FOXP3在胃癌细胞中的表达及其相关功能的初步研究
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摘要
目的:FOXP3(Transcription factor forkhead box protein 3)因具有重要的免疫抑制调节功能目前被认为是调节性T细胞(Regulatory T cells;以下简称:Tregs)功能特异性转录因子。最近,人们采用肿瘤组织显微切割等技术研究发现胰腺癌及其它肿瘤细胞本身也存在FOXP3的表达。虽然,经过统计学分析并没有发现FOXP3的表达与肿瘤组织的分化有相关性,但研究人员使用抗体中和TGF-β2后可以抑制胰腺癌细胞中FOXP3的表达,同时,应用干扰的方法抑制FOXP3的表达后可以上调IL-6和IL-8的分泌。以上这些发现提示FOXP3有可能在肿瘤免疫逃逸中占有重要地位。目前尚未见FOXP3在胃癌肿瘤细胞中的表达及其生物学功能研究的相关报道。故本课题通过研究分析胃癌细胞和组织中FOXP3的表达和定位来探讨FOXP3的表达与胃癌肿瘤组织生长和分化之间的关系;建立稳转过表达FOXP3的SGC-7901胃癌细胞系,初步研究FOXP3的高表达对胃癌细胞免疫耐受和免疫逃逸等生物学活性的影响。方法:首先,我们通过以下的实验方法分别对胃癌细胞和组织中FOXP3的表达进行了检测和分析:(1)通过RT-PCR、免疫印迹、流式细胞术和激光共聚焦等实验技术在不同水平上检测FOXP3在胃癌细胞中的表达和定位;(2)采用免疫组化方法研究FOXP3在胃癌组织中的表达和定位;(3)经统计学分析验证FOXP3的表达与胃癌组织分化之间的关系。其次,我们采用稳定转染SGC-7901胃癌细胞系的方法建立过表达FOXP3的肿瘤细胞模型,通过以下实验初步研究胃癌细胞高表达FOXP3后相关生物学功能的变化:(1)扫描电镜(Scanning Electron Microscope,SEM)和透射电镜(Transmission Electron Microscope,TEM)从形态学上观察过表达FOXP3对胃癌细胞的影响;(2)MTT实验检测过表达FOXP3后,胃癌细胞的增殖变化;(3)流式细胞术检测过表达FOXP3后,胃癌细胞的周期变化;(4)RT-PCR方法检测过表达FOXP3后,胃癌细胞中免疫抑制性膜表面分子的转录;(5)ELISA实验检测高表达FOXP3后,胃癌细胞分泌免疫抑制分子TGF-β的变化;(6)通过与外周血混合单核淋巴细胞共培养的方法研究过表达FOXP3后,胃癌细胞对外周血单核淋巴细胞的抑制作用(;7)使用化疗药物杀伤过表达FOXP3的胃癌细胞,检测胃癌细胞耐药性的变化。
结果:通过以上实验技术和方法我们获得了以下的实验结果:我们通过胃癌细胞系和胃癌组织的研究证实了胃癌细胞和组织内均存在不同水平的FOXP3的表达:(1)在多个胃癌细胞系中发现了Foxp3的mRNA转录及其蛋白的表达;(2)激光共聚焦实验证实,胃癌细胞中FOXP3蛋白的表达主要分布在肿瘤细胞核以及核周的胞浆内;(3)通过免疫组化染色发现胃癌细胞中FOXP3蛋白的表达主要分布在肿瘤细胞核,少数分布在胞浆内;(4)经过统计学分析胃癌组织切片和组织芯片的免疫组化染色结果,我们发现56%以上的胃癌患者肿瘤细胞中存在FOXP3的表达,且胃癌组织的分化与FOXP3蛋白表达的强度呈线性相关,同时,在正常胃组织和癌旁组织细胞内(不包括Tregs)未发现FOXP3的表达。随后,我们通过构建稳定过表达FOXP3的胃癌细胞系对FOXP3在胃癌细胞中的功能进行了初步的研究(:1)通过RT-PCR的方法,我们从外周血单核细胞中获得了Foxp3的cDNA片段,构建了pRc/CMV-Foxp3表达载体,转染SGC-7901细胞,经G418筛选后,成功建立稳定过表达FOXP3的胃癌细胞株;(2)扫描电镜检测发现与对照组细胞相比较,过表达FOXP3的胃癌细胞表面突起回缩,与周围基质之间的连接减少,透射电镜检测发现过表达FOXP3组细胞内出现腺管样结构,30%-40%出现自噬泡,并且,其胆固醇代谢发生障碍;(3)MTT检测发现过表达FOXP3的胃癌细胞增殖速度减慢;(4)细胞周期实验证实过表达FOXP3的胃癌细胞生长与对照相比5%以上阻止在G1期;(5)经RT-PCR鉴定,FOXP3的转录显著升高;(6)半定量PCR实验证实,肿瘤免疫抑制分子B7-H1和TGF-β的转录均明显升高;(7)通过ELISA检测发现过表达FOXP3的胃癌细胞培养上清中TGF-β1和TGF-β2的表达量升高;(8)体外混合外周血单核淋巴细胞共培养实验发现过表达FOXP3的胃癌细胞显著抑制了外周血单核淋巴细胞的增殖;(9)化疗药物5-氟脲嘧啶杀伤实验发现过表达FOXP3的胃癌细胞耐药性显著提高。讨论:(1)胃癌细胞和组织中普遍存在FOXP3的表达;(2)过表达FOXP3的胃癌细胞表面突起回缩,与周围基质之间的连接减少,生长过程中易于脱落,生长速度减慢,少部分细胞阻滞在G1期,FOXP3的过表达可以诱导肿瘤细胞发生自噬和脂类代谢障碍;(3)这种过表达FOXP3的胃癌细胞具有上调肿瘤免疫抑制和肿瘤免疫逃逸相关蛋白分子表达的功能;(4)虽然,过表达FOXP3的胃癌细胞生长速度缓慢,但与对照组相比,其具有更强的抑制淋巴细胞增殖和耐受化疗药物5-氟脲嘧啶的杀伤功能。综上所述,我们发现FOXP3可能在胃癌的免疫逃逸过程中发挥重要功能。本课题初步研究了FOXP3过表达后胃癌细胞SGC-7901的生长、形态以及免疫抑制功能的变化,为以后FOXP3在胃癌细胞中的作用机理研究奠定基础,从而为胃癌的临床治疗提供新的治疗靶位和免疫治疗策略。
Purpose: Transcription factor forkhead box protein 3 (FOXP3) specifically characterizes the naturally occurring regulatory T cells (Tregs). Recently, people found that pancreatic and other cancer cells could express FOXP3 by itself in microdissection. Although they didn’t find the relationship between the expression of FOXP3 and tumor differentiation, they identified that the expression of FOXP3 was induced by transforming growth factor-β2 (TGF-β2). According to these results, some reports implicated that FOXP3 might represent a tumor escape mechanism. But less is known about the role and biology of FOXP3 in gastric cancer. Our aim was to analyze the expression and initial function of FOXP3 in gastric cancer tissues and cell lines to evaluate the clinical character of FOXP3 in human gastric cancers. Experimental Design: Firstly, we detect the expression of FOXP3 in gastric cancer cells and tissues by methods, listing as below: (1) Detection of Foxp3 mRNA was performed using conventional RT-PCR, while protein expression was assessed by western blot, flow cytometry and confocal in gastric cancer cell lines. (2) Sections of resected human gastric cancer specimens were stained with anti human FOXP3 antibody. (3) FOXP3 staining of the gastric cancer was assessed to analyze the relationship between expression of FOXP3 and tumor differentiation. Secondly, we established FOXP3 stable overexpression SGC-7901 cell line to research the biologic character changes initially. (1) Observation of the morphology modification of FOXP3 overexpression gastric cancer cells was performed by scanning electron microscope (SEM)and transmission electron microscope (TEM). (2) MTT assay and flowcytometry was used to evaluate the proliferation. (3) PI staining to identify the cell cycles of FOXP3 overexpression gastric cancer cells. (4) We detected the inhibitory molecules expression of cancer cells by semi-PCR. (5) ELISA Kits was used to detect the inhibitory molecules of TGF-β. (6) Mixed peripheral blood monocyte and lymphocyte (PBMC) reaction was used to detect the proliferation inhibition effect of cancer cells. (7) Tolerance of FOXP3 overexpression gatric cancer cells to 5- fluorouracil was evaluated by MTT. Results: By previously methods and assays, we got results as below: (1) Foxp3 mRNA and protein was detected in all gastric cancer cell lines. (2) The location of FOXP3 expression was in nuclear and paranuclear of cytoplasm of gastric cancer cell line by confocal. (3) FOXP3 expression was mainly located in nuclear and seldom in cytoplasm in gastric cancer tissues. (4) Immunohistochemistry analysis of gastric cancer sections revealed that >56% of gastric cancer displayed nucleus FOXP3 staining. And it showed a linear relationship between differentiations of gastric cancer tissues and FOXP3 expression intensity. We did not find any FOXP3 staining in normal gastric tissues and para-tumor tissues. After that, we established FOXP3 overexpression gastric cell lines to explore the initial function of FOXP3. (1) After RT-PCR, we acquired the full length of Foxp3 cDNA, and inserted it into pRc/CMV plasmid to establish the vector of pRc/CMV-Foxp3. Screening by G418, we established stable FOXP3 high expression gastric cancer cell lines, SGC-7901. (2) Ecphyma retracted and the connection between cancer cells and ground substance was less than negative control cancer cells. We found glandular tube and autophagic vacuole in FOXP3 overexpression cells. And metabolism of cholesterol is obstructed in these cells. (3) The proliferation was down-regulated in FOXP3 overexpression cancer cells. (4) Few of those cells (5%) were held on G1 stage. (5) RT-PCR identified that Foxp3 was up-regulated in transcription level. (6) Semi-quantity PCR showed that the inhibitory molecules, such as B7-H1 and TGF-β, were up-regulated in FOXP3 overexpression cancer cells. (7) ELISA assay find that TGF-β1 and TGF-β2 were also higher in supernatant of culture medium of FOXP3 overexpression cancer cells. (8) Cocultured with mixed PBMC, the proliferation of lymphocytes was down regulated. (9) The tolerance of FOXP3 overexpression gatric cancer cells to 5- fluorouracil (5-Fu) was stronger than negative control cells. Conclusions: (1) FOXP3 nuclear staining seems to be associated with risk of poor tumor differentiation. The high frequency of FOXP3 localization in gastric cancer tissues indicates that FOXP3 warrants further study to understand its association with gastric cancer differentiation. (2) The results of FOXP3 overexpressed gastric cancer cells implicated that the expression of FOXP3 could inhibit the proliferation of gastric cancer cells. Those cells were easily fall off from ground substance. And few of FOXP3 overexpressed cancer cells were held on G1 stage. Overexpression of FOXP3 could promote autophagy and inhibit metabolism of lipid. (3) Those inhibitory membrane molecule and cytokines were up regulated, so we think that FOXP3 could promote gastric cancer cells to express many inhibitive factors to promote tumor escape. (4) Although the proliferation of FOXP3 overexpression gastric cancer cells was slower, these cells could strongly inhibit the growth of PBMC and tolerate the effect of 5-Fu. So, we could hypothesis that FOXP3 might play an inhibitory role in gastric cancer immune escape. Here is the beginning of FOXP3 function exploring in gastric cancer. It would be a new focus in researching of the mechanisms of FOXP3 in cancers cells. And perhaps these findings could provide many effective strategies to clinical therapeutics.
结果:通过以上实验技术和方法我们获得了以下的实验结果:我们通过胃癌细胞系和胃癌组织的研究证实了胃癌细胞和组织内均存在不同水平的FOXP3的表达:(1)在多个胃癌细胞系中发现了Foxp3的mRNA转录及其蛋白的表达;(2)激光共聚焦实验证实,胃癌细胞中FOXP3蛋白的表达主要分布在肿瘤细胞核以及核周的胞浆内;(3)通过免疫组化染色发现胃癌细胞中FOXP3蛋白的表达主要分布在肿瘤细胞核,少数分布在胞浆内;(4)经过统计学分析胃癌组织切片和组织芯片的免疫组化染色结果,我们发现56%以上的胃癌患者肿瘤细胞中存在FOXP3的表达,且胃癌组织的分化与FOXP3蛋白表达的强度呈线性相关,同时,在正常胃组织和癌旁组织细胞内(不包括Tregs)未发现FOXP3的表达。随后,我们通过构建稳定过表达FOXP3的胃癌细胞系对FOXP3在胃癌细胞中的功能进行了初步的研究(:1)通过RT-PCR的方法,我们从外周血单核细胞中获得了Foxp3的cDNA片段,构建了pRc/CMV-Foxp3表达载体,转染SGC-7901细胞,经G418筛选后,成功建立稳定过表达FOXP3的胃癌细胞株;(2)扫描电镜检测发现与对照组细胞相比较,过表达FOXP3的胃癌细胞表面突起回缩,与周围基质之间的连接减少,透射电镜检测发现过表达FOXP3组细胞内出现腺管样结构,30%-40%出现自噬泡,并且,其胆固醇代谢发生障碍;(3)MTT检测发现过表达FOXP3的胃癌细胞增殖速度减慢;(4)细胞周期实验证实过表达FOXP3的胃癌细胞生长与对照相比5%以上阻止在G1期;(5)经RT-PCR鉴定,FOXP3的转录显著升高;(6)半定量PCR实验证实,肿瘤免疫抑制分子B7-H1和TGF-β的转录均明显升高;(7)通过ELISA检测发现过表达FOXP3的胃癌细胞培养上清中TGF-β1和TGF-β2的表达量升高;(8)体外混合外周血单核淋巴细胞共培养实验发现过表达FOXP3的胃癌细胞显著抑制了外周血单核淋巴细胞的增殖;(9)化疗药物5-氟脲嘧啶杀伤实验发现过表达FOXP3的胃癌细胞耐药性显著提高。讨论:(1)胃癌细胞和组织中普遍存在FOXP3的表达;(2)过表达FOXP3的胃癌细胞表面突起回缩,与周围基质之间的连接减少,生长过程中易于脱落,生长速度减慢,少部分细胞阻滞在G1期,FOXP3的过表达可以诱导肿瘤细胞发生自噬和脂类代谢障碍;(3)这种过表达FOXP3的胃癌细胞具有上调肿瘤免疫抑制和肿瘤免疫逃逸相关蛋白分子表达的功能;(4)虽然,过表达FOXP3的胃癌细胞生长速度缓慢,但与对照组相比,其具有更强的抑制淋巴细胞增殖和耐受化疗药物5-氟脲嘧啶的杀伤功能。综上所述,我们发现FOXP3可能在胃癌的免疫逃逸过程中发挥重要功能。本课题初步研究了FOXP3过表达后胃癌细胞SGC-7901的生长、形态以及免疫抑制功能的变化,为以后FOXP3在胃癌细胞中的作用机理研究奠定基础,从而为胃癌的临床治疗提供新的治疗靶位和免疫治疗策略。
Purpose: Transcription factor forkhead box protein 3 (FOXP3) specifically characterizes the naturally occurring regulatory T cells (Tregs). Recently, people found that pancreatic and other cancer cells could express FOXP3 by itself in microdissection. Although they didn’t find the relationship between the expression of FOXP3 and tumor differentiation, they identified that the expression of FOXP3 was induced by transforming growth factor-β2 (TGF-β2). According to these results, some reports implicated that FOXP3 might represent a tumor escape mechanism. But less is known about the role and biology of FOXP3 in gastric cancer. Our aim was to analyze the expression and initial function of FOXP3 in gastric cancer tissues and cell lines to evaluate the clinical character of FOXP3 in human gastric cancers. Experimental Design: Firstly, we detect the expression of FOXP3 in gastric cancer cells and tissues by methods, listing as below: (1) Detection of Foxp3 mRNA was performed using conventional RT-PCR, while protein expression was assessed by western blot, flow cytometry and confocal in gastric cancer cell lines. (2) Sections of resected human gastric cancer specimens were stained with anti human FOXP3 antibody. (3) FOXP3 staining of the gastric cancer was assessed to analyze the relationship between expression of FOXP3 and tumor differentiation. Secondly, we established FOXP3 stable overexpression SGC-7901 cell line to research the biologic character changes initially. (1) Observation of the morphology modification of FOXP3 overexpression gastric cancer cells was performed by scanning electron microscope (SEM)and transmission electron microscope (TEM). (2) MTT assay and flowcytometry was used to evaluate the proliferation. (3) PI staining to identify the cell cycles of FOXP3 overexpression gastric cancer cells. (4) We detected the inhibitory molecules expression of cancer cells by semi-PCR. (5) ELISA Kits was used to detect the inhibitory molecules of TGF-β. (6) Mixed peripheral blood monocyte and lymphocyte (PBMC) reaction was used to detect the proliferation inhibition effect of cancer cells. (7) Tolerance of FOXP3 overexpression gatric cancer cells to 5- fluorouracil was evaluated by MTT. Results: By previously methods and assays, we got results as below: (1) Foxp3 mRNA and protein was detected in all gastric cancer cell lines. (2) The location of FOXP3 expression was in nuclear and paranuclear of cytoplasm of gastric cancer cell line by confocal. (3) FOXP3 expression was mainly located in nuclear and seldom in cytoplasm in gastric cancer tissues. (4) Immunohistochemistry analysis of gastric cancer sections revealed that >56% of gastric cancer displayed nucleus FOXP3 staining. And it showed a linear relationship between differentiations of gastric cancer tissues and FOXP3 expression intensity. We did not find any FOXP3 staining in normal gastric tissues and para-tumor tissues. After that, we established FOXP3 overexpression gastric cell lines to explore the initial function of FOXP3. (1) After RT-PCR, we acquired the full length of Foxp3 cDNA, and inserted it into pRc/CMV plasmid to establish the vector of pRc/CMV-Foxp3. Screening by G418, we established stable FOXP3 high expression gastric cancer cell lines, SGC-7901. (2) Ecphyma retracted and the connection between cancer cells and ground substance was less than negative control cancer cells. We found glandular tube and autophagic vacuole in FOXP3 overexpression cells. And metabolism of cholesterol is obstructed in these cells. (3) The proliferation was down-regulated in FOXP3 overexpression cancer cells. (4) Few of those cells (5%) were held on G1 stage. (5) RT-PCR identified that Foxp3 was up-regulated in transcription level. (6) Semi-quantity PCR showed that the inhibitory molecules, such as B7-H1 and TGF-β, were up-regulated in FOXP3 overexpression cancer cells. (7) ELISA assay find that TGF-β1 and TGF-β2 were also higher in supernatant of culture medium of FOXP3 overexpression cancer cells. (8) Cocultured with mixed PBMC, the proliferation of lymphocytes was down regulated. (9) The tolerance of FOXP3 overexpression gatric cancer cells to 5- fluorouracil (5-Fu) was stronger than negative control cells. Conclusions: (1) FOXP3 nuclear staining seems to be associated with risk of poor tumor differentiation. The high frequency of FOXP3 localization in gastric cancer tissues indicates that FOXP3 warrants further study to understand its association with gastric cancer differentiation. (2) The results of FOXP3 overexpressed gastric cancer cells implicated that the expression of FOXP3 could inhibit the proliferation of gastric cancer cells. Those cells were easily fall off from ground substance. And few of FOXP3 overexpressed cancer cells were held on G1 stage. Overexpression of FOXP3 could promote autophagy and inhibit metabolism of lipid. (3) Those inhibitory membrane molecule and cytokines were up regulated, so we think that FOXP3 could promote gastric cancer cells to express many inhibitive factors to promote tumor escape. (4) Although the proliferation of FOXP3 overexpression gastric cancer cells was slower, these cells could strongly inhibit the growth of PBMC and tolerate the effect of 5-Fu. So, we could hypothesis that FOXP3 might play an inhibitory role in gastric cancer immune escape. Here is the beginning of FOXP3 function exploring in gastric cancer. It would be a new focus in researching of the mechanisms of FOXP3 in cancers cells. And perhaps these findings could provide many effective strategies to clinical therapeutics.
引文
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