HBV阳性血清诱导人外周血单个核细胞凋亡的检测及意义

目的研究乙型肝炎病毒(HBV)对外周血单个核淋细胞(PBMC)凋亡的作用.方法利用10名健康献血员外周血分离PBMC,分为加HBV阳性血清组和加健康人血清对照组,经培养72h后,采用PI染色法在流式细胞仪上检测细胞的凋亡情况.结果加HBV阳性血清组培养细胞的凋亡率为(39.56±7.03)%,明显高于加健康人血清组培养细胞的凋亡率(27.57±7.78)%,两组间具有非常显著的差异(P＜0.02).结论HBV可能具有诱导PBMC凋亡的能力,这可能是形成HBV慢性感染免疫耐受的一个重要机制.     

ABCB1基因多态性对肝移植后他克莫司用量的影响

背景：钙神经素抑制剂他克莫司被广泛用于移植后预防排斥反应的发生,但其治疗窗狭窄,且药代动力学个体差异较大。 目的：观察ABCB1基因多态性对肝移植患者移植后早期免疫抑制剂他克莫司的用量及C/D比值的影响。 方法：选择2008-01/2010-09-31在昆明市第一人民医院暨昆明医学院附属甘美医院肝胆外科接受原位肝移植患者67例,通过检测67例肝移植受者移植后不同时间他克莫司的血药浓度及其用量,并利用DNA直接测序检测受体ABCB1的基因多态性,分析肝移植后免疫抑制剂他克莫司的用量及其血药浓度与ABCB1基因多态性的关系。 结果与结论：肝移植后他克莫司的口服需药量在个体间存在很大差异,67例肝移植患者中,ABCB1不同位点的基因多态性分布不同,其中仅ABCB1 3435C>T基因多态性与他克莫司用量有关。提示ABCB1的基因多态性可能是患者肝移植后他克莫司药代动力学显著个体差异的重要因素,ABCB1 3435C>T野生型的患者需要更高剂量的他克莫司便可达到目标血药浓度水平,检测ABCB1的基因多态性可以优化肝移植后免疫抑制剂的个体化治疗方案。     

他克莫司血药浓度的方法学评价

评价德灵公司均相酶法他克莫司（Tacrolimus）试剂盒检测他克莫司血药浓度的可靠性。用本方法在日立7600分析仪上对其检测他克莫司血药浓度的精密度、线性、回收率、相关性等指标进行评价。本方法的评价表明,在他克莫司质控浓度为5.0ng/mL和15.0ng/mL时,批内变异系数分别为14.17%、10.66%,批间变异系数分别为12.56%、9.92%。平均回收率95.98%,在1.5~30ng/mL范围内,线性良好。与微粒子酶联免疫吸附法测定相关性良好（r=0.93）。结论：该方法操作简便、快速、准确,多通道检测,适用于临床上对肝肾移植患者他克莫司药物谷值浓度的监测。     

基于SVR的他克莫司血药浓度的预测模型

为了准确预测肾移植患者服用他克莫司之后的血药浓度,本研究利用LIBSVM软件包建立了以性别、年龄、体重、移植天数、相关生化指标、药剂量等作为输入因子的支持向量机回归预测模型.同时结合收集整理的服用他克莫司的肾移植患者相关数据进行实验,最终结果显示支持向量回归预测模型的精确度能达到75.00%,表明支持向量机回归模型在血药浓度上能取得良好的预测效果,有很好的应用前景.     

HCV感染外周血单个核细胞及其对T淋巴细胞亚群的影响

目的：研究丙型肝炎病毒（ＨＣＶ）感染外周血单个核细胞（ＰＢＭＣ）的情况及其对Ｔ淋巴细胞亚群的影响。方法：运用非同位素原位杂交（ＮＩＳＨ）法和链酶亲和素－生物素（ＳＡＢＣ）法分别检测２０例慢性丙型肝炎患者ＰＢＭＣ中的ＨＣＶ－ＲＮＡ和非结构（Ｎｏｎｓｔｒｕｃｔｕｒａｌ,ＮＳ）蛋白ＮＳ５抗原,同时用ＳＡＢＣ法检测其Ｔ淋巴细胞亚群。结果：８例（４０．０％）患者的ＰＢＭＣ中ＨＣＶ－ＲＮＡ呈构（Ｎｏｎｓｔｒｕ     

外周血单个核细胞中乙型肝炎病毒感染的研究进展

乙型肝炎病毒（ＨＢＶ）感染乙肝（ＨＢ）患者外周血单个核细胞（ＰＢＭＣｓ），ＨＢＶ－ＤＮＡ以游离和整合型两种形式存在于ＰＢＭＣｓ内。在ＰＢＭＣｓ内复制与表达。研究ＰＢＭＣｓ内ＨＢＶ感染对于探讨ＨＢ的临床进程、治愈和指导治疗等均具有重要的意义。最近提出了一种与上述相反的观点，ＰＢＭＣｓ内有关的ＨＢＶ－ＤＮＡ和ＲＮＡ是吸附的结果，而非病毒的复制。     

他克莫司用于治疗口腔扁平苔癣的疗效观察

目的：观察他克莫司用于治疗有症状的口腔扁平苔癣(OLP)的疗效。方法选取我院就诊OLP患者40例，局部应用他克莫司后了解用药后病损的症状，病变改善的时间，当前用药情况和副作用。结果40例患者中35例(87.5%)在局部应用他克莫司后症状有所改善，32(80%)例在使用后局部病变几近痊愈，3例(7.5%)认为病变没有变化，1(2.5%)人反应病变增多，1例(2.5%)反应症状加重，10(25%)人报告副作用与之前的报道一致(灼烧感，刺激疼，麻刺感等)。结论局部应用他克莫司对于OLP的治疗有效，大部分患者在用药1个月后感觉症状有所改善，但疗效不持久，停药后容易复发。     

他克莫司在体外对HepG2.2.15细胞增殖及乙型肝炎病毒复制的影响

背景：肝癌复发与乙肝病毒再感染两者关系尚不明确,有人认为与肝移植后免疫抑制剂应用有关。 目的：观察他克莫司在体外对肝癌HepG2.2.15细胞增殖及对细胞内乙肝病毒复制的影响。 方法：选择肝癌HepG2.2.15细胞进行体外培养,第3代细胞培养24 h后加他克莫司进行干预,0 g/L他克莫司作为对照组,50 g/L 他克莫司为低浓度组,100,500 g/L他克莫司为中浓度组,1 000,3 000 g/L他克莫司为高浓度组。 结果与结论：①中、高浓度的他克莫司对HepG2.2.15细胞有增殖抑制作用,低浓度无抑制作用,且有相关性。②高浓度他克莫司作用时使HepG2.2.15细胞停止在G0/G1期。③他克莫司可以使HepG2.2.15细胞中CyclinA表达降低,且呈浓度依赖性,他克莫司浓度越高,CyclinA表达越少。④他克莫司作用HepG2.2.15细胞,对HBV的复制无影响。结果说明他克莫司在体外对HepG2.2.15增殖有抑制作用,其中CyclinA可能发挥一定的作用,而对乙肝病毒复制没有影响。     

慢性乙型肝炎患者外周血单个核细胞基因表达谱的研究

目的 分析慢性乙型肝炎患者外周血单个核细胞差异表达基因，探索慢性乙型肝炎形成的分子机制。方法 应用含14000条人体cDNA的微阵列芯片和来自外周血单个核细胞的标记cDNA，分析了10例慢性乙型肝炎患者和10例健康人基因表达谱。通过应用GenePix 4000B扫描仪和ImaGene3．0分析软件比较cy5标记的慢性乙型肝炎来源cDNA与Cy3标记的健康人来源cDNA的杂交结果，获得个体基因的相对表达比值。结果 在分析的14000条基因中，差异表达的基因有92条，占0．66％。其中51条基因表达水平显著上调，41条基因表达水平显著下调。这些差异表达的基因主要为细胞信号转导，细胞周期和代谢，凋亡及炎症相关类基因。结论 在乙型肝炎病毒致慢性乙型肝炎过程中，涉及到了众多基因的差异表达，为进一步阐明慢性乙型肝炎形成的分子机制提供基础。     

他克莫司与环孢菌素A对肝移植受者CD4/CD8 T淋巴细胞亚群及共刺激分子的调节作用

目的:探讨他克莫司(Tacrolimus, FK506)与环孢菌素A(CsA)对肝移植受者T淋巴细胞亚群共刺激分子的调节作用.方法:采用荧光标记单克隆抗体(mAb)结合流式细胞技术, 测定移植术后使用FK506或CsA治疗2月末的肝移植受者外周血T细胞亚群及其表面共刺激分子CD28、CD152 和ICOS的表达情况.以健康志愿者(健康对照组)和患终末期肝脏疾病拟进行肝移植者(疾病对照组)为对照.结果:疾病对照组T细胞亚群平衡紊乱、共刺激分子表达异常(P＜0.05).治疗组肝移植受者T淋巴细胞亚群表达恢复至健康对照水平, T细胞表面CD28和ICOS分子表达显著降低(P＜0.05)而CD152分子表达明显升高(P＜0.05).比较不同药物治疗组:CsA治疗组CD4 T细胞表达和CD8 T细胞表面CD28、CD152分子表达均明显高于FK506治疗组(P＜0.05);其他指标无统计学意义(P＞0.05).结论:在常规血药浓度条件下FK506和CsA的对CD4/CD8T细胞亚群及共刺激分子的免疫调节作用存在差异.FK506对T细胞亚群的调节作用强于CsA.FK506可同时抑制正性共刺激分子CD28和ICOS表达并促进负性共刺激分子CD152表达, 而CsA对T细胞免疫抑制作用主要是通过促进CD152分子的高表达介导.     

Hepatitis B virus DNA in liver, serum, and peripheral blood mononuclear cells after the clearance of serum hepatitis B virus surface antigen

The integration of hepatitis B virus (HBV) DNA in the liver of chronic HBV carriers has been documented extensively. However, the status of the viral genome during acute infection has not been assessed conclusively. While HBV DNA sequences are detected often in serum, liver, and peripheral blood mononuclear cells (PBMCs) after the clearance of serum the hepatitis B virus surface antigen (HBsAg), the precise status of the viral genome, and in particular the possible persistence of integrated genomes in PBMCs, has not been established. A highly sensitive PCR-derived assay (Alu-PCR) was employed to re-examine liver and PBMC specimens obtained from patients with acute (n = 19) and chronic (n = 22) hepatitis in whom serum HBsAg was present (n = 12) (HBV-related chronic active hepatitis) or absent with anti-HCV (n = 10) (HCV-related chronic active hepatitis). Viral integration was demonstrated in 3 out of 19 liver specimens from patients with acute hepatitis and 12 out of 12 specimens from patients with chronic hepatitis. Viral integration was also observed in 4 out of 7 PBMC samples from HBV-related chronic active hepatitis patients and 2 out of 10 liver and PBMC samples from HCV-related chronic active hepatitis patients. In one liver specimen from an acute hepatitis patient, HBV DNA was found integrated in the intronic sequence of the tumour necrosis factor (TNF)-induced protein gene; viral integration into cellular sequences was also found in the PBMCs of four HBV-related chronic active hepatitis and two HCV-related chronic active hepatitis. The results demonstrate the early integration of HBV genome during acute viral infections and the persistence of the viral genome in an integrated form in PBMCs.     

乙肝病毒相关肝病患者肝移植后外周血单个核细胞及肝组织中乙型肝炎病毒cccDNA的检测

背景：肝移植后受者体内绝大部分病毒负荷被清除,植入新肝后其复发性肝炎病原体从肝外进入肝内的途径及其复制规律目前尚无定论。 目的：检测肝移植前后外周血单个核细胞和肝组织中乙型肝炎病毒cccDNA及血清中乙型肝炎病毒DNA的表达。 方法：采用淋巴细胞分离液从乙肝病毒相关终末期肝病37例患者外周血中分离出单个核细胞,采用荧光定量PCR检测肝移植前后及移植后乙肝复发3个时期外周血单个核细胞和肝组织中cccDNA及血清乙型肝炎病毒DNA表达。 结果与结论：肝移植前,单个核细胞cccDNA阳性12例,肝组织cccDNA阳性6例,检出率分别为32%和16%,单个核细胞、肝组织中cccDNA拷贝范围分别为(3.028~6.508)×10⁴,(4.158~6.234)×10⁴ 拷贝/mL。肝移植后,单个核细胞cccDNA阳性1例,无血清乙型肝炎病毒DNA检测阳性病例。6例肝移植后乙肝复发病例中外周血单个核细胞cccDNA阳性4例,肝组织活检cccDNA阳性1例,6例血清乙型肝炎病毒 DNA均为阳性。提示乙肝病毒相关终末期肝病患者肝移植后乙肝复发途径可能是残留乙肝病毒在外周单个核细胞中以cccDNA为模板复制,然后再迁移到肝脏。     

Effects of alpha-interferon on gamma-interferon production of peripheral blood mononuclear cells in hepatitis B virus carriers

We studied gamma-interferon production of phytohemagglutinin-stimulated peripheral blood mononuclear cells in response to alpha-interferon in hepatitis B virus carriers and healthy individuals. The magnitude of gamma-interferon production was significantly higher in patients with anti-HBe antibody than in patients with HBe antigen and healthy individuals. Furthermore, alpha-interferon augmented the production of gamma-interferon of peripheral blood mononuclear cells from patients with active liver injury [serum alanine aminotransferase (ALT), >40 U/L], but not that from patients with inactive liver injury (serum ALT, <40 U/L) or healthy individuals. These results suggested that alpha-interferon could enhance the cellular immune response against hepatitis B virus by augmenting the endogenous production of gamma-interferon in patients with active liver injury, implying that the responsiveness to alpha-interferon might be responsible for liver cell injury.     

Different forms of hepatitis B virus DNA and expression of HBV antigens in peripheral blood mononuclear cells in chronic hepatitis B

The presence of both hepatitis B virus (HBV) DNA and HBV antigens (HBsAg, HBeAg) was assessed in peripheral blood mononuclear cells (PBMC) from 32 patients chronically infected with HBV. Three different molecular forms of HBV DNA were observed: free monomers (5), high-molecular-weight free concatemers (11), and integrated HBV DNA (9). The HBV DNA patterns in the PBMC were different from those found in liver and did not correlate with any specific profile of serum HBV markers. When the same PBMC were assayed for HBsAg, 22 of the 25 HBV DNA positive samples, but only three of the seven HBV DNA negative samples, were positive. By contrast, none of the PBMC samples from five healthy HBV vaccine recipients gave any positive signal in the HBV DNA or HBsAg assays. In some patients, T and B cells, monocytes, and polymorphonuclear (PMN) cells were assayed separately, showing that the DNA pattern was similar for these different leucocytes subsets and ruling out the possibility that these patterns might reflect PMN cell contamination. Thus, in chronic HBV infection, 87.5% (28/32) of patients were found to contain at least one HBV marker in their PBMC, and a strong correlation was found between the presence of HBV DNA and viral antigens, suggesting a specific expression of HBV encoded proteins.     

Real-time PCR quantitation of hepatitis B virus total DNA and covalently closed circular DNA in peripheral blood mononuclear cells from hepatitis B virus-infected patients

It remains unclear whether hepatitis B virus (HBV) replicates in extrahepatic tissues, and particularly in peripheral blood mononuclear cells (PBMCs), which may serve as a reservoir for the maintenance of infection. A real-time PCR assay for the quantitation of total and covalently closed circular (ccc) HBV DNA in serum and in PBMCs was developed. This assay was highly sensitive (detection limit: 27 IU/mL), linear over a wide range (9 log10), and was displayed high inter- and intra-assay reproducibility for the quantitation of total DNA. Genotypes A to E were detected and the results were consistent with those obtained with the COBAS Amplicor HBV Monitor Test. The specificity of the methodology was increased by prior treatment with an enzyme that digests relaxed circular DNA, and the elimination of background signals from virus adsorbed to the surface of PBMCs. HBV DNA was detected in the serum and PBMCs of 12 HBsAg-positive patients, with less than 1% in the cccDNA form. In conclusion, the quantitation of total and ccc HBV DNA in PBMCs is potentially useful as a non-invasive marker, and may help to increase our knowledge of the natural history of hepatitis B.     

外周血单个核细胞在乙肝相关性肾炎发病机制中的作用研究

目的通过观察外周血单个核细胞（PBMc）对体外培养的肾小管上皮细胞凋亡的影响,探讨PBMC在乙肝相关性肾炎发病机制中的作用。方法用流式细胞仪检测HBVDNA阳性血清与人近端肾小管上皮细胞（HK-2）培养时HK．2凋亡率作为对照,分别观察健康人及HBVDNA阳性患者PBMC对HK-2凋亡的影响。结果健康人PBMC组HK-2凋亡率明显低于对照组和HBVDNA阳性患者PBMC组（P〈0．01）。结论健康人PBMC能抑制HK-2凋亡,而HBVDNA阳性患者PBMC此抑制作用降低,提示HBVDNA阳性患者PBMCs功能缺陷,可能是乙型肝炎相关肾炎（HBV—GN）的发病机制之一。     

The effect of interleukin-6 on hepatitis B virus replication in peripheral blood mononuclear cells in vitro

Although the major target organ for hepatitis B virus (HBV) is the liver, the possibility of infection of peripheral blood mononuclear cells (PBMCs) with HBV has also been reported. This study was performed to analyze the course of HBV infection of PBMCs and to investigate the influence of interleukin-6 (IL-6) on the efficiency of infection of PBMCs with HBV in vitro. PBMCs isolated from a healthy donor were infected by exposing to a HBsAg-, HBeAg-positive serum in the presence or absence of exogenous IL-6. The efficiency of infection was estimated by HBV DNA determination in the cells and medium in the course of infection. The results of this study show that the presence of IL-6 during the PBMCs infection with HBV increased the efficiency of this infection.     

Quantitation of hepatitis C virus in liver and peripheral blood mononuclear cells from patients with chronic hepatitis C virus infection

Since the natural history of hepatitis C virus-associated liver disease and the therapeutic responsiveness might vary according to liver and blood mononuclear cells viral levels, it may be important to quantitate viral RNA in liver, blood mononuclear cells and serum, and to compare these data with genotype, biochemical and histologic data. A polymerase chain reaction-based assay available for serum hepatitis C virus RNA quantitation has been optimized to quantitate viral genomes in liver and peripheral blood mononuclear cells from 47 chronic hepatitis C patients. The procedure permitted hepatitis C virus RNA quantitation in freshly isolated mononuclear cells and in total RNA extracted from frozen mononuclear cells and liver tissue. The intrahepatic viral amount (median: 2.6 × 10³ copies/μg RNA; range: 0 to 3.6 × 10⁴ copies/μg RNA) correlated significantly with the hepatitis C virus RNA concentration in serum (r = 0.76, P < .001) but not in mononuclear cells. Viral RNA concentrations in liver (P < .001), serum (P < 0.01) and PBMC (P < 0.05) were significantly higher in hepatitis C virus genotype 1 patients (essentially type 1b) than in non-1 type cases, but were unrelated to biochemical or histologic indexes of disease activity. In conclusion, the optimized assay permit HCV RNA quantitation in liver and peripheral blood mononuclear cells, suggesting that serum viral level is an accurate measurement of intrahepatic viral burden. J. Med. Virol. 54:265–270, 1998. © 1998 Wiley-Liss, Inc.     

沙利度胺对慢性乙型肝炎患者外周血单个核细胞的影响

目的探讨沙利度胺（Thalidomide,Thal）对慢性乙型肝炎（Chronic hepatitis B,CHB）外周血单个核细胞（Peripheral blood mononuelear cells,PBMC）增殖、克隆形成、分泌TNFα和IFNγ的影响。方法选择慢性乙型肝炎患者23例,常规方法分离PBMC,体外扩增,用细胞计数法观察Thal对PBMC增殖的影响,胎盘蓝染色法观察其对细胞存活的影响,琼脂糖凝胶法观察Thal对PBMC克隆形成的影响,酶联免疫吸附法（ELISA）观察Thal对CHB PBMC分泌TNFα和IFNγ的影响。结果Thal对PBMC的增殖具有促进作用,而无毒性影响（P〈0．05）;抑制PBMC克隆的形成（P〈0．05）;抑制TNFα的产生而促进IFNγ的产生（P〈0．05）。结论Thal对CHB PBMC的免疫功能具有调节作用,可望用于临床上慢性乙型肝炎的治疗。     

Divergent evolution of hepatitis C virus in liver and peripheral blood mononuclear cells of infected patients

In infected individuals, hepatitis C virus (HCV) exists as a variably complex population of related genetic variants known as quasispecies. The quasispecies of HCV were studied previously in 10 chronically infected patients by single-strand conformation polymorphism analysis of a segment of the envelope gene E2/NS1 containing the hypervariable region 1 and it was found that certain variants (LC variants) were present both in the liver and in peripheral blood mononuclear cells (PBMC), others (L variants) were present in the liver but not in the PBMC, and still others (C variants) showed the opposite distribution. The sequence data obtained from nine such patients are reported, indicating that, within individual subjects, L and C variants are distinct phylogenetically. Results are described on the growth of HCV in stimulated healthy donor PBMC cultures supporting the concept that genetic divergence might stem, at least in part, from virus adaptation to growth in different cell types. This information may help to understand how HCV persists and produces disease in infected patients, especially with regard to extrahepatic pathology. J. Med. Virol. 57:57–63, 1999. © 1999 Wiley-Liss, Inc.