Patient demographics and disease variables correlate with distinct cytokine patterns in mitogen-stimulated peripheral blood mononuclear cells from rheumatoid arthritis patients

There is paucity of literature on the association of peripheral blood cytokine patterns with patient demographics and disease variables in rheumatoid arthritis (RA). We test the hypothesis that there may be differences in peripheral blood levels of inflammatory cytokines in RA subjects according to various disease variables. In this case, we could identify peripheral blood cytokine markers that correlate with different disease variables. Forty-two seropositive RA patients were characterized according to the age at onset, gender, disease duration, severity, activity and ACR functional class. The production levels in mitogen-stimulated PBMCs of five pro-inflammatory cytokines (IFNγ, TNFα, TNFβ, IL-8, IL-18) and three anti-inflammatory cytokines (IL-4, IL-10, IL-13) were evaluated in these patients and in healthy controls. Several new findings emerge: (1) higher levels of IL-4 correlate with female gender, milder disease, non-erosive disease, and earlier age at onset; (2) higher levels of IL-10 correlate with the requirement of ≤2 DMARDs; (3) higher levels of IL-18 correlate with non-erosive disease and younger age at onset; (4) higher TNFβ levels correlate with older present age of patients; and (5) higher IL-8 levels correlate with established/late disease. There are several interesting differences in cytokine patterns with respect to age at onset, current age, disease severity, and the number of DMARDs the patients require.     

Mitochondrial dysfunction in peripheral blood mononuclear cells in early experimental and clinical acute pancreatitis

Background/objectivesMitochondrial dysfunction occurs in vital organs in experimental acute pancreatitis (AP) and may play an important role in determining severity of AP. However, obtaining vital organ biopsies to measure mitochondrial function (MtF) in patients with AP poses considerable risk of harm. Being able to measure MtF from peripheral blood will bypass this problem. Furthermore, whether mitochondrial dysfunction is detectable in peripheral blood in mild AP is unknown. Therefore, the objective was to evaluate peripheral blood MtF in experimental and clinical AP.MethodMitochondrial respiration was measured using high resolution oxygraphy in an experimental study in caerulein induced AP and in a separate study, in patients with mild AP. Superoxide, cytochrome c, mitochondrial membrane potential (ΔΨ) and adenine triphosphate (ATP) were also measured as other markers of MtF.ResultsEven though some states of mitochondrial respiration were increased in both experimental and clinical AP, this did not lead to an increase in net ATP in patients with AP. The increased leak respiration in both studies was further proof of dyscoupled mitochondria. In the clinical study there were also features of mitochondrial dysfunction with increased leak flux control ratio, superoxide, ΔΨ and decreased cytochrome c.ConclusionThere is evidence of mitochondrial dysfunction with dyscoupled mitochondria, increased superoxide and decreased cytochrome c in patients with mild acute pancreatitis. Further studies should now determine whether mitochondrial function alters with severity in AP and whether mitochondrial dysfunction responds to treatments.     

系统性红斑狼疮患者外周血单个核细胞T细胞受体重组删除环水平

目的 通过检测系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMC)T细胞受体重组删除环(TREC)水平,探讨其与临床表现间的关系及SLE的发病机制.方法 收集21例SLE组患者和22例年龄、性别相匹配的健康对照组一般临床资料;采集2组受试者外周静脉血,分离PBMC,实时荧光定量PCR检测TREC水平,结果以"TREC拷贝数/1000 PBMC"表示.结果 SLE组患者外周血PBMC中TREC水平为(9.6±7.5)拷贝/1000 PBMC,明显低于健康对照组[(16.1±11.1)拷贝/1000 PBMC,P=0.033].健康对照组PBMC中TREC水平与年龄呈负相关(r=-0.614,P=0.002),而SLE组无这种相关性.SLE组患者PBMC中TREC水平与SLEDAI评分呈负相关(r=-0.656,P=0.001),与皮肤黏膜受累旱负相关(r=-0.620,P=0.003),与其他临床表现及实验室指标无明显关联.结论 SLE患者外周血TREC水平降低提示SLE患者胸腺近期输出产物比例减少,这可能与该疾病造成胸腺近期输出功能减弱和(或)T细胞外周增殖增加有关.胸腺近期输出产物在外周T细胞池巾的含量减少,可能埘SLE患者免疫功能异常产生影响.     

白细胞介素-15对脐血和成人外周血单个核细胞抗肿瘤活性的影响

目的：探讨白细胞介素—15(1L—15)对脐血单个核细胞(CBMNC)和成人外周血单个核细胞(PBMNC)抗肿瘤活性的影响。方法：采用MTT法分别检测静止的CBMNC和PBMNC对K562和HL—60细胞株的杀伤活性、经不同浓度的IL—15及不同效靶比条件下激活的CBMNC和PBMNC抗瘤活性，采用ELISA法测定培养上清中IFN—r及TNF—a水平，采用APAAP检测CD56、CD16表面抗原。结果：①静止CBMNC抗肿瘤活性显著低于PBMNC，经IL—15活化后，脐血NK活性显著增强，可达成人水平，脐血LAK活性显著高于成人血LAK活性。②活化后培养上清中的IFN—r及TNF—a水平均显著增强，但脐血培养上清中两种细胞因子的水平仍低于成人血。③活化前后脐血NK细胞CD56表达显著增强。结论：IL—15能显著提高CBMNC的抗肿瘤活性，为今后优选IL—15活化脐血进行肿瘤过继免疫治疗提供了理论依据。     

Correlation of interleukin-6 serum levels with bone density in postmenopausal women

Summary In order to identify possible correlations between interleukin-6 (IL-6) and hormonal and biochemical parameters of bone metabolism, or bone density, 24 postmenopausal women were studied. Serum IL-6, estradiol, calcium, phosphorus, osteocalcin, alkaline phosphatase, the urinary secretion of calcium, phosphoru and hydroxyproline, and bone density of the lumbar spine, femur and radius were measured. No significant correlation was found between IL-6 and the biochemical parameters. A negative correlation was found between IL-6 and serum estradiol, as well as between IL-6 and bone density in 5 out of 6 sites studied. It is possible that women with high IL-6 levels, may develop lower bone mass.     

Dynamic changes of HBV DNA in serum and peripheral blood mononuclear cells of chronic hepatitis patients after lamivudine treatment

AIM: To study the dynamic changes of hepatits B virus (HBV) DNA in serum and peripheral blood mononuclear cells (PBMCs) of patients after lamivudine therapy. METHODS: A total of 72 patients with chronic HBV infection were included in this study. All patients were confirmed to have the following conditions: above 16 years of age, elevated serum alanine amonotransferase (ALT), positive hepatitis B e antigen (HBeAg), positive HBV DNA in serum and PBMCs, negative antibodies against HAV, HCV, HDV, HEV. Other possible causes of chronic liver damages, such as drugs, alcohol and autoimmune diseases were excluded. Seventy-two cases were randomly divided into lamivudine treatment group (n=42) and control group (n=30). HBV DNA was detected both in serum and in PBMCs by fluorescence quantitative polymerase chain reaction (PCR), during and after lamivudine treatment. RESULTS: In the treatment group, HBV DNA became negative both in serum and in PBNIC, of 38 and 25 out of 42 cases respectively during the 48 wk oflamivudine treatment, the negative rate was 90.5% and 59.5% respectively. In the control group, the negative rate was 23.3% and 16.7% respectively. It was statistically significant at 12, 24 and 48 wk as compared with the control group (P < 0.005). The average conversion period of HBV DNA was 6 wk (2-8 wk) in serum and 16 wk (8-24 wk) in PBMC. CONCLUSION: Lamivudine has remarkable inhibitory effects on HBV replication both in serum and in PBMCs. The inhibitory effect on HBV DNA in PBMCs is weaker than that in serum.     

Effects of oestrogen deprivation on interleukin-6 production by peripheral blood mononuclear cells of postmenopausal women

Various hormones can influence the expression of interleukin-6 (IL-6) and oestrogens are the most extensively studied. There is, however, controversy about the nature of the IL-6 secreted by human cells and its regulation by 17beta-oestradiol. The aim of this work was to clarify whether oestrogen deprivation after menopause may contribute to an enhanced IL-6 production by peripheral blood mononuclear cells (PBMC) in postmenopausal women. Twenty-two healthy postmenopausal women, age range 45-63 years, with clinical symptoms of oestrogen deficiency were enrolled in the study. The control group consisted of 16 healthy young women, age range 22-31 years, with regular menses and who were not taking oral contraceptives. Levels of IL-6 in the sera and PBMC culture supernatants were measured by the biological B9 cell-proliferation assay and expression of the IL-6 gene in non-stimulated PBMC was detected by RT-PCR. The effect of 17beta-oestradiol on spontaneous IL-6 production by the PBMC of postmenopausal women was also studied in vitro and in vivo. Seventeen out of the twenty-two postmenopausal women were given hormonal replacement therapy of 50 microg 17beta-oestradiol/day transdermally and the spontaneous production of IL-6 by the PBMC was analysed after 6 and 12 months of treatment. The postmenopausal women had significantly higher serum levels of IL-6 than the young controls. The spontaneous production of IL-6 by non-stimulated PBMC into the culture supernatants was also significantly higher in the postmenopausal women compared with the young. We also found that IL-6 gene expression was present in the non-stimulated PBMC isolated directly from the venous blood of the majority of the postmenopausal women. Women with IL-6 gene expression in the non-stimulated PBMC had significantly lower serum levels of 17beta-oestradiol compared with those where the IL-6 gene was not expressed in the PBMC. Our in vitro experiments showed that 17beta-oestradiol at concentrations of 10(-9) M and 10(-10) M decreased spontaneous IL-6 production by the PBMC of postmenopausal women. In vivo treatment with 17beta-oestradiol transdermally also significantly decreased spontaneous IL-6 production by the PBMC of postmenopausal women after 12 months of the therapy. Our results indicate that oestrogen deprivation after menopause may enhance IL-6 production by the PBMC of postmenopausal women. We suspect that the late complications of oestrogen deficiency, such as osteoporosis, coronary heart disease and Alzheimer's disease, may be mediated by an exaggerated production of IL-6 - a cytokine which seems to play a pivotal role in the pathogenesis of these age-related diseases.     

Assay of IL-22 and IL-25 in serum,whole blood,and peripheral blood mononuclear cell cultures of patients with severe asthma

BackgroundAlthough some studies show that IL-22 and IL-25 play critical roles in the pathogenesis of asthma, little is known about the systemic production of these cytokines. The aim of this study was to assay IL-22 and IL-25 in serum, in mitogen-activated whole blood (WB), and in mitogen-activated peripheral blood mononuclear cell (PBMC) cultures of patients with severe asthma.MethodsIn this cross-sectional study, a questionnaire was prepared to determine the severity of asthma. Through the questionnaire, information including clinical signs, clinical symptoms, and past medical history were acquired. Information collected allowed all patients who were active or ex-smokers to be excluded. A trained observer assessed airway reversibility, peak flowmetry, and spirometry in the remaining patients. Twenty-one patients and simultaneously, twenty age- and sex-matched healthy controls were selected. Sterile blood (10 ml) was taken from each study participant. Sera were isolated and anticoagulant blood used for WB and PBMC cultures and haematological tests. Phytohaemagglutinin and lipopolysaccharide (LPS) were used to activate WB and PBMC. The data from these two groups were compared using Student's t-test.ResultsAlthough the total white blood cell count was elevated in the asthmatic group, other haematological indices, including IL-22 and IL-25 levels in the asthmatic group were not significantly (p > 0.05) different from controls.ConclusionsThe levels of IL-22 and IL-25 in patients with severe asthma are no higher than those of non-asthmatic individuals. Any major role for IL-22 and IL-25 in severe asthma is likely to be localised to the lungs and bronchial tissues.     

Immune phenotype and intracellular cytokine production of peripheral blood mononuclear cells from postmenopausal patients with osteoporotic fractures

A number of factors with known effects on bone turnover are also immune regulatory factors. Disturbances of bone remodeling thus may be a consequence of altered local immune reactivity. We therefore determined surface markers and intracellular cytokine production of peripheral blood mononuclear cells by four-color flow cytometry in 19 postmenopausal patients with established osteoporosis and a control group of 11 postmenopausal women without fragility fractures. No significant differences in bone mineral density as assessed by dual energy X-ray absorptiometry were observed between the two groups. The following surface markers and cytokines were studied: CD3, CD4, CD8, CD16, CD19, CD29, CD45RA, CD56, CD57, HLA-DR, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-13, tumor necrosis factor (TNF)-, interferon (IFN)-γ and granulocyte macrophage colony stimulating factor. In the fracture patients, the percentage of CD8⁺ cells co-expressing CD57 was increased (14±2 vs. 8±1%; p=0.03). Moreover, the proportion of CD8⁺ cells co-expressing TNF- (47±5 vs. 33±4; p=0.05) and both TNF- and IFN-γ was significantly higher in the patients than the controls (41±6 vs. 22±3%; p=0.04). IL-1β expression tended to be increased in monocytes from patients with established osteoporosis. Distinct subsets of CD8⁺ cells thus appear to contribute to the development of osteoporotic fractures.     

外周血单个核细胞诱导为肝细胞样细胞及初步探讨对乙酰氨基酚作用下的细胞损伤反应

目的:建立外周血单个核细胞诱导肝细胞样细胞的方法,初步探讨其在对乙酰氨基酚（APAP）作用下的细胞损伤反应。方法:流式细胞术和免疫荧光法检测外周血分离的单个核细胞表面标志CD45;针对诱导的肝细胞样细胞,倒置显微镜观察细胞形态,实时荧光定量PCR（RT-PCR）检测肝细胞特异性基因细胞色素P450（CYP）1A2、CY...     

扩张型心肌病患者外周血单个核细胞白细胞介素-35表达及分泌的初步研究

目的:观察白细胞介素-35(IL-35)在扩张型心肌病(DCM)患者外周血单个核细胞(PBMCs)中表达和血浆水平变化,探讨其在DCM发病中的作用。方法:选择对象为30例DCM的患者(DCM组),24例心力衰竭的患者(HF组)及30例健康体检者为正常对照组。用逆转录-聚合酶链反应(RT-PCR)测定PBMCS的IL-35特异性亚基EB病毒诱导基因3(EBI3)mRNA的表达。用酶联免疫吸附法(ELISA)检测血浆中IL-35水平。结果:①DCM组PBMCs表达IL-35的EBI3mRNA水平(0.15±0.03)明显降低于HF组(0.34±0.08)及正常对照组(0.33±0.07)(均P0.01),而HF组及正常对照组间差异无统计学意义(P0.05);②DCM组(24.08±4.40)血浆IL-35的水平明显低于HF组(53.03±5.04)和正常对照组(43.89±8.01)(均P0.01),而HF组及正常对照组间差异无统计学意义(P0.05);③DCM组血浆中IL-35含量与射血分数呈正相关(r=0.449,P0.05),与心功能NYHA分级呈负相关(r=-0.839,P0.01)。结论:DCM患者的IL-35表达下调,提示IL-35可能在DCM的免疫发病机制中起了一定作用。     

The influence of uremic serum on interleukin-1beta and interleukin-1 receptor antagonist production by peripheral blood mononuclear cells

We investigated whether or not uremic serum has an influence on IL-1beta and IL-1Ra production by normal peripheral blood mononuclear cells (PBMC). Four groups of subjects were divided into healthy controls and non-dialyzed patients with chronic renal failure (CRF), patients undergoing continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis (HD) patients. We examined cytokine concentrations and cytokine production by PBMC from a normal subject at the density of 2.5 x 10(6) cells/mL incubated with 25% serum in the medium and serum containing polymyxin-B or lipopolysaccharides (LPS). IL-1Ra concentrations in the serum of the uremic groups were significantly higher than those of the controls. In IL-1beta production by PBMC in medium with both serum and serum containing polymyxin-B, these values in the uremic groups were significantly higher than in the controls. In IL-1Ra production with serum containing polymyxin-B, these values in the uremic groups were significantly higher than in the controls. In contrast, in IL-1Ra production by PBMC in medium with serum containing LPS, these values in the uremic groups were significantly lower than in the controls. It was concluded that uremic serum not only contains nonendotoxemic cytokine-inducing substances, but also shows impaired cytokine production by PBMC in response to LPS.     

慢性乙型肝炎病人血清及外周血单个核细胞培养上清液白细胞介素-10的检测及其临床意义

目的探讨慢性乙型肝炎（ＣＨＢ）病人血清及外周血单个核细胞（ＰＢＭＣ）培养上清液白细胞介素一１０（ＩＬ－１０）检测的临床意义。方法分离１５例ＣＨＢ病人及６名正常人血清及ＰＢＭＣ。ＰＢＭＣ体外单独或与金黄色葡萄球菌场毒素Ｂ（ＳＥ）或重组ＨＢｃＡｇ（ｒＨＢｃＡ）共培养４８小时后,用双抗体夹心ＥＬＩＳＡ方法检测血清及ＰＢＭＣ培养上清液ＩＬ－１０水平。结果ＣＨＢ病人血清ＩＬ－１０水平为（１２３．１１±１３．８９）ｎｇ／Ｌ,正常对照组平均为（９５．９７±１１．６８）ｎｇ／Ｌ,两者之间差异有非常显著性,Ｐ＜０．０１。ＰＢＭＣ体外培养４８小时,培养上清液中ＩＬ－１０水平均明显高于正常对照组,Ｐ＜０．０１。其中ｒＨＢＣＡｄｅ导病人ＰＢＭＣ产生的ＩＬ－１０水平最高,达（３６９．５±３０．５２）ｎｇ／Ｌ。ＨＢＶＤＮＡ阳性病人血清及ＰＢＭＣ培养上清液ＩＬ－１０水平均明显高于ＨＢＶＤＮＡ阴性组病人。慢性轻度和中度肝炎病人血清及ＰＢＭＣ培养上清液ＩＬ－１０水平又高于慢性重度或慢性重型肝炎病人。结论ＣＨＢ病人血清及ＰＢＭＣ培养上清液ＩＬ－１０水平升高可能与ＨＢＶ持续感染有关。     

2型糖尿病患者外周血单个核细胞组蛋白乙酰化研究

目的 本研究探讨了2型糖尿病患者外周血单个核细胞( PBMC)组蛋白乙酰化修饰状态.方法 收集12例2型糖尿病患者及12名健康对照者的PBMC,H3/H4乙酰化检测试剂盒提取组蛋白,检测H3/H4乙酰化水平.实时定量PCR检测2型糖尿病PBMC组蛋白修饰基因mRNA表达水平.结果 与健康对照组比较,2型糖尿病组PBMC组蛋白H3/H4乙酰化水平升高(H3:0.134±0.035对0.181±0.023,P＜0.05;H4:0.266±0.070对0.324 ±0.062,P＜0.01).2型糖尿病组PBMC的p300、CREBBP、HDAC2、HDAC7、SIRT1基因表达分别是健康对照组的3.11±0.38、2.78±0.45、3.55±0.30、0.77±0.37、0.40±0.25倍.结论 2型糖尿病患者外周血PBMC组蛋白乙酰化修饰呈现异常.     

类风湿关节炎患者外周血单个核细胞蛋白质组学研究

目的 运用蛋白质组学的方法,比较正常人及早期活动性类风湿关节炎(RA)患者外周血单个核细胞(PBMCs)蛋白质的差异表达,寻找RA疾病相关致病蛋白质.方法 选取9例早期活动期RA患者以及9名健康成年志愿者,用淋巴细胞分离液分离PBMCs,抽提PBMCs中的蛋白,采用同相pH梯度(IPG)双向凝胶电泳(2-DE)分离正常人及RA患者PBMCs总蛋白质.凝胶经考马斯亮蓝染色显色后,PDQuest图像分析软件进行比较分析、识别差异表达的蛋白质,对差异蛋白质点用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)进行鉴定,运用反转录-聚合酶链反应(RT-PCR)方法验证部分差异蛋白质.结果 获得RA患者及正常人PBMCs蛋白质双向凝胶电泳图谱,平均蛋白质点数分别为556和579,匹配率分别为89.4%和 88.5%,通过比较分析,差异表达蛋白质点数为23,选取18个点进行质谱鉴定,成功鉴定14个蛋白质,其中a-肌动蛋白、纤维蛋白素原a-链、载脂蛋白A-I(ApoA-I)等9个蛋白质点在RA中表达上调,硫氧还蛋白-2、谷胱甘肽S-转移酶等6个蛋白质点在RA中表达下调,这些差异蛋白质的功能涉及物质代谢、抗氧化、信号传导、能量产生及细胞骨架.并用RT-PCR方法验证差异蛋白质ApoA-I.其结果与上述蛋白质差异表达结果相符.结论 在RA患者PBMCs中存在着差异表达蛋白质,这些差异蛋白质可能是RA发病的内在因素.其RT-PCR结果与蛋白质差异表达相符,证明蛋白质组研究的可靠性.     

重症肌无力患者外周血单个核细胞中糖皮质激素受体不同亚型表达和意义

目的探讨重症肌无力（MG）患者外周血单个核细胞（PBMC）中两种糖皮质激素受体（GR）亚型表达水平及其与糖皮质激素疗效的关系。方法在糖皮质激素治疗之前留取MG患者PBMC并涂片,根据糖皮质激素治疗MG的不同疗效分组,通过免疫细胞化学方法观察各组GRα和GRβ阳性细胞比例并评分,分析GRα和GRβ表达量与糖皮质激素疗效的关系。结果对照组、糖皮质激素治疗敏感组和依赖组PBMC中GRα阳性细胞计分差异无统计学意义,3组计分明显高于糖皮质激素治疗抵抗组（P〈0．01）;对照组、糖皮质激素治疗敏感组和糖皮质激素治疗抵抗组PBMC中GRβ计分差异无统计学意义,3组计分明显低于糖皮质激素治疗依赖组（P〈0．01）。结论MG患者PBMC中GRα表达降低和GRβ表达增高均与糖皮质激素疗效降低有关。     

不同浓度甲氨蝶呤对外周血单个核细胞表达和分泌白细胞介素-17的影响

目的 体外观察不同浓度的甲氨蝶呤(MTX)对外周血单个核细胞(PBMCs)表达和分泌白细胞介素(IL)-17的影响,探究MTX有效治疗类风湿关节炎(RA)的可能机制.方法 分离RA患者和健康人PBMCs,予不同浓度MTX预处理,抗人CD3和抗人CD28刺激后,37 ℃体积分数为0.05的CO2培养箱培养.分别于培养的不同阶段收集细胞或上清液,采用反转录聚合酶链反应(RT-PCR)技术分析IL-17mRNA的表达水平;酶联免疫吸附试验(ELISA)法检测细胞培养液中IL-17的浓度;荧光抗体标记细胞表面抗原CD4和胞内蛋白 IL-17,流式细胞仪分析CD4+IL-17+细胞的比例.结果 比较采用两两配对t检验或独立样本t检验.结果 浓度为0.1、1.0、5.0、25.0μg/ml MTX组IL-17mRNA/GAPDH比值分别为(0.58±0.09、0.48±0.11、0.50±0.09、0.51±0.14),与无药抗体组(0.76±0.08)组间两两比较差异有统计学意义(P＜0.01);IL-17分泌水平则分别为(121±54)、(104±45)、(90±36)、(115±41)pg/ml,与无药抗体组(370±187)pg/ml组间两两比较差异有统计学意义(P＜0.01);MTX处理组CD4+IL-17+细胞比例的平均值下降,但统计学检验差异无统计学意义(P＜0.05).结论 MTX可以有效抑制PBMCs合成和分泌IL-17,而且在一定的剂量下可发挥显著作用,加大剂量并不能增强该作用.     

外周血单个核细胞介导乙型肝炎病毒感染的transwell 小室体外模型研究

目的：观察感染 HBV 的 PBMC 经人绒毛膜癌滋养层细胞（Bewo 细胞）构建的胎盘屏障迁移情况,探讨 PBMC 作为载体转运 HBV 的生物学作用。方法培养并用细胞计数试剂盒-8检测培养 Bewo 细胞和 PBMC 的增殖和活性。用 HBV DNA 含量为5×106拷贝／mL 的乙型肝炎患者血清100μL 感染 PBMC 后,用羟基荧光素二醋酸盐琥珀酰亚胺脂（CFSE）荧光染料标记感染的细胞, transwell 小室建立 Bewo 细胞和感染 HBV 的 PBMC 共培养模型。用流式细胞仪检测感染 HBV 的PBMC 迁移情况,荧光定量 PCR 法检测 transwell 下室中 PBMC HBV DNA 含量。结果 PBMC、Bewo细胞均在接种24 h 左右开始增殖,120 h 左右开始进入生长停滞期。transwell 小室共培养0、12、24和48 h,下室中绿色荧光标记的 PBMC 量分别为（0．445±0．021）％、（21．180±4．653）％、（34．830±7．156）％和（64．185±3．161）％,随着培养时间的延长,下室中检测到的标有绿色荧光的 PBMC 量越多（F ＝68．983,P ＝0．001）。共培养24、48 h 时,下室 PBMC HBV DNA 分别为（1．925±0．431）×103、（2．565±0．361）×103拷贝／mL,即下室中的 PBMC 发生感染。结论 PBMC可以作为 HBV 肝外感染的靶细胞,利用 transwell 小室构建胎盘滋养层屏障可以为体外研究 HBV 跨胎盘传播提供新思路。     

原位杂交法检测外周血单个核细胞中HCV RNA

目的比较慢性丙型肝炎患者用干扰素治疗前以及治疗后３个月ＰＢＭＣ中ＨＣＶＲＮＡ。方法应用地高辛素标记ＨＣＶＲＮＡ正链及负链探针，建立原位杂交方法检测外周血单个核细胞（ＰＭＢＣ）中的ＨＣＶＲＮＡ。结果治疗前１９例患者正链ＨＣＶＲＮＡ阳性，８例负链ＨＣＶＲＮＡ阳性，用正链探针杂交在较多的细胞中出现杂交信号，负链探针杂交仅在少数细胞中出现杂交信号，ＨＣＶＲＮＡ在ＰＢＭＣ胞浆中呈均质性分布。用干扰素治疗结束后３个月２０例患者中９例ＨＣＶＲＮＡ转阴性，近期治愈率４５％。结论原位杂交技术的敏感性及特异性较高，且重复性较好，是研究ＨＣＶＲＮＡ在组织中定位分布和病毒复制场所一种切实可行的方法