构建高生物相容性干细胞心脏补片的研究

目的:去细胞化处理脐动脉构建生物支架,将小鼠脐带胶样组织间充质干细胞(MCS)种植到生物支架上构建心脏补片,移植到小鼠心肌梗死区域观察其生物相容性。方法:1联合应用胰酶和胶原蛋白酶处理小鼠脐带动脉组织构建生物支架;2体外分离并培养小鼠脐带MCS至第三代,种植到生物支架构建心脏补片;3移植到野生型小鼠皮下3天后观察免疫反应;4移植到小鼠心肌梗死区域,观察其与原位心肌的融合和血管新生。结果:脱细胞化处理后的脐带动脉组织在电镜观察下显示为富含多孔隙的纤维支架;荧光染色观察MSC种植到支架上深入生长到支架的内部并黏附生长;去细胞化处理未明显改变脐动脉组织的含水比例[(95.3±1)vs.(94.9±0.6),P>0.05];与对比组比较,去细胞化处理导致组织支架的断裂牵张力显著降低[(0.24±0.0)vs.(0.15±0.06)m Pa,P<0.05]。但是去细胞化处理未明显改变脐动脉组织的断裂变形率[(45±15)vs.(53±10)%,P>0.05];将裸支架和种植了干细胞的组织补片分别种植于C57野生小鼠的皮下,3日后切片染色,荧光显微镜下观察裸支架移植后局部CD+4淋巴细胞为(15±2.4)%,而干细胞补片内仅见少量CD+4的细胞浸润[(1±0.4)%,P<0.05]。将干细胞补片移植到小鼠急性心肌梗死区1周后,发现补片与原位心肌的融合性良好,补片内部有大量的新生血管生成。结论:成功构建心脏补片;移植到小鼠具有较高的生物相容性,并与原位心肌融合性好并有新生血管形成,为急性心肌梗死的干细胞治疗提供了实验依据。     

丝素蛋白/壳聚糖生物支架复合骨髓间质干细胞修复老年兔软骨缺损

目的 探讨丝素蛋白(SF)/壳聚糖(CS)生物支架复合诱导后骨髓间质干细胞(BMSCs)修复老年兔膝关节软骨缺损的可行性. 方法 分离培养及诱导BMSCs,将成功诱导后的BMSCs接种在SF-CS生物支架上构成修复体.54只16～18月龄兔,随机分为支架修复体组、单纯支架组和对照组,每组18只.采用右膝关节制备软骨缺损模型并植入支架.术后4、8、12周取材进行大体观察,组织学染色和改良Wakitani法组织学评分. 结果 SF-CS支架为相通性好的多孔结构,孔径平均151.72 μm,孔隙率为(92.72±4.78)％,吸水膨胀率为(141.10±6.87)％.BMSCs诱导后在SF-CS支架上生长良好,增殖活跃.12周时,支架修复体组软骨缺损基本修复,Ⅱ型胶原明显的阳性反应,生物材料基本吸收;单纯支架组以纤维样组织修复为主,Ⅱ型胶原阳性反应弱,未见支架残留;空白对照组修复不良;改良Wakitani评分显示支架修复体组优于单纯支架组和对照组(P＜0.05).结论 SF-CS生物支架可以作为BMSCs载体修复老年兔膝关节软骨缺损.     

PLGA—Ⅰ型胶原-壳聚糖复合人工硬脊膜生物相容性的研究

目的 观察聚乳酸-聚乙醇酸共聚物(PLGA)-Ⅰ型胶原-壳聚糖复合人工硬脊膜的生物相容性.方法 制作PLGA膜(膜Ⅰ)、PLGA-Ⅰ型胶原复合膜(膜Ⅱ)、PLGA-Ⅰ型胶原-壳聚糖(9∶ 1)复合膜(膜ⅢA)、PLGA-Ⅰ型胶原-壳聚糖(5∶ 5)复合膜(膜ⅢB),对其行接触角、吸水率测定及细胞毒性实验.结果 吸水率:膜Ⅰ＜膜ⅢB＜膜ⅢA＜膜Ⅱ,P均＜0.01;接触角:膜Ⅱ＜膜ⅢA ＜膜ⅢB＜膜Ⅰ,P均＜0.01;细胞毒性实验:第1天,各膜间OD值比较,P＞0.05;第3、7天,膜Ⅰ与膜Ⅱ、膜ⅢA,膜ⅢA与膜ⅢB比较,P均＜0.05.结论 PLGA膜经Ⅰ型胶原和壳聚糖改性后,可以促进细胞在膜上的黏附、贴壁能力.膜ⅢA在生物相容性方面基本符合人工硬脊膜的要求.     

泡球蚴蛋白促进脂肪间充质干细胞向肝细胞分化的研究北大核心CSCD

目的 探讨泡球蚴蛋白(Echinococcus multilocularis protein,EmP)促脂肪间充质干细胞(Adipose tissue Derived Mesenchymal Stem Cells,ADSCs)向肝细胞分化的作用。方法 体外分离人脂肪组织来源的ADSCs,第三代(P3)ADSCs分为对照组(HDM肝细胞诱导分化组)和Em蛋白处理组(HDM+EmP组),经无血清肝细胞诱导培养基培养7、14和21 d,检测对照组和EmP处理组肝细胞样细胞(iHep)百分比、糖原染色面积、肝细胞标志物白蛋白(Albumin,ALB)的蛋白及基因表达水平。Western-Blot法检测p-GSK-3β的表达情况。结果 P3代ADSCs具有成脂及成骨分化能力并表达干细胞标志物(CD29、CD90和CD105等)。在ADSCs向肝细胞诱导分化14 d,EmP处理组iHep细胞百分比显著高于对照组(P<0.05),EmP处理组糖原染色面积显著高于对照组(P<0.05)。免疫荧光染色结果显示两组细胞在诱导分化7 d开始表达ALB,荧光强度随诱导时间增加呈增强趋势。qPCR结果显示在诱导分化后第14 d,EmP处理组ALB表达水平显著高于对照组,两组间具有统计学差异(P<0.05)。Western-Blot结果显示,诱导分化第7 d和14 d,EmP处理组p-GSK-3β蛋白表达水平显著高于对照组(P<0.01和P<0.05)。结论 Em蛋白可能通过激活GSK-3β而具有促进ADSCs向肝细胞分化的作用,本研究为临床应用ADSCs移植治疗泡型棘球蚴病奠定了基础。     

C1q/TNF相关蛋白3减轻氧化应激诱导的间充质干细胞衰老

目的建立间充质干细胞(MSCs)衰老模型,探讨C1q/TNF相关蛋白3(CTRP3)对衰老MSCs的作用并初步探讨机制。方法用过氧化氢(H_2O_2)诱导小鼠MSCs衰老,CCK8实验和β-半乳糖苷酶染色验证模型的建立。外源性给予CTRP3处理MSCs 24 h,CCK8实验检测增殖活性,β-半乳糖苷酶染色检测MSCs衰老,分化诱导实验检测MSCs分化能力,Western blotting法检测MSCs凋亡。RT-PCR检测MSCs中衰老相关基因P16、P21以及抗衰老基因SIRT1的mRNA表达变化。采用GraphPad Prism 6进行统计分析。组间比较采用方差分析或LSD两两比较。结果 H_2O_2可抑制MSCs的增殖活性,且呈浓度依赖性。与正常对照组相比,200μmol/L H_2O_2作用4 h,细胞立体感消失,细胞形态不规则;CCK8染色结果显示细胞增殖活性显著下降;β-半乳糖苷酶阳性细胞百分比显著升高。继续CTRP3处理24 h后,衰老MSCs增殖和分化能力增强。Western blotting结果表明CTRP3可显著减低衰老MSCs的凋亡。RT-PCR检测发现CTRP3上调MSCs抗衰老基因SIRT1 mRNA表达,并且下调衰老相关基因P16、P21 mRNA的表达。结论 CTRP3可抑制MSCs衰老,SIRT1基因表达水平上调,及P16、P21基因表达水平下调可能是其重要机制。     

壳聚糖/肝素共价包被先天性心脏病介入封堵材料生物相容性的体外实验研究

目的体外评价壳聚糖/肝素(Chi/Hep)共价包被先天性心脏病介入封堵材料的生物相容性。方法在南京医科大学第一附属医院心血管内科于2005年11月至2006年10月,通过体外实验检测Chi/Hep共价包被镍钛合金片的血液相容性和细胞相容性。实验分成血液对照组、未包被组、基础包被组、Chi/Hep包被组,其中按肝素质量浓度又分为低(0.1mg.mL-1)、中(1mg.mL-1)、高(10mg.mL-1)3个剂量组。Chi/Hep共价交联于试验用镍钛金属表面,(1)血液相容性实验:通过血液溶血试验,动态人全血接触试验评价其红细胞毒性及抗栓性能,扫描电子显微镜观察镍钛金属表面血小板粘附、聚集。(2)细胞相容性:利用纤维连接蛋白、Ki67免疫荧光标记人脐静脉内皮细胞(HUVECs)观察其细胞毒性。结果(1)血液相容性实验:实验各组红细胞、白细胞、血小板计数均在正常范围内,且组间比较差异无显著性意义,各实验组溶血率均小于5%。Chi/Hep包被组部分凝血活酶活化时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT)均明显延长,血液对照组、未包被组及基础包被组两两比较差异无显著性意义,不同质量浓度肝素包被之间差异亦无显著性意义(APTT、PT方差均齐LSD、SNK法检验,TT方差不齐Dunnetts法检验P<0.05)。扫描电镜结果显示,Chi/Hep包被组金属表面少量血小板粘附。(2)细胞相容性实验:经过与HUVECs72h孵育,金属片边缘细胞生长、移行良好,无细胞变形;Fn免疫荧光标记HUVECs,发现不同包被镍钛金属片表面的HUVECs粘附顺序如下:基础包被组>未包被组>Chi/Hep组;Ki67免疫荧光标记HUVECs,随机双盲法计数细胞发现不同包被镍钛金属片表面的HUVECs增殖顺序如下:基础包被组=未包被组>Chi/Hep组(P<0.01)。结论体外观察Chi/Hep共价包被镍钛金属有良好抗血栓作用,但抑制内皮细胞粘附、增殖。     

Cloning of the silk fibroin gene and its flanking sequences.

Thirteen Escherichia coli clones containing the whole or a part of the fibroin gene (16 kilobases long) have been isolated. The starting material was DNA extracted from the posterior silk glands of Bombyx mori. Most clones were obtained from sheared DNA fragments linked by poly(dA)-poly(dT) joints to the plasmid pMB9. One of them includes the 5' end of the fibroin gene with a flanking sequence of 12 kilobases, and another includes the 3' end of the gene with a flanking sequence of about 1 kilobase. One clone was obtained by ligation, to pMB9, of a fragment generated by endodeoxy-ribonuclease EcoRI. This clone has a 21-kilobase insertion that probably includes the entire fibroin gene with flanking sequences at both ends. The cleavage sites for endodeoxyribonucleases EcoRI, HindIII, and BamHI have been established for the cloned sequences.     

Discontinuous translation of silk fibroin in a reticulocyte cell-free system and in intact silk gland cells.

Silk fibroin mRNA was translated in a rabbit reticulocyte cell-free system. Addition of tRNA from silk glands was essential for complete translation of the fibroin polypeptide. (Mr approximately 400,000). Synthesis of full-sized product took at least 85 min. In addition to full-size product, a large number of smaller polypeptides were observed upon analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Evidence is presented that these smaller polypeptides are growing fibroin chains that transiently accumulate as discrete size classes due to discontinuities in the translation process. These discontinuities, or pauses, occur at specific sites in the fibroin mRNA template. The relative duration of the pauses can be experimentally modulated by changing the source of the supplementary tRNA added to the in vitro system. Silk glands were incubated in organ culture under conditions where essentially exclusive labeling of newly synthesized fibroins was attained. Analysis in sodium dodecyl sulfate gels showed that the labeling pattern of nascent silk fibroins is similar to the pattern observed in the reticulocyte cell-free system. This result suggests that discontinuities or pauses in polypeptide chain elongation also occur in vivo under conditions of organ culture.     

In vitro and in vivo characterization of silk fibroin/gelatin composite scaffolds for liver tissue engineering

OBJECTIVE: To investigate the cytotoxicity of silk fibroin/gelatin (SF/G) composite scaffolds in vitro as well as their biocompatibility and degradation in vivo. METHODS: The proliferation and relative growth rate of human hepatic QZG cells grown on different blends of two‐dimensional (2‐D) SF/G scaffolds were assessed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Flow cytometry was used to evaluate apoptotic rate of QZG cells on different blends of 2‐D SF/G scaffolds. The effect of silk protein materials on cell growth was observed by scanning electron microscopy. Three‐dimensional (3‐D) SF/G scaffolds of three different ratios (diameter 10 mm, thickness 1 mm) were implanted into subcutaneous pockets on male Sprague–Dawley (SD) rats. On the 7th, 14th and 30th day post‐implantation, the rats were sacrificed. The scaffold area including the surrounding tissues was retrieved. Hematoxylin and eosin staining was performed for observation under a light microscope. RESULTS: Significant cell attachment and proliferation on the SF/G scaffolds were observed. As the increased gelatin concentration, SF/G scaffolds became more amenable to cell adhesion. After the subcutaneous implantation of the SF/G scaffolds in SD rats, immunological rejection tests showed only slight inflammation, measured by the presence of inflamed cells on day 7 and 14. By day 30, each scaffold had been completely infiltrated and organized by fibroblasts and inflamed cells. The greater the gelatin concentration in the scaffold, the faster the degradation rate. CONCLUSION: Composite SF/G scaffolds are a promising candidate matrix for implantable bio‐artificial livers.     

Segmented nanofibers of spider dragline silk: atomic force microscopy and single-molecule force spectroscopy

Despite its remarkable materials properties, the structure of spider dragline silk has remained unsolved. Results from two probe microscopy techniques provide new insights into the structure of spider dragline silk. A soluble synthetic protein from dragline silk spontaneously forms nanofibers, as observed by atomic force microscopy. These nanofibers have a segmented substructure. The segment length and amino acid sequence are consistent with a slab-like shape for individual silk protein molecules. The height and width of nanofiber segments suggest a stacking pattern of slab-like molecules in each nanofiber segment. This stacking pattern produces nano-crystals in an amorphous matrix, as observed previously by NMR and x-ray diffraction of spider dragline silk. The possible importance of nanofiber formation to native silk production is discussed. Force spectra for single molecules of the silk protein demonstrate that this protein unfolds through a number of rupture events, indicating a modular substructure within single silk protein molecules. A minimal unfolding module size is estimated to be around 14 nm, which corresponds to the extended length of a single repeated module, 38 amino acids long. The structure of this spider silk protein is distinctly different from the structures of other proteins that have been analyzed by single-molecule force spectroscopy, and the force spectra show correspondingly novel features.     

Ad-VEGF165转基因成纤维细胞修饰的再生丝素膜诱导血管形成效应及其分子机制研究

目的研究经腺病毒介导的人血管内皮生长因子165（VEGF165）转基因细胞修饰的再生丝素膜（SF）诱导血管形成活性及其分子机制。方法将WI-38人胚肺成纤维细胞分别培养在有（SFC组）或无（PBS组）再生丝素膜的24孔培养板上,24h后用带绿色荧光蛋白（GFP）基因的空载体腺病毒Ad-GFP（Ad-GFP组/SFCG组）或含有VEGF165目的基因的重组腺病毒Ad-VEGF165（Ad-VEGF165组/SFCV组）感染培养细胞。通过形态学观察和ELISA法检测各组细胞培养上清液中VEGF165目的基因表达及其对细胞自分泌的血管生成素-1（Ang-1）、碱性成纤维细胞生长因子（FGF2）、血小板衍化生长因子（PDGF）表达的影响,并用鸡胚绒毛尿囊膜（CAM）和在兔眼角膜上进行新生血管诱导实验。结果形态学观察发现WI-38细胞在再生丝素膜上不仅贴壁生长良好,而且易被Ad-GFP、Ad-VEGF165腺病毒感染,呈现强荧光。再生丝素膜利于Ad-VEGF165组中VEGF165目的基因在细胞中的高表达（P〈0.05）,而且还可使细胞自分泌的Ang-1表达水平明显升高（P〈0.05）,同时可维持细胞自分泌的FGF2及PDGF稳定表达。Ad-VEGF165组（SFCV组）具有促进CAM上血管生长活性和诱导兔眼角膜组织新生血管生成作用。结论 Ad-VEGF165感染WI-38成纤维细胞所修饰的再生丝素膜,具有诱导血管新生的功能。     

Adverse effects of permanent cardiac internal defibrillator patches on external defibrillation

At the time of left ventricular aneurysm resection, antiarrhythmic operations or other open-heart operative procedures in patients with ventricular dysrhythmia, permanent internal defibrillator patches may be inserted. Insertion of the energy source may be delayed due to its unavailability or to a desire for postoperative electrophysiologic study before its insertion. To assess the effects of permanent internal defibrillator patches on external defibrillation, 7 anesthetized calves were studied. Fibrillation-defibrillation studies were performed before and after insertion of permanent internal defibrillator patches (model L67, 27 cm2, Intec Systems), one on each ventricle. The values of percent successful defibrillation obtained before insertion of the patches, although much lower than values that would be expected in humans, are consistent with the results of an extensive earlier study involving this calf model. Similar values obtained after insertion of the patches are appreciably lower than the values obtained before implantation of the patches, and appreciably lower than the results predicted by the earlier study. A significant decrease in the percent of successful defibrillations (p less than 0.001) was observed for a shock intensity of approximately 400 J. Permanent internal cardiac defibrillator patches on the right and left ventricles reduce the probability of achieving successful defibrillation externally with unidirectional shocks. The wisdom of implanting permanent large internal cardiac defibrillator patches without the energy source is questioned.