【作者】 林丹；

【导师】 曾晓娟；

【作者基本信息】 广西医科大学 ， 口腔临床医学（专业学位）， 2025， 博士

【摘要】 目的和意义:头颈部肿瘤放射治疗常导致唾液腺（Salivary Glands,SGs）不可逆损伤,引发严重口干症,显著降低患者生存质量。传统治疗手段对腺体功能恢复效果有限,而基因疗法因其作用持久、靶向性强及毒副作用低等优势,成为SGs放射损伤修复的研究热点。研究表明,由DEFB1基因编码的人β防御素1（h BD-1）作为兼具抗菌、免疫调节及组织修复功能的多肽分子,在干燥综合征患者SGs及放射后皮肤创面中表达下调,提示DEFB1可能参与放射损伤病理进程。因此,外源性补充h BD-1或将成为改善放射后腺体功能损伤的新策略。目前,Defb1在放射应激中的分子机制尚未明确。本研究通过构建小鼠颌下腺（Submandibular Gland,SMG）体内外放射模型,分别结合Defb1基因敲除(Defb1^-/-)及腺病毒过表达技术,系统阐述Defb1在放射应激中的功能及分子调控机制,为开发靶向Defb1的SGs放射损伤修复策略开辟新路径,为改善头颈癌患者生存质量提供理论基础。方法:1.探究Defb1在小鼠SMG放射后的早期表达变化。（1）选取8周龄雌性C57BL/6J小鼠36只,分为对照组（WT,6只）和放射组（30只）,放射组接受15 Gy X线照射后于1 h、2 h、3 h、4 h、6 h各处死6只小鼠并取材。（2）利用IHC、ELISA、RT-PCR、Western blot技术检测放射后不同时间点SMG组织中Defb1基因及蛋白的表达变化。2.Defb1^-/-小鼠颌下腺放射后功能及凋亡、自噬相关因子的检测。（1）采用PCR技术鉴定Defb1^-/-小鼠的基因型。（2）将32只小鼠按基因型分为四组,对照组（WT）和Defb1基因敲除组（KO）,放射组（RWT）和基因敲除放射组（RKO）,每小组8只。（3）放射后8W,收集唾液测定唾液流率,通过组织病理学（透射电镜、H&E染色、PAS染色）观察SMG组织损伤程度,运用TUNEL染色观察小鼠SMG凋亡情况,利用IHC、RT-PCR及Western blot检测颌下腺功能代表基因AQP5、Defb1以及凋亡因子Bcl-2,Bax,Caspase-3,自噬因子LC3-Ⅱ,Beclin-1的表达。3.Defb1过表达SMGECs放射后功能及凋亡、自噬相关因子的检测。（1）体外培养SMGECs,通过IHC、IF和PAS染色进行鉴定。通过CCK-8实验、IF和流式细胞术,分别检测细胞活力、形态及凋亡情况,最终确定细胞放射剂量、时间以及Defb1基因腺病毒过表达最佳剂量。（2）将SMGECs细胞分为不放射组:WT、基因过表达组（OE）、病毒载体阴性对照组（NC）,以及放射组:RWT,病毒载体放射组（RNC）,基因过表达放射组（ROE）。给予放射组10 Gy放射治疗,48 h后收集细胞。（3）采用PAS染色法,评估Defb1过表达SMGECs放射后唾液分泌能力;利用RT-PCR及Western blot技术检测Defb1、AQP5的表达;通过JC-1检测线粒体膜电位,ROS检测活性氧水平,MDC染色观察自噬,并结合流式细胞术,评估SMGECs的损伤情况。通过RT-PCR及Western blot检测过表达Defb1的SMGECs中凋亡因子Bcl-2,Bax,Caspase-3以及自噬因子LC3-Ⅱ,Beclin-1表达。4.MAPK/mTOR-自噬/凋亡通路相关因子的验证。（1）利用CCK-8法测定SMGECs中3-甲基腺嘌呤（3-MA）和雷帕霉素（Rapa）的IC50值。（2）SMGECs细胞分为Defb1非过表达组及过表达组,非过表达组分为RWT组,IR及Rapa联合干预组（IR+Rapa）;过表达组分为:ROE组,ROE+3-MA联合干预组（ROE+3-MA）,ROE+3-MA+Rapa联合干预组（ROE+3-MA+Rapa）。各组给予10 Gy放射治疗,48 h后收集细胞,进行检测。（3）通过流式细胞术检测各组细胞凋亡情况,MDC染色观察细胞自噬率。通过Western blot检测各组细胞凋亡因子Bcl-2,Bax,Caspase-3以及自噬因子LC3-Ⅱ,Beclin-1表达。通过Western blot技术检测p-p38MAPK/p38 MAPK以及p-mTOR/mTOR蛋白的磷酸化水平变化。结果:1.放射后Defb1动态表达特征:放射后约3 h Defb1表达达峰值（p<0.01）,8周后显著低于正常水平。2.Defb1缺失对小鼠颌下腺放射损伤功能及凋亡、自噬的影响:（1）RKO组唾液流率、糖原含量及AQP5表达较RWT组显著降低（p<0.05）,转录组富集于MAPK、mTOR、自噬及凋亡等通路。（2）RKO组小鼠SMG组织凋亡率较RWT增高,促凋因子Bax,Caspase-3表达明显增高,抑凋因子Bcl-2表达降低（p<0.05）。（3）RKO组自噬因子LC3-Ⅱ,Beclin-1表达较RWT组降低（p<0.05）。3.Defb1高表达对SMGECs功能及凋亡、自噬的影响:（1）成功获得Defb1高表达SMGECs,10 Gy为SMGECs合适的放射剂量。MOI=100为最佳Defb1腺病毒转染浓度。（2）ROE组细胞糖原含量较RWT组高。ROE组JC-1和ROS水平,凋亡率较RWT组下降更为明显,MDC染色增强较WT组有统计学意义（p<0.05）。（3）ROE组Defb1、AQP5表达较RWT组升高（p<0.05）。（4）ROE组SMGECs凋亡率及促凋因子表达较RWT组降低,抑凋因子及自噬因子表达升高（p<0.05）,提示高表达Defb1抵御细胞放射损伤。4.Defb1通过MAPK/mTOR-自噬/凋亡通路调控SMG放射损伤。（1）加入3-MA,ROE组的自噬活性降低,细胞凋亡增加,同时自噬相关蛋白表达降低;加入Rapa后,这些作用被部分逆转:（ROE+3-MA+Rapa）组自噬活性恢复,凋亡表达减少,自噬蛋白表达回升（p<0.05）,提示自噬与凋亡相互制衡。（2）放射后,ROE组MAPK活化水平（p-p38 MAPK/p38 MAPK比值）较RWT组增高,mTOR活性水平（p-mTOR/mTOR比值）降低（p<0.05）,说明MAPK/mTOR可能参与Defb1介导的放射损伤;（3）而（ROE+3-MA）组较ROE组MAPK活化水平降低,mTOR活化水平升高（p<0.05）,提示自噬抑制剂部分阻断了Defb1对p38MAPK/mTOR信号轴的激活作用,进一步同时加入Rapa后,（ROE+3-MA+Rapa）组自噬活性升高,凋亡表达减少,自噬表达回升（p<0.05）,MAPK/mTOR信号通路轴活性增高,提示MAPK/mTOR参与Defb1调控的放射损伤自噬凋亡通路。结论:1.Defb1基因具有放射敏感性,Defb1基因表达水平是SMG放射损伤积极调控因子和潜在标志物之一。2.Defb1缺失加重小鼠SMG放射功能损伤:Defb1缺失SMG唾液分泌功能下降,组织自噬水平降低,凋亡水平升高。3.Defb1过表达抵御SMGECs放射功能损伤:Defb1过表达,SMGECs通过降低细胞活性氧,降低线粒体膜电位,提高自噬水平,减少细胞凋亡,从而抵御放射功能损伤。4.MAPK/mTOR通路的核心作用:Defb1通过激活p38 MAPK,并抑制mTOR,协调自噬与凋亡,维护SMG功能。更多还原

【Abstract】 ObjectiveRadiotherapy for head and neck tumors often causes irreversible damage to salivary glands（SGs）,leading to severe xerostomia and significantly compromising patients’quality of life.Conventional therapies have limited efficacy in restoring glandular function,while gene therapy has emerged as a promising approach for SGs radioprotection due to its sustained effects,precise targeting,and low toxicity.Studies have shown that humanβ-defensin 1（h BD-1）,encoded by the DEFB1 gene,is a multifunctional peptide with antimicrobial,immunomodulatory,and tissue-repair properties.Its expression is downregulated in SGs of Sj（?）gren’s syndrome and in irradiated skin wounds,suggesting a potential role of DEFB1 in radiation-induced tissue injury.Therefore,exogenous supplementation of h BD-1 may represent a novel strategy for mitigating radiation-induced SGs dysfunction.The molecular mechanisms of Defb1 in radiation stress remain poorly understood.In this study,we established in vivo and ex vivo radiation models of mouse submandibular glands（SMGs）.By combining Defb1 knockout(Defb1^-/-)techniques with adenovirus-mediated overexpression approaches,we systematically elucidated the functional roles and molecular regulatory mechanisms of Defb1 in radiation stress response.The findings pave a novel pathway for developing Defb1-targeted repair strategies against radiation-induced SG damage,while providing crucial theoretical foundations for improving quality of life in head and neck cancer patients.Methods1.To investigate the early expression dynamics of Defb1 in murine SMGs following irradiation.（1）Thirty-six 8-week-old female C57BL/6J mice were divided into a control group（WT,n=6）and a radiation group（n=30）.The radiation group received 15 Gy X-ray irradiation,6 mice were sacrificed at 1 h,2 h,3 h,4 h and 6 h respectively.（2）IHC,ELISA,RT-PCR,and Western blot were used to assess the temporal changes in Defb1 gene and protein expression in SMG tissues after irradiation.2.Assessment of functional parameters and detection of apoptosis-and autophagy-associated factors in irradiated submandibular glands of Defb1^-/-mice.（1）PCR was used to genotype Defb1^-/-mice.（2）Thirty-two mice were divided into four groups according to genotype:control group（WT）and Defb1 knockout group（KO）,radiation group（RWT）and gene knockout radiation group（RKO）,with 8 mice in each group.（3）At 8 weeks post-irradiation,saliva was collected for salivary flow rate measurement.Histopathological evaluation of SMG tissue damage was performed through TEM,H&E staining,and PAS staining.Apoptotic activity was assessed via TUNEL assay.Molecular analyses including IHC,RT-PCR,and Western blot were conducted to detect expression levels of salivary functional markers（AQP5 and Defb1）,apoptotic regulators（Bcl-2,Bax,and cleaved Caspase-3）,and autophagy-related proteins（LC3-II and Beclin-1）in SMGs.3.Impact of Defb1 overexpression on SMGECs functionality and apoptosis/autophagy regulation:（1）SMGECs were authenticated via IHC,IF,and periodic acid-Schiff（PAS）staining.Cell viability,morphology,and apoptosis were assessed using CCK-8 assays,IF,and flow cytometry,respectively,to determine the optimal radiation dosage,exposure time,and adenovirus-mediated Defb1 overexpression efficiency.（2）SMGECs were divided into non-irradiated groups:WT,OE,and NC,and irradiated groups:RWT,RNC,and ROE.Irradiated groups received 10 Gy X-ray exposure,and cells were harvested 48 hours post-irradiation.（3）Salivary secretory capacity of Defb1-overexpressing SMGECs post-irradiation was evaluated via PAS staining.Defb1 and AQP5 expression levels were quantified by RT-PCR and Western blot.Mitochondrial membrane potential was measured using JC-1 staining,reactive oxygen species（ROS）levels were assessed via fluorescence probes,and autophagic activity was monitored by MDC staining combined with flow cytometry.Apoptotic regulators（Bcl-2,Bax,cleaved Caspase-3）and autophagy-related proteins（LC3-II,Beclin-1）were further analyzed by RT-PCR and Western blot in Defb1-overexpressing SMGECs.4.Validation of MAPK/mTOR-autophagy/apoptosis signaling pathway-associated factors.（1）CCK-8 assay was used to determine the IC50of 3-MA and Rapa in SMGECs.（2）SMGECs were divided into overexpression（ROE,ROE+3-MA,ROE+3-MA+Rapa）and non-overexpression（RWT,IR+Rapa）groups.All groups received 10 Gy irradiation,and cells were collected after 48 h.（3）Flow cytometry and MDC staining were used to assess apoptosis and autophagy rates,respectively.Western blot was used to detect apoptosis（Bcl-2,Bax,Caspase-3）and autophagy（LC3-II,Beclin-1）markers.Western blot was used to measure phosphorylation levels of p-p38 MAPK/p38MAPK and p-mTOR/mTOR.Results1.Dynamic Expression of Defb1 Post-Irradiation:Defb1 expression peaked at approximately 3 h post-irradiation（p<0.01）and was significantly lower than baseline levels at 8 weeks.2.Impact of Defb1 deficiency on radiation-induced functional impairment and apoptosis/autophagy in murine submandibular glands:（1）The RKO group（radiation-treated Defb1 knockout）exhibited significantly reduced salivary flow rate,glycogen content,and AQP5 expression compared to the RWT group（radiation-treated wild-type）（p<0.05）.Transcriptomic enrichment analysis highlighted perturbations in MAPK,mTOR,autophagy,and apoptosis pathways.（2）SMG tissues from RKO mice showed elevated apoptosis rates compared to RWT controls,with upregulated pro-apoptotic factors（Bax,cleaved Caspase-3）and downregulated anti-apoptotic protein Bcl-2（p<0.05）.（3）Autophagy-related proteins LC3-II and Beclin-1 were significantly reduced in the RKO group relative to RWT（p<0.05）.3.Impact of Defb1 overexpression on submandibular gland epithelial cells（SMGECs）functionality and apoptosis/autophagy regulation:（1）Defb1-overexpressing SMGECs were successfully established,with 10 Gy identified as the optimal radiation dosage and MOI=100 determined as the ideal adenoviral transduction concentration.（2）The ROE group exhibited higher glycogen content compared to the RWT group.JC-1 staining revealed improved mitochondrial membrane potential,while ROS levels and apoptosis rates were significantly reduced in ROE versus RWT（p<0.05）.Enhanced autophagic flux in ROE cells was confirmed by intensified MDC fluorescence relative to WT controls（p<0.05）.（3）Both Defb1 and AQP5 expression levels were markedly elevated in the ROE group compared to RWT（p<0.05）.（4）ROE SMGECs demonstrated reduced apoptosis rates and downregulated pro-apoptotic factors（Bax,cleaved Caspase-3）,alongside upregulated anti-apoptotic protein Bcl-2and autophagy markers（LC3-II,Beclin-1）compared to RWT（p<0.05）,collectively indicating that Defb1 overexpression confers radioprotection by mitigating apoptosis and enhancing autophagy4.Defb1 regulates radiation-induced SMG damage through MAPK/mTOR-autophagy/apoptosis signaling axis.（1）Administration of3-methyladenine（3-MA）in the ROE group suppressed autophagic activity,increased apoptosis,and reduced autophagy-related protein expression.These effects were partially reversed by co-treatment with rapamycin（Rapa）:the ROE+3-MA+Rapa group exhibited restored autophagy flux,reduced apoptosis,and recovered autophagy protein levels（p<0.05）,indicating a counterbalancing interplay between autophagy and apoptosis.（2）Post-irradiation,the ROE group demonstrated elevated MAPK activation（p-p38 MAPK/p38 MAPK ratio）and reduced mTOR activity（p-mTOR/mTOR ratio）compared to RWT（p<0.05）,suggesting MAPK/mTOR signaling involvement in Defb1-mediated radioprotection.（3）The ROE+3-MA group showed diminished MAPK activation and enhanced mTOR activity relative to ROE alone（p<0.05）,implying that autophagy inhibition partially abrogated Defb1-driven p38 MAPK/mTOR axis activation.Subsequent Rapa addition（ROE+3-MA+Rapa）restored autophagic activity,reduced apoptosis,and upregulated autophagy markers（p<0.05）,accompanied by increased MAPK/mTOR pathway activation.These data collectively demonstrate that Defb1 coordinates autophagy-apoptosis equilibrium via MAPK/mTOR signaling to mitigate radiation damage.Conclusions1.Radiosensitivity of Defb1 Gene:Defb1 exhibited radiation-responsive dynamics:These findings position Defb1 as both a positive regulator and a potential biomarker of radiation-induced SMG injury.2.Defb1 Deficiency Exacerbates Radiation-Induced SMG Dysfunction:Defb1^-/-mice displayed aggravated post-irradiation SMG dysfunction,characterized by reduced salivary secretion,suppressed autophagic flux,and elevated apoptosis compared to WT3.Defb1 Overexpression Confers Radioprotection in SMGECs:Defb1-overexpressing SMGECs mitigated radiation damage through ROS scavenging,mitochondrial membrane potential stabilization,enhanced autophagy activation,and apoptosis suppression,collectively improving cellular radioresistance.4.Central Role of MAPK/mTOR Signaling Axis:Defb1 orchestrates autophagy-apoptosis crosstalk by activating p38 MAPK and inhibiting mTOR activity,thereby maintaining SMG functional homeostasis under radiation stress.更多还原