人发角蛋白人工腱材料体内降解及生物相容性研究

目的研究人发角蛋白人工腱(humanhairkeratinartificaltendon,HHKAT)材料在体内的可降解性及其生物相容性.方法对12只日本大耳白兔随机分组,在脊旁肌埋藏不同处理时间的人发角蛋白人工腱试件F及Z,用正常人发O做对照,分别在2、6、12、24周取材,观察人发角蛋白的降解吸收过程及其周围的组织反应.结果动物植入实验中发现不同时间处理的材料其降解速度不同,其中降解最快的F组在24周已完全吸收,而Z组在24周只有部分降解,O组未见降解.HHKAT及人发在肌肉组织内无明显的炎症排斥反应,随着HHKAT材料的降解吸收,其周围的组织反应逐渐降低.结论本研究表明人发角蛋白人工腱材料具有良好的生物相容性,在体内能够被降解吸收,可根据不同的需要调节其降解速度,是良好的肌腱替代材料.     

适量运动对大鼠跟腱复健有促进效应

本研究为比较不同运动量对大鼠跟腱修复的影响,采用54只8周大,体重200～250 g的SD雄性大鼠为实验对象。根据随机分组方式平均分成3组,每组18只:控制组、行走15 min组(15分组)、行走30 min组(30分组)。将54大鼠的右后脚跟腱切断,控制组不予任何运动干预,其余两组在手术处理后的第5天起,进行运动干预。每天运动1次,运动时间分别为15 min和30 min;前2天行走速度4.0 m/min;之后12.0m/min。分别在第5天、第12天、第19天、第28天评估跟腱功能指数(achilles function index, AFI);手术后第28天处死大鼠,取出跟腱,观察外观、胶原蛋白生长情况、断裂强度(最大破坏力量)(breaking strength, BS)、拉伸应力(tensile stress, TS)及恢复指数(recovery index, RI)。AFI值:结果显示,第12天30分组显著优于控制组和15分组,有统计学差异(p0.05);第28天,30分组显著优于控制组,有统计学差异(p0.05);而15分组无明显差异,不具有统计学意义(p0.05)。跟腱直径与胶原蛋白含量方面:各组受伤肢直径均显著大于自身正常肢,胶原蛋白含量更高;在各组小鼠的受伤肢中,30分组的跟腱直径显著小于控制组,差异具有统计学意义(p0.05);参与行走运动的组别受伤跟腱中胶原蛋白排列较整齐。断裂强度方面:控制组受伤肢显著大于正常肢,差异具有统计学意义(p0.05);受伤肢间均未达显著差异(p0.05)。拉伸应力方面:控制组受伤肢的跟腱断裂强度显著小于正常肢,差异具有统计学意义(p0.05);各组受伤肢间跟腱断裂强度均未有显著差异(p0.05)。恢复指数方面:3组间均未达显著差异,无统计学差异(p0.05)。本研究表明,在设定的模式下进行走路运动,可以促进新生胶原蛋白排列整齐,从而提升修复后的跟腱功能;虽然增加运动量并不能增强断裂强度,也不能提升拉伸应力,但可能会使大鼠提早适应受伤后的行动过程。     

柱层析法纯化人血纤维蛋白原及其药理功能研究

目的:探讨层析法新工艺和原有的低温乙醇工艺制备纤维蛋白原在大鼠跟腱断裂模型中促恢复的效果差异。方法:构建大鼠跟腱部位断裂模型,将其分为空白组(未剪跟腱)、模型组(跟腱断裂未给药)、实验组(自制纤维蛋白原2 mg/mL)与对照组(市售纤维蛋白原2 mg/mL),观察各组大鼠在手术后三周跟腱部位的最大滑动距离、弹性模量和最大抗拉力差异。结果:采用冷沉淀溶解、酸沉除杂、S/D灭活病毒、MacroCap Q柱层析、过滤等流程可从人血浆冷沉淀组分Ⅰ中成功分离纯度为90.9%纤维蛋白原。试验中构建的大鼠跟腱部位断裂模型无感染、且均存活至试验终止。生物力学结果显示,四组大鼠跟腱在最大滑行距离上无明显统计学差异(P0.05);在跟腱部位弹性模量及最大抗压力比较上,实验组及对照组均优于模型组,但与空白组仍有一定差距,且差异均具有统计学意义(P0.05)。结论:采用柱层析法分离人纤维蛋白原,不仅能有效提高分离效率,减少蛋白损失,还可增强纤维蛋白原在断裂跟腱中的促恢复效果。     

脂肪基质血管组分对兔跟腱损伤修复的研究

目的 探讨肌腱内注射脂肪来源的基质血管组分(SVF)对兔跟腱损伤的修复作用.方法 采用前列腺素E2诱导兔跟腱损伤模型,造模组注射前列腺素E2,空白对照组注射等剂量的生理盐水,超声检查、HE染色和Masson染色评估造模情况.损伤模型成模后,将造模组随机分为2组,SVF治疗组于跟腱局部注射兔子来源的脂肪SVF,模型对照组...     

外源DNA在体内的吸收与降解

外源ＤＮＡ在动物体内的吸收与降解研究已经受到了关注[1、2 ] 。由于基因治疗与ＤＮＡ疫苗的飞速发展 ,经常要通过一定的方式将外源的ＤＮＡ导入到动物体 ,然而对于它们的吸收与降解机制到目前为止仍然不是十分清楚。用基因治疗疾病时 ,须解决外源基因在动物体内的吸收与降解问题 ,尤其是大量外源ＤＮＡ质粒在动物体内的吸收与降解问题。外源ＤＮＡ的吸收与降解研究已成为一个热点 ,目前主要集中在肠胃道吸收降解和细胞吸收降解两个方面。1 .外源ＤＮＡ在肠胃道中的吸收与降解最有可能让外源ＤＮＡ进入动物体的就是肠胃道 ,在每天的进食中 …     

同种异体肌腱不同时段深低温保存对肌腱愈合影响的实验研究

目的:探讨深低温(-80℃)保存异体肌腱的最佳时间段.方法:体重250-300g的雌雄不分的SD大鼠144只,随机分为Ⅰ、Ⅱ两组.每组72只.Ⅰ组为供腱组,提供144条供移植用的肌腱.Ⅱ组又随机分为A、B、C、D四组,每组18只,分别接受由Ⅰ组提供的经深低温保存1天、10天、1个月、3个月的肌腱.术后3天时、10天时分别从A组、B组、C组、D组随机抽取3只,近端自肌肉肌腱移行处,远端自跟腱附着处切断(包括近、远端吻合口),进行大体观察,组织学观察;术后4W、8W时分别从A组、B组、C组、D组随机抽取6只,进行大体观察,组织学观察,最大载荷测定.结果:保存10天以上的三个时间段的肌腱移植后和保存1天的比较,保存10天以上的肌腱术后粘连较轻,肌腱抗拉力强度降低.保存10天以上三个时间段的肌腱移植后大体观察、组织学观察无明显差异;最大载荷测定,结果:分析无统计学意义(P＞0.05).结论:肌腱在深低温(-80℃)环境中长时间保存后,其用于功能修复,愈合情况无明显差别.     

节肢动物肠道及腹神经索的降解实验*

节肢动物的内部器官组织, 如消化系统和神经系统等精细结构在寒武纪化石中的保存特征对探索早期生命和地球环境的演化具有重要意义。近年来, 其原始形态与保存机理被广泛讨论, 说明此类型软躯体组织的保存行为还需多方面的实验和理论探索。实验埋藏学作为埋藏学的分支, 为解释生物化石化过程中解剖结构的变化及相应特异埋藏化石库的形成提供了重要理论支撑和实验技术手段。本文以节肢动物南美白对虾的肠道和腹神经索为材料, 在不同温度及组合条件下进行埋藏学实验, 以观察此类生物软组织的降解过程。实验结果显示, 肠道和腹神经索在不同温度下的降解趋势相似, 但降解速率不同: 肠道和腹神经索在25℃条件下的降解速率和降解程度明显大于其在10℃条件。同一温度条件下, 肠道与腹神经索的降解程度存在早期差异; 10℃条件下, 反应产生的微生物膜形态及规模有所不同, 肠道-腹神经索组合产生的微生物膜规模远大于肠道、腹神经索的单独处理组, 且难以辨认。由此可见, 温度是影响肠道和腹神经索降解速度和程度的重要因素。此外, 微生物在生物内部器官组织降解过程中起到了重要的生化作用。生物器官组织的降解过程, 以及不同条件下微生物膜的形成对化石的形态特征尤其是内部精细结构的保存解释具有重要影响。     

β-淀粉样蛋白的体内清除机制

阿尔茨海默病(alzheimer disease,AD)是一种神经退行性疾病,β-淀粉样蛋白(amyloid-β,Aβ)被认为是其发病的中心分子.体内Aβ产生和清除的平衡在阿尔茨海默病的病理过程中扮演了重要的角色.人体清除Aβ的机制包括多种途径:通过血脑屏障、血脑脊液屏障等转运出脑;在脑脊液及血液等外周系统降解;在脑内通过水解等方式清除.细胞外Aβ既可在胞外通过水解酶的降解作用被降解,也可被吞噬入细胞后通过自噬作用、泛素-蛋白酶体途径被最终水解.能够增强体内固有的Aβ清除机制的药物和方法将对AD的治疗起到积极作用.     

外源DNA在动物体内的吸收代谢

综述了外源DNA在动物体内组织和细胞的代谢命运。外源DNA经核酸酶不完全降解后可以通过胃肠道被哺乳动物吸收,外源DNA片段能够被哺乳动物细胞吸收并整合到宿主细胞基因组中。     

胶原/纤维蛋白止血效果观察

目的研究胶原/纤维蛋白对新西兰兔的止血作用,并与胶原蛋白海绵止血效果作比较。方法选用胶原/纤维蛋白止血贴,对新西兰兔耳部动、静脉出血、耳表创面、股动脉割伤、肝损伤、体表创面进行止血试验,同时与胶原蛋白海绵止血效果作比较,观察其止血时间、失血量、敷料与创面的粘合等情况,并定期观察创面愈合、体内吸收和抗炎情况。结果胶原/纤维蛋白复合止血贴组、胶原蛋白海绵组新西兰兔耳动、静脉、耳表创面、股动脉割伤、标准肝创伤的止血时间与对照组比较,差异显著（P〈0.05）。胶原/纤维蛋白复合止血贴组新西兰兔耳动、静脉、耳表创面的止血时间与胶原蛋白海绵组,差异显著（P〈0.05）。胶原/纤维蛋白复合止血贴组、胶原蛋白海绵组动物的耳表创面、标准肝创伤失血量与对照组比较,差异显著（P〈0.05）。体内标准肝创伤、体表创伤后期观察,胶原/纤维蛋白复合止血贴与胶原蛋白海绵均能在21d内完全吸收,未见炎症反应。结论胶原/纤维蛋白复合止血贴对新西兰兔耳动脉割伤、耳静脉割伤、耳标创伤、股动脉割伤和标准肝损伤模型都具有明显的止血作用,体表创面伤口恢复良好,体内吸收速度快,具有一定的抗炎作用,而且在新西兰兔耳动脉、耳静脉割伤和耳表创伤的止血效果明显优于胶原蛋白海绵。胶原/纤维蛋白复合止血贴是一种较安全有效的局部止血生物材料。     

γ射线照射对人肌腱组织形态学及羟脯氨酸含量的影响(英文)

目的:研究2.87Mrad剂量γ射线照射后,肌腱组织形态学及羟脯氨酸含量的变化。方法:选取40根人新鲜上肢掌深屈肌腱,-80℃深低温冷冻6周,等分为二:实验组(A组),对照组(B组)。A组肌腱在干冰低温保存条件下行γ射线照射,检测肌腱最终吸收剂量为2.87Mrad。分别行HE、VG染色,在普通光镜和透射电镜下观察其组织形态学变化。用柱前衍生高效液相色谱检测肌腱胶原蛋白水解液中羟脯氨酸含量。结果:①形态学观察发现,同非照射组肌腱相比,照射组肌腱胶原纤维束间隙较大,纤维排列卷曲紊乱,并可见部分断裂。电镜下胶原纤维横纹模糊消失,腱细胞膜溶解消失,核崩解,细胞器减少。②在同等水解条件下,胶原蛋白水解液中照射组羟脯氨酸含量与非照射组相比,有显著差异(P0.05)。结论:2.87Mrad剂量γ射线照射可引起肌腱组织形态结构发生显著改变。在同等水解条件处理下,照射组肌腱胶原蛋白水解液中羟脯氨酸含量显著高于非照射组。     

Plyometric exercise increases serum indices of muscle damage and collagen breakdown

The aim of the present study was to examine the effect of acute plyometric exercise on indices of muscle damage and collagen breakdown. Nine untrained men performed an intense bout of plyometric jumping exercises (experimental group) and nine men remained at rest (control group). Seven days before and 24, 48, and 72 hours after plyometric exercise or rest, several physiological and biochemical indices of muscle damage and two biochemical indices of collagen damage were determined. No significant changes in concentric and eccentric peak torque of knee extensors and flexors or flexion and extension range of motion were found after the plyometric exercise. Delayed-onset muscle soreness increased 48 hours after exercise. Creatine kinase increased 48 and 72 hours post exercise, whereas lactate dehydrogenase increased 24, 48, and 72 hours post exercise. Serum hydroxyproline increased 24 hours post exercise, peaked at 48 hours, and remained elevated up to 72 hours post exercise. Hydroxylysine (which was measured only before exercise and at 48 hours) was found increased 48 hours post exercise. No differences were found in any physiological or biochemical index in the control group. Intense plyometric exercise increased muscle damage, delayed-onset muscle soreness, and serum indices of collagen breakdown without a concomitant decrease in the functional capacity of muscles. Hydroxyproline and hydroxylysine levels in serum seem promising measures for describing exercise-induced collagen degradation. Coaches need to keep in mind that by using plyometric activities, despite the increased muscle damage and collagen turnover that follow, it is not necessarily accompanied by decreases in skeletal muscle capacity.     

Effect of disuse on the ultrastructure of the achilles tendon in rats

We examined the influence exerted, through disuse of the hindlimb, on the collagen fibres of the achilles tendon in rats. With disuse the body mass decreased by 28%, and the mass of soleus muscle decreased by 20%. A decrease in the surface area and diameter was observed in the experimental group when compared to the control group. A histogram of the collagen fibres showed a decrease of the thick fibres in the experimental group. The maximum surface area of collagen fibres in the experimental group was seen to be only 43% of that of the control group. These results showed a decrease in the thickness of the collagen fibres of the achilles tendon through disuse. This seemed to suggest that resistance to tension is decreased by disuse.     

Effect of exercise training on passive stiffness in locomotor skeletal muscle: role of extracellular matrix

The purpose ofthis study was to evaluate the effect of endurance exercise training onboth locomotor skeletal muscle collagen characteristics and passivestiffness properties in the young adult and old rat. Young(3-mo-old) and senescent (23-mo-old) male Fischer 344 rats wererandomly assigned to either a control or exercise training group[young control (YC), old control (OC), young trained (YT), oldtrained (OT)]. Exercise training consisted of treadmill runningat ~70% of maximal oxygen consumption (45 min/day, 5 days/wk, for 10 wk). Passive stiffness (stress/strain) of the soleus (Sol) muscle fromall four groups was subsequently measured in vitro at 26°C.Stiffness was significantly greater for Sol muscles in OC rats comparedwith YC rats, but in OT rats exercise training resulted in muscles withstiffness characteristics not different from those in YC rats. Solmuscle collagen concentration and the level of the nonreduciblecollagen cross-link hydroxylysylpyridinoline (HP) significantlyincreased from young adulthood to senescence. Although training had noeffect on Sol muscle collagen concentration in either age group, itresulted in a significant reduction in the level of Sol muscle HP in OTrats. In contrast, exercise had no effect on HP in the YT animals.These findings indicate that 10 wk of endurance exercise significantlyalter the passive viscoelastic properties of Sol muscle in old but notin young adult rats. The coincidental reduction in the principalcollagen cross-link HP also observed in response to training in OTmuscle highlights the potential role of collagen in influencing passivemuscle viscoelastic properties.

     

Loss of Function Mutation in the Palmitoyl-Transferase HHAT Leads to Syndromic 46,XY Disorder of Sex Development by Impeding Hedgehog Protein Palmitoylation and Signaling

The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development.     

A microstructural model of elastostatic properties of articular cartilage in confined compression

A microstructural model of cartilage was developed to investigate the relative contribution of tissue matrix components to its elastostatic properties. Cartilage was depicted as a tensed collagen lattice pressurized by the Donnan osmotic swelling pressure of proteoglycans. As a first step in modeling the collagen lattice, two-dimensional networks of tensed, elastic, interconnected cables were studied as conceptual models. The models were subjected to the boundary conditions of confined compression and stress-strain curves and elastic moduli were obtained as a function of a two-dimensional equivalent of swelling pressure. Model predictions were compared to equilibrium confined compression moduli of calf cartilage obtained at different bath concentrations ranging from 0.01 to 0.50 M NaCl. It was found that a triangular cable network provided the most consistent correspondence to the experimental data. The model showed that the cartilage collagen network remained tensed under large confined compression strains and could therefore support shear stress. The model also predicted that the elastic moduli increased with increasing swelling pressure in a manner qualitatively similar to experimental observations. Although the model did not preclude potential contributions of other tissue components and mechanisms, the consistency of model predictions with experimental observations suggests that the cartilage collagen network, prestressed by proteoglycan swelling pressure, plays an important role in supporting compression.     

Electrical stimulation inhibits neointimal hyperplasia after abdominal aorta balloon injury through the PTEN/p27Kip1 pathway

Electric fields (EFs) exert biological effects on promoting wound healing by facilitating cell division, cell proliferation, and cell directional migration toward the wound. In this study, we examined the inhibitory effect of direct-current (DC) EFs on the formation of neointimal hyperplasia and the possible mechanism in an abdominal aorta balloon injury rabbit model. Sixty rabbits were divided into normal, control, and experimental groups. After establishment of the abdominal aorta balloon injury model, electrodes were implanted into the bilateral psoas major muscle in control and experimental groups. Only the experimental group received electric stimulation (EFs applied at 3 or 4 V/cm for 30 min/day) for 1, 2, and 4 weeks, respectively. Neointimal hyperplasia of the abdominal aorta and proliferation of vascular smooth muscle cells (VSMCs) were measured. Expressions of collagen, p27(Kip1), and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) were detected. Results showed that the ratio of the tunica intima area to the tunica media area, the expression of type-I collagen in the neointimal, and the proliferating cell nuclear antigen index in experimental groups were significantly less than those in control groups 2 weeks post-operation (P< 0.01). Expressions of p27(Kip1) and PTEN were increased in experimental groups compared with control groups (P< 0.01). In conclusion, our results suggested that the application of DC EFs could inhibit neointimal hyperplasia and reduce collagen expression after abdominal aorta balloon injury. This was probably induced by upregulation of PTEN/p27(Kip1) expression, thereby inhibiting VSMC proliferation.     

microRNA-15a模拟物对人关节软骨细胞增殖与凋亡的影响

目的：近年来研究表明,关节软骨细胞凋亡在骨关节炎发病过程中起到了重要的作用,本文旨在探讨microma-15a模拟物对于原代人膝关节软骨细胞增殖与凋亡的影响。方法：取人外伤性截肢后的膝关节软骨,采用双酶消化法分离获得人膝关节软骨细胞,并进行体外培养,通过甲苯胺蓝染色和II型胶原免疫细胞化学染色进行软骨细胞鉴定。将培养的软骨细胞传代后取第l代细胞,分为实验组和对照组,实验组采用mir．15a模拟物（has．mir-15amimics）转染软骨细胞,上调软骨细胞内mir-15a的表达量;对照组分为阴性对照组、空白对照组。采用MTT法测定细胞增殖曲线,流式细胞仪测定细胞凋亡率。结果：原代细胞中细胞呈多角形、圆形与梭型,贴壁生长;甲苯胺蓝染色胞质呈深蓝色,II型胶原染色胞质呈黄褐色,为特异性染色。经统计学分析,实验组与对照组相比增殖速率明显下降（P〈0．05）。实验组凋亡率（7．13％±0．57）与阴性对照组凋亡率（2．66％±0．15）相比明显增高（P〈0．05）。结论：采用双酶消化法成功分离并培养具有生物学特性的原代人膝关节软骨细胞,通过转染mir-15a模拟物外源性增加关节软骨细胞内mir．15a表达量可显著促进其凋亡并抑制其增殖,为阐明骨关节炎发病机制提供了新的理论依据,为’临床治疗提供了新的靶点。     

Attenuation of acute and chronic liver injury in rats by iron-deficient diet

Oxidative stress due to iron deposition in hepatocytes or Kupffer cells contributes to the initiation and perpetuation of liver injury. The aim of this study was to clarify the association between dietary iron and liver injuries in rats. Liver injury was initiated by the administration of thioacetamide or ligation of the common bile duct in rats fed a control diet (CD) or iron-deficient diet (ID). In the acute liver injury model induced by thioacetamide, serum levels of aspartate aminotransferase and alanine aminotransferase, as well as hepatic levels of lipid peroxide and 4-hydroxynonenal, were significantly decreased in the ID group. The expression of 8-hydroxydeoxyguanosine and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling positivity showed a similar tendency. The expression of interleukin-1beta and monocyte chemotactic protein-1 mRNA was suppressed in the ID group. In liver fibrosis induced by an 8-wk thioacetamide administration, ID suppressed collagen deposition and smooth muscle alpha-actin expression. The expressions of collagen 1A2, transforming growth factor beta, and platelet-derived growth factor receptor beta mRNA were all significantly decreased in the ID group. Liver fibrosis was additionally suppressed in the bile-duct ligation model by ID. In culture experiments, deferoxamine attenuated the activation process of rat hepatic stellate cells, a dominant producer of collagen in the liver. In conclusion, reduced dietary iron is considered to be beneficial in improving acute and chronic liver injuries by reducing oxidative stress. The results obtained in this study support the clinical usefulness of an iron-reduced diet for the improvement of liver disorders induced by chronic hepatitis C and alcoholic/nonalcoholic steatohepatitis.