| [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 cellsChapter 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...
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