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。     

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.     

人硫氧还蛋白cDNA的克隆和表达

我们在本研究中克隆出hTrx成熟区cDNA序列并在原核中高效表达出hTrx蛋白,现将结果报道如下。一、材料和方法14 3 (TK-)人骨肉瘤细胞、pBV2 2 0由西安华广生物工程公司馈赠。pGEM TEasy为Promega公司产品。AMV逆转录酶、Oligo(dT) 15等试剂均购自华美生物工程公司。收集铺满度为80 %的14 3 (TK-)人骨肉瘤细胞2×10 7,异硫氢酸胍一步法提取RNA。根据Genbank提供的已知序列(J0 40 2 6) ,设计下列引物:上游引物5’CGGAATTCATGGT GAAGCAGATCGAGAGC 3’,5’端设有EcoRI酶切位点;下游引物:5’CGGGATCCGATTAGACTAAT…     

Expression of Inflammatory and Regenerative Genes in a Model of Liver Ischemia/Reperfusion and Partial Hepatectomy

Purpose/Aim: Hepatic ischemia/reperfusion (I/R) describes the paradox of additional tissue injury caused by reperfusion. The aim of this survey was to investigate the mRNA expression of genes exerting their inflammatory and regenerative reaction in a porcine model of I/R and extended hepatectomy.

Material and methods: Twelve pigs were used, weighing 30–35 kg in average, which were allocated in two groups: the I/R group with eight pigs and the sham-operated (control) one with four pigs. The I/R group underwent portacaval anastomosis and Pringle maneuver followed by extended hepatectomy. The hepatoduodenal ligament was occluded for 150 min and the liver remnant was reperfused for 24 hours. Blood samples were steadily received throughout the surgical procedure, where hepatic biopsies were taken for pathological evaluation. Animals were sacrificed in 24 hours after the onset of reperfusion.

Results: Between the two groups, statistically significant differences were noticed in serum values of AST, ALT, ALP, and total bilirubin in the early and late phase of reperfusion. The mRNA expression of iNOS, IL-1b, and TGF-a did not increase significantly in the I/R group. Conversely, the mRNA modification of IL-6, STAT-3, and E-selectin demonstrated significantly increased expression in I/R animals.

Conclusions: In the present survey, a new I/R swine model was proposed and specific parameters were analyzed, revealing differences between the study groups.     

Expression of Mesenchymal, Hematopoietic, and Biliary Cell Markers in Adult Rat Hepatocytes After Partial Hepatectomy

Background and Purpose

It has been suggested that liver regeneration can occur either by differentiated adult hepatocytes which retain the capability for several rounds of replication or by hepatic progenitor cells, depending on the number of hepatocytes lost. We sought to study the differentiation potential of hepatocytes following partial hepatectomy (PH) in rats.

Methods

Using immunohistochemistry and confocal microscopy we studied the distribution of cytokeratin 7 (CK7), CK19, vimentin, desmin, CD34, and c-kit among adult rat liver hepatocytes after PH at various times just after hepatectomy and after 8, 16, 24, 36, 48, and 60 hour and 6 and 16 days.

Results

Vimentin, c-kit, and desmin positivity were observed in regenerating hepatocytes in the early stages. Desmin and vimentin staining were also demonstrated in stellate cells. Staining enhancement in stellate cells progressed from day 3 to day 6. No liver sections were stained positive for CD34, CK19, or CK7.

Conclusion

After PH, mature hepatocytes revealed their potential to regain the markers that they do not express when they are quiescent. This result supported the plasticity and differentiation potential of adult hepatocytes during regeneration.     

原癌基因在生精细胞中的表达及其作用

睾丸生精细胞中有许多原癌基因特异表达。在正常睾丸生精过程中,原癌基因的正常表达对精原细胞的有丝分裂、精母细胞的减数分裂及精子变形、成熟有重要的调控作用。     

晚期糖基化终末产物对人肾小管上皮细胞表达结缔组织生长因子和纤维连接蛋白的影响

目的：观察AGE-BSA认对体外培养的人近端肾小管上皮细胞株HK-2表达结缔组织生长因子（CTGF）和纤维连接蛋白（FN）的影响。方法：将HK-2细胞在体外与不同浓度的AGE—BSA和BSA共同培养。以酶联免疫法（ELISA）检测转化生长因子-β1（TGF-β1）的分泌，以逆转录-聚合酶联反应法（RT—PCR）测定CTGF mRNA和FN mRNA的表达。结果：100μg／ml的AGE—BSA刺激HK-2细胞24h，细胞上清中TGF-β1的浓度是对照组的2.37倍。AGE—BSA刺激HK-2细胞48h后，CTGF mRNA的表达开始出现显著升高（P〈0.01）。AGE—BSA能够剂量依赖性的上调HK-2细胞CTGF mRNA和FN mRNA的表达。BSA刺激组没有以上作用。结论：AGE—BSA可上调HK-2细胞CTGF和FN的表达，这可能在糖尿病肾病肾小管间质纤维化形成中起到一定作用。     

丹参对马兜铃酸肾损害大鼠肾组织病理学改变及ACE、ACE2表达的影响

目的：观察丹参对马兜铃酸诱导的大鼠肾组织病理改变、血管紧张素转化酶（ACE）7L其同源物AC1、2以及细胞因子表达的影响。方法：建立马兜铃酸肾损害大鼠模型,并灌服不同剂量丹参水溶液（5、10、15g·kg^-1·d^-1）,于实验第12、16、20、24周留取肾组织标本,HE、P／kS、Masson观察肾组织病理改变,免疫印迹检测肾组织ACE、ACE2的蛋白表达量,RT—PCR检测AngⅡ、TGF—β1、PAI-1mRNA水平。结果：第12周开始,模型大鼠出现肾小管结构损伤,表现为刷状缘消失、细胞空泡变性、脱落,并出现裸膜,伴炎细胞浸润,实验后期出现局部动脉管壁增厚及轻度纤维化表现。模型大鼠肾组织ACE的表达升高,并随实验进展而明显;第16周开始艘的表达降低。AngⅡ、TGF—β1、PAI-1mRNA表达上调,以AngⅡ mRNA上调明显。丹参干预后肾组织病变有所减轻,未出现纤维化表现;丹参可降低肾组织ACE的表达,并下调AngⅡ mRNA水平。结论：丹参可能通过降低ACE的表达、促进ACE2的合成,抑制了AngⅡ的活性,并下调TGF-β1、PAI-1的过高表达,从而减轻马兜铃酸诱导的肾小管间质病变的发生发展。     

体外肝切除自体余肝原位再植术并门腔-心房转流对血流动力学的影响

为研究体外肝切除自体余肝原位再植术中采用门腔－心房体外转流技术对无肝期血流动力学的影响，９只健康幼猪（体重１８～３０ｋｇ）麻醉后经脾静脉，右股静脉置管；全肝血流阻断后，经体外循环泵将血液转流至右心房。左颈外静脉置Ｓｗａｎ～Ｇａｎｚ导管监测血流动力学参数。全肝血流阻断时间为１．１±０．２小时。结果表明：体外肝切除自体余肝原位再植术中存在严重血流动力学紊乱，无肝期采用门腔－心房转流技术能改善其紊乱。术中发生的代谢性酸中毒和低温，可通过补碱和保温得到纠正     

肝卵圆细胞在进行性肝损伤过程中分布及迁移的实验研究

目的 探讨卵圆细胞在进行性肝损伤过程中的分布及迁移规律。方法 清洁型SD大鼠60只随机分为对照组(20只)和实验组(40只)，两组均于肝癌造模后的不同时相切取肝组织标本进行形态学观察、常规病理和原癌基因编码蛋白(C-kit)免疫组化检测。结果 对照组大鼠肝脏表面光滑，组织学形态正常，偶见C-kit阳性细胞。实验组于肝癌造模后的第2周，首先于汇管区发现卵圆细胞沿胆管上皮依次排列增生，这些卵圆细胞呈C-kit阳性表达。随着肝损伤进行性加重，卵圆细胞以汇管区为中心向肝小叶穿插、迁移。肝癌形成期，以混合型癌多见，癌结节内外均见有卵圆细胞，此期C-kit阳性细胞仍集中于汇管区。结论 卵圆细胞为肝组织内对损伤反应最敏感的一种细胞；肝卵圆细胞的无序迁移参与假小叶形成；卵圆细胞与肝癌的发生密切相关。     

食管癌细胞诱导裸鼠肝血管内皮细胞E-选择素的表达

目的探讨食管癌细胞诱导裸鼠肝血管内皮细胞E-选择素(E-selectin)的表达,及其在食管癌肝转移中的作用。方法 Balb/c裸鼠12只,鼠龄6～8周,重量20～25g。将12只鼠随机分为对照组和实验组,每组6只。实验组鼠用食管癌细胞株EC9706悬液(5×106/0.02ml)注入脾包膜下,1h后切除脾;对照组同样行开腹手术,注射磷酸盐缓冲液(PBS)。注射8h后处死鼠取肝,用逆转录-聚合酶链式反应(RT-PCR)和免疫组织化学链霉亲和素生物素标记(LsAB)法检测裸鼠肝血管内皮细胞E-选择素的表达。结果实验组食管癌细胞株EC9706(5×106个)注射到裸鼠脾内,8h后用RT-PCR可在肝脏检测到E-选择素mRNA的表达;免疫组织化学检测显示:肝窦血管胞浆和胞膜E-选择素蛋白表达呈阳性。而对照组肝脏未能检测到E-选择素mRNA表达,E-选择素蛋白表达亦呈阴性。结论食管癌细胞株EC9706能诱导裸鼠肝脏E-选择素mRNA及蛋白表达,表明食管癌细胞株EC9706可能在肝脏中停留并形成转移灶。     

Enhancing Survival of Human Hepatocytes by Neonatal Thymectomy and Partial Hepatectomy in Micro-miniature Pigs

Background

With the goal of in vivo cultivation of human hepatocytes that have not been sufficient in full differentiation in vitro, the advantage of neonatal thymectomy was verified on expansion of xenogeneic human hepatocyte in the micro-miniature pig (MMP).