逆转录—套式—聚合酶链反应检测HCV RNA正链和负链及与肝细胞…

为了解ＨＣＶ与肝癌的关系，采用ＨＣＶ５′－ＮＣＲ正相引物和反相引物逆转录－套式－聚合酶链反应（ＲＴ－Ｎｅｓｔｅｄ－ＰＣＲ）检测了２２例肝癌手术患者血清，癌组织和癌旁组织ＨＣＶＲＮＡ正链和负链，用ＨＢＶＣ保守区引物ＰＣＲ法检测了ＨＢＶＤＮＡ，结果发现，５例抗－ＨＣＶ阳性的肝癌患者中，３例血清，癌组织和癌旁组织ＨＣＶＲＮＡ正链扩增试验均阳性，１例三者均阴性，１例血清和癌旁组织阳性。３例正链扩增试验阳性     

肝癌组织中丙型和乙型肝炎病毒基因状况研究

采用逆转录巢式ＰＣＲ技术检测了５１例ＨＣＣ患者的肝癌及癌周肝组织中ＨＣＶＲＮＡ正负链，同时以巢式ＰＣＲ技术检测了这些组织中的ＨＢＶＤＮＡ。结果，１１．８％（６／５１）检出组织中ＨＣＶＲＮＡ正链：其中５例在癌周肝组织，２例在癌组织中检测出ＨＣＶＲＮＡ负链，由于负链ＲＮＡ为ＨＣＶ的复制中间体，不释放到细胞外，其检出ＨＣＶ感染与ＨＣＣ关系的研究提供了进一步的病因证据，组织中ＨＢＶＤＮＡ检出率高达９２．２     

肝癌及癌旁组织中丙型肝炎病毒基因负链的研究

肝癌及癌旁组织中丙型肝炎病毒基因负链的研究查文章，等．中华医学杂志，１９９４，７４：２０５运用巢式聚合酶链反应（ＰＣＲ）等方法检测了５例丙型肝炎病毒核糖核酸（ＨＣＶＲＮＡ）阳性患者的肝癌、癌旁组织和周围血清中ＨＣＶＲＮＡ正链和负链，其中男４例，女１例...     

原位聚合酶链技术检测肝细胞癌组织内丙型肝炎病毒核酸及其意义

应用一步反转录。聚合酶链原位扩增法（ＯＲＴ－ＰＣＲＩＳ）检测肝细胞癌（ＨＣＣ）及癌周组织内丙型肝炎病毒核糖核酸（ＨＣＶＲＮＡ），同时以一步反转录－ＰＣＲ（ＯＲＴ－ＰＣＲ）法检测血清及肝组织匀浆内ＨＣＶＲＮＡ。发现癌和癌周组织经原位扩增后ＨＣＶＲＮＡ检出率为７７．８％（２／２７）及８１．５％（２２／２７），而血清及组织匀浆内仅为２９．６％及３７．Ｏ％，两者与癌及癌周组织相比差异均有显著性（Ｐ＜０．０１）。ＯＲＴ－ＰＣＲＩＳ示在癌组织中ＨＣＶＲＮＡ以核型为主（１２／２１），阳性细胞点样分布，细胞内信号强度多为＋（５７．１％）；而癌周组织以核浆型为主（１４／２２），阳性细胞弥漫分布，信号强度多为＋＋＋（５０．０％）。无论在癌还是在癌周组织均未发现ＨＣＶｃＤＮＡ存在。结果提示ＯＲＴ－ＰＣＲＩＳ优于ＲＴ－ＰＣＲ，ＨＣＶ对本地区ＨＣＣ发生、发展起着极为重要的作用。癌周ＨＣＶ复制较癌组织内活跃，癌和癌周细胞核阳性表明ＨＣＶＲＮＡ在核内整合或复制，这种整合或复制对肝细胞基因组ＤＮＡ产生的影响可能与乙型肝炎病毒（ＨＢＶ）相类似。     

外周血单核细胞中丙型肝炎病毒RNA正负链检测的临床意义

目的通过对外周血单核细胞（ＰＢＭＣ）中ＨＣＶＲＮＡ正负链的检测,来探讨其与丙型肝炎慢性化及干扰素治疗的关系．方法慢性丙型肝炎患者４０例,其中干扰素治疗者１０例,分离血清及ＰＢＭＣ．异硫氰酸胍－酚－氯仿抽提法提取ＨＣＶＲＮＡ,应用逆转录－巢式ＰＣＲ技术检测ＨＣＶＲＮＡ正负链．结果血清正链ＨＣＶＲＮＡ阳性率为６７５％,但负链均为阴性,ＰＢＭＣ中正链ＨＣＶＲＮＡ阳性率为５７５％,负链的阳性率为３５０％．其中３例患者血清中正链ＨＣＶＲＮＡ为阴性,而ＰＢＭＣ中为阳性．１０例干扰素治疗者在治疗结束时血清正链ＨＣＶＲＮＡ６０％转阴,ＰＢＭＣ中负链ＨＣＶＲＮＡ８０％转阴,而正链仅３７５％转阴．结论ＨＣＶ能在ＰＢＭＣ中存在和复制,这可能是导致丙型肝炎易发生慢性化的原因之一．ＰＢＭＣ中ＨＣＶＲＮＡ正负链的检测对于临床判断干扰素的疗效及预后有重要意义     

周围血白细胞的复制型丙型肝炎病毒RNA的检测及临床意义

用简并引物作套式反转录。聚合酶链反应检测正、负链丙型肝炎病毒（ＨＣＶ）ＲＮＡ。显示３０例急、慢性丙型肝炎患者的血清及血浆中，７例无症状抗－ＨＣＶ阳性者的血清、血浆及周围血白细胞（ＰＢＬ／Ｃ）中，均未检出负链ＨＣＶＲＮＡ。慢性丙型肝炎者ＰＢＩ（中正、负链ＨＣＶＲＮＡ的检出率高于急性丙型肝炎及无症状抗－ＨＣＶ阳性者（Ｐ＜０．０５～０．００１）。１７例经肝组织学检查的患者中，急性肝炎（ＡＨ）者ＰＢＬ／Ｃ的正、负链ＨＣＶＲＮＡ检出率低于慢性活动性肝炎（ＣＡＨ）者（Ｐ＜０．０５）。１例ＡＨ及６例ＣＡＨ患者肝组织内正、负链ＨＣＶＲＮＡ全部阳性。证实丙型肝炎患者的ＰＢＬ／Ｃ确可被ＨＣＶ感染，病程越长，被ＨＣＶ感染的可能性越大；病情活动者，ＰＢＬ／Ｃ中负链ＨＣＶＲＮＡ的检出率越高。提示ＨＣＶ不仅可以感染ＰＢＬＣ，而且可在其中复制；负链ＨＣＶＲＮＡ的出现与病情活动有关。     

原发性肝癌患者血清和肝组织中庚型肝炎病毒基因的PCR检测

用逆转录－聚合酶链反应法（ＲＴ－ＰＣＲ）检测了原发性肝癌（ＰＬＣ）患者血清及肝癌和癌旁肝组织中的庚型肝炎病毒（ＨＧＶ）ＲＮＡ，以ＰＣＲ－双脱氧末端终止法测定了ＰＣＲ产物的核苷酸序列。结果显示，血清和肝组织中ＨＧＶＲＮＡ的检出率分别为１９．４％（１３／６７）和２５．７％（９／３５），且ＨＧＶＲＮＡ在肝癌组织呼吕旁肝组织中同时存在，血清和肝组织中ＨＧＶ ＲＮＡ检测结果的符合率为８５％；５′非编码区（５     

肝细胞癌组织中HCV复制中间体的研究

本文报道在逆转录反应时仅加入与负链（复制中间体）ＨＣＶＲＮＡ互补的有义外引物，待将逆转录酶变性灭活后，再加入反义外引物，然后进行两轮聚合酶链反应（ＰＣＲ）扩增。用这种逆转录反应和套式ＰＣＲ方法，检测了ＨＣＣ组织和对照血清中负链ＨＣＶＲＮＡ的存在状况。结果负链ＨＣＶＲＮＡ在１０例（其中３例为已测血清总ＨＣＶＲＮＡ阳性，余７例为随机选择）ＨＣＣ组织中检出５例，而在５例（其中３例与组织配对）总ＨＣＶＲＮＡ的ＨＣＣ阳性病人血清则均阴性。表明ＨＣＣ组织中确有ＨＣＶ复制，从而进一步提示ＨＣＶ感染与我国ＨＣＣ的发生密切相关。     

肝癌及癌旁组织HBV DNA定点PCR研究

目的采用定点ＰＣＲ技术，研究ＨＢＶ的Ｓ和Ｃ基因在肝癌与癌旁组织的存在状态，探讨癌旁ＨＢｓＡｇ表达与Ｓ基因的关系。方法福尔马林固定，石蜡包埋肝癌及癌旁组织２８例，ＨＢｓＡｇ免疫组化后的切片分别切割癌组织、癌旁ＨＢｓＡｇ强阳性区（Ｐ２）及癌旁ＨＢｓＡｇ阴性区（Ｐ１）各１个低倍视野，作Ｓ及Ｃ基因ＰＣＲ。结果癌组织Ｓ基因较Ｃ基因检出率高（１８／２８ｖｓ１０／２８，Ｐ＜０．０５），癌旁二者无差异；癌旁中Ｐ１与Ｐ２的Ｓ及Ｃ基因检出率无显著性差异；高分化肝癌Ｓ和／或Ｃ基因检出率较中分化者低（２／５ｖｓ２０／２８，Ｐ＜０．０５）。结论癌组织中整合的ＨＢＶＤＮＡ为残缺不全的，ＨＢｓＡｇ强阳性与阴性区的Ｓ基因检出无显著性差异。     

巢式PCR证明肝癌细胞有HBV─DNA复制

目的研究肝癌细胞和癌旁组织ＨＢＶ＿ＤＮＡ复制状况。方法３２例手术肝癌标本作癌及癌旁组织巢式ＰＣＲ，用特制引物，目的基因为ＨＢＶ的ｃｃｃＤＮＡ。同时作ＨＢＡｇ免疫组化。结果５／３２（例）的肝癌组织，１０／３２的癌旁组织呈ｃｃｃＤＮＡ（＋）。阳性者其ＨＢＡｇ阳性率亦较高。结论首次证明肝癌细胞有ＨＢＶ复制模板－ｃｃｃＤＮＡ，是肝癌细胞有ＨＢＶ复制的可靠证据。     

静脉应用毒品致脓毒性肺栓塞的临床特点与治疗

目的探讨静脉应用毒品致脓毒性肺栓塞（SPE）的临床特点和诊治经过，提高对该病的诊断和治疗水平。方法回顾性分析1994年1月至2006年12月收治的22例静脉应用毒品致脓毒性肺栓塞患者的临床表现、胸部影像学特点、血培养结果、超声心动图结果和诊治经过。结果临床表现为发热（22／22）、呼吸困难（20／22）、咳嗽（18／22）、胸痛（10／22）和咯血（8／22）。X线胸片和CT表现为结节（15／22）、局灶浸润影（12／22）、楔型阴影（5／22）、气囊（18／22）、空洞（11／22）及胸膜病变（11／22）。病灶分布在外周或胸膜下（20／22），CT较X线胸片可更清晰地显示病灶。血标本可培养出金黄色葡萄球菌（22／22）。超声心动图可见三尖瓣赘生物（22／22）。所有患者静脉使用2—4周耐酶青霉素、丁胺卡那霉素、万古霉素或左氧氟沙星，同时分别给予呼吸、循环支持和胸腔闭式引流。14例患者痊愈出院，8例好转出院。结论病情隐匿，缺乏特异性。高危人群如静脉吸毒者出现发热、影像学表现为多发和散在的胸膜下或周边气囊、结节影伴或不伴有空洞形成提示本病。早期诊断、及时给予抗生素治疗、控制肺外感染灶、给予呼吸支持，多数患者预后良好。     

Do the ethanol metabolizing enzymes modify the relationship between alcohol consumption and blood pressure?

BACKGROUND: Several cross-sectional studies have examined whether the relationship between alcohol consumption and blood pressure (alcohol-BP relationship) differs among individuals with different aldehyde dehydrogenase-2 (ALDH2) genotypes, but few studies have examined the association with alcohol dehydrogenase-2 (ADH2), and those have yielded inconsistent results. We examined the potential modulatory effects of ADH2 and ALDH2 genotypes on the alcohol-BP relationship in a cross-sectional sample of a Japanese rural community. METHODS AND RESULTS: The study subjects were 335 randomly selected men aged 40-69 years, who lived in Shiso, a Japanese rural county, in 1999 or 2000. The genetic polymorphisms of ADH2 and ALDH2 were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. The frequencies of ADH21/21 (wild-type), 21/22 (superactive heterozygotes), and 22/22 (superactive homozygotes) were 8.4, 34.9 and 56.7%, respectively; and those of ALDH21/21 (wild-type), 21/22 (inactive heterozygotes), and 22/22 (inactive homozygotes) were 52.8, 40.9, and 6.3%, respectively. A multiple linear regression analysis showed that the relationship between alcohol consumption and diastolic blood pressure was significantly stronger in men with ADH21/21 than those with ADH21/22 or 22/22 (adjusted regression coefficient = 0.0392 versus 0.0113 mmHg for + 1 g ethanol/week, P for difference in slope = 0.018). The strength of the alcohol-BP relationship was similar in all of the ALDH2 genotype groups. CONCLUSION: The alcohol-BP relationship was significantly stronger in men with ADH21/21 than in men with ADH21/22 or 22/22 in this Japanese rural population. This finding was exactly the opposite of what one previous study suggested.     

光学相干断层扫描比较不稳定性和稳定性心绞痛患者粥样硬化斑块特征

目的 应用光学相干断层成像(OCT)技术比较不稳定性心绞痛(UAP)和稳定性心绞痛(SAP)患者冠状动脉粥样硬化斑块特征.方法 对临床诊断的23例UAP和24例SAP患者,在完成冠状动脉造影并确诊冠心病后进行OCT检查.根据OCT结果 回顾性比较分析UAP和SAP患者冠状动脉粥样硬化斑块特征,包括富含脂质斑块(≥2个象限的脂质斑块)、斑块纤维帽厚度、薄纤维帽粥样斑块(TCFA)、斑块破裂、钙化和血栓等.结果 47例患者中有44例成功进行OCT检查,包括22例UAP和22例SAP患者.UAP患者冠状动脉富含脂质斑块为91%(20/22),多于SAP患者的73%(16/22),但差异无统计学意义(P=0.741).UAP患者冠状动脉脂质斑块表面纤维帽厚度明显小于SAP患者[(69.5±34.7)μm比(141.1±68.5)μm,P=0.000],纤维帽侵蚀比例为59%(13/22),明显多于SAP患者的9%(2/22,P=0.000);TCFA[73%(16/22)比14%(3/22),P=0.000]和斑块破裂[50%(11/22)比9%(2/22),P=0.003]多于SAP患者.UAP患者冠状动脉斑块表而可见血栓形成多于SAP患者,但差异无统计学意义[27%(6/22)比9%(2/22),P=0.761].在斑块钙化方面,UAP与SAP患者之间差异无统计学意义.结论 OCT技术可清晰显示冠状动脉粥样斑块特征.与SAP患者比较,UAP患者冠状动脉粥样硬化斑块表现为纤维帽更薄、更多的纤维帽侵蚀、更多的破裂斑块和TCFA.     

Time of exposure as a prognostic factor in avian hypersensitivity pneumonitis

Spirometric values were subsequently evaluated in 22 patients suffering from hypersensitivity pneumonitis caused by avian problems. First spirometric values were abnormal in 18/22 (82%) of patients. A restrictive pattern was observed in 16/22 (72%) of patients and an obstructive pattern in 6/22 (27%). The TLCO was reduced in all cases (12/12). Improvement or normalization of the respiratory function occurred 3.4 +/- 2.4 months after the avian contact had ceased. At the end of the follow-up, parameters were normal in 13/22 (59%) of patients. The restrictive pattern remained unchanged in 7/22 (32%), and the obstructive pattern persisted in 4/22 (18%) of the patients. The TLCO was normal in 6/12 (50%) of patients. Neither age nor treatment with corticosteroids (13 patients) had a significant influence upon the evolution of the lung function. However, total recovery or significant improvement was observed in 12/12 (100%) of patients who had been in contact with birds less than 2 years, in contrast to 6/10 (60%) of patients with more than 2 years of contact (P = 0.002).     

Loss of heterozygosity on chromosome 10q22-10q23 and 22q11.2-22q12.1 and p53 gene in primary hepatocellular carcinoma

AIM: To analyze loss of heterozygosity (LOH) and homozygous deletion on p53 gene (exon2-3, 4 and 11), chromosome 10q22-10q23 and 22q11.2 -22q12.1 in human hepatocellular carcinoma (HCC). METHODS: PCR and PCR-based microsatellite polymorphism analysis techniques were used. RESULTS: LOH was observed at D10S579 (10q22-10q23) in 4 of 20 tumors (20%), at D22S421 (22q11.2-22q12.1) in 3 of 20(15%), at TP53.A (p53 gene exon 2-3) in 4 of 20(20%), at TP53.B (p53 gene exon 4) in 6 of 20(30%), and at TP53.G (p53 gene exon 11) in 0 of 20(0%). Homozygous deletion was detected at 10q22-10q23(8/20; 40%), 22q11.2-22q12.1(8/20; 40%), p53 gene exon 2-3(0/20;0%), p53 gene exon 4(6/20; 30%), and p53 gene exon 11(2/20; 10%). CONCLUSION: There might be unidentified tumor suppressor genes on chromosome 10q22-10q23 and 22q11.2-22q12.1 that contribute to the pathogenesis and development of HCC.     

Expression of interleukin-22/STAT3 signaling pathway in ulcerative colitis and related carcinogenesis

AIM:To investigate the expression of interleukin (IL)-22 and its related proteins in biopsy specimens from patients with ulcerative colitis (UC) and UC-related carcinogenesis. METHODS:Biopsy specimens were obtained from patients with inactive (n = 10), mild-to-moderately active (n = 30), severely active (n = 34), initial (n = 30), and chronic UC (n = 44), as well as UC patients with dysplasia (n = 10). Specimens from patients without colonic abnormalities (n = 20) served as controls. Chronic colitis in experimental mice was induced by 2.5% dextran sodium sulfate. The expression levels of IL-22, IL-23, IL-22R1 and phosphorylated STAT3 (p- STAT3) were determined by immunohistochemistry. Bcl-2, cyclin D1 and survivin expression was detected by Western blotting. RESULTS:Patients with active UC had significantly more IL-22, IL-23, IL-22R1 and p-STAT3-positive cells than the patients with inactive UC and normal controls. Furthermore, IL-22 and related proteins were closely related to the severity of the colitis. The expression of IL-22 and IL-22R1 in the tissue of initial UC was stronger than in that of chronic UC, whereas the expression of p-STAT3 was significantly increased in chronic UC tissues. In dysplasia tissues, the expression level of IL-22 and related proteins was higher compared with controls. Mouse colitis model showed that expression of IL-22, IL-22R1 and IL-23 was increased with time, p-STAT3 and the downstream gene were also remarkably upregulated.CONCLUSION:IL-22/STAT3 signaling pathway may be related to UC and UC-induced carcinogenesis and IL-22 can be used as a biomarker in judging the severity of UC.     

In-vitro activation of cytotoxic T lymphocytes by fusion of mouse hepatocellular carcinoma cells and lymphotactin gene-modified dendritic cells

AIM： To investigate the in-vitro activation of cytotoxic T lymphocytes （CTLs） by fusion of mouse hepatocellular carcinoma （HCC） ceils and lymphotactin gene-modified dendritic cells （DCs）.
METHODS： Lymphotactin gene modified DCs （DCLptn） were prepared by lymphotactin recombinant adenovirus transduction of mature DCs which differentiated from mouse bone marrow cells by stimulation with granulocyte/macrophage colony-stimulating factor （GM- CSF）, interleukin-4 （IL-4） and tumor necrosis factor alpha （TNF-α）. DCLptn and H22 fusion was prepared using 50% PEG. Lymphotactin gene and protein expression levels were measured by RT-PCR and ELISA, respectively. Lymphotactin chemotactic responses were examined by in-vitro chemotaxis assay. In-vitro activation of CTl_s by DCLptn/H22 fusion was measured by detecting CD25 expression and cytokine production after autologous T cell stimulation. Cytotoxic function of activated T lymphocytes stimulated with DCLptn/H22 cells was determined by LDH cytotoxicity assay. RESULTS： Lymphotactin gene could be efficiently transduced to DCs by adenovirus vector and showed an effective biological activity. After fusion, the hybrid DCLptn/H22 cells acquired the phenotypes of both DCLptn and H22 cells. In T cell proliferation assay, flow cytometry showed a very high CD25 expression, and cytokine release assay showed a significantly higher concentration of IFN-α, and IL-2 in DCLptn/H22 group than in DCLptn, DCLptn＋H22, DC/H22 or H22 groups. Cytotoxicity assay revealed that T cells derived from DCLptn/H22 group had much higher anti-tumor activity than those derived from DCLptn, H22, DCLptn ＋ H22, DC/H22 groups. CONCLUSION： Lymphotactin gene-modified dendritoma induces T-cell proliferation and strong CTL reaction against allogenic HCC cells. Immunization-engineered fusion hybrid vaccine is an attractive strategy in prevention and treatment of HCC metastases.     

A multi-district audit of the management of HIV infection in pregnant women and the prevention of mother-to-child transmission: Yorkshire HIV pregnancy audit

A regional audit was performed to compare clinical practice against the British HIV Association guidelines for the management of HIV infection in pregnancy. Data were collected from 2000 to 2002 from eight clinics across Yorkshire using a questionnaire. There were 22 live births to 22 HIV + pregnant women. There were no cases of transmission of HIV from mother to child. The majority (20/22) of mothers received therapy as recommended in the guidelines, with only two initially receiving inappropriate dual therapy. In all, 16/22 (73%) had elective caesarean sections, 5/22 (23%) emergency sections after the onset of labour and one had a vaginal delivery. Also, 12/22 (55%) definitely received intravenous zidovudine during delivery. Added to these, 19/22 infants received appropriate antiretroviral medication for four to six weeks after birth. No mothers were known to have breast-fed.