【作者】 李洋；

【导师】 刘颖斌；

【作者基本信息】 上海交通大学 ， 外科学（普通外科学）， 2020， 博士

【摘要】 背景胆囊癌是胆道系统肿瘤中最为常见的一类恶性肿瘤,其发病率位于消化道肿瘤的第七位,胆囊癌因为其早期难以发现故而其预后较差。Sciellin(SCEL)是一种角质层前体蛋白,在多种肿瘤中发现其高表达并促进肿瘤的发生发展,其具体分子机制各不相同。中性粒细胞胞外陷阱(NETs)是中性粒细胞在受到一刺激后释放染色质形成的胞外结构,在炎症中其可以捕获并杀灭细菌,在肿瘤中可以促进肿瘤细胞的转移,并加速血栓的形成。血栓是导致肿瘤患者死亡的第二大因素,但血栓形成的机制还并不清楚。目的本研究的目的包括:1.研究SCEL促进胆囊癌细胞增殖的分子机制;2.探究SCEL对于促进中性粒细胞胞外陷阱形成的机制;3.探究胆囊癌高表达SCEL对于血栓形成的影响,并试图为胆囊癌患者发生血栓提出可能的预测标志物。方法和结果(1)收集已经确诊的胆囊癌和胆囊炎手术标本病理切片,对其进行SCEL的免疫组化,之后根据其染色面积和染色强度给与评分,并根据评分对样本进行生存预后,临床病理信息的分析。结果发现,与胆囊炎相比SCEL在胆囊癌中呈现高表达,并且SCEL在胆囊癌中的表达情况和患者的生存预后呈现负相关性,同时SCEL与患者的T分期,TNM分期呈现正相关。(2)利用RT-PCR,Western Blot技术检测五株胆囊癌细胞系中SCEL的相对表达情况,对其中相对表达量高的两株细胞系利用RNAi进行SCEL的敲减,同时对其中相对表达量较低的两株细胞系转染SCEL质粒增加其表达量,RT-PCR及Western Blot检测敲减及过表达效率,并用CCK8检测其细胞活性,克隆实验检测器增殖能力;结果显示SCEL高表达可以增加胆囊癌细胞的细胞活性与增殖能力,反之敲减SCEL后胆囊癌细胞活性和增殖能力下降。(3)采用Western-Blot,RT-PCR以及COIP技术探究SCEL可以促进胆囊癌细胞增殖的可能分子机制。结果显示,SCEL可以与EGFR分子相结合,并防止EGFR的降解从而激活PI3K/AKT突进促进细胞增殖。(4)采用细胞共培养技术将过表达或敲减SCEL的胆囊癌细胞与中性粒细胞共培养之后利用Western-Blot与免疫荧光技术检测中性粒细胞胞外陷阱形成的情况,另外同时利用液体芯片技术检测过表达或敲减SCEL的胆囊癌细胞对于一些炎性因子的形成状况。结果显示,肿瘤源性SCEL的表达情况与其诱导中性粒细胞胞外陷阱的能力呈正相关。(5)建立SCEL过表达及对照病毒,构建过表达稳系,裸鼠皮下成瘤,定期观察测量肿瘤大小,并在肿瘤到达一定大小后,采用深静脉血栓构建模型技术检测胆囊癌细胞系在过表达SCEL后对于血栓形成的影响,结果显示,胆囊癌过表达SCEL后其形成的肿瘤体积更大,增长速度更快,而且SCEL过表达胆囊癌细胞更容易形成血栓。结论综合以上实验结果:1、SCEL在胆囊癌中高表达并且其对生存预后呈现负相关;2、SCEL可以增加胆囊癌细胞活性并且促进其增殖;3、SCEL通过与EGFR结合从而降低EGFR降解速率进而激活PI3K/AKT途径;4、SCEL同影响IL8的分泌量从而影响中性粒细胞胞外陷阱的形成;5、SCEL通过影响中性粒细胞胞外陷阱从而影响血栓的形成更多还原

【Abstract】 Background: Gallbladder cancer is the most common type of malignant tumor in biliary system.The incidence of gallbladder cancer is in the seventh place of digestive tract tumor.The prognosis of gallbladder cancer is poor because it is difficult to diagnose in the early stage.Scierlin(SCEL)is a kind of keratinocyte precursor protein,which is found to be highly expressed in a variety of tumors and promotes the development of tumors.Its specific molecular mechanisms are different.Neutrophil extracellular traps(NETs)are extracellular structures formed by neutrophils releasing chromatin after being stimulated.In inflammation,NETs can catch and kill bacteria,promote tumor cell metastasis and accelerate thrombosis.Thrombus is the second leading cause of death in cancer patients,but the mechanism of thrombus formation is not clear.Objective: The purpose of this study includes: 1.To study the molecular mechanism of SCEL in promoting the proliferation of gallbladder cancer cells;2.To explore the mechanism of SCEL in promoting the formation of neutrophil extracellular trap;3.To explore the effect of overexpression of scel in gallbladder cancer on thrombosis,and try to provide a possible prediction marker for the occurrence of thrombosis in patients with gallbladder cancer.Methods and Results:(1)The pathological sections of the confirmed gallbladder cancer and cholecystitis were collected,the SCEL immunohistochemistry was carried out,and then the score was given according to the staining area and staining intensity,and the survival prognosis and clinicopathological information of the samples were analyzed according to the score.The results showed that SCEL was highly expressed in gallbladder cancer compared with cholecystitis,and the expression of SCEL in gallbladder cancer was negatively correlated with the survival and prognosis of patients,while scel was positively correlated with T stage and TNM stage of patients.(2)RT-PCR and Western blot were used to detect the relative expression of SCEL in five gallbladder cancer cell lines.RNAi was used to knock down SCEL in two of the cell lines with high relative expression,while transfection of scel plasmid in two of the cell lines with low relative expression increased the expression.RT-PCR and Western blot were used to detect the knock down and over expression efficiency,and CCK8 was used to detect the cell activity The results showed that the overexpression of SCEL could increase the cell activity and proliferation ability of gallbladder cancer cells,whereas the decrease of SCEL could decrease the cell activity and proliferation ability of gallbladder cancer cells..(3)Western blot,RT-PCR and Co IP were used to explore the possible molecular mechanism of scel promoting the proliferation of gallbladder cancer cells.The results showed that SCELcould combine with EGFR and prevent EGFR degradation,thus activating PI3 K / Akt to promote cell proliferation.(4)After co culture of overexpressed or knockdown SCEL gallbladder cancer cells with neutrophils,Western blot and immunofluorescence were used to detect the formation of extracellular traps of neutrophils.In addition,liquid chip technology was used to detect the formation of some inflammatory factors in overexpressed or knockdown SCEL gallbladder cancer cells.The results showed that the expression of tumor derived SCELwas positively correlated with its ability to induce neutrophil extracellular trap.(5)To establish overexpression and control virus of SCEL,construct stable overexpression line,subcutaneous tumorigenesis of nude mice,regularly observe and measure tumor size,and after the tumor reaches a certain size,use deep vein thrombus construction model technology to detect the influence of overexpression SCEL of gallbladder cancer cell line on thrombosis.The results show that overexpression SCELof gallbladder cancer results in larger tumor volume and faster growth rate Faster,and SCEL overexpression of gallbladder cancer cells more prone to thrombosis.Conclusion: Based on the above experimental results: 1.SCEL is highly expressed in gallbladder cancer and has a negative correlation with survival and prognosis;2.SCEL can increase the activity of gallbladder cancer cells and promote their proliferation;3.SCEL can reduce the degradation rate of EGFR and activate the PI3 K / Akt pathway by combining with EGFR;4.SCEL can affect the secretion of IL8 and the formation of neutrophil extracellular trap;5.SCEL Influence the extracellular trap of neutrophils and the formation of thrombus更多还原