照射后成纤维细胞促进食管鳞癌EMT及HDGF表达的实验研究

发布时间：2018-06-20 10:07

本文选题：食管鳞癌 + 肝癌衍生生长因子　；参考：《山东大学》2015年硕士论文

【摘要】：研究背景食管鳞癌(Esophageal squamous cell carcinoma, ESCC)患者五年生存率仍然低于20%,放射治疗是ESCC患者一项重要的治疗方法。最近,有研究表明活化地肿瘤相关成纤维细胞(cancer-associated fibroblasts, CAFs)与ESCC患者的差的预后及同步放化疗后局部复发密切相关。越来越多的研究亦显示放射线可以通过作用肿瘤间质微环境影响肿瘤发生发展,由此丰富了放射线诱发肿瘤的证据。近几年来肝癌衍生生长因子(Hepatoma-derived growth factor, HDGF)引起了学术界广泛关注,它既是具有促成纤维细胞有丝分裂活性的酸性多肽,也与多种肿瘤差的预后密切相关：同时其表达变化与多种肿瘤的转化、凋亡、血管生成以及转移密切相关。HDGF与ESCC患者的放疗敏感性以及肿瘤复发亦有紧密联系。CAFs与肿瘤转移的多种信号转导通路相关联,包括可以促进肿瘤细胞的上皮间质转化(Epithelial-mesenchymal transition, EMT)过程,而大量研究表明EMT可以赋予细胞迁移、侵袭能力以及诱导干细胞特性。并且已有多篇文献证明间质成纤维细胞可以通过EMT促进肿瘤细胞侵袭与转移。与此同时,最近越来越多的研究表明γ射线源照射成纤维细胞可以通过肿瘤间质相互作用促进肿瘤发生以及侵袭生长。而X射线源照射成纤维细胞是否促进ESCC侵袭转移及其机制尚有待于进一步研究。目的1.本实验旨在研究X射线对间质成纤维细胞的作用以及照射后CAFs对ESCC细胞的影响。2.探讨在放射线对ESCC与间质成纤维细胞相互作用中的EMT相关蛋白以及HDGF表达变化。方法1.我们分离及培养原代CAFs和正常成纤维细胞(Normal Fibroblasts, NFs)来研究ESCC肿瘤微环境内的间质成纤维细胞特点。为了证明CAFs没有其他细胞成分混杂及其纯度,利用成纤维细胞标记蛋白纤连蛋白(fibronectin)区分成纤维细胞与ESCC细胞。Western blotting验证原代成纤维细胞高表达a-平滑肌肌动蛋白(a-SMA)和不表达E-钙粘蛋白(E-cadherin),体现原代成纤维细胞的细胞特性。2.在室温条件下,用X射线源以不同剂量照射原代成纤维细胞,剂量率为400cGy/min。照射后收集条件培养液留待后续培养ESCC细胞试验用。3.采用侵袭实验检测与照射后(4Gy和8Gy)或未照射成纤维细胞(NFs和CAFs)共培养的ESCC细胞的侵袭能力。4.采用Scattering实验检测照射后成纤维细胞的条件培养液对ESCC细胞的Scattering能力的影响。5. ESCC细胞和4Gy照射后成纤维细胞或未照射成纤维细胞均匀混合后种植于裸鼠右背侧皮下,每组六只。每3天用游标卡尺测量肿瘤大小一次并记录,肿瘤体积计算公式为：肿瘤体积(V)=(长径×宽径2)/2。6.为了研究照射后成纤维细胞促进ESCC细胞的迁移及侵袭与EMT有关,western blotting检测照射后成纤维细胞条件培养液和未照射成纤维细胞条件培养液培养24小时的ESCC细胞中上皮细胞标记物E-cadherin和间质细胞标记物波纹蛋白(vimentin)表达情况。并用免疫组化检测p-链蛋白(p-catenin)在照射组裸鼠移植瘤内的表达变化,未照射组为对照组。7.为探讨HDGF在放射线照射成纤维细胞促进ESCC细胞迁移与侵袭中的作用,采用western blotting检测照射后成纤维细胞条件培养液和未照射成纤维细胞条件培养液培养24小时的ESCC细胞中HDGF表达情况。并用免疫组化检测HDGF在照射组裸鼠移植瘤内的表达变化,未照射组为对照组。结果1.成纤维细胞的分离,提纯及特点结果表明从ESCC组织分离及培养的原代成纤维细胞保持成纤维细胞特性。用于本实验的所有成纤维细胞均为10代以内并表现为梭形细胞形态。3代以后的成纤维细胞E-cadherin未见表达,表明早期代数的成纤维细胞保持成纤维细胞特性。2.照射后成纤维细胞条件培养液增强ESCC细胞Scattering能力由于上皮细胞EMT的时候Scattering能力会有所增强,具体表现为细胞与细胞之间的黏附作用消失并获得较高活性的间质细胞表型,Scattering实验已被用于研究细胞的EMT和检测细胞迁移诱导能力。照射后成纤维细胞条件培养液增强ESCC细胞的Scattering能力并呈剂量依赖性。3.照射后成纤维细胞增强ESCC细胞的侵袭能力我们的研究表明ESCC细胞与未照射成纤维细胞共培养侵袭能力提高,当其与照射后成纤维细胞共培养其侵袭能力会得到进一步的增强。4.体内体外实验表明照射后成纤维细胞促进ESCC细胞EMT结果显示照射组中ESCC细胞E-cadherin呈剂量依赖性降低,vimentin随剂量依赖性升高并在所有组中高表达。体内体外实验表明P-catenin在照射组中都表达升高,免疫组化结果显示其在照射组肿瘤组织中的细胞核高表达及核转位。5.体内体外实验表明照射后成纤维细胞增强照射组中肿瘤细胞及成纤维细胞中HDGF的表达照射后成纤维细胞促进ESCC生长,特别是在Eca-9706肿瘤细胞组中。体内体外实验表明照射后成纤维细胞增强照射组中肿瘤细胞及成纤维细胞中HDGF的表达。值得注意的是,与间质成纤维细胞相邻的肿瘤细胞HDGF在细胞核中有相对更高的表达,显示成纤维细胞的旁分泌效应。结论1.照射后CAFs促进ESCC细胞侵袭及EMT,增强其Scattering能力。2.照射后成纤维细胞诱导ESCC细胞β-catenin核转位。3.照射后成纤维细胞促进ESCC肿瘤生长及提高HDGF蛋白表达。
[Abstract]:The five year survival rate of patients with Esophageal squamous cell carcinoma (ESCC) is still lower than 20%. Radiation therapy is an important treatment for ESCC patients. Recent studies have shown that the poor prognosis and concurrent chemo chemotherapy of activated tumor related fibroblasts (cancer-associated fibroblasts, CAFs) and ESCC patients Local recurrence is closely related. More and more studies have also shown that radiation can affect the development of tumor by acting on the interstitial microenvironment of tumor, which enriches the evidence of radiation induced tumor. In recent years, Hepatoma-derived growth factor (HDGF) has attracted extensive attention in the academic world. Acid peptides that contribute to mitosis of fibrous cells are also closely related to the prognosis of a variety of tumors: at the same time, the expression changes are closely related to the transformation, apoptosis, angiogenesis and metastasis of various tumors. The sensitivity of.HDGF to ESCC and the recurrence of tumor are closely related to the multiple signal transduction of.CAFs and tumor metastasis Pathways are associated with the process of promoting Epithelial-mesenchymal transition (EMT) in tumor cells, and a large number of studies have shown that EMT can endow cell migration, invasion and stem cell characteristics. And there have been many documents that interstitial fibroblasts can promote tumor cell invasion and transformation through EMT. At the same time, more and more recent studies have shown that gamma ray sources irradiate fibroblasts can promote tumorigenesis and invasion through the interaction of tumor stroma. And whether X ray sources irradiate fibroblasts to promote ESCC invasion and metastasis and its mechanism remain to be further studied. Objective 1. experiments were aimed at studying X ray pairs. Effect of interstitial fibroblasts and the effect of CAFs on ESCC cells after irradiation.2. explored the changes of EMT related protein and HDGF expression in the interaction between ESCC and interstitial fibroblasts. Methods 1. we isolated and cultured primary CAFs and normal fibroblasts (Normal Fibroblasts, NFs) to study the ESCC tumor microloop. The characteristics of interstitial fibroblasts. In order to prove that CAFs has no other cell components and its purity, fibroblast labeled protein fibronectin (fibronectin) is used to differentiate fibroblasts from ESCC cells and.Western blotting to verify the high expression of a- smooth muscle myoprotein (a-SMA) and non E- cadherin (E-cadherin) in primary fibroblasts (E-cadherin). The cell characteristics of the primary fibroblasts,.2., were irradiated with X ray sources at different doses at room temperature, and the dose rate was 400cGy/min. irradiated with the conditioned medium for subsequent culture of ESCC cells..3. was detected and irradiated by.3. (4Gy and 8Gy) or unirradiated fibroblasts (NFs and CAF). S) the invasiveness of co cultured ESCC cells.4. used Scattering test to detect the Scattering ability of ESCC cells after irradiation of the conditioned medium of the irradiated fibroblasts..5. ESCC cells and 4Gy irradiated fibroblasts or unirradiated fibroblasts were mixed evenly and planted in the right dorsum of nude mice, each group was six. Every 3 days, the vernier was used. The size of the tumor was measured once and recorded by the caliper. The calculation formula of tumor volume was: tumor volume (V) = (length diameter * 2) /2.6. in order to study the migration and invasion of ESCC cells to promote the migration and invasion of EMT after irradiated fibroblasts. Western blotting was used to detect the conditioned medium of fibroblasts and the conditioned medium of unirradiated fibroblasts after irradiation. The expression of epithelial cell marker E-cadherin and interstitial cell marker bellow (vimentin) in the ESCC cells of 24 hours was expressed. The expression of p- chain protein (P-catenin) in the transplanted tumor of irradiated group was detected by immunohistochemistry. The unirradiated group was the control group.7. to explore the HDGF in the radiation irradiated fibroblasts to promote ESCC cells. The role of migration and invasion, Western blotting was used to detect the expression of HDGF in ESCC cells cultured for 24 hours after irradiated fibroblast conditioned medium and unirradiated fibroblast conditioned medium, and the expression of HDGF in the transplanted tumor of irradiated group was detected by immunohistochemistry. The unirradiated group was the control group. The results were 1. fibroblasts. The isolation, purification and characteristics of vascular cells showed that the primary fibroblasts isolated and cultured from ESCC tissues maintained fibroblast properties. All fibroblasts used in this experiment were within 10 generations and showed spindle cell morphology after.3 generation, E-cadherin was not expressed, indicating that the early generation of fibroblasts was thin. After.2. irradiation, the condition culture fluid of fibroblasts enhanced the ability of ESCC cells to enhance the ability of Scattering to be enhanced at the time of epithelial cell EMT. The specific expression is that the adhesion between cells and cells disappears and obtains the highly active interstitial cell phenotype. The Scattering experiment has been used for research. Investigate cell EMT and detect cell migration and induction ability. After irradiation, fibroblast conditioned medium enhanced the Scattering ability of ESCC cells and increased the invasion ability of ESCC cells after dose dependent.3. irradiation. Our study showed that the invasiveness of ESCC cells and unirradiated fibroblasts increased. After irradiation, the invasion ability of the fibroblasts could be further enhanced in.4. in vitro and in vitro experiments. The results showed that the EMT results of ESCC cells in the irradiated group showed that the E-cadherin of ESCC cells in the irradiated group decreased in a dose-dependent manner and the vimentin increased with a dose-dependent manner and expressed high expression in all groups. In vitro and in vitro experiments showed that P The expression of -catenin was elevated in the irradiated group. The results of immunohistochemistry showed that the cell nucleus was highly expressed in the tumor tissue of the irradiated group and the nuclear transposition of.5. in vitro and in vitro experiments showed that the expression of HDGF in the tumor cells and fibroblasts in the fibroblast enhanced irradiation group irradiated the cells to promote the growth of ESCC, especially in Eca-9. 706 in the tumor cell group. In vivo and in vitro experiments showed the expression of HDGF in the tumor cells and fibroblasts in the irradiated group after irradiation. It is worth noting that the tumor cells adjacent to the interstitial fibroblasts, HDGF, have a relatively higher expression in the nucleus, showing the paracrine effect of the fibroblasts. Conclusion 1. irradiation. CAFs promoted ESCC cell invasion and EMT, enhanced its Scattering ability, and.2. irradiation induced ESCC cells to induce ESCC cell beta -catenin nuclear transposition.3. to promote the growth of ESCC tumor and increase the expression of HDGF protein.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.1

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