本文選題：食管鱗癌 + 肝癌衍生生長(zhǎng)因子��； 參考：《山東大學(xué)》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(xiàng)重要的治療方法。最近,有研究表明活化地腫瘤相關(guān)成纖維細(xì)胞(cancer-associated fibroblasts, CAFs)與ESCC患者的差的預(yù)后及同步放化療后局部復(fù)發(fā)密切相關(guān)。越來(lái)越多的研究亦顯示放射線(xiàn)可以通過(guò)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(xiàn)誘發(fā)腫瘤的證據(jù)。近幾年來(lái)肝癌衍生生長(zhǎng)因子(Hepatoma-derived growth factor, HDGF)引起了學(xué)術(shù)界廣泛關(guān)注,它既是具有促成纖維細(xì)胞有絲分裂活性的酸性多肽,也與多種腫瘤差的預(yù)后密切相關(guān)：同時(shí)其表達(dá)變化與多種腫瘤的轉(zhuǎn)化、凋亡、血管生成以及轉(zhuǎn)移密切相關(guān)。HDGF與ESCC患者的放療敏感性以及腫瘤復(fù)發(fā)亦有緊密聯(lián)系。CAFs與腫瘤轉(zhuǎn)移的多種信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān)聯(lián),包括可以促進(jìn)腫瘤細(xì)胞的上皮間質(zhì)轉(zhuǎn)化(Epithelial-mesenchymal transition, EMT)過(guò)程,而大量研究表明EMT可以賦予細(xì)胞遷移、侵襲能力以及誘導(dǎo)干細(xì)胞特性。并且已有多篇文獻(xiàn)證明間質(zhì)成纖維細(xì)胞可以通過(guò)EMT促進(jìn)腫瘤細(xì)胞侵襲與轉(zhuǎn)移。與此同時(shí),最近越來(lái)越多的研究表明γ射線(xiàn)源照射成纖維細(xì)胞可以通過(guò)腫瘤間質(zhì)相互作用促進(jìn)腫瘤發(fā)生以及侵襲生長(zhǎng)。而X射線(xiàn)源照射成纖維細(xì)胞是否促進(jìn)ESCC侵襲轉(zhuǎn)移及其機(jī)制尚有待于進(jìn)一步研究。目的1.本實(shí)驗(yàn)旨在研究X射線(xiàn)對(duì)間質(zhì)成纖維細(xì)胞的作用以及照射后CAFs對(duì)ESCC細(xì)胞的影響。2.探討在放射線(xiàn)對(duì)ESCC與間質(zhì)成纖維細(xì)胞相互作用中的EMT相關(guān)蛋白以及HDGF表達(dá)變化。方法1.我們分離及培養(yǎng)原代CAFs和正常成纖維細(xì)胞(Normal Fibroblasts, NFs)來(lái)研究ESCC腫瘤微環(huán)境內(nèi)的間質(zhì)成纖維細(xì)胞特點(diǎn)。為了證明CAFs沒(méi)有其他細(xì)胞成分混雜及其純度,利用成纖維細(xì)胞標(biāo)記蛋白纖連蛋白(fibronectin)區(qū)分成纖維細(xì)胞與ESCC細(xì)胞。Western blotting驗(yàn)證原代成纖維細(xì)胞高表達(dá)a-平滑肌肌動(dòng)蛋白(a-SMA)和不表達(dá)E-鈣粘蛋白(E-cadherin),體現(xiàn)原代成纖維細(xì)胞的細(xì)胞特性。2.在室溫條件下,用X射線(xiàn)源以不同劑量照射原代成纖維細(xì)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(xì)胞試驗(yàn)用。3.采用侵襲實(shí)驗(yàn)檢測(cè)與照射后(4Gy和8Gy)或未照射成纖維細(xì)胞(NFs和CAFs)共培養(yǎng)的ESCC細(xì)胞的侵襲能力。4.采用Scattering實(shí)驗(yàn)檢測(cè)照射后成纖維細(xì)胞的條件培養(yǎng)液對(duì)ESCC細(xì)胞的Scattering能力的影響。5. ESCC細(xì)胞和4Gy照射后成纖維細(xì)胞或未照射成纖維細(xì)胞均勻混合后種植于裸鼠右背側(cè)皮下,每組六只。每3天用游標(biāo)卡尺測(cè)量腫瘤大小一次并記錄,腫瘤體積計(jì)算公式為：腫瘤體積(V)=(長(zhǎng)徑×寬徑2)/2。6.為了研究照射后成纖維細(xì)胞促進(jìn)ESCC細(xì)胞的遷移及侵襲與EMT有關(guān),western blotting檢測(cè)照射后成纖維細(xì)胞條件培養(yǎng)液和未照射成纖維細(xì)胞條件培養(yǎng)液培養(yǎng)24小時(shí)的ESCC細(xì)胞中上皮細(xì)胞標(biāo)記物E-cadherin和間質(zhì)細(xì)胞標(biāo)記物波紋蛋白(vimentin)表達(dá)情況。并用免疫組化檢測(cè)p-鏈蛋白(p-catenin)在照射組裸鼠移植瘤內(nèi)的表達(dá)變化,未照射組為對(duì)照組。7.為探討HDGF在放射線(xiàn)照射成纖維細(xì)胞促進(jìn)ESCC細(xì)胞遷移與侵襲中的作用,采用western blotting檢測(cè)照射后成纖維細(xì)胞條件培養(yǎng)液和未照射成纖維細(xì)胞條件培養(yǎng)液培養(yǎng)24小時(shí)的ESCC細(xì)胞中HDGF表達(dá)情況。并用免疫組化檢測(cè)HDGF在照射組裸鼠移植瘤內(nèi)的表達(dá)變化,未照射組為對(duì)照組。結(jié)果1.成纖維細(xì)胞的分離,提純及特點(diǎn)結(jié)果表明從ESCC組織分離及培養(yǎng)的原代成纖維細(xì)胞保持成纖維細(xì)胞特性。用于本實(shí)驗(yàn)的所有成纖維細(xì)胞均為10代以?xún)?nèi)并表現(xiàn)為梭形細(xì)胞形態(tài)。3代以后的成纖維細(xì)胞E-cadherin未見(jiàn)表達(dá),表明早期代數(shù)的成纖維細(xì)胞保持成纖維細(xì)胞特性。2.照射后成纖維細(xì)胞條件培養(yǎng)液增強(qiáng)ESCC細(xì)胞Scattering能力由于上皮細(xì)胞EMT的時(shí)候Scattering能力會(huì)有所增強(qiáng),具體表現(xiàn)為細(xì)胞與細(xì)胞之間的黏附作用消失并獲得較高活性的間質(zhì)細(xì)胞表型,Scattering實(shí)驗(yàn)已被用于研究細(xì)胞的EMT和檢測(cè)細(xì)胞遷移誘導(dǎo)能力。照射后成纖維細(xì)胞條件培養(yǎng)液增強(qiáng)ESCC細(xì)胞的Scattering能力并呈劑量依賴(lài)性。3.照射后成纖維細(xì)胞增強(qiáng)ESCC細(xì)胞的侵襲能力我們的研究表明ESCC細(xì)胞與未照射成纖維細(xì)胞共培養(yǎng)侵襲能力提高,當(dāng)其與照射后成纖維細(xì)胞共培養(yǎng)其侵襲能力會(huì)得到進(jìn)一步的增強(qiáng)。4.體內(nèi)體外實(shí)驗(yàn)表明照射后成纖維細(xì)胞促進(jìn)ESCC細(xì)胞EMT結(jié)果顯示照射組中ESCC細(xì)胞E-cadherin呈劑量依賴(lài)性降低,vimentin隨劑量依賴(lài)性升高并在所有組中高表達(dá)。體內(nèi)體外實(shí)驗(yàn)表明P-catenin在照射組中都表達(dá)升高,免疫組化結(jié)果顯示其在照射組腫瘤組織中的細(xì)胞核高表達(dá)及核轉(zhuǎn)位。5.體內(nèi)體外實(shí)驗(yàn)表明照射后成纖維細(xì)胞增強(qiáng)照射組中腫瘤細(xì)胞及成纖維細(xì)胞中HDGF的表達(dá)照射后成纖維細(xì)胞促進(jìn)ESCC生長(zhǎng),特別是在Eca-9706腫瘤細(xì)胞組中。體內(nèi)體外實(shí)驗(yàn)表明照射后成纖維細(xì)胞增強(qiáng)照射組中腫瘤細(xì)胞及成纖維細(xì)胞中HDGF的表達(dá)。值得注意的是,與間質(zhì)成纖維細(xì)胞相鄰的腫瘤細(xì)胞HDGF在細(xì)胞核中有相對(duì)更高的表達(dá),顯示成纖維細(xì)胞的旁分泌效應(yīng)。結(jié)論1.照射后CAFs促進(jìn)ESCC細(xì)胞侵襲及EMT,增強(qiáng)其Scattering能力。2.照射后成纖維細(xì)胞誘導(dǎo)ESCC細(xì)胞β-catenin核轉(zhuǎn)位。3.照射后成纖維細(xì)胞促進(jìn)ESCC腫瘤生長(zhǎng)及提高HDGF蛋白表達(dá)。
[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(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R735.1

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