本文選題：食管鱗癌 + 肝癌衍生生長因子　； 參考：《山東大學》2015年碩士論文

【摘要】：研究背景食管鱗癌(Esophageal squamous cell carcinoma, ESCC)患者五年生存率仍然低于20%,放射治療是ESCC患者一項重要的治療方法。最近,有研究表明活化地腫瘤相關成纖維細胞(cancer-associated fibroblasts, CAFs)與ESCC患者的差的預后及同步放化療后局部復發(fā)密切相關。越來越多的研究亦顯示放射線可以通過作用腫瘤間質(zhì)微環(huán)境影響腫瘤發(fā)生發(fā)展,由此豐富了放射線誘發(fā)腫瘤的證據(jù)。近幾年來肝癌衍生生長因子(Hepatoma-derived growth factor, HDGF)引起了學術界廣泛關注,它既是具有促成纖維細胞有絲分裂活性的酸性多肽,也與多種腫瘤差的預后密切相關：同時其表達變化與多種腫瘤的轉化、凋亡、血管生成以及轉移密切相關。HDGF與ESCC患者的放療敏感性以及腫瘤復發(fā)亦有緊密聯(lián)系。CAFs與腫瘤轉移的多種信號轉導通路相關聯(lián),包括可以促進腫瘤細胞的上皮間質(zhì)轉化(Epithelial-mesenchymal transition, EMT)過程,而大量研究表明EMT可以賦予細胞遷移、侵襲能力以及誘導干細胞特性。并且已有多篇文獻證明間質(zhì)成纖維細胞可以通過EMT促進腫瘤細胞侵襲與轉移。與此同時,最近越來越多的研究表明γ射線源照射成纖維細胞可以通過腫瘤間質(zhì)相互作用促進腫瘤發(fā)生以及侵襲生長。而X射線源照射成纖維細胞是否促進ESCC侵襲轉移及其機制尚有待于進一步研究。目的1.本實驗旨在研究X射線對間質(zhì)成纖維細胞的作用以及照射后CAFs對ESCC細胞的影響。2.探討在放射線對ESCC與間質(zhì)成纖維細胞相互作用中的EMT相關蛋白以及HDGF表達變化。方法1.我們分離及培養(yǎng)原代CAFs和正常成纖維細胞(Normal Fibroblasts, NFs)來研究ESCC腫瘤微環(huán)境內(nèi)的間質(zhì)成纖維細胞特點。為了證明CAFs沒有其他細胞成分混雜及其純度,利用成纖維細胞標記蛋白纖連蛋白(fibronectin)區(qū)分成纖維細胞與ESCC細胞。Western blotting驗證原代成纖維細胞高表達a-平滑肌肌動蛋白(a-SMA)和不表達E-鈣粘蛋白(E-cadherin),體現(xiàn)原代成纖維細胞的細胞特性。2.在室溫條件下,用X射線源以不同劑量照射原代成纖維細胞,劑量率為400cGy/min。照射后收集條件培養(yǎng)液留待后續(xù)培養(yǎng)ESCC細胞試驗用。3.采用侵襲實驗檢測與照射后(4Gy和8Gy)或未照射成纖維細胞(NFs和CAFs)共培養(yǎng)的ESCC細胞的侵襲能力。4.采用Scattering實驗檢測照射后成纖維細胞的條件培養(yǎng)液對ESCC細胞的Scattering能力的影響。5. ESCC細胞和4Gy照射后成纖維細胞或未照射成纖維細胞均勻混合后種植于裸鼠右背側皮下,每組六只。每3天用游標卡尺測量腫瘤大小一次并記錄,腫瘤體積計算公式為：腫瘤體積(V)=(長徑×寬徑2)/2。6.為了研究照射后成纖維細胞促進ESCC細胞的遷移及侵襲與EMT有關,western blotting檢測照射后成纖維細胞條件培養(yǎng)液和未照射成纖維細胞條件培養(yǎng)液培養(yǎng)24小時的ESCC細胞中上皮細胞標記物E-cadherin和間質(zhì)細胞標記物波紋蛋白(vimentin)表達情況。并用免疫組化檢測p-鏈蛋白(p-catenin)在照射組裸鼠移植瘤內(nèi)的表達變化,未照射組為對照組。7.為探討HDGF在放射線照射成纖維細胞促進ESCC細胞遷移與侵襲中的作用,采用western blotting檢測照射后成纖維細胞條件培養(yǎng)液和未照射成纖維細胞條件培養(yǎng)液培養(yǎng)24小時的ESCC細胞中HDGF表達情況。并用免疫組化檢測HDGF在照射組裸鼠移植瘤內(nèi)的表達變化,未照射組為對照組。結果1.成纖維細胞的分離,提純及特點結果表明從ESCC組織分離及培養(yǎng)的原代成纖維細胞保持成纖維細胞特性。用于本實驗的所有成纖維細胞均為10代以內(nèi)并表現(xiàn)為梭形細胞形態(tài)。3代以后的成纖維細胞E-cadherin未見表達,表明早期代數(shù)的成纖維細胞保持成纖維細胞特性。2.照射后成纖維細胞條件培養(yǎng)液增強ESCC細胞Scattering能力由于上皮細胞EMT的時候Scattering能力會有所增強,具體表現(xiàn)為細胞與細胞之間的黏附作用消失并獲得較高活性的間質(zhì)細胞表型,Scattering實驗已被用于研究細胞的EMT和檢測細胞遷移誘導能力。照射后成纖維細胞條件培養(yǎng)液增強ESCC細胞的Scattering能力并呈劑量依賴性。3.照射后成纖維細胞增強ESCC細胞的侵襲能力我們的研究表明ESCC細胞與未照射成纖維細胞共培養(yǎng)侵襲能力提高,當其與照射后成纖維細胞共培養(yǎng)其侵襲能力會得到進一步的增強。4.體內(nèi)體外實驗表明照射后成纖維細胞促進ESCC細胞EMT結果顯示照射組中ESCC細胞E-cadherin呈劑量依賴性降低,vimentin隨劑量依賴性升高并在所有組中高表達。體內(nèi)體外實驗表明P-catenin在照射組中都表達升高,免疫組化結果顯示其在照射組腫瘤組織中的細胞核高表達及核轉位。5.體內(nèi)體外實驗表明照射后成纖維細胞增強照射組中腫瘤細胞及成纖維細胞中HDGF的表達照射后成纖維細胞促進ESCC生長,特別是在Eca-9706腫瘤細胞組中。體內(nèi)體外實驗表明照射后成纖維細胞增強照射組中腫瘤細胞及成纖維細胞中HDGF的表達。值得注意的是,與間質(zhì)成纖維細胞相鄰的腫瘤細胞HDGF在細胞核中有相對更高的表達,顯示成纖維細胞的旁分泌效應。結論1.照射后CAFs促進ESCC細胞侵襲及EMT,增強其Scattering能力。2.照射后成纖維細胞誘導ESCC細胞β-catenin核轉位。3.照射后成纖維細胞促進ESCC腫瘤生長及提高HDGF蛋白表達。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R735.1

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