胃癌相关成纤维细胞源生长因子和趋化因子的高通量筛选和验证
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摘要
胃癌是我国发病率和死亡率最高的消化系统肿瘤,在世界范围内发病率并不平衡,但病人总体5年死亡率超过80%,每年因胃癌死亡的人数居世界癌症死亡病人的第二位,严重威胁着人类健康。新近,肿瘤研究中出现了一些新的思路和技术,例如对肿瘤微环境的再认识以及蛋白质组学的迅猛发展,将为胃癌发病机理和防治研究带来新的契机。肿瘤组织微环境由功能性上皮、细胞外基质(Extracellular matrix, ECM)和间质细胞三部分组成,后者主要包括成纤维细胞、各种免疫细胞和血管／淋巴管内皮细胞；此外,肿瘤细胞还可以合成、分泌到细胞外间隙中新的基质成分。目前对肿瘤微环境的认识主要包括：1.微环境是细胞基因不稳定性的来源,可以诱发基因突变和DNA损伤,并导致DNA修复通路失调；2.许多有明确致癌作用的理化因素(慢性炎症、化学致癌物等)是通过破坏微环境的组织结构、改变间质细胞因子表达谱并重塑细胞外基质,从而刺激功能细胞发生恶变的；3.肿瘤细胞生长、侵袭、转移所需的氧、营养成分和生长刺激信号被认为是来自于周围的间质细胞；4.在适当的微环境中,恶性细胞可以被诱导恢复分化状态。5.ECM中存在的抗肿瘤成分,例如：肿瘤抑素(tumstatin)、内皮抑素(endostatin),已经得到成功分离正在进行临床试验。现有的一些研究已经表明,成纤维细胞在肿瘤微环境中的研究有着重要意义和良好前景：1.成纤维细胞组织微环境中主要的间质细胞,也是产生ECM(例如Ⅰ、Ⅱ、Ⅳ型胶原与纤维连接蛋白)和组织蛋白酶的主要细胞,处于“基质母细胞”的地位；2.成纤维细胞是正常组织结构的“守护者”,存在抑制周围上皮细胞发生恶变的机制,已发生癌变的细胞在正常成纤维细胞环境下也可以向正常细胞分化；3.成纤维细胞还是上皮细胞增生、分化的“调控者”,成纤维细胞发生转化后可以表达多种细胞因子、粘附分子直接作用于上皮细胞,促进肿瘤细胞的发生、生长、血管生成、浸润与转移。因此,对肿瘤微环境中成纤维细胞进行深入研究必将有助于对肿瘤发生机制以及浸润、转移等生物学行为的理解以及新的治疗靶点的发现。
鉴于成纤维细胞具有组织特异性,哺乳动物成纤维细胞具有高度异质性,不同部位分离出来的成纤维细胞反映出明显的部位多样性。最近的研究表明,和不同谱系的白细胞的基因表达模式一样,从16个不同部位分离出来的50个人成纤维细胞种群的基因表达模式也有部位的特异性,存在于不同部位的成纤维细胞分泌不同的胞外基质成分、生长因子和分化因子。那么胃癌发生过程中是否存在组织特异的分泌因子或信号蛋白?是否还存在未知的、与胃癌发生密切相关的成纤维细胞生长因子?本项目拟从胃癌相关成纤维细胞切入胃癌微环境的研究,体外培养和鉴定人胃粘膜成纤维细胞(HGMF)和胃癌相关成纤维细胞(GCAF),接着对从HGMF和GCAF从形态学、细胞学和生物学功能各方面进行对比,证实二者确实存在显著的生物学行为方面的差异,进而利用人细胞因子芯片技术对HGMF和GCAF的培养上清液进行分析,筛选出差异因子,选择其中表达量较高,差异较明显,且文献报道与肿瘤密切相关的生长因子以及趋化因子用ELISA法在HGMF-GCAF上清液之间及正常-胃癌血清间进行验证。这些胃癌成纤维细胞来源的生长因子和趋化因子的表达差异提示胃癌微环境中的成纤维细胞在胃癌的发生、发展过程中起重要作用,进一步探讨它们与胃癌类型、分期的关系,可能寻找GCAF表达和分泌的、与胃癌发生、发展密切相关的关键蛋白。HGMF和GCAF功能上的差异研究将为下一步对两者进行蛋白质组学分析,建立HGMF和GCAF各自的蛋白表达谱并筛选出二者之间的差异蛋白提供实验基础。此外,对HGMF和GCAF之间的差异蛋白进行深入的研究和分析,可能会发现新的、与胃癌密切相关的成纤维细胞源因子。
第一部分胃粘膜成纤维细胞的原代培养、鉴定和培养条件的优化
目的
原代培养和鉴定人胃粘膜成纤维细胞,并对培养条件进行优化,提高培养成功率,使其适用于进一步研究需要。
方法
1.人胃粘膜成纤维细胞的原代分离和培养采用植块法。
2.人胃粘膜成纤维细胞的鉴定：分别用免疫组化方法和流式细胞术分别检测角蛋白(keratin)、波形丝蛋白(vimentin)和结蛋白(Desmin)表达情况。
3.人胃成纤维细胞的原代培养条件的优化；选择培养表面、培养基种类和培养液PH值三个因素,每个因素设三个水平,观察不同组合条件下原代培养成纤维细胞的游出情况。
结果
1.完成人胃粘膜成纤维细胞的原代分离和培养,对临床标本的取材、运输、培养时间等摸索出特定的方法和条件。
2.免疫组化染色方法和流式细胞术鉴定结果相吻合,原代培养的人胃粘膜成纤维细胞符合角蛋白(keratin)表达阴性,波形丝蛋白(vimentin)表达阳性,结蛋白(Desmin)表达阴性。
3.含20%胎牛血清RPMI1640培养液,PH值为7.4是人胃粘膜成纤维细胞的原代培养的适宜培养条件。
结论
1.人胃粘膜成纤维细胞的原代从取材、运输、培养时间、培养条件等都有其特殊要求,原代培养的条件优化后可行性和可重复性较高。
2.通过原代培养可以获得高纯度的人胃黏膜成纤维细胞。
第二部分体外胃癌相关成纤维细胞(GCAF)和人胃粘膜成纤维细胞(HGMF)形态学、生物学特性和功能差异分析
目的
探讨胃癌相关成纤维细胞(GCAF)和人胃粘膜成纤维细胞(HGMF)在生物学特性和功能活性方面的差异。
方法
1.细胞HE染色光镜观察和透射电镜观察HGMF和GCAF形态学上的差异。
2.CCK-8法检测两者的增殖活性并绘制生长曲线。
3.BCA法检测两者的总蛋白。
4.流式细胞术检测HGMF和GCAF的细胞周期。
5.分别用CCK-8法、Tanswell小室检测及流式细胞检测GCAF条件培养基对胃癌
细胞系BGC-823增殖、侵袭能力和细胞周期的影响。
结果
1. GCAF和HGMF形态学对比：HGMF光镜下呈梭长形,大小一致,可见少量双核细胞；GCAF胞体变大,细胞不规则,且大小不一,多见大胞体细胞、双核及多核细胞；透射电镜观察GCAF内可见畸形核,多个核仁且核仁边集,核浆比增加,线粒体、滑面内质网增多,可见到高尔基复合体及较多低电子密度块状物,且线粒体肿胀,粗面内质网轻度扩张。
2. GCAF和HGMF细胞增殖能力对比：CCK-8所测细胞生长曲线显示与HGMF对比,GCAF增殖活性明显增强,差异有统计学意义。
3. GCAF和HGMF细胞总蛋白测定：测得1×106个GCAF蛋白浓度明显高于HGMF,分别为1.0018±0.0940mg/ml和0.7688±0.0441 mg/ml(t=5.0155, P=0.0010)。
4. GCAF和HGMF细胞周期检测：GCAF及HGMF的增殖指数分别为42.0200±2.7314%和35.2520±4.4368%,差异有统计学意义P= 0.0198)。GCAF及HGMF的SPF分别为19.5040±2.7944%和9.3180±2.2068%,差异有统计学意义(P=0.0002)。
5. GCAF条件培养基对胃癌细胞系BGC-823增殖和侵袭能力的影响：培养24h、48h及72h, GCAF-CM组(蛋白浓度分别为0.25ug/uL、0.5ug/uL、1.0ug/uL)胃癌细胞增殖活性均比纯RPMI1640培养液组增高(P值均<0.05),且胃癌细胞的增殖能力与GCAF-CM的蛋白含量成正相关；最高蛋白浓度GCAF-CM组(1.0ug/uL)在24h、48h及72h细胞增殖活性均与10%FCS组相当,差异无统计学意义(P值分别=0.36、0.79、0.75)；蛋白浓度0.5ug/uL的GCAF-CM组观察到的侵袭细胞数量比纯RPMI1640培养液组增多(7.33±1.2比2.33±1.85,P=0.0335),且胃癌细胞BGC-823的侵袭数量与GCAF-CM的蛋白浓度呈正相关；而蛋白浓度1.0ug/uL的GCAF-CM组与10%FCS组的侵袭细胞数量相当,差异无统计学意义(20±2.78比22.33±3.7, P=0.239); GCAF-CM组及RPMI1640培养液组的增殖指数分别为46.09±1.19%和39.98±3.07%,差异有统计学意义(P=0.002); GCAF-CM组及RPMI1640培养液组的SPF分别为17.88±0.99%和14.43±0.87%,差异有统计学意义(P=0.0199)。
结论原代培养的GCAF和HGMF在形态学、细胞增殖能力、总蛋白含量、细胞周期及其对胃癌细胞增殖和侵袭的影响等方面存在显著差异。
第三部分GCAF和HGMF培养上清液细胞因子芯片检测及ELISA验证
目的
筛选GCAF和HGMF源差异细胞因子并进行验证。
方法
1细胞培养上清液制备：取1×106个生长良好的第3代GCAF和HGMF,以纯RPMI1640培养液培养48h后收集细胞培养上清备用。
2 RayBio(?)人细胞因子抗体芯片检测GCAF和HGMF上清液中120种因子的表达量,采集图象和数据,计算各抗体表达的标准值。
3根据细胞因子芯片筛选结果选择肝细胞生长因子(HGF)、血管生成素(Angiogenin)、转化生长因子-β1 (TGF-β1)、人细胞间粘附分子-1(sICAM-1)和人胰岛素样生长因子-1(IGF-1)、RANTES (Regulated upon activation, normal T cell expressed and secreted)、单核细胞趋化蛋白-1(monocyte chemotactic protein 1, MCP-1)、CCL16/HCC-4(Hemofiltrate CC Chemokine 4), CXCL9/MIG,人粘膜相关上皮趋化因子(MEC/CCL28)、GROα/CXCL1 (growth-related oncogene),用ELISA法在扩大样本量的HGMF-GCAF上清液之间及正常-胃癌血清间进行验证。
结果
1两组细胞因子芯片中高表达的因子共67个,GCAF-CM组中细胞因子表达量比值≥1.3的有23个,其中细胞因子6个,生长因子4个,信号蛋白7个,趋化因子6个。与HGMF-CM组相比；
2 GCAF和HGMF培养上清液中差异基因的ELISA验证
GCAF和HGMF培养上清液中表达HGF分别为448.32±100.13pg/ml和101.78±16.35pg/ml (P=0.0000), TGF-β1分别为191.41±21.04pg/ml和109.45±23.84pg/ml (P=0.0000), Angiogenin分别为67.35±17.17pg/ml和43.53±16.01pg/ml(P=0.0088), IGF-1分别为16.24±3.54ng/ml和12.30±2.62ng/ml (P=0.0111), CCL5/RANTES分别为487.15±117.54pg/ml和271.05±78.94pg/ml (P=0.0002), MCP-1/CXCL2分别为63.47±15.24pg/ml和51.46±11.33pg/ml(P=0.0524),CCL16／HCC-4分别为16.19±5.51pg/ml和8.66±3.43pg/ml(P=0.0018),CXCL9／MIG分别为278.36±59.45pg/ml和171.58±58.48pg/ml(P=0.0008),MEC／CCL28分别为219.51±64.61pg/ml和114.32±22.13pg/ml(P=0.0000),GROα／CXCL1分别为16.98±6.78pg/ml和10.91±3.58pg/ml (P=0.0221).
胃癌血清和正常血清表达HGF分别为687.52±127.37pg/ml和211.02±38.36pg/ml(P=0.0000),TGF-β1分别为219.51±64.61pg/ml和114.32±22.13pg／ml (P=0.0000),Angiogenin分别为63.76±15.22ng/ml和38.42±9.80ng/ml(P=0.0000), IGF-1分别为59.14±14.52ng/ml和41.56±12.72ng／ml(P=0.0000),CCL5／RANTES分别为5.21±0.97ng/ml和3.88±0.83ng／ml(P=0.0000),MCP-1／CXCL2分别为188.07±45.23pg/ml和128.76±30.68pg／ml(P=0.0000),CCL16／HCC-4分别为35.78±10.36pg／ml和11.48±4.01pg／ml(P=0.0000),CXCL9/MIG分别为758.67±184.41pg/ml和480.14±149.88pg／ml(P=0.0000),MEC／CCL28分别为886.46±255.51pg/ml和432.45±171.63pg/ml(P=0.0000),GROα／CXCL1分别为42.47±8.41pg/ml和18.54±8.47pg/ml(P=0.0000).
结论
GCAF与HGMF在部分细胞因子分泌水平上存在显著差异,可能是两者功能活性差异的重要生物学基础。
Recently, a lot of new treament and measurment have been applied to gastric cancer, such as recognition of tumor microenvironment and rapid development of proteomeology which can improve the investigation of pathogenesis and treatment of gastric cancer. The tumor microenvironment is consisted of three parts:functional endepidermis, extracellular matrix and mesenchymal cells. The mesenchymal cells include fibroblasts,various immunocytes and vascular/lympgatic vessel endotheliocytes.Tumour cells can also synthesize and secrete new matrix contents into extracellular space. Tumor microenvironment plays a very important role in the process of the tumor genesis and development:(1)Gene instability caused by microenvironment may induce gene mutation and DNA damage; furthermore, it can also lead to disorder of DNA repair pathways.(2)Many physical and chemical factors play a significant role in the carcinogenicity of functional cells by damaging tissue structure of microenvironment, alterating mesenchymal cytokine expression spectra and remodeling extracellular matrix.(3) The oxygen, nutrients and growth stimulation signal which are need by tumor cells for growth, invasion and metastasis are considered to come from surrounding stromal cells.(4)At the appropriate microenvironment, malignant tumor cells can be induced back to the differential state. (5)A few anti-tumor components in the ECM,such as tumstatin and endostatin, have been abstracted successfully in clinical trials.
Fibroblasts are widely used as model cells in many researches, which may be used as tissue engineering seed cells for clinical research in the future. Some former studies have shown that fibroblasts in the tumor microenvironment are very important and prospectful.Fibroblasts are main stromal cells of tissue microenvironment. They are also the main cells which produce ECM and cathepsin, occupying "maternal cell matrix" status. Fibroblasts are thought as "guardian" of normal tissue structure because they can inhibit periphery cellula epithelialis from being canceration. What is more, malignant tumor cells in normal fibroblasts environment can differentiate into normal cells.Fibroblasts are the "controlers" of epithelial cell proliferation and differentiation. Transformed fibroblasts can express a variety of cytokines, adhesion molecules, and then the genesis, growth, angiogenesis, invasion and metastasis of the epithelial and tumor cells will be enhanced. Accordingly, furhter study of fibroblasts in the tumor microenvironment will contribute to understanding biological behavior of the tumorigenesis mechanism and tumor invasion and metastasis to discovery new therapeutic target.
For fibroblasts have tissue specificity, does any tissue specific excreted factors or signal protein work during the procedure of gastric tumor carcinogenesis? Whether there are any unknown fibroblast growth factors that have close relation with gastric cancer? There are few cytological studies involving establishment and analysis of gastric cancer stromal fibroblasts model or special analysis of gastric cancer microenvironment and the important stromal cells. To invesgate the microenvironment of gastric cancer, we are going to begin with investigating gastric cancer stromal fibroblasts. Firstly, gastric cancer-asociated fibroblast (GCAF) and their couterpart normal human gastric mucosa fibroblast (HGMF) were isolated from 10 resected gastric cancer samples and primary cultured. We observed the diffrences of morphology, bioloagical feature and impact on gastirc cancer cell growth between GCAF and HGMF. Moreover, using Human chemokine antibody array, we compared the relative expression levels of cytokines in their conditioned medium. Some high expressed and significantly different growth factors and chemokines were determined as key different mediators secreted by GCAF and HGMF. Finally, we confirmed the different expression levels of these key cytokines in serum of gastric cancer patient and health controls by enzyme-linked immunosorbent assay (ELISA). Our findings indicate that GCAF and HGMF have different expression spectrums of cytokines, some of which may contribute to their different impact on gastric cancer cell growth and involve in promotion of gastric cancer progression.
Part 1 Primary culture and identification of HGMF and optimization of culture condition
Objective
Primary culture and identification of HGMF, and then optimize the culture condition in order to improve the success culture rate to make it meet the needs of research.
Methods
1. HGMF Primary separate and culture:obtained normal gastric mucosa tissue from resected gastric cancer specimens, using outgrowth method primary culture HGMF.
2. Identification of HGMF:Immunohistochemical and FCM method were used to identifica primary cultured HGMF.
3. Optimize culture conditions:compared original generation rate of HGMF under various culture conditions,such as culture surface, types and PH value of culture medium.
Results
1. We primarily separate and culture HGMF and found out ways and specific conditions of taking clinical specimens, transportationas as well as the culture time.
2. Optimized culture conditions:RPMI-1640 mixes with 20% FCS, plastic culture surface and PH=7.4 were ideal culture conditions.
3. Immunohistochemical staining and FCM method indicated primarily cultured HGMF negatively expressed keratin and desmin and positively expressed vimentin.
Conclusion
1. Primary culturation of HGMF had special requirement;optimized conditions may improve the repeatability and successful rate of primary culture.
2. Immunohistochemical staining and FCM method proved that the primarily cultured cells come from mesoderm.
Part 2 Differences of morphology, biological characteristic and function between human gastric mucosa fibroblasts and gastric cancer associated fibroblasts
Objective
Pairs of primary cultured GCAF and HGMF from the morphology, cell proliferation, total protein content, cell cycle and cell culture supernatant effect on gastric cancer cells and note the differences between the two kinds of cells. And to provide experimental basis for further research.
Methods
1.Observed HE staining cells under light microscope and transmission electron microscopy observation morphological differences between HGMF and GCAF.
2.CCK-8 method were used to detected their proliferative activity and draw a growth curve.
3.BCA was used to assay total protein of HGMF and GCAF.
4.Cell cycle of HGMF and GCAF were detected by flow cytometry.
5.CCK-8 method, tanswell small chamber testing and flow cytometry were used to conditioned media of GCAF effect on proliferation, invasion capacity and cell cycle of human gastric cancer cell line BGC-823,respectively.
Results
1.Morphology contrast:HGMF had long shuttle-shaped, consistent-size, showing a small amount of double-nucleated cells under light microscope;otherwise.GCAF had larger cell body, different sizes and irregular cell shape.And large cells, dual-core or multi-core cell were more common.
2.Contrast of cell proliferation capability:conpared with HGMF,GCAF had stronger proliferative activity.
3.Protein concentration of 1×106 GCAF and HGMF was 1.0018±0.0940mg/ml and 0.7688±0.0441mg/ml,respectively.GCAF's protein concentration significantly higher than that of HGMF's,
4.Proliferation index of GCAF and HGMF was 42.0200±2.7314%和35.2520±4.4368%,respectively.And SPF of the two was 19.5040±2.7944%和9.3180±2.2068%,respectively.
5.GCAF-CM enhanced the proliferation and invasive ability of gatric cancer cell line BGC-823.
Conclusion
There were significant differences of morphology, biological features and functions between primary cultured GCAF and HGMF.
Part 3 Cytokine microarray of culture supernatant between human gastric mucosa fibroblasts and gastric cancer associated fibroblasts and ELISA authentication
Objective
To explore expression levels of specific cytokines in culture supernatant of GCAF and HGMF,and then select some relevant different factors.Investigated their expression laws in large sample of GCAF-HGMF culture supernatant and gastric cancer-normal serum.To screening clinical diagnosis and treatment fibroblast derived targets of gastric cancer
Methods
1.Preparation of cell culture supernatant:1×106 well growing GCAF and HGMF of the third-generation,cultued with RPMI1640, culture supernatant were collected back after 48 hours later.
2. RayBio(?) Cytokine microarray was used to detect expression of 120 factors in GCAF and HGMF supernatant,and then images and data were collected and standard values expression of every antibody were calculated.
3.According to the results of cytokine microarray screening,we chose some high expressed and significantly different growth factors and chemokines. Study their expression laws in large sample of GCAF-HGMF culture supernatant and gastric cancer-normal serum by use of ELISA method.
Results
1.There wre a total of 67 high expression cytokines in the two groups,including 23 cytokines,8 growth factors,20 decapentaplegics and 16 chemotactic factors.Compared with the HGMF-CM,there are 22 cytokines in GCAF-CM higher expression in the value of≥1.3.
2. The expression levels of HGF in GCAF and HGMF supernatant is 448.32± 100.13pg/ml and 101.78±16.35pg/ml (P=0.0000),TGF-(31 is 191.41±21.04pg/ml and 109.45±23.84pg/ml (P=0.0000),Angiogenin is 67.35±17.17pg/ml and 43.53±16.01 pg/ml (P=0.0088),IGF-1 is 16.24±3.54ng/ml and 12.30±2.62ng/ml (P=0.0111),CCL5/RANTES is 487.15±117.54pg/ml and 271.05±78.94pg/ml (P=0.0002),MCP-1/CXCL2 is 63.47±15.24pg/ml and 51.46±11.33pg/ml (P=0.0524),CCL16/HCC-4 is 16.19±5.51pg/ml and 8.66±3.43pg/ml (P=0.0018), CXCL9/MIG is 278.36±59.45pg/ml and 171.58±58.48pg/ml (P=0.0008), MEC/CCL28 is 219.51±64.61pg/ml and 114.32±22.13pg/ml (P=0.0000), GROa/CXCL1 is 16.98±6.78pg/ml and 10.91±3.58pg/ml (P=0.0221).The expression levels of HGF in gastric cancer and normal serum is 687.52±127.37pg/ml and 211.02±38.36pg/ml,TGF-β1 is 219.51±64.61pg/ml and 114.32±22.13pg/ml (P=0.0000),Angiogenin is 63.76±15.22ng/ml and 38.42±9.80ng/ml (P=0.0000), ICAM-1 is 3.44±0.65ng/ml and 2.75±0.64ng/ml (P=0.0000),IGF-1 is 59.14±14.52ng/ml and 41.56±12.72ng/ml (P=0.0000), CCL5/RANTES is 5.21±0.97ng/ml and 3.88±0.83ng/ml (P=0.0000),MCP-1/CXCL2 is 188.07±45.23pg/ml and 128.76±30.68pg/ml (P=0.0000),CCL16/HCC-4 is 35.78±10.36pg/ml and 11.48±4.01pg/ml (P=0.0000),CXCL9/MIG is 758.67±184.41pg/ml and 480.14±149.88pg/ml (P=0.0000),MEC/CCL28 is 886.46±255.51pg/ml and 432.45±171.63pg/ml (P=0.0000),GROa/CXCL1 is 142.47±8.41pg/ml and 18.54±8.47 (P=0.0000)
Conclusion
The ability to secrete cytokines between GCAF and HGMF were significantly different.The expression level of HGF,Angiogenin,TGF-β1,IGF-1,CCL5/ RANTES,MCP-1/CXCL2,CCL16/HCC-4,CXCL9/MIG,MEC/CCL28 and GROα/CXCL1 of gastric cancer serum was significantly higher than that of normal serum. Differenct factors secreted by fibroblasts may serve as indicators of of diagnosis and prognosis in gastric cancer.
鉴于成纤维细胞具有组织特异性,哺乳动物成纤维细胞具有高度异质性,不同部位分离出来的成纤维细胞反映出明显的部位多样性。最近的研究表明,和不同谱系的白细胞的基因表达模式一样,从16个不同部位分离出来的50个人成纤维细胞种群的基因表达模式也有部位的特异性,存在于不同部位的成纤维细胞分泌不同的胞外基质成分、生长因子和分化因子。那么胃癌发生过程中是否存在组织特异的分泌因子或信号蛋白?是否还存在未知的、与胃癌发生密切相关的成纤维细胞生长因子?本项目拟从胃癌相关成纤维细胞切入胃癌微环境的研究,体外培养和鉴定人胃粘膜成纤维细胞(HGMF)和胃癌相关成纤维细胞(GCAF),接着对从HGMF和GCAF从形态学、细胞学和生物学功能各方面进行对比,证实二者确实存在显著的生物学行为方面的差异,进而利用人细胞因子芯片技术对HGMF和GCAF的培养上清液进行分析,筛选出差异因子,选择其中表达量较高,差异较明显,且文献报道与肿瘤密切相关的生长因子以及趋化因子用ELISA法在HGMF-GCAF上清液之间及正常-胃癌血清间进行验证。这些胃癌成纤维细胞来源的生长因子和趋化因子的表达差异提示胃癌微环境中的成纤维细胞在胃癌的发生、发展过程中起重要作用,进一步探讨它们与胃癌类型、分期的关系,可能寻找GCAF表达和分泌的、与胃癌发生、发展密切相关的关键蛋白。HGMF和GCAF功能上的差异研究将为下一步对两者进行蛋白质组学分析,建立HGMF和GCAF各自的蛋白表达谱并筛选出二者之间的差异蛋白提供实验基础。此外,对HGMF和GCAF之间的差异蛋白进行深入的研究和分析,可能会发现新的、与胃癌密切相关的成纤维细胞源因子。
第一部分胃粘膜成纤维细胞的原代培养、鉴定和培养条件的优化
目的
原代培养和鉴定人胃粘膜成纤维细胞,并对培养条件进行优化,提高培养成功率,使其适用于进一步研究需要。
方法
1.人胃粘膜成纤维细胞的原代分离和培养采用植块法。
2.人胃粘膜成纤维细胞的鉴定：分别用免疫组化方法和流式细胞术分别检测角蛋白(keratin)、波形丝蛋白(vimentin)和结蛋白(Desmin)表达情况。
3.人胃成纤维细胞的原代培养条件的优化；选择培养表面、培养基种类和培养液PH值三个因素,每个因素设三个水平,观察不同组合条件下原代培养成纤维细胞的游出情况。
结果
1.完成人胃粘膜成纤维细胞的原代分离和培养,对临床标本的取材、运输、培养时间等摸索出特定的方法和条件。
2.免疫组化染色方法和流式细胞术鉴定结果相吻合,原代培养的人胃粘膜成纤维细胞符合角蛋白(keratin)表达阴性,波形丝蛋白(vimentin)表达阳性,结蛋白(Desmin)表达阴性。
3.含20%胎牛血清RPMI1640培养液,PH值为7.4是人胃粘膜成纤维细胞的原代培养的适宜培养条件。
结论
1.人胃粘膜成纤维细胞的原代从取材、运输、培养时间、培养条件等都有其特殊要求,原代培养的条件优化后可行性和可重复性较高。
2.通过原代培养可以获得高纯度的人胃黏膜成纤维细胞。
第二部分体外胃癌相关成纤维细胞(GCAF)和人胃粘膜成纤维细胞(HGMF)形态学、生物学特性和功能差异分析
目的
探讨胃癌相关成纤维细胞(GCAF)和人胃粘膜成纤维细胞(HGMF)在生物学特性和功能活性方面的差异。
方法
1.细胞HE染色光镜观察和透射电镜观察HGMF和GCAF形态学上的差异。
2.CCK-8法检测两者的增殖活性并绘制生长曲线。
3.BCA法检测两者的总蛋白。
4.流式细胞术检测HGMF和GCAF的细胞周期。
5.分别用CCK-8法、Tanswell小室检测及流式细胞检测GCAF条件培养基对胃癌
细胞系BGC-823增殖、侵袭能力和细胞周期的影响。
结果
1. GCAF和HGMF形态学对比：HGMF光镜下呈梭长形,大小一致,可见少量双核细胞；GCAF胞体变大,细胞不规则,且大小不一,多见大胞体细胞、双核及多核细胞；透射电镜观察GCAF内可见畸形核,多个核仁且核仁边集,核浆比增加,线粒体、滑面内质网增多,可见到高尔基复合体及较多低电子密度块状物,且线粒体肿胀,粗面内质网轻度扩张。
2. GCAF和HGMF细胞增殖能力对比：CCK-8所测细胞生长曲线显示与HGMF对比,GCAF增殖活性明显增强,差异有统计学意义。
3. GCAF和HGMF细胞总蛋白测定：测得1×106个GCAF蛋白浓度明显高于HGMF,分别为1.0018±0.0940mg/ml和0.7688±0.0441 mg/ml(t=5.0155, P=0.0010)。
4. GCAF和HGMF细胞周期检测：GCAF及HGMF的增殖指数分别为42.0200±2.7314%和35.2520±4.4368%,差异有统计学意义P= 0.0198)。GCAF及HGMF的SPF分别为19.5040±2.7944%和9.3180±2.2068%,差异有统计学意义(P=0.0002)。
5. GCAF条件培养基对胃癌细胞系BGC-823增殖和侵袭能力的影响：培养24h、48h及72h, GCAF-CM组(蛋白浓度分别为0.25ug/uL、0.5ug/uL、1.0ug/uL)胃癌细胞增殖活性均比纯RPMI1640培养液组增高(P值均<0.05),且胃癌细胞的增殖能力与GCAF-CM的蛋白含量成正相关；最高蛋白浓度GCAF-CM组(1.0ug/uL)在24h、48h及72h细胞增殖活性均与10%FCS组相当,差异无统计学意义(P值分别=0.36、0.79、0.75)；蛋白浓度0.5ug/uL的GCAF-CM组观察到的侵袭细胞数量比纯RPMI1640培养液组增多(7.33±1.2比2.33±1.85,P=0.0335),且胃癌细胞BGC-823的侵袭数量与GCAF-CM的蛋白浓度呈正相关；而蛋白浓度1.0ug/uL的GCAF-CM组与10%FCS组的侵袭细胞数量相当,差异无统计学意义(20±2.78比22.33±3.7, P=0.239); GCAF-CM组及RPMI1640培养液组的增殖指数分别为46.09±1.19%和39.98±3.07%,差异有统计学意义(P=0.002); GCAF-CM组及RPMI1640培养液组的SPF分别为17.88±0.99%和14.43±0.87%,差异有统计学意义(P=0.0199)。
结论原代培养的GCAF和HGMF在形态学、细胞增殖能力、总蛋白含量、细胞周期及其对胃癌细胞增殖和侵袭的影响等方面存在显著差异。
第三部分GCAF和HGMF培养上清液细胞因子芯片检测及ELISA验证
目的
筛选GCAF和HGMF源差异细胞因子并进行验证。
方法
1细胞培养上清液制备：取1×106个生长良好的第3代GCAF和HGMF,以纯RPMI1640培养液培养48h后收集细胞培养上清备用。
2 RayBio(?)人细胞因子抗体芯片检测GCAF和HGMF上清液中120种因子的表达量,采集图象和数据,计算各抗体表达的标准值。
3根据细胞因子芯片筛选结果选择肝细胞生长因子(HGF)、血管生成素(Angiogenin)、转化生长因子-β1 (TGF-β1)、人细胞间粘附分子-1(sICAM-1)和人胰岛素样生长因子-1(IGF-1)、RANTES (Regulated upon activation, normal T cell expressed and secreted)、单核细胞趋化蛋白-1(monocyte chemotactic protein 1, MCP-1)、CCL16/HCC-4(Hemofiltrate CC Chemokine 4), CXCL9/MIG,人粘膜相关上皮趋化因子(MEC/CCL28)、GROα/CXCL1 (growth-related oncogene),用ELISA法在扩大样本量的HGMF-GCAF上清液之间及正常-胃癌血清间进行验证。
结果
1两组细胞因子芯片中高表达的因子共67个,GCAF-CM组中细胞因子表达量比值≥1.3的有23个,其中细胞因子6个,生长因子4个,信号蛋白7个,趋化因子6个。与HGMF-CM组相比；
2 GCAF和HGMF培养上清液中差异基因的ELISA验证
GCAF和HGMF培养上清液中表达HGF分别为448.32±100.13pg/ml和101.78±16.35pg/ml (P=0.0000), TGF-β1分别为191.41±21.04pg/ml和109.45±23.84pg/ml (P=0.0000), Angiogenin分别为67.35±17.17pg/ml和43.53±16.01pg/ml(P=0.0088), IGF-1分别为16.24±3.54ng/ml和12.30±2.62ng/ml (P=0.0111), CCL5/RANTES分别为487.15±117.54pg/ml和271.05±78.94pg/ml (P=0.0002), MCP-1/CXCL2分别为63.47±15.24pg/ml和51.46±11.33pg/ml(P=0.0524),CCL16／HCC-4分别为16.19±5.51pg/ml和8.66±3.43pg/ml(P=0.0018),CXCL9／MIG分别为278.36±59.45pg/ml和171.58±58.48pg/ml(P=0.0008),MEC／CCL28分别为219.51±64.61pg/ml和114.32±22.13pg/ml(P=0.0000),GROα／CXCL1分别为16.98±6.78pg/ml和10.91±3.58pg/ml (P=0.0221).
胃癌血清和正常血清表达HGF分别为687.52±127.37pg/ml和211.02±38.36pg/ml(P=0.0000),TGF-β1分别为219.51±64.61pg/ml和114.32±22.13pg／ml (P=0.0000),Angiogenin分别为63.76±15.22ng/ml和38.42±9.80ng/ml(P=0.0000), IGF-1分别为59.14±14.52ng/ml和41.56±12.72ng／ml(P=0.0000),CCL5／RANTES分别为5.21±0.97ng/ml和3.88±0.83ng／ml(P=0.0000),MCP-1／CXCL2分别为188.07±45.23pg/ml和128.76±30.68pg／ml(P=0.0000),CCL16／HCC-4分别为35.78±10.36pg／ml和11.48±4.01pg／ml(P=0.0000),CXCL9/MIG分别为758.67±184.41pg/ml和480.14±149.88pg／ml(P=0.0000),MEC／CCL28分别为886.46±255.51pg/ml和432.45±171.63pg/ml(P=0.0000),GROα／CXCL1分别为42.47±8.41pg/ml和18.54±8.47pg/ml(P=0.0000).
结论
GCAF与HGMF在部分细胞因子分泌水平上存在显著差异,可能是两者功能活性差异的重要生物学基础。
Recently, a lot of new treament and measurment have been applied to gastric cancer, such as recognition of tumor microenvironment and rapid development of proteomeology which can improve the investigation of pathogenesis and treatment of gastric cancer. The tumor microenvironment is consisted of three parts:functional endepidermis, extracellular matrix and mesenchymal cells. The mesenchymal cells include fibroblasts,various immunocytes and vascular/lympgatic vessel endotheliocytes.Tumour cells can also synthesize and secrete new matrix contents into extracellular space. Tumor microenvironment plays a very important role in the process of the tumor genesis and development:(1)Gene instability caused by microenvironment may induce gene mutation and DNA damage; furthermore, it can also lead to disorder of DNA repair pathways.(2)Many physical and chemical factors play a significant role in the carcinogenicity of functional cells by damaging tissue structure of microenvironment, alterating mesenchymal cytokine expression spectra and remodeling extracellular matrix.(3) The oxygen, nutrients and growth stimulation signal which are need by tumor cells for growth, invasion and metastasis are considered to come from surrounding stromal cells.(4)At the appropriate microenvironment, malignant tumor cells can be induced back to the differential state. (5)A few anti-tumor components in the ECM,such as tumstatin and endostatin, have been abstracted successfully in clinical trials.
Fibroblasts are widely used as model cells in many researches, which may be used as tissue engineering seed cells for clinical research in the future. Some former studies have shown that fibroblasts in the tumor microenvironment are very important and prospectful.Fibroblasts are main stromal cells of tissue microenvironment. They are also the main cells which produce ECM and cathepsin, occupying "maternal cell matrix" status. Fibroblasts are thought as "guardian" of normal tissue structure because they can inhibit periphery cellula epithelialis from being canceration. What is more, malignant tumor cells in normal fibroblasts environment can differentiate into normal cells.Fibroblasts are the "controlers" of epithelial cell proliferation and differentiation. Transformed fibroblasts can express a variety of cytokines, adhesion molecules, and then the genesis, growth, angiogenesis, invasion and metastasis of the epithelial and tumor cells will be enhanced. Accordingly, furhter study of fibroblasts in the tumor microenvironment will contribute to understanding biological behavior of the tumorigenesis mechanism and tumor invasion and metastasis to discovery new therapeutic target.
For fibroblasts have tissue specificity, does any tissue specific excreted factors or signal protein work during the procedure of gastric tumor carcinogenesis? Whether there are any unknown fibroblast growth factors that have close relation with gastric cancer? There are few cytological studies involving establishment and analysis of gastric cancer stromal fibroblasts model or special analysis of gastric cancer microenvironment and the important stromal cells. To invesgate the microenvironment of gastric cancer, we are going to begin with investigating gastric cancer stromal fibroblasts. Firstly, gastric cancer-asociated fibroblast (GCAF) and their couterpart normal human gastric mucosa fibroblast (HGMF) were isolated from 10 resected gastric cancer samples and primary cultured. We observed the diffrences of morphology, bioloagical feature and impact on gastirc cancer cell growth between GCAF and HGMF. Moreover, using Human chemokine antibody array, we compared the relative expression levels of cytokines in their conditioned medium. Some high expressed and significantly different growth factors and chemokines were determined as key different mediators secreted by GCAF and HGMF. Finally, we confirmed the different expression levels of these key cytokines in serum of gastric cancer patient and health controls by enzyme-linked immunosorbent assay (ELISA). Our findings indicate that GCAF and HGMF have different expression spectrums of cytokines, some of which may contribute to their different impact on gastric cancer cell growth and involve in promotion of gastric cancer progression.
Part 1 Primary culture and identification of HGMF and optimization of culture condition
Objective
Primary culture and identification of HGMF, and then optimize the culture condition in order to improve the success culture rate to make it meet the needs of research.
Methods
1. HGMF Primary separate and culture:obtained normal gastric mucosa tissue from resected gastric cancer specimens, using outgrowth method primary culture HGMF.
2. Identification of HGMF:Immunohistochemical and FCM method were used to identifica primary cultured HGMF.
3. Optimize culture conditions:compared original generation rate of HGMF under various culture conditions,such as culture surface, types and PH value of culture medium.
Results
1. We primarily separate and culture HGMF and found out ways and specific conditions of taking clinical specimens, transportationas as well as the culture time.
2. Optimized culture conditions:RPMI-1640 mixes with 20% FCS, plastic culture surface and PH=7.4 were ideal culture conditions.
3. Immunohistochemical staining and FCM method indicated primarily cultured HGMF negatively expressed keratin and desmin and positively expressed vimentin.
Conclusion
1. Primary culturation of HGMF had special requirement;optimized conditions may improve the repeatability and successful rate of primary culture.
2. Immunohistochemical staining and FCM method proved that the primarily cultured cells come from mesoderm.
Part 2 Differences of morphology, biological characteristic and function between human gastric mucosa fibroblasts and gastric cancer associated fibroblasts
Objective
Pairs of primary cultured GCAF and HGMF from the morphology, cell proliferation, total protein content, cell cycle and cell culture supernatant effect on gastric cancer cells and note the differences between the two kinds of cells. And to provide experimental basis for further research.
Methods
1.Observed HE staining cells under light microscope and transmission electron microscopy observation morphological differences between HGMF and GCAF.
2.CCK-8 method were used to detected their proliferative activity and draw a growth curve.
3.BCA was used to assay total protein of HGMF and GCAF.
4.Cell cycle of HGMF and GCAF were detected by flow cytometry.
5.CCK-8 method, tanswell small chamber testing and flow cytometry were used to conditioned media of GCAF effect on proliferation, invasion capacity and cell cycle of human gastric cancer cell line BGC-823,respectively.
Results
1.Morphology contrast:HGMF had long shuttle-shaped, consistent-size, showing a small amount of double-nucleated cells under light microscope;otherwise.GCAF had larger cell body, different sizes and irregular cell shape.And large cells, dual-core or multi-core cell were more common.
2.Contrast of cell proliferation capability:conpared with HGMF,GCAF had stronger proliferative activity.
3.Protein concentration of 1×106 GCAF and HGMF was 1.0018±0.0940mg/ml and 0.7688±0.0441mg/ml,respectively.GCAF's protein concentration significantly higher than that of HGMF's,
4.Proliferation index of GCAF and HGMF was 42.0200±2.7314%和35.2520±4.4368%,respectively.And SPF of the two was 19.5040±2.7944%和9.3180±2.2068%,respectively.
5.GCAF-CM enhanced the proliferation and invasive ability of gatric cancer cell line BGC-823.
Conclusion
There were significant differences of morphology, biological features and functions between primary cultured GCAF and HGMF.
Part 3 Cytokine microarray of culture supernatant between human gastric mucosa fibroblasts and gastric cancer associated fibroblasts and ELISA authentication
Objective
To explore expression levels of specific cytokines in culture supernatant of GCAF and HGMF,and then select some relevant different factors.Investigated their expression laws in large sample of GCAF-HGMF culture supernatant and gastric cancer-normal serum.To screening clinical diagnosis and treatment fibroblast derived targets of gastric cancer
Methods
1.Preparation of cell culture supernatant:1×106 well growing GCAF and HGMF of the third-generation,cultued with RPMI1640, culture supernatant were collected back after 48 hours later.
2. RayBio(?) Cytokine microarray was used to detect expression of 120 factors in GCAF and HGMF supernatant,and then images and data were collected and standard values expression of every antibody were calculated.
3.According to the results of cytokine microarray screening,we chose some high expressed and significantly different growth factors and chemokines. Study their expression laws in large sample of GCAF-HGMF culture supernatant and gastric cancer-normal serum by use of ELISA method.
Results
1.There wre a total of 67 high expression cytokines in the two groups,including 23 cytokines,8 growth factors,20 decapentaplegics and 16 chemotactic factors.Compared with the HGMF-CM,there are 22 cytokines in GCAF-CM higher expression in the value of≥1.3.
2. The expression levels of HGF in GCAF and HGMF supernatant is 448.32± 100.13pg/ml and 101.78±16.35pg/ml (P=0.0000),TGF-(31 is 191.41±21.04pg/ml and 109.45±23.84pg/ml (P=0.0000),Angiogenin is 67.35±17.17pg/ml and 43.53±16.01 pg/ml (P=0.0088),IGF-1 is 16.24±3.54ng/ml and 12.30±2.62ng/ml (P=0.0111),CCL5/RANTES is 487.15±117.54pg/ml and 271.05±78.94pg/ml (P=0.0002),MCP-1/CXCL2 is 63.47±15.24pg/ml and 51.46±11.33pg/ml (P=0.0524),CCL16/HCC-4 is 16.19±5.51pg/ml and 8.66±3.43pg/ml (P=0.0018), CXCL9/MIG is 278.36±59.45pg/ml and 171.58±58.48pg/ml (P=0.0008), MEC/CCL28 is 219.51±64.61pg/ml and 114.32±22.13pg/ml (P=0.0000), GROa/CXCL1 is 16.98±6.78pg/ml and 10.91±3.58pg/ml (P=0.0221).The expression levels of HGF in gastric cancer and normal serum is 687.52±127.37pg/ml and 211.02±38.36pg/ml,TGF-β1 is 219.51±64.61pg/ml and 114.32±22.13pg/ml (P=0.0000),Angiogenin is 63.76±15.22ng/ml and 38.42±9.80ng/ml (P=0.0000), ICAM-1 is 3.44±0.65ng/ml and 2.75±0.64ng/ml (P=0.0000),IGF-1 is 59.14±14.52ng/ml and 41.56±12.72ng/ml (P=0.0000), CCL5/RANTES is 5.21±0.97ng/ml and 3.88±0.83ng/ml (P=0.0000),MCP-1/CXCL2 is 188.07±45.23pg/ml and 128.76±30.68pg/ml (P=0.0000),CCL16/HCC-4 is 35.78±10.36pg/ml and 11.48±4.01pg/ml (P=0.0000),CXCL9/MIG is 758.67±184.41pg/ml and 480.14±149.88pg/ml (P=0.0000),MEC/CCL28 is 886.46±255.51pg/ml and 432.45±171.63pg/ml (P=0.0000),GROa/CXCL1 is 142.47±8.41pg/ml and 18.54±8.47 (P=0.0000)
Conclusion
The ability to secrete cytokines between GCAF and HGMF were significantly different.The expression level of HGF,Angiogenin,TGF-β1,IGF-1,CCL5/ RANTES,MCP-1/CXCL2,CCL16/HCC-4,CXCL9/MIG,MEC/CCL28 and GROα/CXCL1 of gastric cancer serum was significantly higher than that of normal serum. Differenct factors secreted by fibroblasts may serve as indicators of of diagnosis and prognosis in gastric cancer.
引文
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