重组沙门氏菌VNP20009-M对骨肉瘤的治疗作用

目的 研究携带甲硫氨酸酶基因的减毒沙门氏菌VNP20009-M对骨肉瘤的治疗作用及其机制。方法 将重组沙门氏菌VNP20009-M与骨肉瘤细胞MNNG-HOS共培养；骨肉瘤细胞MNNGHOS、U2OS及SaoS-2均高表达甲硫氨酸酶基因后探索细胞增殖、迁移及凋亡能力的变化；构建MNNG-HOS裸鼠皮下荷瘤模型，评价不同剂量的VNP20009-M在动物模型中的治疗效果。结果 通过质粒PCR验证甲硫氨酸酶基因只存在于目的菌株VNP20009-M中，且具有较高的甲硫氨酸酶活性，重组沙门氏菌构建成功。VNP20009-M显著诱导骨肉瘤细胞MNNG-HOS的凋亡。相比对照组，甲硫氨酸酶基因过表达的骨肉瘤细胞增殖、迁移能力被显著抑制。VNP20009-M治疗后小鼠皮下肿瘤生长被显著抑制。结论 VNP20009-M可诱导骨肉瘤细胞凋亡并抑制癌细胞增殖和迁移，可以作为一种新型高效的药物为临床提供新的治疗方式。     

自噬基因Atg5的缺失对精子成熟的影响

目的探讨自噬蛋白Atg5在维持附睾生理功能及精子的成熟过程中发挥的作用。方法通过基因敲除技术构建附睾头4-5段主细胞Atg5特异性敲除小鼠模型,根据基因型分为三组:对照组(野生型)、杂合敲除组以及纯合敲除组。8周龄时,做合笼实验并记录致孕率和产仔数,14周龄时通过精子动力分析仪检测小鼠精子活力和数量的变化,并通过病理组织染色观察小鼠附睾组织的形态学变化,以及通过Western blot和免疫荧光技术检测三组模型鼠中ATG5蛋白表达和自噬通路变化情况。结果与对照组、杂合敲除组相比,纯合敲除组小鼠的附睾头4-5段主细胞Atg5特异性敲除后,自噬标记蛋白LC3-Ⅰ以及p62得到积累,自噬过程成功阻断;但纯合敲除组小鼠的附睾组织形态、精子活力、精子数目、产仔率和致孕率均无变化。结论在正常条件下(无外界刺激),Atg5特异性敲除导致附睾头4-5段的自噬过程阻断后,对小鼠附睾的生理功能和精子成熟过程没有影响。     

TRPM8通道激活剂薄荷醇对肺动脉高压大鼠的治疗作用

目的 探讨瞬时受体电位M8(transient receptor potential melastatin-8,TRPM8)通道激活剂薄荷醇(menthol)对野百合碱(monocrotaline, MCT)诱导的肺动脉高压(pulmonary arterial hypertension, PAH)大鼠的治疗作用。方法 SPF级雄性Sprague-Dawley大鼠随机分为6组：正常对照组、MCT组、MCT+menthol(1 mg·kg^-1·d^-1)治疗组、MCT+menthol(2 mg·kg^-1·d^-1)治疗组、MCT+menthol(5 mg·kg^-1·d^-1)治疗组、MCT+menthol(10 mg·kg^-1·d^-1)治疗组。腹腔注射MCT(50 mg·kg^-1)构建PAH大鼠模型。造模1周后治疗组分别给予不同剂量的menthol灌胃治疗,持续2周。通过血流动力学检测、肺组织病理学...     

新型磷酸二酯酶5抑制剂CPD1对肺动脉高压大鼠血管收缩的影响

目的探讨新型磷酸二酯酶5抑制剂CPD1对肺动脉高压(pulmonary arterial hypertension,PAH)大鼠肺动脉和主动脉收缩效应的影响。方法一次性腹腔注射野百合碱(monocrotaline,MCT,50 mg·kg^-1),制备PAH大鼠模型,造模7 d后给予CPD1(10 mg·kg^-1·d^-1)灌胃治疗,持续14 d。通过血管环张力检测技术观察CPD1对MCT致PAH大鼠血管收缩效应的作用。结果成功制备PAH大鼠模型;CPD1治疗可显著降低PAH大鼠右心室收缩压和右心质量指数,改善肺小动脉内膜增生情况,抑制苯肾上腺素(phenylephrine,Phen)和内皮素-1(endothelin-1,ET-1)诱导的PAH大鼠肺动脉和主动脉的收缩效应,而对KCl诱导的血管收缩效应无影响。结论磷酸二酯酶5抑制剂CPD1干预能抑制PAH大鼠模型,其机制可能是CPD1抑制PAH大鼠非电压依赖性钙通道功能,引起血管收缩力降低、血管平滑肌增殖减弱和血管重塑减轻。     

新型磷酸二酯酶5抑制剂CPD1预防性治疗对博来霉素诱导肺纤维化大鼠的保护作用

目的 探讨新型磷酸二酯酶5抑制剂CPD1预防性治疗对博来霉素(bleomycin,BLM)诱导的肺纤维化大鼠肺组织病理结构损伤和肺间质纤维化的影响。方法 大鼠随机分为假手术组(n=10)、模型组(n=14)、CPD1治疗组(n=13)和尼达尼布阳性对照药治疗组(n=13)。模型组和治疗组大鼠左侧主支气管内缓慢滴注BLM(3 mg/kg),2 h后分别灌胃CPD1(20 mg·kg^–1·d^–1)、阳性对照药尼达尼布(50 mg·kg^–1·d^–1),持续2周。观察CPD1治疗对肺纤维化大鼠患侧肺组织病理结构损伤、胶原沉积、肺组织中纤维连接蛋白(fibronectin,Fn)、α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原蛋白(Collagen Ⅰ)及E-钙粘蛋白(E-Cadherin,E-Cad)的影响。并进一步检测CPD1干预对腺癌性人肺泡基底上皮细胞A549中转化生长因子-β1(transforming growth factor-β1,TGF-β1)、Smad...     

ω-3多不饱和脂肪酸促进1型糖尿病模型小鼠胰岛β细胞再生的作用研究

目的 探讨ω-3多不饱和脂肪酸(ω-3 PUFAs)促进胰岛α细胞向β细胞转分化的作用。方法 在小鼠胰岛α细胞系αTC1培养基中添加不同配比的ω-3 PUFAs, 4周后，利用荧光染色观察胰岛素和胰高血糖素的表达，并通过荧光定量PCR(qRT-PCR)检测胰岛α细胞和胰岛β细胞特异性基因表达情况。野生型(WT)组和mfat-1转基因组小鼠连续5天腹腔注射60 mg·kg^-1链脲佐霉素(streptozocin, STZ)。选取注射STZ 7周后的WT小鼠，给予97%EPA和75%鱼油(50%EPA和25%DHA)饮食干预，每周检测随机血糖。8周后，通过免疫荧光染色观察小鼠胰腺中胰岛β细胞再生情况。结果 在DHA与EPA混合液培养以及与单独EPA培养后，胰岛α细胞可分泌胰岛素，而对照组细胞没有胰岛素染色阳性；qRT-PCR结果显示，经过ω-3 PUFAs处理的细胞中，胰岛β细胞特异性基因明显上调。注射STZ后，mfat-1组和饮食干预组小鼠不仅随机血糖水平明显低于WT组，而且免疫荧光结果显示mfat-1组和饮食干预组小鼠胰岛中出现胰岛素和胰高血糖素共定位的细胞。结论...     

FKBP38蛋白对子宫内膜癌前期病变的影响及其机制研究北大核心CSCD

目的构建子宫特异敲除FKBP38小鼠模型,探究FKBP38对于小鼠子宫内膜癌前期病变的影响及其作用机制。方法通过胚胎注射法使小鼠FKBP38基因上携带loxP位点,构建转基因小鼠模型。在此基础上,以在子宫特异表达的孕酮受体启动子Pgr-Cre小鼠介导FKBP38条件性敲除,以获得FKBP38子宫特异敲除小鼠模型Pgr-Cre;FKBP38^(fl/fl)。通过PCR及Western blot对子宫特异敲除FKBP38小鼠进行鉴定;通过苏木精-伊红染色法(hematoxylin-eosin staining,HE)检测小鼠子宫敲除FKBP38后组织病理性变化;通过Western blot技术检测小鼠子宫敲除FKBP38后对mTOR通路的影响。结果小鼠子宫特异敲除FKBP38后子宫中FKBP38蛋白表达水平明显降低,与同年龄同窝小鼠相比,差异有统计学意义(P<0.01)。在己烯雌酚刺激下,小鼠18月龄时,子宫特异敲除FKBP38小鼠中16.7%(1/6)发生复杂性非典型性增生(complex atypical hyperplasia,CAH),33.3%(2/6)发生简单性增生(simple hyperplasia,SH)。此外,子宫特异敲除FKBP38小鼠子宫中AKT、S6及4E-BP-1蛋白的磷酸化水平明显升高。结论子宫特异敲除FKBP38小鼠子宫中FKBP38蛋白明显降低,表明小鼠子宫特异敲除模型构建成功。此外,子宫特异敲除FKBP38后,小鼠发生SH以及CAH,表明FKBP38可能与子宫内膜癌癌前病变相关。这可能与FKBP38调控mTOR通路关键性蛋白Akt、S6及4E-BP-1蛋白的磷酸化相关。     

Effect of a novel phosphodiesterase type 5 inhibitor,CPD1,on unilateral renal interstitial fibrosis caused by ureteric obstruction in mice北大核心CSCD

Aim To investigate the effects of CPD1,a novel phosphodiesterase 5 inhibitor,on renal pathological phenotype and fibrotic protein expression in renal fibrosis model mice. Methods Male C57BL/6 J mice were divided into three groups randomly(sham group,UUO group and UUO+CPD1 group). Unilateral ureteric obstruction model was constructed by surgery,and CPD1(5 mg·kg-1·d-1)was administered by intragastric administration two hours after the modeling for seven days. HE and Sirius Red staining were used to observe the distribution of tissue structural lesions and fibrosis. Immunohistochemical staining and Western blot were used to detect the expression of fibronectin(FN),α-SMA,collagen-I and kidney injury molecule-1(Kim-1). Results Compared with sham operation group,the renal tubules of mice were dilated and accompanied by a large amount of inflammatory infiltration. Moreover,the expressions of FN,α-SMA,collagen-I and Kim-1 proteins increased significantly(P<0.05)in UUO group. CPD1 treatment improved the kidney structure and decreased the expression of collagen fibers. Furthermore,CPD1 inhibited the expression of FN,α-SMA,collagen-I and Kim-1 markedly(P<0.05). Conclusions Phosphodiesterase 5 inhibitor CPD1 alleviates the progression of renal fibrosis induced by unilateral ureteral obstruction through down-regulating ECM deposition in the extracellular matrix and expression of Kim-1. The specific mechanism remains to be further studied. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Effect of a novel phosphodiesterase type 5 inhibitor,CPD1, on carbon tetrachloride-induced liver fibrosis in mice北大核心CSCD

Aim To investigate the effects of CPD1, a novel phosphodiesterase 5 inhibitor, on liver pathological phenotype and hepatic stellate cells (HSCs) activation in hepatic fibrosis model mice caused by carbon tetrachloride ( CCl4). Methods Male C57BL/6 J mice were divided into four groups randomly ( control group, CCl4group, CCl4+ CPD1 group and CCl4+ tadalafil group) . Hepatic fibrosis model was construc¬ted by intraperitoneal injection of CCl4( twice a week) . Four weeks after CCl4injection, the mice were treated with CPD1 (2 mg kg-1• d-1) , or Tadalafil (10 mg • kg-1• d-1) by intragastric administration, respec¬tively, for four weeks. Hematoxylin-eosin staining and Sirius Red staining were used to observe the distribu¬tion of liver tissue structural lesions and fibrosis. Im-munohistochemical staining was used to detect the ex¬pression of a-smooth muscle actin ( a-SMA) and fi-bronectin. Results Compared with control group, the liver tissue structure was seriously damaged in CCl4group with many hepatocytes necrosis and inflammatory cell infiltration, indicating that liver injury occurred in the CCl4-induced hepatic fibrosis model mice. Moreo¬ver, the expressions of a-SMA increased significantly in CCl4group. Compared to CCl4group, the liver tissue damage was significantly improved in PDE5 inhibitors group,most notably, CPD1 had a better curative effect than tadalafil did. Furthermore, CPD1 inhibited the ex¬pression of a-SMA markedly and reduced the expres-sion of ECM-related proteins induced by transforming growth factor pi ( TGF-f31 ) in Lieming Xu-2 ( LX-2 ) cells. Conclusions Phosphodiesterase 5 inhibitor CPD1 strongly alleviates CCl4-induced hepatic damage by inhibiting the activation of HSCs and expression of collagen fibers. © 2023 Publication Centre of Anhui Medical University. All rights reserved.