到6孔板中,细胞贴壁后以无血清RPMI1640培养液培养24h后进行干预,分为正常对照组和AGEs组(200ug/ml),继续培养24h后消化收集细胞,预冷PBS洗两遍,70%预冷乙醇4℃固定过夜,固定后细胞用PBS洗两遍,离心弃PBS,加入PI染液(碘化丙啶及RNase A终浓度均为50ug/ml)避光染色30 min,进行流式细胞仪检测。
5、实时荧光定量PCR法测定Cyclin D1 mRNA和RAGE mRNA的表达：收取对照组及200ug/mlAGEs组干预24h后的细胞。Trizol法提取细胞总RNA,实时定量PCR检测细胞中RAGE mRNA和cyclin D1 mRNA表达情况。以β-actin作为内参。
6、数据以均数±标准差表示,应用SPSS 13.0软件进行统计分析。多组间数据的比较采用单向方差分析,若方差不齐采用Welch近似方差分析；多重比较采用LSD法(Least-significant difference test),如果方差不齐采用Dunnett's T3检验。两个独立样本比较采用t检验。P<0.05被认为差异具有统计学意义。
[结果]
1、细胞活力的比较：与正常对照组比较,100ug/ml、200ug/ml、500ug/ml AGEs分别作用SW-480细胞24h后均可以显著促进人结肠癌细胞SW-480的增殖(F=42.007,P<0.05),这种促进作用呈浓度依赖性(P<0.05)。
2、细胞周期的比较：流式细胞术结果示,200ug/ml AGEs干预SW-480细胞24h后,正常对照组和AGEs组G1期百分率分别为43.558±1.089、36.640±1.068,两组比较有统计学差异(t=10.143,P<0.001)；S期百分率分别为33.960±1.701、44.146±5.440,两组间有统计学差异(t=-3.996,P=0.011)；G2／M期百分率分别为22.482±0.946、19.214±4.393,差异无统计学意义(t=1.626,P=0.173)。
3、Cyclin D1 mRNA的表达：实时荧光定量PCR示：Cyclin D1 mRNA表达量为正常对照组的1.585倍(P<0.05)。
4、RAGE mRNA的表达：实时荧光定量PCR示：RAGE mRNA表达量为正常对照组的1.94倍(P<0.05)。
[结论]
1、AGEs可以促进SW-480细胞的增殖,促进作用呈浓度依赖性。
2、AGEs可以促进SW-480细胞RAGE和Cyclin D1的表达,加速细胞G1期向S期转换,进而促进了SW-480细胞的增殖。
第二章罗格列酮对人结肠癌细胞SW-480增殖的影响及机制研究[目的]
观察不同浓度罗格列酮对人结肠癌细胞SW-480细胞增殖的影响,并探讨其可能机制。
[研究对象和方法]
1、以人结肠癌SW-480细胞为实验对象,培养方法同前；
2、罗格列酮母液的配制方法：取10mg罗格列酮粉末,加入560ul DMSO中溶解,以50mmol/L浓度-20℃保存。
3、MTT测细胞增殖情况：实验分组为：正常对照组、罗格列酮组(0.1umol/L、1umol/L、10umol/L)组。具体检测方法同第一章。
4、流式细胞术测细胞周期：实验分组为：正常对照组、罗格列酮组(1umol/L)组。具体检测方法同第一章。
5、实时荧光定量PCR法测定Cyclin D1 mRNA表达：实验分组为：正常对照组、罗格列酮组(1umol/L)组。具体检测方法同第一章。
6、数据以均数±标准差表示,应用SPSS13.0软件进行统计分析。多组间数据的比较采用单向方差分析,若方差不齐采用Welch近似方差分析；多重比较采用LSD法,如果方差不齐采用Dunnett's T3检验。两个独立样本比较采用t检验。P<0.05被认为差异具有统计学意义。
[结果]
1、细胞活力的比较：0.1umol/L、1umol/L、10umol/L罗格列酮分别作用于SW-480细胞24h后均可以抑制人结肠癌细胞的活力,这种抑制作用呈浓度依赖性(F=34.339,P<0.05)。其中0.1umol/L罗格列酮对细胞的抑制作用与正常对照组比较无统计学差异(P=0.395)；1umol/L和10umol/L罗格列酮组与正常对照组比较均有统计学差异(P<0.05)
2、细胞周期的比较：流式细胞术结果示,1umol/L罗格列酮干预SW-480细胞24h后,正常对照组和罗格列酮组G1期百分率分别为43.558±1.089、50.488±1.773,两组间有统计学差异(t=-7.448,P<0.001)；S期百分率分别为33.96±1.701、26.594±2.162,两组间有统计学差异(t=5.988,P<0.001)；G2／M期百分率分别为22.482±0.946、22.918±0.728,差异无统计学意义(t=-8.17,P=0.438)。
3、CyclinD1 mRNA的表达：实时荧光定量PCR示：Cyclin D1 mRNA表达量为正常对照组的0.825倍。(P<0.05)
4、RAGE mRNA的表达：实时荧光定量PCR示：RAGE mRNA表达量为正常对照组的0.809倍(P<0.05)。
[结论]
1、罗格列酮可以抑制SW-480细胞的增殖,抑制作用呈浓度依赖性。
2、罗格列酮可以抑制SW-480细胞Cyclin D1的表达,减慢细胞G1期向S期转换,进而抑制了SW-480细胞的增殖。
第三章罗格列酮对AGEs诱导的人结肠癌细胞SW-480增殖的影响及机制研究
[目的]
观察罗格列酮对AGEs诱导的人结肠癌细胞SW-480细胞增殖的影响,并探讨其可能机制。
[研究对象和方法]
1、以SW-480为实验对象,培养方法同前；
2、体外制备AGEs修饰牛血清白蛋白(AGE-BSA)同第一章。
3、实验分组为：正常对照组、200ug/mlAGEs组,200ug/ml AGEs+1umol/L罗格列酮组。
4、各组在干预24h后,MTT法测定细胞活力,流式细胞术检测细胞周期,实时荧光定量PCR法测定CyclinD1 mRNA、RAGE mRNA的表达,检测方法同第一章；
5、数据以均数±标准差表示,应用SPSS13.0软件进行统计分析。多组间数据的比较采用单向方差分析,若方差不齐采用Welch近似方差分析；多重比较采用LSD法,如果方差不齐采用Dunnett's T3检验。P<0.05被认为差异具有统计学意义。
[结果]
1、细胞活力的测定：与正常对照组比较,200ug/mlAGEs能显著促进SW-480细胞的增殖(P<0.05)；1umol/L罗格列酮+200ug/mlAGEs组与AGEs组比较可以显著抑制SW-480细胞的增殖(P<0.05)。
2、细胞周期的测定：正常对照组、200 ug/mlAGEs;组、1umol/L罗格列酮+200ug/mlAGEs组培养24h后G1期百分率分别为55.81±0.63,36.640-±1.068,40.366±1.281,三组间存在统计学差异(F=45.329,P<0.05),其中AGEs组与正常对照组比较有显著统计学差异(P<0.05),罗格列酮+AGEs组与AGEs组比较有显著统计学差异(P<0.05)；S期百分率分别为33.960±1.701,44.146±5.440,35.340±3.472,三组间存在显著统计学差异(F=10.282,P<0.05)；其中罗格列酮+AGEs组与AGEs组比较有显著统计学差异(P<0.05)；G2／M期百分率分别为22.482±0.946,19.214±4.393,23.334±2.981,组间无统计学差异(F=2.194,P=0.192)。
3、Cyclin D1 mRNA:AGEs组Cyclin D1 mRNA的表达量约为正常对照组的1.588倍(P<0.05)；罗格列酮+AGEs组Cyclin Dl mRNA的表达量约为正常对照组的1.242倍(P<0.05)；罗格列酮+AGEs组与AGEs组Cyclin D1 mRNA的表达量比较具有统计学差异(t=6.752,P<0.001)。
4、RAGE mRNA:AGEs组RAGE mRNA的表达量约为正常对照组的1.937倍(P<0.05)；罗格列酮+AGEs组RAGE mRNA的表达量约为正常对照组的1.518倍(P<0.05)；罗格列酮+AGEs组与AGEs组RAGE mRNA的表达量比较具有统计学差异(t=7.510,P<0.001)。
[结论]
1、罗格列酮可以抑制AGEs诱导的人结肠癌细胞SW-480的促增殖作用。
2、罗格列酮可以抑制AGEs诱导的Cyclin D1和RAGE的上调作用,进而通过细胞周期调控,减慢细胞G1期向S期转换,最终抑制了SW-480细胞的增殖。
[BACKGROUND]
With the development of socio-economic and people's living standards, the prevalence of diabetes increased Gradually. Diabetes lead to a series of acute and chronic complications of diabetes significantly reduces the quality of life and survival. Currently, the global number of diabetics has reached 250 million, a figure in the next 20 years will rise to 380 million.Therefore, diabetes has become a serious health problem threat to the world.
In recent years, a large number of epidemiological studies found that diabetes incidence rates of colorectal cancer was significantly higher than non-diabetic population. Diabetes and the occurrence of colorectal cancer may have close ties. Some scholars believe that advanced glycation end products (AGEs) interaction with its receptor RAGE is one of the most important mechanisms to increase the incidence of cancer in diabetic patients.
AGEs are reducing sugar such as glucose and protein carbonyl, lipid free N-terminal, through non-enzymatic glycosylation (Mailard reaction) to form a reversible Schiff base and through a series of molecular rearrangements to form stable ammonia ketone compounds (Amadori products), further dehydration and condensation, the formation of irreversible AGEs. When the body aging or high blood sugar diabetes glycosylation state of the process will lead to accelerated, leading to accumulation of AGEs in vivo. In the body's tissues and cells exist in both AGEs receptors, including RAGE is the most in-depth study of AGEs receptor. Previous studies that, AGEs and RAGE may start a series of combined receptor signaling pathway, leading to a variety of cytokines and growth factor synthesis and release, causing endothelial damage, changes in hemodynamics and blood rheology, cell pathological changes such as abnormal proliferation of matrix, thus contributing to the occurrence of chronic complications of diabetes and development.
Current studies suggest that, AGEs-RAGE pathway may also be associated with tumor formation, the Mechanism may be that AGEs interaction with RAGE first and promote the generation of oxidative stress, leading to DNA damage, and oxidative stress, in turn, promote the formation of AGEs cause RAGE expression increased; addition, AGEs-RAGE-mediated increase in ROS levels can activate a number of cell proliferation and apoptosis related signal pathway, ultimately promote the growth of cancer cells. Currently, the research on relationship between AGEs-RAGE pathway and colorectal cancer are seldom, so it need for more and more in-depth study.
In recent years, although the view of the treatment of diabetes may lead to suffer cancer is caused some controversy, even epidemiological studies suggest that insulin and sulfonylureas may increase cancer risk, but other studies have found that: Rosiglitazone as an insulin sensitizer in addition to hypoglycemic can have anticancer effects.
Rosiglitazone is a synthetic PPAR-γligands as PPAR-γagonists, which not only can increase insulin sensitivity, regulating blood fat, anti-inflammatory and other effects. The role of Rosiglitazone and other PPAR-γligands on cancer developing being a concern. In a variety of malignant cells (lung, stomach, breast, liver, pancreas, etc.) experimental study, found that PPAR-y ligands can promote cell differentiation, cell cycle arrest, inhibition of tumor cell proliferation and induce apoptosis and inhibit tumor angiogenesis and reduced matrix metalloproteinases to inhibit tumor cell invasiveness to antitumor. Currently, PPAR-y on the relationship between colorectal cancer and the remaining differences. Although most studies confirmed that PPAR-y in the inhibition of colorectal cancer, but there are some experiments obtained with completely opposite results. Therefore, more and more detailed studies need to verify the PPAR-y in colorectal carcinogenesis.
Previous studies suggested that in vascular smooth muscle cells, umbilical vein endothelial cells, PPAR-y agonists can down the expression of RAGE and other ways to inhibit the AGEs-RAGE system to play the role of anti-atherosclerosis; in human monocyte-derived dendritic cells, rosiglitazone can reduce AGEs in promoting the role of T lymphocyte proliferation, inhibition of the DC cytokines IL-10, IL-12, INF-y secretion, which play a role in the anti-inflammatory. Therefore, is rosiglitazone can suppress the proliferation effect which AGEs-induced in colon cancer cells? These study is seldom.
This study used human colon cancer cells SW-480, observate the proliferation of SW-480 cell when treated with rosiglitazone and AGEs, and to explore its possible mechanism. The search will provide a scientific basis for the prevention and treatment of colon cancer in diabetes.
The research content includes three aspects:
Chapter 1 Effects and mechanism of AGEs on proliferation of human colon carcinoma cell line SW-480
[Objective]
To observe the effects of different concentration of AGEs on proliferation of SW-480 cells, and to explore its possible mechanism.
[Methods]
1、The experiment object was human colon carcinoma cell line SW-480.SW-480 cells were cultured in RPMI1640 containing 10% fetal bovine serum (FBS),100ug/ml penicillin and 100ug/ml streptomycin at 37℃,the cells were passaged every other day.
2、Preparation of BSA and AGEs:AGEs were prepared according to previously described methods. Briefly,BSA and AGEs were prepare by incubation of 3.55mg/ml BSA in the presence or absence of the glucose and 0.5mmol/L sodium azide in PBS(PH7.4) at 37℃for 8 weeks. And then they were dialyzed against 1×PBS for 24h to remove unbound sugars,at last they were identified by spectrofluorometer. The specimen of AGEs was shown 102.67U/mg protein, while that of BSA was shown as 12.84U/mg protein.
3、Cell proliferation assay Cells were seeded at 3000 per well in 96-well plates, after the cells'adhesion to wall, they were incuated at RPMI1640 midium for 24 hours, then they were classed into five groups:blank group, control group, AGEs groups(100ug/ml、200ug/ml、500ug/ml), BSA group(500ug/ml)。every group contains seven wells and corresponding medium, the medium was changed every day. Every 24 hours,the cells of one 96-well plate were added MTT, after incubating for four hours, removed the medium and added DMSO, then measure the OD after ten minute's vibration. Draw the growth curve according the average OD of every day.
4、Cell cycle analyzed by FCM cells were seeded at six-well plates, after the cells'adhesion to wall, they were incuated at RPMI1640 midium for 24 hours,then the cells were incubated at midum with 200ug/ml AGEs and midum with the same volume of complete medium for 24h, after the incubation,the cells were digested into monoplasts, after being washed twice with PBS,fixed the cells in cold 70% alchhol overnight, then removed the alchhol and washed the cells with PBS twice, added the PI staining solution(the final concentration of PI and RNAase A were both 50ug/ml), after 30 minutes away from the light, analyze the results with FCM.
5、Real-time quantitative PCR method and CyclinD1 mRNA expression of RAGE mRNA:the control group and 200ug/mlAGEs group received intervention in the cells after 24h. Trizol extraction of total cellular RNA, real-time quantitative PCR detection of cells in RAGE mRNA and cyclin D1 mRNA expression. Toβ-actin as internal.
6、All values were represented by means±standard deviation. Statistics analysis was performed by using analysis of variance of factorial design in SPSS 13.0 software. The differences among groups were analyzed by One-ANOVA or t-test.The received level of significance is p<0.05.
[Results]
1、MTT comparison:100ug/ml,200ug/ml,500ug/ml AGEs were stimulated 24h after SW-480 cells were significantly promote the proliferation of human colon cancer cells(F=42.007, P＜0.05), This promotion effect is concentration dependent (P<0.05).
2、The proportion of the cells at G0/G1 stage of control group and expriment group(200ug/ml AGEs 24h) were 43.558±1.089,36.640±1.068 respectively (t=10.143, P＜0.001); while the cells at S stage were 33.960±1.701, 44.146±5.440 respctively (t=-3.996, P=0.011) and at G2/M stage were 22.482±0.946,19.214±4.393 respectively (t=1.626, P=0.173)
3、CyclinD1 mRNA expression:Real-time quantitative PCR said:Cyclin D1 mRNA expression level was 1.585 times than the normal control group (P <0.05).
4、RAGE mRNA expression:Real-time quantitative PCR said:RAGE mRNA expression was 1.94 times than the normal control group (P<0.05).
[Conclusion]
1、AGEs was significantly promote the proliferation of SW-480 cells with concentration dependence.
2、AGEs can significantly increases the expression of RAGE mRNA and CyclinDl mRNA on SW-480 cell, and then accelerate SW-480 cells in G1 phase to S phase transition so that promote the proliferation of SW-480 cells
Chapter 2 Effects and mechanism of rosiglitazone on proliferation of human colon carcinoma cell line SW-480
[Objective]
To observe the effects of different concentration of rosiglitazone on proliferation of SW-480 cells, and to explore its possible mechanism.
[Methods]
1、The experiment object was human colon carcinoma cell line SW-480.SW-480 cells were cultured in RPMI1640 containing 10% fetal bovine serum (FBS),100ug/ml penicillin and 100ug/ml streptomycin at 37℃,the cells were passaged every other day.
2、Rosiglitazone liquor of the preparation method:Take 10mg rosiglitazone powder, dissolved in 560ul DMSO added to 50mmol/L concentration of-20℃save.
3、Cell proliferation assay they were classed into two groups:control group, rosiglitazone groups(lummol/L)。The specific detection methods are the same as first chapter.
4、Cell cycle analyzed by FCM they were classed into two groups:control group, rosiglitazone groups(lummol/L)。The specific detection methods are the same as first chapter.
5、Real-time quantitative PCR method and the expression of CyclinD 1 mRNA they were classed into two groups:control group, rosiglitazone groups(1ummol/L)。The specific detection methods are the same as first chapter.
6、All values were represented by means±standard deviation. Statistics analysis was performed by using analysis of variance of factorial design in SPSS 13.0 software. The differences among groups were analyzed by One-ANOVA or t-test.The received level of significance is p<0.05.
[Results]
1、MTT comparison:0.1umol/L, lumol/L, 10umol/L rosiglitazone were on the SW-480 cells after 24h were significantly inhibit the activity of human colon cancer cells, this inhibition was concentration-dependent (F=34.339, P<0.05). O.lumol/L of rosiglitazone on cell line compared with the control group no significant statistical difference (P=0.395); lumoll/L and 10umol/L of rosiglitazone have the significant inhibition compared with the control group (P <0.05).
2、The proportion of the cells at G0/G1 stage of control group and expriment group (lumoll/L of rosiglitazone,24h) were 43.558±1.089,50.488±1.773 respectively(t=-7.448, P＜0.001); while the cells at S stage were 33.96±1.701, 26.594±2.162 respctively (t=5.988, P＜0.001) and at G2/M stage were 22.482±0.946,22.918±0.728respectively. (t=-8.17, P=0.438)
3、CyclinD 1 mRNA expression:Real-time quantitative PCR said:Cyclin D1 mRNA expression level was 0.825 times than the normal control group (P <0.05).
4、RAGE mRNA expression:Real-time quantitative PCR said:RAGE mRNA expression level was 0.809 times than the normal control group (P<0.05).
[Conclusion]
1、rosiglitazone was significantly inhibit the proliferation of SW-480 cells with concentration dependence.
2、rosiglitazone can significantly inhibit the expression of CyclinD1 mRNA on SW-480 cell, and then Slow down SW-480 cells in G1 phase to S phase transition so that inhibit the proliferation of SW-480 cells.
Chapter 3 Effects and mechanism of rosiglitazone on proliferation of colon carcinoma cell line SW-480 cells induced by AGEs
[Objective]
To observe the effects of rosiglitazone on proliferation of SW-480 cells induced by AGEs, and to explore its possible mechanism.
[Methods]
1、The experiment object was SW-480 cells, they were cultured like the chapter1.
2、prepared AGEs modified bovine serum albumin (AGE-BSA) in vitro which is the same as chapter 1.
3、There had three experimental groups, normal control group,200ug/mlAGEs group,200ug/mlAGEs+1 umol/L rosiglitazone groups at the same time for 24 hours.
4、MTT for cell viability, cell cycle, CyclinD1 mRNA, RAGE mRNA detection, detection is the same as chapter 1.
5、All data are present as means and standard error(SE) of multiple measurements. Statistical analyses were carried out with the SPSS 13.0. One-way ANOVA was used for the differences between the groups,and LSD was used for the differences of the two groups. The received level of significance is P<0.05.
[Results]
1、According to the results of cell viability, comparied with normal control group, 200ug/mlAGEs group can significantly promote the proliferation of SW-480 cells (P<0.05); comparied with AGEs group, lumol/L rosiglitazone+200 ug/ mlAGEs group can significantly inhibit the proliferation of SW-480 cells (P <0.05).
2、According to the results of the cell cycle:after culture for 24h, the percentage of G1 phase of control group,200ug/ml AGEs group, lumol/L rosiglitazone+200 ug/mlAGEs group were 55.81±0.63,36.640±1.068,40.366±1.281, respectively. among the three groups were significantly different (F=45.329, P<0.05), which AGEs group compared with the control group there was a significant difference (P<0.05),.comared to AGEs group, rosiglitazone+AGEs group had significant statistical difference (P＜0.05); the percentage of S phase were 33.960±1.701,44.146±5.440,35.340±3.472, among the three groups were statistically significant difference(F= 10.282, P<0.05);which rosiglitazone +AGEs group compared with AGEs group had significant statistical difference (P＜0.05); the percentage of G2/M phase were 22.482±0.946,19.214±4.393,23.334±2.981, there was no significant difference (F=2.194, P=0.192).
3、According to the results of Cyclin D1 mRNA:AGEs group of Cyclin D1 mRNA expression was about 1.588 fold to the normal control group (P<0.05); rosiglitazone+AGEs group of Cyclin D1 mRNA expression was about 1.242 fold to the normal control group (P<0.05); the expression of Cyclin Dl in rosiglitazone+AGEs group and AGEs group had significantly difference (t= 6.752, P＜0.001).
4、According to the results of RAGE mRNA:AGEs group of RAGE mRNA expression was about 1.937 fold to the normal control group (P<0.05); rosiglitazone+AGEs group of RAGE mRNA expression was about 1.518 fold to the normal control group (P＜0.05); the expression of RAGE in rosiglitazone+AGEs group and AGEs group had significantly difference (t= 7.510, P＜0.001).
[Conclusion]
1、Rosiglitazone can inhibit the proliferation of human colon cancer SW-480 cells induced by AGEs.
2、rosiglitazone can significantly inhibit the expression of CyclinDl mRNA and RAGE mRNA on SW-480 cell induced by AGEs, and then Slow down SW-480 cells in G1 phase to S phase transition so that inhibit the proliferation of SW-480 cells
随着社会经济的发展和人民生活水平的提高,糖尿病的患病率日渐增高。糖尿病所引起的一系列急、慢性并发症极大地降低了糖尿病患者的生活质量和生存率。目前,全球糖尿病患者人数已达到2.5亿,而这个数字在未来的20年内将上升至3.8亿。因此,糖尿病已成为一种严重威胁全球人类健康的疾病。
近年来,大量流行病学研究发现,糖尿病患者结直肠癌的发病率显著高于非糖尿病人群。糖尿病与结直肠癌的发生可能存在着密切联系。有学者认为晚期糖基化终产物(advanced glycation end products, AGEs)与其受体RAGE的相互作用可能是糖尿病患者包括结直肠癌在内的各种恶性肿瘤发病率增加的重要机制之一。
AGEs为还原糖如葡萄糖等的羰基与蛋白质、脂质的游离氨基端,通过非酶糖基化作用(Mailard反应)形成可逆的Schiff碱,并经一系列的分子重排形成酮氨类化合物(Amadori products),再进一步脱水和凝聚,形成不可逆的终产物。糖尿病的高血糖状态会促使糖基化进程加快,导致体内AGEs蓄积。在机体的组织和细胞中均存在AGEs受体,其中RAGE是研究最为深入的AGEs受体。以往研究认为,AGEs与RAGE结合后可启动一系列受体后信号转导通路,导致多种细胞因子与生长因子的合成与释放,引起血管内皮损伤、血流动力学和血液流变学改变、细胞基质异常增生等病理变化,从而参与了糖尿病慢性并发症的发生和发展。
目前研究认为,AGEs-RAGE途径还与肿瘤形成相关,其可能机制是AGEs通过与RAGE相互作用促进氧化应激的产生,导致DNA损伤,而氧化应激反过来又促进AGEs的形成并上调RAGE的表达；另外,AGEs-RAGE介导的ROS水平增多可以激活多条与细胞增殖和凋亡相关的信号通路,最终促进了癌细胞的生长。目前,关于AGEs-RAGE途径与结直肠癌之间的研究较少,因此有必要进行更多、更深入的研究。
近年来,尽管糖尿病的治疗和癌症的关系引发了一些争议,甚至有流行病学研究提示胰岛素和磺脲类药物可能增加患癌症风险。但是,另外一些研究却发现：罗格列酮作为一种胰岛素增敏剂除了可以降血糖外,还具备抑制肿瘤细胞增殖的作用。
罗格列酮是PPAR-γ的一种合成配体,作为PPAR-γ激动剂,它除了可以增加胰岛素敏感性、调节血脂、抗炎等作用外,它和其他PPAR-γ配体在肿瘤发生、发展中的作用受到了广泛的关注。在多种恶性肿瘤细胞(肺癌、胃癌、结肠癌、乳腺癌、肝癌、胰腺癌等)实验研究中,发现PPAR-γ配体可以通过促进细胞分化、细胞周期阻滞,诱导细胞凋亡,抑制肿瘤血管形成和下调基质金属蛋白酶以抑制肿瘤细胞的侵袭性等机制发挥抗癌作用。目前,关于PPAR-γ与结直肠癌的关系尚存分歧。尽管大多数研究肯定了PPAR-γ在结直肠肿瘤的抑制作用,但也有部分实验得出了与之相反的结果。因此,需要更多、更细致的研究来证实PPAR-γ在结直肠肿瘤中的作用。
既往研究提示,PPAR-γ激动剂可以抑制.AGEs诱导的促动脉粥样硬化和促炎作用。在血管平滑肌细胞、脐静脉内皮细胞的研究中,有学者发现PPAR-γ激动剂可以通过下调RAGE的表达来抑制AGEs-RAGE途径,从而减轻AGEs引起的促动脉粥样硬化作用；在人单核细胞源树突状细胞(DC)中,罗格列酮可以减轻AGEs的促T淋巴细胞增殖作用,抑制了DC细胞因子IL-10、IL-12、INF-γ的分泌,从而发挥了抗炎作用。那么,在结肠癌细胞中,罗格列酮是不是也可以抑制.AGEs诱导的促增殖作用呢?目前鲜见报导。
因此,本研究拟采用人结肠癌细胞SW-480,观察罗格列酮和AGEs对其增殖的影响,并探讨其可能机制。为糖尿病患者结肠癌的防治提供理论依据。
本研究包括以下三部分：
第一章AGEs对人结肠癌细胞SW-480增殖的影响及机制研究
[目的]
观察不同浓度AGEs对人结肠癌细胞SW-480细胞增殖的影响,并探讨其可能机制。
[研究对象和方法]
1、以人结肠癌细胞株SW-480细胞为实验对象；SW-480细胞株培养于含37℃、5%CO2、10%胎牛血清、1%青链霉素的RPMI 1640完全培养基中,隔天传代1次。
2、体外制备AGEs修饰牛血清白蛋白(AGE-BSA)和BSA：将BSA与葡萄糖混合于37℃孵育60天,4℃透析24h以去除未结合的葡萄糖。体外制备的AGE-BSA经荧光分光光度计鉴定,AGEs含量为102.67U／mg蛋白,BSA对照为12.84U／mg。
3、MTT测细胞活力情况：取生长对数期的细胞消化成单细胞,以细胞数3000个／孔接种至96孔培养板,每孔100ul。细胞贴壁后以无血清培养基继续培养24h,然后进行分组干预,分为正常对照组、AGEs组(100ug/ml、200ug/ml、500ug/ml)组、BSA组(500ug/ml)组,并设置空白对照组(培养液中无细胞),每组7复孔,孵育24h后,每孔加MTT (4mg/ml) 20ul,继续孵育4h,弃上清,加150ulDMSO,振荡10分钟后用酶标仪上测各孔490nm处吸光值(OD值)。
4、流式细胞术测细胞周期：取生长对数期的细胞消化,接种到6孔板中,细胞贴壁后以无血清RPMI1640培养液培养24h后进行干预,分为正常对照组和AGEs组(200ug/ml),继续培养24h后消化收集细胞,预冷PBS洗两遍,70%预冷乙醇4℃固定过夜,固定后细胞用PBS洗两遍,离心弃PBS,加入PI染液(碘化丙啶及RNase A终浓度均为50ug/ml)避光染色30 min,进行流式细胞仪检测。
5、实时荧光定量PCR法测定Cyclin D1 mRNA和RAGE mRNA的表达：收取对照组及200ug/mlAGEs组干预24h后的细胞。Trizol法提取细胞总RNA,实时定量PCR检测细胞中RAGE mRNA和cyclin D1 mRNA表达情况。以β-actin作为内参。
6、数据以均数±标准差表示,应用SPSS 13.0软件进行统计分析。多组间数据的比较采用单向方差分析,若方差不齐采用Welch近似方差分析；多重比较采用LSD法(Least-significant difference test),如果方差不齐采用Dunnett's T3检验。两个独立样本比较采用t检验。P<0.05被认为差异具有统计学意义。
[结果]
1、细胞活力的比较：与正常对照组比较,100ug/ml、200ug/ml、500ug/ml AGEs分别作用SW-480细胞24h后均可以显著促进人结肠癌细胞SW-480的增殖(F=42.007,P<0.05),这种促进作用呈浓度依赖性(P<0.05)。
2、细胞周期的比较：流式细胞术结果示,200ug/ml AGEs干预SW-480细胞24h后,正常对照组和AGEs组G1期百分率分别为43.558±1.089、36.640±1.068,两组比较有统计学差异(t=10.143,P<0.001)；S期百分率分别为33.960±1.701、44.146±5.440,两组间有统计学差异(t=-3.996,P=0.011)；G2／M期百分率分别为22.482±0.946、19.214±4.393,差异无统计学意义(t=1.626,P=0.173)。
3、Cyclin D1 mRNA的表达：实时荧光定量PCR示：Cyclin D1 mRNA表达量为正常对照组的1.585倍(P<0.05)。
4、RAGE mRNA的表达：实时荧光定量PCR示：RAGE mRNA表达量为正常对照组的1.94倍(P<0.05)。
[结论]
1、AGEs可以促进SW-480细胞的增殖,促进作用呈浓度依赖性。
2、AGEs可以促进SW-480细胞RAGE和Cyclin D1的表达,加速细胞G1期向S期转换,进而促进了SW-480细胞的增殖。
第二章罗格列酮对人结肠癌细胞SW-480增殖的影响及机制研究[目的]
观察不同浓度罗格列酮对人结肠癌细胞SW-480细胞增殖的影响,并探讨其可能机制。
[研究对象和方法]
1、以人结肠癌SW-480细胞为实验对象,培养方法同前；
2、罗格列酮母液的配制方法：取10mg罗格列酮粉末,加入560ul DMSO中溶解,以50mmol/L浓度-20℃保存。
3、MTT测细胞增殖情况：实验分组为：正常对照组、罗格列酮组(0.1umol/L、1umol/L、10umol/L)组。具体检测方法同第一章。
4、流式细胞术测细胞周期：实验分组为：正常对照组、罗格列酮组(1umol/L)组。具体检测方法同第一章。
5、实时荧光定量PCR法测定Cyclin D1 mRNA表达：实验分组为：正常对照组、罗格列酮组(1umol/L)组。具体检测方法同第一章。
6、数据以均数±标准差表示,应用SPSS13.0软件进行统计分析。多组间数据的比较采用单向方差分析,若方差不齐采用Welch近似方差分析；多重比较采用LSD法,如果方差不齐采用Dunnett's T3检验。两个独立样本比较采用t检验。P<0.05被认为差异具有统计学意义。
[结果]
1、细胞活力的比较：0.1umol/L、1umol/L、10umol/L罗格列酮分别作用于SW-480细胞24h后均可以抑制人结肠癌细胞的活力,这种抑制作用呈浓度依赖性(F=34.339,P<0.05)。其中0.1umol/L罗格列酮对细胞的抑制作用与正常对照组比较无统计学差异(P=0.395)；1umol/L和10umol/L罗格列酮组与正常对照组比较均有统计学差异(P<0.05)
2、细胞周期的比较：流式细胞术结果示,1umol/L罗格列酮干预SW-480细胞24h后,正常对照组和罗格列酮组G1期百分率分别为43.558±1.089、50.488±1.773,两组间有统计学差异(t=-7.448,P<0.001)；S期百分率分别为33.96±1.701、26.594±2.162,两组间有统计学差异(t=5.988,P<0.001)；G2／M期百分率分别为22.482±0.946、22.918±0.728,差异无统计学意义(t=-8.17,P=0.438)。
3、CyclinD1 mRNA的表达：实时荧光定量PCR示：Cyclin D1 mRNA表达量为正常对照组的0.825倍。(P<0.05)
4、RAGE mRNA的表达：实时荧光定量PCR示：RAGE mRNA表达量为正常对照组的0.809倍(P<0.05)。
[结论]
1、罗格列酮可以抑制SW-480细胞的增殖,抑制作用呈浓度依赖性。
2、罗格列酮可以抑制SW-480细胞Cyclin D1的表达,减慢细胞G1期向S期转换,进而抑制了SW-480细胞的增殖。
第三章罗格列酮对AGEs诱导的人结肠癌细胞SW-480增殖的影响及机制研究
[目的]
观察罗格列酮对AGEs诱导的人结肠癌细胞SW-480细胞增殖的影响,并探讨其可能机制。
[研究对象和方法]
1、以SW-480为实验对象,培养方法同前；
2、体外制备AGEs修饰牛血清白蛋白(AGE-BSA)同第一章。
3、实验分组为：正常对照组、200ug/mlAGEs组,200ug/ml AGEs+1umol/L罗格列酮组。
4、各组在干预24h后,MTT法测定细胞活力,流式细胞术检测细胞周期,实时荧光定量PCR法测定CyclinD1 mRNA、RAGE mRNA的表达,检测方法同第一章；
5、数据以均数±标准差表示,应用SPSS13.0软件进行统计分析。多组间数据的比较采用单向方差分析,若方差不齐采用Welch近似方差分析；多重比较采用LSD法,如果方差不齐采用Dunnett's T3检验。P<0.05被认为差异具有统计学意义。
[结果]
1、细胞活力的测定：与正常对照组比较,200ug/mlAGEs能显著促进SW-480细胞的增殖(P<0.05)；1umol/L罗格列酮+200ug/mlAGEs组与AGEs组比较可以显著抑制SW-480细胞的增殖(P<0.05)。
2、细胞周期的测定：正常对照组、200 ug/mlAGEs;组、1umol/L罗格列酮+200ug/mlAGEs组培养24h后G1期百分率分别为55.81±0.63,36.640-±1.068,40.366±1.281,三组间存在统计学差异(F=45.329,P<0.05),其中AGEs组与正常对照组比较有显著统计学差异(P<0.05),罗格列酮+AGEs组与AGEs组比较有显著统计学差异(P<0.05)；S期百分率分别为33.960±1.701,44.146±5.440,35.340±3.472,三组间存在显著统计学差异(F=10.282,P<0.05)；其中罗格列酮+AGEs组与AGEs组比较有显著统计学差异(P<0.05)；G2／M期百分率分别为22.482±0.946,19.214±4.393,23.334±2.981,组间无统计学差异(F=2.194,P=0.192)。
3、Cyclin D1 mRNA:AGEs组Cyclin D1 mRNA的表达量约为正常对照组的1.588倍(P<0.05)；罗格列酮+AGEs组Cyclin Dl mRNA的表达量约为正常对照组的1.242倍(P<0.05)；罗格列酮+AGEs组与AGEs组Cyclin D1 mRNA的表达量比较具有统计学差异(t=6.752,P<0.001)。
4、RAGE mRNA:AGEs组RAGE mRNA的表达量约为正常对照组的1.937倍(P<0.05)；罗格列酮+AGEs组RAGE mRNA的表达量约为正常对照组的1.518倍(P<0.05)；罗格列酮+AGEs组与AGEs组RAGE mRNA的表达量比较具有统计学差异(t=7.510,P<0.001)。
[结论]
1、罗格列酮可以抑制AGEs诱导的人结肠癌细胞SW-480的促增殖作用。
2、罗格列酮可以抑制AGEs诱导的Cyclin D1和RAGE的上调作用,进而通过细胞周期调控,减慢细胞G1期向S期转换,最终抑制了SW-480细胞的增殖。
[BACKGROUND]
With the development of socio-economic and people's living standards, the prevalence of diabetes increased Gradually. Diabetes lead to a series of acute and chronic complications of diabetes significantly reduces the quality of life and survival. Currently, the global number of diabetics has reached 250 million, a figure in the next 20 years will rise to 380 million.Therefore, diabetes has become a serious health problem threat to the world.
In recent years, a large number of epidemiological studies found that diabetes incidence rates of colorectal cancer was significantly higher than non-diabetic population. Diabetes and the occurrence of colorectal cancer may have close ties. Some scholars believe that advanced glycation end products (AGEs) interaction with its receptor RAGE is one of the most important mechanisms to increase the incidence of cancer in diabetic patients.
AGEs are reducing sugar such as glucose and protein carbonyl, lipid free N-terminal, through non-enzymatic glycosylation (Mailard reaction) to form a reversible Schiff base and through a series of molecular rearrangements to form stable ammonia ketone compounds (Amadori products), further dehydration and condensation, the formation of irreversible AGEs. When the body aging or high blood sugar diabetes glycosylation state of the process will lead to accelerated, leading to accumulation of AGEs in vivo. In the body's tissues and cells exist in both AGEs receptors, including RAGE is the most in-depth study of AGEs receptor. Previous studies that, AGEs and RAGE may start a series of combined receptor signaling pathway, leading to a variety of cytokines and growth factor synthesis and release, causing endothelial damage, changes in hemodynamics and blood rheology, cell pathological changes such as abnormal proliferation of matrix, thus contributing to the occurrence of chronic complications of diabetes and development.
Current studies suggest that, AGEs-RAGE pathway may also be associated with tumor formation, the Mechanism may be that AGEs interaction with RAGE first and promote the generation of oxidative stress, leading to DNA damage, and oxidative stress, in turn, promote the formation of AGEs cause RAGE expression increased; addition, AGEs-RAGE-mediated increase in ROS levels can activate a number of cell proliferation and apoptosis related signal pathway, ultimately promote the growth of cancer cells. Currently, the research on relationship between AGEs-RAGE pathway and colorectal cancer are seldom, so it need for more and more in-depth study.
In recent years, although the view of the treatment of diabetes may lead to suffer cancer is caused some controversy, even epidemiological studies suggest that insulin and sulfonylureas may increase cancer risk, but other studies have found that: Rosiglitazone as an insulin sensitizer in addition to hypoglycemic can have anticancer effects.
Rosiglitazone is a synthetic PPAR-γligands as PPAR-γagonists, which not only can increase insulin sensitivity, regulating blood fat, anti-inflammatory and other effects. The role of Rosiglitazone and other PPAR-γligands on cancer developing being a concern. In a variety of malignant cells (lung, stomach, breast, liver, pancreas, etc.) experimental study, found that PPAR-y ligands can promote cell differentiation, cell cycle arrest, inhibition of tumor cell proliferation and induce apoptosis and inhibit tumor angiogenesis and reduced matrix metalloproteinases to inhibit tumor cell invasiveness to antitumor. Currently, PPAR-y on the relationship between colorectal cancer and the remaining differences. Although most studies confirmed that PPAR-y in the inhibition of colorectal cancer, but there are some experiments obtained with completely opposite results. Therefore, more and more detailed studies need to verify the PPAR-y in colorectal carcinogenesis.
Previous studies suggested that in vascular smooth muscle cells, umbilical vein endothelial cells, PPAR-y agonists can down the expression of RAGE and other ways to inhibit the AGEs-RAGE system to play the role of anti-atherosclerosis; in human monocyte-derived dendritic cells, rosiglitazone can reduce AGEs in promoting the role of T lymphocyte proliferation, inhibition of the DC cytokines IL-10, IL-12, INF-y secretion, which play a role in the anti-inflammatory. Therefore, is rosiglitazone can suppress the proliferation effect which AGEs-induced in colon cancer cells? These study is seldom.
This study used human colon cancer cells SW-480, observate the proliferation of SW-480 cell when treated with rosiglitazone and AGEs, and to explore its possible mechanism. The search will provide a scientific basis for the prevention and treatment of colon cancer in diabetes.
The research content includes three aspects:
Chapter 1 Effects and mechanism of AGEs on proliferation of human colon carcinoma cell line SW-480
[Objective]
To observe the effects of different concentration of AGEs on proliferation of SW-480 cells, and to explore its possible mechanism.
[Methods]
1、The experiment object was human colon carcinoma cell line SW-480.SW-480 cells were cultured in RPMI1640 containing 10% fetal bovine serum (FBS),100ug/ml penicillin and 100ug/ml streptomycin at 37℃,the cells were passaged every other day.
2、Preparation of BSA and AGEs:AGEs were prepared according to previously described methods. Briefly,BSA and AGEs were prepare by incubation of 3.55mg/ml BSA in the presence or absence of the glucose and 0.5mmol/L sodium azide in PBS(PH7.4) at 37℃for 8 weeks. And then they were dialyzed against 1×PBS for 24h to remove unbound sugars,at last they were identified by spectrofluorometer. The specimen of AGEs was shown 102.67U/mg protein, while that of BSA was shown as 12.84U/mg protein.
3、Cell proliferation assay Cells were seeded at 3000 per well in 96-well plates, after the cells'adhesion to wall, they were incuated at RPMI1640 midium for 24 hours, then they were classed into five groups:blank group, control group, AGEs groups(100ug/ml、200ug/ml、500ug/ml), BSA group(500ug/ml)。every group contains seven wells and corresponding medium, the medium was changed every day. Every 24 hours,the cells of one 96-well plate were added MTT, after incubating for four hours, removed the medium and added DMSO, then measure the OD after ten minute's vibration. Draw the growth curve according the average OD of every day.
4、Cell cycle analyzed by FCM cells were seeded at six-well plates, after the cells'adhesion to wall, they were incuated at RPMI1640 midium for 24 hours,then the cells were incubated at midum with 200ug/ml AGEs and midum with the same volume of complete medium for 24h, after the incubation,the cells were digested into monoplasts, after being washed twice with PBS,fixed the cells in cold 70% alchhol overnight, then removed the alchhol and washed the cells with PBS twice, added the PI staining solution(the final concentration of PI and RNAase A were both 50ug/ml), after 30 minutes away from the light, analyze the results with FCM.
5、Real-time quantitative PCR method and CyclinD1 mRNA expression of RAGE mRNA:the control group and 200ug/mlAGEs group received intervention in the cells after 24h. Trizol extraction of total cellular RNA, real-time quantitative PCR detection of cells in RAGE mRNA and cyclin D1 mRNA expression. Toβ-actin as internal.
6、All values were represented by means±standard deviation. Statistics analysis was performed by using analysis of variance of factorial design in SPSS 13.0 software. The differences among groups were analyzed by One-ANOVA or t-test.The received level of significance is p<0.05.
[Results]
1、MTT comparison:100ug/ml,200ug/ml,500ug/ml AGEs were stimulated 24h after SW-480 cells were significantly promote the proliferation of human colon cancer cells(F=42.007, P＜0.05), This promotion effect is concentration dependent (P<0.05).
2、The proportion of the cells at G0/G1 stage of control group and expriment group(200ug/ml AGEs 24h) were 43.558±1.089,36.640±1.068 respectively (t=10.143, P＜0.001); while the cells at S stage were 33.960±1.701, 44.146±5.440 respctively (t=-3.996, P=0.011) and at G2/M stage were 22.482±0.946,19.214±4.393 respectively (t=1.626, P=0.173)
3、CyclinD1 mRNA expression:Real-time quantitative PCR said:Cyclin D1 mRNA expression level was 1.585 times than the normal control group (P <0.05).
4、RAGE mRNA expression:Real-time quantitative PCR said:RAGE mRNA expression was 1.94 times than the normal control group (P<0.05).
[Conclusion]
1、AGEs was significantly promote the proliferation of SW-480 cells with concentration dependence.
2、AGEs can significantly increases the expression of RAGE mRNA and CyclinDl mRNA on SW-480 cell, and then accelerate SW-480 cells in G1 phase to S phase transition so that promote the proliferation of SW-480 cells
Chapter 2 Effects and mechanism of rosiglitazone on proliferation of human colon carcinoma cell line SW-480
[Objective]
To observe the effects of different concentration of rosiglitazone on proliferation of SW-480 cells, and to explore its possible mechanism.
[Methods]
1、The experiment object was human colon carcinoma cell line SW-480.SW-480 cells were cultured in RPMI1640 containing 10% fetal bovine serum (FBS),100ug/ml penicillin and 100ug/ml streptomycin at 37℃,the cells were passaged every other day.
2、Rosiglitazone liquor of the preparation method:Take 10mg rosiglitazone powder, dissolved in 560ul DMSO added to 50mmol/L concentration of-20℃save.
3、Cell proliferation assay they were classed into two groups:control group, rosiglitazone groups(lummol/L)。The specific detection methods are the same as first chapter.
4、Cell cycle analyzed by FCM they were classed into two groups:control group, rosiglitazone groups(lummol/L)。The specific detection methods are the same as first chapter.
5、Real-time quantitative PCR method and the expression of CyclinD 1 mRNA they were classed into two groups:control group, rosiglitazone groups(1ummol/L)。The specific detection methods are the same as first chapter.
6、All values were represented by means±standard deviation. Statistics analysis was performed by using analysis of variance of factorial design in SPSS 13.0 software. The differences among groups were analyzed by One-ANOVA or t-test.The received level of significance is p<0.05.
[Results]
1、MTT comparison:0.1umol/L, lumol/L, 10umol/L rosiglitazone were on the SW-480 cells after 24h were significantly inhibit the activity of human colon cancer cells, this inhibition was concentration-dependent (F=34.339, P<0.05). O.lumol/L of rosiglitazone on cell line compared with the control group no significant statistical difference (P=0.395); lumoll/L and 10umol/L of rosiglitazone have the significant inhibition compared with the control group (P <0.05).
2、The proportion of the cells at G0/G1 stage of control group and expriment group (lumoll/L of rosiglitazone,24h) were 43.558±1.089,50.488±1.773 respectively(t=-7.448, P＜0.001); while the cells at S stage were 33.96±1.701, 26.594±2.162 respctively (t=5.988, P＜0.001) and at G2/M stage were 22.482±0.946,22.918±0.728respectively. (t=-8.17, P=0.438)
3、CyclinD 1 mRNA expression:Real-time quantitative PCR said:Cyclin D1 mRNA expression level was 0.825 times than the normal control group (P <0.05).
4、RAGE mRNA expression:Real-time quantitative PCR said:RAGE mRNA expression level was 0.809 times than the normal control group (P<0.05).
[Conclusion]
1、rosiglitazone was significantly inhibit the proliferation of SW-480 cells with concentration dependence.
2、rosiglitazone can significantly inhibit the expression of CyclinD1 mRNA on SW-480 cell, and then Slow down SW-480 cells in G1 phase to S phase transition so that inhibit the proliferation of SW-480 cells.
Chapter 3 Effects and mechanism of rosiglitazone on proliferation of colon carcinoma cell line SW-480 cells induced by AGEs
[Objective]
To observe the effects of rosiglitazone on proliferation of SW-480 cells induced by AGEs, and to explore its possible mechanism.
[Methods]
1、The experiment object was SW-480 cells, they were cultured like the chapter1.
2、prepared AGEs modified bovine serum albumin (AGE-BSA) in vitro which is the same as chapter 1.
3、There had three experimental groups, normal control group,200ug/mlAGEs group,200ug/mlAGEs+1 umol/L rosiglitazone groups at the same time for 24 hours.
4、MTT for cell viability, cell cycle, CyclinD1 mRNA, RAGE mRNA detection, detection is the same as chapter 1.
5、All data are present as means and standard error(SE) of multiple measurements. Statistical analyses were carried out with the SPSS 13.0. One-way ANOVA was used for the differences between the groups,and LSD was used for the differences of the two groups. The received level of significance is P<0.05.
[Results]
1、According to the results of cell viability, comparied with normal control group, 200ug/mlAGEs group can significantly promote the proliferation of SW-480 cells (P<0.05); comparied with AGEs group, lumol/L rosiglitazone+200 ug/ mlAGEs group can significantly inhibit the proliferation of SW-480 cells (P <0.05).
2、According to the results of the cell cycle:after culture for 24h, the percentage of G1 phase of control group,200ug/ml AGEs group, lumol/L rosiglitazone+200 ug/mlAGEs group were 55.81±0.63,36.640±1.068,40.366±1.281, respectively. among the three groups were significantly different (F=45.329, P<0.05), which AGEs group compared with the control group there was a significant difference (P<0.05),.comared to AGEs group, rosiglitazone+AGEs group had significant statistical difference (P＜0.05); the percentage of S phase were 33.960±1.701,44.146±5.440,35.340±3.472, among the three groups were statistically significant difference(F= 10.282, P<0.05);which rosiglitazone +AGEs group compared with AGEs group had significant statistical difference (P＜0.05); the percentage of G2/M phase were 22.482±0.946,19.214±4.393,23.334±2.981, there was no significant difference (F=2.194, P=0.192).
3、According to the results of Cyclin D1 mRNA:AGEs group of Cyclin D1 mRNA expression was about 1.588 fold to the normal control group (P<0.05); rosiglitazone+AGEs group of Cyclin D1 mRNA expression was about 1.242 fold to the normal control group (P<0.05); the expression of Cyclin Dl in rosiglitazone+AGEs group and AGEs group had significantly difference (t= 6.752, P＜0.001).
4、According to the results of RAGE mRNA:AGEs group of RAGE mRNA expression was about 1.937 fold to the normal control group (P<0.05); rosiglitazone+AGEs group of RAGE mRNA expression was about 1.518 fold to the normal control group (P＜0.05); the expression of RAGE in rosiglitazone+AGEs group and AGEs group had significantly difference (t= 7.510, P＜0.001).
[Conclusion]
1、Rosiglitazone can inhibit the proliferation of human colon cancer SW-480 cells induced by AGEs.
2、rosiglitazone can significantly inhibit the expression of CyclinDl mRNA and RAGE mRNA on SW-480 cell induced by AGEs, and then Slow down SW-480 cells in G1 phase to S phase transition so that inhibit the proliferation of SW-480 cells
引文
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[2]Larsson S C, Orsini N, Wolk A. Diabetes mellitus and risk of colorectal cancer:a meta-analysis.[J]. J Natl Cancer Inst,2005,97(22):1679-1687.
[3]Michels K B, Solomon C G, Hu F B, et al. Type 2 diabetes and subsequent incidence of breast cancer in the Nurses'Health Study.[J]. Diabetes Care,2003,26(6):1752-1758.
[4]Wang F, Herrington M, Larsson J, et al. The relationship between diabetes and pancreatic cancer.[J]. Mol Cancer,2003,2:4.
[5]El-Serag H B, Hampel H, Javadi F. The association between diabetes and hepatocellular carcinoma:a systematic review of epidemiologic evidence.[J]. Clin Gastroenterol Hepatol,2006,4(3):369-380.
[6]Ren X, Zhang X, Zhang X, et al.Type 2 diabetes mellitus associated with increased risk for colorectal cancer:evidence from an international ecological study and population-based risk analysis in China. Public Health. 2009123(8):540-4.
[7]Seow A, Yuan J M, Koh W P, et al. Diabetes mellitus and risk of colorectal cancer in the Singapore Chinese Health Study.[J]. J Natl Cancer Inst,2006,98(2):135-138.
[8]Larsson S C, Giovannucci E, Wolk A. Diabetes and colorectal cancer incidence in the cohort of Swedish men.[J]. Diabetes Care,2005, 28(7):1805-1807.
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