罗格列酮对裸鼠移植性人肝癌细胞生长的抑制作用

目的:研究罗格列酮(rosiiglitazone,ROZ)对裸鼠体内人肝癌SMMC7721细胞增殖的影响.方法:建立人肝癌裸鼠移植瘤模型,实验动物分为罗格列酮治疗组及对照组.观察罗格列酮对移植瘤体积和重量的变化,采用光镜观察移植瘤细胞形态学变化,流式细胞术检测移植瘤细胞周期分布,免疫组织化学和蛋白印迹检测瘤组织内增殖细胞核抗原(proliferating cellnuclear antigen,PCNA)蛋白表达变化.结果:罗格列酮在体内能明显抑制移植瘤的生长,抑瘤率为50.81%;组织学显示治疗组瘤细胞异型性降低,核质比下降;流式细胞术分析显示,对照组移植瘤细胞处于G2/M期细胞为13.1%,与对照组比较,治疗组处于G2/M期细胞显著增多(29.1%).免疫组织化学和蛋白印迹检测均显示检测显示,经罗格列酮处理后,裸鼠移植瘤组织PCNA蛋白表达明显下调(P＜0.05).结论:罗格列酮可体内抑制人肝癌SMMC7721细胞增殖,并使癌细胞阻滞于G2/M期.     

人肝癌细胞与肝星形细胞裸鼠皮下共培养体系的建立

目的:建立裸鼠皮下共培养人肝癌细胞与肝星形细胞模型,观察人肝癌细胞与肝星形细胞间相互作用后超微结构的改变.方法:将16只裸鼠分为两组,肝癌细胞单独培养组和癌细胞与肝星形细胞共培养组,40天后将荷瘤组织切片行光镜及透射电镜观察.结果:肝癌细胞单独培养组中可观察到肝癌细胞的胞质液化及早期细胞凋亡现象,而共培养组中可见肝星形细胞时肝癌细胞的趋化现象,可观察到肝癌细胞结构完整且有增殖趋势.结论:裸鼠皮下荷瘤三维立体模型建立成功,该模型能够模拟肝癌微环境中肝癌细胞与肝星形细胞问的作用,为进一步研究肝癌细胞与肝星形细胞间的相互作用奠定了基础.     

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.     

ROCK Is Involved in Vasculogenic Mimicry Formation in Hepatocellular Carcinoma Cell Line

Ras homolog family member A (RhoA) and Rho-associated coiled coil-containing protein kinases 1 and 2 (ROCK1 and 2) are key regulators of focal adhesion, actomyosin contraction and cell motility. RhoA/ROCK signaling has emerged as an attractive target for the development of new cancer therapeutics. Whether RhoA/ROCK is involved in regulating the formation of tumor cell vasculogenic mimicry (VM) is largely unknown. To confirm this hypothesis, we performed in vitro experiments using hepatocellular carcinoma (HCC) cell lines. Firstly, we demonstrated that HCC cells with higher active RhoA/ROCK expression were prone to form VM channels, as compared with RhoA/ROCK low-expressing cells. Furthermore, Y27632 (a specific inhibitor of ROCK) rather than exoenzyme C3 (a specific inhibitor of RhoA) effectively inhibited the formation of tubular network structures in a dose-dependent manner. To elucidate the possible mechanism of ROCK on VM formation, real-time qPCR, western blot and immunofluorescence were used to detect changes of the key VM-related factors, including VE-cadherin, erythropoietin-producing hepatocellular carcinoma-A2 (EphA2), phosphoinositide 3-kinase (PI3K), matrix metalloproteinase (MMP)14, MMP2, MMP9 and laminin 5γ2-chain (LAMC2), and epithelial-mesenchymal-transition (EMT) markers: E-cadherin and Vimentin. The results showed that all the expression profiles were attenuated by blockage of ROCK. In addition, in vitro cell migration and invasion assays showed that Y27632 inhibited the migration and invasion capacity of HCC cell lines in a dose-dependent manner markedly. These data indicate that ROCK is an important mediator in the formation of tumor cell VM, and suggest that ROCK inhibition may prove useful in the treatment of VM in HCC.     

p16在HBsAg和HBx转基因小鼠肝脏肿瘤中的表达

目的:探讨p16基因在由乙型肝炎病毒基因整合引起的小鼠肝细胞癌发生发展中的表达变化规律。方法:以乙肝病毒表面抗原基因(HBsAg)及X基因(HBx)定位整合转基因小鼠及对照小鼠的肝脏组织为标本,利用North-ern印迹、Western印迹及免疫组织化学检测p16在乙肝病毒基因定位整合转基因小鼠肝脏正常组织与肿瘤组织中的差异表达。结果:p16主要在小鼠胚胎期的肝脏中表达,在新生小鼠和成年小鼠的肝脏组织中几乎检测不到其表达;在HBsAg转基因小鼠和HBx转基因小鼠的肝脏肿瘤中,p16的表达明显升高。结论:p16基因在HBsAg或HBx诱导的肝细胞癌发生过程中被重新激活,也许发挥重要的作用。     

A Collision Probability Model of Portal Vein Tumor Thrombus Formation in Hepatocellular Carcinoma

Hepatocellular carcinoma is one of the most common malignancies worldwide, with a high risk of portal vein tumor thrombus (PVTT). Some promising results have been achieved for venous metastases of hepatocellular carcinoma; however, the etiology of PVTT is largely unknown, and it is unclear why the incidence of PVTT is not proportional to its distance from the carcinoma. We attempted to address this issue using physical concepts and mathematical tools. Finally, we discuss the relationship between the probability of a collision event and the microenvironment of the PVTT. Our formulae suggest that the collision probability can alter the tumor microenvironment by increasing the number of tumor cells.     

Resveratrol Ameliorates Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice

BackgroundThe polyphenol resveratrol has anti-inflammatory effects in various cells, tissues, animals and human settings of low-grade inflammation. Psoriasis is a disease of both localized and systemic low-grade inflammation. The Sirtuin1 enzyme thought to mediate the effects of resveratrol is present in skin and resveratrol is known to down regulate NF-κB; an important contributor in the development of psoriasis. Consequently we investigated whether resveratrol has an effect on an Imiquimod induced psoriasis-like skin inflammation in mice and sought to identify candidate genes, pathways and interleukins mediating the effects.MethodsThe study consisted of three treatment groups: A control group, an Imiquimod group and an Imiquimod+resveratrol group. Psoriasis severity was assessed using elements of the Psoriasis Area Severity Index, skin thickness measurements, and histological examination. We performed an RNA microarray from lesional skin and afterwards Ingenuity pathway analysis to identify affected signalling pathways. Our microarray was compared to a previously deposited microarray to determine if gene changes were psoriasis-like, and to a human microarray to determine if findings could be relevant in a human setting.ResultsImiquimod treatment induced a psoriasis-like skin inflammation. Resveratrol significantly diminished the severity of the psoriasis-like skin inflammation. The RNA microarray revealed a psoriasis-like gene expression-profile in the Imiquimod treated group, and highlighted several resveratrol dependent changes in relevant genes, such as increased expression of genes associated with retinoic acid stimulation and reduced expression of genes involved in IL-17 dependent pathways. Quantitative PCR confirmed a resveratrol dependent decrease in mRNA levels of IL-17A and IL-19; both central in developing psoriasis.ConclusionsResveratrol ameliorates psoriasis, and changes expression of retinoic acid stimulated genes, IL-17 signalling pathways, IL-17A and IL-19 mRNA levels in a beneficial manner, which suggests resveratrol, might have a role in the treatment of psoriasis and should be explored further in a human setting.     

Bevacizumab 联合吉西他滨对荷肝癌裸鼠移植瘤生长的抑制作用

目的:探讨抗血管生成药物Bevacizumab联合吉西他滨对人肝癌裸鼠皮下移植瘤生长的抑制作用。方法:构建人肝癌细胞HepG2裸鼠皮下移植瘤模型,随机分为空白对照组、Bevacizumab组、吉西他滨组和联合用药组。观察用药前后肿瘤体积,绘制肿瘤生长曲线;应用免疫组化检测肿瘤微血管密度(MVD);Western Blot检测Bcl-2蛋白的表达。结果:Bevacizumab和吉西他滨单药均能抑制肿瘤生长,两药联合疗效明显增强(P=0.000)。与对照组和吉西他滨组相比,Bevacizumab组和联合用药组能明显抑制肿瘤血管生成,MVD值均明显降低,以联合用药组最为明显(P均0.000)。Bevacizumab和吉西他滨单药均能下调Bcl-2的表达,两药联合下调作用明显增强。结论:Bevacizumab联合吉西他滨能增强对人肝癌裸鼠移植瘤的生长及微血管生成的抑制作用,其机制可能与调控Bcl-2的表达有关。     

An RGD-Modified MRI-Visible Polymeric Vector for Targeted siRNA Delivery to Hepatocellular Carcinoma in Nude Mice

RNA interference (RNAi) has significant therapeutic promise for the genetic treatment of hepatocellular carcinoma (HCC). Targeted vectors are able to deliver small interfering RNA (siRNA) into HCC cells with high transfection efficiency and stability. The tripeptide arginine glycine aspartic acid (RGD)-modified non-viral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles (RGD-PEG-g-PEI-SPION), was constructed as a magnetic resonance imaging (MRI)-visible nanocarrier for the delivery of Survivin siRNA targeting the human HCC cell line Bel-7402. The biophysical characterization of the RGD-PEG-g-PEI-SPION was performed. The RGD-modified complexes exhibited a higher transfection efficiency in transferring Survivin siRNA into Bel-7402 cells compared with a non-targeted delivery system, which resulted in more significant gene suppression at both the Survivin mRNA and protein expression levels. Then, the level of caspase-3 activation was significantly elevated, and a remarkable level of tumor cell apoptosis was induced. As a result, the tumor growth in the nude mice Bel-7402 hepatoma model was significantly inhibited. The targeting ability of the RGD-PEG-g-PEI-SPION was successfully imaged by MRI scans performed in vitro and in vivo. Our results strongly indicated that the RGD-PEG-g-PEI-SPION can potentially be used as a targeted non-viral vector for altering gene expression in the treatment of hepatocellular carcinoma and for detecting the tumor in vivo as an effective MRI probe.     

Liraglutide Ameliorates Cerebral Ischemia in Mice via Antipyroptotic Pathways

Neurochemical Research - It was recently shown that pyroptosis, an inflammatory form of programmed cell death, is critically involved in the pathogenesis of ischemic stroke. Liraglutide (Lg) is a...     

直接注射法制作ICR 小鼠肝癌原位移植瘤模型

目的:培养鼠肝癌H22细胞,直接注射法制作ICR小鼠肝癌原位移植瘤,为后续实验奠定基础。方法:鼠肝癌H22细胞体外培养,将调整好的对数生长期的肝癌细胞直接注射小鼠肝脏,2周后解剖观察,并进行组织HE染色。结果:所有实验小鼠均可见肿瘤生长,HE染色示肝细胞肝癌。结论:直接注射法制作ICR小鼠肝癌原位移植瘤模型简便易行,值得推广应用。     

直接注射法制作ICR小鼠肝癌原位移植瘤模型

目的：培养鼠肝癌H22细胞,直接注射法制作ICR小鼠肝癌原位移植瘤,为后续实验奠定基础。方法：鼠肝癌H22细胞体外培养,将调整好的对数生长期的肝癌细胞直接注射小鼠肝脏,2周后解剖观察,并进行组织HE染色。结果：所有实验小鼠均可见肿瘤生长,HE染色示肝细胞肝癌。结论：直接注射法制作ICR小鼠肝癌原位移植瘤模型简便易行,值得推广应用。     

Arginase Inhibition Ameliorates Hepatic Metabolic Abnormalities in Obese Mice

Objectives

We examined whether arginase inhibition influences hepatic metabolic pathways and whole body adiposity in diet-induced obesity.

Methods and Results

After obesity induction by a high fat diet (HFD), mice were fed either the HFD or the HFD with an arginase inhibitor, N^ω-hydroxy-nor-L-arginine (nor-NOHA). Nor-NOHA significantly prevented HFD-induced increases in body, liver, and visceral fat tissue weight, and ameliorated abnormal lipid profiles. Furthermore, nor-NOHA treatment reduced lipid accumulation in oleic acid-induced hepatic steatosis in vitro. Arginase inhibition increased hepatic nitric oxide (NO) in HFD-fed mice and HepG2 cells, and reversed the elevated mRNA expression of hepatic genes in lipid metabolism. Expression of phosphorylated 5′ AMPK-activated protein kinase α was increased by arginase inhibition in the mouse livers and HepG2 cells.

Conclusions

Arginase inhibition ameliorated obesity-induced hepatic lipid abnormalities and whole body adiposity, possibly as a result of increased hepatic NO production and subsequent activation of metabolic pathways involved in hepatic triglyceride metabolism and mitochondrial function.     

Cellulose Supplementation Early in Life Ameliorates Colitis in Adult Mice

Decreased consumption of dietary fibers, such as cellulose, has been proposed to promote the emergence of inflammatory bowel diseases (IBD: Crohn disease [CD] and ulcerative colitis [UC]) where intestinal microbes are recognized to play an etiologic role. However, it is not known if transient fiber consumption during critical developmental periods may prevent consecutive intestinal inflammation. The incidence of IBD peaks in young adulthood indicating that pediatric environmental exposures may be important in the etiology of this disease group. We studied the effects of transient dietary cellulose supplementation on dextran sulfate sodium (DSS) colitis susceptibility during the pediatric period in mice. Cellulose supplementation stimulated substantial shifts in the colonic mucosal microbiome. Several bacterial taxa decreased in relative abundance (e.g., Coriobacteriaceae [p = 0.001]), and other taxa increased in abundance (e.g., Peptostreptococcaceae [p = 0.008] and Clostridiaceae [p = 0.048]). Some of these shifts persisted for 10 days following the cessation of cellulose supplementation. The changes in the gut microbiome were associated with transient trophic and anticolitic effects 10 days following the cessation of a cellulose-enriched diet, but these changes diminished by 40 days following reversal to a low cellulose diet. These findings emphasize the transient protective effect of dietary cellulose in the mammalian large bowel and highlight the potential role of dietary fibers in amelioration of intestinal inflammation.     

透射电镜观察p21-（HBsAg/HBsAg）转基因小鼠肝癌细胞凋亡

目的观察p21HBsAg/HBsAg转基因小鼠肝癌细胞凋亡。方法用透射电镜观察p21HBsAg/HBsAg转基因小鼠肝癌细胞凋亡的形态学改变。结果细胞凋亡早期,细胞核染色体发生边集,核形不规整,核膜表面凹凸;凋亡中期,核内染色质凝聚,趋边呈月牙状,核膜孔消失,核膜呈波纹状皱缩;凋亡晚期,核固缩,细胞膜出芽形成小泡,可见凋亡小体。结论p21HBsAg/HBsAg转基因小鼠肝癌细胞凋亡具有典型性病变特征,是研究HBV感染诱发肝癌发病机理的合适动物模型。     

A Mitochondrion-Targeted Antioxidant Ameliorates Isoflurane-Induced Cognitive Deficits in Aging Mice

Isoflurane possesses neurotoxicity and can induce cognitive deficits, particularly in aging mammals. Mitochondrial reactive oxygen species (mtROS) have been linked to the early pathogenesis of this disorder. However, the role of mtROS remains to be evaluated due to a lack of targeted method to treat mtROS. Here, we determined in aging mice the effects of the mitochondrion-targeted antioxidant SS-31, on cognitive deficits induced by isoflurane, a general inhalation anesthetic. We further investigated the possible mechanisms underlying the effects of SS-31 on hippocampal neuro-inflammation and apoptosis. The results showed that isoflurane induced hippocampus-dependent memory deficit, which was associated with mitochondrial dysfunction including reduced ATP contents, increased ROS levels, and mitochondrial swelling. Treatment with SS-31 significantly ameliorated isoflurane-induced cognitive deficits through the improvement of mitochondrial integrity and function. Mechanistically, SS-31 treatment suppressed pro-inflammatory responses by decreasing the levels of NF-κB, NLRP3, caspase 1, IL-1β, and TNF-α; and inhibited the apoptotic pathway by decreasing the Bax/Bcl-2 ratio, reducing the release of cytochrome C, and blocking the cleavage of caspase 3. Our results indicate that isoflurane-induced cognitive deficits may be attenuated by mitochondrion-targeted antioxidants, such as SS-31. Therefore, SS-31 may have therapeutic potentials in preventing injuries from oxidative stresses that contribute to anesthetic-induced neurotoxicity.     

Spontaneous Rupture of Hepatocellular Carcinoma

Forty-two cases of ruptured hepatoma with intra-abdominal haemorrhage were seen over a period of eight years in the professorial surgical unit at the Queen Mary Hospital, Hong Kong. In all, 207 cases of liver cancer were seen during this period, giving an incidence of rupture of 14·5%. There were 37 men and five women. The clinical features were the sudden onset of pain with shock and the presence of blood in the peritoneal cavity. The diagnosis was made before operation on suspicion and by a process of elimination. In cases of doubt paracentesis abdominis was performed to determine whether there was blood in the peritoneal cavity. Treatment was directed to control of haemorrhage and resection.The prognosis, although extremely poor, is no worse than the hepatocellular carcinoma that has not ruptured. There was one long-term survivor—a patient who had undergone an extensive resection. The man was alive and well after more than five years.