【作者】 李萌；

【导师】 柳增善；

【作者基本信息】 吉林大学 ， 兽医公共卫生， 2022， 博士

【摘要】 作为严重威胁公共卫生安全的一种疾病,癌症正在逐渐改变现代人的生存方式。胃癌作为其中死亡率极高的一种恶性肿瘤,全球每年新增病例达上百万,其中大约40%来自中国。外科切除术辅助化疗和放疗是目前治疗胃癌的常规手段,其中手术仍是根治胃癌的最佳选择。但不是所有的胃癌患者都有机会接受切除术,且切除术后易转移和复发。常规治疗后晚期胃癌患者预后较差,平均5年存活率不到30%;辅助疗法对胃癌患者的治愈意义重大,但副作用巨大且会给患者带来较大痛苦;因此,分子靶向疗法成为肿瘤治疗领域的一大热点。与传统放化疗手段相比,分子靶向疗法基于肿瘤细胞的异质性,通过靶向调节肿瘤细胞的特异性分子及其下游信号转导,最终杀死肿瘤细胞。本研究旨在通过分析CCKBR在胃癌中的表达水平、所发挥的生物学功能及其介导的靶向重组毒素的特异性杀伤活性,综合评价CCKBR作为胃癌靶向重组毒素治疗性靶标的临床应用价值。本研究分别利用线上表达量数据库、RT-PCR方法和Western-blotting技术对泛癌、30株肿瘤细胞系、2016年至2017年采集自吉林大学中日联谊医院的59组临床胃癌肿瘤样本及其对应正常胃组织样本进行了CCKBR的相对表达量检测;利用重组慢病毒技术构建CCKBR敲降及过表达细胞系,并利用细胞毒性实验、克隆形成实验、划痕愈合实验、Transwell小室实验、活体成瘤实验等分析了CCKBR表达水平对胃癌细胞增殖、迁移侵袭及肿瘤形成能力等特性的影响;利用CCKBR与其天然配体胃泌素间的互作关系构建了以胃泌素17肽(G17)为导向单元的重组毒素,经表达纯化后通过细胞增殖实验、流式细胞术、裸鼠荷瘤胃癌模型等方法,评估了CCKBR介导的该重组毒素对胃癌细胞的靶向杀伤活性;最后,根据CCKBR表达水平对胃癌临床组织样本进行分类,结合患者临床诊断统计分析过表达CCKBR的相关病理因素,并在此基础上建立CCKBR靶向疗法临床适用病例的筛选策略。结果显示,CCKBR在多种消化道肿瘤细胞系中过表达,其中胃癌细胞系BGC-823和SGC-7901显著过表达;无论是m RNA转录水平(P=0.007)还是蛋白质表达水平(P=0.037),胃癌组织样本的CCKBR水平均显著高于正常胃组织样本;预后分析结果显示,胃癌患者的预后风险随CCKBR表达水平的上调而升高,CCKBR表达量较低胃癌患者的总生存期显著延长(4.5年vs 1.6年,P=0.005)。通过敲降及过表达胃癌细胞系分析CCKBR在胃癌中的生物学功能,结果表明,CCKBR敲降后胃癌细胞BGC-823和SGC-7901的细胞增殖、单克隆形成率、迁移率和侵袭率均显著降低(P<0.05),而对CCKBR低表达胃癌细胞系MKN-1进行过表达处理后,其增殖、单克隆形成率、迁移率和侵袭率均呈现相反趋势(P<0.05);通过流式细胞术检测发现,BGC-823和SGC-7901细胞在CCKBR敲降后分别发生了G2/M期阻滞(P<0.001)和S期阻滞(P<0.001);此外,CCKBR表达量的下调显著抑制了BGC-823细胞的活体成瘤能力。以上结果证实了CCKBR在胃癌发生发展及预后中扮演的重要角色,而这也为胃癌靶向CCKBR新疗法的建立提供了理论基础。制备的CCKBR靶向重组毒素FQ17P可以特异性结合胃癌细胞表面的CCKBR,并呈现CCKBR表达量依赖的靶向杀伤;FQ17P处理可以引起胃癌细胞BGC-823(P<0.0001)、SGC-7901(P<0.0001)以及MGC-803(P<0.01)的凋亡率显著升高;此外,FQ17P还可以杀伤CCKBR高表达胃癌原代细胞,并通过腹腔注射体内抑制肿瘤的生长,2 mg/kg FQ17P注射给药10天后肿瘤重量显著降低(P<0.001),8 mg/kg FQ17P注射10天后肿瘤重量显著低于2 mg/kg(P<0.001)。根据CCKBR水平对所检临床样本进行分类并分析与临床病理因素间的相关性;结果显示,FQ17P临床适用的蛋白相对表达量(CCKBR/GAPDH)的筛选阈值为0.493;样本的FQ17P适用性与肿瘤EGFR表达水平(χ~2=13.26,P=0.004)及分化程度(χ~2=13.59,P=0.001)密切相关;血清CCKBR微创检测方法的灵敏度低但准确性高,ROC曲线下面积为0.7108,P<0.001,可以作为CCKBR靶向潜在适用病例的初筛辅助手段;结合以上结果建立了一套临床适用病例的筛选策略。总之,我们分别从CCKBR表达丰度、过表达CCKBR的生物学功能及其介导的重组毒素靶向杀伤作用这三个方面评价了CCKBR作为胃癌重组毒素靶向治疗靶标的临床应用价值,并结合临床胃癌组织及血清样本建立了靶向疗法适用病例筛选策略。该研究不仅为胃癌的抗CCKBR靶向疗法提供参考依据,而且为其靶向重组毒素的进一步应用提供了宝贵的临床数据支撑,有望改善胃癌患者的治疗现状。更多还原

【Abstract】 As a serious threaten to public health,cancer is gradually changing the way modern people live.As one of the most deadly malignancies,gastric cancer(GC)accounts for millions of new cases every year,and 40% of these cases came from China.Surgical resection combined with chemotherapy and radiotherapy is the conventional therapy strategy of GC,among which surgery is still the best choice for radical treatment.However,not all GC patients have the opportunity to receive surgery,and metastasis and recurrence are easy after resection.The prognosis of advanced GC patients after conventional treatment is poor,and the average 5-year survival rate is less than 30%.Although adjuvant therapy is of great significance for cure of GC patients,it has huge side effects and can cause great pain for patients.Therefore,molecular targeted therapy has become a hot topic in the field of tumor therapy.Compared with chemoradiotherapy,molecular targeted therapy kills tumor cells based on the heterogeneity of tumor by targeting specific molecules and their downstream signal transduction.This study aimed to comprehensively evaluate the clinical application value of CCKBR as a therapeutic target of recombinant toxin in GC by analyzing its expression level,biological function and specific killing activity of recombinant toxin which mediated by CCKBR.The relative expression levels of CCKBR were detected by online database,RT-PCR and Western-blotting methods in pan-cancer,30 tumor cell lines,and 59 groups of GC tumor samples and their corresponding normal stomach tissue samples which were collected from China-Japan Union Hospital of Jilin University from 2016 to 2017;Recombinant lentivirus was used to construct CCKBR knockdown and overexpression GC cell lines,and the effects of CCKBR expression level on proliferation,migration and invasion and tumorigenesis of GC cells were analyzed by cytotoxicity assay,clone formation assay,scratch healing assay,Transwell chamber assay and tumorigenesis assay;Recombinant toxins which have a gastrin 17 peptide(G17)as the targeting domain were constructed based on the interaction between CCKBR and its natural ligand.CCK-8,flow cytometry,nude mice model of GC and other methods were used to evaluate targeted killing activity of recombinant toxins against GC cells followed by expression and purification;At last,clinical samples were classified according to CCKBR expression level,and the relationship between CCKBR level and clinical pathological characteristics was analyzed statistically in combination with clinical diagnosis of the patients,and a screening strategy for clinical potential cases for CCKBR targeted therapy was established on this basis.The results showed that CCKBR was overexpressed in a variety of gastrointestinal tumor cells,especially in GC cell lines BGC-823 and SGC-7901;The CCKBR level of GC tissue samples was significantly higher than that of normal tissue samples in both m RNA transcription level(P=0.007)and protein expression level(P=0.037);Prognostic analysis results showed that the prognostic risk of GC patients increased with the up-regulation of CCKBR level,and the overall survival of GC patients with low CCKBR expression level significantly prolonged(4.5years vs 1.6 years,P=0.005).Biological functions of CCKBR were analyzed using knockdown and overexpression of GC cell lines,the results showed that proliferation,monoclonal formation,migration and invasion rate of GC cell lines BGC-823 and SGC-7901 were significantly decreased after CCKBR knockdown(P<0.05);After overexpression of CCKBR in GC cell line MKN-1,opposite trends were observed in the proliferation,monoclonal formation rate,migration rate and invasion rate(P<0.05);The results of flow cytometry showed that BGC-823 and SGC-7901 cells exhibited G2/M phase arrest(P < 0.001)and S phase arrest(P < 0.001)after CCKBR knockdown,respectively;In addition,the down-regulation of CCKBR level significantly inhibited the tumorigenicity of BGC-823 cells in vivo.These results confirmed the important role of CCKBR in the pathogenesis,development and prognosis of GC,which also provided a theoretical basis for the establishment of new therapy targeting CCKBR for GC.The prepared recombinant toxin FQ17 P could specifically bind to CCKBR on the surface of GC cells,and exhibited CCKBR expression dependent targeted activity;FQ17P treatment could significantly increase the apoptosis rate of GC cell lines BGC-823(P<0.0001),SGC-7901(P<0.0001)and MGC-803(P<0.01);In addition,FQ17 P can kill primary GC cells with high CCKBR expression,and inhibit tumor growth by intraperitoneal injection in vivo.After 10 days of 2 mg/kg FQ17 P treatment,tumor weight was significantly reduced(P < 0.001);and the tumor weight of mice in 8 mg/kg FQ17 P group was significantly lower than that in 2 mg/kg FQ17 P group(P<0.001).The clinical samples were classified according to CCKBR level and their correlation with clinicopathological factors was analyzed.The results showed that the screening threshold of CCKBR/GAPDH of samples for FQ17 P was 0.493;The applicability of FQ17 P was closely correlated with EGFR expression level(χ2=13.26,P=0.004)and differentiation degree(χ2=13.59,P=0.001).The serum minimally invasive CCKBR assay has low sensitivity but high accuracy,with an area under curve 0.7108 of ROC(P<0.001),which means it can be used as a preliminary aid for screening potential cases for CCKBR-targeted treatment.Combined with the above results,a screening strategy for potential clinical cases was established.In conclusion,we evaluated the clinical application value of CCKBR as a target of recombinant toxins therapy for GC from three aspects,the CCKBR expression abundance,biological function of overexpressed CCKBR and targeted killing activity of recombinant toxins mediated by CCKBR,and established a potential case screening strategy for CCKBR targeted therapy by combining with clinical GC tissue and serum samples.This study not only provides a reference for anti-CCKBR targeted therapy for GC,but also provides valuable clinical data support for the further application of CCKBR-targeted recombinant toxin,which is expected to improve the treatment status of GC patients.更多还原