Plasma Metabonomics of Human Adenovirus-infected Patients with Pneumonia and Upper Respiratory Tract Infection

Objective Human adenovirus(HAdV)infection is common and can develop to serious conditions with high mortality,yet the mechanism of HAdV infection remains unclear.In the present study,the serum metabolite profiles of HAdV-7-infected patients with pneumonia or upper respiratory tract infection(URTI)were explored.Methods In total,35 patients were enrolled in the study following an outbreak of HAdV-7 in the army,of whom 14 had pneumonia and 21 had URTI.Blood samples were collected at the acute stage and at the recovery stage and were analyzed by untargeted metabolomics.Results Over 90% of the differential metabolites identified between the pneumonia patients and URTI patients were lipids and lipid-like molecules,including glycerophospholipids,fatty acyls,and sphingolipids.The metabolic pathways that were significantly enriched were primarily the lipid metabolism pathways,including sphingolipid metabolism,glycerophospholipid metabolism,and linoleic acid metabolism.The sphingolipid metabolism was identified as a significantly differential pathway between the pneumonia patients and URTI patients and between the acute and recovery stages for the pneumonia patients,but not between the acute and recovery stages for the URTI patients.Ceramide and lactosylceramide,involved in sphingolipid metabolism,were significantly higher in the pneumonia patients than in the URTI patients with good discrimination abilities[area under curve(AUC)0.742 and 0.716,respectively;combination AUC 0.801].Conclusion Our results suggested that HAdV modulated lipid metabolism for both the patients with URTI and pneumonia,especially the sphingolipid metabolism involving ceramide and lactosylceramide,which might thus be a potential intervention target in the treatment of HAdV infection.

Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication

Human adenovirus B7(HAdV-B7)causes severe acute lower respiratory tract infections in children.However,neither the child-specific antivirals or vaccines are available,nor the pathogenesis is clear.Autophagy,as part of innate immunity,plays an important role in resistance to viral infection by degrading the virus and promoting the development of innate and adaptive immunity.This study provided evidence that HAdV-B7 infection induced complete autophagic flux,and the pharmacological induction of autophagy decreased HAdV-B7 replication.In this process,the host protein Bcl2-associated athanogene 3(BAG3)mediated autophagy to inhibit the replication of HAdV-B7 by binding to the PPSY structural domain of viral protein pVI through its WW structural domain.These findings further our understanding of the host immune response during viral infection and will help to develop broad anti-HAdV therapies.

Analysis of severe human adenovirus infection outbreak in Guangdong Province,southern China in 2019

During 2018–2019,a severe human adenovirus(HAdV)infection outbreak occurred in southern China.Here,we screened 18 respiratory pathogens in 1704 children(≤14 years old)hospitalized with acute respiratory illness in Guangzhou,China,in 2019.In total,151 patients had positive HAdV test results;34.4%(52/151)of them exhibited severe illness.HAdV infection occurred throughout the year,with a peak in summer.The median patient age was 3.0(interquartile range:1.1–5.0)years.Patients with severe HAdV infection exhibited increases in12 clinical indexes(P≤0.019)and decreases in four indexes(P≤0.007),compared with patients exhibiting nonsevere infection.No significant differences were found in age or sex distribution according to HAdV infection severity(P>0.05);however,the distributions of comorbid disease and HAdV co-infection differed according to HAdV infection severity(P<0.05).The main epidemic types were HAdV-3(47.0%,71/151)and HAdV-7(46.4%,70/151).However,the severe illness rate was significantly higher in patients with HAdV-7(51.4%)than in patients with HAdV-3(19.7%)and other types of HAdV(20%)(P<0.001).Sequencing analysis of genomes/capsid genes of 13 HAdV-7 isolates revealed high similarity to previous Chinese isolates.A representative HAdV-7isolate exhibited a similar proliferation curve to the curve described for the epidemic HAdV-3 strain Guangzhou01(accession no.DQ099432)(P>0.05);the HAdV-7 isolate exhibited stronger virulence and infectivity,compared with HAdV-3(P<0.001).Overall,comorbid disease,HAdV co-infection,and high virulence and infectivity of HAdV-7 were critical risk factors for severe HAdV infection;these data can facilitate treatment,control,and prevention of HAdV infection.

Current status of human adenovirus infection in China

Background Outbreaks of severe,acute hepatitis among children have recently attracted global attention.The pathogen causing the outbreak remains unknown,but there is growing evidence that it may be associated with human adenovirus(HAdV).Data sources A review of adenovirus-related clinical studies,epidemiological studies,etiological studies,and case reports was conducted by reviewers independently.Results HAdV can cause a wide variety of clinical symptoms.In the Mainland of China,HAdV infection accounts for 5.8%–13%of patients with acute respiratory infections,and these infections are mainly caused by species B,C,and E of HAdV.For acute conjunctivitis,39.8%–74.9%of sporadic cases were infected by B and D species of HAdV.Outbreaks of keratoconjunctivitis and pharyngoconjunctival fever related to HAdV infection could be found throughout the country.In pediatric patients with acute gastroenteritis,HAdV-41 was the predominant HAdV type,followed by HAdV species B and C.Several types of HAdV,including HAdV-5,HAdV-7,HAdV-1,and HAdV-2,have previously been reported as potential pathogens associated with HAdV hepatitis in immunocompromised patients.However,few HAdV-related hepatitis cases have been reported in China to date.Conclusions There are no systematic surveillance and clinical studies on HAdV hepatitis in China.Therefore,it is imperative to establish a nationwide HAdV virological surveillance system to collect relevant clinical,epidemiological and virological surveillance data and risk factor information as soon as possible to assess the potential risk of HAdV hepatitis among children.

Human adenovirus(HAdV)infection in children with acute respiratory tract infections in Guangzhou,China,2010–2021:a molecular epidemiology study

Background Human adenovirus(HAdV)infection can cause a variety of diseases.It is a major pathogen of pediatric acute respiratory tract infections(ARIs)and can be life-threatening in younger children.We described the epidemiology and subtypes shifting of HAdV among children with ARI in Guangzhou,China.Methods We conducted a retrospective study of 161,079 children diagnosed with acute respiratory illness at the Guangzhou Women and Children’s Medical Center between 2010 and 2021.HAdV specimens were detected by real-time PCR and the hexon gene was used for phylogenetic analysis.Results Before the COVID-19 outbreak in Guangzhou,the annual frequency of adenovirus infection detected during this period ranged from 3.92%to 13.58%,with an epidemic peak every four to fve years.HAdV demonstrated a clear seasonal distribution,with the lowest positivity in March and peaking during summer(July or August)every year.A signifcant increase in HAdV cases was recorded for 2018 and 2019,which coincided with a shift in the dominant HAdV subtype from HAdV-3 to HAdV-7.The latter was associated with a more severe disease compared to HAdV-3.The average mortality proportion for children infected with HAdV from 2016 to 2019 was 0.38%but increased to 20%in severe cases.After COVID-19 emerged,HAdV cases dropped to 2.68%,suggesting that non-pharmaceutical interventions probably reduced the transmission of HAdV in the community.Conclusion Our study provides the foundation for the understanding of the epidemiology of HAdV and its associated risks in children in Southern China.

Seroprevalence of Neutralizing Antibodies against Six Human Adenovirus Types Indicates the Low Level of Herd Immunity in Young Children from Guangzhou, China

Human adenoviruses(HAdVs) commonly cause many diseases such as respiratory diseases, gastroenteritis, cystitis worldwide. HAdV-3,-7,-4 and emergent HAdV-55 and HAdV-14 are the most important types causing severe respiratory diseases. There is no effective drug available for clinical treatment, and no vaccine available for the general population.Therefore, it is important to investigate the seroprevalence against HAdV for developing novel vaccines and vectors. In this study, we investigated the seroprevalence and titer levels of neutralizing antibodies(NAb) against HAdV-3,-4,-7,-14,-55,and-11 in total 278 healthy populations between 0 months and 49 years of age(228 children and 50 adults) from Guangzhou. In children under the age of 18 years, the seropositive rates were significantly increased against HAdV-3 at12.07%, 33.96%, and 64.29% and against HAdV-7 at 0%, 18.87%, and 19.05% in age groups of 1–2, 3–5, and 6–17 years,respectively. The seroprevalence was very low(0% - 8.1%) for all other four types. In adults aged between 18 and49 years, HAdV-3,-4, and-7(> 50.00%) were the most common types, followed by HAdV-14(38.00%),-55(34.00%),and-11(24.00%). Adults tended to have high NAb titers against HAdV-4 and-55. HAdV-55-seropositive donors tended to be HAdV-11-and HAdV-14-seropositive. These results indicated the low level of herd immunity against all six HAdV types in young children, and HAdV-14,-55,-11 in adults from Guangzhou City. Our findings demonstrate the importance of monitoring HAdV types and developing vaccines against HAdV for children and adults.

Application of Human Adenovirus Genotyping by Phylogenetic Analysis in an Outbreak to Identify Nosocomial Infection

Nosocomial infections are common in pediatric patients and can be fatal in infants and immunocompromised patients. In September 2018, a high positive rate of human adenovirus HAdV was occurred among hospitalized children in the Children's Hospital Affiliated to the Capital Institute of Paediatrics in Beijing. To investigate whether this outbreak of HAdV was related to nosocomial infections or the result of community infections, we collected respiratory specimens from patients with acute respiratory infections in a respiratory ward during June to December 2018, and screened for respiratory viruses. Among 1,840 cases included, 95(5.2%, 95/1840) were positive for HAdV and 81 were genotyped based on phylogenetic analysis, including seven as HAdV-1(8.6%), 30 HAdV-3(37.0%), two HAdV-6(2.5%), and 42 HAdV-7(51.9%). More HAdV-positive samples were collected in August(4.7%, 12/255), September(15.0%, 41/274) and October(6.9%, 17/247), with a peak in September 2018. By combining the results of HAdV phylogenetic analysis with clinical data of patients, there were 77 cases(4.2%, 77/1840;81.1%, 77/95) excluded from nosocomial infections, eight cases representing possible infections transmitted by visitors or attending parents, three cases without sequences that might have been due to infection transmitted by roommates positive for HAdV, one case of a roommate without an HAdV sequence, and six cases that shared highly homologous sequences with those of their roommates, for which nosocomial infections might be considered. In conclusion, genotyping of HAdVs based on phylogenetic analysis combined with clinical information provides a powerful method to distinguish nosocomial infections from community acquired infection, especially when tracing the origins of nosocomial infections.

Desmoglein 2(DSG2) Is A Receptor of Human Adenovirus Type 55 Causing Adult Severe Community-Acquired Pneumonia

Human adenovirus type 55(HAdV-B55) is a re-emergent acute respiratory disease pathogen that causes adult communityacquired pneumonia(CAP). Previous studies have shown that the receptor of HAdV-B14, which genome is highly similar with HAdV-B55, is human Desmoglein 2(DSG2). However, whether the receptor of HAdV-B55 is DSG2 is undetermined because there are three amino acid mutations in the fiber gene between HAdV-B14 and HAdV-B55. Here, firstly we found the 3T3 cells, a mouse embryo fibroblast rodent cell line which does not express human DSG2, were able to be infected by HAdV-B55 after transfected with pcDNA3.1-DSG2, while normal 3T3 cells were still unsusceptible to HAdV-B55 infection. Next, A549 cells with h DSG2 knock-down by siRNA were hard to be infected by HAdV-B3/-B14/-B55, while the control siRNA group was still able to be infected by all these types of HAdVs. Finally, immunofluorescence confocal microscopy indicated visually that Cy3-conjugated HAdV-B55 viruses entered A549 cells by binding to DSG2 protein.Therefore, DSG2 is a major receptor of HAdV-B55 causing adult CAP. Our finding is important for better understanding of interactions between adenoviruses and host cells and may shed light on the development of new drugs that can interfere with these processes as well as for the development of potent prophylactic vaccines.

Construction and Characterization of a Novel Recombinant Attenuated and Replication-Deficient Candidate Human Adenovirus Type 3 Vaccine:"Adenovirus Vaccine Within an Adenovirus Vector"

Human adenoviruses(HAd Vs)are highly contagious and result in large number of acute respiratory disease(ARD)cases with severe morbidity and mortality.Human adenovirus type 3(HAd V-3)is the most common type that causes ARD outbreaks in Asia,Europe,and the Americas.However,there is currently no vaccine approved for its general use.The hexon protein contains the main neutralizing epitopes,provoking strong and lasting immunogenicity.In this study,a novel recombinant and attenuated adenovirus vaccine candidate against HAd V-3 was constructed based on a commercially-available replication-defective HAd V-5 gene therapy and vaccine vector.The entire HAd V-3 hexon gene was integrated into the E1 region of the vector by homologous recombination using a bacterial system.The resultant recombinants expressing the HAd V-3 hexon protein were rescued in AD293 cells,identified and characterized by RT-PCR,Western blots,indirect immunofluorescence,and electron microscopy.This potential vaccine candidate had a similar replicative efficacy as the wild-type HAd V-3 strain.However,and importantly,the vaccine strain had been rendered replication-defective and was incapable of replication in A549 cells after more than twentygeneration passages in AD293 cells.This represents a significant safety feature.The mice immunized both intranasally and intramuscularly by this vaccine candidate raised significant neutralizing antibodies against HAd V-3.Therefore,this recombinant,attenuated,and safe adenovirus vaccine is a promising HAd V-3 vaccine candidate.The strategy of using a clinically approved and replication-defective HAd V-5 vector provides a novel approach to develop universal adenovirus vaccine candidates against all the other types of adenoviruses causing ARDs and perhaps other adenovirus-associated diseases.

Genetic Analysis of Human Adenovirus Type 7 Strains Circulating in Different Parts of China

To investigate the molecular epidemiology and genetic variation of human adenovirus type 7(HAdV-7)in children with acute respiratory infections(ARI)in China.HAdV-7-positive respiratory samples collected from children with ARI in Beijing,Shijiazhuang,Wenzhou and Guangzhou from 2014–2018 were selected for gene amplification and sequence analysis.Fifty-seven HAdV-7 clinical strains with hexon,penton base and fiber gene sequences were obtained.Meanwhile17 strains were selected randomly from different cities for whole genome sequencing.Phylogenetic and variation analyses were performed based on the obtained sequences,HAdV-7 prototype strain Gomen(AY594255),vaccine strains(AY495969 and AY594256)and representative sequences of strains.The phylogenetic trees constructed based on whole genome sequences,major capsid protein genes(hexon,penton base and fiber)and the early genes(E1,E2,E3 and E4)were not completely consistent.The HAdV-7 strains obtained in this study always clustered with most of the circulating strains worldwide from the 1980 s to the present.Compared with the HAdV-7 prototype strain Gomen(AY594255),some amino acid mutations in loop1 and loop2 of hexon and the RGD loop region of the penton base gene were observed.Recombination analysis showed that partial regions of 55 k Da protein and 100 kDa hexon-assembly associated protein genes among all HAdV-7 strains in this study were from HAdV-16 and HAdV-3,respectively.Our study demonstrated the molecular evolution characteristics of HAdV-7 strains circulating in China and provided basic reference data for the prevention,control and vaccine development of HAdV-7.

Construction of recombinant adeno-associated viral vectors in human neurenergen-3 gene

BACKGROUND： Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene. OBJECTIVE： To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN： Gene directed cloning. SETTING： Central Laboratory of Northern China Coal Medical College. MATERIALS： DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University. METHODS： NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid （pAAV-NT-3）. pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10：1 and the mixture was used to infect HT 1080 cells. X-gal stain was used to measure virus titer. MAIN OUTCOME MEASURES： Size of targeted gene fragments, validity of vehicle construction and virus titer. RESULTS： Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified. β-galactoside staining in situ indicated that LacZ genes were expressed in human fibrosarcoma cells （HT1080） and the recombinant virus titer was measured as 1 ×10^12 virus particles per milliliter. CONCLUSION： Total-length cDNA fragment of NT-3 gene, which is obtained from pCDNA3-NT-3 plasmids, is closely matched to polyclone enzyme-shearing sites of adeno-associated viral vectors, while the combination can be used to construct recombinant adeno-associated viral vectors expression in hNT-3 gene.

Adenovirus F40/41 Gastroenteritis-Associated Emphysematous Gastritis:A Case Report

Emphysematous gastritis(EG)is a rare form of gastritis characterized by gastric wall pneumatosis.It is associated with a high mortality rate.Our case report describes a patient with a history of atrial fibrillation treated with apixaban,prior stroke,and schizoaffective disorder who showed epigastric tenderness on examination.However,no peritonitis signs were recorded,and laboratory values revealed high lactate levels and the presence of adenovirus F40/41.Abdominal computed tomography scan showed extensive portal venous gas in the left hepatic lobe of the liver,as well as pneumatosis involving the entire stomach,indicating EG.The diagnosis was histologically confirmed and initially managed medically,which proved inefficacious,necessitating surgical intervention in the form of a laparotomy and esophagojejunostomy with Roux-en-Y reconstruction.This case report suggests a potential association between EG and adenovirus F40/41–induced gastroenteritis,thereby emphasizing the challenging nature of EG.It underscores the need for comprehensive research to enhance our understanding of its underlying causes and effective treatment strategies.

热毒宁注射液对腺病毒-3的体外抑制作用

目的:研究中草药热毒宁注射液对腺病毒-3(ADV_3)的抑制作用。方法:以阿昔洛韦为阳性对照药,采用细胞培养技术及MTT比色法,观察在热毒宁干预下,转染腺病毒E1A基因人胚肾上皮细胞(293细胞)的细胞病变效应(CPE)及光密度值(D),计算各组细胞的病毒抑制率。比较不同药物剂量及不同给药方式下热毒宁对ADV_3的抑制作用效果。结果:热毒宁注射液半数中毒浓度TC_(50)为15.58 g·L^(-1)。预防、直接灭活及治疗给药方式下,CPE法测定的半数有效浓度EC_(50)分别为1.452、1.321、1.193 g·L^(-1),治疗指数TI分别为10.73、11.79、13.06;MTT法测定的EC_(50)分别为2.356、1.494、1.144g·L^(-1),TI分别为6.61、10.43和13.62。结论:热毒宁对ADV_3有直接灭活及抑制增殖作用,对其感染靶细胞有阻断作用,且呈明显量效关系。热毒宁对ADV_3的抑制作用,以预防给药更明显。

人血管形成素-1重组腺病毒高效制备

目的 :制备人血管形成素 - 1重组腺病毒 ,为基因转染心肌缺血区促血管新生研究作准备。方法 :人血管形成素 - 1重组粘粒与DNA末端蛋白复合物混合后以磷酸钙共沉淀法转染 2 93细胞。获得的重组腺病毒经扩增 ,并进行酶切鉴定。结果 :酶切结果显示 ,重组粘粒中人血管形成素 - 1基因插入方向正确 ,所获重组腺病毒带有此基因。重组腺病毒滴度达 5 .6× 10 11pfu/L。结论 :所获人血管形成素 - 1重组腺病毒为E1、E3缺陷型重组子 ,可用于动物体内基因治疗研究。

小儿肺炎的病毒性病原学研究

目的:探讨小儿肺炎的病毒性病原学特点.方法:应用直接免疫荧光法,连续3年对13 642例住院肺炎患儿呼吸道分泌物中呼吸道合胞病毒(RSV),副流感病毒(PIV) 1、2、3型,流感病毒(IV)A型、B型和腺病毒(ADV)抗原进行检测.结果:病毒性肺炎占34.3%,其中RSV占25.8%,PIV4.7%,IV A 2.4%,IV B 0.2%,ADV 1.3%.RSV在≤1岁组检出率 33.1%,>1～3岁组19.7%,>3岁组5.1%(趋势χ2=763.4,P<0.001).ADV肺炎,>1～3岁组与>3岁组分别为2.3%及2.5%,均高于≤1岁组的0.7%(P<0.01).>1～3岁组IV A检出率3.4%,高于≤1岁组(χ2=18.2,P<0.01).>1～3岁组PIV 1肺炎占1.2%,较其它年龄组高(P<0.05).PIV 3,≤1岁组检出率 4.7%,>1～3岁组3.2%,>3岁组1.4%(趋势χ2=52.4,P<0.01).RSV感染率11月开始明显增高,持续到次年的3～4月份,但每年仍有差别.肺炎中RSV月感染率最高62.8%,IV A达15.7%.结论:小儿肺炎的病毒性病原,感染率高低依次为RSV、PIV、IV和ADV.RSV、PIV 3,年龄越大检出率越低.≤1岁婴儿ADV感染较少.幼儿较婴儿易致IV A感染.RSV流行见于冬春季,但存在变化.

腺病毒介导的白细胞介素24基因对人肺腺癌细胞的放疗增敏作用

目的研究腺病毒介导的白细胞介素24基因(Ad-IL-24)对人肺腺癌细胞SPC-A1体外抑癌效应、放疗增敏作用。方法将Ad-IL-24感染的SPC-A1细胞,用逆转录-聚合酶链反应(RT-PCR)及蛋白印迹(Western blot)法检测IL-24目的基因在SPC-A1细胞中的转录和表达;四甲基偶氮唑蓝(MTT)法检测磷酸盐缓冲液(PBS)组、腺病毒(Ad-GFP)组、Ad-IL-24组、放疗组、Ad-GFP联合放疗组(Ad+放疗组)及Ad-IL-24联合放疗组(Ad-IL-24+放疗组)对SPC-A1细胞生长抑制作用,流式细胞术(FCM)检测各组SPC-A1细胞生长周期和凋亡率。RT-PCR法检测SPCA1细胞中生存素、半胱氨酸蛋白酶3(Caspase-3)、B细胞淋巴瘤/白血病基因伴随蛋白X(bax)、B细胞淋巴瘤/白血病基因-2(bcl-2)凋亡相关因子的表达。结果 Ad-IL-24作用SPC-A1细胞后,目的基因在SPC-A1细胞中成功转录及表达;Ad-IL-24能显著抑制SPC-A1细胞的生长且呈现时间依赖性,Ad-IL-24组(31.1±0.9)%、放疗组(44.4±2.3)%、Ad-IL-24+放疗组(72.4±1.5)%的生长抑制率分别与Ad-GFP组(2.7±1.1)%比较,均差异有统计学意义(P<0.01),且Ad-IL-24+放疗组优于Ad-IL-24组、放疗组(F=314.613,P<0.01),呈现放疗增敏的协同作用(Q=1.172);Ad-IL-24可诱导SPC-A1细胞G2/M期阻滞和细胞凋亡,Ad-IL-24+放疗组(40.6±3.3)%的细胞凋亡率与Ad-IL-24组(15.4±1.1)%、放疗组(26.3±1.7)%比较,差异有统计学意义(F=87.194,P<0.01),且具有放疗增敏协同作用(Q=1.162)。Ad-IL-24能明显上调促细胞凋亡相关因子bax、Caspase-3,并下调细胞抗凋亡因子bcl-2、生存素的表达,Ad-IL-24+放疗组作用优于Ad-IL-24组、放疗组。结论 Ad-IL-24有放疗增敏的作用,是理想的放疗增敏剂,该作用机制可能与诱导肿瘤细胞阻滞在G2/M期以及促进肿瘤细胞凋亡有关。

H101溶瘤腺病毒联合长春瑞滨/顺铂一线治疗晚期非小细胞肺癌

目的:探讨溶瘤腺病毒H101瘤内注射联合长春瑞滨/顺铂(NP)治疗晚期非小细胞肺癌(NSCLC)的疗效与安全性。方法:病理学或细胞学确诊的初治NSCLC患者随机接受经皮肺穿剌瘤内注射H1011.5×1012病毒颗粒联合NP方案化疗(A组)或单纯NP方案化疗(B组)。治疗2个周期后进行一次评价疗效,随访无进展生存时间(TTP)和生存期。结果:A组19例可评价患者中,总体疗效部分缓解(PR)5例,疾病稳定(SD)10例,疾病进展(PD)4例;B组可评价17例中,PR3例,SD9例和PD5例。第1次评价疗效时,A组PD1例,而B组PD5例,B组治疗失败率显著高于A组(P<0.05)。2组生存曲线几乎相似,但A组6月、9月和1年生存率均要稍高于B组;A组中位TTP时间也较B组有所延长。A组副反应中,除了非感染发热外,其他不良反应与B组相似。A组有2例发生轻度气胸。结论:经皮肺穿剌瘤内注射H101联合NP方案治疗晚期NSCLC是可行、安全与有效的。

人骨形态发生蛋白-2腺病毒表达载体转染人骨髓基质干细胞及对其增殖的影响

目的 :用人骨形态发生蛋白 - 2腺病毒表达载体 ( Ad- BMP- 2 )转染人骨髓基质干细胞( h BMSC) ,以探讨基因转染对 h BMSC增殖的影响。方法 :将 Ad- BMP- 2转染体外培养的成人骨髓基质干细胞 ,利用免疫组化、原位杂交染色方法检测细胞内骨形态发生蛋白 - 2 ( BMP- 2 )的表达 ,蛋白印迹法检测转染后细胞培养液中 BMP- 2分泌蛋白表达。然后通过流式细胞仪分析其对细胞增殖的影响。结果 :转染后 ,h BMP- 2基因在 m RNA水平和蛋白水平均有表达。蛋白印迹法检测到培养液中有 BMP- 2蛋白阳性表达。转染 h BMP- 2基因后 ,细胞经流式细胞仪分析 ,S期细胞比例增多 ,说明细胞 DNA的合成增加。结论 :Ad- BMP- 2可高效转染 h BMSC。

重组Ad-Cp-CDglyTK双自杀基因腺病毒载体构建及体外抑制鼻咽癌细胞的实验研究

目的:构建含EB病毒Cp启动子的CDglyTK双自杀基因靶向腺病毒载体Ad-Cp-CDglyTK,探讨Cp启动子能否特异性调控自杀基因在转染鼻咽癌细胞中表达及靶向性治疗鼻咽癌的可行性。方法:采用pDC316穿梭质粒系统,高保真PCR扩增tk、cd、Cp等基因序列,采用定向克隆方法构建含Cp启动子的双自杀基因pDC316-CP-CDglyTK重组质粒,经DNA测序、酶切法鉴定所构建质粒,在293细胞中进行重组腺病毒Ad-Cp-CDglyTK包装、扩增、纯化与病毒滴度测定,体外转染鼻咽癌细胞株CNE1与正常鼻咽NP69细胞株,RT-PCR法检测转染细胞中CDglyTK基因的表达,MTT法观察Ad-Cp-CDglyTK/GCV+5-FC系统对CNE1细胞株的体外杀伤作用。结果:经DNA测序、限制性酶切法分析显示pDC316-Cp-CDglyTK含完整正确的tk、cd、Cp基因序列,在293细胞中包装扩增后病毒滴度为5.6×1012TC ID50/L,体外转染鼻咽癌CNE1细胞株与正常鼻咽NP69细胞株后,采用RT-PCR法从CNE1细胞株总RNA中扩出Cp片段,而NP69细胞株未检测到相应基因mRNA表达,MTT结果显示经前体药物处理转染后CNE1细胞株与NP69细胞株,5-FC+GCV联合用药较单一前体药物对CNE1细胞具有更强的抑制作用(P<0.05),联合用药对NP69细胞株未见明显杀伤作用。结论:Cp启动子可以特异性调控CDglyTK融合自杀基因在鼻咽癌细胞株(CNE1)中表达,融合自杀基因/前药系统较单一基因对鼻咽癌细胞具有更强的杀伤作用。

内耳转基因表达的实验研究

目的 了解内耳转基因表达的可行性。方法 将人复制缺陷重组腺病毒基因(Adenoviruses ,Ad ,含大肠杆菌 β 半乳糖苷酶基因LacZ基因 ,Ad5 LacZ)经豚鼠耳蜗蜗窗接种到鼓阶外淋巴后 ,观察在不同时间、不同内耳组织中LacZ基因的表达 (X Gal染色 )及Ad对豚鼠声反应 (听性脑干反应 )、听毛细胞 (扫描电镜 )的影响。结果 Ad介导的LacZ基因在内耳组织中的表达至少可持续 4周 ,其中在螺旋神经节细胞表达稳定 ,Corti器、前庭囊斑、壶腹嵴的毛细胞等也有较强的表达。Ad未对豚鼠声反应 (≤ 80 0 0Hz)造成明显的损伤 ,除耳蜗底回外 ,其余各回未见明显的毛细胞缺失。结论 腺病毒载体可成功地将LacZ基因转导致豚鼠内耳组织中 ,并且未对豚鼠声反应 (低、中频 )及听毛细胞造成明显损伤 ,这对未来的内耳基因治疗研究可能具有重要的参考价值。