人CD52基因的克隆及稳定转染细胞系的建立

目的克隆人CD52基因,构建真核表达载体,并在CHO细胞中稳定表达。方法提取人Hut-78细胞总RNA,采用RT-PCR法扩增CD52基因,定向克隆至真核表达载体pcDNA3.1(+),构建重组表达质粒pcDNA3.1(+)/CD52,通过脂质体法转染CHO细胞,建立稳定转染的细胞系CHO-CD52。采用RT-PCR、免疫荧光组化技术及流式细胞术检测目的基因和蛋白的表达。结果 RT-PCR扩增得到186bp的DNA片段。重组表达质粒pcDNA3.1(+)/CD52经PCR、双酶切及测序证明构建正确。CHO-CD52细胞经RT-PCR分析,可见186bp的目的基因条带;经免疫荧光检测,可见绿色荧光分布;经流式细胞术分析,阳性细胞百分率及荧光平均值分别为98.18和193.56。结论已成功克隆了人CD52基因,并建立了稳定转染的CHO细胞系,为CD52单克隆抗体的制备及抗体药物的开发奠定了基础。     

Mesenchymal Stem Cells Can Both Enhance and Inhibit the Cellular Response to DNA Immunization by Genes of Nonstructural Proteins of the Hepatitis C Virus

Despite extensive research, there is still no vaccine against the hepatitis C virus (HCV). The aim of this study was to investigate whether MSCs can exhibit adjuvant properties during DNA vaccination against hepatitis C. We used the pcNS3-NS5B plasmid encoding five nonstructural HCV proteins and MSCs derived from mice bone marrow. Five groups of DBA mice were immunized with the plasmid and/or MSCs in a different order. Group 1 was injected with the plasmid twice at intervals of 3 weeks; Group 2 with the plasmid, and after 24 h with MSCs; Group 3 with MSCs followed by the plasmid the next day; Group 4 with only MSCs; and Group 5 with saline. When the MSCs were injected prior to DNA immunization, the cell immune response to HCV proteins assessed by the level of IFN-γ synthesis was markedly increased compared to DNA alone. In contrast, MSCs injected after DNA suppressed the immune response. Apparently, the high level of proinflammatory cytokines detected after DNA injection promotes the conversion of MSCs introduced later into the immunosuppressive MSC2. The low level of cytokines in mice before MSC administration promotes the high immunostimulatory activity of MSC1 in response to a DNA vaccine. Thus, when administered before DNA, MSCs are capable of exhibiting promising adjuvant properties.     

HIV-1表面糖蛋白gp120 DNA疫苗质粒的构建与表达

目的 构建含HIV-1表面糖蛋白基因的真核表达质粒,并在Hela细胞内表达。方法 在核酸疫苗载体质粒 pVAX1中插入gp120基因,构建真核表达质粒pVAXGP。在体外用脂质体法将重组质粒转染Hela细胞,并用间接免疫荧光试验、免疫印迹试验和Dot-ELISA对表达产物进行检测。结果 间接免疫荧光试验结果显示,转染重组质粒的细胞表面有绿色荧光。免疫印迹试验和Dot-ELISA结果均显示,重组质粒转染细胞的裂解物中存在表达的gp120蛋白。结论 已成功地构建了真核表达质粒,且表达的蛋白具有良好的反应原性和特异性。     

Hypoxia-Induced FAM13A Regulates the Proliferation and Metastasis of Non-Small Cell Lung Cancer Cells

Hypoxia in non-small cell lung cancer (NSCLC) affects cancer progression, metastasis and metabolism. We previously showed that FAM13A was induced by hypoxia in NSCLC but the biological function of this gene has not been fully elucidated. This study aimed to investigate the role of hypoxia-induced FAM13A in NSCLC progression and metastasis. Lentiviral shRNAs were used for FAM13A gene silencing in NSCLC cell lines (A549, CORL-105). MTS assay, cell tracking VPD540 dye, wound healing assay, invasion assay, BrdU assay and APC Annexin V staining assays were performed to examine cell proliferation ability, migration, invasion and apoptosis rate in NSCLC cells. The results of VPD540 dye and MTS assays showed a significant reduction in cell proliferation after FAM13A knockdown in A549 cells cultured under normal and hypoxia (1% O₂) conditions (p < 0.05), while the effect of FAM13A downregulation on CORL-105 cells was observed after 96 h exposition to hypoxia. Moreover, FAM13A inhibition induced S phase cell cycle arrest in A549 cells under hypoxia conditions. Silencing of FAM13A significantly suppressed migration of A549 and CORL-105 cells in both oxygen conditions, especially after 72 and 96 h (p < 0.001 in normoxia, p < 0.01 after hypoxia). It was showed that FAM13A reduction resulted in disruption of the F-actin cytoskeleton altering A549 cell migration. Cell invasion rates were significantly decreased in A549 FAM13A depleted cells compared to controls (p < 0.05), mostly under hypoxia. FAM13A silencing had no effect on apoptosis induction in NSCLC cells. In the present study, we found that FAM13A silencing has a negative effect on proliferation, migration and invasion activity in NSCLC cells in normal and hypoxic conditions. Our data demonstrated that FAM13A depleted post-hypoxic cells have a decreased cell proliferation ability and metastatic potential, which indicates FAM13A as a potential therapeutic target in lung cancer.     

携带人血管抑制因子K1-5基因的腺相关病毒载体的构建及其对血管内皮细胞的抑制作用

目的构建含人血管抑制因子K15基因的重组腺相关病毒载体rAAVK5,研究其在体外表达及对血管内皮细胞的抑制作用。方法将K15基因插入通用型AAV载体质粒pSNAV中,构建重组质粒pSNAVK5。用pSNAVK5转染BHK21细胞,经G418选择培养基培养载体细胞株BHKK5。用辅助病毒感染BHKK5细胞包装出重组病毒rAAVK5。研究rAAVK5感染BHK细胞获得的培养上清对血管内皮细胞的抑制作用。结果已获得了重组病毒rAAVK5,滴度达0.5×1012v.g.ml。免疫斑点印迹实验表明,rAAVK5可介导人血管抑制因子K15的体外表达,表达产物对血管内皮细胞增殖具有抑制作用。结论重组病毒rAAVK5在体外对血管内皮细胞的增殖具有抑制作用。为进一步用rAAV病毒载体进行抗血管生成基因治疗的动物实验及临床应用打下了基础。     

RhoC基因沉默对肝癌细胞增殖的影响

目的构建靶向RhoC基因的RNA干扰(RNAi)载体,并研究RhoC基因沉默对肝癌细胞增殖的影响。方法将合成的寡核苷酸片段克隆入RNAi载体,并转染肝癌细胞,以沉默RhoC基因表达,RT-PCR检测基因抑制效果。绘制生长曲线,检测转染细胞的增殖情况,FCM检测细胞周期变化。结果构建的RNAi载体有效地抑制了肝癌细胞RhoC基因的表达,RhoC基因沉默显著地抑制肿瘤细胞增殖,增殖期细胞百分数显著下降,而静止期细胞百分数显著升高。结论RhoC基因沉默能够抑制肝癌细胞的增殖,为靶向RhoC的肿瘤基因治疗奠定了基础。     

Influence of Polydatin on the Tumor Microenvironment In Vitro: Studies with a Colon Cancer Cell Model

The tumor microenvironment of colon carcinoma, the site at which tumor cells and the host immune system interact, is influenced by signals from tumor cells, immunocompetent cells, and bacterial components, including LPS. A large amount of LPS is available in the colon, and this could promote inflammation and metastasis by enhancing tumor cell adhesion to the endothelium. Polydatin (PD), the 3-β-D-glucoside of trans-resveratrol, is a polyphenol with anti-cancer, anti-inflammatory, and immunoregulatory effects. This study was designed to explore whether PD is able to produce antiproliferative effects on three colon cancer lines, to reduce the expression of adhesion molecules that are upregulated by LPS on endothelial cells, and to decrease the proinflammatory cytokines released in culture supernatants. Actually, we investigated the effects of PD on tumor growth in a coculture model with human mononuclear cells (MNCs) that mimics, at least in part, an in vitro tumor microenvironment. The results showed that PD alone or in combination with MNC exerts antiproliferative and proapoptotic effects on cancer cells, inhibits the production of the immunosuppressive cytokine IL-10 and of the proinflammatory cytokines upregulated by LPS, and reduces E-selectin and VCAM-1 on endothelial cells. These data provide preclinical support to the hypothesis that PD could be of potential benefit as a therapeutic adjuvant in colon cancer treatment and prevention.     

Homophilic Interaction of CD147 Promotes IL-6-Mediated Cholangiocarcinoma Invasion via the NF-κB-Dependent Pathway

Cholangiocarcinoma (CCA), an aggressive cancer of bile ducts, is a well-known chronic inflammation-related disease. The major impediment in CCA treatment is limited treatment options for advanced disease; hence, an alternative is urgently required. The role of CD147 on cytokine production has been observed in inflammation-related diseases, but not in CCA. Therefore, this study was focused on CD147-promoting proinflammatory cytokine production and functions. Proinflammatory cytokine profiles were compared between CD147 expressing CCA cells and CD147 knockout cells (CD147 KO). Three cytokines, namely interleukin (IL)-6, IL-8, and granulocyte–monocyte colony-stimulating factor (GM-CSF), were dramatically diminished in CD147 KO clones. The involvement of the CD147-related cytokines in CCA invasion was established. CD147-promoted IL-6, IL-8, and GM-CSF secretions were regulated by NF-κB nuclear translocation, Akt activation, and p38 phosphorylation. CD147-fostering IL-6 production was dependent on soluble CD147, CD147 homophilic interaction, and NF-κB function. The overexpression of specific genes in CCA tissues compared to normal counterparts emphasized the clinical importance of these molecules. Altogether, CD147-potentiated proinflammatory cytokine production leading to CCA cell invasion is shown for the first time in the current study. This suggests that modulation of CD147-related inflammation might be a promising choice for advanced CCA treatment.     

Brain-Derived Neurotrophic Factor Suppressed Proinflammatory Cytokines Secretion and Enhanced MicroRNA(miR)-3168 Expression in Macrophages

We investigated the role of brain-derived neurotrophic factor (BDNF) and its signaling pathway in the proinflammatory cytokines production of macrophages. The effects of different concentrations of BDNF on proinflammatory cytokines expression and secretion in U937 cell-differentiated macrophages, and human monocyte-derived macrophages were analyzed using enzyme-linked immunosorbent assay and real-time polymerase chain reaction. The CRISPR-Cas9 system was used to knockout p75 neurotrophin receptor (p75NTR), one of the BDNF receptors. Next-generation sequencing (NGS) was conducted to search for BDNF-regulated microRNA. A very low concentration of BDNF (1 ng/mL) could suppress the secretion of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 in lipopolysaccharide (LPS)-stimulated macrophages but did not change their mRNA expression. BDNF suppressed IL-1β and IL-6 secretion in human monocyte-derived macrophages. In U937 cells, BDNF suppressed the phosphorylation of JNK and c-Jun. The p75NTR knockout strongly suppressed IL-1β, IL-6, and TNF-α secretion in macrophages and LPS-stimulated macrophages. BDNF regulated the expression of miR-3168 with Ras-related protein Rab-11A as its target. In conclusion, BDNF suppressed proinflammatory cytokines secretion in macrophages and inhibited the phosphorylation of JNK. Knockout of p75NTR suppressed proinflammatory cytokines expression and secretion. BDNF upregulated the expression of miR-3168. The inhibition of p75NTR could be a potential strategy to control inflammation.     

Tonabersat Inhibits Connexin43 Hemichannel Opening and Inflammasome Activation in an In Vitro Retinal Epithelial Cell Model of Diabetic Retinopathy

This study was undertaken to evaluate the connexin hemichannel blocker tonabersat for the inhibition of inflammasome activation and use as a potential treatment for diabetic retinopathy. Human retinal pigment epithelial cells (ARPE-19) were stimulated with hyperglycemia and the inflammatory cytokines IL-1β and TNFα in order to mimic diabetic retinopathy molecular signs in vitro. Immunohistochemistry was used to evaluate the effect of tonabersat treatment on NLRP3, NLRP1, and cleaved caspase-1 expression and distribution. A Luminex cytokine release assay was performed to determine whether tonabersat affected proinflammatory cytokine release. NLRP1 was not activated in ARPE-19 cells, and IL-18 was not produced under disease conditions. However, NLRP3 and cleaved caspase-1 complex formation increased with hyperglycemia and cytokine challenge but was inhibited by tonabersat treatment. It also prevented the release of proinflammatory cytokines IL-1β, VEGF, and IL-6. Tonabersat therefore has the potential to reduce inflammasome-mediated inflammation in diabetic retinopathy.     

复合通用CD4~+T辅助细胞表位基因克隆载体的构建及测序

目的构建复合通用CD4+T辅助细胞表位基因克隆载体,并进行测序。方法由DNA work2.0软件设计并人工合成20条55个碱基的寡核苷酸序列,利用套叠PCR技术人工合成全基因序列,并克隆至pUC19载体,转化大肠杆菌DH5α,提取质粒,酶切鉴定并进行测序。结果经PCR扩增出645bp的目的DNA片段。酶切鉴定筛选出8个阳性克隆,经测序获得一个序列完全正确的克隆。结论已成功构建了复合通用CD4+T辅助细胞表位基因克隆载体,为研究表位疫苗和细菌多糖结合疫苗提供了新的载体表位。     

鸡贫血病毒VP3基因与人IL-18基因的共表达对人肝癌细胞BEL-7402的作用

目的利用凋亡素和IL18基因的抗肿瘤优势,设计构建共表达凋亡素和hIL-18基因的真核重组子,检测其在体外对人肝癌细胞BEL7402的作用效果,旨在探索新的肿瘤基因治疗方法。方法将凋亡素VP3基因与hIL-18基因一同克隆到真核表达载体pVAX1上,构建重组质粒pVVP3IL-18,经脂质体介导,转染人肝癌细胞BEL7402,AO/EB染色,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法和流式细胞仪分别分析凋亡细胞的形态学变化、DNA断裂情况和不同细胞周期的DNA含量变化,透射电镜分析凋亡细胞超微结构变化。结果随着转染时间的延长,重组质粒pVVP3IL18所表达的目的蛋白对BEL7402细胞的杀伤作用逐渐增强,作用48h,形态学观察表明BEL7402细胞发生明显的凋亡,凋亡率可达(56.5±11.9)%,并且大部分细胞为TUNEL阳性着色,流式细胞术分析可见明显的亚二倍体凋亡峰,透射电镜观察可见细胞核固缩、染色质聚集等典型的凋亡超微结构变化。结论联合应用凋亡素与hIL18基因,在体外对人肝癌细胞BEL7402具有明显的凋亡诱导作用。     

ASC and SVF Cells Synergistically Induce Neovascularization in Ischemic Hindlimb Following Cotransplantation

Previously, we reported the angio-vasculogenic properties of human stromal vascular fraction (SVF) and adipose tissue-derived mesenchymal stem cells (ASCs). In this study, we investigated whether the combination of ASCs and SVF cells exhibited synergistic angiogenic properties. We conducted quantitative (q)RT-PCR, Matrigel plug, tube formation assays, and in vivo therapeutic assays using an ischemic hind limb mouse model. Immunohistochemical analysis was also conducted. qRT-PCR results revealed that FGF-2 was highly upregulated in ASCs compared with SVF, while PDGF-b and VEGF-A were highly upregulated in SVF. Conditioned medium from mixed cultures of ASCs and SVF (A+S) cells showed higher Matrigel tube formation and endothelial cell proliferation in vitro. A+S cell transplantation into ischemic mouse hind limbs strongly prevented limb loss and augmented blood perfusion compared with SVF cell transplantation. Transplanted A+S cells also showed high capillary density, cell proliferation, angiogenic cytokines, and anti-apoptotic potential in vivo compared with transplanted SVF. Our data indicate that A+S cell transplantation results in synergistic angiogenic therapeutic effects. Accordingly, A+S cell injection could be an alternative therapeutic strategy for treating ischemic diseases.     

Electrochemical high-content screening of nitric oxide release from endothelial cells

Release of nitric oxide (NO) is of high importance for regulating endothelial cell functions during vasodilatation, vascular remodeling, and angiogenesis. Thus, a direct and reliable real-time method for NO detection that takes into account time-dependent variations of the NO concentration in the complex reaction within the diffusion zone above the cells is vital for obtaining information about the role of NO in intracellular endothelial signal transduction and its impact on the surrounding cells. In this study, the time course of vascular endothelial growth factor E (VEGF-E) stimulated NO release from transformed human umbilical vein endothelial cells (T-HUVEC) was investigated by means of metalloporphyrin-based NO sensors employed in an electrochemical robotic system. The NO sensor was obtained by electrochemically induced deposition of Ni(II) tetrakis(p-nitrophenylporphyrin) on a 50-microm diameter platinum disk electrode which was integrated, together with a 25-microm diameter platinum disk, in a double-barrel electrode arrangement. The second electrode was used as a guidance sensor for the automatic and highly reproducible positioning of the NO sensor at a known distance from a layer of adherently growing cells by using z-approach curves in the negative feedback mode of scanning electrochemical microscopy (SECM). The electrochemical robotic system allows the fully automated detection of NO with high sensitivity and selectivity to be performed in real time within 96-well microtiter plates. A functional cell assay was established to allow the standardized detection of NO released upon stimulation from T-HUVEC with a sensor positioned at a known distance above the endothelial cells. The overall system was evaluated by automatic detection of NO release from T-HUVEC upon stimulation with VEGF-E after incubation with a variety of drugs that are known to act on different sites in the complex signal-transduction pathway that finally invokes NO release.     

Ipr1和GFP基因真核共表达穿梭质粒的构建及其在人肺癌细胞中的表达

目的构建胞内病原体抗性基因1(Ipr1)和绿色荧光蛋白(GFP)基因真核共表达穿梭质粒,并在人肺腺癌细胞A549中表达。方法采用PCR方法,分别从质粒pEGFP-C1-Ipr1和pEGFP-C1中扩增Ipr1和GFP基因,将GFP基因、分枝杆菌复制子OriM和Ipr1基因同时克隆入多启动子真核共表达载体pBudCE4.1中,构建pBud-GFP-OriM-Ipr1穿梭质粒,脂质体法转染A549细胞,荧光显微镜观察GFP的表达,免疫组化方法检测Ipr1蛋白的表达。结果酶切和测序分析表明,pBud-GFP-OriM-Ipr1真核共表达穿梭质粒构建正确。转染A549细胞后,荧光显微镜下可观察到转染细胞中有GFP表达,免疫组化法可检测到Ipr1蛋白的表达,且定位于细胞核内。结论已成功构建Ipr1和GFP基因真核共表达穿梭质粒,为进一步研究Ipr1抗结核的功能奠定了基础。     

Exploring the Potential Use of a PBMC-Based Functional Assay to Identify Predictive Biomarkers for Anti-PD-1 Immunotherapy

The absence of reliable, robust, and non-invasive biomarkers for anti- Programmed cell death protein 1 (PD-1) immunotherapy is an urgent unmet medical need for the treatment of cancer patients. No predictive biomarkers have been established based on the direct assessment of T cell functions, the primary mechanism of action of anti-PD-1 therapy. In this study, we established a model system to test T cell functions modulated by Nivolumab using anti-CD3 monoclonal antibody (mAb)-stimulated peripheral blood mononuclear cells (PBMCs), and characterized T cell functions primarily based on the knowledge gained from retrospective observations of patients treated with anti-PD-1 immunotherapy. During a comprehensive cytokine profile assessment to identify potential biomarkers, we found that Nivolumab increases expression of T helper type 1 (Th1) associated cytokines such as interferon-γ (IFN-γ) and interleukin-2 (IL-2) in a subset of donors. Furthermore, Nivolumab increases production of Th2, Th9, and Th17 associated cytokines, as well as many proinflammatory cytokines such as IL-6 in a subset of donors. Conversely, Nivolumab treatment has no impact on T cell proliferation, expression of CD25, CD69, or Granzyme B, and only modestly increases in the expansion of regulatory T cells. Our results suggest that assessment of cytokine production using a simple PBMC-based T cell functional assay could be used as a potential predictive marker for anti-PD-1 immunotherapy.