Kuzbanian基因非放射性原位杂交探针制备

目的研究制备地高辛(DIG)标记的Kuzbanian(KUZ)基因非放射性原位杂交探针,用于检测转基因动物、正常组织KUZ基因的表达。方法用体外转录法依自构建质粒pcDNA-KUZFL(质粒:6.0kb;探针:623bp)和pcDNA-KUZDN(质粒:5.8kb;探针:360bp)制备反义链与正义链DIG标记cRNA探针。结果用转基因模型阳性与阴性对照品系动物小鼠胰岛组织非放射性原位杂交实验,证实制备探针具有较高的特异性和敏感性。结论DIG标记KUZ基因杂交探针的成功制备,为进一步研究有关转基因动物的KUZ基因的分布和表达以及为正常组织KUZ基因的有关研究提供了实验基础。     

地高辛标记的RNA探针原位杂交检测急性白血病多药耐基因表达

目的：将简便可靠的非同位素原位杂交方法应用于急性白血病多药耐药基因（Ｍｄｒ１）临床检测。方法：自行构建ＲＮＡ探针，经地高辛标记，直接在骨髓涂片上进行原位杂交，检测４５例急性白血病骨髓细胞Ｍｄｒ１ｍＲＮＡ。对部分Ｍｄｒ１高表达者高速治疗。结果：杂交信号清晰，重现性好。复治组中Ｍｄｒ１阳性８８．２％，初治组４２．８％，差异有极显著性。经标准化疗２疗程后初治组中Ｍｄｒ１阳性组完全缓解（ＣＲ）率２２．２％     

DNA探针原位杂交对念珠菌病的诊断研究

本文报道以蜗牛酶破壁、氯化铯密度梯度超速离心的初步纯化白念珠菌 DNA 制备探针以 Southern 转膜,点杂交及原位杂交法与标准念珠菌属各种及临床分离 11 株酵母样菌(初步疑为念珠菌)作分子杂交,结果仅有白念珠菌阳性,热带念珠菌为弱阳性,其它念珠菌及红色毛癣菌均为阴性,与常规鉴定法相同。提示念珠菌探针可以作为临床快速诊断鉴定菌种的一个方法。     

地高辛标记的RNA探针原位杂交检测急性白血病多药耐药基因表达

目的：将简便可靠的非同位素原位杂交方法应用于急性白血病多药耐药基因（Ｍｄｒ１）的临床检测。方法：自行构建ＲＮＡ探针,经地高辛标记,直接在骨髓涂片上进行原位杂交,检测４５ 例急性白血病骨髓细胞Ｍｄｒ１ ｍ ＲＮＡ。对部分Ｍｄｒ１ 高表达者调整临床治疗。结果：杂交信号清晰,重现性好。复治组中Ｍｄｒ１ 阳性８８．２％ ,初治组４２．８％ ,差异有极显著性。经标准化疗２ 疗程后初治组中Ｍｄｒ１ 阳性组完全缓解（ＣＲ）率２２．２％ ,阴性组６３．６％ （ Ｐ＜ ０．００１）。对Ｍｄｒ１ 阳性病例调整化疗方案或加用逆转剂可改善预后。结论：Ｍｄｒ１ 高表达确为白血病不良预后的主要指标。本方法操作简便,结果可靠,可用于急性白血病Ｍｄｒ１ 常规检测,辅助临床治疗并及断预后。     

非同位素标记脆弱类杆菌DNA探针的研究

为探索应用分子生物学技术检测厌氧菌的方法，选择脆弱类村抽提其染色体ＤＮＡ，用生物素和地高辛配基标记制备ＤＮＡ探针，检测厌氧和需氧菌９４株，临床标本４５份，并和＾３２Ｐ标记探针及培养生化鉴定比较，该探针可检出临床上常见的类杆菌属细菌与其它细菌无交叉反应，结果：地高辛配基标记探针的灵敏度（１０ｐｇＤＮＡ和１０＾３ＣＦＵ／ｍｌ）接近＾３２Ｐ标记探针的灵敏度（１ｐｇＤＮＡ和１０＾３ＣＦＵ／ｍｌ）。地高辛配     

地高辛素标记探针原位杂交法检测肝组织中庚型肝炎病毒RNA

目的自从建立起甲、乙、丙、丁、戊5种肝炎病毒的病原学诊断之后,仍有少部分肝炎患者的病因得不到明确,因此不少学者试图探索是否还有新型肝炎病毒的存在,并进行了大量的流行病学和实验诊断的研究,认为的确存在可经肠道外传播并引起人类肝炎的致病因子.目前关于庚型肝炎病毒(HGV/GBV-C)的致病性和组织嗜性尚无结论性资料.本文目的是研究HGV/GBV-C RNA在肝组织中表达并进一步探讨HGV/GBV-C引起肝脏损害的可能机制.方法应用地高辛素标记HGV/GBV-C cDNA探针原位杂交法检测肝组织中庚型肝炎病毒RNA.结果检测196例肝炎患者肝组织切片中的HGV/GBV-CRNA阳性率为37.24%;在血清中HGV/GBV-CRNA呈阳性患者肝组织中该病毒RNA检出率为48.94%;单一HGV/GBV-C感染者肝组织中检出率为58.33%;阳性信号仅为胞质型.原位杂交信号阳性细胞与肝细胞变性、瘀胆、炎性细胞浸润及细胞坏死程度等并不相关.本文原位杂交与免疫组化两种检测方法对比研究结果表明,两项技术有较高的符合率(86.74%),说明这两项技术具有相辅相成的作用.结论提示HGV/GBV-CRNA并不直接损害肝细胞.原位杂交和免疫组化两项技术成功地应用于石蜡包埋切片,为回顾性研究HGV/GBV-C的致病性及致病机制提供了有价值的实验手段.     

双探针原位杂交法检测慢性肝病和肝细胞癌患者肝组织TTV DNA的感染状况

目的 探讨慢性肝病和肝细胞癌患者肝组织TIV DNA的感染状况。方法采用PCR扩增法分别合成G1a、G2b两种亚型的双链TTV DNA探针。应用两型探针对45例肝组织标本进行TTV DNA原位杂交检测，巢氏PCR法检测血清TTV DNA。结果31例血清TTTV DNA阳性患者的肝组织TTV DNA均为阳性(100％)。14例血清TTV DNA阴性的患者肝组织中TTV DNA阳性者7例(50%)。慢性肝病患者的肝组织中TTV DNA散在分布在汇管区周围的肝细胞核内，肝癌患者TTV DNA则集中分布在肝癌细胞核内及癌组织周围的肝细胞核内。结论慢性肝病与肝癌患者肝组织中TTV DNA的感染状态存在一定差异。     

用DNA探针技术鉴定鼠疫菌的研究

本试验采用的鼠疫菌ｆｒａ基因经ＰＣＲ扩增地高辛标记方法制备基因探针，通过菌落原位杂交试验进行部分鼠疫菌、假结核菌和大肠杆菌的鉴定、被试菌中ＥＶ、ＥＶ７６ｐａｒｉｓ、６１、Ｍ２３四株鼠疫菌呈现阳性反应，假结核菌和大肠菌均为阴性。     

聚合酶链反应评价原位杂交检测慢性HBV感染者肝内HBVDNA的结果

用地高辛素探针原位杂交检测慢性ＨＢＶ感染者肝内ＨＢＶＤＮＡ，聚合酶链反应（ＰＣＲ）检测血清ＨＢＶＤＮＡ评价病毒复制状态。发现肝内ＨＢＶＤＮＡ阳性肝细胞，在１４例肝组织病变不活动的ＨＢｅＡｇ阳性慢性无症状ＨＢＶ携带者（ＡｓＣ）呈弥漫性分布；而在ＨＢｅＡｇ阳性或阴性活动性肝病病人均聚合分布于肝细胞坏死区。上述慢性感染者９８％血清ＨＢＶＤＮＡ阳性。说明ＨＢＶ复制与肝组织病变有关但非肝损伤直接原因。１６例ＨＢｅＡｇ阴性ＡｓＣ中，９例肝内仅见局灶性ＨＢＶＤＮＡ阳性肝细胞，其中４例血清ＨＢＶＤＮＡ阳性，说明部分ＨＢｅＡｇ阴性ＡｓＣ也存在极低水平病毒复制。     

Risk of Hepatocellular Carcinoma in Patients with Chronic Hepatitis B Virus Infection

Abstract

Objective. To determine the risk factors for the occurrence of hepatocellular carcinoma (HCC) in patients with hepatitis B virus (HBV) infection. Material and methods. A total of 620 patients who tested positive for hepatitis B surface antigen and were referred to Chiba University Hospital between February 1985 and March 2008 were included in the study and the following characteristics were analyzed: age, gender, status of hepatitis B e antigen, alanine aminotransferase level, HBV DNA level, and number of platelets (PLTs). Results. HCC was detected in 30 cases during the follow-up period (5.4 ± 5.1 years). Multivariate analysis revealed that age >?40 years [compared with patients aged <?40 years; odds ratio (OR) = 4.28; 95% confidence interval (CI) = 1.68–10.9] and PLT level <?206,000/μl (compared with patients with a higher PLT level; OR = 8.50; 95% CI = 1.98–36.2) were predictive factors for HCC occurrence. In patients aged >?40 years, the HBV DNA level (compared with <?5.0 log copies/ml; OR = 4.22, 95% CI = 1.13–15.8) and PLT level (compared with patients with >?196,000/μl PLTs; OR = 15.6, 95% CI = 2.06–118.3) were predictive factors for HCC occurrence. Conclusions. Advanced age and low PLT level were risk factors for HCC occurrence in patients with HBV infection. In patients aged >?40 years, viral load was also a risk factor for HCC.     

原发性肝癌内HBV DNA及HBAg的分布与意义

采用原位分子杂交和免疫组化及双标记技术,对44例原发性肝细胞肝癌及癌旁肝组织进行了乙型肝炎病毒DNA及其表达产物X抗原,核心抗原检测。癌组织中HBV DNA,HBxAg,HBcAg检出率分别为70．5％,65．9％及20．5％,癌旁组织依次为76．5％,76．5％,29．4％。HBV DNA及HBxAg主要位于肝细胞浆中,在HBcAg阳性组中的检出率高于HBcAg阴性组（P＜0．05）。9例枯否细胞胞浆有HBxAg检出。HBV DNA在癌及癌旁组织中的检出有“伴随现象”。HBcAg多位于核内,部分为核浆型,且总是伴随HBV DNA和（或)HBxAg检出,无一例单独检出HBcAg者。此外,HBxAg可由肝内游离型HBV DNA所表达;肝内核型或浆型HBc Ag均可反映HBV的复制。因此,肝内HBxAg检测可作为HBV感染的灵敏指标之一,进一步证实HBV感染与HCC的发生有密切关系。     

Hepatitis B Virus Gene in Liver Tissue Promotes Hepatocellular Carcinoma Development in Chronic Hepatitis C Patients

The hepatitis B virus (HBV) gene has been detected in hepatocellular carcinoma (HCC) tissue negative for the hepatitis B surface antigen and positive for the hepatitis C virus (HCV) antibody, but the precise role of the HBV gene in hepatocarcinogenesis has yet to be clarified. We studied the HBV gene in liver tissue several years before the emergence of HCC. Eleven patients diagnosed with HCV-positive chronic liver disease and who developed HCC were assigned to group A. HBV DNA was detected in 8 of the 11 patients (73%). Twenty-five patients, who did not develop HCC, were selected as group B. Six of the group B patients were classified as DNA-positive (24%). The HBV DNA in liver tissue was found to be significantly related to HCC development (P < 0.01). Thus, the presence of the HBV gene in patients with chronic HCV associated-liver injury appears to promote hepatocarcinogenesis, although prospective studies are needed to confirm this result.     

HCV HBV感染与肝细胞性肝癌

调查了肝癌高发地区不同肝病患者中丙型肝炎病毒(HCV)感染率。慢性肝病患者绝大多数已被乙型肝炎病毒(HBV)感染。HCV第二代抗体阳性率,肝癌7.3％,肝硬化6.6％,慢性肝炎6.6％和急性肝炎3.4％。两种病毒的复合感染率,肝癌5.1％,肝硬化1.7％,慢性肝炎3.9％和急性肝炎1.1％。在38例HCV抗体阳性的慢性肝病患者中,ALT异常84.2％,有输血史者占57.9％,HCV-RNA阳性率为71.1％。本研究的资料分析提示,在肝癌高发地区尽管HCV抗体阳性率较低,但HCV感染也是肝癌发生的重要病因之一。     

48例原发性肝癌患者血清丙型肝炎抗体检测

为探讨PHC与丙型肝炎的关系,采用ELISA方法检测48例PHC及106例非肝病患者的扰HCV抗体及HBV-M。结果显示PHC组中抗HCV抗体阳性21例(43.8％),对照组14例(13.2％,P<0.01);HBV-M阳性45例(93.8％),对照组50例(47.2％,P<0.01)。HBV-M阳性PHC病人中,HBsAg、HBeAb及HBcAb三项阳性21例(46.7％),HBsAg、HBeAg及HBcAb三项阳性7例(15.6％),HBsAg及HBcAb二项阳性5例(11.1％)。HBV-M阴性PHC患者中,抗HCV抗体阳性2/3例(66.7％),HBV-M阳性PHC病人中,抗HCV抗体阳性19/45例(42.2％,P<0.05);HBsAg阳性PHC病人中,抗HCV抗体阳性14/38例(36.8％);HBsAg阴性病人中,抗HCV抗体阳性7/10例(70.0％,P<0.05)。在抗HCV抗体阳性PHC患者中,3个月前有明确输血史8例(38.1％)。以上结果提示在我国,PHC与丙型肝炎有关,在HBV-M阳性患者中。HBsAg、HBeAb及HBcAb三项阳性患者发生PHC可能性最大。     

乙肝病毒e抗原系统与肝癌的关系

本文报道了98例肝癌患者和70倒慢性肝病患者 HBV 抗原抗体系统的检测情况检查结果表明。①HCC 患者,HBeAg 阳性率为11.2％,慢性肝病组为34.3％(P<0.01);②抗-HBe 阳性率:慢性肝病组为38.5％而中晚期 HCC 为50％,小肝癌为61.1％(P<0.01),揭示肝癌患者 HBeAs 转为抗-HBe 阳性才增加,故慢性肝病患者 HBekAg 转为抗-HBe 阳性之后,必须定期随访,作 AFP 及 B 超监测,警惕 HCC。     

肝病患者肝组织内CMV感染的初步研究

本文应用免疫组织化学方法检测140例急、慢性肝炎患者肝组织内CMV抗原,发现15例阳性(10.7%),有9例同时存在HBV感染,其中3例为三重感染。15例CMV抗原阳性肝炎中,11例为慢性肝炎。以同法检测20例肝细胞性肝癌患者肝组织中CMV抗原,发现1例阳性。结果提示CMV感染为病毒性肝炎的病因之一;CMV肝炎与其他病毒性肝炎及慢性肝脏疾病可同时存在;CMV肝炎可能存在慢性化倾向。     

Hepatocellular carcinoma and hepatitis B virus infection: molecular evidence for monoclonal origin and expansion of malignantly transformed hepatocytes

Summary The clonality of tumor cells was studied in a patient with metastasizing hepatocellular carcinoma (HCC). Using hepatitis B virus (HBV) DNA as a genetic marker, the pattern of integration of viral DNA into the tumor cell genome was determined by Southern blot analyses of DNAs extracted from different HCC lesions in the liver and both lungs. All tumor tissues examined were found to have viral DNA integrated into the same site(s) of the cellular genome. This finding provides direct molecular evidence for a monoclonal origin and expansion of malignantly transformed hepatocytes during tumor growth and metastasis. This characteristic is similar to other human cancers associated with viral infections, such as adult T-cell leukemia, Burkitt's lymphoma, or cervical cancer, and is important für our understanding of viral oncogenesis in man.     

Comparisons of Viral Etiology and Outcomes of Hepatocellular Carcinoma Undergoing Liver Resection between Taiwan and Vietnam

Epidemiologic data have suggested that etiologic variations of hepatocellular carcinoma (HCC) exist in different geographic areas, and might be associated with different outcomes. We compared the viral etiology, clinicopathological characteristics and surgical outcomes between 706 Taiwanese and 1704 Vietnamese patients with HCC undergoing liver resection. Vietnamese patients had a significantly higher ratio of hepatitis B virus (HBV) (p < 0.001) and a lower ratio of hepatitis C virus (HCV) (p < 0.001) and non-B non-C than Taiwanese patients. Among patients with HBV or non-B non-C, the mean age was younger in Vietnam than in Taiwan (p < 0.001, p = 0.001, respectively). The HCC patients in Vietnam had significantly higher serum alpha-fetoprotein (AFP) levels (p < 0.001), larger tumors (p < 0.001), and a higher ratio of macrovascular invasion (p < 0.001) and extrahepatic metastasis (p < 0.001), compared to those in Taiwan. Patients treated in Vietnam had a higher tumor recurrent rate (p < 0.001), but no difference in overall survival was found between both groups. In subgroup analysis, the recurrent rate of HCC was the highest in patients with dual HBV/HCV, followed by HCV or HBV, and non-B non-C (p < 0.001). In conclusion, although the viral etiology and clinicopathological characteristics of HCC differed, postoperative overall survival was comparable between patients in Taiwan and Vietnam.