成体小鼠肝脏卵圆细胞的分离及培养

目的:建立一种稳定的成体小鼠卵圆细胞的分离和培养方法。方法:采用喂饲2-乙酰氨基芴（AAF）加2/3肝切除方法诱导肝脏卵圆细胞的增殖。经门静脉灌注消化法和等密度离心法分离卵圆细胞，并在体外进行长期培养。免疫荧光和免疫双标法对培养的卵圆细胞加以鉴定。结果:体外培养的卵圆细胞呈集落样生长，稳定传代并已培养至3个月。免疫荧光和免疫双标法证实培养的细胞为卵圆细胞，并显示该细胞具有分化潜能。结论:该方法是一种稳定的成体小鼠卵圆细胞的分离和培养方法，为肝脏干细胞的相关研究和应用奠定基础。     

大鼠肝卵圆细胞的分离培养和体外分化研究

目的 观察大鼠肝卵圆细胞的活化，为进一步研究肝卵圆细胞特征建立简单的分离培养方法。方法 利用2-乙酰氨基笏／部分肝切除建立肝卵圆细胞活化的大鼠模型，采用选择性消化法从该模型中分离纯化肝卵圆细胞，并做免疫细胞荧光鉴定。用丁酸钠诱导其分化。结果 成功地建立了肝卵圆细胞活化模型，并从中分离培养了表达OV6、CK19、AFP、白蛋白、c-kit、Thy1、CD45和CD34的卵圆细胞。在丁酸钠刺激下它可向肝细胞分化。结论 利用选择性消化法可以分离到肝卵圆细胞，并且其具有肝干细胞的特征。     

大鼠肝卵圆细胞的分离与鉴定

目的：分离、培养和鉴定大鼠卵圆细胞。方法：以3'-me-DAB刺激卵圆细胞迅速增生,改良梯度离心法分离、培养大鼠卵圆细胞,观察其形态及增值特点并进行细胞免疫组织化学鉴定。结果：改良的梯度离心的方法能够有效地分离大鼠肝卵圆细胞,卵圆细胞胞体为卵圆形,代谢缓慢,具有干细胞的特点,可以形成集落,并且表达OV6、CD34及Nestin。结论：改良梯度离心法分离的细胞具有干细胞的特点,可以表达卵圆细胞特异性的表面标记OV6、造血干细胞表面标记CD34和神经中间丝蛋白Nestin,具有横向分化的可塑性,为进一步实验研究提供参考依据。     

ABCG2/Bcrpl基因在大鼠肝脏卵圆细胞中的表达

目的探讨ABCG2／Bcrp1基因在大鼠肝脏卵圆细胞中的表达及意义。方法建立大鼠2-乙酰氨基芴／三分之二肝切除模型，二步胶原酶灌注结合Percoll密度梯度离心分离大鼠肝脏卵圆细胞，采用荧光定量PCR技术检测ABCG2／Bcrp1基因在大鼠肝脏卵圆细胞和肝细胞中的表达水平，免疫组织化学、免疫荧光双标染色和Westernblot方法检测该转运蛋白的表达。结果大鼠肝卵圆细胞中ABCG2／Bcrp1基因的表达水平是肝细胞的13．8倍，ABCG2／Bcrp1蛋白相对分子质量大小为72000，定位于卵圆细胞膜。其表达位于门脉区周围，并向肝小叶延伸。免疫荧光双标染色显示与OV-6共表达。结论大鼠肝卵圆细胞表达高水平的ABCG2／Bcrp1，后者参与肝干细胞免受外源性化学物质损伤的自我保护机制。     

端粒酶在大鼠肝卵圆细胞中表达的意义

目的 检测大鼠肝卵圆细胞端粒酶活性,探讨端粒酶表达与卵圆细胞增殖分化的关系.方法 采用2-AAF/PH模型诱导大鼠卵圆细胞增殖,改良的胶原酶灌注结合密度梯度离心法分离,用细胞免疫荧光和电镜鉴定卵圆细胞.采用免疫组织化学、RT-PCR、荧光定量PCR等方法检测卵圆细胞端粒酶的表达,组间比较采用t检验分析其意义.结果 采用2-AAF/PH模型成功诱导大鼠卵圆细胞增殖.分离的卵圆细胞核大,卵圆形,胞质少,呈铺路石样生长.电镜发现细胞核质比大,胞质内细胞器少且发育不成熟.细胞免疫荧光结果表达OV-6、AFP、CK-19、albumin、c-kit.端粒酶逆转录酶(TERT)表达在门静脉周围增殖的卵圆细胞核内,随着卵圆细胞逐渐向肝细胞方向分化,TERT阳性细胞数逐渐减少.大鼠正常肝组织TERT mRNA表达水平最高,为LE-6卵圆细胞的2.27倍;分离的卵圆细胞TERT mRNA表达水平为LE.6卵圆细胞的1.26倍;采用2μg和4μg细胞提取物分析细胞的端粒酶活性,随着LE-6卵圆细胞传代次数的增加,由24代传至40代,端粒酶活性由△A=1.05、1.15降低到△A=0.25、0.45(t=17.74,12.38,P<0.05).结论 卵圆细胞具有端粒酶活性,端粒酶可能是卵圆细胞维持其增殖能力和多分化潜能的一个必要条件.     

硫氧还蛋白2在2-乙酰氨基芴加肝切除诱导的卵圆细胞中的表达及意义

目的 观察硫氧还蛋白2(Trx2)在2-乙酰氨基芴(2-AAF)加肝切除诱导的卵圆细胞增殖中的表达,并探讨其表达的意义.方法 Balb/c小鼠喂饲2-AAF加2/3肝切除方法诱导肝脏卵圆细胞的增殖.经门静脉灌注消化法和等密度离心法分离卵圆细胞,并在体外进行原代培养.免疫荧光和双标法对培养的细胞进行鉴定.免疫组化法检测卵圆细胞的增殖,免疫荧光法检测Trx2在卵圆细胞和肝实质细胞中的表达,TUNEL法检测卵圆细胞和肝实质细胞的凋亡.结果 喂饲2-AAF后引起肝脏损伤,与肝切除前相比,肝切除后肝脏损伤明显加重(P＜0.01),同时有明显的卵圆细胞增殖.卵圆细胞中Trx2的表达明显高于肝实质细胞,同时其凋亡率明显低于肝实质细胞(P＜0.01).结论 喂饲2-AAF加2/3肝切除可以引起严重的肝损伤,促进卵圆细胞增殖,卵圆细胞高表达Trx2可以抑制自身凋亡和损伤,在肝脏功能的恢复过程中起着非常重要的作用.     

大鼠肝卵圆细胞的体外分离、培养及脾内移植

我们在成功建立和优化大鼠肝卵圆细胞增殖模型的基础上[1,2 ] ,在体外对大鼠肝卵圆细胞进行分离与培养 ,并将其移植入同种异体大鼠脾脏内 ,观察其在脾内的演变结果 ,为肝干细胞移植的临床应用提供实验依据。一、材料与方法1.大鼠肝卵圆细胞分离 :采用本室建立和改进的体外两步灌流法分离大鼠肝实质细胞和非实质细胞[3 ] ;所获细胞先用 5 0g离心 ,沉淀即为肝实质细胞 ,上清液中富含肝卵圆细胞 ,上清液采用 5 0 0g进行离心 ,沉淀物中富含肝卵圆细胞 ,加F12培养液悬浮细胞 ,再用相同条件离心 ,反复 3次 ,将沉淀物用F12培养液制备成细胞悬液 ;…     

大鼠胚胎来源肝干细胞分离纯化方法的比较

目的 观察比较机械分离法和酶消化法分离纯化孕鼠胚胎来源肝干细胞的优劣.方法 取孕13.5 d SD大鼠胚胎肝后分别用机械分离法和酶消化法分离纯化肝干细胞,比较两种方法所得细胞总量和存活率的差异,用免疫荧光染色法鉴定细胞,并比较原代培养前后肝干细胞存活率的变化.结果两种方法分离培养出的细胞形似卵圆形,经免疫荧光染色法检测表达干细胞标志物CD34和胆管细胞标志物CK19,机械分离法分离的肝干细胞数量(57.49±14.25)×106与酶消化法(63.36±12.67)× 106相当,两者比较差异无统计学意义(P>0.05),但机械分离法所得肝干细胞的存活率(96.37±0.82)%高于酶消化法(93.53±0.63)%(P<0.05),并且培养前后细胞存活率也明显高于酶消化法,两两比较差异有统计学意义(P<0.05).结论 机械分离法是一种简单、经济、实用的胚胎来源肝干细胞分离纯化方法.     

大鼠骨髓间充质干细胞的体外培养及初步鉴定

目的 探讨体外培养大鼠骨髓间充质干细胞的可行性,分析其部分表型特点,为细胞移植治疗中枢神经系统疾病奠定基础.方法 采用贴壁筛选法分离纯化大鼠MSCs,进行培养,传代扩增,免疫组化鉴定细胞是否表达具有干细胞特性的标记抗原-神经巢蛋白(nestin),并用流式细胞仪进行荧光检测初步鉴定.结果 分离后的MSCs出现增殖性生长,免疫组化显示巢蛋白(nestin)表达阳性,经流式细胞仪检测显示CD44,CD90阳性,CD45阴性.结论 在体外可培养出较大丰度大鼠骨髓间充质干细胞,而且此方法简单易行.     

大鼠肝卵圆细胞中matrilin-2的表达及其意义

目的探讨大鼠肝脏再生过程中肝卵圆细胞matrilin-2的表达及其意义。方法利用大鼠部分肝切除(PH)或2-乙酰氨基芴灌胃+/部分肝切除(2-AAF/PH)建立大鼠肝脏再生模型,并设正常对照组。应用免疫组织化学和RT-PCR方法研究肝脏组织中matrilin-2表达情况。结果免疫组化发现正常对照组及PH组肝脏组织中只有极少量的matrilin-2表达位于胆管血管周围及肝窦状隙内,而在2-AAF/PH模型中可见matrilin-2表达位于门静脉周围的肝窦状隙内,RT-PCR发现2-AAF/PH组肝卵圆细胞中matrilin-2mRNA高表达,而PH组及正常对照组成熟肝细胞中无matrilin-2mRNA表达。结论matrilin-2是肝卵圆细胞在肝脏再生过程中产生的重要的细胞外基质蛋白,与肝卵圆细胞的增殖分化过程有密切关系。     

牙体牙弓颌骨阻力中心及其临床意义

牙体、牙弓及颌骨的阻力中心在正畸矫治力系统中具有重要的意义,也是正畸学领域争论较多的一个问题。Dermaut等研究表明,当力作用于物体阻力中心时,物体将发生平动,否则将发生平动和转动的复合运动。目前,国内外多数学者认为牙体、牙弓及颌骨存在阻力中心,但其位置存在争议。本文就牙体、牙弓及颌骨的阻力中心及其临床意义作一综述。     

Hazards and complications of endoscopic transurethral ureterolithotripsy and lithoextraction and means of prevention

Complications related to ureterolithotomy and ultrasonic ureterolithotripsy performed under the control of visual endoscope were analyzed in 86 ureterolithiasis patients, methods of their prevention discussed. All the aforementioned complications were distributed into three groups: inapplicability of surgery due to anatomic and functional defects of lower and upper urinary tracts, intraoperative, and postoperative complications. The commonest ones were ureteral abruption and perforation, acute pyelonephritis, temporary vesicoureteral reflux. Their control measures were considered as relative methods of treatment: immediate surgical intervention in case of ureteral abruption, renal catheterization in patients with insignificant ureteral perforation or acute pyelonephritis. Adequate ureteroscopy, careful consideration of pro- and contraindications, catheterization of renal pelvis and urinary bladder performed within 2-3 days after the surgery and adequate antibacterial therapy are the most decisive steps in the control of aforementioned complications.     

Histochemical and contractile properties of striated muscles of urethra and levator ani of dogs and sheep

AIMS: To understand their possible importance in long- and short-term control of continence, some properties of the striated muscles of the urethra and pelvic floor (levator ani) of dogs and sheep were investigated, especially fiber types and contractile characteristics. MATERIALS AND METHODS: Striated muscles of urethra and levator ani of 29 male and 6 female dogs and 11 male and 6 female sheep were removed and cut into strips. Some strips were frozen and stained for ATPase at pH 9.4 and 4.3 for fiber typing; others were set up in an organ bath to study contractile responses to nerve stimulation. RESULTS: All muscles contained both type I (slow) and type II fibers, ranging from 97% type II in female greyhound urethra to 60% in female sheep levator ani. For each muscle, there were fewer type II muscles in sheep than in dog. The diameters of the urethral fibers were about 60% of the levator ani in dogs and 34% in sheep. Contraction of the urethral muscle was faster than for levator ani and declined to about 80% of the peak, 500 msec after the beginning of stimulation at 20 Hz. The levator ani contraction rose to a steady level as long as stimulation continued. CONCLUSIONS: Both the levator ani and urethral striated muscles contain slow and fast fiber types. The levator ani muscles are capable of sustained contraction with rapid onset which will produce long-term closure of the urethra. The circular urethral muscle contraction was faster but less well maintained.     

Characterisation and differentiation of chondroblastomas and chondromyxoidfibromas - presence and expression of collagen types I and II

AIM: Chondroblastomas and chondromyxoidfiibromas are rare benign skeletal neoplasms with reported overlapping histology. Aim of this study was to analyse the biochemical composition of the matrix of these tumour entities in order to further characterise the cellular phenotypes of these neoplasms using typical cell biological marker genes. METHODS: The matrix compositions of chondroblastomas and chondromyxoidfibromas were analyzed by HE-histology, histochemistry, and immunolocalization techniques. Cellular gene expression patterns were detected by mRNA in situ hybridization. RESULTS: Chondroblastomas are rich in collagen type I and show foci of an osteoid-like matrix, whereas collagen type II as a typical marker of chondrocytic differentiation was not detected in any of the specimens. Chondromyxoidfiibromas had foci of chondroid appearance with chondroblastic cellular differentiation characterised by collagen type II expression. CONCLUSION: These results characterise chondroblastomas and chondromyxoidfiibromas as skeletal neoplasms that have a different biology and which can be distinguished by matrix protein expression products: collagen type II, the typical marker of chondroblast differentiation, could only be detected in chondromyxoidfibromas, but not in chondroblastomas. Thus, both neoplasms are clearly different on the cell biological level.     

Effect of smoking and abstention on oxidative burst and reactivity of neutrophils and monocytes

BACKGROUND: Smoking is associated with surgical wound infections, impaired wound healing, and tissue-destructive disorders. The mechanisms are largely unknown, but changes in the function and activity of inflammatory cells may be involved. METHODS: Seventy healthy volunteers (54 smokers and 16 never smokers) were included. The smokers were studied while they smoked and after 20 days of abstinence. After the first 10 days of abstinence, they were randomized to double-blind treatment with transdermal nicotine patch 25 mg per day or placebo. Venous blood neutrophils and monocytes were sampled and isolated. In 22 randomly selected smokers and in all never smokers, the oxidative burst and chemotaxis were determined by a chemiluminescence response assay and a modified Boyden chamber technique, respectively. Stimulants were opsonized zymosan, formyl-Met-Leu-Phe, and zymosan-activated serum. RESULTS: The neutrophil and monocyte oxidative burst was 50% and 68% lower, respectively, in smokers compared to never smokers (P < .05). Neutrophil chemotaxis was 93% higher in smokers (P < .05). Monocyte chemotaxis was lower in smokers compared to never smokers (P < .05). After 20 days of abstinence, neutrophil oxidative burst increased to the level of never smokers (P < .05); monocyte oxidative burst increased by 50% (P < .05). Chemotaxis was only marginally affected. The changes induced by abstinence were less pronounced in the transdermal nicotine patch group compared to the placebo group. CONCLUSIONS: Smoking attenuates the oxidative burst of inflammatory cells and increases chemotaxis. Three weeks of abstinence normalize the oxidative burst, but affect chemotaxis only marginally.