去细胞猪主动脉瓣的构建及性能鉴定

目的　研究猪主动脉瓣 (猪瓣 )去细胞方法,观察埋藏前后猪瓣内源性逆转录病毒(PERV)和瓣膜支架变化。方法　①采用去垢剂、核酸酶和改变溶液渗透压方法制备去细胞猪瓣支架。②应用聚合酶链式反应(PCR)和逆转录 聚合酶链式反应(RT PCR)等方法检测去细胞前后新鲜猪瓣和去细胞猪瓣以及供体和受体外周血PERV。③去细胞瓣叶埋藏家兔皮下 4、6、8、10周后分别取材,应用苏木素 伊红和免疫组织化学染色,Instron1122型万能强力仪、分光光度计、光镜和电镜进行检查。结果　①经去细胞处理后猪瓣内细胞成分完全去除,可溶性蛋白明显丢失 [ ( 0 24±0 04 )%vs(0 48±0 12)% ],瓣叶含水量增加[ (92 16±1 48)% vs(89 20±1 55)% ],P均 <0. 01,但去细胞猪瓣仍保持了良好纤维支架结构,热皱缩温度 [ ( 72 0±0 7 )℃vs( 71 2±0 8 )℃]和断裂强度[ (449±19)g/mm2 vs(541±19)g/mm2 ]均无明显变化。②新鲜猪瓣和猪外周血标本经PCR和RT PCR检测,于琼脂糖凝胶电泳内均出现 219bp扩增带,经测序这一产物与Medline公布的PERV C型病毒有 90% ~97%同源性;去细胞猪瓣,及其移植于犬体内一个月后,抽取犬的外周血标本检测, 219bp扩增带均消失。③埋藏于兔皮下的去细胞猪主动脉瓣有轻微组织反应, 4周时可见中性粒细胞、淋巴细胞和浆细     

猪去细胞主动脉瓣组织学及生物力学特征

目的　观察猪主动脉瓣叶去细胞后的组织学和理化性能变化 ,探讨其作为未来“杂种”瓣膜支架的可能性 .方法　新鲜采集的猪主动脉瓣叶经表面活性剂、核酸酶、渗透压变化作用 ,去除瓣叶组织的细胞成分 ,在光镜、电镜下观察组织学变化 ,测定瓣叶组织的含水量、可溶性蛋白含量、热皱缩温度、瓣叶厚度、描计了应力应变曲线 .结果　成功地去除了猪主动脉瓣组织中的细胞成分 ;瓣叶去细胞后组织含水量明显增高 [φ( 94.6± 0 .3) % vs ( 91.2± 2 .0 ) % ,P<0 .0 1],可溶性蛋白含量明显降低 [ω( 0 .0 41± 0 .0 0 8) % vs( 0 .0 5 6±0 .0 0 6 ) % ,P<0 .0 5 ];热皱缩温度 [( 72 .0± 0 .7)℃ vs( 71.2±0 .8)℃ ,P>0 .0 5 ],瓣叶厚度 [( 0 .8± 0 .3) m m vs ( 0 .7± 0 .3)mm,P>0 .0 5 ],断裂强度 [( 6 46± 133) g.mm- 2 vs( 6 5 0±10 5 ) g.mm- 2 ,P>0 .0 5 ]和断裂伸长率 [( 5 7± 14) % vs( 6 5±18) % ,P>0 .0 5 ]均无明显差别 .结论　采用表面活性剂、核酸酶结合渗透压变化的方法 ,能够成功地去除猪主动脉瓣组织的细胞成分 ,且不影响组织的理化性能 ,有可能作为纤维支架 ,用于构建“杂种”生物瓣膜     

猪主动脉组织工程瓣膜制备的初步研究

目的：初步探索猪主动脉组织工程瓣膜的制备方法。方法：经胰酶－EDTA、表面活性剂、核酸酶处理，去除猪主动脉瓣的细胞成分，测定瓣叶去细胞前、后的生物力学特性，并在其表面种植BAECs。结果：猪主动脉瓣膜中的细胞成分能完全被去除，获得完整无细胞的纤维网状支架，瓣叶去细胞前、后的断裂强度和断裂伸长率无明显变化，瓣膜近端存留的主动脉壁中的细胞一半被去除；种植的BAECs在去细胞瓣膜表面、主动脉内膜形成一连续的细胞层。结论：猪主动脉瓣膜去细胞后获得的纤维支架适宜血管内皮细胞的生长，可以用来构建组织工程瓣膜。     

三种去细胞方法构建的猪主动脉瓣膜支架的性能比较

目的:评价不同去细胞方法制备的猪主动脉瓣支架的组织学、生物力学特性及组织相容性,寻找更合适的去细胞心脏瓣膜支架制备方法.方法:分别采用3种不同的去细胞方法对新鲜猪主动脉瓣叶及带瓣主动脉管道进行脱细胞处理:0.01%胰蛋白酶 1%Triton 核酸酶组用含有0.01%胰蛋白酶-1%Triton和核酸酶的去细胞溶液37℃持续震荡24 h;0.01%胰蛋白酶8 h 1%脱氧胆酸(DCA) 核酸酶组先用 0.01%胰蛋白酶37℃持续震荡消化处理8 h,终止胰酶后加入含有1?A和核酸酶的去细胞溶液37℃持续震荡24 h;1?A 核酸酶32 h组:用含有1?A和核酸酶的去细胞溶液37℃持续震荡32 h;同时以PBS漂洗的新鲜瓣叶作为对照组.通过H-E染色、扫描电镜和透射电镜观察评价去细胞效果.测定瓣叶弹性模量、最大抗张强度、应力伸长比及断裂伸长比以评价各组瓣叶的生物力学特性.大鼠皮下包埋实验评价去细胞支架的免疫源性、体内炎性反应及改建情况.结果:0.01%胰蛋白酶8 h 1?A 核酸酶组细胞去除完全,优于0.01%胰蛋白酶 1%Triton 核酸酶组和1?A 核酸酶32 h组.所制备瓣膜支架的弹性模量及最大抗张强度大于0.01%胰蛋白酶 1%Triton 核酸酶组.应力伸长比及断裂伸长比则小于0.01%胰蛋白酶 1%Triton 核酸酶组,与新鲜瓣叶无明显差异.体内埋植实验炎症反应轻微,宿主成纤维细胞能够长入支架并对其进行改建.结论:短时间低浓度胰蛋白酶与1?A和核酸酶相结合的去细胞法方便有效,既能完全去除猪带瓣主动脉管道的细胞成分,使免疫源性显著下降;同时瓣叶的生物力学特性保持稳定,可为受体细胞化组织工程心脏瓣膜的提供可靠的支架材料.     

环氧氯丙烷处理对生物瓣膜及组织工程瓣膜材料表面性状的影响

目的:研究环氧氯丙烷(EC)处理对生物瓣膜及组织工程瓣膜去细胞支架材料表面性状的影响,探究EC处理生物瓣膜防钙化的机制. 方法: 通过血浆吸附试验和血小板吸附试验测定EC处理的猪主动脉瓣以及经去细胞处理的组织工程猪瓣支架材料对血浆中蛋白质和血小板的吸附程度,与戊二醛处理的生物材料进行对照研究,评价EC处理对生物材料的表面性状的影响. 结果: EC处理的生物瓣膜较戊二醛处理的生物瓣膜对血浆蛋白质吸附率减少[(0.120±0.008) g/kg vs (0.183±0.017) g/kg, P＜0.05],血小板黏附率减少[(1.158±0.120) ×1012/kg vs (1.737±0.093)×1012/kg, P＜0.05],对猪主动脉瓣去细胞处理过程能够减少血浆蛋白质吸附率[(0.116±0.013) g/kg vs (0.134±0.011) g/kg, P＜0.05]和血小板黏附率[(0.769±0.136) ×1012/kg vs (1.052±0.185)×1012/kg, P＜0.05],EC处理能够进一步减少血浆蛋白质吸附率[(0.102±0.004) g/kg vs (0.116±0.013) g/kg, P＜0.05]和血小板黏附率[(0.744±0.097) ×1012/kg vs (0.769±0.136)×1012/kg, P＜0.05]. 结论: EC处理能够进一步改善生物瓣膜和组织工程瓣膜去细胞支架材料的表面性能,有利于提高植入循环系统后的血液相容性和减缓瓣膜钙化.     

胰蛋白酶法和Triton—X100法去除瓣膜细胞的效果比较

目的比较胰蛋白酶法和Triton—X100法去除异种或同种心脏瓣膜上细胞的效果。方法选取新鲜猪主动脉瓣膜片分别置入0．05％胰酶溶液和0．25％Triton-X100溶液中处理48h，并以未经处理的新鲜瓣膜片作对照，行光镜及电镜观察．并作热皱缩温度、断裂强度和断裂拉伸率测定。结果光镜和电镜下均显示，胰酶法和Triton-X100法均彻底去除了主动脉瓣膜上的细胞成分，但Triton—X100法对瓣膜支架结构的损伤较大。热皱缩温度、断裂强度和断裂拉伸率在新鲜瓣膜组和胰酶组间无显著性差异，但Triton—X100组与新鲜瓣膜组及胰酶组相比均有下降。结论胰酶法去除猪主动脉瓣膜细胞彻底．保持了瓣叶支架结构，优于Triton—X100法。     

胰蛋白酶法和Triton-X100法去除瓣膜细胞的效果比较

目的 比较胰蛋白酶法和Triton-X100法去除异种或同种心脏瓣膜上细胞的效果。方法 选取新鲜猪主动脉瓣膜片分别置入0.05%胰酶溶液和0.25%Triton-X100溶液中处理48h,并以未经处理的新鲜瓣膜片作对照,行光镜及电镜观察,并作热皱缩温度、断裂强度和断裂拉伸率测定。结果 光镜和电镜下均显示,胰酶法和Triton-X100法均彻底去除了主动脉瓣膜上的细胞成分,但Triton-X100法对瓣膜支架结构的损伤较大。热皱缩温度、断裂强度和断裂拉伸率在新鲜瓣膜组和胰酶组间无显著性差异,但Triton-X100组与新鲜瓣膜组及胰酶组相比均有下降。结论 胰酶法去除猪主动脉瓣膜细胞彻底,保持了瓣叶支架结构,优于Triton-X100法。     

改良去细胞方法对猪源性逆转录病毒的影响

目的：观察改良的去细胞方法对猪主动脉瓣膜的猪源性逆转录病毒的影响．方法：用0．1％胰酶消化猪主动脉瓣后,再用Triton X-100-DNA酶／RNA酶洗脱消化,结合溶液渗透压改变等去除细胞;取新鲜猪瓣、猪外周血标本和去细胞猪瓣各20只,降主动脉内移植去细胞猪瓣后的犬外周血10例,以PK15细胞系为对照,采用PCR和RT-PCR法检测猪源性逆转录病毒．结果：经去细胞处理后猪瓣内的细胞成分完全去除,纤维支架和力学性能保持良好;新鲜猪瓣及猪外周血标本均检测到219bp的扩增条带,与Medline公布的猪源性逆转录病毒有90％-95％同源性;去细胞猪瓣及移植了去细胞猪瓣犬的外周血,均未测到该病毒．结论：改良的去细胞方法可以去除猪心瓣膜的猪源性逆转录病毒,能够有效预防该病毒物种间的交叉感染．     

改良去细胞方法对猪源性逆转录病毒的影响

目的观察改良的去细胞方法对猪主动脉瓣膜的猪源性逆转录病毒的影响。方法用0．1％胰酶消化猪主动脉瓣后,再用TritonX-100-DNA酶／RNA酶洗脱消化,结合溶液渗透压改变等去除细胞;取新鲜猪瓣、猪外周血标本和去细胞猪瓣各20只,降主动脉内移植去细胞猪瓣后的犬外周血10例,以PK15细胞系为对照,采用PCR和RT-PCR法检测猪源性逆转录病毒。结果经去细胞处理后猪瓣内的细胞成分完全去除,纤维支架和力学性能保持良好;新鲜猪瓣及猪外周血标本均检测到219bp的扩增条带,与Medline公布的猪源性逆转录病毒有90％～95％同源性;去细胞猪瓣及移植了去细胞猪瓣犬的外周血,均未测到该病毒。结论改良的去细胞方法可以去除猪心瓣膜的猪源性逆转录病毒,能够有效预防该病毒物种间的交叉感染。     

血红素加氧酶-2基因敲除对Fe2+诱导的小鼠脑氧化应激损伤中基底节细胞的保护作用

目的:以人体骨髓间充质干细胞(BMSCs)为种子细胞,以去细胞猪主动脉瓣为支架,体外三维构建组织工程心脏瓣膜(TEHV).方法:以胸外科术中切除的肋骨为骨髓来源,采用1.073 g/ml的Percoll梯度分离液离心法获取BMSCs,体外培养扩增、鉴定;以0.05%胰酶 0.02?TA消化法制备去细胞猪主动脉瓣叶支架,将体外培养扩增的BMSCs种植于支架上,体外三维培养,应力环境下构建TEHV,观察TEHV细胞分泌的一氧化氮(NO)及内皮素(ET-1)水平、免疫组化特征及光镜和电镜下的结构.结果:人BMSCs为梭形,α-SMA及Vimentin染色阳性,CD-31及Ⅷ因子染色阴性,其分泌NO及ET-1分别为(104.1±5.9)μmol/L及(40.9±7.6)ng/L,明显较正常人血管内皮细胞低(P＜0.01);猪去细胞瓣膜支架去细胞完全,弹力纤维及胶原纤维保持完整;应力条件下体外三维构建的TEHV上活细胞数较静态下构建明显增多(P＜0.01),H-E染色示人BMSCs在支架上生长良好,形成完整的细胞层,扫描电镜示瓣膜表面细胞层完整,细胞呈梭形生长.结论:人BMSCs作为种子细胞已初步具有内皮细胞功能,在去细胞猪主动脉瓣膜支架上生长良好,可作为一种有前途的种子细胞构建自体TEHV;体外三维应力条件下构建TEHV效果明显优于静态培养.     

"Not thrush again!" Women's experience of post-antibiotic vulvovaginitis

OBJECTIVES: To examine the frequency of post-antibiotic vulvovaginitis (PAV); describe how women prevent and treat PAV; and determine whether concern about PAV affects their decisions about taking antibiotics. DESIGN: Cross-sectional survey using a written questionnaire. SETTING AND PARTICIPANTS: Five general practice waiting rooms in north-western Melbourne, in February 2000. 1298 women aged 18-70 years were surveyed. MAIN OUTCOME MEASURES: Self-reported symptoms and management of vulvovaginitis and PAV. RESULTS: The response rate was 86%. Thirty-five per cent of women reported ever having PAV and 73% reported ever having symptoms suggestive of vulvovaginal candidiasis. Antifungal medications and lactobacillus products or yoghurt were most popular for both prevention (49%, 40%) and treatment (63%, 43%) of PAV. Other home remedies such as tea tree oil, vinegar, and dietary and clothing modification were infrequently used by the women surveyed. Twenty-three per cent of women who had taken antibiotics in the previous month had experienced symptoms of vulvovaginitis. Of women who had ever had vulvovaginitis, 35% were moderately to very concerned about developing PAV when prescribed antibiotics. Because of this concern, around a fifth of these women would not take prescribed antibiotics. CONCLUSIONS: Concern about PAV affects women's decision-making regarding antibiotic use. Many women use unproven complementary therapies to prevent or treat PAV. When prescribing antibiotics, doctors should discuss the risks of PAV and its management with patients.     

指尖光学体积描记法检测血管内皮功能在冠心病患者筛查中的应用

目的：探讨光学体积描记法在指尖脉搏容积（PAV）评价内皮功能在冠心病诊断中的应用,阐明其与心血管危险因素的关系。方法：选择409例行冠状动脉造影术（CAG）的胸痛患者,依据造影结果分为冠状动脉造影阳性（CAG+）组288例与冠状动脉造影阴性（CAG-）组121例。应用内皮功能诊断仪,采用光学体积描记法分析反应性缺血后的PAV,并分析PAV值与冠心病危险因素的相关性,确定PAV作为预测冠心病指标的临界参考值。分析各组受试者年龄、性别、血清总胆固醇（TC）、低密度脂蛋白（LDL）、高密度脂蛋白（HDL）、非高密度脂蛋白（non-HDL）、甘油三酯（TG）、高血压、糖尿病、吸烟、冠心病家族史和体质量指数（BMI）等危险因素,将PAV与冠心病危险因素的关系进行Logistic回归分析。结果：CAG+组患者PAV值明显低于CAG-组（P<0.01）;PAV预测冠心病的临界点为1.37,如以PAV值<1.37来预测冠心病,其敏感度为74.65%,特异度为44.63%,阳性预测值为76.24%,阴性预测值为42.52%;Logistic回归分析,PAV与高血压、吸烟史和TG呈负相关关系（OR=1.476,OR=2.002,OR=1.844,P<0.01）。结论：PAV与冠心病及其危险因素高血压、吸烟史和TG相关,PAV=1.37可作为预测冠心病的临界参考值。     

PAV对COPD并发呼吸衰竭患者血流动力学的影响

目的比较成比例辅助通气(PAV)与双水平气道正压(BiPAP)通气对慢性阻塞性肺疾病(COPD)合并呼吸衰竭患者血流动力学参数的影响。方法COPD合并呼吸衰竭患者31例,给予常规内科治疗的同时,进行PAV和BiPAP通气各60min,监测患者在鼻导管吸氧自主呼吸以及接受不同通气模式支持时血流动力学参数、通气参数和动脉血气的变化。结果与自主呼吸相比,患者在BiPAP通气后心率(HR)和平均动脉压(MAP)略有下降,而在PAV通气时呈明显下降(P0.05)。BiPAP通气时患者的心输出量(CO)、心脏指数(CI)、搏出量(SV)和每搏指数(SI)与PAV时相近,但低于未进行PAV时。PAV通气后射血前时间(PEP)明显缩短,对左室射血时间(LVET)则影响不大。PAV通气时的浅快呼吸指数(RR/VT)为81.2±4.7,显著高于BiPAP时的53.6±2.9(P0.05)。PAV支持时的PaO2/FiO2较鼻导管吸氧时显著增高,PaCO2则有所降低。结论COPD呼吸衰竭患者接受PAV通气后,患者的血流动力学参数中CO、CI、SV等均明显下降,心输出量、心脏指数、搏出量和每搏指数明显改善,患者的心脏收缩能力得到增强。     

成比例辅助机械通气治疗慢阻肺合并高碳酸血症的研究

目的探讨成比例辅助机械通气（PAV）对慢性阻塞性肺疾病（COPD）合并高碳酸血症患者呼吸力学的影响。方法在常规药物治疗的基础上,应用无创成比例辅助机械通气（PAV）治疗COPD合并高碳酸血症患者40例,观察并比较不同模式下动脉血气分析指标（Pa02、PaC02）及呼吸力学变化,包括潮气量（VT）、每分通气量（MV）、呼吸频率（RR）、气道峰压（PUP））和吸气／呼吸周期时间比（Ti/Ttot）,并计算平均吸气流速（VT/Ti）。结果COPD合并高碳酸血症患者PAV通气时的RR、PIP和VT/Ti与BiPAP（S/T）相比有显著降低（P〈0．05）,Ti/Ttotal明显增加（P〈0．05）,MV有所增加,夜间连续通气时间显著延长。结论与BiPAP相比,无创成比例辅助机械通气（PAV）应用于慢性阻塞性肺病并高碳酸血症患者时气道峰压低、吸气,呼吸周期时间比明显增加,通气依存性好。PAV适宜的辅助比例在40％-60％。     

Proportional assist ventilation: methodology and therapeutics on COPD patients compared with pressure support ventilation

Objective To investigate the impact of proportional assist ventilation (PAV) on tolerance and breathlessness in ventilated chronic obstructive pulmonary disease (COPD), and to describe the patient-ventilator interaction, hemodynamic state, breathing pattern and work of breath during PAV and pressure support ventilation (PSV).Methods Ten intubated COPD patients on weaning from mechanical ventilation were studied. Elastance and resistance were measured by both the inspiratory-hold technique during a brief period of volume control ventilation and runaway technique during PAV. Each assistance level of PAV (80%, 60% and 40%) and PSV was selected randomly. Patients’ response, hemodynamics, blood gas and lung mechanics were monitored. Results Tidal volume and respiratory rate didn’t change in a consistent manner as the level of assist was decreased ( P &gt;0.05). With the level of assist increasing, peak inspiratory pressure was increasing significantly ( P &lt;0.05), while patients’ work of breath had the tendency to decrease ( P &lt;0.05). A significant difference in the Borg Category Scale was observed between PAV and PSV (0.50［1.50］ vs 0.75［2.00］, P &lt;0.05) at the same degree of respiratory muscle unloading. PaCO(2) was significantly higher on PAV (54［23］mmHg) than on PSV (48［23］mmHg) ( P &lt;0.05). Peak inspiratory pressure on PAV was significantly lower than on PSV （16±4cmH(2)O vs 21±3cmH(2)O, respectively, P &lt;0.05). Hemodynamics and oxygenation remained unchanged.Conclusions PAV is a feasible method for supporting ventilator-dependent patients and was well tolerated. It can improve the breathing pattern and reduce inspiratory effort. At the same degree of respiratory muscle unloading, PAV can be implemented at much lower peak inspiratory pressure than PSV. It can also apply proportional pressure support according to the patients’ ventilatory demand.     

Distribution of ventilation and hemodynamic effects of different ventilatory patterns

Objective To compare the influence of cardiac-pulmonary function on clinical acute respiratory failure patients using Proportional assist ventilation (PAV), Pressure support ventilation (PSV) and intermittent positive pressure ventilation (IPPV). Here, we also describe some our experience with the clinical use of PAV.Methods Using the IPPV mode in ten acute respiratory failure patients, calculate Elastance (Ers) and Resistance (Rrs), then change to PSV, set inspiratory positive airway pressure (IPAP) according to IPPV, so that tidal volume (V(T)) is the same as that of IPPV. We then changed the mode into PAV and set the assist ratio according to PSV, so that V(T) and Ppeak were the same as that of PSV. Then we observed the changes of respiratory mechanics, blood gas levels and hemodynamics during ventilation.Results Compared with PSV and IPPV, peak pressure (Ppeak) of PAV was markedly lower while V(T) was similar; work of breathing of patient (WOBp), and work of breathing of ventilation (WOBv) were also lower; center vein pressure (CVP) and pulmonary capillary wedge pressure (PCWP) of PAV were markedly lower than that of IPPV while V(T) were similar. Compared with PSV, V(T), mean blood pressure (mBP) and cardiac output (CO) of PAV were higher. Mean pulmonary artery pressure (mPAP) and WOBp of PAV were lower while Ppeak was similar; the differences in WOBp were notable. Conclusions For clinical acute respiratory failure patients, compared with PSV and IPPV, PAV has lower airway pressure、less WOBp and less influence on hemodynamics.     

病窦综合征患者双腔起搏器植入术后PAV间期的优化

目的研究病态窦房结综合征患者植入双腔起搏器后,心房起搏至心房除极波延迟时间对设置起搏器PAV间期的作用。方法通过随访2008年1月～2013年6月在惠州市中心人民医院安装双腔心脏永久起搏器的病态窦房结综合征的65例患者,调整PAV设置,对比出厂PAV设置,观察右心室起搏百分比变化,同时检测血浆BNP浓度。结果延长PAV间期后,与出厂设置PAV对比,心室起搏百分比可显著减少,同时BNP浓度也随之下降。结论表明S85患者双腔起搏器植入术后,设置起搏器PAV间期要将心房起搏至心房除极波延迟时间计算在内,降低心室起搏比例同时降低患者心衰发生率。