课题一、雌激素通过激活CD38／SIRT1信号途径促进低氧诱导小鼠气道平滑肌细胞凋亡的机制研究 研究背景及研究目的： 支气管哮喘是临床上常见的呼吸道疾病，是一种由气道炎性细胞及结构细胞等多种细胞及细胞组分参与，以气道炎症、气道重塑和气道高反应性为特征的异质性疾病。缺氧被认为是肺部疾病进展的关键因素，例如支气管哮喘、气道阻塞和肺动脉高压等都存在不同程度的缺氧。缺氧会刺激气道炎症和气道重塑，并在气道重塑期间诱导气道平滑肌细胞(ASMC)凋亡。CD38是体内NAD⁺降解的关键酶，在调节细胞内的NAD⁺水平以及NAD⁺依赖的SIRT1活性中发挥重要作用。另有证据表明肺部疾病的发病率和进展存在性别差异，雌激素在这些病理过程中发挥着至关重要的作用；雌激素可通过CD38影响平滑肌收缩，但其调控的具体分子机制尚不清楚。本研究旨在探讨CD38缺失对于低氧状态下小鼠气道平滑肌细胞凋亡的影响，以及雌激素对CD38／SIRT1信号轴的调控作用，并在整体动物模型中验证平滑肌CD38缺失对小鼠哮喘的保护作用，为哮喘等呼吸系统疾病的治疗提供重要靶点和实验依据。 实验方法： 1.雌激素(E2)对气道平滑肌细胞CD38／SIRT1表达调控分析：分离和体外培养原代气道平滑肌细胞(ASMCs)，采用不同浓度E2及不同时间处理ASMCs，利用荧光定量PCR检测CD38和SIRT1的mRNA水平；Western blot检测CD38和SIRT1的蛋白表达水平，明确E2调控CD38表达的量效和时效变化；比较野生型(WT)及CD38敲除(CD38 KO)的ASMCs中CD38、 SIRT1、乙酰化p53(Ac-p53)和总p53表达水平，以及E2对其表达的干预作用。 2.CD38对低氧条件下ASMCs凋亡的影响及E2的干预作用分析：建立体外低氧模型，荧光定量PCR检测低氧条件下E2对WT和CD38 KO的ASMCs中CD38和SIRT1 mRNA水平的影响，Western blot检测E2对于低氧刺激后CD38／SIRT1／p53通路的影响；通过Hoechst33258染色、凋亡相关蛋白Bax和Bcl-2表达分析和Caspase-3活性检测，明确CD38缺失对低氧诱导ASMCs凋亡的保护作用以及E2的干预作用。 3.E2调控ASMCs凋亡的分子机制分析：观察在SIRT1特异性活性激动剂白藜芦醇(RSV)存在情况下E2对ASMCs凋亡的影响；检测在雌激素受体特异性抑制剂(ICI 182，780)存在时E2对CD38 mRNA和蛋白表达的影响，明确E2是否通过雌激素受体调控CD38表达。通过上述实验，以期阐明E2通过调控CD38／SIRT1信号影响ASMCs凋亡的分子机制。 4.卵清蛋白(OVA)诱导小鼠哮喘模型的构建：比较野生型和平滑肌CD38敲除小鼠的哮喘程度；检测两组小鼠肺泡灌洗液中的白细胞数；HE染色观察炎症细胞浸润和肺部损伤，在整体动物水平上验证平滑肌CD38缺失对于小鼠哮喘的保护作用。 实验结果： 1.量效实验显示，ASMCs的CD38 mRNA和蛋白表达随着E2浓度的增加而升高，而SIRT1的mRNA和蛋白表达则下降；时效实验显示，CD38 mRNA和蛋白表达在48 h内持续增加，SIRT1 mRNA和蛋白表达水平在24 h时间点降低，在48 h时部分恢复，二者的表达呈现显著的负相关。因此，选用10 nM E2，且预处理48 h后用于随后的细胞实验。 2.E2处理可使野生型ASMCs的CD38蛋白表达增加，SIRT1蛋白表达降低，同时促进乙酰化p53(Ac-p53)的表达；CD38 KO导致ASMCs的SIRT1蛋白水平显著增加，Ac-p53蛋白水平降低，E2处理后，ASMCs的SIRT1和Ac-p53表达水平不变。 3.低氧处理可使野生型ASMCs的CD38和SIRT1 mRNA表达明显下调， CD38蛋白水平增加，SIRT1表达减少，Ac-p53表达增加。E2可抑制低氧导致的CD38 mRNA表达下调，进而促进SIRT1的mRNA表达下调；低氧情况下E2可以进一步促进CD38蛋白表达、下调SIRT1蛋白水平和促进p53乙酰化。敲除CD38基因可使E2在ASMCs的上述作用消失。 4.E2处理可显著增加低氧诱导的野生型ASMCs凋亡，Caspase-3活性增加，Bax／Bcl-2比值显著增高；敲除CD38基因可显著抑制低氧导致的ASMCs凋亡；SIRT1激动剂RSV则可显著抑制E2诱导的ASMCs凋亡。 5.雌激素受体抑制剂(ICI 182，780)可显著降低ASMCs的CD38 mRNA和蛋白表达，在ICI 182，780存在的情况下，可抵消E2促进CD38表达的作用。 6.在整体动物模型中，与对照小鼠相比较，平滑肌特异性敲除CD38小鼠对哮喘敏感性下降，气道高反应性降低，支气管肺泡灌洗液中白细胞减少；肺部组织病理学和肺部炎症显著改善。 结论： E2通过雌激素受体上调CD38基因表达，导致SIRT1表达减少，p53乙酰化增加，促进低氧诱导的ASMCs凋亡，而CD38缺失可抵消E2的这一作用。平滑肌特异性敲除CD38小鼠可减轻小鼠哮喘症状和肺部损伤。上述结果表明平滑肌细胞CD38是哮喘发病过程中的关键靶点，通过对其的调控可能为哮喘等呼吸系统疾病的治疗提供新的策略。 关键词：哮喘；CD38；雌激素；低氧；细胞凋亡 课题二、人羊膜上皮干细胞对小鼠急性肺损伤的治疗作用及其机制研究 研究背景及研究目的： 急性肺损伤(Acute lung injury，ALI)／急性呼吸窘迫综合征(Acute respiratory distress syndrome，ARDS)，是由于重症肺炎、吸入性肺炎、重度脓毒血症、严重创伤、休克等非心源性疾病引起的肺毛细血管内皮细胞和肺泡上皮细胞损伤造成弥漫性肺间质水肿及肺泡水肿，导致急性低氧性呼吸功能不全或呼吸衰竭，伴随失控性全身炎症反应(systemic inflammatory response syndrome，SIRS)以及机体代偿性抗炎反应(CARS，Compensatory antiinflammatory response syndrome)。ARDS由于其复杂的发病机制导致病死率高，临床上对于该病尚无有效的治疗方法。近年来，许多研究发现干细胞拥有多向分化潜能，在应用于常规治疗难以解决的疑难复杂病症中获得了甚为满意的疗效。人羊膜上皮干细胞(hAESCs)是源自孕妇所娩出胎盘上的羊膜组织，具有广泛的分化潜能，研究表明hAESCs对急性肺损伤的修复作用是通过免疫调节功能实现的，但具体的作用机制尚不清楚。本课题拟通过构建急性肺损伤模型，通过体内外实验证实hAESCs对于肺损伤的修复作用，并尝试寻找到其相关的作用机制，为hAESCs应用于该疾病的治疗提供科学参考和实验依据。 实验方法： 1.hAESCs的分离、鉴定及安全性评价：分离和体外培养hAESCs，获取hAESCs来源的条件培养液(CM)；利用RT-PCR、免疫荧光和流式细胞技术对hAESCs进行鉴定，并对其分化潜能和免疫原性进行评价；对hAESCs体内外致瘤性进行分析；采用小动物活体成像仪观察hAESCs的组织分布。 2.hAESCs对小鼠急性肺损伤的保护作用研究：分离与体外培养人羊膜上皮干细胞(hAESCs)，制备细胞悬液及hAESCs来源的CM。建立经气道给予LPS诱导的小鼠急性肺损伤模型，设立正常对照组、LPS组、LPS+Cell组(尾静脉注射hAESCs)和LPS+CM组(尾静脉注射CM)，于造模后3天和7天分别检测各组小鼠肺泡灌洗液中的炎症细胞数；ELISA检测IL-6、TNF-α、IL-1β和IL-10水平；HE染色观察组织病理学改变和炎症细胞浸润；天狼星红染色检测肺部纤维化程度，评价hAESCs或CM对小鼠急性肺损伤的保护作用。 3.hAESCs或CM体外抗炎作用分析：体外培养人肺上皮细胞(BEAS2B)，建立hAESCs或CM与BEAS-2B共培养体系，LPS诱导体外炎症反应模型，荧光定量PCR检测LPS刺激后BEAS-2B中IL-6、TNF-α、IL-1β及IL-10 mRNA水平的改变，体外验证hAESCs或CM的抗炎作用。 实验结果： 1.hAESCs具有多分化潜能及较低的免疫原性，hAESCs在体内外均无致瘤性，主要在肺内聚集。 2.与模型组比较，尾静脉注射hAESCs及CM可显著降低肺泡灌洗液中白细胞数量，减少促炎因子IL-6、TNF-α、IL-1β释放，同时增加抗炎因子IL-10水平；hAESCs或CM注射可显著改善LPS诱导的肺部炎症和肺部损伤，肺损伤评分显著下降，同时可有效减轻小鼠肺部纤维化。 3.在体外炎症反应模型中，hAESCs或CM可显著抑制BEAS-2B中促炎基因IL-6、TNF-α及IL-1β的表达。 实验结论： 人羊膜上皮干细胞(hAESCs)对LPS诱导的小鼠急性肺损伤具有保护作用，这一保护作用可能与hAESCs调节肺部炎症反应相关。hAESCs具有多分化潜能和低免疫原性，安全性高，易于靶向于肺部，显示出在急性肺损伤治疗中应用的良好潜力。 关键词：急性肺损伤；人羊膜上皮干细胞；人肺上皮细胞；炎症

 Background and objective: Bronchial asthma is a common clinical respiratory disease, which is a kind of heterogeneous disease characterized by airway inflammation, airway remodeling and airway hyper-responsiveness, involving a variety of cells and cell components, such as airway inflammatory cells and structural cells.Hypoxia is believed to be a key factor in the progression of lung diseases, such as bronchial asthma, airway obstruction, and pulmonary hypertension. Hypoxia stimulates airway inflammation and airway remodeling, and induces the apoptosis of airway smooth muscle cell (ASMC) during airway remodeling. CD38 is a key enzyme for NAD⁺ degradation in vivo and plays an important role in regulating NAD⁺ levels and SIRT1 activity indispensable to NAD⁺ in cells. Evidences suggest that there are gender differences in the incidence and progression of lung diseases, and estrogen plays a crucial role in these pathological processes. Estrogen can affect smooth muscle contraction through CD38, but the specific molecular mechanism of its regulation is still unclear.This study aims to investigate the effect of CD38 deficiency on the apoptosis of mouse airway smooth muscle cells under hypoxic condition, and the regulation effect of estrogen on the CD38/SIRT1 signal axis, and to verify the protective effect of CD38 deficiency on asthma in mice in an overall animal model, providing an important target and experimental basis for the treatment of asthma and other respiratory diseases. Methods: 1.Estrogen (E2) regulation of CD38/SIRT1 expression in airway smooth muscle cells:isolation and in vitro culture of primary airway smooth muscle cells (ASMCs), treatment of ASMCs with different concentrations and different time of E2, detection of CD38 and SIRT1 by real-time PCR mRNA levels; Western blot analysis of CD38 and SIRT1 protein expression levels, clear dose- and time-dependent changes in E2 regulation of CD38 expression; comparing CD38, SIRT1, acetylated p53 (Ac-p53) and total p53 expression levels of wild-type (WT) and CD38 knockout (CD38 KO) in ASMCs, as well as the intervention of E2 on their expression. 2.Effect of CD38 on apoptosis of ASMCs under hypoxic conditions and intervention of E2: an in vitro hypoxic model was established, and the effect of E2 on CD38 and SIRT1 mRNA levels in ASMCs of WT and CD38 KO under hypoxic conditions was detected by real-time PCR; Western blot was used to detect the effect of E2 on CD38/SIRTl/p53 pathway after hypoxia stimulation; assayingapoptosisrelated protein Bax and Bcl-2 expression analysis and Caspase-3 activity by Hoechst33258 staining;clarifying the protective effect of CD38 deletion on hypoxiainduced apoptosis of ASMCs and the intervention of E2. 3.Molecular mechanism analysis of E2 regulation of apoptosis in ASMCs: observing of the effect of E2 on apoptosis of ASMCs in the presence of SIRT1-specific active agonist resveratrol (RSV); examining the effect of E2 on CD38 mRNA and protein expression in the presence of the estrogen receptor-specific inhibitor ICI 182,780 to determine whether E2 regulates CD38 expression via estrogen receptors; elucidating through the above experiments the molecular mechanism of E2 affecting apoptosis of ASMCs by regulating CD38/SIRT1 signaling. 4.Construction of the mouse asthma model induced by ovalbumin (OVA): comparison the degree of asthma in wild-type with smooth muscle CD38 KO mice; detecting of white blood cell count in alveolar lavage fluid of two groups of mice; HE staining was used to observe inflammatory cell infiltration and lung injury, and the protective effect of smooth muscle CD38 deletion on asthma in mice was verified at the overall animal level. Results: 1.The results of dose-effect experiments showed that the expression of CD38 mRNA and protein in ASMCs increased with the increase of E2 concentration, while the mRNA and protein expression of SIRT1 decreased. In time course experiments ,CD38 mRNA and protein expression continued to increase within 48 h, SIRT1 mRNA and the expression level of protein decreased at 24 h and partially recovered at 48 h, and the protein levels of CD38 and SIRT1 showed a significant negative correlation at 24 and 48 h. Therefore, pre-treatment with 10 nM of E2 for 48 h were selected for the subsequent experiments. 2.E2 treatment can increase the expression of CD38 protein in wild-type ASMCs, decrease the expression of SIRT1 protein, and promote the expression of acetylated p53 (Ac-p53). CD38 KO resulted in a significant increase in SIRT1 protein levels in ASMCs and a decrease in Ac-p53 protein levels. After E2 treatment, SIRT1 and Ac-p53 expression levels in ASMCs were unchanged. 3.Hypoxia treatment can significantly down-regulate the expression of CD38 and SIRT1 mRNA in wild-type ASMCs, increase the level of CD38 protein, decrease the expression of SIRT1 and increase the expression of Ac-p53. E2 can inhibit the down-regulation of CD38 mRNA expression induced by hypoxia, and then promote the down-regulation of SIRT1 mRNA expression; E2 can further promote CD38 protein expression, down-regulate SIRT1 protein level and promote p53 acetylation under hypoxia. CD38 KO can make the above effects of E2 disappear in ASMCs. 4.E2 treatment significantly increased hypoxia-induced apoptosis of wild-type ASMCs, increased Caspase-3 activity, and significantly increased Bax/Bcl-2 ratio; CD38 KO significantly inhibited apoptosis of ASMCs induced by hypoxia; SIRT1 agonist RSV can significantly inhibit E2-induced apoptosis of ASMCs. 5.Estrogen receptor inhibitors (ICI182, 780) significantly reduce the expression of CD38 mRNA and protein in ASMCs, and in the presence of ICI 182,780, counteract the role of E2 in promoting CD38 expression. 6.In the overall animal model, smooth muscle-specific knockout CD38 mice had decreased sensitivity to asthma, decreased airway hyperresponsiveness, and decreased leukopenia in bronchoalveolar lavage fluid compared with control mice; significant improvement in lung histopathology and lung inflammation. Conclusions: E2 up-regulates CD38 gene expression through the estrogen receptor, acting on the downstream SIRTl/p53 signaling pathway, resulting in decreased expression of SERT1, increased p53 acetylation, and promotion of hypoxia-induced apoptosis of ASMCs. This effect disappeared in ASMCs where CD38 deletion can eliminate E2. Smooth muscle-specific knockout of CD38 mice alleviates asthma symptoms and lung damage in mice. These results indicate that smooth muscle cell CD38 is a key target in the pathogenesis of asthma, and its regulation may provide new strategies for the treatment of respiratory diseases such as asthma. Keywords: asthma, CD38, 17β-estradiol, hypoxia, apoptosis Background and objective: Acute lung injury (ALI)/Acute Respiratory Distress Syndrome (ARDS) is the damage of pulmonary capillary endothelial cells and alveolar epithelial cell damage caused by non-cardiac diseases, such as severe pneumonia, aspiration pneumonia, severe sepsis, severe trauma, and shock, which result in diffuse pulmonary interstitial edema and alveolar edema, leading to acute hypoxic respiratory insufficiency or respiratory failure with an uncontrolled systemic inflammatory response syndrome (SIRS) and the compensatory anti-inflammatory response (CARS). Due to its complex pathogenesis, ARDS has a high mortality rate, and there is no effective clinic treatment for this disease. In recent years, many studies have found that stem cells have multiple differentiation potentials and have achieved satisfactory results in difficult and complicated diseases that are difficult to solve with conventional treatment. At present, studies have suggested that the repair effect of human amniotic membrane-derived epithelial stem cells (hAESCs) on lung injury is achieved by immunomodulation, but the specific mechanism is still unclear. This study intends to establish a model of acute lung injury, confirming the repair effect of hAESCs on lung injury in vitro and in vivo, and try to find its related mechanism, providing scientific reference and experimental basis for the application of hAESCs in the treatment of this disease. Methods: 1.Isolation, identification, and safety evaluation of hAESCs: hAESCs were isolated and cultured in vitro, and conditioned medium (CM) derived from hAESCs was obtained; hAESCs were identified by RT-PCR, immunofluorescence and flow cytometry, and their differentiation potential was identified, and the immunogenicity was evaluated; the tumorigenicity of hAESCs in vitro and in vivo was analyzed; the tissue distribution of hAESCs was observed using a small animal in vivo imager. 2.Protective effect of hAESCs on acute lung injury in mice: human amniotic epithelial stem cells (hAESCs) were isolated and cultured in vitro to prepare cell suspensions and CMs derived from hAESCs; establish a model of acute lung injury induced by LPS in the airway, establish normal control group, LPS group, LPS+Cell group (tail vein injection hAESCs) and LPS+CM group (tail vein injection CM);the number of inflammatory cells in alveolar lavage fluid of each group was detected at 3 and 7 days after model establishment; IL-6, TNF-α, IL-1β and IL-10 levels were detected by ELISA; histopathological changes and inflammatory cell infiltration were observed by HE staining; Sirius red staining to detect the degree of pulmonary fibrosis, evaluation of the protective effect of hAESCs or CM on acute lung injury in mice. 3.In vitro anti-inflammatory effect analysis of hAESCs or CM: human lung epithelial cells (BEAS-2B) were cultured in vitro, hAESCs or CM and BEAS-2B coculture system were established, LPS induced in vitro inflammatory response model,and real-time PCR was used to detect the changes of IL-6,TNF-α,IL-1βand IL10 mRNA levels in BEAS-2B after LPS stimulation,and the anti-inflammatory effects of hAESCs or CM were verified in vitro. Results: 1.hAESCs have multi-differentiation potential and low immunogenicity. hAESCs have no tumorigenicity in vivo and in vitro, mainly aggregating in the lung. 2.Compared with the model group, tail vein injection of hAESCs and CM can significantly reduce the number of white blood cells in alveolar lavage fluid, reduce the release of pro-inflammatory factors IL-6, TNF-α, IL-1β, and increase the level of anti-inflammatory factor IL-10; hAESCs and CM injection can significantly improve LPS-induced lung inflammation and lung injury, lung injury scores are significantly reduced, and can effectively reduce lung fibrosis in mice. 3.In the inflammatory response model, hAESCs or CM significantly inhibited the expression of pro-inflammatory genes IL-6, TNF-α and IL-1β in BEAS-2B. Conclusions: Human amniotic epithelial stem cells (hAESCs) have protective effects on LPS-induced acute lung injury in mice, and this protective effect may be related to hAESCs regulating lung inflammation. With multi-differentiation potential and low immunogenicity, hAESCs are highly safe, and easy to target in the lungs, showing good potential for use in the treatment of acute lung injury. Keywords: acute lung injury; human amnion epithelial stem cells; human lung epithelial cells; inflammation