Objective:To evaluate the effects of different anticoagulant preservation solutions on the proliferation of natural killer (NK) cells cultured in umbilical cord blood and their killing effects on liver cancer cells.Methods:A portion of 20 ml umbilical cord blood was aseptically collected from 10 healthy pregnant women and transferred into anticoagulation tubes containing heparin sodium and blood preservation solution, respectively. Cord blood mononuclear cells (CBMCs) were isolated by using lymphocyte solutions. NK cells were proliferated and cultured in vitro, and cell count was carried out at 0, 3, 6, 9, 12, 15, 18 and 21 d after culture, respectively. The percentage of CD3-CD56+, CD16+NK cells and the apoptosis rate were detected by flow cytometry at 15 and 21 d. At 15 d, CCK8 assay was utilized to detect the cytotoxic effects of CBMCs collected from two anticoagulant solutions and NK cells obtained from in vitro proliferation on human liver cancer cell lines (SMMC7721, BEL7402, Sk-hep1, HepG2, Hep3B, and Huh7) according to the effect-target ratio of 5∶1, 10∶1 and 20∶1, respectively. Cell count between two groups was compared by t test. Multi-group comparison was performed by one-way ANOVA. Two-group comparison was conducted by LSD-t test.Results:NK cells grew logarithmically from 9 d of in vitro culture, and the proliferation capability was declined and the number of cells was decreased at 15 d. Cell growth curve showed that the number of NK cells at 9, 15 and 21 d in the heparin sodium group was (1.8±0.8)×108, (6.2±1.9)×108 and (4.9±1.3)×108, significantly higher than (1.0±0.5)×108, (4.3±2.0)×108 and (3.4±1.9)×108 in the blood preservation solution group (t=2.797, 2.198, 2.177; P<0.05). The cytotoxic effects of NK cells on 6 human liver cancer cells were strengthened with the increase of effect-target ratio, and the killing efficiency was the highest at an effect-target ratio of 20∶1, especially for SMMC7721. However, no significant difference was observed in the killing efficiency between two groups (P>0.05).Conclusions:Both heparin sodium and blood preservation solution can be used as anticoagulants for NK cells cultured in umbilical cord blood. Heparin sodium can enhance the proliferation of NK cells, whereas it does not increase the killing effect on liver cancer cells.

目的 探讨体外扩增的外周血NK细胞对不同肿瘤细胞系的细胞毒作用.方法 人肝癌细胞株BEL-7402、HepG2、SMCC-7721,人肺癌细胞株A549和人宫颈癌细胞株Hela均为中山大学附属第三医院岭南医院生物治疗中心实验室保存并传代.10例健康志愿者外周血均采集自中山大学附属第三医院岭南医院,其中男5例,女5例;平均年龄(52±13)岁.所有受试者均签署知情同意书,符合医学伦理学规定.分离人外周血单核细胞,培养NK细胞,并采用流式细胞术鉴定.采用CCK-8法检测体外扩增的人外周血CD56+CD3-NK细胞对肿瘤细胞毒作用;观察不同波长、培养时间、靶细胞数和不同效靶比等条件下吸光度(A)值.结果 人外周血培养14 d后,CD56+CD3-NK细胞占(61±24)％.靶细胞最佳吸收波长为450 nm,最佳测定时间1～4 h,最佳细胞数量(1～4)x 104个/ml.NK细胞对不同肿瘤细胞系的细胞毒作用不同,其中在效靶比40∶ 1时,对BEL7402作用最强,其后依次为A549、SMMC7721、HepG2,作用最弱为Hela;在20 ∶ 1、10 ∶ 1时,对BEL7402作用最强,其后依次为SMMC7721、A549、HepG2,作用最弱为Hela.NK细胞对3株肝癌细胞的细胞毒作用也不同,其中对BEL7402作用最强,SMMC7721次之,HepG2最弱.NK细胞在效靶比40∶1时对 BEL-7402、SMCC-7721、A549、Hela 细胞的杀伤率明显高于 10∶1 时(LSD-t=2.10,2.37,2.26,5.61;P＜0.05);NK细胞在效靶比40∶1时对A549细胞的杀伤率明显高于20∶1时(LSD-t=1.14,P＜0.05).结论 效靶比40∶1时NK细胞对各肿瘤细胞系的细胞毒作用最强.NK细胞对不同肿瘤细胞系的细胞毒作用不同,对同一种肿瘤不同细胞株的细胞毒作用也不同.

目的 比较双色法和三色法检测自然杀伤细胞(NK cell)的体外杀伤功能.方法 选取20例自2019年至2021年于中山大学附属第三医院献血者外周血,体外诱导扩增NK细胞(效应细胞),在培养第14天按10:1、20:1效靶比分别与K562细胞(靶细胞)混合共培养,且在1 h、2 h、3 h、4 h共4个时间组取样,分别采用CFSE/PI双色法和CFSE/Annexin?V/7?AAD三色法进行流式染色,检测K562细胞的凋亡.结果 在不同效靶比实验组,两种染色方法都能检测出NK细胞在效靶比20:1杀伤功能显著高于效靶比10:1,差异有统计学意义(P<0.05),在4个时间点实验组中,效靶细胞共培养4 h时NK细胞杀伤功能最强.其中,三色法检测出细胞杀伤功能显著高于双色法,差异有统计学意义(P<0.05).结论 三色荧光染色法比双色法能更灵敏更特异地检测NK细胞杀伤功能,结果更为可靠.

目的 流式细胞术和乳酸脱氢酶(LDH)释放法检测自然杀伤细胞(natural killer cell,NK细胞)体外杀伤功能的比较和优化.方法 以人外周血PBMCS经体外诱导扩增的NK细胞为效应细胞,K562细胞作为靶细胞,按5:1、10:1、20:1、40:1效靶细胞比共培养,分别采用LDH法和流式细胞术检测靶细胞死亡率.结果 在4个效靶细胞比实验组中,5:1效靶细胞比条件下LDH释放法检测的靶细胞死亡率显著低于CFSE/PI标记法,其他3个效靶细胞比组的两种方法检测结果无显著性差异.高效靶比、细胞毒作用较强时,LDH释放法的结果误差较大,可重复性不强.结论 流式细胞术和LDH释放法检测NK细胞体外杀伤效能的最佳效靶细胞比为10:1.流式细胞术比LDH法灵敏度更高,稳定性更好;在效靶细胞比为10:1的实验条件下,LDH法可作为流式细胞术的替代方法.

目的:观察样本保存时间对流式细胞术分析健康人外周血淋巴细胞亚群结果的影响.方法:随机选取健康人外周血标本23例,分别在采血后立即检测(D0组)、4℃保存1 d(D1组)、2 d(D2组)、3 d(D3组)和5 d(D5组)后用流式细胞仪分析淋巴细胞亚群.结果:D5组淋巴细胞的物理参数发生变化;D5组CD3+细胞百分比明显降低(P<0.05);D3组和D5组CD3-CD19+细胞百分比明显升高(P<0.05),CD3+CD56+细胞百分比明显降低(P<0.05).结论:外周血标本在单核细胞分离后,于PBS保存于4℃条件下,处理后的标本3 d内检测健康人外周血淋巴细胞亚群结果比较稳定.

目的:研究外周血自然杀伤(NK)细胞培养的扩增效率及杀伤活性的变化趋势.方法:采集10份健康供血者的外周血,采用无血清培养法对NK细胞进行扩增培养,并分别在培养的第0天、第4天、第8天、第12天、第16天、第20天进行细胞观察及计数、细胞表型分析和杀伤活性检测,总结外周血NK细胞培养的扩增效率及杀伤活性的变化趋势.结果:外周血单个核细胞(PBMCs)经诱导培养2～3 d即出现细胞集落.随着培养时间的延长,NK细胞的数量逐渐增加,不同培养时间点(第4天、第8天、第12天、第16天和第20天)NK细胞的数量相比,P<0.05;NK细胞的杀伤活性从培养第8天开始升高,至第12天达到峰值后开始下降,不同培养时间点NK细胞的杀伤活性相比,P<0.05;NK细胞的比例先增加后下降(在培养至16 d达到峰值后开始下降),其比例的下降趋势较杀伤活性的下降趋势滞后,不同培养时间点NK细胞的比例相比,P<0.05;各时间点NK细胞的存活率均大于95％.结论:采用无血清培养法对外周血NK细胞进行培养时,随着培养时间的延长,外周血NK细胞的数量呈逐渐增加的趋势.外周血NK细胞的杀伤活性在培养12 d后出现下降的趋势.在培养的第12天～第16天,NK细胞的杀伤活性较强,细胞数量较多,细胞纯度较高.采用无血清培养法体外扩增NK细胞的效率较高.

Hepatic stellate cell (HSC) activation is important in the pathogenesis of liver fibrosis. However, the molecular mechanism of HSC activation is not completely understood. In the present study, it was demonstrated that interleukin-32γ (IL-32γ) is capable of enhancing intefgrin αvβ6 expression by inducing integrin αvβ6 promoter activity in a dose-dependent manner in HSCs. Furthermore, it was determined that nuclear factor κB (NF-κB) activation is required for IL-32γ-induced integrin αvβ6 expression. Increased integrin αvβ6 expression is then able to activate HSCs. These results indicate that NF-κB activation is required for IL-32γ to induce integrin αvβ6 expression and consequently promote HSC activation. Therefore, IL-32γ activates HSCs and therefore may be associated with hepatic fibrogenesis. These results may enable the development of novel effective strategies to treat hepatic fibrosis.

More than 170 million individuals worldwide are infected with hepatitis C virus (HCV), and up to an estimated 30% of chronically infected individuals will go on to develop progressive liver disease. Despite the recent advances in antiviral treatment of HCV infection, it remains a major public health problem. Thus, development of an effective vaccine is urgently required. In this study, we constructed novel adeno-associated virus (AAV) vectors expressing the full-length NS3 or NS3/4 protein of HCV genotype 1b. The expression of the NS3 or NS3/4 protein in HepG2 cells was confirmed by western blotting. C57BL/6 mice were intramuscularly immunised with a single injection of AAV vectors, and the resultant immune response was investigated. The AAV2/rh32.33.NS3/4 vaccine induced stronger humoral and cellular responses than did the AAV2/rh32.33.NS3 vaccine. Our results demonstrate that AAV-based vaccines exhibit considerable potential for the development of an effective anti-HCV vaccine.

目的:研究天晴甘美注射液对慢性乙型肝炎(慢乙肝)患者外周血单个核细胞(PBMC)细胞周期的影响.方法:采用凯基细胞周期检测法检测天晴甘美对细胞周期的影响.结果:天晴甘美注射液呈剂量依赖性抑制外周血单个核细胞z增殖,使细胞阻滞于G0/G1期,细胞DNA合成减少,但细胞无凋亡.结论:天晴甘美注射液对PBMC具有一定抑制作用,可能使细胞阻滞于G0/G1期,导致细胞DNA合成减少.

Objective To study influence of magnesium isoglycyrrhizinate(MIG) on Toll-like receptor 3pathway in PBMC cells in chronic hepatitis B patients.Methods Inhibitory effect of MIG injection on PBMC proliferation were examined by MTT.Semi-quantitative RT-PCR examined expression of TLR3,TRIF,TNFα and IFNβ1 in PBMC cells post-treated with Poly I: C and MIG.Results MIG injection inhibited proliferation of PBMC cells significantly in dose-dependent manner,except for the comparison between 12.5 and 6.25 μg / ml group.The inhibition rates were 58%,52%,40%,30%,23%,17%,8%,and 6%,repectively in the eight groups,800,400,200,100,50,25,12.5 and 6.25 μg / ml.Expression of interferon(IFN) β1 was significantly higher in 200 μg / ml of MIG group compared with blank control group(14.6 ± 2.43 and 9.79 ± 3.02,respectively),while expression of TNFα was significantly downregulated in 200 μg / ml and 100 μg / ml group compared with blank control group(10.59± 1.67,13.3 ± 2.07 and 15.7 ± 1.59,respectively).Conclusion MIG might play a role in immunoregulation by promoting IFNβ1 and inhibiting TNFα in PBMC cells.

The envelope protein of dengue virus is involved in host cell attachment for entry and induction of protective immunity. Current efforts are focused on producing a tetravalent vaccine by mixing four monovalent vaccine components. In this work, we developed a genetic vaccine based on a novel adeno-associated viral (AAV) vector expressing the carboxy-terminal truncated envelope protein (79E) of dengue virus. The expression of the recombinant 79E protein in HEK 293 cells was confirmed by Western blot. Vectors packaged with novel AAV capsids (AAV2/8 or AAV2/rh32.33) were injected into C57BL/6 mice intramuscularly. Dengue virus antigen was produced in the mice and induced long-lasting antibody responses against the dengue virus still detectable 20 weeks after immunization. AAV2/8 vaccine induced higher anti-dengue virus antibody levels than AAV2/rh32.33 vaccine or AAV plasmid. Furthermore, the anti-dengue antibodies could neutralize homogeneous dengue virus. These results demonstrated that the AAV vaccines possessed appropriate immunogenicity and could be used for the development of an effective dengue vaccine.

objectives The role of interleukin-32 (IL-32) in chronic hepatitis B (CHB) remains unclear. In order to identify the role of IL-32 in CHB, we detected the expression levels of IL-32 in liver samples of CHB patients and analyzed the correlation between IL-32 and liver inflammation/fibrosis. Methods Real-time PCR and immunohistochemistry were used to detect the expression levels of IL-32 in liver tissues of patients with CHB. The correlation between hepatic IL-32 expression and the severity of liver inflammation/fibrosis was assessed using Spearman's correlation. Results Hepatic IL-32 expression was increased in CHB patients and increased with the severity of liver inflammation/fibrosis. Moreover, hepatic IL-32 expression was significantly positively correlated with serum ALT level and negatively related with serum ALB level. Conclusions Our results suggest that IL-32 could be implicated in HBV-related liver inflammation/fibrosis. We believe that IL-32 might play important roles in the pathogenesis of CHB.

OBJECTIVE:In an attempt to study the moleculr characterization and epidemiology of simian adenoviruses in nonhuman primate (NHP) populations.METHODS:We examined a colony of captively bred rhesus macaques (Macaca mulatta) in China for the presence of adenoviral DNA in stool samples. This was done by using the PCR method that targeted the adenovirus polymerase gene, and the PCR positive fragments were cloned for sequencing and phylogenetic analyses.RESULTS:Among the 57 animals analyzed, fecal samples from 12 animals were positive for the presence of adenoviral DNA. The results suggested that the viral DNA clones were primarily segregated into two large groups: SAdV-6 (2 non-redundant sequences) and SAdV-7 (9 non-redundant sequences). In addition, there were three clones with more similarity to SAdV-1, SAdV-3 and HAdV-52 respectively.CONCLUSION:Our data confirmed the prevalence of adenoviral DNA in the feces of NHPs and revealed the adenoviruses in the gastrointestinal tract of the study animals. heterogeneity and phylogenetics of the adenoviruses in the gastrointestinal tract of the study animals.

Objective To estimate the epidemic situation of preexisting neutralizing antibody of human adenovirus type 5(HAdV-5) in Guangzhou population.Methods β-galactosidase(LacZ) as reporter gene,CMV promoter driven recombinant HAdV-5 vector and chemiluminescence method were used to investigate the present of neutralizing antibodies against HAdV-5 in the serum of 209 immunocompetent individuals.Results We found that the positive rate of HAdV-5 neutralizing antibodies was 82.3%(172:209).The HAdV-5 neutralizing antibodies could be detected at low(1:20),medium(1:40-1:160),high(1:320-1:1 280) and ultra-high(1:1 280) titers.The positive rate of HAdV-5 neutralizing antibodies of the 20-40 year old age group was the highest,while the rate of 20 year old age group was the lowest.Conclusion The positive rate of HAdV-5 neutralizing antibodies was high in Guangzhou.So there are some limitations of using this virus vector for gene vaccination and gene therapy research.

目的:探讨TMX基因exon6上的多态性位点rs7161242[c.492T>G]及exon7上的多态性位点rs7160810[c.648G>A]与CHPS发病易感性的相关性.方法:采集27个汉族核心家系(CHPS患者及父母双亲)的血样,提取基因组DNA,进行PCR扩增,测序后进行基因分型,应用传递不平衡检验来分析基因多态性与CHPS发病的相联性.结果:①序列分析显示未发现新的突变位点.②患儿及父母组内rs7161242 及rs7160810这两个多态性位点的Hardy-Weinberg平衡检验均P>0.05,TDT检验提示多态性位点rs7161242的G等位基因及rs7160810的A等位基因的均与CHPS发病相关(P均<0.001);连锁不平衡分析结果提示:这两个位点的r2=0.757,D'=0.893,成紧密连锁关系.结论:TMX基因的多态性位点rs7161242及rs7160810与中国汉族人群CHPS发病密切相关.

Objective To search and classify genotyping method of gastrointestinal adenoviruses conveniently and quickly.Methods Stool samples were collected from a colony of captively bred rhesus macaques and two methods were used to figure out the presence and genotypes of adenovirus.Method 1:Stool samples were suspended in Herpes buffer and filtered.The filtration was used to infect BSC-1 cell.Genomic DNA was extracted from the infected cells and used as PCR template,and the PCR positive fragments were cloned for sequencing and phylogenetic analyses;method 2:DNA extracted directly from fecal samples was used as the template for nested PCR.The PCR product was also cloned and submitted for sequencing.Results 57 stool samples were detected with the two methods.The PCR positive rate was the same 21.1%(12/57),as well as the genotypes identified by the subsequent sequencing analysis.The results suggested that the viral DNA clones were primarily segregated into two large groups:SAdV-6(2 non-redundant sequences) and SAdV-7(9 non-redundant sequences).In addition,there were three clones with more similarity to SAdV-1,SAdV-3 and HAdV-52 respectively.Conclusion Although the sensitivity and specificity of two methods is the same,but method 2 saved time and cost,especially avoided the possible contamination that may occur during the process of in vitro infection,so method 2 is superior to method 1.

Objective To evaluate the positive ratio of preexisting neutralizing antibodies of simian adenovirus type 7 (SAdV-7) in Guangzhou.Methods The simian recombinant adenovirus type 7 vector that was driven by CMV promoter and used β-galactosidase (LacZ) as reporter gene was constructed and the positive ratio of neutralizing antibodies of SAdV-7 in the serum of 209 immunocompetent individuals was evaluated using chemiluminescence method.Results Among the studied population,the ratio of individuals with SAdV-7 neutralizing antibodies was 27.8% (58/209).The SAdV-7 neutralizing antibodies were highly prevalent in low titer,the prevalence decreased as the titer increased.The positive ratio of SAdV-7 neutralizing antibodies were highest in the individuals who were at the age of 20 to 40.While SAdV-7 neutralizing antibodies in the individuals younger than 20 was not detected.Conclusion As the SAdV-7 neutralizing antibody is lowly prevalent in population of Guangzhou,it is a promising candidate vector for gene therapy and gene vaccine.

Objective To estimate the epidemic situation of preexisting neutralizing antibody of human adenovirus type 5(HAdV-5) in Guangzhou.Method β-galactosidase(LacZ) was used as the reporter gene,combined with the genome of HAdV-5 driven by CMV promoter.Then chemiluminescence method was used to investigate the ratio of positive neutralizing antibodies of HAdV-5 in the sera of 209 immunocompetent individuals.Result The ratio of positive neutralizing antibodies of HAdV-5 was 82.3%(172/209).The HAdV-5 neutralizing antibodies were found at low(1/20),medium(1/40-1/160),high(1/320-1/1280) and ultra-high(1/1280) titers.And the ratio of positive gradually increased as the titer increased.The antibodies were highly prevalent at high and ultra-high titers.The highest rate of HAdV-5 neutralizing antibodies appeared at the ages of 20 to 40,while the lowest rate was seen in ages under 20.Conclusion The neutralizing antibodies against HAdV-5 have a high positive ratio in Guangzhou population.So there are some limitations using this virus vector for gene vaccine and gene therapy research.

OBJECTIVE:To analyze the gene expression level of fibroblast activation protein in HBV related hepatocellular carcinoma patients and discuss its clinical significance.METHODS:FAP gene expression in 33 hepatocellular carcinoma patients cancer tissues, peficancerous tissues, distant relative normal liver tissues and 13 normal liver tissues were examined by reverse transcription PCR; and real-time fluorescent quantitative PCR (qRT-PCR) was used to quantify their expression.RESULTS:FAP were expressed in all the tissues,the relative expression values in cancer tissues, peficancerous tissues and distant relative normal liver tissues were 5.14 +/- 6.69, 1.58 +/- 0.96, 1.63 +/- 0.94, respectively, the differences were statistically significant (F = 4.401, P < 0.05); and in TNM stage I, II, IIII, they were 2.89 +/- 3.35, 4.15 +/- 4.69, 10.09 +/- 9.51 respectively; in well-differentiated, differentiated and poorly differentiated hepatocellular carcinoma were 1.62 +/- 1.74, 3.84 +/- 3.79, 1.26 +/- 13.34 respectively. The differences were all statistically significant (P < 0.05).CONCLUSION:FAP may play an important role in the occurrence and development of HBV related hepatocellular carcinoma.