Generating Customized Transgene Landing Sites and Multi-Transgene Arrays in Drosophila Using phiC31 Integrase

Transgenesis in numerous eukaryotes has been facilitated by the use of site-specific integrases to stably insert transgenes at predefined genomic positions (landing sites). However, the utility of integrase-mediated transgenesis in any system is constrained by the limited number and variable expression properties of available landing sites. By exploiting the nonstandard recombination activity exhibited by a phiC31 integrase mutant, we developed a rapid and inexpensive method for isolating landing sites that exhibit desired expression properties. Additionally, we devised a simple technique for constructing arrays of transgenes at a single landing site, thereby extending the utility of previously characterized landing sites. Using the fruit fly Drosophila melanogaster, we demonstrate the feasibility of these approaches by isolating new landing sites optimized to express transgenes in the nervous system and by building fluorescent reporter arrays at several landing sites. Because these strategies require the activity of only a single exogenous protein, we anticipate that they will be portable to species such as nonmodel organisms, in which genetic manipulation is more challenging, expediting the development of genetic resources in these systems.     

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。     

Oral Administered Particulate Yeast-Derived Glucan Promotes Hepatitis B Virus Clearance in a Hydrodynamic Injection Mouse Model

Hepatitis B virus (HBV) persistent infection is associated with ineffective immune response for the clearance of virus. Immunomodulators represent an important class of therapeutics, which potentially could be beneficial for the treatment of HBV infection. The particulate yeast-derived glucan (PYDG) has been shown to enhance the innate and adaptive immune responses. We therefore, assessed the efficacy of PYDG in enhancing HBV specific immune responses by employing the hydrodynamic injection-based (HDI) HBV transfection mouse model. Mice were intragatric administered PYDG daily for 9 weeks post pAAV/HBV1.2 hydrodynamic injection. PYDG treatment significantly promoted HBV DNA clearance and production of HBsAb compared to control mice. PYDG treatment resulted in recruitment of macrophages, dendritic cells (DCs) and effector T cells to the liver microenvironment, accompanied by a significantly augmented DCs maturation and HBV-specific IFN-γ and TNF-α production by T cell. In addition, enhanced production of Th1 cytokines in liver tissue interstitial fluid (TIF) was associated with PYDG administration. Live imaging showed the accumulation of PYDG in the mouse liver. Our results demonstrate that PYDG treatment significantly enhances HBV-specific Th1 immune responses, accompanied by clearance of HBV DNA, and therefore holds promise for further development of therapeutics against chronic hepatitis B.     

Characterization of the Treg Response in the Hepatitis B Virus Hydrodynamic Injection Mouse Model

Regulatory T cells (Tregs) play an important role in counter-regulating effector T cell responses in many infectious diseases. However, they can also contribute to the development of T cell dysfunction and pathogen persistence in chronic infections. Tregs have been reported to suppress virus-specific T cell responses in hepatitis B virus (HBV) infection of human patients as well as in HBV animal models. However, the phenotype and expansion of Tregs has so far only been investigated in other infections, but not in HBV. We therefore performed hydrodynamic injections of HBV plasmids into mice and analyzed the Treg response in the spleen and liver. Absolute Treg numbers significantly increased in the liver but not the spleen after HBV injection. The cells were natural Tregs that surprisingly did not show any activation or proliferation in response to the infection. However, they were able to suppress effector T cell responses, as selective depletion of Tregs significantly increased HBV-specific CD8+ T cell responses and accelerated viral antigen clearance. The data implies that natural Tregs infiltrate the liver in HBV infection without further activation or expansion but are still able to interfere with T cell mediated viral clearance.     

Radiofrequency Ablation Is Superior to Ethanol Injection in Early-Stage Hepatocellular Carcinoma Irrespective of Tumor Size

Background

Randomized trials suggest that radiofrequency ablation (RFA) may be more effective than percutaneous ethanol injection (PEI) in the treatment of hepatocellular carcinoma (HCC). However, the survival advantage of RFA needs confirmation in daily practice.

Methods

We conducted a population-based cohort study using the Taiwan Cancer Registry, National Health Insurance claim database and National Death Registry data from 2004 through 2009. Patients receiving PEI or RFA as first-line treatment for newly-diagnosed stage I-II HCC were enrolled.

Results

A total of 658 patients receiving RFA and 378 patients receiving PEI treatment were included for final analysis. The overall survival (OS) rates of patients in the RFA and PEI groups at 5-year were 55% and 42%, respectively (p < 0.01). Compared to patients that received PEI, those that received RFA had lower risks of overall mortality and first-line treatment failure (FTF), with adjusted hazard ratios (HRs) [95% confidence interval (CI)] of 0.60 (0.50-0.73) for OS and 0.54 (0.46-0.64) for FTF. The favorable outcomes for the RFA group were consistently significant for patients with tumors > 2 cm as well as for those with tumors < 2 cm. Consistent results were also observed in other subgroup analyses defined by gender, age, tumor stage, and co-morbidity status.

Conclusion

RFA provides better survival benefits than PEI for patients with unresectable stage I-II HCC, irrespective of tumors > 2 cm or ≤ 2 cm, in contemporary clinical practice.     

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.     

Germline Genetic Variation Modulates Tumor Progression and Metastasis in a Mouse Model of Neuroendocrine Prostate Carcinoma

Neuroendocrine (NE) differentiation has gained increased attention as a prostate cancer (PC) prognostic marker. The aim of this study is to determine whether host germline genetic variation influences tumor progression and metastasis in C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of aggressive NEPC. TRAMP mice were crossed to the eight progenitor strains of the Collaborative Cross recombinant inbred panel to address this. Tumor growth and metastasis burden were quantified in heterozygous transgene positive F1 male mice at 30 weeks of age. Compared to wild-type C57BL/6J-Tg(TRAMP)824Ng/J males, TRAMP x CAST/EiJ, TRAMP x NOD/ShiLtJ and TRAMP x NZO/HlLtJ F1 males displayed significant increases in tumor growth. Conversely, TRAMP x WSB/EiJ and TRAMP x PWK/PhJ F1 males displayed significant reductions in tumor growth. Interestingly, despite reduced tumor burden, TRAMP x WSB/EiJ males had an increased nodal metastasis burden. Patterns of distant pulmonary metastasis tended to follow the same patterns as that of local dissemination in each of the strains. All tumors and metastases displayed positive staining for NE markers, synaptophysin, and FOXA2. These experiments conclusively demonstrate that the introduction of germline variation by breeding modulates tumor growth, local metastasis burden, and distant metastasis frequency in this model of NEPC. These strains will be useful as model systems to facilitate the identification of germline modifier genes that promote the development of aggressive forms of PC.     

Hepatocyte-Specific Arid1a Deficiency Initiates Mouse Steatohepatitis and Hepatocellular Carcinoma

ARID1A, encoding a subunit of chromatin remodeling SWI/SNF complexes, has recently been considered as a new type of tumor suppressor gene for its somatic mutations frequently found in various human tumors, including hepatocellular carcinoma (HCC). However, the role and mechanism of inactivated ARID1A mutations in tumorigenesis remain unclear. To investigate the role of ARID1A inactivation in HCC pathogenesis, we generated hepatocyte-specific Arid1a knockout (Arid1a ^LKO) mice by crossing mice carrying loxP-flanked Arid1a exon 8 alleles (Arid1a ^f/f) with albumin promoter-Cre transgenic mice. Significantly, the hepatocyte-specific Arid1a deficiency results in mouse steatohepatitis and HCC development. In Arid1a ^LKO mice, we found that innate immune cells, including F4/80+ macrophages and CD11c+ neutrophil cells, infiltrate into the liver parenchyma, accompanied by the increased tumor necrosis factor (TNF)-α and interleukin (IL)-6, and activation of STAT3 and NF-κB pathways. In conclusion, hepatocyte-specific Arid1a deficiency could lead to mouse steatohepatitis and HCC development. This study provides an alternative mechanism by which Arid1a deficiency contributes to HCC tumorigenesis.     

Immunosuppressive Drugs Modulate the Replication of Hepatitis B Virus (HBV) in a Hydrodynamic Injection Mouse Model

Hepatitis B virus (HBV) reactivation and recurrence are common in patients under immunosuppression and can be controlled by hepatitis B immunoglobulin, antivirals, and hepatitis B vaccine. However, the detailed analysis of HBV infection under immunosuppression is essential for the prophylaxis and therapy for HBV reactivation and recurrence. In this study, HBV replication and T cell responses were analyzed in a HBV-transfected mouse model under immunosuppressive therapy. During the treatment, HBV replication was at a high level in mice treated with dexamethasone, cyclosporine, and cyclophosphamide, whereas was terminated in mice treated with mycophenolate mofetil. After the withdrawal, HBV replication was at low or high levels in the dexamethasone-treated mice or in both cyclosporine- and cyclophosphamide-treated mice. The early withdrawal of cyclosporine allowed the recovery of suppressed T cell responses and led to subsequent HBV clearance, while the adoptive immune transfer to the mice with HBV persistence led to HBV suppression. Taken together, long-term HBV persistence under immunosuppression depends on the immunosuppressive drugs used and on the treatment duration and is mediated by the suppressed intrahepatic CD8 T cell response. These data may be helpful for individualized immunosuppressive therapy in patients with high risk of HBV reactivation and recurrence, and the mouse system is suitable for studying HBV reactivation and recurrence under immunosuppression.     

肝细胞癌的微环境研究进展

越来越多的研究表明,肿瘤细胞与其周围微环境的交互作用是肿瘤发生、上皮间质转化、肿瘤浸润和转移的关键调节因素．肝细胞癌的微环境可以分为细胞组分和非细胞组分．主要的细胞组分包含：肝星形细胞、肿瘤相关的纤维母细胞、免疫细胞和肝窦内皮细胞等．非细胞组分包含：胞外基质蛋白、酶类、各种生长因子和炎症因子等．综述了近年来肝细胞癌的微环境研究进展,分别从细胞组分和非细胞组分及其之间的相互作用角度对肝细胞癌微环境作一介绍．     

Activated Human Hepatic Stellate Cells Promote Growth of Human Hepatocellular Carcinoma in a Subcutaneous Xenograft Nude Mouse Model

Tumor cell microenvironment defines cancer development, also in hepatocellular carcinoma (HCC). Hepatic stellate cells (HSCs) are believed to be the key contributors to tumor microenvironment in HCC, yet their precise role in cancer progression is still unclear. The aim of this study was to determine the effect of human HSCs on progression of HCC using a subcutaneous xenograft nude mouse model. Nude mice were stratified to receive subcutaneous injections of human HCC cell line HepG2 and human HSC line LX-2 (HepG2 + LX-2), HepG2 alone, LX-2 alone, or phosphate-buffered saline. Tumor growth was assessed by measuring tumor size. After 30 days, final tumor size, weight, and histology were assessed. Compared with mice that were only injected HepG2 cells, mice injected with HepG2 + LX-2 exhibited more rapid tumor growth, increased tumor size and weight, higher tumor cell numbers due to increased proliferation and reduced apoptosis, increased fibrotic bands containing LX-2 cells, and increased tumor angiogenesis. In conclusion, HSCs play a significant role in promotion of HCC growth.     

半乳糖基壳聚糖/5-氟尿嘧啶纳米粒抑制小鼠原位肝癌移植模型的实验研究

目的:观察半乳糖基壳聚糖(GC)/5-氟尿嘧啶(5-FU)纳米粒抑制小鼠原位肝癌移植模型的疗效及机制.方法:合成GC/5-FU纳米粒,建立小鼠原位肝癌移植模型,通过动物实验观察GC/5-FU纳米粒治疗小鼠原位肝癌移植模型的疗效,通过原位末端标记法(TUNEL法)检测肝癌的凋亡.结果:GC/5-FU纳米粒成功合成,呈规则的球形,表面光滑,大小均匀,纳米粒间无粘连.实验结束时,小鼠的肝癌组织称重,进行方差分析发现GC/5-Fu的瘤重为(0.4361±0.1153)g,5-Fu组为(0.7932±0.1283)g,GC为(1.3989±0.2125)g,和对照组为(1.5801±0.2821)g,4组瘤重之间差异有显著的统计学意义(P＜0.01).GC/5-FU组和5-FU组的瘤重明显小于GC组及对照组,差异有统学意义(P＜0.01);GC/5-FU的瘤重又显著小于5-Fu组(P＜0.01).观察各组小鼠的生存期发现对照组的中位生存时间为12d,GC组为13d,5-FU为17d,而GC/5-FU组最高为30d.GC/5-FU组的生存时间最长.通过TUNEL法观察肝癌细胞的凋亡发现GC/5-FU组的平均凋亡指数(AI)为21.34％较5-FU组的14.74％明显增高(P＜0.05),均较GC组和对照组明显增高,而GC组和对照组间的AI无明显差异(P＞0.05).结论:GC/5-FU纳米粒在体内对小鼠原位肝癌具有明显抑制作用,较5-FU明显增强.其机理可能与GC能通过细胞膜将5-FU从细胞外转移到细胞内,增强5-FU对肝癌细胞的凋亡.     

Differential Impact of Tumor Suppressor Pathways on DNA Damage Response and Therapy-Induced Transformation in a Mouse Primary Cell Model

The RB and p53 tumor suppressors are mediators of DNA damage response, and compound inactivation of RB and p53 is a common occurrence in human cancers. Surprisingly, their cooperation in DNA damage signaling in relation to tumorigenesis and therapeutic response remains enigmatic. In the context of individuals with heritable retinoblastoma, there is a predilection for secondary tumor development, which has been associated with the use of radiation-therapy to treat the primary tumor. Furthermore, while germline mutations of the p53 gene are critical drivers for cancer predisposition syndromes, it is postulated that extrinsic stresses play a major role in promoting varying tumor spectrums and disease severities. In light of these studies, we examined the tumor suppressor functions of these proteins when challenged by exposure to therapeutic stress. To examine the cooperation of RB and p53 in tumorigenesis, and in response to therapy-induced DNA damage, a combination of genetic deletion and dominant negative strategies was employed. Results indicate that loss/inactivation of RB and p53 is not sufficient for cellular transformation. However, these proteins played distinct roles in response to therapy-induced DNA damage and subsequent tumorigenesis. Specifically, RB status was critical for cellular response to damage and senescence, irrespective of p53 function. Loss of RB resulted in a dramatic evolution of gene expression as a result of alterations in epigenetic programming. Critically, the observed changes in gene expression have been specifically associated with tumorigenesis, and RB-deficient, recurred cells displayed oncogenic characteristics, as well as increased resistance to subsequent challenge with discrete therapeutic agents. Taken together, these findings indicate that tumor suppressor functions of RB and p53 are particularly manifest when challenged by cellular stress. In the face of such challenge, RB is a critical suppressor of tumorigenesis beyond p53, and RB-deficiency could promote significant cellular evolution, ultimately contributing to a more aggressive disease.     

流体动力学法表达乙肝病毒X蛋白小鼠模型的建立

目的建立表达乙肝病毒X蛋白的小鼠模型。方法实验动物分成模型组和对照组,模型组利用已构建的含有HBX基因的真核表达质粒pcDNA3.1（＋）-HBX,对照组以等量生理盐水代替,采用流体动力学法将质粒经尾静脉高压注入小鼠体内,24h后取小鼠肝组织,行免疫荧光、RT-PCR和Western blot法从不同水平检测HBX在小鼠肝组织内的表达情况。结果模型组小鼠RT-PCR显示肝组织内有HBX mRNA的存在,免疫荧光和Western blot检测均有HBX蛋白的表达;对照组小鼠则无HBX表达。结论成功建立了表达乙肝病毒X蛋白小鼠模型,为进一步探讨HBX蛋白在动物体内的生物学作用提供实验基础。     

PARP2表达对肝细胞癌模型小鼠肿瘤生长及化疗敏感性影响及其机制研究

摘要 目的：研究聚[ADP-核糖]聚合酶2（Poly[ADP-ribose]polymerase 2, PAPR2）表达对干细胞癌模型小鼠肿瘤生长和对化疗药物敏感性的影响。方法：未转染的（对照组）、空载质粒转染（空载组）和si-PARP2转染（si-PARP2组）的Huh7细胞作为研究对象,比较体外化疗药物对不同Huh7细胞克隆形成数目和凋亡率。通过腋下皮下注射不同Huh7建立肝细胞癌模型小鼠,采用结晶紫染色观察并计数克隆形成数目;Annexin V-FITC细胞凋亡检测试剂盒检测Huh7细胞凋亡率;排水法测定肿瘤组织体积;免疫印迹法检测PARP2, B淋巴细胞瘤-2（B-cell lymphoma-2, Bcl2）和Bcl-2-Associated X蛋白（Bcl-2-Associated X, Bax）蛋白表达。结果：在体内外,转染si-PARP2均显著降低肝癌细胞中PARP2蛋白表达（P<0.05）。在体外,对照组和空载组Huh7细胞形成的细胞克隆数目和化疗药物诱导的凋亡率比较无显著差异（P>0.05）;si-PARP2组Huh7细胞形成的细胞克隆数目显著低于对照组和空载组（P<0.05）,而化疗药物引起的细胞凋亡率显著高于空白组和对照组（P<0.05）。在体内,si-PARP2组小鼠肝癌移植瘤重量和体积均显著低于对照组和空载组（P<0.05）。此外,与对照组和空载组相比,肝癌移植瘤组织内细胞经阿霉素治疗后细胞凋亡率、Bax和cleaved-caspase 3蛋白表达水平显著增加（P<0.05）,而Bcl2蛋白表达水平显著降低（P<0.05）。结论：PAPR2基因敲低可以显著抑制肝癌模型肿瘤生长和增强肝癌细胞对化疗药物的敏感性,其机制可能与PAPR2基因敲低促进肝癌细胞凋亡有关。     

荧光标记技术在肿瘤模型研究中的应用

目的利用绿色荧光小鼠和红色荧光蛋白标记肿瘤细胞,建立荧光标记的小鼠肿瘤模型,并建立活体荧光成像和荧光显微镜成像在整体和细胞水平直接观察肿瘤的技术。方法将小鼠B16黑色素瘤细胞接种到绿色荧光蛋白转基因小鼠皮下,建立GFP小鼠肿瘤模型。以红色荧光蛋白作为标记基因导入小鼠黑色素瘤细胞B16细胞,建立稳定表达红色荧光蛋白的细胞株。将表达红色荧光蛋白B16细胞接种到绿色荧光转基因小鼠皮下,建立双荧光小鼠肿瘤模型。用荧光显微镜和活体荧光成像系统检测小鼠肿瘤的发生发展。结果分别建立了GFP小鼠肿瘤模型和双色荧光小鼠肿瘤模型。利用活体荧光影像仪可以观察双色荧光小鼠模型中受体绿色荧光组织和红色荧光移植肿瘤相互融合。利用荧光显微镜,可以观察到肿瘤内绿色荧光标记的来源于受体小鼠的血管和免疫细胞。经香菇多糖刺激的GFP小鼠肿瘤模型的移植瘤组织中,来源于受体小鼠绿色荧光标记的免疫细胞明显多于经生理盐水刺激的对照小鼠。结论利用绿色荧光小鼠和红色荧光RFP标记肿瘤细胞建立荧光标记的小鼠肿瘤模型,采用活体荧光成像仪和荧光显微镜可在整体和细胞水平直接观察肿瘤的生长以及肿瘤与宿主的相互作用。