BALB/c鼠枯否细胞的分离培养与鉴定

目的 :肝脏是肿瘤转移的好发部位 ,而肝脏含有丰富的免疫细胞 -枯否细胞 (Kupffer Cell,KC)。为研究枯否细胞的抗癌活性 ,旨在探讨 BAL B/ c鼠枯否细胞的分离培养方法。方法 :选用健康雄性 8～ 10周龄 BAL B/ c鼠 ,麻醉无菌取肝 ,注射冲洗去血并剪去部分结缔组织 ,小玻璃瓶中剪碎肝组织 ,加入 0 .0 4 %的 EDTA和 0 .0 5 %链霉蛋白酶 E溶液 ,交替轻轻吹打消化。不锈钢筛网滤过 ,用 0 .0 0 4 % Dnase 的 16 4 0液清洗细胞液 ,加 Tris-氯化胺溶解红细胞。 30 %～ 70 % Percoll梯度离心 ,10 % FBS贴壁培养 4～ 6 h洗去非贴壁细胞。通过对比体内、体外枯否细胞吞噬墨水和体外吞噬聚苯乙烯乳珠 (latexbeads,D1.1μm )鉴定枯否细胞。结果 :Kupffer细胞的得率为 (7.5 7± 1.92 )× 10 6 个 /鼠肝 ,即 6 .15× 10 6 个 / g,贴壁率为4 0 .18%。用 0 .4 %台盼蓝染色鉴定 ,细胞存活率在 95 %以上 ,吞噬鉴定发现 Kupffer细胞的纯度可达 90 %。贴壁 Kupffer细胞的形态多样 ,可见不规则 ,多边形 ,多角伪足 ,典型的星形及多角形。体外培养枯否细胞存活 2周后未见再有吞噬功能。结论 :酶消化法结合梯度离心和贴壁培养是分离 BAL B/ c鼠枯否细胞的可靠方法 ,为进一步实验打下了基础     

肝脏神经、枯否细胞对鼠移植肝内皮素-1基因的影响

肝移植是治疗终末期肝病的主要方法,如何降低缺血再灌注损伤,改善移植肝的肝功能受到了越来越多的重视。肝窦内皮细胞是内皮素(ET)合成、清除和作用的主要场所,而内皮素是已知最强的血管收缩剂,其三种异构体ET-1、ET-2、ET-3、ET-1的生物活性最强,多位作者均报道ET在肝硬化门脉高压的发病机制中起重要作用,但其在移植肝缺血再灌注损伤中的作用．尚无定论。本实验通过研究大鼠肝移植术后移植肝内皮素-1基因mRNA表达．为移植肝再灌注损伤的发生机制提供实验依据。     

大鼠及人肝枯否细胞的分离纯化和培养

目的 改进大鼠肝枯否细胞分离、纯化和培养技术,探讨从人体肝脏手术标本中分离、纯化和培养枯否细胞的可行有效方法.方法 32只SD大鼠肝脏及9例人体肝脏手术标本用于本实验.大鼠肝脏经门静脉、人体肝组织通过肝静脉灌注预灌流液,灌注后肝脏标本采用离体胶原酶灌注消化获得细胞悬液,差速离心获得富含KC的肝非实质细胞.改传统PBS重悬肝非实质细胞为25%percoll重悬,形成新percoll梯度由上向下为:PBS、含肝非实质细胞的25%percoll、50%percoll,不连续密度梯度离心分离获得枯否细胞,最后选择性贴壁进一步纯化.应用吞噬功能实验对KC进行鉴定,台盼蓝拒染实验判定细胞活力.结果 最终获得的枯否细胞的产量分别为:大鼠3.1±0.5×106/g肝脏,人体肝脏组织为2.3±0.4×106/g肝脏,细胞活力和纯度均达90%.结论 本实验建立的大鼠及人体肝脏枯否细胞分离方法,简便经济,效果稳定,获得的枯否细胞可用于进一步的实验研究.     

大蒜油激活鼠肝枯否细胞抗癌作用的实验研究

目的：体外实验明确大蒜油是否能增强枯否细胞（Kupffer Cell，KC）的抗肿瘤活性。方法：酶消化法提取小鼠肝脏枯否细胞，分别以含大蒜油浓度为1200、600、300、150、75、37．5μg／mL的培养液培养枯否细胞24h。换洗培养液后，按10：1效靶比加人小鼠结肠癌细胞CT26，共同培养24h。用MTT法测定活细胞数量。以未加药的KC／CT26共同培养和KC、CT26单独培养为对照组。结果：KC的杀癌率为10．49％。经一定浓度大蒜油激活后，KC的杀癌率时显增强，并且杀癌率随大蒜油浓度的升高而增强，大蒜油浓度分别为1200、600、300、150、75、37．5μg／mL的杀癌率分别为91.91％、75．92％、73．71％、42．38％、37．94％和18．07％。300μg／mL组与600μg／mL组差异不明显，37．5μg／mL组与不用大蒜油激活组亦无差异。结论：大蒜油能激活枯否细胞，从而增强其抗癌活性。300μg／mL的大蒜油浓度可能是一个恰当的选择。     

枯否细胞和肝脏缺血再灌注损伤

目的 了解枯否细胞在肝脏缺血再灌注损伤中的作用。方法 采用文献回顾的方法对枯否细胞在肝脏缺血再灌注损伤中的作用加以综述。结果 活化后的枯否细胞可产生和释放多种介质直接或间接地影响肝脏微循环。结论 枯否细胞在肝缺血再灌注损伤中发挥了重要作用。     

不饱和脂肪酸对枯否细胞功能的影响

大鼠饲喂4周鱼油后，盲肠结扎穿孔，术后12h，摘取肝脏，分离枯否细胞并培养，检测培养液中细胞因子炎性介质、抗氧化酶和自由基产物、分析枯否细胞膜磷脂和花生四烯酸。结果表明，术前喂鱼油的大鼠，枯否细胞培养液中TNF，IL-l、IL-6、TXB2、6-kPGFt。明显降低，SOD明显升高，MDA明显减少。与此同时，枯否细胞膜PL及AA的古量明显增加、提示鱼油有修复脓毒症大鼠枯否细胞膜PL成分，调节枯否细胞功能的作用。     

枯否细胞与肝脏缺血再灌注损伤

休克、肝脏外伤及肝移植所致肝功能衰竭都与肝脏缺血再灌注损伤有关，其中枯否细胞（ＫＣ）的作用不容忽视。ＫＣ可因补体系的激活和钙超载或噬作用而激活，活化后的ＫＣ除主要释放活性氧外，还可入蛋白酶及各种细胞因子，介导对肝细胞的毒性作用，导致严重的肝脏再灌注损伤。干扰ＫＣ功能的药物可能具有重要的临床应用价值。     

老化对肝脏枯否细胞吞噬功能及受损易感性的影响

以聚苯乙烯乳珠的吞噬数量为指标研究了老化对大鼠肝脏枯否细胞（ＫＣ）吞噬功能及其对内毒素（ＬＰＳ）刺激的剂量耐受性和时间耐受性的影响。结果表明，①ＫＣ吞噬功能随年龄的增加呈现明显的降低。②各组ＫＣ对ＬＰＳ的反应性随ＬＰＳ剂量的增加均有一从增强到减弱的过程，并以老化ＫＣ为最显著。③随着月龄的增吕，ＫＣ对ＬＰＳ刺激的时间耐受性逐渐降低。结果提示，随着机体的老化，ＫＣ亦呈现明显的老化状态，其受损易感性明显     

冷保存鼠肝枯否细胞的变化及氯化钆的作用

目的 研究枯否细胞抑制剂氯化钆在大鼠肝脏冷保存时对估否细胞（ｋｕｐｆｆｅｒ ｃｅｌｌ,以下简称ＫＣ）的影响。方法 将Ｗｉｓｔａｒ大鼠随机分成８组,每组５只。Ａ、Ｂ、Ｃ、Ｄ组为对照组,肝脏保存时间分别为１、２、３、４小时,其余４组为相应的实验组,各组大鼠经手术灌注、取肝后于０～４℃生理盐水中保存。测定各组动物肝组织脂质过氧化物（ＬＰＯ）含量,做组织学和超微结构的观察。结果 随着保存时间的延长,ＫＣ呈     

脂质体包裹氯膦酸二钠剔除大鼠肝脏枯否细胞作用的研究

目的探讨脂质体包裹氯膦酸二钠剔除大鼠肝脏枯否细胞的作用。方法实验组大鼠给予脂质体包裹的氯膦酸二钠,对照组给予等量生理盐水。给药后不同时间点计数ED1、ED2阳性细胞数目;尾静脉注射印度墨汁判断枯否细胞吞噬碳素颗粒的情况;RT-PCR检测枯否细胞受体mRNA的表达情况。结果给药后2d大鼠肝脏吞噬碳素颗粒的枯否细胞消失,ED1、ED2阳性细胞基本消失,PCR检测不到肝脏枯否细胞受体mRNA的表达。给药后第8天ED1阳性细胞开始明显增多,至第11天其数目基本恢复正常。ED2阳性细胞至第11天开始增加,但平均每中倍视野仍仅有(4.8±1.7)个细胞,明显少于对照组的(17.7±2.1)个细胞,差异具有统计学意义(P0.01)。结论静脉单次注射脂质体包裹氯膦酸二钠至少在1周内能有效剔除肝脏枯否细胞。     

The role of p65 NF-kB/RelA in pancreatitis-induced kupffer cell apoptosis

Acute pancreatitis induces liver injury by upregulating Kupffer cell-derived Fas/FasL; on the other hand, acute pancreatitis induces apoptosis of Kupffer cells via NF-kB-dependent pathways. The balance between upregulation of Fas/FasL and Fas/FasL-induced apoptosis of its originator cell may determine the severity of pancreatitis-related liver injury. The aim of our study was to determine the role of p65 NF-kB/RelA in pancreatitis-induced Kupffer cell apoptosis. Acute pancreatitis was induced in NIH Swiss mice by a choline-deficient ethionine-supplement (CDE) diet. In vitro mouse Kupffer cell line was transfected with p65 siRNA and treated with pancreatic elastase to mimic pancreatitis. CDE pancreatitis upregulated nuclear translocation of p65 NF-kB/RelA, Fas/FasL, caspase-3, and DNA fragmentation in mice livers (all P<0.001). In vitro, pancreatic elastase mimicked CDE-pancreatitis by upregulating nuclear translocation of p65 NF-kB/RelA, Fas/FasL, caspase-3, DNA fragmentation, and apoptosis in Kupffer cells (all P<0.001). Transfection with p65 siRNA attenuated the elastase-induced nuclear translocation of p65 NF-kB/RelA, upregulation of Fas/FasL, caspase-3, DNA fragmentation, and apoptosis in Kupffer cells (all P<0.001). Acute pancreatitis activates p65 NF-kB/RelA and induces apoptosis of Kupffer cells. Inhibition of p65NF-kB/RelA attenuates elastase-induced upregulation of proapoptotic pathways and apoptosis in Kupffer cells. The ability of Kupffer cells to autoregulate their stress response by inducing self-apoptosis warrants further investigation. Presented at the Forty-Sixth Annual Meeting of The Society for Surgery of the Alimentary Tract, Chicago, Illinois, May 14–19, 2005 (poster presentation). Supported by VA Merit Award (M.M.) and Dr. Bob Haines Pancreatitis Research Fund (M.M.).     

小鼠肝星状细胞分离纯化和体外培养模型的建立

目的 建立小鼠肝星状细胞(m-HSCs)分离纯化的高效稳定方案,并通过原代和传代培养观察其生物学特性.方法 依次应用预灌注液、0.1% Pronase E、0.075% Collagenase NB4G对在体肝脏原位灌注消化.肝脏离体后,在0.02%DNaseI液中磁力搅拌消化10 min,低速离心(25g,5 min)去除残余肝实质细胞,进一步用Optiprep密度梯度液获得纯化的m-HSCs.采用锥虫蓝染色法评估细胞产量及活力;采用自发荧光及油红O染色鉴定细胞纯度;采用光镜、电镜以及Desmin/α-SMA免疫荧光双染色观察细胞形态和生物学特征.结果 分离得到的原代m-HSCs数量为(1.4±0.3)×106/g肝脏,细胞活率＞95%;原代培养24 b后,细胞纯度＞90%,传1代后细胞纯度接近100%.静止期和不同活化阶段的m-HSCs在亚显微结构以及α-SMA表达强度等方面存在差异.结论 建立的m-HSCs分离和纯化方法及体外细胞培养模型具有稳定性和高纯度性及高活率性.

Abstract：

Objective To establish a high-performance and stable model for isolation and purification of mouse hepatic stellate cells, and investigate their biological phenotypes by primary culture and subculture. Methods The liver was digested by in situ perfusion of pre-perfusion solution, 0.1% pronase E and 0.075% collagenase NB4G in turn. The liver was continued to digest using 0.02% DNase Ⅰ liquid with magnetic stirring for 10 min in vitro. After low-speed centrifugation used to remove residual hepatocytes, cells were treated with Optiprep density gradient solution to obtain the purified m-HSCs. Trypan blue staining method was used to calculate cell production and viability. The cell purity was identified by mHSCs autofluorescence and oil red O staining. Light microscopy, electronic transmission microscopy and double immunofluorescence staining of Desmin and α-SMA were done to observe morphological characteristics and biological phenotypes of m-HSCs. Results The yeild rate of m-HSCs was (1.4±0.3)×106/g of liver tissue, the cell viability was more than 95%, the cell purity was more than 90% after 24 h of primary cluture and close to 100% after the first cell passage. The activated m-HSCs had different characteristics of submicroscopic structures and expression level of α-SMA. Conclusion This study established a stable model for isolation, purification and culture of m-HSCs, which obtained the high purity and high-living rate of m-HSCs.     

实验性自身免疫性睾丸炎病理变化及肥大细胞改变

目的 观察和分析BALB/c小鼠自身免疫性睾丸炎后病理改变及肥大细胞(MC)的数量、分布等变化.方法 BALB/c雄性小鼠72只,随机分为6组,每组12只,其中5组为模型组,行单侧睾丸动脉结扎,在结扎后2h开通血管,分别在开通后12h、1周、2周、3周、4周处死,取睾丸和附睾;另一组为对照组,不做任何处理,直接取睾丸和附睾进行对比.结果 TB染色显示对照组侧睾丸MC基本上只见于白膜下面,睾丸间质中几乎不存在或仅偶见MC.模型组侧小鼠睾丸白膜、睾丸间质、睾丸纵隔以及附睾中均可见MC.其中附睾中MC的数量最多,依次是纵隔、白膜、睾丸间质.模型组MC的数量和分布与对照组比较差异具有显著性.HE染色显示:随着缺血后再灌注的时间延长,组织病理变化逐渐减轻.结论 自身免疫性睾丸炎模型组MC数量高于对照组,睾丸组织病理变化随时间延长逐渐减轻,具有相对的可恢复性.     

膜滤过分离肿瘤细胞技术在泌尿系肿瘤患者临床治疗中的应用

目的 探讨膜滤过分离肿瘤细胞技术(ISET)检测泌尿系肿瘤患者外周血循环肿瘤细胞(CTC)的临床效果,分析对泌尿系肿瘤患者应用ISET检测的意义.方法 选择2018年4月至2019年4月在本院治疗的98例肾癌患者为实验组,将同期选取的24例健康志愿者作为对照组进行研究.实验组进行术前采血,对照组行空腹采血,采用CTC-...     

Cytokines in the BALB／c mouse testis in various conditions

Aim: To investigate whether testosterone, estrogens, vasectomy, experimental cryptorchidism, varicocele or aging would induce changes in the cytokine environment of the mouse testis, Methods: In adult male BALB/c mice, testosterone implants, estradiol benzoate, vasectomy, unilateral cryptorchidism, unilateral varicocele were administered/performed. The mice were followed up for different periods of time and were then sacrificed with testes incised for examination. The control mice received the vehicle or sham-operation. Results: IL-10 was present in Leydig cells of nearly every testis and IL-10 macrophages in 39% of testes. IL-6 was found in the testes of intact adult mice, mice treated with testosterone for 70 days, cryptorchid testes and sham-operated testes. Conclusion: Results suggest that IL-10 might be involved in the generation of the immunologically privileged microenvironment in the testis.     

Functional heterogeneity of the kupffer cell population is involved in the mechanism of gadolinium chloride in rats administered endotoxin

BACKGROUND: The purpose of this study was to determine if evidence of functional heterogeneity between subtypes of the Kupffer cell (KC) may be involved in the mechanism of the protective effect of gadolinium chloride (GdCl3) in endotoxemia. METHODS: Rats pretreated with or without GdCl3 were administered lipopolysaccharide (LPS) or vehicle. Serum and liver tissues were collected after LPS administration for cytokine measurements and pathological and immunohistochemical evaluation. RESULTS: After LPS administration, increases in expression of TNF-alpha and IL-6 mRNA in the liver were blunted significantly by GdCl3. In control liver tissue, ED2-positive cells were a predominant fraction, with a few ED1-positive cells, and GdCl3 eliminated only ED2-positive cells. Further, ED2-positive cells were larger in size than ED1-positive ones. Importantly, the number of ED1-positive cells in the liver was increased about threefold in the control group but not in the GdCl3 group after LPS injection. Intermediate or large KCs isolated by counterflow centrifugal elutriation showed greater capacity for phagocytosis and production of superoxide and TNF-alpha than small ones. In contrast, IL-6 production was increased to a greater extent in small than in intermediate or large cells. GdCl3 eliminated the intermediate or large KC subpopulation predominantly. CONCLUSION: Collectively, functional heterogeneity of the KC population was involved in the mechanism of the protective effects of GdCl3 in endotoxemia. TNF-alpha derived from activated intermediate or large KCs may activate small KCs and the latter may be recruited to other organs, such as lungs and kidneys, and produce a large amount of IL-6, leading to multiple organ failure.     

BALB/c小鼠精原干细胞长期培养和移植的实验研究

目的 建立小鼠精原干细胞(SSC)长期培养体系,探讨SSC体外增殖分化的关键因子.方法 收集出生4～6 d BALB/c绿色荧光小鼠睾丸,采用改良两步消化法获得细胞悬液,3次差速贴壁去除体细胞获得富集的精原细胞,采用添加生长因子的无血清基础培养液重悬,种植到小鼠胚胎成纤维细胞饲养层上培养.基础培养液为StemPro-34 SFM干细胞培养基并补充15种添加成分;生长因子为10 ng/ml碱性成纤维细胞因子、20 ng/ml胶质细胞源性神经营养因子和200 ng/mlGDNF家族受体a1.取4～5周龄BALB/c雄性小鼠15只,腹腔注射40 mg/kg的白消安建立受体模型,采用三维显微注射系统将培养的SSC移植到受体左侧睾丸精曲小管内,右侧睾丸作为自身对照;分别采用体视荧光显微镜观察和HE染色检测细胞移植后睾丸生精功能恢复情况.结果 改良消化富集法消化后细胞活性>98%,SSC富集约18.5倍.饲养层培养1～2 d后细胞成对称或线形排列,细胞间可见明显的胞质桥连接.3～4 d后精原细胞增殖形成典型的克隆,为边缘不清楚的团块;小鼠SSC能在该培养体系中稳定培养、传代3个月.移植后2个月,体视荧光显微镜下受体睾丸内可见明显绿色阳性克隆,HE染色证实移植的SSC在受体睾丸内克隆增殖并分化产生成熟的精子.结论 成功建立了BALB/c小鼠SSC培养体系,为研究SSC增殖分化调控机制及SSC移植治疗男性不育提供了实验依据.     

ERp29蛋白在BALB／c小鼠附睾头部和尾部精子中的鉴定和定位

目的 对在BALB/c小鼠附睾精子中内质网蛋白29(endoplasmic reticulum protein 29,ERp29)进行鉴定以及定位.方法 应用免疫印迹方法鉴定BALB/c小鼠附睾头、尾部精子ERp29蛋白,同时利用激光共聚焦显微镜和间接免疫荧光定位的方法研究ERp29在附睾头、尾部精子上的定位.结果 证实ERp29蛋白存在于BALB/c小鼠附睾头部和尾部精子中,且其在尾部精子中含量较头部明显增高.ERp29蛋白主要定位于BALB/c小鼠附睾头部精子的头部前端,而在附睾尾部精子则主要定位于头部和尾部主段.结论 通过对ERp29蛋白在BALB/c小鼠附睾头部和尾部精子上的鉴定和定位,可以帮助进一步研究该蛋白对小鼠精子的作用.     

生精障碍BALB/c小鼠睾丸组织的体外培养研究

目的:探寻生精障碍小鼠睾丸组织生精能力的体外发育与成熟方法。方法:8周龄BALB/c雄性小鼠68只,随机分为4组,每组17只。对照组为正常同龄BALB/c雄性小鼠睾丸组织,实验组分别采用注射40 mg/kg白消安后第4周睾丸组织为SCOS组,第6周睾丸组织为重度H-S(H-S1)组,第8周睾丸组织为轻度H-S(H-S2)组。琼脂糖凝胶法体外培养3种生精障碍小鼠睾丸组织及对照组睾丸组织至第4周,通过免疫组化检测减数分裂过程中标记蛋白[减数分裂启动:维甲酸诱导蛋白8(STRA8);减数分裂中:联会复合蛋白3(SCP3);减数分裂后:转换蛋白1(TNP1)]的表达情况,判断体外培养过程中生殖细胞的发育与成熟能力; TUNEL法检测培养前后生殖细胞凋亡情况。结果:①培养前与对照组相比,睾丸组织损伤越严重其STRA8的表达越少(P0.05);随着培养时间的延长,在培养4周后3个实验组中STRA8的表达呈上升趋势(P0.05)。②培养前与对照组相比,SCOS组SCP3表达最少、H-S2表达最多(P0.05);培养4周后SCP3表达增加,但仍未达到对照组表达水平,且损伤越严重,表达越少;③培养4周后TNP1在对照组有阳性表达,H-S1、H-S2组可见个别阳性表达(P0.05),SCOS组无表达。④与培养前相比,培养4周后SCOS组凋亡增加,H-S组凋亡减少(P0.05)。结论:琼脂糖凝胶体外培养可以诱导生精障碍的BALB/c小鼠睾丸组织进行减数分裂,生精功能损伤较轻者的体外培养效果好。