阳离子脂质体介导的基因转移机制

背景：与病毒载体相比,许多天然与合成的阳离子类脂以脂质体的形式用于基因转移,具有无免疫原性、易生产、质粒免受核酸酶降解和无致瘤性等优点,并且作为病毒载体的有效替代物,阳离子脂质体能用于细胞的体内和体外转染。 目的：介绍阳离子脂质体介导的基因转移机制研究进展。 方法：由第一作者用计算机检索中国期刊全文数据库(CNKI：1987/2010)和PubMed (1987/2010)数据库,检索词分别为“基因治疗、阳离子脂质体、基因转移、机制”和“gene therapy, cationic liposome, gene transfer, mechanism”,语言分别设定为中文和英文。从阳离子脂质体基因转染和基因转移机制进行总结,综述了阳离子脂质体介导的基因转移机制。 结果与结论：共检索到108篇,按纳入和排除标准对文献进行筛选,共纳入20篇文章。综述了阳离子脂质体介导的基因转移机制,包括阳离子脂质体/DNA复合物的形成、细胞吸收、内含体释放和复合物解体以及细胞核摄入等方面的研究内容。结果提示,对类脂构效关系和基因转移机制的研究,是提高阳离子脂质体转染效率和优化基因治疗的关键。     

脂质体介导IFNγ基因治疗小鼠肝癌的研究

以大肠杆菌β－ｇａｌ基因为报告基因，优化了３种阳离子脂质本的转染条件，评估了其转染效率。用构建的含腺相关病毒反向末端重复序列的质粒表达载体，经Ｄｏｓｐｅｒ介导将小鼠ＩＦＮγ基因导入ＭＭ４５Ｔ．Ｌｉ细胞，体外有效地表达ＩＦＮγ。瘤体内注射Ｄｏｓｐｅｒ－ｐＡＩ－ｍＩＦＮγ复合物可明显报制肿瘤生长，荷瘤小鼠生存期延长。     

脂质体介导RNAi的研究

本文选用阳离子脂质体Lipofectamine 2000为基因载体,使用沉默荧光素酶基因的小干扰RNA(siRNA)进行体外RNA干扰(RNAi),对阳离子脂质体转运siRNA的效率进行研究。首先以阳离子脂质体运载质粒DNA转染细胞,并以延滞实验对阳离子脂质体与siRNA的结合能力进行检测;然后通过基因载体将荧光素酶基因载入细胞,转运不同浓度的siRNA对其进行沉默,应用酶标仪对荧光素酶基因的表达进行分析,并用MTT法对转染后细胞存活率进行检测。结果表明:阳离子脂质体Lipofectamine 2000可以高效转运质粒DNA,并可与siRNA很好地结合。在较低的siRNA浓度下,对荧光素酶基因的沉默率达到较高的水平,转运siRNA转染细胞对细胞活性的影响很小,但细胞存活率随着siRNA浓度的增加而逐渐降低。通过优化实验条件,阳离子脂质体转运较低浓度的siRNA即可高效沉默目的基因。     

上调RI对小鼠黑色素瘤B16-F10细胞EMT及转移的影响

目的研究核糖核酸酶抑制因子基因表达对小鼠黑色素瘤B16-F10细胞EMT及转移的影响。方法构建RI真核表达质粒p IRES2-EGFP-RI,稳定转染B16-F10细胞。RT-PCR、Western blot和免疫荧光检测RI的表达;HE染色及相差显微镜观察细胞形态;FITC标记的鬼笔环肽染色,激光共聚焦观察细胞骨架;黏附实验、划痕实验和Transwell法检测细胞黏附、迁移和侵袭能力的变化;Western blot检测EMT及转移相关蛋白的表达;分别将各组B16-F10细胞眼眶静脉注射到c57/BL小鼠,建立肺转移动物模型,注射3周后处死小鼠。取肺称重,在解剖镜下计数肺转移结节数;肺组织切片HE染色观察肿瘤细胞转移;免疫组化检测肺转移瘤组织中转移及EMT相关蛋白的表达。结果 RI表达上调后,细胞由间质型向上皮型转换,细胞骨架重排;B16-F10-RI细胞组黏附、迁移和侵袭能力下降;与对照组相比,B16-F10-RI细胞中MMP2、MMP9、snail、slug、vimentin、twist和N-cadherin的表达明显降低,而E-cadherin,nm23-H1蛋白的表达明显增加(P0.01或P0.01)。实验组与对照组比小鼠肺的转移结节明显减少,同时EMT及转移相关蛋白在瘤组织中表达与体外细胞一致。结论上调核糖核酸酶抑制因子能够显著抑制B16-F10细胞EMT及侵袭、转移,RI可望作为治疗黑色素瘤的靶蛋白。     

气管内吸入IL-12重组腺病毒对实验性肺转移癌的治疗作用

为研究气道内应用IL 12重组腺病毒 (AdIL 12 )对实验性肺转移癌的治疗作用。我们在小鼠气管内滴入AdIL 12 ,以ELISA法测定局部及外周血IL 12及相关因子水平 ,在C5 7BL/ 6小鼠尾静脉注射Lewis肺癌 3LL细胞建立肺转移癌模型 ,观察气管内应用AdIL 12对实验性肺转移癌的治疗作用 ,包括肺转移结节、动物存活期、生存率变化等 ,比较自然杀伤活性(NK )和特异性细胞毒 (CTL )活性的差异。结果显示 :(1)气管应用AdIL 12后肺灌洗液中测到IL 12 ,且浓度高于外周血水平 (P <0 0 1) ,对照组肺灌洗液及外周血中均未检出 ;(2 )气道应用AdIL 12对实验性肺转移癌有治疗作用 ,可使肺转移结节减少、动物的生存期延长及存活率升高 ,并增强了NK、CTL活性。本研究表明气道内应用AdIL 12对实验性肺转移癌有治疗作用。     

电脉冲介导hIL-10基因转移治疗骨关节炎的实验研究

目的 研究电脉冲介导人白细胞介素10裸质粒肌肉注射对骨关节炎的治疗作用。方法 兔右膝关节髌旁内侧切口,行内侧半月板切除术制作骨关节炎模型,4周后按分组于右侧股四头肌注射pcDI或pcDI-IL10质粒,然后立即于注射部位施以电压200v(电极间距离为1cm),波宽40ms,脉冲次数6次和频率1Hz的电脉冲。共注射2次,每次间隔2周。按分组情况分别于术后4周、8周处死动物,行解剖显微镜观察及关节软骨、滑膜病理检查。结果 pcDI-IL10质粒注射组的关节软骨破坏程度较注射pcDI组轻(P<0.01),而较4周处死组稍重。结论 电脉冲介导肌肉pcDI-IL10转移可以延缓骨关节炎的发展。     

脂质体介导的IL-2基因疗法增强机体巨噬细胞免疫功能的研究

本文报道了脂质体包裹的ＩＬ－２基因直接腹腔内注射后，巨噬细胞在介导该基因疗法中的作用及其功能变化。结果发现巨噬细胞是ＩＬ－２基因表达的主要靶细胞；巨噬细胞的吞噬功能、抗原提呈能力、Ｆｃ受体和Ｉａ抗原表达以及分泌ＩＬ－１、ＴＮＦ水平及杀伤肿瘤细胞的活性均明显提高。巨噬细胞功能的上述变化表明　，通过腹腔途径应用脂质体介导的ＩＬ－２基因疗法治疗腹部肿瘤可能具有一定的可行性。     

基因治疗骨骼肌注射方法的实验研究

骨骼肌具有合成蛋白的强大能力,并且合成的蛋白可进入血循环.它不仅可以表达外源性基因,而且表达时间长.近年来,基因治疗的骨骼肌注射方法是研究热点之一.随着研究的深入,此方法有可能成为一种实用、具有前景性的基因治疗方法.     

变应性鼻炎和哮喘患者血清IL-5、IL-15及IL-18的水平研究

目的探讨IL-5、IL-15和IL-18在变应性鼻炎、支气管哮喘、变应性鼻炎合并哮喘疾病中的作用。方法采用双抗体夹心ELISA测定法对33例支气管哮喘患者、35例变应性鼻炎患者、35例变应性鼻炎合并哮喘的患者及35例正常健康查体者血清中IL-5、IL-15和IL-18的水平进行检测。结果支气管哮喘、变应性鼻炎、变应性鼻炎合并哮喘的患者血清中IL-5、IL-15和IL-18水平较正常对照组升高（P〈0．01）,IL-5、IL-15,IL-18水平在变应性鼻炎合并哮喘组均高于鼻炎组与和哮喘组;鼻炎组IL-5水平高于哮喘组（P=0．003）,哮喘组IL-18水平高于鼻炎组（P=0．001）。结论IL-5、IL-15和IL-18参与了过敏性鼻炎和哮喘的发病过程;变应性鼻炎合并哮喘的炎症程度较高;哮喘和鼻炎因发病部位不同炎症反应也有不同。     

Novel murine tumour models depend on strain and route of inoculation

This study describes variations in tumour growth patterns which occur when changes in the routes of inoculation and mouse strain are used to introduce tumours into established murine model systems that are known to vary in location and aggression. Intraperitoneal, subcutaneous, intravenous and hydrodynamic inoculations of B16F10 cells were compared among CD‐1, C57BL/6 and Balb/c mice. Most surprisingly, allogeneic tumour growth in Balb/c mice after intravenous and hydrodynamic inoculation of B16F10 cells was faster than tumour growth in the syngeneic C57BL/6 mice. These and other variations in the tumour growth patterns described here can help provide the researcher with more experimental control when planning to use the optimal tumour model for any particular study.     

B16F10 melanoma cell colonization of mouse lung is enhanced by partial pneumonectomy

Surgical resection of lung tissue is employed clinically as a therapy for pulmonary metastases; however, local cancer recurrence is a frequent post-surgical complication. In a variety of small mammals, left pneumonectomy (PNX) initiates rapid compensatory hyperplasia of the remnant lung lobes restoring normal tissue mass, structure and function. Post-PNX compensatory lung growth is known to promote lung tumor formation in carcinogen-treated mice. The present study tests the hypothesis that PNX enhances experimental metastasis to lung. C57Bl/6 mice subjected to PNX were given an intravenous injection of B16F10 melanoma cells at various stages of compensatory lung growth. Animals injected with B16F10 cells during the linear phase of the response had 77% to 260% more pulmonary metastases than mice subjected to thoracotomy (P<0.01). Moreover, measurements of tumor area (mm²) revealed that PNX mice harbored a substantially larger lung tumor burden than control animals. Normalization of the tumor cell inoculum to lung mass yielded similar results. PNX had no effect on growth of sub-cutaneous B16F10 melanoma tumors, suggesting that experimental melanoma metastasis was enhanced by local alterations in the lung microenvironment. These results suggest (1) that PNX is a relevant model in which to investigate mechanisms of local cancer recurrence and, (2) melanoma cell metastatic potential is influenced, at least in part, by local factors modified during post-PNX compensatory lung growth. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of piperine on the inhibition of lung metastasis induced B16F-10 melanoma cells in mice

The effect of piperine on the inhibition of lung metastasis induced by B16F-10 melanoma cells was studied in C57BL/6 mice. Simultaneous administration of the compound with tumor induction produced a significant reduction (95.2%) in tumor nodule formation. Increased lung collagen hydroxyproline (22.37 μg/mg protein) in the metastasized lungs of the control animals compared to normal animals (0.95 μg/mg protein) was significantly reduced (2.59 μg/mg protein) in the piperine-treated animals. The high amount of uronic acid (355.83 μg/100 mg tissue) in the metastasized control animals was significantly reduced (65 μg/100 mg tissue) in the animals treated with piperine. Lung hexosamine content was also significantly reduced in the piperine-treated animals (0.98 mg/100 mg lyophilized tissue) compared to the untreated tumor-bearing animals (4.2 mg/100 mg lyophilized tissue). The elevated levels of serum sialic acid and serum gamma glutamyl transpeptidase activity in the untreated control animals was significantly reduced in the animals treated with piperine. The piperine-treated animals even survived the experiment (90 days). Histopathology of the lung tissue also correlated with the lifespan of the drug-treated animals. Our results demonstrate the antimetastatic activity of piperine, an alkaloid present in plants such as Piper nigrum and Piper longum. This revised version was published online in July 2006 with corrections to the Cover Date.     

Bile acids cycle disruption in patients with nasopharyngeal carcinoma promotes the elevation of interleukin-10 secretion

Background

Unclear pathogenesis existed for nasopharyngeal carcinoma.

Aims

to analyze the role of bile acids in the pathogenesis of nasopharyngeal carcinoma.

Methods

20 healthy volunteers and 20 patients with nasopharyngeal carcinoma were enrolled between January 1^st, 2013 and December 31^st, 2014. ESI-QTOF-MS analysis of serum was performed to find altered bile acids components. The biological function of changed bile acids was investigated using in vitro experiment.

Results

Compared with healthy volunteers, the level of DCA and GDCA exhibited higher abundance in patients with nasopharyngeal carcinoma (p<0.01). Furthermore, the biological function was investigated for the inhibition of DCA and GDCA towards the secretion of IL-10 by CD4+CD25− T cells. Both DCA and GDCA significantly inhibited the secretion of IL-10 by CD4+CD25− T cells. Furthermore, DCA+GDCA can show stronger inhibition towards the secretion of IL-10 than DCA and GDCA.

Conclusion

The inhibition of IL-10 secretion by elevated DCA and GDCA components in nasopharyngeal carcinoma patients is the inducer for nasopharyngeal carcinoma.     

气管内应用白介素2重组腺病毒免疫治疗肺转移癌

目的：研究气管内应用白介素2（IL－2）重组腺病毒（IL－2 gene recombinant adenovirus,AdIL-2)对实验性肺转移癌的治疗作用。方法：①小鼠气管内滴入AdIL-2,ELISA法测定局部及外周血IL－2及相关因子的水平。②C57BL／6小鼠尾静脉注射Lewis肺癌3LL细胞建立肺转移癌模型，观察气管内应用AdIL-2对实验性肺转移癌的治疗作用，包括肺转移结节、动物存活期、生存率变化等，并比较自然杀伤活性（Natural killer,NK)和特异性细胞毒（Cytotoxic T lymphocytes,CTL)活性的变化。结果：①气管应用AdIL-2后肺灌洗液中测到IL－2且高于外周血水平，对照组肺灌洗液及外周血中的水平明显低于实验组（P＜0．01）。②气管内滴入AdIL-2对实验性肺转移癌有治疗作用，可使肺转移结节减少、动物的生存期延长及存活率升高，同时对NK、CTL活性有增强作用（P＜0．01）。结论：气管内应用IL－2重组腺病毒可有效表达并对实验性肺转移癌有治疗作用，为肺部肿瘤的治疗提供了一种新方法。     

The inhibition of lung colonization of B16-F10 melanoma cells in EFA-deficient animals is related to enhanced apoptosis and reduced angiogenesis

Previous studies conducted in our laboratory showed that the reproduction of spontaneous and experimental metastases was reduced in host animals deprived of essential fatty acids (EFA). In the present study, we have explored the possibility whether apoptosis, proliferation, and angiogenesis might be involved in the antimetastatic effect of EFA deficiency. To this aim, in pulmonary colonies developed from B16-F10 cells in EFA-deficient animals or in animals fed a 5% corn oil diet, we performed an immunohistochemical analysis of bcl-2/bax proteins, PCNA, and VEGF and von Willebrand Factor (vWF), typical markers of apoptosis, proliferation, and angiogenesis, respectively. Apoptosis was also evaluated by detecting DNA fragments in metastatic cells. We found that the reduction of pulmonary colonies grown in EFA-deficient animals was associated with a high expression of apoptotic activity as revealed by the presence of apoptotic nuclei and a high immunoreactivity for bax. Cell proliferation seemed not to be influenced by EFA deficiency in view of the observation that PCNA was highly expressed in pulmonary colonies of control as well as EFA-deficient animals. Pulmonary colonies developed in EFA- deficient animals showed a lower expression of VEGF and a decreased microvessel density, indicating that a reduced angiogenesis contributes to the antimetastatic effects of EFA deficiency. Our analysis of the results invokes the possibility that a relationship between angiogenesis and apoptosis may account for the diminution of the development of experimental metastases in the lungs of EFA-deficient animals.     

Recombinant avian adenovirus CELO expressing the human interleukin-2: characterization in vitro, in ovo and in vivo

In our study, a recombinant adenovirus based on the avian adenovirus CELO genome, has been constructed that contains the human interleukin-2 gene. We have shown the production of biologically active recombinant interleukin-2 in vitro (LMH and 293 cells) and in ovo (chicken embryos) infected with recombinant virus CELO-IL2. An increase in the median survival time of C57BL/6 mice carrying B16 melanoma tumors has been demonstrated after multiple intra-tumors injections of the recombinant adenovirus CELO-IL2.     

高低转移肺腺癌细胞系中肿瘤抑制基因p16、p21和p53表达研究

目的 探讨肿瘤抑制基因对肺腺癌细胞生长的抑制作用。方法 利用FuGene转染方式分别将p21和p16基因的表达质粒转入－对肺腺癌细胞系Anip973和AGZY83－a中,同时用含野生型p53 基因的腺病毒感染p16基因转染前后的这一对细胞系。对P16和P21蛋白过表达的细胞系进行了细胞生长曲线、克隆形成率、原位末端标记分析和流式细胞仪分析。结果 p16基因的过表达只能使细胞系的G1期细胞比例提高,但细胞生长曲线,克隆形成率均未出现改变,未检测到凋亡信号。P21蛋白过表达的一对细胞系细胞生长曲线斜率降低,克隆形成能力下降,并出现明显的G1期阻滞,但未检测到凋亡信号。p53基因感染AGZY83－a,Anip973及经过p16基因转染的细胞AGZY83－ap16和Anip973p16后呈现时间依赖性表达,细胞生长曲线和四唑盐比色法分析提高,野生型p53基因的大量表达明显抑制以上4种细胞的生长,Anip973和Anip973p16的生长抑制率高于AGZY83－a和AGZY83－ap16;Anip973p16和AGZY83-ap16的生长抑制率高于Anip973和AAGZY83－a。这4种细胞在感染p53后出现典型的凋亡信号。结论p16基因的过表达并不能抑制细胞系的生长,而p21基因的过表达通过G1期阻滞抑制这1对肺腺癌细胞的生长;野生型p53基因在AGZY83－a和Anip973中高效表达可产生明显的细胞生长抑制效应;野生型p53基因对肺腺癌高转移细胞系Anip973抑制作用更为明显。     

乳腺癌患者手术治疗前后血浆leptin和血清CA15-3、IL-8、hs-CRP检测的临床意义

目的:探讨乳腺癌患者手术治疗前后血浆leptin和血清CA15-3、IL-8、hs-CRP水平的变化及临床意义。方法:应用放射免疫分析和免疫比浊法对31例乳腺癌患者进行了手术治疗前后血浆leptin和血清CA15-3、IL-8、hs-CRP检测,并与35名正常人作比较。结果:乳腺癌患者在手术治疗前血浆leptin和血清CA15-3、IL-8、hs-CRP水平非常显著地高于正常人组(P<0.01),手术治疗后3个月则与正常人组比较无显著性差异(P>0.05)。结论:血浆leptin和血清CA15-3、IL-8、hs-CRP水平的变化在乳腺癌的发生和发展中相互作用,观察其浓度的变化对探讨乳腺癌的发病机理、预防和治疗均有重要的临床价值。