小鼠IDO基因重组腺病毒载体的构建表达和鉴定

目的 构建携带小鼠IDO基因的重组腺病毒载体,以期进一步进行研究其免疫学效应.方法 酶切含有小鼠全长IDOcDNA的PCOUS-2质粒,亚克隆至穿梭质粒pAdtrack-CMV上,在BJ5183细菌中和AdEasy-1进行同源重组,筛选阳性克隆,酶切、测序鉴定正确后,脂质体法转染AD-293细胞进行包装,扩增,氯化铯密度梯度离心纯化病毒并检测其滴度.RT-PCR和荧光显微镜鉴定和检测重组腺病毒转染AD-293细胞后IDO的表达,同时进行野生型腺病毒的检测.结果 经酶切及测序证实IDO基因重组腺病毒载体构建成功,RT-PCR检测到转染后AD-293细胞内IDO的表达,且无野生型腺病毒的产生.结论 成功构建了含小鼠IDO基因的重组腺病毒载体,为探讨IDO的生物学功能奠定了基础.     

重视儿童腺病毒肺炎规范诊疗与临床研究

人腺病毒（HAdV）是一种无包膜双链DNA病毒,是引起6月龄至2岁婴幼儿重症肺炎最常见的病毒病原体。2019年以来,中国多个地区发现多例重症HAdV肺炎患儿,对儿童生命健康造成严重威胁。该文就HAdV的基因型、流行趋势、疾病严重度、致病机制以及儿童HAdV肺炎的早期诊断和干预、治疗方法等方面进行介绍。     

The Effect of PEI-Mediated E1A on the Radiosensitivity of Hepatic Carcinoma Cells

Objective: The study was undertaken to investigate the effects of polyethyleneimine (PEI)-mediated adenovirus 5 early region 1A (E1A) on radiosensitivity of human hepatic carcinoma cell in vitro and to disclosure the underlying mechanism. Materials and Methods: Human hepatic carcinoma SMMC-7721 cell line was transfected with E1A gene using PEI vector. Untransfected cells (SMMC-7721 group), cells transfected with blank-vector (SMMC-7721-vect group), and cells transfected with E1A gene (SMMC-7721-E1A group) were treated with 6 MV X-ray irradiation at doses of 0, 1, 2, 4, 8 and Gy, respectively. Radiosensitivity was determined by MTT assay and quantified by calculating the cell survival rate. Cell-cycle distribution and apotosis rate were monitored by flow cytometry. Results: The survival rate of SMMC-7721-E1A was significantly lower than that of SMMC-7721 cell. Apoptosis rate of SMMC-7721-E1A group was significantly higher than that of SMMC-7721group (P<0.01).The ratio of S stage in cell cycle of SMMC-7721-E1A was significantly lower than that in SMMC-7721 cell. The ratio of G2/M stage in cell cycle of SMMC-7721-E1A was significantly higher than that in SMMC-7721 cell (P<0.01). Conclusion: PEI could transfect E1A gene into hepatic carcinoma cells PEI-mediated E1A could effectively enhance radiosensitivity of hepatic carcinoma cells which may be related to its effects on apoptosis promoting leading to S phase suppression and G2/M phase arrest.     

紫外线和过氧乙酸对腺病毒灭活效果的观察

目的：寻找对腺病毒灭活的有效方法,来预防大规模生产腺病毒时所致的生产细胞的污染。方法：采用紫外线照射和不同浓度过氧乙酸的灭活方法灭活腺病毒,并将处理后的病毒加入到正常人胚肾293细胞中培养,观察细胞是否出现病变作为灭活效果判断标准。结果：我们发现一定强度紫外线照射至少2 h以上才能完全灭活腺病毒,过氧乙酸达到1%的浓度和至少作用30 min,才能将完全病毒灭活。结论：紫外线照射2 h以上能有效灭活腺病毒,我们可以用于操作台的腺病毒灭活;1%浓度的过氧乙酸作用30 min以上能有效将腺病毒灭活,我们可以运用于实验室大空间的腺病毒灭活。     

Recombinant adenovirus containing hyper-interleukin-6 and hepatocyte growth factor ameliorates acute-on-chronic liver failure in rats

AIM: To investigate the protective efficacy of recombinant adenovirus containing hyper-interleukin-6(HyperIL-6, HIL-6) and hepatocyte growth factor(HGF)(AdHGF-HIL-6) compared to that of recombinant adenovirus containing either HIL-6 or HGF(Ad-HIL-6 or Ad-HGF) in rats with acute-on-chronic liver failure(ACLF).METHODS: The recombinant adenoviruses containing HIL-6 and/or HGF were constructed. We established an ACLF model, and rats were randomly assigned to control, model, Ad-GFP, Ad-HIL-6, Ad-HGF or AdHGF-HIL-6 group. We collected serum and liver tissue samples to test pathological changes, biochemical indexes and molecular biological indexes.RESULTS: Attenuated alanine aminotransferase, prothrombin time, high-mobility group box 1(HMGB1), endotoxin, tumour necrosis factor(TNF)-α and interferon-γ were observed in the Ad-HGF-, Ad-HIL-6- and Ad-HGFHIL-6-treated rats with ACLF. Likewise, reduced hepaticdamage and apoptotic activity, as well as reduced HMGB1 and Bax proteins, but raised expression of Ki67 and Bcl-2 proteins and Bcl-2/Bax ratio were also observed in the Ad-HGF-, Ad-HIL-6- and Ad-HGF-HIL-6-treated rats with ACLF. More significant changes were observed in the Ad-HGF-HIL-6 treatment group without obvious side effects. Furthermore, caspase-3 at the protein level decreased in the Ad-HIL-6 and Ad-HGFHIL-6 treatment groups, more predominantly in the latter group.CONCLUSION: This study identifies that the protective efficacy of Ad-HGF-HIL-6 is more potent than that of Ad-HGF or Ad-HIL-6 in ACLF rats, with no significant side effects.     

Transfer of Hexon‐ and Penton‐selected adenovirus‐specific T cells for refractory adenovirus infection after haploidentical stem cell transplantation

Adenovirus (HAdV) infections confer a high risk of morbidity and mortality for immunocompromised patients after stem cell transplantation (SCT). Treatment with standard antiviral drugs is of limited efficacy and associated with a high rate of adverse effects. HAdV‐specific T cells are crucial for sustained viral elimination and the efficacy of adoptive T‐cell therapy with donor‐derived HAdV‐specific T cells has been reported by several investigators. Here, we report our experience with the transfer of HAdV‐specific T cells specific for penton, which was recently identified as an immunodominant target of T cells, and hexon in a 14‐year‐old boy after T‐cell‐depleted haploidentical SCT for myelodysplastic syndrome (MDS). He developed severe HAdV‐associated enteritis complicated by acute graft‐versus‐host disease (GvHD). The patient received ten infusions of allogeneic HAdV‐specific T cells manufactured from the haploidentical stem cell donor using the CliniMacs Interferon‐γ (IFN‐γ) cytokine capture and immunomagnetic selection. Initially, T cells were generated against the immunodominant target hexon and in subsequent transfers dual antigen‐specific T cells against hexon and penton were applied. T‐cell transfers were scheduled individually tailored to current immunosuppressive treatment. Each transfer was followed by reduction of HAdV load in peripheral blood and clinical improvement. Importantly, T‐cell responses to both penton and hexon pools emerged in patient blood after repetitive transfers. Unfortunately, the patient experienced bacterial sepsis, and in this context, severe GvHD requiring intensive immunosuppression followed by secondary progression of HAdV infection. The patient succumbed to multiorgan failure 283 days after SCT. This case demonstrates the feasibility of HAdV‐specific T‐cell transfer even in the presence of immunosuppressive treatment. Targeting of multiple immunodominant viral proteins may prove valuable in patients with complicated HAdV infections.     

人微小RNA-16（miR-16）重组腺病毒的制备和鉴定

目的制备人微小RNA-16（miR-16）重组腺病毒,研究其在人骨髓间充质干细胞（hMSCs）中的表达及对细胞周期素依赖蛋白激酶6（CDK6）表达的影响。方法从人基因组DNA中扩增miR-16前体的DNA,定向插入腺病毒穿梭载体pAdTrack-CMV。将经PmeⅠ线性化的重组穿梭载体pAdTrack-CMV-miR-16与腺病毒骨架质粒pAdEasy-1共转化感受态大肠杆菌BJ5183,在细菌内同源重组获得重组腺病毒质粒rAdTrack-miR-16。将经PacⅠ线性化的rAdTrack-miR-16在人胚肾293细胞（HEK293）中包装并扩增出miR-16的重组腺病毒rAd-miR-16。将重组腺病毒感染hMSCs,分别检测miR-16前体和miR-16靶基因CDK6的表达。结果 pAdTrack-CMV-miR-16构建正确,在HEK293细胞中成功包装并扩增出重组腺病毒rAd-miR-16。rAd-miR-16感染的hMSCs中miR-16前体水平显著升高（P〈0.05）,CDK6mRNA的表达降低不明显,CDK6蛋白表达水平显著降低（P〈0.01）。结论成功制备了表达人miR-16的重组腺病毒,并能有效介导miR-16在hMSCs中的表达,为进行miR-16的功能研究创造了条件。     

大鼠siTLR4重组腺病毒载体的构建及功能鉴定

目的构建特异性针对大鼠TLR4基因的siRNA重组腺病毒载体,为进一步研究TLR4在不同疾病中的免疫调节机制奠定重要基础。方法设计并合成3对大鼠TLR4基因的siRNA序列,退火处理后定向克隆到pSES-HUS穿梭质粒中,获得pSES-HUS-siTLR4,经Pme I线性化后与pAdEasy-1骨架质粒在BJ5183细菌中进行同源重组,从而构建pAd-siTLR4质粒,通过脂质体转染,在HEK293细胞中包装形成Ad-siTLR4腺病毒颗粒,用该病毒感染PC12细胞,从基因和蛋白质水平分别检测3对siTLR4的抑制效果,同时从蛋白质水平检测TLR4下游关键分子NF-κB的表达。结果 PCR、酶切和测序均证实目的基因正确克隆到所构建的pAd-siTLR4重组腺病毒载体中;经包装获得的Ad-siTLR4病毒颗粒在PC12细胞中能够有效地抑制TLR4 mRNA和蛋白水平的表达,并显著地降低了NF-κB的表达。结论成功构建了pAd-siTLR4重组腺病毒质粒,同时包装获得了Ad-siTLR4重组腺病毒,转染PC12细胞后不仅能明显降低TLR4的表达,而且有效地抑制了TLR4通路下游关键分子NF-κB的表达。