目的:制备甲基丙烯酰化明胶/丝胶(GelMA/SS)互穿网络水凝胶支架，构建胰岛(Islets)仿生微环境。方法:从6周龄美国癌症研究所(ICR)小鼠提取胰岛并经双硫腙染色鉴定。设置组织培养板(TCP)培养为对照组，单独GelMA以及不同质量比GelMA/SS(1∶1、1∶2、1∶4、1∶8)培养为实验组，分别记为G1S1、G1S2、G1S4和G1S8。扫描电镜下观察微观形态，流变学评价支架黏弹性，支架浸提液培养L929细胞评估生物相容性。钙黄绿素-乙酰甲氧基甲酯(AM)/碘化丙锭染色评价胰岛存活，活性氧(ROS)试剂盒评价拮抗ROS产生情况，酶联免疫吸附试验(ELISA)检测葡萄糖刺激胰岛素释放量。两组数据间比较采用 t检验，多组数据组间比较采用方差分析。 结果:分离胰岛呈类圆形；水凝胶弹性随丝胶含量增加而下降，至GelMA/SS＝1∶8时已不能成胶(G">G’)；噻唑蓝(MTT)结果显示实验组均具有良好的生物相容性，且G1S4组显示出最高的细胞活力[(120.67±3.33)%]和显著拮抗ROS产生能力(5.18±0.48)；活/死细胞染色显示实验组能够促进胰岛存活；葡萄糖刺激胰岛素释放(GSIS)结果显示在低糖刺激下G1S4具有最高的胰岛素分泌量[(7.73±0.52) μg/L]，而高糖刺激下实验组与对照组差异无统计学意义( F＝0.395， P>0.05)。 结论:GelMA/SS水凝胶具有良好的生物相容性和葡萄糖刺激响应能力，是构建仿生微环境，用于胰岛培养及移植的理想载体。

目的:探讨丝胶对糖尿病(DM)合并动脉粥样硬化(AS)大鼠认知障碍的影响.方法:采用链脲佐菌素(STZ)+维生素D3建立DM+AS大鼠模型;48只DM+AS大鼠随机分为模型组,400 mg/kg、800 mg/kg丝胶组和抑制剂组(800 mg/kg丝胶+ LY294002组),每组12只,另选12只大鼠设为对照组;丝胶组和800 mg/kg丝胶+LY294002组分别给予相应剂量丝胶溶液灌胃42 d,对照组和模型组给予等容量蒸馏水;800 mg/kg丝胶+LY294002组于实验结束前1周侧脑室注射PI3K/AKT抑制剂LY294002溶液;实验结束用Morris水迷宫检测大鼠学习记忆能力,并检测大鼠糖耐量;酶联免疫吸附试验(ELISA)法检测大鼠血清AS指标[C反应蛋白(CRP)、基质金属蛋白酶-9(MMP-9)]及海马炎症因子[白介素(IL)-1β、IL-18、肿瘤坏死因子-α(TNF-α)];苏木精—伊红(HE)染色观察大鼠胸主动脉和海马组织形态,免疫荧光法检测海马小胶质细胞活化标志物Iba-1荧光强度;蛋白质免疫印迹(western blotting)法检测PI3K/AKT通路蛋白表达量.结果:与对照组相比,模型组大鼠血糖值、血清CRP和MMP-9含量显著增加(均P＜0.05),胸主动脉受损;游泳轨迹更加复杂,逃避潜伏期延长,穿越平台次数减少(均P＜0.05);海马损伤,IL-18、IL-1β、TNF-α含量升高,Iba-1荧光强度升高(均P＜0.05),PI3K、p-PI3K和p-AKT表达降低(均P＜0.05).与模型组相比,丝胶组大鼠血糖降低,血清CRP和MMP-9含量下降(均P＜0.05),胸主动脉受损得到缓解;大鼠海马损伤改善,炎症因子含量下降,Iba-1荧光强度下降(均P＜0.05);海马PI3K、p-PI3K和p-AKT表达升高,大鼠学习记忆能力改善(均P＜0.05).抑制PI3K/AKT后,丝胶对海马损伤的改善作用、抗炎作用及大鼠学习记忆的改善作用均下降(均P＜0.05).结论:丝胶可能通过PI3K/AKT通路抑制神经炎症改善DM+AS大鼠认知障碍.

背景 内质网应激(ERS)特异性caspase-12凋亡途径在细胞凋亡过程中发挥重要作用,细胞凋亡是糖尿病视网膜神经退行性病变的重要特征.研究证实,丝胶对视网膜神经细胞的凋亡具有保护作用,但其对糖尿病视网膜病变(DR)过程中与caspase-12凋亡途径相关的视网膜神经细胞是否具有保护作用仍有待研究证实. 目的 探讨丝胶是否能够通过影响ERS特异性caspase-12凋亡途径对糖尿病大鼠视网膜细胞凋亡发挥抑制作用.方法 采用高脂高糖饲料喂养联合链脲佐菌素(STZ)连续腹腔内注射3d对30只2～3月龄SPF级SD大鼠制备糖尿病模型,以大鼠空腹血糖≥16.7 mmol/L及出现多饮、多食、多尿特点为造模成功.将24只造模成功的糖尿病模型大鼠按照计算机数字随机分配法分为丝胶治疗组和糖尿病模型组,每组12只,另取同周龄12只正常大鼠作为正常对照组.丝胶治疗组大鼠于成模后给予丝胶溶液2.4 g/(kg·d)灌胃,连续35 d.各组大鼠采用过量麻醉法处死并制备视网膜标本,采用TUNEL法检测并计算各组大鼠视网膜神经细胞的凋亡指数(AI);分别采用Western blot法和逆转录PCR技术检测大鼠视网膜中ERS标志物葡萄糖调节蛋白78(GRP78)、ERS特异性caspase-12凋亡途径和easpase-3蛋白及其mRNA的相对表达量,并对各组检测结果进行比较. 结果 大鼠糖尿病造模成功率为80％(24/36).正常对照组、糖尿病模型组和丝胶治疗组大鼠均可见视网膜神经细胞凋亡,阳性产物主要位于视网膜神经节细胞(RGCs)层和内核层,AI分别为0.028 4±0.002 3、0.215 1±0.020 9和0.1150±0.018 1,糖尿病模型组大鼠视网膜AI明显高于对照组,丝胶治疗组AI明显低于糖尿病模型组,差异均有统计学意义(均P＜0.05).丝胶治疗组大鼠视网膜中GRP78、caspase-12和easpase-3蛋白的相对表达量分别为0.523±0.029、1.118±0.051和0.315±0.024,较糖尿病模型组的0.924±0.039、1.468±0.037和0.554±0.032均明显下降,差异均有统计学意义(均P＜0.05),丝胶治疗组大鼠视网膜中GRP78、easpase-12和caspase-3 mRNA的相对表达量分别为0.816±0.022、0.216±0.023和0.322±0.022,较糖尿病模型组的1.218±0.033、0.407±0.012和0.531±0.029均明显下降,差异均有统计学意义(均P＜0.05). 结论 丝胶可以通过下调糖尿病模型大鼠视网膜中GRP78、caspase-12和caspase-3的表达改善内质网ERS,减少视网膜神经细胞的凋亡.

目的 探索丝胶蛋白对胃癌MKN45细胞增殖活性的影响及作用机制.方法 用LC3双荧光自噬病毒转染胃癌MKN45细胞,用嘌呤霉素筛选LC3双荧光自噬病毒的MKN45稳转株.实验分为3组,空白对照组(Blank)、丝胶蛋白阳性组(Sericin)、丝胶蛋白加自噬抑制剂组(Sericin+3-MA).培育48 h后用cck-8试剂测定细胞增殖活性,根据所测数据得出丝胶蛋白半数致死浓度IC50,按此浓度处理细胞并培育48h,用流式细胞仪分别检测凋亡、周期,电镜检测细胞自噬,Western blotting检测LC3、p62、Beclin蛋白表达.将种植胃癌的裸鼠分为2组,即对照组(Saline)、丝胶蛋白阳性组(Sericin),每组5只;分别注射生理盐水和丝胶蛋白,测量肿瘤体积和质量.结果 丝胶蛋白阳性组(Sericin)与空白对照组(Blank)相比,MKN45细胞增殖活性明显受到抑制,且细胞凋亡增加(P<0.01),细胞阻滞于G2/M期(P<0.01).相对于空白组,丝胶蛋白处理后细胞在电镜观察到自噬小体明显增多;Western blotting检测到LC3-2表达上调,Beclin表达增加,p62表达逐渐下调;丝胶蛋白加自噬抑制剂组(Sericin+3-MA)实验结果则介于两者之间.动物实验中,丝胶蛋白阳性组(Sericin)与对照组(Saline)相比,肿瘤体积和质量有显著下降.结论 丝胶蛋白可通过调控胃癌MKN45细胞自噬影响细胞增殖活性.

目的 探讨丝胶对糖尿病大鼠视网膜氧化应激和微炎症状态的改善作用.方法 采用高脂高糖饲料喂养联合链脲佐菌素腹腔注射法建立糖尿病大鼠模型,将24只成模大鼠随机分为丝胶治疗组和糖尿病模型组,每组12只,另取同周龄12只正常大鼠作为正常对照组,成模后丝胶治疗组给予丝胶溶液空腹灌胃、糖尿病模型组及正常对照组给予等体积生理盐水灌胃每天1次,共35 d.药物干预后,检测3组视网膜组织中丙二醛(malondialdehyde,MDA)、还原型谷胱甘肽(glutathione,GSH)的含量;采用Western blot法检测视网膜核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)、血红素氧合酶1(heme oxygenase 1,HO-1)、核因子-κB (nuclear factor kappa-B,NF-κB)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)蛋白的表达,苏木素-伊红染色法观察视网膜形态结构.结果 各指标三组间整体比较差异显著(均为P＜0.01).丝胶治疗组大鼠视网膜中MDA含量、NF-κB和TNF-oα蛋白表达水平分别为(4.145±0.282) mmol·gprot-1、0.232±0.027和0.761±0.058,较糖尿病模型组的(6.813±0.446)mmol·gprot-1、0.334±0.024、0.994±0.084均显著降低(均为P＜0.05).丝胶治疗组大鼠视网膜中还原型GSH含量、Nrf2和HO-1蛋白表达水平分别为(78.518±4.317) mg·gprot-1、0.591±0.054和0.954±0.091,较糖尿病模型组的(59.890±5.932) mg ·gprot-1、0.351±0.044、0.585±0.054均显著升高(均为P＜0.05).糖尿病模型组大鼠视网膜各层细胞排列紊乱,内界膜肿胀,神经节细胞可见空泡、水肿样改变,丝胶治疗组大鼠视网膜各层细胞形态较规则、排列轻度紊乱,病理变化较糖尿病模型组明显减轻.结论 丝胶可改善糖尿病视网膜氧化应激和炎症介质的损伤,延缓糖尿病视网膜病变发展.

目的:探讨丝胶通过调节c-Jun氨基末端激酶(JNK)信号通路抑制糖尿病大鼠视网膜神经细胞凋亡的作用.方法:高脂高糖饲料喂养联合链脲佐菌素腹腔内注射制备糖尿病大鼠模型,SD 大鼠随机分为丝胶治疗组、糖尿病模型组和正常对照组.丝胶治疗组大鼠于成模后给予丝胶溶液2.4g· kg-1·d-1灌胃,连续35d.各组大鼠采用过量麻醉法处死并制备视网膜标本;采用Western blot法检测大鼠视网膜中磷酸化c-Jun 氨基末端激酶(p-JNK)、细胞色素 C(Cyt-c)和半胱氨酸天冬氨酸激酶-9 (caspase-9)蛋白的相对表达量,分光光度法检测视网膜组织中半胱氨酸天冬氨酸激酶-3(caspase-3)活性,采用TUNEL法检测并计算大鼠视网膜神经细胞的凋亡指数(AI),并对各组检测结果进行比较.结果:丝胶治疗组大鼠视网膜中p-JNK、Cyt-c、caspase-9蛋白的相对表达量,caspase-3活性,AI较糖尿病模型组均明显下降,差异均有统计学意义(均 P<0.05).结论:丝胶可以通过调节 JNK 信号通路,减少DR视网膜神经细胞的凋亡.

Silkworm cocoon was recorded to cure carbuncle in the Compendium of Materia Medica.Previous studies have demonstrated that the supplemental silk protein sericin exhibits anticancer activity.In the present study,we investigated the effects of silk fibroin peptide (SFP) extracted from silkworm cocoons against human lung cancer cells in vitro and in vivo and its possible anticancer mechanisms.SFP that we prepared had high content of glycine (～ 30％) and showed a molecular weight of ～ 10 kDa.Intragastric administration of SFP (30 g/kg/d) for 14 days did not affect the weights,vital signs,routine blood indices,and blood biochemical parameters in mice.MTT assay showed that SFP dose-dependently inhibited the growth of human lung cancer A549 and H460 cells in vitro with ICs0 values of 9.921 and 9.083 mg/mL,respectively.SFP also dose-dependently suppressed the clonogenic activity of the two cell lines.In lung cancer H460 xenograft mice,intraperitoneal injection of SFP (200 or 500 mg/kg/d) for 40 days significantly suppressed the tumor growth,but did not induce significant changes in the body weight.We further examined the effects of SFP on cell cycle and apoptosis in H460 cells using flow cytometry,which revealed that SFP-induced cell cycle arrest at the S phase,and then promoted cell apoptosis.We demonstrated that SFP (20-50 mg/mL) dose-dependently downregulates Bcl-2 protein expression and upregulates Bax protein in H460 cells during cell apoptosis.The results suggest that SFP should be studied further as a novel therapeutic agent for the treatment of lung cancer.

Lepidopteran insects produce cocoons with unique properties.The cocoons are made of silk produced in the larval tissue silk gland and our understanding of the silk genes is still very limited.Here,we investigated silk genes in the bagworm moth Eumeta variegata,a species that has recently been found to produce extraordinarily strong and tough silk.Using short-read transcriptomic analysis,we identified a partial sequence of the fibroin heavy chain gene and its product was found to have a C-terminal structure that is conserved within nonsaturniid species.This is in accordance with the presence of fibroin light chain/fibrohexamerin genes and it is suggested that the bagworm moth is pro-ducing silk composed of fibroin ternary complex.This indicates that the fibroin structure has been evolutionarily conserved longer than previously thought.Other than fibroins we identified candidates for sericin genes,expressed strongly in the middle region of the silk gland and encoding serine-rich proteins,and other silk genes,that are structurally con-served with other lepidopteran homologues.The bagworm moth is thus considered to be producing conventional lepidopteran type of silk.We further found a number of genes ex-pressed in a specific region of the silk gland and some genes showed conserved expression with Bombyx mori counterparts.This is the first study allowing comprehensive silk gene identification and expression analysis in the lepidopteran Psychidae family and should contribute to the understanding of silk gene evolution as well as to the development of novel types of silk.

Cardiac valve replacement is an effective method to treat valvular heart disease.Artificial valves used routinely in clinic still have defects.In our study,we explored a novel method to modify the performance of Decellularized Heart Valve(DHV)scaffold.The decellularized porcine aortic valve was prepared using sequential hydrophile and lipophile solubilization method.The sericin was extracted from silk fibroin-deficient silkworm cocoon by lithium bromide method.First,DHV was immersed in sericin solution to produce the sericin-DHV composite scaffold.Then,we modified the DHV by mak-ing a Polydopamine(PDA)coating on the DHV first and then binding the sericin.The physical properties and biological compatibility of our composite scaffold were assessed in vitro and in vivo.Sericin were successfully prepared,combined to DHV and improved its biocompatibility.PDA coating further promoted the combination of sericin on DHV and improved the physical properties of scaffolds.The decay rate of our modified valve scaffold was decreased in vivo and it showed good compatibility with blood.In conclusion,our modification improved the physical properties and biocompatibility of the valve scaffold.The combination of PDA and sericin promoted the recellularization of decellularized valves,showing great potential to be a novel artificial valve.