兰州大学机构库
LIF对食管鳞癌中不同LET射线辐射应答的作用及机制研究
Alternative TitleEffect and mechanism of LIF in radiation response to different LET rays in esophageal squamous cell carcinoma
罗宏涛
Subtype博士
Thesis Advisor王小虎
2020-11-23
Degree Grantor兰州大学
Place of Conferral兰州
Degree Discipline内科学
Keyword食管鳞癌 LIF 碳离子 蛋白组学 STAT3信号通路
Abstract背景与目的 食管鳞状细胞癌(Esophageal squamous cell carcinoma,ESCC)对低线性能量传递(Linear energy transfer,LET)X射线中度敏感,放疗后局部控制率低,极易出现局部复发和远处转移。碳离子为高LET射线,具有优于低LET射线的物理学和放射生物学特性,对低LET X射线辐射不敏感或辐射抗拒肿瘤具有较好的治疗效果。白血病抑制因子(Leukaemia inhibitory factor,LIF)是一种多功能细胞因子,通过白血病抑制因子受体(LIFR)和糖蛋白(gp)-130传递信号,激活STAT3信号通路,刺激肿瘤的生长、增殖和转移,与化疗耐药和放疗抗拒相关。本研究拟采用低LET X射线和高LET碳离子射线辐照食管鳞癌细胞,分析LIF对食管鳞癌细胞经不同LET射线辐照后生物学行为的影响并探讨其发挥作用的分子机制,进一步为高LET碳离子治疗ESCC提供理论基础。 方法 1. X射线对ESCC细胞辐射生物学行为影响的研究:0、1、2和4 Gy X射线辐照ECA109和KYSE150细胞,采用克隆存活实验检测各剂量X射线对两种细胞存活情况的影响,CCK8法检测各剂量X射线对两种细胞在辐照后24、48和72 h增殖活性的影响,流式细胞术检测各剂量X射线对ECA109和KYSE150细胞辐照后24和48 h凋亡和细胞周期的影响,划痕和Transwell实验检测2 Gy X射线辐照ECA109[i1]细胞后48 h对细胞迁移和侵袭的影响,免疫荧光共聚焦实验检测2 Gy X射线辐照ECA109细胞后48 h对γH2AX蛋白的表达的影响,Western blot检测2 Gy X射线辐照ECA109细胞后48 h对Bax,Bcl-2和γH2AX蛋白表达的影响。 2. ECA109细胞中辐射抗性差异蛋白分子的筛选:根据上一步实验结果,选用2 Gy X射线辐照后培养48 h和未被辐照过的ECA109细胞两组样本,利用TMT蛋白定量技术进行蛋白组学检测,结合生物信息学方法对所得的结果经过差异分析,筛选得到显著差异的LIF蛋白分子。 3. ESCC放疗患者血清中LIF的临床意义:采用ELISA检测60例ESCC患者放疗前后血清LIF浓度变化,并与30例健康者血清LIF浓度对照,观察血清LIF浓度变化与ESCC患者临床病理特征、疾病预后之间的关系。 4. LIF通过靶向STAT3在食管鳞癌细胞中生物学效应的探索:采用siRNA技术下调ECA109细胞中LIF分子后采用克隆形成实验,CCK8试验、凋亡实验、 迁移和侵袭实验,观察LIF对ECA109 细胞克隆形成、增殖、凋亡和转移生物学行为的影响;利用 RT-PCR技术和Western blot检测ECA109细胞中LIF和 STAT3的表达情况。 5. LIF通过STAT3信号通路调控食管鳞癌细胞对碳离子的辐射应答效应及分子机制研究:0、1、2和4 Gy碳离子辐照ECA109细胞,采用克隆存活实验检测各剂量碳离子对ECA109细胞存活情况的影响,CCK8法检测各剂量碳离子辐照ECA109细胞后24、48和72 h对细胞增殖活性的影响,流式细胞术检测各剂量碳离子辐照ECA109细胞后24和48 h对细胞凋亡和细胞周期的影响,划痕和Transwell实验检测碳离子辐照ECA109细胞后48 h对迁移和侵袭的影响,实时荧光定量PCR检测碳离子辐照后ECA109细胞LIF,STAT3,MMP2,和E-cadherin mRNA表达情况,Western blot检测碳离子辐照后ECA109细胞LIF,STAT3,MMP2和E-cadherin蛋白表达情况。 结果 1. 不同剂量X射线对ECA109和KYSE150细胞的生物学行为影响具有差异,与0 Gy组比较:2 Gy X射线辐照后48 h,ECA109细胞的增殖无显著抑制(p>0.05),而KYSE150细胞增殖被显著抑制(p<0.05);ECA109细胞在4 Gy辐照后48h细胞周期发生阻滞(p<0.05),而KYSE150细胞在2和4 Gy辐照后48 h均出现细胞周期阻滞(p<0.05);ECA109细胞经4 Gy辐照后48 h细胞凋亡显著增加(p<0.05),而KYSE150细胞在2和4 Gy辐照后48 h细胞凋亡均显著增加(p<0.05);2 Gy X射线对ECA109细胞侵袭和迁移的影响无显著变化(p>0.05);2 Gy组ECA109细胞γH2AX 焦点差异有统计学意义(p<0.05);Bax,Bcl-2和STAT3蛋白表达量变化无明显差异(p>0.05)。 2. 本研究选用2 Gy X射线辐照后48 h的ECA109细胞行蛋白组学检测分析,获得差异表达蛋白399个(215个下调,184个上调),其中LIF的表达差异显著(p=0.0003),并且通过GO和KEGG 通路富集分析显示LIF参与STAT3信号通路。 3. 临床血清样本检测结果显示:ESCC患者血清LIF浓度放疗前后分别为0.5224 ± 0.1983μg/ml和0.3861 ± 0.1506μg/ml,均高于健康对照者(0.3233 ± 0.0985μg/ml)(p<0.05);ESCC患者血清LIF浓度与肿瘤T分期、N分期、临床分期、组织学分级具有相关性(p<0.05);13例患者放疗后血清LIF浓度较放疗前升高,47例患者放疗后血清LIF浓度较放疗前降低,血清LIF浓度升高组和下降组在随访期内(36个月)局部复发率分别为69.2%(9/13)和34.0%(16/47),(p=0.023);两组患者肿瘤远处转移率分别为53.8% (7/13)和23.4%(11/47)(p=0.034)。两组患者1、2、3生存率分别为76.9%、46.2%、30.7%和85.1%、66.0%、46.8%(p=0.048)。 4. 敲低ECA109细胞中LIF结果显示:下调LIF可诱导ECA109细胞凋亡,抑制细胞增殖、迁移和侵袭能力,与对照组比较,差异显著(p<0.05)。同时,下调LIF可降低ECA109细胞中STAT3的表达量(p<0.05)。 5. 不同剂量碳离子辐照ECA109细胞结果显示:与0 Gy组比较,2和4 Gy碳离子可诱导ECA109细胞凋亡,阻滞细胞周期在G2期,抑制ECA109细胞增殖、迁移和侵袭能力(p<0.05)。转录和翻译水平检测结果显示:2和4 Gy碳离子可显著下调ECA109细胞中LIF、p-STAT3、STAT3和MMP2表达量,上调E-cadherin表达量(p<0.05)。 结论 1. 2 Gy X射线对食管鳞癌ECA109细胞增殖和转移的抑制作用不显著,辐照后细胞中γH2AX 表达显著上调,STAT3表达变化不显著。 2. LIF在2 Gy X射线辐照后的食管鳞癌ECA109细胞中表达下调;血清LIF浓度与ESCC患者局部复发、远处转移和预后具有相关性。 3. 食管鳞癌ECA109细胞中低表达的LIF通过靶向STAT3分子,介导STAT3信号通路调控食管鳞癌ECA109细胞的生物学行为。 4. 高LET碳离子通过下调食管鳞癌ECA109细胞中的LIF,诱导STAT3信号通路中相关分子的表达,从而调控食管鳞癌ECA109细胞的增殖和转移。
Other AbstractBackground and purpose Esophageal squamous cell carcinoma (ESCC) is moderately sensitive to low linear energy transfer (LET) X-ray, and is prone to local recurrence and distant metastasis after radiotherapy, with the worse clinical prognosis.Carbon ion is high LET ray, which have better physical and radiobiological properties than low LET rays, and have better therapeutic effects for tumor which is insensitive or resistant to low LET X-ray.The leukemia inhibitory factor(LIF) is a pluripotent cytokine that activates the STAT3 signaling pathway through the leukemia suppressor receptor (LIFR) and glycoprotein (GP-130), which stimulates tumor growth, proliferation and metastasis, and is associated with chemoradiotherapy resistance.In this study, low LET X-ray and high LET carbon ion were used to irradiate ESCC cells, then analyze the effects of LIF on the biological behavior of ESCC cells irradiated by different LET rays and to explore the molecular mechanism of its action, so as to provide a theoretical basis for the treatment of ESCC with high LET carbon ion. Method 1. Study on radiation biological behavior of ESCC cells: ECA109 and KYSE150 cells were irradiated by 0, 1, 2 and 4 Gy X-rays. Colony formation assay was used to detect the effects of different doses of X-rays on the survival of ECA109 and KYSE150 cells. CCK8 method was used to detect the effects of different doses of X-rays on the proliferation of ECA109 and KYSE150 cells at 24, 48 and 72 h after irradiation. Flow cytometry was used to detect the effects of various doses of X-rays on apoptosis and cell cycle of ECA109 and KYSE150 cells at 24 and 48 h after irradiation. Scratches and Transwell experiments were used to detect the effects of 2 Gy X-rays on the migration and invasion of ECA109 cells at 48 h. The effect of 2 Gy X-ray irradiation on the expression of γ-H2AX protein in ECA109 cells was detected by immunofluorescence confocal assay., Western blot was used to detect the effect of 2-Gy X-ray irradiation on the expression of Bax, Bcl-2 and γ H2AX protein in ECA109 cells. 2. Screening of differential protein molecules of radiation resistance in ECA109 cells: According to the results of the previous experiment, ECA109 cells which one were cultured for 48 h after 2 Gy X-ray irradiation and the another were unirradiated were selected for proteomic detection by TMT protein quantitative technology. Combined with bioinformatics methods, the results were analyzed by difference analysis, and the extremely significant differences of LIF protein molecules were obtained. 3. Clinical significance of serum LIF in patients with ESCC: ELISA was used to detect the changes of serum LIF concentration before and after radiotherapy in 60 patients with ESCC, and compared with 30 healthy controls. Then observe the relationship between serum LIF concentration and clinicopathological features and disease prognosis. 4. The exploration of biological effects of LIF targeting STAT3 in esophageal squamous cell carcinoma: After down-regulating LIF molecule in ECA109 cells by siRNA technique, the biological effects of LIF in ESCC were detected by clone formation test, CCK8 test and flow cytometry, and the metastatic changes of ESCC cells were detected by Transwell chamber. RT-PCR and Western blot were used to verify the expression of LIF in ESCC and its regulation of STAT3 signal pathway. 5. LIF regulates the radiation response of ESCC cells to carbon ion through STAT3 signal pathway and its mechanism: ECA109 cells were irradiated by carbon ion with 0,1,2 and 4 Gy, and the radiation effects of different doses of carbon ion were detected by colony formation test. CCK8 method was used to detect the effects of different doses of carbon ion on the proliferation of ESCC cells in different time periods. Flow cytometry was used to detect the effects of different doses of carbon ion radiation on apoptosis and cycle of ECA109 cells at 24 and 48 h. Scratches and Transwell experiments were used to detect the migration and invasion ability of ECA109 cells after carbon ion irradiation. RT-PCR was used to detect the relative expression of LIF, STAT3, MMP2 and E-cadherin mRNA, Western blot was used to detect the expression of LIF, STAT3, MMP2 and E-cadherin protein after carbon ion irradiation. Result 1. The biological behavior of ECA109 and KYSE150 cells was significantly affected by different doses of X rays. Compared with the 0 Gy group, the proliferation of ECA109 cells at 48 h after 2 Gy X rays was not significantly inhibited (p > 0.05), while the proliferation of KYSE150 cells was significantly inhibited (p< 0.05).ECA109 cells showed cell cycle arrest at 48h after 4 Gy irradiation (p < 0.05), while KYSE150 cells showed cell cycle arrest at 48h after 2 and 4 Gy irradiation (p < 0.05).Apoptosis of ECA109 cells was significantly increased 48 h after irradiation with 4 Gy (p < 0.05), while apoptosis of KYSE150 cells was significantly increased 48 h after irradiation with 2 and 4 Gy (p < 0.05).The effect of 2 Gy X ray on the invasion and migration of ECA109 cells did not change significantly (p > 0.05). There was significant difference in γ-H2AX focus of ECA109 cells in 2 Gy group(p < 0.05), but there was no significant difference in the expression of Bax, Bcl-2 and STAT3 protein (p > 0.05). 2. In this study, proteomic analysis was performed on ECA109 cells at 48 h after 2 Gy X-ray irradiation. 399 differentially expressed proteins were obtained (215 down-regulated and 184 up-regulated), of which the expression of LIF was significantly different (p = 0.0003). Enrichment analysis of GO and KEGG pathway showed that LIF was involved in STAT3 signal pathway. 3.The serum LIF concentrations of ESCC patients before and after radiotherapy were 0.5224 ± 0.1983μg/ml and 0.3861 ± 0.1506μg/ml, respectively, which were significantly higher than those of healthy controls (0.3233 ± 0.0985μg/ml) (p<0.05). The serum LIF concentration of ESCC was correlated with tumor T stage, lymph node stage, clinical stage and histological grade (p<0.05). The serum LIF concentration was increased in 13 patients after radiotherapy, and 47 patients was decreased than that before radiotherapy. The local recurrence rates during the follow-up period (36 months) were 69.2%(9/13)in the increased LIF group and 34.0%(16/47)in the decreased group, respectively(p= 0.023).The distant metastasis rates in the two groups were 53.8% and 23.4%, respectively (p= 0.034).The 1 -, 2-and 3-survival rates of the two groups were 76.9%, 46.2%, 30.7% and 85.1%, 66.0%, 46.8%, respectively (p= 0.048). 4. After knocking down the LIF in ECA109 cells, the proliferation ability of ECA109 cells decreased, the apoptosis rate increased, and the migration and invasion ability of ECA109 cells were inhibited, which was significantly different from that of the control group (p<0.05). Down-regulation of LIF could reduce the expression of STAT3 in ECA109 cells. (p < 0.05). 5. The results of ECA109 cells irradiated by different doses of carbon ion showed that compared with 0 Gy group, 2 and 4 Gy carbon ion rays could induce apoptosis of ECA109 cells, block the cell cycle in G2 ~ M phase, and inhibit the proliferation, migration and invasion of ECA109 cells (p<0.05). The results of transcription and translation showed that 2 and 4 Gy carbon ion could significantly down-regulate the expression of LIF, p-STAT3, STAT3 and MMP2 and up-regulate the expression of E-cadherin in ECA109 cells(p<0.05). Conclusion 1. 2 Gy X-ray had no significant inhibitory effect on the proliferation and metastasis of esophageal squamous cell carcinoma ECA109 cells.The expression of γH2AX was significantly up-regulated after irradiation, while the expression of STAT3 was not significant. 2. The expression of LIF in esophageal squamous cell carcinoma ECA109 cells was down-regulated after 2 Gy X-ray irradiation, and the concentration of serum LIF in ESCC patients was correlated with local recurrence, distant metastasis and prognosis of diseases. 3. The low expression of LIF regulates the biological effects of esophageal squamous cell carcinoma ECA109 cells by targeting STAT3 molecules and mediating STAT3 signal pathway. 4. Carbon ions induce the expression of relevant molecules in the STAT3 signaling pathway by down-regulating LIF in esophageal squamous cell carcinoma ECA109 cells, thus inhibiting the proliferation and metastasis of ESCC cells.
Pages117
URL查看原文
Language中文
Document Type学位论文
Identifierhttp://ir.lzu.edu.cn/handle/262010/448666
Collection兰州大学
Affiliation第一临床医学院
First Author AffilicationFirst Clinical School
Recommended Citation
GB/T 7714
罗宏涛. LIF对食管鳞癌中不同LET射线辐射应答的作用及机制研究[D]. 兰州. 兰州大学,2020.
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