调节性T细胞对肾癌局部免疫微环境的负性调控作用及其机制研究
摘要
第一部分肾癌肿瘤局部Treg与肿瘤预后关系及与肿瘤COX-2表达的相关性
目的本部分旨在从细胞水平了解肾癌局部免疫微环境中T淋巴细胞的亚型、数目与肾癌各项临床指标及预后的关系,并通过与肿瘤中COX-2表达的相关性分析,探讨肿瘤局部调节性T细胞增多的可能机制。
方法本研究收集1999年1月～2005年12月在复旦大学附属中山医院行根治性肾切除或部分肾切除的肾透明细胞癌患者资料,随机选取其中有完整随访资料的125例肾癌石蜡标本,借助于高通量的组织微阵列及免疫组化染色,研究肿瘤中与癌旁微环境中CD4~+T细胞、CD8~+T细胞、FOXP3~+调节性T细胞、GranzymeB~+活化的细胞毒性T细胞的数目、COX-2的表达情况与肿瘤各项临床指标及预后的关系,并探讨COX-2表达与调节性T细胞的相关性。
结果(1)不同患者免疫细胞浸润程度不同,肾癌的淋巴细胞以分散形式或聚集体形式存在,以癌旁淋巴细胞浸润更加明显。(2)单因素生存分析显示患者年龄、性别与肾癌预后无关,肿瘤中及癌旁CD4~+T细胞、CD8~+T细胞、Granzyme B~+活化的细胞毒性T细胞表达与肾癌预后无关;而癌旁调节性T细胞增多与总生存率及疾病无进展生存率呈显著负相关(P＜0.001,P＜0.001),肿瘤中COX-2高表达也与总生存率及疾病无进展生存率呈显著负相关(P=0.021,P=0.027)。但肿瘤中调节性T细胞、癌旁COX-2表达与肾癌预后无关。肿瘤TNM分期、肿瘤核分级和肿瘤大小均与肾癌预后相关。(3)多因素Cox生存分析显示癌旁调节性T细胞增多、肿瘤TNM分期增加(Ⅲ、Ⅳ期)、肿瘤核分级增加(3、4级)和肿瘤直径增大(≥7cm)提示肾癌预后不良。相关性分析显示癌旁调节性T细胞数目与肿瘤TNM分期及肿瘤直径呈正相关(P=0.001,P=0.002),肿瘤中Granzyme B~+T细胞数目与肿瘤核分级呈正相关(P=0.024),而COX-2表达程度与肿瘤TNM分期和肿瘤核分级呈正相关(P=0.018,P=0.013);癌旁调节性T细胞与肿瘤中COX-2表达呈正相关(P＜0.001)。
结论结果表明,肾癌癌旁调节性T细胞增多与肿瘤TNM分期增加(Ⅲ、Ⅳ期)、肿瘤核分级增加(3、4级)和肿瘤直径增大(≥7cm)均可作为肾癌不良预后的一个独立指标。癌旁调节性T细胞与肿瘤中COX-2表达呈正相关,基于COX-2来源的PGE_2可诱导非调节性T细胞向调节性T细胞转化,因此肾癌可能通过相同机制诱导调节性T细胞生成,进而促进肿瘤发生免疫逃逸。
第二部分肾癌局部Treg来源及其机制研究
2.1 pcDNA3.1-hCOX-2真核表达质粒的构建
目的构建pcDNA3.1-hCOX-2真核表达质粒。
方法以pSG5-COX-2为模板,PCR获得人COX-2cDNA全长,EcoRI、BamHI双酶切后定向克隆到真核细胞表达载体pcDNA3.1中,限制性内切酶酶切分析和DNA序列分析鉴定重组质粒。
结果重组真核表达质粒pcDNA3.1-hCOX-2经限制性内切酶酶切,电泳后显示1800bp左右的hCOX-2目的片段和5.4kb的pcDNA3.1载体片段,测序证实1800bp的酶切片段与Gene-bank登记的COX-2序列相同。
结论成功构建pcDNA3.1-hCOX-2真核表达质粒并可用于后续转染。
2.2 pcDNA3.1-hCOX-2转染对肾癌细胞株COX-2表达及上清中PGE_2生成的增强作用
目的测定常见肾癌细胞株COX-2表达,挑选COX-2高表达的肾癌细胞株进入后续转染实验;将pcDNA3.1-hCOX-2真核表达质粒通过脂质体lipofectamine 2000转染COX-2高表达的肾癌细胞株,了解对COX-2 mRNA及蛋白影响,尤其对肿瘤细胞上清中PGE_2分泌影响;转染组中分别加入不同剂量NS-398,了解其对上清中PGE_2分泌影响。
方法(1)引入两种常见肾癌细胞株:786-0和OS-RC-2,分别进行体外培养。半定量RT-PCR方法测定两种肾癌细胞株COX-2 mRNA的表达水平;Western-blot方法测定两种肾癌细胞株COX-2蛋白的表达水平。(2)将pcDNA3.1-hCOX-2真核表达质粒通过脂质体lipofectamine2000转染COX-2高表达的肾癌细胞株OS-RC-2;转染72小时后,半定量RT-PCR测定肾癌细胞株OS-RC-2中COX-2 mRNA的表达水平,Western-blot测定COX-2蛋白表达水平,确定转染效果。(3) ELISA方法检测转染对肾癌细胞株OS-RC-2的PGE_2分泌影响,转染组分别加入12.5μM、25μM、50μM、100μM NS-398,ELISA方法检测各组上清中PGE_2含量变化。
结果(1)两种肾癌细胞株COX-2mRNA和蛋白表达水平均不同。半定量RT-PCR测定结果:以两种肾癌细胞株COX-2/beta-actin作比较,786-0和OS-RC-2的比值分别为0.2756±0.01318、0.4667±0.02159,前者为后者表达量的59.1%,结果在统计学上有显著性差异(P＜0.05)。Western-blot测定结果:以两种肾癌细胞株COX-2/GAPDH作比较,786-0和OS-RC-2的比值分别为0.2333±0.02183、0.4686±0.04294,前者为后者表达量的50%,结果在统计学上有显著性差异(P＜0.05)。后续转染实验选择肾癌细胞株OS-RC-2。(2)肾癌细胞株OS-RC-2转染pcDNA3.1-hCOX-2真核表达质粒后COX-2mRNA和蛋白表达水平均明显升高。半定量RT-PCR结果:转染72小时后COX-2 mRNA在转染组、空转组和对照组COX-2/beta-actin比值分别为0.7922±0.07139、0.4325±0.02045、0.4739±0.02377,两两比较结果显示转染组明显高于空转组和对照组,统计学有明显差异(P＜0.05);后两者比较统计学无明显差异(P＞0.05)。Western-blot测定结果:转染72小时后COX-2蛋白在转染组、空转组和对照组COX-2/GAPDH比值分别为0.8593±0.1332、0.4321±0.03362、0.4533±0.05883,两两比较结果显示转染组明显高于空转组及对照组,统计学有明显差异(P＜0.05);后两者比较统计学无明显差异(P＞0.05)。(3)ELISA检测发现转染组肿瘤细胞上清中PGE_2分泌明显高于空转组和对照组,分别为6.330±1.181、0.2817±0.03181、0.3030±0.03246,两两比较结果显示转染组明显高于空转组和对照组,统计学有明显差异(P＜0.05);后两者比较统计学无明显差异(P＞0.05)。转染组应用NS-39812.5μM、25μM、50μM、100μM之后,PGE_2含量分别为2.906±0.5892、0.6484±0.09880、0.4189±0.06513、0.2221±0.04094 ng/ml,两两比较结果显示转染组明显高于转染组+12.5μMNS-398组、转染组+25μMNS-398组、转染组+50μMNS-398组、转染组+100μMNS-398组,统计学上有明显差异(P＜0.05)。
结论(1)肾癌细胞株786-0和OS-RC-2均有COX-2表达,但前者COX-2表达无论在mRNA水平或蛋白水平均低于后者,后续转染实验选择OS-RC-2作为靶细胞株。(2)所构建pcDNA3.1-hCOX-2真核表达质粒可通过脂质体lipofectamine 2000转染肾癌细胞株OS-RC-2,增加OS-RC-2中COX-2表达水平。(3) pcDNA3.1-hCOX-2真核表达质粒转染可增强转染肾癌细胞株OS-RC-2上清中PGE_2分泌,而应用NS-398可以剂量依赖性地降低转染OS-RC-2上清中PGE_2分泌。
2.3 pcDNA3.1-hCOX-2转染肾癌细胞株上清诱导CD4~+FOXP3~-T细胞向Treg转化
目的了解转染肾癌细胞株OS-RC-2上清是否可诱导CD4~+FOXP3T细胞向CD4~+FOXP3~+调节性T细胞转化。
方法密度梯度离心法获得外周血单个核细胞,免疫磁珠细胞分选获得CD4~+CD25~-效应T细胞和CD4~+CD25~+调节性T细胞,RT-PCR及FACS检测分选细胞的纯度。anti-CD3/CD28抗体和灭活APC细胞活化条件下,将分选的CD4~+CD25~-T细胞分别与PGE_2阳性对照组、转染组、转染+100μM对照抗体、转染+100μMNS-398、阴性对照组的上清共孵育96小时,FACS检测各组细胞中CD4~+FOXP3~+调节性T细胞比例。
结果(1)免疫磁珠细胞分选可稳定获得CD4~+CD25~-效应T细胞和CD4~+CD25~+调节性T细胞,二者纯度均大于90%,后者特异性表达FOXP3基因和蛋白。(2)转染组上清可明显诱导CD4~+FOXP3~-T细胞向CD4~+FOXP3~+调节性T细胞转化,96小时后CD4~+FOXP3~+调节性T细胞占CD4~+T细胞比率为30.00±2.618%,而与转染+100μMNS-398组上清共孵育后CD4~+FOXP3~+调节性T细胞生成明显降低,96小时后CD4~+FOXP3~+调节性T细胞占CD4~+T细胞比率仅为7.990±1.227%。
结论(1)免疫磁珠细胞分选可稳定获得CD4~+CD25~-效应T细胞和CD4~+CD25~+调节性T细胞,由于CD4~+CD25~+调节性T细胞特异性表达FOXP3基因和蛋白,后续部分以CD4~+FOXP3~+调节性T细胞代表CD4~+CD25~+调节性T细胞,CD4~+CD25~-效应T细胞中FOXP3表达阴性,后续部分以CD4~+FOXP3~-T细胞代表CD4~+CD25~-效应T细胞。(2)转染肾癌细胞株上清可诱导CD4~+FOXP3~-T细胞向CD4~+FOXP3~+调节性T细胞转化,而NS-398可以减弱转染肾癌细胞上清所诱导的调节性T细胞生成,提示COX-2抑制剂可通过减少调节性T细胞生成,诱发局部抗肿瘤免疫反应来辅助治疗肾癌。
PartⅠThe prognostic value of peritumoral regulatory T cell and its correlation with intratumoral cyclooxygenase-2 expression in renal cell carcinoma
Objective This study aims to investigate the prognostic value of various tumorinfiltrating lymphocytes(TILs) in renal cell carcinoma(RCC).Furthermore,we intend to know whether they are also associated with the clinical parameters in RCC.In addition,by elucidating the prognostic value of regulatory T cell(Treg) and its correlation with cyclooxygenase-2(COX-2) expression,we try to know the possible mechanism of aberrant gathering of Treg in RCC.
Methods In this study we identified 125 patients with RCC treated with radical nephrectomy or nephron-sparing surgery for unilateral,sporadic,clear cell RCC at Zhong Shan Hospital of Fudan University from Jan 1999 to Dec 2005.Then CD4~+, CD8~+,Granzyme B~+,FOXP3~+ TILs and tumour COX-2 expression were assessed by immunohistochemistry in tissue microarrays.The prognostic effects of low and high expression were evaluated and the expression of various immunohistochemical parameters were compared with the clinicopathological variables.In addition,Treg and its correlation with COX-2 expression was also analysed.
Results The results showed that(1) immune cell infiltration varied substantially among different patients.Lymphocytes infiltrated RCC in a diffuse manner or in lymphoid aggregates,with more abundant cells in peritumoral areas.(2) On univariate analysis,age and sex had no prognostic significance for overall survival nor disease-free survival.CD4~+,CD8~+,Granzyme B~+ T cells were associated with neither overall survival nor disease-free survival,while peritumoral Treg is an prognostic factor for both overall survival and disease-free survival(P＜0.001,P＜0.001).Similarly, high intratumoral COX-2 expression was also considered to be prognostic for both reduced overall survival and disease-free survival(P=0.021,P=0.027).Nevertheless, both peritumoral COX-2 and intratumoral Treg had no prognostic value in RCC. Tumor TNM stage,tumor nuclear grade and tumor size could also be prognostic in RCC.(3) Using multivariate analysis,increased peritumoral Treg,higher TNM stage (Ⅲ,Ⅳ),higher nuclear grade(3,4) and larger tumour size(≥7cm) were independent predictors for significant shorter overall survival and disease-free survival.In addition, peritumoral Treg were positively associated with TNM stage and tumor size(P=0.001, P=0.002),granzyme B~+ TILs were positively associated with nuclear grade(P=0.024), while intratumoral COX-2 expression were positively associated with TNM stage and nuclear grade(P=0.018,P=0.013).Peritumoral Treg were positively correlated with intratumoral COX-2 expression(P＜0.001).
Conclusions These results suggested that increased peritumoral Treg,higher TNM stage(Ⅲ,Ⅳ),higher nuclear grade(3,4) and larger tumour size(≥7cm) were independent predictors for significantly shorter overall survival and disease-free survival.In the present study peritumoral Treg was positively correlated with intratumoral COX-2 expression.In addition,a previous report in lung cell carcinoma showed the important role of COX-2 derived prostaglandins E_2(PGE_2) in the transformation of Treg.Similarly,the overexpression of COX-2 in RCC could be the underlying reason for the aberrant gathering of Treg.Therefore,we should further elucidate the possible mechanism of the aberrant gathering of Treg in RCC.
PartⅡThe origin of regulatory T cell in renal cell carcinoma and its possible undelying mechanism
2.1 Construction of eukaryotic expression vector pcDNA3.1-hCOX-2
Objective To construct eukaryotic expression vector of pcDNA3.1-hCOX-2.
Methods The hCOX-2 cDNA fragment was extracted from plasmid pSG5-COX-2,amplified by PCR,and inserted into pcDNA3.1 vector before the double digestion with restriction enzymes EcoRI and BamHI.Then the vector was identified by the double digestion with restriction enzymes and was sequenced by the Sanger-dideoxy-mediated chain termination.
Results The recombinant eukaryotic expression vector for COX-2 was digested with EcoRI and BamHI,and the electrophoresis of the digested products showed two fragments:1800 bp fragmant and 5.4 kb fragment.The sequence of 1800 bp fragment was identical to COX-2 sequence published in GeneBank.
Conclusions The recombinant plasmid pcDNA3.1-hCOX-2 was constructed successfully and can be applicated into subsequent transfection experiment.
2.2 The increased COX-2 expression and PGE_2 production in the transfected RCC cell line after pcDNA3.1-hCOX-2 transfection
Objective To detect the expression of COX-2 gene and protein in RCC cell lines and pick out RCC cell line with higher expression of COX-2 for the following transfection experiment.Next we transfect pcDNA3.1-hCOX-2 plasmid vector into the targeted RCC cell line,then we analyze its effect to the expression of COX-2 gene and protein,especially we tend to know its effect to the production of PGE_2 in the RCC cell line supematants.In addition,we add different doses of NS-398 into the transfection group to know their effect to the production PGE_2 in the supernatants.
Methods(1) Two RCC cell lines 786-0 and OS-RC-2,were cultured in vitro. The expressions of COX-2 mRNA were assessed by RT-PCR,and its protein expressions were determined by Western-blot.The results were compared with each other to determine which cell line can be used in the next part of experiments.(2) To transfect pcDNA3.1-hCOX-2 plasmid vector into OS-RC-2 using lipofectamine 2000,COX-2 mRNA were detected 72h after transfection by RT-PCR and COX-2 protein were analysed by Western-blot 72h after transfection.(3) The different levels of PGE_2 in the transfected OS-RC-2 cell line supernatants and control groups were analysed by ELISA.In addition,we added 12.5μM,25μM,50μM,100μM NS-398 among the supernatants respectively and detected the PGE_2 levels by ELISA.
Results(1) The two RCC cell lines had different COX-2 mRNA and protein expression.RT-PCR:The COX-2/beta-actin ratio was used to represent the expression of COX-2 mRNA.The ratio of 786-0 and OS-RC-2 cell lines were 0.2756±0.01318 and 0.4667±0.02159 respectively.It represented that the expression of COX-2 mRNA of 786-0 was about 59.1%compared with that of OS-RC-2 cell lines.The results between 786-0 and OS-RC-2 cell lines had significant difference(p＜0.05). Western-blot:The COX-2/GAPDH ratio was used to represent the expression of COX-2 protein.The ratio of 786-0 and OS-RC-2 cell lines were 0.2333±0.02183 and 0.4686±0.04294 respectively.It represented that the expression of COX-2 protein of 786-0 was about 50%compared with that of OS-RC-2 cell lines.The results between 786-0 and OS-RC-2 cell lines had significant difference(p＜0.05).Next we chose OS-RC-2 as the targeted cell line for the next transfection experiment.(2) The expression of COX-2 mRNA and protein in the transfected OS-RC-2 cell line have increased significantly after pcDNA3.1-hCOX-2 plasmid vector transfection.We divided into three groups:pcDNA3.1-hCOX-2,pcDNA3.1 and control group.Results of RT-PCR:COX-2 mRNA was detected 72h after transfection.The COX-2mRNA in the above three groups were 0.7922±0.07139、0.4325±0.02045、0.4739±0.02377 respectively.Compared with pcDNA3.1 and control groups,the transfection group had higher COX-2mRNA expression(p＜0.05),while the COX-2 mRNA between pcDNA3.1 and control group had no significant difference(p＞0.05).Results of Western-blot:COX-2 protein was detected 72h after transfection.The COX-2 protein in the above three groups were 0.8593±0.1332、0.4321±0.03362、0.4533±0.05883 respectively.Compared with pcDNA3.1 and control groups,the transfection group had higher COX-2 protein expression(p＜0.05),while the COX-2 protein between pcDNA3.1 and control group had no significant difference(p＞0.05).(3)The result of ELISA experiment indicated that PGE_2 levels varied significantly among pcDNA3.1-hCOX-2,pcDNA3.1 and control group.The PGE_2 levels in the above three groups were 6.330±1.181、0.2817±0.03181、0.3030±0.03246 respectively. Compared with pcDNA3.1 and control groups,the transfection group had higher PGE_2 production(p＜0.05),while PGE_2 production between pcDNA3.1 and control group had no significant difference(p＞0.05).When applicating NS-398 at 12.5μM、25μM、50μM、100μM respectively in pcDNA3.1-hCOX-2 transfection group,the PGE_2 levels were 2.906±0.5892、0.6484±0.09880、0.4189±0.06513、0.2221±0.04094 respectively.Thus,the PGE_2 production between these groups and pcDNA3.1-hCOX-2 transfection group had significant difference(p＜0.05).
Conclusions(1) The two RCC cell lines had COX-2 mRNA and protein expression.Since OS-RC-2 cell line had higher COX-2 expression,we chose OS-RC-2 as target cell line for the following transfection experiment.(2) The constructed pcDNA3.1-hCOX-2 could be successfully transfected into OS-RC-2 cell line using lipofectamine 2000,and subsequently increased the COX-2 expression.(3) The production of PGE_2 in the transfected OS-RC-2 cell line increased significantly. The application of NS-398 could reduce PGE_2 production at the supernatants in a dose-dependent manner.
2.3 The increased conversion from CD4~+FOXP3~- T cell to CD4~+FOXP3~+ Treg in the supernatants of pcDNA3.1-hCOX-2 transfection group
Objective To investigate whether the supernatants from cultured transfected OS-RC-2 cell line could convert peripheral CD4~+FOXP3~- T cell into CD4~+FOXP3~+ Treg.
Methods We obtained peripheral blood mononuclear cells by density gradient centrifugation method.Subsequently we isolated CD4~+CD25~- T cell and CD4~+ CD25~+ Treg by MACS and analysed the purity of isolated cells by RT-PCR and FACS respectively.Under the stimulation of anti-CD3/CD28 antibody and APC cells, isolated CD4~+FOXP3~- T cells were cocultured with transfected OS-RC-2 supernatants and different control supernatants respectively,96 hours later,the proportion of CD4~+FOXP3~+ Treg in each group were detected by FACS.
Results(1) MACS could obtain CD4~+CD25- T cell and CD4~+CD25~+ Treg steadily and the purity of two groups were over 90%respectively.The latter characteristically expressed FOXP3 gene and protein.(2) The transfected OS-RC-2 cell line supernatants could obviously induce the production of CD4~+FOXP3~+Treg, the proportion of CD4~+FOXP3~+Treg reached 30.00±2.618%after 96 hours coculture, while NS-398 could markedly reduce the above effect,the proportion of CD4~+ FOXP3~+ Tregs was only 7.990±1.227%in the transfection+100μMNS-398 group.
Conclusions(1) MACS could obtain CD4~+CD25~-T cell and CD4~+CD25~+ Treg steadily.Since CD4~+CD25~+ Treg specifically express FOXP3 gene and protein,we regard CD4~+CD25~+Treg as CD4~+FOXP3~+Treg,while CD4~+CD25~- T cell do not express FOXP3 gene and protein,we regard CD4~+CD25~-T cell as CD4~+FOXP3~- T cell in the following experiment.(2) The transfected RCC cell line supernatants could induce the transformation from CD4~+FOXP3~- T cells to CD4~+FOXP3~+ Treg,while NS-398 could reduce the proportion of induced CD4~+FOXP3~+Treg.Therefore, COX-2 inhibitors may induce the local anti-tumor effect and in turn contribute to eradicating RCC.
目的本部分旨在从细胞水平了解肾癌局部免疫微环境中T淋巴细胞的亚型、数目与肾癌各项临床指标及预后的关系,并通过与肿瘤中COX-2表达的相关性分析,探讨肿瘤局部调节性T细胞增多的可能机制。
方法本研究收集1999年1月～2005年12月在复旦大学附属中山医院行根治性肾切除或部分肾切除的肾透明细胞癌患者资料,随机选取其中有完整随访资料的125例肾癌石蜡标本,借助于高通量的组织微阵列及免疫组化染色,研究肿瘤中与癌旁微环境中CD4~+T细胞、CD8~+T细胞、FOXP3~+调节性T细胞、GranzymeB~+活化的细胞毒性T细胞的数目、COX-2的表达情况与肿瘤各项临床指标及预后的关系,并探讨COX-2表达与调节性T细胞的相关性。
结果(1)不同患者免疫细胞浸润程度不同,肾癌的淋巴细胞以分散形式或聚集体形式存在,以癌旁淋巴细胞浸润更加明显。(2)单因素生存分析显示患者年龄、性别与肾癌预后无关,肿瘤中及癌旁CD4~+T细胞、CD8~+T细胞、Granzyme B~+活化的细胞毒性T细胞表达与肾癌预后无关;而癌旁调节性T细胞增多与总生存率及疾病无进展生存率呈显著负相关(P＜0.001,P＜0.001),肿瘤中COX-2高表达也与总生存率及疾病无进展生存率呈显著负相关(P=0.021,P=0.027)。但肿瘤中调节性T细胞、癌旁COX-2表达与肾癌预后无关。肿瘤TNM分期、肿瘤核分级和肿瘤大小均与肾癌预后相关。(3)多因素Cox生存分析显示癌旁调节性T细胞增多、肿瘤TNM分期增加(Ⅲ、Ⅳ期)、肿瘤核分级增加(3、4级)和肿瘤直径增大(≥7cm)提示肾癌预后不良。相关性分析显示癌旁调节性T细胞数目与肿瘤TNM分期及肿瘤直径呈正相关(P=0.001,P=0.002),肿瘤中Granzyme B~+T细胞数目与肿瘤核分级呈正相关(P=0.024),而COX-2表达程度与肿瘤TNM分期和肿瘤核分级呈正相关(P=0.018,P=0.013);癌旁调节性T细胞与肿瘤中COX-2表达呈正相关(P＜0.001)。
结论结果表明,肾癌癌旁调节性T细胞增多与肿瘤TNM分期增加(Ⅲ、Ⅳ期)、肿瘤核分级增加(3、4级)和肿瘤直径增大(≥7cm)均可作为肾癌不良预后的一个独立指标。癌旁调节性T细胞与肿瘤中COX-2表达呈正相关,基于COX-2来源的PGE_2可诱导非调节性T细胞向调节性T细胞转化,因此肾癌可能通过相同机制诱导调节性T细胞生成,进而促进肿瘤发生免疫逃逸。
第二部分肾癌局部Treg来源及其机制研究
2.1 pcDNA3.1-hCOX-2真核表达质粒的构建
目的构建pcDNA3.1-hCOX-2真核表达质粒。
方法以pSG5-COX-2为模板,PCR获得人COX-2cDNA全长,EcoRI、BamHI双酶切后定向克隆到真核细胞表达载体pcDNA3.1中,限制性内切酶酶切分析和DNA序列分析鉴定重组质粒。
结果重组真核表达质粒pcDNA3.1-hCOX-2经限制性内切酶酶切,电泳后显示1800bp左右的hCOX-2目的片段和5.4kb的pcDNA3.1载体片段,测序证实1800bp的酶切片段与Gene-bank登记的COX-2序列相同。
结论成功构建pcDNA3.1-hCOX-2真核表达质粒并可用于后续转染。
2.2 pcDNA3.1-hCOX-2转染对肾癌细胞株COX-2表达及上清中PGE_2生成的增强作用
目的测定常见肾癌细胞株COX-2表达,挑选COX-2高表达的肾癌细胞株进入后续转染实验;将pcDNA3.1-hCOX-2真核表达质粒通过脂质体lipofectamine 2000转染COX-2高表达的肾癌细胞株,了解对COX-2 mRNA及蛋白影响,尤其对肿瘤细胞上清中PGE_2分泌影响;转染组中分别加入不同剂量NS-398,了解其对上清中PGE_2分泌影响。
方法(1)引入两种常见肾癌细胞株:786-0和OS-RC-2,分别进行体外培养。半定量RT-PCR方法测定两种肾癌细胞株COX-2 mRNA的表达水平;Western-blot方法测定两种肾癌细胞株COX-2蛋白的表达水平。(2)将pcDNA3.1-hCOX-2真核表达质粒通过脂质体lipofectamine2000转染COX-2高表达的肾癌细胞株OS-RC-2;转染72小时后,半定量RT-PCR测定肾癌细胞株OS-RC-2中COX-2 mRNA的表达水平,Western-blot测定COX-2蛋白表达水平,确定转染效果。(3) ELISA方法检测转染对肾癌细胞株OS-RC-2的PGE_2分泌影响,转染组分别加入12.5μM、25μM、50μM、100μM NS-398,ELISA方法检测各组上清中PGE_2含量变化。
结果(1)两种肾癌细胞株COX-2mRNA和蛋白表达水平均不同。半定量RT-PCR测定结果:以两种肾癌细胞株COX-2/beta-actin作比较,786-0和OS-RC-2的比值分别为0.2756±0.01318、0.4667±0.02159,前者为后者表达量的59.1%,结果在统计学上有显著性差异(P＜0.05)。Western-blot测定结果:以两种肾癌细胞株COX-2/GAPDH作比较,786-0和OS-RC-2的比值分别为0.2333±0.02183、0.4686±0.04294,前者为后者表达量的50%,结果在统计学上有显著性差异(P＜0.05)。后续转染实验选择肾癌细胞株OS-RC-2。(2)肾癌细胞株OS-RC-2转染pcDNA3.1-hCOX-2真核表达质粒后COX-2mRNA和蛋白表达水平均明显升高。半定量RT-PCR结果:转染72小时后COX-2 mRNA在转染组、空转组和对照组COX-2/beta-actin比值分别为0.7922±0.07139、0.4325±0.02045、0.4739±0.02377,两两比较结果显示转染组明显高于空转组和对照组,统计学有明显差异(P＜0.05);后两者比较统计学无明显差异(P＞0.05)。Western-blot测定结果:转染72小时后COX-2蛋白在转染组、空转组和对照组COX-2/GAPDH比值分别为0.8593±0.1332、0.4321±0.03362、0.4533±0.05883,两两比较结果显示转染组明显高于空转组及对照组,统计学有明显差异(P＜0.05);后两者比较统计学无明显差异(P＞0.05)。(3)ELISA检测发现转染组肿瘤细胞上清中PGE_2分泌明显高于空转组和对照组,分别为6.330±1.181、0.2817±0.03181、0.3030±0.03246,两两比较结果显示转染组明显高于空转组和对照组,统计学有明显差异(P＜0.05);后两者比较统计学无明显差异(P＞0.05)。转染组应用NS-39812.5μM、25μM、50μM、100μM之后,PGE_2含量分别为2.906±0.5892、0.6484±0.09880、0.4189±0.06513、0.2221±0.04094 ng/ml,两两比较结果显示转染组明显高于转染组+12.5μMNS-398组、转染组+25μMNS-398组、转染组+50μMNS-398组、转染组+100μMNS-398组,统计学上有明显差异(P＜0.05)。
结论(1)肾癌细胞株786-0和OS-RC-2均有COX-2表达,但前者COX-2表达无论在mRNA水平或蛋白水平均低于后者,后续转染实验选择OS-RC-2作为靶细胞株。(2)所构建pcDNA3.1-hCOX-2真核表达质粒可通过脂质体lipofectamine 2000转染肾癌细胞株OS-RC-2,增加OS-RC-2中COX-2表达水平。(3) pcDNA3.1-hCOX-2真核表达质粒转染可增强转染肾癌细胞株OS-RC-2上清中PGE_2分泌,而应用NS-398可以剂量依赖性地降低转染OS-RC-2上清中PGE_2分泌。
2.3 pcDNA3.1-hCOX-2转染肾癌细胞株上清诱导CD4~+FOXP3~-T细胞向Treg转化
目的了解转染肾癌细胞株OS-RC-2上清是否可诱导CD4~+FOXP3T细胞向CD4~+FOXP3~+调节性T细胞转化。
方法密度梯度离心法获得外周血单个核细胞,免疫磁珠细胞分选获得CD4~+CD25~-效应T细胞和CD4~+CD25~+调节性T细胞,RT-PCR及FACS检测分选细胞的纯度。anti-CD3/CD28抗体和灭活APC细胞活化条件下,将分选的CD4~+CD25~-T细胞分别与PGE_2阳性对照组、转染组、转染+100μM对照抗体、转染+100μMNS-398、阴性对照组的上清共孵育96小时,FACS检测各组细胞中CD4~+FOXP3~+调节性T细胞比例。
结果(1)免疫磁珠细胞分选可稳定获得CD4~+CD25~-效应T细胞和CD4~+CD25~+调节性T细胞,二者纯度均大于90%,后者特异性表达FOXP3基因和蛋白。(2)转染组上清可明显诱导CD4~+FOXP3~-T细胞向CD4~+FOXP3~+调节性T细胞转化,96小时后CD4~+FOXP3~+调节性T细胞占CD4~+T细胞比率为30.00±2.618%,而与转染+100μMNS-398组上清共孵育后CD4~+FOXP3~+调节性T细胞生成明显降低,96小时后CD4~+FOXP3~+调节性T细胞占CD4~+T细胞比率仅为7.990±1.227%。
结论(1)免疫磁珠细胞分选可稳定获得CD4~+CD25~-效应T细胞和CD4~+CD25~+调节性T细胞,由于CD4~+CD25~+调节性T细胞特异性表达FOXP3基因和蛋白,后续部分以CD4~+FOXP3~+调节性T细胞代表CD4~+CD25~+调节性T细胞,CD4~+CD25~-效应T细胞中FOXP3表达阴性,后续部分以CD4~+FOXP3~-T细胞代表CD4~+CD25~-效应T细胞。(2)转染肾癌细胞株上清可诱导CD4~+FOXP3~-T细胞向CD4~+FOXP3~+调节性T细胞转化,而NS-398可以减弱转染肾癌细胞上清所诱导的调节性T细胞生成,提示COX-2抑制剂可通过减少调节性T细胞生成,诱发局部抗肿瘤免疫反应来辅助治疗肾癌。
PartⅠThe prognostic value of peritumoral regulatory T cell and its correlation with intratumoral cyclooxygenase-2 expression in renal cell carcinoma
Objective This study aims to investigate the prognostic value of various tumorinfiltrating lymphocytes(TILs) in renal cell carcinoma(RCC).Furthermore,we intend to know whether they are also associated with the clinical parameters in RCC.In addition,by elucidating the prognostic value of regulatory T cell(Treg) and its correlation with cyclooxygenase-2(COX-2) expression,we try to know the possible mechanism of aberrant gathering of Treg in RCC.
Methods In this study we identified 125 patients with RCC treated with radical nephrectomy or nephron-sparing surgery for unilateral,sporadic,clear cell RCC at Zhong Shan Hospital of Fudan University from Jan 1999 to Dec 2005.Then CD4~+, CD8~+,Granzyme B~+,FOXP3~+ TILs and tumour COX-2 expression were assessed by immunohistochemistry in tissue microarrays.The prognostic effects of low and high expression were evaluated and the expression of various immunohistochemical parameters were compared with the clinicopathological variables.In addition,Treg and its correlation with COX-2 expression was also analysed.
Results The results showed that(1) immune cell infiltration varied substantially among different patients.Lymphocytes infiltrated RCC in a diffuse manner or in lymphoid aggregates,with more abundant cells in peritumoral areas.(2) On univariate analysis,age and sex had no prognostic significance for overall survival nor disease-free survival.CD4~+,CD8~+,Granzyme B~+ T cells were associated with neither overall survival nor disease-free survival,while peritumoral Treg is an prognostic factor for both overall survival and disease-free survival(P＜0.001,P＜0.001).Similarly, high intratumoral COX-2 expression was also considered to be prognostic for both reduced overall survival and disease-free survival(P=0.021,P=0.027).Nevertheless, both peritumoral COX-2 and intratumoral Treg had no prognostic value in RCC. Tumor TNM stage,tumor nuclear grade and tumor size could also be prognostic in RCC.(3) Using multivariate analysis,increased peritumoral Treg,higher TNM stage (Ⅲ,Ⅳ),higher nuclear grade(3,4) and larger tumour size(≥7cm) were independent predictors for significant shorter overall survival and disease-free survival.In addition, peritumoral Treg were positively associated with TNM stage and tumor size(P=0.001, P=0.002),granzyme B~+ TILs were positively associated with nuclear grade(P=0.024), while intratumoral COX-2 expression were positively associated with TNM stage and nuclear grade(P=0.018,P=0.013).Peritumoral Treg were positively correlated with intratumoral COX-2 expression(P＜0.001).
Conclusions These results suggested that increased peritumoral Treg,higher TNM stage(Ⅲ,Ⅳ),higher nuclear grade(3,4) and larger tumour size(≥7cm) were independent predictors for significantly shorter overall survival and disease-free survival.In the present study peritumoral Treg was positively correlated with intratumoral COX-2 expression.In addition,a previous report in lung cell carcinoma showed the important role of COX-2 derived prostaglandins E_2(PGE_2) in the transformation of Treg.Similarly,the overexpression of COX-2 in RCC could be the underlying reason for the aberrant gathering of Treg.Therefore,we should further elucidate the possible mechanism of the aberrant gathering of Treg in RCC.
PartⅡThe origin of regulatory T cell in renal cell carcinoma and its possible undelying mechanism
2.1 Construction of eukaryotic expression vector pcDNA3.1-hCOX-2
Objective To construct eukaryotic expression vector of pcDNA3.1-hCOX-2.
Methods The hCOX-2 cDNA fragment was extracted from plasmid pSG5-COX-2,amplified by PCR,and inserted into pcDNA3.1 vector before the double digestion with restriction enzymes EcoRI and BamHI.Then the vector was identified by the double digestion with restriction enzymes and was sequenced by the Sanger-dideoxy-mediated chain termination.
Results The recombinant eukaryotic expression vector for COX-2 was digested with EcoRI and BamHI,and the electrophoresis of the digested products showed two fragments:1800 bp fragmant and 5.4 kb fragment.The sequence of 1800 bp fragment was identical to COX-2 sequence published in GeneBank.
Conclusions The recombinant plasmid pcDNA3.1-hCOX-2 was constructed successfully and can be applicated into subsequent transfection experiment.
2.2 The increased COX-2 expression and PGE_2 production in the transfected RCC cell line after pcDNA3.1-hCOX-2 transfection
Objective To detect the expression of COX-2 gene and protein in RCC cell lines and pick out RCC cell line with higher expression of COX-2 for the following transfection experiment.Next we transfect pcDNA3.1-hCOX-2 plasmid vector into the targeted RCC cell line,then we analyze its effect to the expression of COX-2 gene and protein,especially we tend to know its effect to the production of PGE_2 in the RCC cell line supematants.In addition,we add different doses of NS-398 into the transfection group to know their effect to the production PGE_2 in the supernatants.
Methods(1) Two RCC cell lines 786-0 and OS-RC-2,were cultured in vitro. The expressions of COX-2 mRNA were assessed by RT-PCR,and its protein expressions were determined by Western-blot.The results were compared with each other to determine which cell line can be used in the next part of experiments.(2) To transfect pcDNA3.1-hCOX-2 plasmid vector into OS-RC-2 using lipofectamine 2000,COX-2 mRNA were detected 72h after transfection by RT-PCR and COX-2 protein were analysed by Western-blot 72h after transfection.(3) The different levels of PGE_2 in the transfected OS-RC-2 cell line supernatants and control groups were analysed by ELISA.In addition,we added 12.5μM,25μM,50μM,100μM NS-398 among the supernatants respectively and detected the PGE_2 levels by ELISA.
Results(1) The two RCC cell lines had different COX-2 mRNA and protein expression.RT-PCR:The COX-2/beta-actin ratio was used to represent the expression of COX-2 mRNA.The ratio of 786-0 and OS-RC-2 cell lines were 0.2756±0.01318 and 0.4667±0.02159 respectively.It represented that the expression of COX-2 mRNA of 786-0 was about 59.1%compared with that of OS-RC-2 cell lines.The results between 786-0 and OS-RC-2 cell lines had significant difference(p＜0.05). Western-blot:The COX-2/GAPDH ratio was used to represent the expression of COX-2 protein.The ratio of 786-0 and OS-RC-2 cell lines were 0.2333±0.02183 and 0.4686±0.04294 respectively.It represented that the expression of COX-2 protein of 786-0 was about 50%compared with that of OS-RC-2 cell lines.The results between 786-0 and OS-RC-2 cell lines had significant difference(p＜0.05).Next we chose OS-RC-2 as the targeted cell line for the next transfection experiment.(2) The expression of COX-2 mRNA and protein in the transfected OS-RC-2 cell line have increased significantly after pcDNA3.1-hCOX-2 plasmid vector transfection.We divided into three groups:pcDNA3.1-hCOX-2,pcDNA3.1 and control group.Results of RT-PCR:COX-2 mRNA was detected 72h after transfection.The COX-2mRNA in the above three groups were 0.7922±0.07139、0.4325±0.02045、0.4739±0.02377 respectively.Compared with pcDNA3.1 and control groups,the transfection group had higher COX-2mRNA expression(p＜0.05),while the COX-2 mRNA between pcDNA3.1 and control group had no significant difference(p＞0.05).Results of Western-blot:COX-2 protein was detected 72h after transfection.The COX-2 protein in the above three groups were 0.8593±0.1332、0.4321±0.03362、0.4533±0.05883 respectively.Compared with pcDNA3.1 and control groups,the transfection group had higher COX-2 protein expression(p＜0.05),while the COX-2 protein between pcDNA3.1 and control group had no significant difference(p＞0.05).(3)The result of ELISA experiment indicated that PGE_2 levels varied significantly among pcDNA3.1-hCOX-2,pcDNA3.1 and control group.The PGE_2 levels in the above three groups were 6.330±1.181、0.2817±0.03181、0.3030±0.03246 respectively. Compared with pcDNA3.1 and control groups,the transfection group had higher PGE_2 production(p＜0.05),while PGE_2 production between pcDNA3.1 and control group had no significant difference(p＞0.05).When applicating NS-398 at 12.5μM、25μM、50μM、100μM respectively in pcDNA3.1-hCOX-2 transfection group,the PGE_2 levels were 2.906±0.5892、0.6484±0.09880、0.4189±0.06513、0.2221±0.04094 respectively.Thus,the PGE_2 production between these groups and pcDNA3.1-hCOX-2 transfection group had significant difference(p＜0.05).
Conclusions(1) The two RCC cell lines had COX-2 mRNA and protein expression.Since OS-RC-2 cell line had higher COX-2 expression,we chose OS-RC-2 as target cell line for the following transfection experiment.(2) The constructed pcDNA3.1-hCOX-2 could be successfully transfected into OS-RC-2 cell line using lipofectamine 2000,and subsequently increased the COX-2 expression.(3) The production of PGE_2 in the transfected OS-RC-2 cell line increased significantly. The application of NS-398 could reduce PGE_2 production at the supernatants in a dose-dependent manner.
2.3 The increased conversion from CD4~+FOXP3~- T cell to CD4~+FOXP3~+ Treg in the supernatants of pcDNA3.1-hCOX-2 transfection group
Objective To investigate whether the supernatants from cultured transfected OS-RC-2 cell line could convert peripheral CD4~+FOXP3~- T cell into CD4~+FOXP3~+ Treg.
Methods We obtained peripheral blood mononuclear cells by density gradient centrifugation method.Subsequently we isolated CD4~+CD25~- T cell and CD4~+ CD25~+ Treg by MACS and analysed the purity of isolated cells by RT-PCR and FACS respectively.Under the stimulation of anti-CD3/CD28 antibody and APC cells, isolated CD4~+FOXP3~- T cells were cocultured with transfected OS-RC-2 supernatants and different control supernatants respectively,96 hours later,the proportion of CD4~+FOXP3~+ Treg in each group were detected by FACS.
Results(1) MACS could obtain CD4~+CD25- T cell and CD4~+CD25~+ Treg steadily and the purity of two groups were over 90%respectively.The latter characteristically expressed FOXP3 gene and protein.(2) The transfected OS-RC-2 cell line supernatants could obviously induce the production of CD4~+FOXP3~+Treg, the proportion of CD4~+FOXP3~+Treg reached 30.00±2.618%after 96 hours coculture, while NS-398 could markedly reduce the above effect,the proportion of CD4~+ FOXP3~+ Tregs was only 7.990±1.227%in the transfection+100μMNS-398 group.
Conclusions(1) MACS could obtain CD4~+CD25~-T cell and CD4~+CD25~+ Treg steadily.Since CD4~+CD25~+ Treg specifically express FOXP3 gene and protein,we regard CD4~+CD25~+Treg as CD4~+FOXP3~+Treg,while CD4~+CD25~- T cell do not express FOXP3 gene and protein,we regard CD4~+CD25~-T cell as CD4~+FOXP3~- T cell in the following experiment.(2) The transfected RCC cell line supernatants could induce the transformation from CD4~+FOXP3~- T cells to CD4~+FOXP3~+ Treg,while NS-398 could reduce the proportion of induced CD4~+FOXP3~+Treg.Therefore, COX-2 inhibitors may induce the local anti-tumor effect and in turn contribute to eradicating RCC.
引文
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