脊索细胞培养基促进BMSCs增殖分化的研究

目的为研究椎间盘组织工程种子细胞,观察脊索细胞培养基对BMSCs增殖分化的影响。方法取4周龄日本大耳白兔胸腰段椎间盘分离培养脊索细胞,取双侧股骨分离培养BMSCs,用含15%FBS的DMEM/F12培养基培养脊索细胞,5 d后制备脊索细胞培养基。实验分为两组,实验组BMSCs中加入脊索细胞培养基培养,对照组BMSCs中加入含15%FBS的DMEM/F12培养基培养。使用细胞活力细胞毒性检测检测细胞增殖情况,采用免疫荧光及实时荧光定量PCR检测BMSCs蛋白多糖及Ⅱ型胶原表达情况。结果成功分离脊索细胞及BMSCs。细胞增殖检测示,培养5、7、9、14 d,实验组细胞数量明显多于对照组(P<0.05)。免疫荧光检测示对照组培养7、14 d细胞内均无或者有较少Ⅱ型胶原及蛋白多糖表达,实验组二者均有较多表达,且培养14 d时表达明显多于7 d。实时荧光定量PCR检测示,培养7、14 d实验组蛋白多糖和Ⅱ型胶原mRNA表达均显著高于对照组(P<0.05);实验组14 d蛋白多糖和Ⅱ型胶原mRNA表达显著高于7 d(P<0.05)。结论脊索细胞培养基可促进BMSCs增殖,并诱导BMSCs向类软骨细胞分化,为脊索细胞和BMSCs作为种子细胞治疗椎间盘退变提供了依据。     

TGF-β3修饰退变髓核细胞移植修复兔退变椎间盘的效果观察

目的探讨TGF-β3基因修饰后退变髓核细胞生物学效应以及植入兔退变椎间盘后对退变椎间盘的影响。方法将重组腺病毒载体Ad-TGF-β3与第2代退变髓核细胞按10∶1比例混合培养转染(Ad-TGF-β3组),待细胞融合后传代,MTT检测转染细胞增殖活性,Western blot检测TGF-β3蛋白含量,免疫细胞化学染色观察对数生长期转染细胞Ⅱ型胶原染色阳性率;采用病毒空载体转染髓核细胞(Adv组)和未经转染髓核细胞(空白组)作为对照。取30只新西兰兔,体重3.2～3.5 kg,雌雄不限,通过针刺L3、4、L4、5和L5、6椎间盘制备椎间盘退变模型。将实验动物按照随机数字法分为3组,转染细胞组(A组,n=12)、退变细胞组(B组,n=12)和空白对照组(C组,n=6)。A、B组将100μL浓度为1×105个/mL对应细胞悬液注射入退变椎间盘,C组同法注入等量PBS。注射后6、10、14周取A、B组各4只、C组2只实验动物处死,取L3、4、L4、5和L5、6椎间盘行组织学观察,RT-PCR检测Ⅱ型胶原和蛋白多糖mRNA表达。结果 Ad-TGF-β3转染后髓核细胞活性明显改善;转染后3、7、14 d,TGF-β3在髓核细胞内表达逐渐升高;Ad-TGF-β3组髓核细胞细胞质内见棕黄色Ⅱ型胶原阳性染色,阳性率显著高于Adv组及空白组(P<0.05)。组织学观察示,A组椎间盘退变程度较B、C组明显减轻。6、10、14周A组Ⅱ型胶原和蛋白多糖mRNA表达显著高于B、C组,差异均有统计学意义(P<0.05)。结论 TGF-β3基因修饰退变髓核细胞后可明显改善细胞生物活性,转染后髓核细胞植入兔体内可明显增加退变椎间盘的基质分泌。     

人唾液对创面愈合影响的实验研究

目的通过与云南白药比较,观察人唾液对创面愈合的作用,以期初步阐明作用机制。方法 3月龄雄性日本大耳白兔6只,体重2.0～2.5 kg;于每只兔脊柱两侧制备深至皮下、大小为2.5 cm×2.5 cm的创面6个。根据处理方法不同,将36个创面随机分为3组(n=12):空白对照组每天涂抹0.4 mL生理盐水;云南白药组每天涂抹0.5 g云南白药粉,唾液组每天涂抹0.4 mL人唾液,连续15 d。观察创面愈合情况,伤后3、5、8、11、15 d测量创面面积,计算创面愈合率;伤后15 d处死动物取创面组织行组织学观察,计数炎性细胞及微血管密度。结果唾液组和云南白药组创面愈合速度明显快于空白对照组,渗液量少,结痂快。伤后5、8、11 d,唾液组创面愈合率均显著高于空白对照组及云南白药组,差异有统计学意义(P<0.05)。组织学观察显示术后15 d,唾液组创面未见明显出血、坏死,创面基本由表皮覆盖,再生表皮向创面中心覆盖生长,唾液组炎性细胞计数及微血管密度均显著低于云南白药组及空白对照组,差异有统计学意义(P<0.05)。结论唾液可明显促进创面愈合,作用机制可能与其减少炎性细胞浸润、防止伤口感染、加速胶原纤维增生及促进创面血管重建有关。     

研究在体关节软骨营养来源的动物模型

目的 建立局部阻断关节软骨营养的动物模型.方法 将40只5月龄雄性新西兰大白兔随机分5组:保持软骨的关节液营养和软骨下骨营养组(Control组,n=8);假手术对照组(Sham组,n=8);阻断软骨的关节液营养组(DNSF组,n=8);阻断软骨的软骨下骨营养组(DNBM 组,n=8);阻断软骨的关节液和软骨下骨营养组(DNSY+ DNBM组,n=8).术后2周每组随机取3只(6膝),关节腔内注射入墨水,自由活动1d,处死动物,观察内植物的阻断效果;术后4周每组5只(10膝),过量麻醉处死动物,取出膝关节,进行大体评分,并对软骨组织进行组织学评分,免疫组织化学染色.结果 术后2周关节腔内墨水未渗入内植物与骨软骨间隙,提示阻断效果确切;术后4周,大体研究和组织学研究提示,与Control组比较,DNSF组软骨退变明显(P＜0.01),DNBM组软骨未见明显退变(P＞0.01);与DNBM组比较,DNSF组软骨退变明显.结论成功建立局部阻断兔关节软骨各种营养途径的动物模型;失去关节液的营养后,关节软骨表现出早期退变迹象.     

一种简易兔心肌梗死模型的建立

目的：探讨一种建立兔急性心肌梗死（MI）模型的简易方法。方法：健康成年雌兔20只,在无气管插管和呼吸机的情况下,正中纵行剪开下段胸骨,同时保持肋骨和胸膜腔完整,在左心耳缘下5~10mm内缝扎左室支（LVB）,建立MI模型,结扎LVB30min后查心电图（ECG）。4周后,行ECG、心脏彩超检查。符合MI心脏彩超和ECG造模成功标准的兔,视为造模成功;并制作心肌病理切片,行HE染色、Masson三色染色,观察病理变化。结果：结扎LVB后,兔ECG出现心肌缺血改变;术后4周动物存活率85%,造模成功率70%,左室舒张末内径（LVEDD）、左室收缩末内径（LVESD）、左室缩短分数（LVFS）与术前差异均有统计学意义（P〈0.05）,病理切片显示心肌梗死。结论：采用纵剪胸骨下段、结扎LVB的方法制作兔MI模型,该方法具有操作简便、模型可靠、成功率高、重复性好、术后能长期存活的优点。     

Evaluation of the protective effects of a nanogel-based vaccine against rabbit hepatitis E virus

Infection with hepatitis E virus (HEV) has raised serious public health concerns worldwide. In this study, a nanogel-based vaccine encapsulating the capsid protein of rabbit HEV was developed and its protective efficacy was compared with a subunit vaccine. A total of 23 rabbits were divided into 5 groups: (1) negative control (n = 4), (2) positive control (n = 4), (3) nanogel control (n = 5), (4) nanogel vaccine (n = 5), and (5) subunit vaccine (n = 5). Rabbits were vaccinated two times, at weeks 0 and 1, with nanogel and subunit vaccines, respectively, and challenged with rabbit HEV at week 4. By week 11, rabbits vaccinated with the nanogel vaccine produced higher antibodies than those vaccinated with the subunit vaccine. Fecal viral shedding and viremia were identified in rabbits of the positive and nanogel control groups at weeks 6–10. However, there was no viral shedding and viremia in rabbits immunized with both the nanogel and subunit vaccines. Alanine aminotransferase and aspartate aminotransferase levels were not elevated in any rabbit. However, histopathological examination revealed much less hepatic inflammation in rabbits of the nanogel vaccine group compared to the positive and nanogel control groups. Significant increases in IL-12 and IFN–γlevels were identified from rabbits immunized with the nanogel vaccine. Collectively, these results indicate that the newly developed nanogel vaccine induced sufficient immunity leading to complete protection from HEV infection in rabbits. Application of this vaccine should be considered as a preventive measure against HEV infection in other animal species and humans.     

Pharmacological postconditioning with the phytoestrogen genistein

Estrogens are known to activate the phosphatidyl-inosityl 3-kinase (PI3K)/Akt pathway, which is central in the cardioprotection afforded by ischemic postconditioning. Therefore, our goal was to investigate whether a phytoestrogen, genistein, could induce a pharmacological postconditioning and to investigate potential mechanisms. We used low doses of genistein in order to avoid tyrosine kinases inhibition. Thus, pentobarbital-anesthetized rabbits underwent a coronary artery occlusion followed by 4 h of reperfusion. Prior to reperfusion, they randomly received an i.v. injection of either saline (Control), 100 or 1000 microg/kg of genistein (Geni(100) and Geni(1000), respectively), and 10 or 100 microg/kg of 17beta-estradiol (17beta(10) and 17beta(100), respectively). Infarct size (IS, % area at risk) was significantly reduced in Gen(100), Gen(1000) and 17beta(100) but not in 17beta(10) (6+/-2, 16+/-5, 12+/-3 and 29+/-7%, respectively) vs. Control (35+/-4%). A significant decrease in the percentage of TUNEL-positive nuclei within infarcted area was observed in Gen(100) and 17beta(100) vs. Controls. The estrogen receptor antagonist fulvestrant (1 mg/kg i.v.) and the PI3K inhibitor wortmaninn (0.6 mg/kg) abolished the cardioprotective effect of genistein. Western blots also demonstrated an increase in Akt posphorylation in Gen(100). In the same group, in vitro mitochondrial swelling studies demonstrated a significant inhibition of calcium-induced opening of mitochondrial transition pore vs. Controls. In conclusion, genistein exerts pharmacological postconditioning with a similar potency as 17beta-estradiol through a pathway involving activation of the estrogen receptor, of PI3K/Akt and mitochondrial preservation. Therefore, genistein should not be only considered as an inhibitor of tyrosine kinase but also as a cardioprotective estrogen.     

氯沙坦对血管球囊损伤后内皮素与一氧化氮的影响

目的　观察血管紧张素Ⅱ受体拮抗剂 (ARB)类药物氯沙坦对动脉球囊损伤后内皮素与一氧化氮的影响。方法　 72只雄性新西兰兔随机分为对照组和药物组。两组又按球囊损伤后采血的时间段分为球囊损伤术前、术后 6小时、术后 1天、术后 3天、术后 1周、术后 2周组。其中药物组动物于术前 1周连续给予氯沙坦 (2 0mg/kg/Day)干预 ,术后坚持服药直到采血 ;对照组动物则只进行球囊损伤 ,不予药物干预。测定血浆内皮素 - 1与一氧化氮含量。结果　与单纯球囊损伤组相比 ,药物组球囊损伤术后血浆内皮素水平 (pg/ml)明显降低 ;血浆一氧化氮水平 (μmol/L)明显增高。 结论　氯沙坦能调节内皮素 /一氧化氮比值平衡 ,保护血管内皮细胞的功能。     

高钾饮食对高脂血症家兔抗氧化作用的研究

目的　观察高钾摄入对脂质过氧化作用和ox LDL的影响。方法　新西兰白兔 3 0只 ,随机分为 3组 :空白组予普通饲料喂养 ;对照组予高脂饲料喂养 ;治疗组予高脂饲料喂养并添加钾剂饲入。将 3组的多项观察指标进行比较分析。结果　对照组和治疗组动物高脂饮食后血TC、TG、LDL c和HLD c水平较空白组显著升高 (P <0 0 5-0 0 1) ,停止高脂饮食后血脂水平下降 (与同组实验前比较P <0 0 5)。治疗组服钾后 7d～ 45d血钾水平较空白组和对照组显著升高 (P <0 0 5) ,但均在生理浓度范围内。对照组高脂饮食 7d和 3 0dLPO的稳定终产物丙二醛 (MDA)水平较空白组和治疗组显著升高 (P <0 0 5-0 0 1) ,停止高脂饮食后 15d其MDA达更高水平 ;治疗组高脂饮食 7dMDA水平与空白组无明显差异 ,高脂饮食 3 0dMDA水平较同期空白组升高 (P <0 0 5) ,但停止高脂饮食后 15d其MDA水平与同期空白组无明显差异 (P >0 0 5) ;对照组和治疗组两组SOD活性在各时点均无显著性差异 ,但高脂饮食 3 0dSOD活性较空白组下降。对照组在 7d～ 45dox LDL水平均较空白组和治疗组显著升高 (P <0 0 5-0 0 1) ;治疗组高脂饮食7dox LDL水平开始上升 ,但与空白组比较未具显著性 (P >0 0 5) ,3 0d后ox LDL水平较同期空白组升高 (P <0 0 5) ,但停止     

兔的心房扑动模型

目的对比犬和兔对心房颤动(Af)和心房扑动(AF)的易患性特点,探讨更理想的房性心律失常建模方法。方法20只新西兰白兔,起搏组(n=11)10Hz快速心房起搏(RAP)2h,对照组(n=9)不起搏,剪下心脏建立Lan-gendorff灌流模型,在左右心房进行电生理扫查。观察心律失常诱发情况;18只健康成年犬,于心耳10HzRAP2h后,在双心房外膜进行电生理扫查,自身前后对照并与兔的起搏组比较心律失常的诱发特点。结果(1)RAP2h后使犬和兔的Af诱发率显著增高,犬的Af比兔的持续时间更长。(2)RAP和对照组新西兰白兔均容易诱发持续AF(7/11比5/9,P=1.000),持续时间在两组间无差异[(39.8±54.4)s比(70.7±62.2)s,P=0.381];18只犬无1例诱发AF。(3)S1S2或S1S1起搏可诱发兔AF,并可被超速起搏终止。结论犬比兔更容易建立Af模型,而新西兰白兔更适于建立AF模型。