阳离子脂质体介导DNA聚合酶α反义寡核苷酸转染并抑制人肝癌细胞的生长

ＤＮＡｐｏｌα是真核生物细胞ＤＮＡ合成的关键酶，与肿瘤的发生及发展密切相关〔１〕。其在肿瘤细胞中含量增加，酶活性增强，ｍＲＮＡ高表达〔２〕。因此以ｐｏｌαｍＲＮＡ为靶核酸而构建的反义寡核苷酸（ＡＳＯＤＮ）可进入细胞内与靶ｍＲＮＡ特异互补结合，抑制其翻译，使ＤＮＡｐｏｌα的表达减少，从而抑制了人胃〔３〕、肝、人卵巢及乳腺等癌细胞的增殖。我们选用阳离子脂质体脂染素（ｌｉｐｏｆｅｃｔｉｏｎ，Ｌｉｐ）与ＡＳＯＤＮ形成复合物，通过Ｌｉｐ介导使靶细胞快速大量摄入ＡＳＯＤＮ，并躲避细胞内外各种酶的降解，…     

空泡型质子泵在骨巨细胞瘤多核巨细胞中表达

通过电镜观察附着于骨片的骨巨细胞瘤多核巨细胞，表明在骨吸收时具有褶皱缘结构。采用可标记破骨细胞褶皱缘的空泡型质子泵３１ＫＤ亚基的抗体，对体外培养的骨巨细胞瘤细胞进行免疫组织化学染色，结果显示多核巨细胞为阳性，间质细胞为阴性，阳性区主要分布于多核巨细胞浆内的空泡区及伪足区。通过ｍＲＮＡ原位杂交方法检测到，多核巨细胞和间质细胞中均有空泡型质子泵的３ｌＫＤ亚基ｍＲＮＡ表达。结果提示多核巨细胞与破骨细胞可能有相似的骨吸收泌酸机制。     

用PCR检测肾移植受者人巨细胞病毒感染

用自行设计的引物，建立了聚合酶链反应（ＰＣＲ）检人巨细胞病毒（ＨＣＭＶ）ＤＮＡ的方法，经实验证明有高度的特异性和敏感性，对其它疱疹病毒和正常人基因组ＤＮＡ无交叉反应，ＨＣＭＶ ＡＤ１６９株ＤＮＡ ＥｃｏＲＩ酶切Ｊ片段的最小检出量为０．１ｆｇ，相当于６个基因拷贝。对２２例肾移植受者的血和尿标本进行ＰＣＲ检测，ＨＣＭＶ ＤＮＡ的阳性率为５９．１％。实验结果表现ＰＣＲ是一种敏感特异、简便快速、能早期诊断     

无环鸟苷引起肾移植病人精神异常一例报告

无环鸟苷引起肾移植病人精神异常一例报告石益民秦万长李冀渝杨渝马晗患者，女性，４３岁。因第二次肾移植术后２年，肾功减退伴反复高热入院。切除一侧移植肾后病人自行停用免疫抑制剂，发热渐加重。血巨细胞病毒ＤＮＡ（ＣＭＶＤＮＡ）阳性。口服无环鸟苷（ａｃｙｃｌ...     

血小板活化因子（PAF）受体mRNA在人睾丸组织和精子的表达

本文应用ＲＴ－ＰＣＲ和序列测定研究了人睾丸组织和精子内血小板因子受体（ＰＡＦ－Ｒ）ｍＲＮＡ的表达。结果证实（１）人睾丸组织和精子表达ＰＡＦ－ＲｍＲＮＡ,（２）ＰＡＦ－ＲｃＤＮＡ序列与人其它组织ＰＡＦ－ＲｃＤＮＡ序列完全一致。结果揭示,人睾丸组织和精子有ＰＡＦ－Ｒ的ｍＲＮＡ表达。     

血小板活化因子及其受体对颈髓损伤后血脊髓屏障损害的分子机制研究

目的：探讨血小板活化因子（ＰＡＦ）及其受体对颈髓损伤后血脊髓屏障损害的分子机制。方法：采用蛛网膜下腔注射ＰＡＦ及静脉注射ＰＡＦ受体拮抗剂ＢＮ５２０２１，应用地高辛标记ｃＤＮＡ探针原位杂交技术检测颈髓损伤后颈髓血管内皮细胞细胞间粘附分子－１ｍＲＮＡ（ＩＣＡＭ－１ｍＲＮＡ）和内皮细胞白细胞粘附分子－１ｍＲＮＡ（ＥＬＡＭ－１ｍＲＮＡ）表达。观察ＰＡＦ及其受体拮抗剂对颈髓损伤后血脊髓屏障、ＩＣＡＭ－１ｍＲＮＡ、ＥＬＡＭ－１ｍＲＮＡ表达的影响。结果：伤后颈髓血管内皮细胞ＩＣＡＭ－１ｍＲＮＡ、ＥＬＡＭ－１ｍＲＮＡ表达、伊文思蓝含量、水含量呈不同程度的增加；ＰＡＦ可使伤后血管内皮细胞ＩＣＡＭ－１ｍＲＮＡ、ＥＬＡＭ－１ｍＲＮＡ表达、伊文思蓝含量、水含量增加更为显著，ＰＡＦ受体拮抗剂可抑制ＩＣＡＭ－１ｍＲＮＡ、ＥＬＡＭ－１ｍＲＮＡ表达，降低颈髓组织伊文思蓝含量及水含量。结论：ＰＡＦ通过增加伤后颈髓血管内皮细胞粘附分子的表达是导致血脊髓屏障损害的重要分子基础。ＰＡＦ受体对伤后血管内皮细胞细胞粘附分子的表达具有调控作用。     

肾脏移植15年回顾

我院１９９２年７月－１９９３年３月共４９５份病理诊断为湿疣（５２份）、鳞状乳头状瘤病和非特异性炎或增生性病变的外阴、阴道及宫颈赘生物的活检标本，分别采用免疫组化ＡＢＣ法和核酸斑点杂交技术检测人乳头状瘤病毒的衣壳抗原和ＤＮＡ。在湿疣、鳞状乳状状瘤病和非特异性炎或增生性病变中，ＨＰＶ－Ａｇ阳性检分别为６９．２３％、０．００％和０．００％；ＨＰＶ－ＤＮＡ阳性检出率分别为８４．６２％、１１．３８％和３．３     

肿瘤坏死因子mRNA在严重烫伤大鼠肝脏中的表达及细胞定位

作者采用原位杂交法检测了大鼠严重烫伤后肿瘤坏死因子ｍＲＮＡ（ＴＮＦｍＲＮＡ）在肝脏中的表达及细胞定位。结果发现，在正常大鼠肝脏中的枯否氏细胞就表达ＴＮＦｍＲＮＡ。烧伤后，ＴＮＦｍＲＮＡ表达阳性细胞数迅速增加。伤后６～１２小时达顶峰，２４小时与正常无明显差异。其动态变化与门静脉血浆内毒素的变化极为相似。同时还发现烧伤后血窦内皮细胞及门静脉周围浸润的炎症细胞ＴＮＦｍＲＮＡ表达也为阳性，其变化以伤后１２～２４小时最为明显。作者认为肠源性内毒素是ＴＮＦｍＲＮＡ表达的主要刺激物。内皮细胞、中性粒细胞表达阳性提示其已被活化。上述三种细胞在内毒素导致的肝脏损害中发挥了重要作用。     

血小板活化因子及其受体对颈髓损伤后血脊髓屏障损害的 …

探讨血小板活化因子及其受体对颈髓损伤后血脊髓屏障损害的的分子机制。方法：采用蛛网膜下腔注射ＰＡＦ及静脉注射ＰＡＦ受体拮抗剂ＢＮ５２０２１，应用地高辛标记ｃＤＮＡ探讨针原交技术检测颈髓损伤后颈髓血管内皮细胞细胞间粘附分子－１ｍＲＮＡ和     

肝硬化大鼠肝组织中组织胺H2受体基因的mRNA表达

目的探讨肝硬化大鼠肝组织中组织胺Ｈ２受体基因的ｍＲＮＡ表达情况。方法逆转录聚合酶链反应方法从Ｗｉｓｔａｒ大鼠脑组织中合成Ｈ２受体的ｃＤＮＡ片段，克隆入载体中测序。用同位素标记ｃＤＮＡ片段后用于Ｎｏｒｔｈｅｒｎ印迹杂交。采用Ｎｏｒｔｈｅｒｎ印迹杂交法检测了１０例肝硬化Ｗｉｓｔａｒ大鼠及１０例正常对照组大鼠肝组织的Ｈ２受体ｍＲＮＡ。结果正常及肝硬化大鼠肝组织中均有Ｈ２受体的ｍＲＮＡ表达，且肝硬化组的表达量显著低于正常对照组。结论肝硬化时肝组织Ｈ２受体基因的转录减少是造成肝硬化时Ｈ２受体蛋白减少的重要原因，在门静脉高压症的形成和维持中起重要作用。     

肾移植术后远期急性排斥反应与巨细胞病毒感染

目的探讨肾移植术后远期急性排斥反应与巨细胞病毒感染的关系。方法检测４５例移植肾有功能存活１年以上后发生急性排斥反应者外周血白细胞中巨细胞病毒ＤＮＡ（ＣＭＶＤＮＡ）,并给予激素冲击治疗,对激素冲击无效的部分患者给予更昔洛韦抗病毒治疗。结果３２例激素冲击有效,１３例无效。无效者外周血白细胞内可测到ＣＭＶＤＮＡ,８例用更昔洛韦治疗后ＣＭＶＤＮＡ转阴,２例肾功能好转,６例恢复正常,另５例未用更昔洛韦者ＣＭＶＤＮＡ持续阳性,肾功能损害加重。结论部分肾移植受者发生的远期急性排斥反应与ＣＭＶ感染有一定关系;临床上对用激素冲击治疗无效的远期急性排斥可给予抗病毒治疗     

Clinical evaluation in organ transplant patients of a polymerase chain reaction test for CMV DNA applied on white blood cells and serum

A polymerase chain reaction (PCR) test for CMV DNA was evaluated for clinical usefulness. Leukocytes and serum were sampled from 36 patients who had recently undergone organ transplantation. Clinical symptoms, virus culture, and IgG and IgM antibodies were used to identify, in retrospect, patients with CMV disease certified beyond all doubt, with probable disease, with asymptomatic infection, or without infection. PCR tests for CMV DNA in leukocytes (BC-PCR) and serum (SE-PCR) were then evaluated. BC-PCR was positive in all patients with certified CMV disease but also in 31% of the samples from patients without infection. SE-PCR was positive in 11/13 patients with certified disease and was concordant with CMV culture in 192/231 tests. Of the 39 discordant cases, 27 had a positive SE-PCR with a negative culture. The effect of ganciclovir treatment could not be predicted by any test. In conclusion, a negative BC-PCR is strong evidence against CMV disease and a positive SE-PCR strongly suggests CMV disease, but the opposite results are of little clinical help.     

A longitudinal prospective study of cytomegalovirus pp65 antigenemia in renal transplant recipients

Cytomegalovirus (CMV)-encoded pp65 antigen in peripheral blood leukocytes (CMV antigenemia) was investigated in 1017 serial samples from 64 patients for 16 weeks after renal transplantation in a prospective study. In 110 samples from 24 patients, at least one antigen-positive leukocyte was identified. The median number of stained cells was 4 (range 1–1000) per 4×10⁵ leukocytes. Twenty-one of 24 patients with serological signs of an active CMV infection were antigen-positive (sensitivity 87.5%), whereas 3 patients with antigenemia did not show serological signs of infection during the observation period (specificity 92.5%). Positive results were obtained 19 days (median) before serological response and 9 days (median) before the onset of CMV syndrome. The sensitivity in defining a CMV syndrome was 100% (n=8). In all patients who presented with CMV syndrome, antigenemia was present prior to the onset of symptoms or on the same day. In contrast, serological monitoring rendered the diagnosis of CMV infection possible at the onset of clinical symptoms in only two of eight patients. We conclude that (1) insufficient results obtained with the CMV antigenemia assay by other investigators are mainly due to technical problems that can easily be overcome by the protocol presented and (2) the detection of CMV pp65 antigen in peripheral blood leukocytes is an excellent tool for rapid and early diagnosis of CMV infection.     

Rapid detection of cytomegalovirus DNA and RNA in blood of renal transplant patients by in vitro enzymatic amplification.

Cytomegalovirus infection causes significant morbidity and mortality in renal transplant patients. The only marker of CMV infection that appears to correlate with the development of symptomatic illness is viremia. However, CMV grows slowly in tissue culture, requiring 2-6 weeks of incubation for detection of characteristic cytopathic effect. The efficacy of antiviral therapy for CMV may be improved by earlier detection of viremia and institution of antiviral therapy. We performed amplification of CMV DNA and RNA from peripheral blood of renal transplant patients using the polymerase chain reaction (PCR) technique. We consistently detected CMV DNA by PCR earlier than CMV was detected by culture. Detection of CMV RNA in one patient confirmed the presence of actively replicating virus in peripheral blood. Amplification of peripheral blood from healthy CMV-seropositive and seronegative individuals, and from seropositive renal transplant patients without evidence of active CMV disease, was consistently negative. These preliminary data indicate that PCR may provide a means for earlier diagnosis of CMV viremia. Future prospective studies should determine if early detection of CMV DNA by PCR in peripheral blood does predict viremia and symptomatic illness, and if earlier institution of antiviral therapy based on PCR results improves outcome for the CMV-infected transplanted patient.     

Herpes virus infection can cause intervertebral disc degeneration: a causal relationship?

It has been proposed that intervertebral disc degeneration might be caused by low-grade infection. The purpose of the present study was to assess the incidence of herpes viruses in intervertebral disc specimens from patients with lumbar disc herniation. A polymerase chain reaction based assay was applied to screen for the DNA of eight different herpes viruses in 16 patients and two controls. DNA of at least one herpes virus was detected in 13 specimens (81.25%). Herpes Simplex Virus type-1 (HSV-1) was the most frequently detected virus (56.25%), followed by Cytomegalovirus (CMV) (37.5%). In two patients, co-infection by both HSV-1 and CMV was detected. All samples, including the control specimens, were negative for Herpes Simplex Virus type-2, Varicella Zoster Virus, Epstein Barr Virus, Human Herpes Viruses 6, 7 and 8. The absence of an acute infection was confirmed both at the serological and mRNA level. To our knowledge this is the first unequivocal evidence of the presence of herpes virus DNA in intervertebral disc specimens of patients with lumbar disc herniation suggesting the potential role of herpes viruses as a contributing factor to the pathogenesis of degenerative disc disease.     

HLA-G与肾移植术后巨细胞病毒活动性感染的相关性研究

目的 研究HLA-G与肾移植术后巨细胞病毒(CMV)活动性感染的相关性,分析其作用机制及意义。方法 初次肾移植受者215例,按照术后是否发生CMV活动性感染将受者分为CMV阳性组和CMV阴性组,采用流式细胞术检测膜结合型HLA-G1 (mHLA-G1)的表达,采用酶联免疫吸附试验检测可溶性HLA-G5 (sHLA-G5)的表达,采用逆转录聚合酶链法检测HLA-G mRNA的表达,采用蛋白质印迹法验证sHLA-G5的表达,采用免疫组织化学法和HE染色观察移植肾组织中HLA-G的表达,采用ROC曲线分析sHLA-G5水平预测CMV活动性感染的Cutoff值。结果 术前两组间HLA-G表达的差异无统计学意义(P＞0.05)。术后两组间外周血淋巴细胞表面mHLA-G1均呈低表达,CMV pp65阳性时亦无明显变化。CMV阳性组外周血中CD14+ mHLA-G1+细胞显著升高(P＜0.05),达到(45.53±17.32)％,转阴后下降至(10.22±5.78)％。CMV阳性组外周血中sHLA-G5表达水平明显升高(P＜0.05),其预测CMV活动性感染的最适Cutoff值为202.9μg/L,具有很高的诊断准确性。CMV阳性组受者外周血中HLA-G mRNA的表达水平均显著高于CMV阴性组(P＜0.05)。12例CMV活动性感染受者移植肾活检样本中,10例肾小管上皮细胞HLA-G表达呈阳性。结论 HLA-G在外周血中的表达显著升高和移植肾肾小管上皮细胞的阳性表达可能是保护移植肾功能的机制之一。以sHLA-G5表达水平202.9μg/L作为Cutoff阈值,具有很好的判断CMV活动性感染的价值。     

Determinants of protracted cytomegalovirus infection in solid-organ transplant patients

BACKGROUND: Recurrent infection frequently follows the response to the initial treatment of cytomegalovirus (CMV) infection in solid-organ transplant (SOT) recipients. The objective of this study was to describe the course of CMV infection in SOT patients and to identify factors that would predict protracted CMV infection with recurrences. METHODS: Quantitative polymerase chain reaction (PCR) assay for CMV DNA in leukocytes and in plasma were used to assess viral load changes retrospectively in consecutive SOT patients, whose CMV infection episodes had been attended therapeutically or preemptively using quantitative blood culture. RESULTS: Among 101 SOT patients, CMV infection occurred in 63, of whom 32 developed recurrent infection after the initial episode. In patients with recurrent infection, PCR indicated that a majority (27) of recipients had high level of CMV DNA in peripheral blood leukocytes and plasma throughout a protracted (>/=1 month) period including after preemptive or therapeutic ganciclovir courses. Predictors of protracted high-level infection were increasing age, CMV donor seropositivity, and all measures of viral load during the initial episode. CMV recipient seropositivity protected strongly against protracted infection. End of treatment plasma CMV DNA best discriminated between patients who did or did not develop protracted infection. CONCLUSIONS: In SOT patients, protracted CMV infection is associated with increasing age, donor seropositivity, recipient seronegativity, and high viral load during the first episode. End of therapy plasma CMV DNA level best predicts the occurrence of protracted infection. In patients with a high risk of protracted infection, prophylaxis is likely to be particularly cost effective.     

肾移植术后的巨细胞病毒感染及其对急性排斥反应的影响

目的 探讨肾移植受者术后活动性巨细胞病毒（ＣＭＶ）感染的发生率、感染的原因以及ＣＭＶ感染对急性排斥反应的影响。方法 检测１８７例肾移植受者和供者术前血清抗－ＣＭＶ抗体;受者术后定期检测体内ＣＭＶ ＤＮＡ、对ＣＭＶ ＤＮＡ阳性的部分患者给予抗ＣＭＶ治疗,并比较各组排斥反应的发生率。结果 无论是供者还是受者,术前如血清抗－ＣＭＶ抗体阳性,受者术后发生活动性ＣＭＶ感染者明显增多,这些患者急性排斥反应的发     

肝移植术后巨细胞病毒感染的防治研究

目的 探讨肝移植术后患者巨细胞病毒(CMV)感染的诊治经验。方法回顾分析我科2001年1月至2002年12月期间进行的96例同种异体肝移植病例的临床资料。结果发生巨细胞病毒感染19例,均检测到CMV抗原IE-E和CMV抗原PP65,其中CMV-IgM( )8例。在CMV感染的患者中,出现呼吸窘迫3例,发热4例,黄疸2例,14例无明显症状。经更昔洛韦治疗后18例患者血CMV抗原IE．E和CMV抗原PP65转变为阴性,1例患者死于间质性肺炎。结论肝移植后CMV感染与多种因素有关,积极预防、早期治疗肝移植术后患者CMV感染至关重要,CMV抗原检测的应用能够对CMV感染患者作出早期诊断并且指导治疗,更昔洛韦能够有效治疗CMV感染。