【作者】 王萍；

【导师】 姚学清；

【作者基本信息】 南方医科大学 ， 外科学， 2025， 硕士

【摘要】 研究背景和目的:肝转移是结直肠癌(Colorectal Cancer,CRC)患者预后不良的重要原因,越来越多的流行病学证据表明,代谢相关脂肪性肝病(metabolic dysfunction-associated steatotic liver disease,MASLD)与结直肠癌肝转移密切相关。种子-土壤学说认为肝脏为转移性肿瘤细胞提供有利的微环境,而MASLD促进CRC肝转移的具体机制未明。因此,揭示MASLD促进CRC肝转移的潜在机制,寻找新的治疗策略具有重要的理论和临床意义。研究方法:1、首先通过对伴及不伴MASLD的肝转移肿瘤组织进行RNA测序,发现关键分子CD36,通过体内外实验验证CD36在肝转移中的作用。2、进一步寻找MASLD微环境中脂质增多促CD36上调的分子机制。通过对伴及不伴MASLD的肝转移癌旁组织进行测序,发现关键分子POLDIP2。构建基因小鼠及脂肪肝小鼠模型,验证POLDIP2与脂肪肝之间的关系。通过对敲降POLDIP2的肝细胞进行RNA测序及脂质组学检测,验证POLDIP2调控脂质代谢紊乱。3、接着通过生物信息学及co-IP寻找POLDIP2的下游分子,进一步验证POLDIP2调控脂质代谢的具体分子机制。4、构建外泌体靶向递送系统Exo-siCD36,小动物活体成像评估其靶向性。构建脂肪肝合并结直肠癌肝转移小鼠模型验证治疗效果,实验终点对小鼠关键脏器解剖验证载体的生物安全性。研究内容及研究结果:1、通过收集200例CRC肝转移患者的临床数据及随访信息评估MASLD对结直肠癌预后的影响,并通过构建脂肪肝小鼠模型、结直肠癌肝转移小鼠模型及CRC细胞-肝细胞共培养模型验证脂肪肝对肠癌肝转移的促进作用。2、通过RNA测序及免疫组化找到促肝转移的关键分子CD36,肿瘤细胞通过上调CD36的表达摄取MASLD微环境中的脂质,增强自身恶性表型。3、收集新鲜伴及不伴MASLD的肠癌肝转移癌旁组织进行RNA-seq测序,寻找MASLD关键分子POLDIP2,检测POLDIP2敲除后肝细胞内及培养上清中脂质含量的改变,结合q-PCR、WB,明确POLDIP2表达下调促肝细胞脂质代谢紊乱,进一步促进脂质外排至微环境中。4、通过对肝细胞进行质谱、IP等检测,明确POLDIP2的下游分子CPT1A,通过生信预测互作位点。进一步通过q-PCR、WB明确POLDIP2对脂肪酸合成及氧化相关分子的调控均基于CPTIA分子。5、通过qPCR、WB、IP及稳定性检测明确POLDIP2对CPT1A的调控在于蛋白表达层面,POlDIP2调控CPT1A的稳定性。6、构建靶向外泌体递送系统Exo-siCD36,通过CCK8、溶血实验、外泌体体外稳定性实验、小动物活体成像及小鼠治疗模型,验证工程化外泌体具有良好生物安全性及靶向性,具有良好的治疗效果,为临床治疗提出新的思路。主要结论:1、CD36是促进CRC肝转移的关键分子,在肝转移肿瘤组织中过表达,促进肿瘤细胞摄取MASLD微环境中的脂质。2、POlDIP2调控肝细胞脂质代谢,在MASLD中,POLDIP2表达下调,促进肝脏脂质沉积。3、POLDIP2通过调控CPT1A的稳定性来调控肝细胞脂肪酸氧化,并负反馈调控肝细胞内脂质合成,使肝细胞脂质沉积。4、工程化外泌体递送系统具有靶向肿瘤细胞的作用,通过抑制肿瘤细胞CD36的表达延缓肿瘤进展。更多还原

【Abstract】 Background and Objective:Liver metastasis is a significant cause of death in patients with colorectal cancer(CRC).An increasing amount of epidemiological evidence suggests that metabolic dysfunction-associated steatotic liver disease(MASLD)is closely related to colorectal cancer liver metastasis.The seed-soil hypothesis posits that the liver provides a favorable microenvironment for metastatic tumor cells,but the specific mechanism by which MASLD promotes CRC liver metastasis remains unclear.Therefore,revealing the potential mechanism by which MASLD promotes CRC liver metastasis and finding new treatment strategies are of significant theoretical and clinical importance.Research Methods:Firstly,RNA sequencing was performed on liver metastasis tissues with and without MASLD to identify the key molecule CD36.The role of CD36 in liver metastasis was verified through in vitro and in vivo experiments.Further investigation was conducted into the molecular mechanism by which the increased lipids in the MASLD microenvironment promote the upregulation of CD36.Sequencing was performed on liver metastasis tissues with and without MASLD to identify the key molecule POLDIP2.Gene knockout mice and fatty liver mouse models were constructed to verify the relationship between POLDIP2 and fatty liver.RNA sequencing and lipidomic detection of hepatocytes with POLDIP2 knockout were performed to verify the role of POLDIP2 in regulating lipid metabolism disorders.Next,IP-MS and co-IP were used to identify downstream molecules of POLDIP2,and rescue experiments further verified the specific molecular mechanism by which POLDIP2 regulates lipid metabolism.An exosome-targeted delivery system Exo-siCD36 was constructed,and its targeting ability was evaluated by small animal in vivo imaging.A mouse model of fatty liver combined with colorectal cancer liver metastasis was constructed to verify the therapeutic effect,and the key organs of the mice were dissected at the experimental endpoint to verify the biosafety of the carrier.Research Content and Results:The impact of MASLD on the prognosis of colorectal cancer was assessed by collecting clinical data and follow-up information from 200 CRC liver metastasis patients.The promoting effect of fatty liver on colorectal cancer liver metastasis was verified by constructing fatty liver mouse models,colorectal cancer liver metastasis mouse models,and CRC cell-hepatocyte co-culture models.Key molecule CD36,which promotes liver metastasis,was identified through RNA sequencing and immunohistochemistry.Tumor cells upregulate the expression of CD36 to absorb lipids from the MASLD microenvironment,enhancing their malignant phenotype.Fresh liver metastasis tissues with and without MASLD were collected for RNA-seq sequencing to identify the key molecule POLDIP2 in MASLD.Changes in lipid content within hepatocytes and in the culture supernatant after POLDIP2 knockout were detected,combined with q-PCR and WB,to clarify that the downregulation of POLDIP2 expression promotes hepatocyte lipid metabolism disorders,further promoting lipid efflux into the microenvironment.Mass spectrometry and IP detection of hepatocytes clarified the downstream molecule CPT1A of POLDIP2,and the interaction sites were predicted by bioinformatics.Further clarification of the regulation of fatty acid synthesis and oxidation-related molecules by POLDIP2 based on CPT1A expression was made through q-PCR,WB,IP,and stability detection.The regulation of CPT1A by POLDIP2 was clarified at the protein expression level through qPCR,WB,IP,and stability detection.POLDIP2 regulates the stability of CPT1A.An engineered exosome delivery system Exo-siCD36 was constructed.Through CCK8,hemolysis experiments,exosome in vitro stability experiments,small animal in vivo imaging,and mouse treatment models,the engineered exosome was verified to have good biosafety and targeting ability,with good therapeutic effects,providing new ideas for clinical treatment.Main Conclusions:1.CD36 is a key molecule that promotes CRC liver metastasis,overexpressed in liver metastasis tumor tissues,and promotes tumor cells to absorb lipids from the MASLD microenvironment.2.POLDIP2 regulates hepatocyte lipid metabolism.In MASLD,the downregulation of POLDIP2 expression promotes liver lipid deposition.3.POLDIP2 regulates hepatocyte fatty acid oxidation by regulating the stability of CPT1A and negatively feeds back to regμlate hepatocyte lipid synthesis,causing hepatocyte lipid deposition.4.The engineered exosome delivery system has a tumor cell-targeting effect,and by inhibiting the expression of tumor cell CD36,it delays tumor progression.更多还原