大鼠创伤性脑损伤后细胞凋亡及NOS阳性细胞的变化

目的：探讨大鼠创伤性脑损伤后不同时相皮质、海马、隔区细胞凋亡及NOS、ChAT阳性细胞的变化。方法：采用大鼠自由落体脑损伤模型，伤后1、2、3、4、5、7、10d取脑切片，经Nissl染色，用TUNEL法检测细胞凋亡，NADPH—d组化染色观察NOS阳性细胞，ChAT免疫组化染色观察隔区ChAT阳性细胞。结果：Nissl染色可见损伤侧海马CA2、CA3区锥体细胞层细胞消失或紊乱。损伤区周围皮质凋亡细胞伤后3d达到高峰；损伤侧海马凋亡细胞伤后5d达到高峰；损伤侧隔区凋亡细胞7d达到高峰。正常侧上述脑区各时相点均未见到凋亡细胞。损伤区周围皮质、损伤侧海马和隔区iNOS阳性细胞数量明显增加。损伤侧隔区ChAT阳性神经元也明显减少。结论：大鼠创伤性脑损伤后损伤区周围皮质和损伤侧海马、隔区细胞凋亡数量的变化与伤后时程有关。伤后细胞iNOS表达增加是导致细胞凋亡的因素。     

猪卵泡闭锁过程中颗粒细胞凋亡现象的检测

目的　验证猪卵泡闭锁过程中是否存在细胞凋亡及其发生的范围。　方法　用透射电镜观察猪各类卵泡的超微结构 ,对颗粒细胞DNA进行琼脂糖凝胶电泳图谱分析 ,对颗粒细胞涂片进行了DNA末端原位标记(TUNEL)及HE染色检测。　结果　猪的闭锁卵泡中存在大量的凋亡颗粒细胞 ,而健康卵泡中凋亡颗粒细胞则很少 ;对颗粒细胞涂片进行DNA末端标记和常规HE染色检测所得到的颗粒细胞凋亡比例分别为 :健康卵泡TUNEL法为 9% ,HE法为 3 6 % ;闭锁卵泡TUNEL法为 39 2 % ,HE法为 34 % ,尽管两种方法检测的结果之间有差异 ,但两者之间的相关系数达到 0 94。　结论　猪卵泡闭锁过程中确实存在细胞凋亡现象 ,并且凋亡细胞主要集中于颗粒层 ;同时DNA末端标记和常规HE染色检测所得到的颗粒细胞凋亡比例结果说明在某些情况下可以采用简单易行的HE法替代TUNEL法进行细胞凋亡趋势判定     

TUNEL法原位检测心肌细胞凋亡的改进

目的：脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）是检测细胞凋亡的常用方法,但以往仍有TUNEL假阳性率高的报道。本实验目的在于探讨应用TUNEL检测心肌细胞凋亡时增强其特异性的方法。方法：缺血再灌注心肌组织的石蜡切片（2 μm）和冰冻切片（3 μm）进行TUNEL染色。分为石蜡切片HE染色组,石蜡切片常规TUNEL组,冰冻切片改良TUNEL组。石蜡切片常规TUNEL组完全按照试剂盒说明书进行,光学显微镜下观察;冰冻切片改良TUNEL组使用荧光素（FITC）标记的dUTP及4’,6-二脒基-2-苯基吲哚(DAPI）双重荧光染色,在共聚焦显微镜下观察。结果：TUNEL改良法凋亡背景清晰,阳性细胞主要分布在缺血再灌注损伤区域,其凋亡检出率与石蜡切片HE染色相比无显著差异（P>0.05）;常规TUNEL组凋亡背景清晰度较差,非缺血再灌注损伤区域亦有大量阳性细胞出现,其凋亡率明显高于石蜡切片HE染色（P<0.01）。结论：冰冻切片改良TUNEL法可能是更适宜研究SD大鼠缺血再灌注心肌细胞凋亡的方法。     

亚低温对细胞凋亡及凋亡相关基因表达影响的实验研究

目的　研究亚低温对新生鼠缺氧缺血性脑损伤细胞凋亡及凋亡相关基因表达的影响。方法　7日龄大鼠行左侧颈总动脉结扎后吸入 8%氧气 2h,制成缺氧缺血性脑损伤模型。动物随机分成三组 :假手术组 (C组 ) ,缺氧缺血正常温度组 (HI组 ) ,缺氧缺血后 30min亚低温治疗组。应用原位缺口末端标记 (TUNEL)检测凋亡细胞 ,用免疫组化SABC法检测表达bax基因阳性细胞数。结果　 30min后给予亚低温组凋亡细胞及表达bax基因细胞数明显少于HI组 ,差异有显著意义 (P <0 0 1)。结论　亚低温可以通过抑制细胞凋亡 ,抑制凋亡相关基因的表达等机制发挥对缺氧缺血性脑损伤的保护作用。     

组织细胞性坏死性淋巴结炎的电镜和原位末端标记观察

目的 从分子生物学角度,探讨组织性坏死性淋巴结炎的组织发生,提高认识,避免误诊。方法 对34例组织细胞性坏死性淋巴结炎标本行HE和原位末端标记TUNEL染色,光镜观察,其中4例做电镜检查。结果 显示多样的组织细胞主要为T淋巴细胞,以T淋巴细胞大量增生和凋亡为其特征。结论 必须抓住组织细胞性坏死性淋巴结炎的T细胞大量增生和凋亡的形态特征,才能确诊。     

大鼠局灶性脑梗死后细胞凋亡及热休克蛋白70表达的变化

目的 探讨大鼠局灶性脑梗死后热休克蛋白70(HSP70)及细胞凋亡的变化。方法采用光化学法诱导制作大鼠局灶性脑梗死模型，冰冻切片行HSP70免疫组化染色，并用TdT-介导duTP-生物缺口末端标记(TUNEL)法检测凋亡细胞。结果免疫组化显示对照组和假手术组动物无HSP70免疫反应；局灶性脑梗死组12h即有：HSP70的表达，48h时HSP70表达至高峰，阳性反应局限于半暗带。TUNEL结果显示，对照组和假手术动物无凋亡细胞；局灶性脑梗死组6h时在半暗区凋亡细胞出现，3d达高峰。结论局灶性脑梗死可诱导HSP70的表达、神经细胞凋亡，两者的分布一致，但HSP70的表达高峰明显早于细胞凋亡。     

大鼠脑损伤后氨基胍的神经保护作用

目的探讨大鼠脑损伤后诱导性一氧化氮合酶（iNOS）抑制剂——氨基胍（AG）的神经保护作用。方法采用大鼠额叶锐器损伤模型,将SD大鼠随机分成氨基胍（AG）治疗组和生理盐水（NS）对照组。用生物化学方法检测NO含量;RT-PCR法及免疫组织化学染色方法观察iNOSmRNA和iNOS阳性细胞表达变化;末端脱氧核苷酸转移酶介导的原位缺1：3末端标记法（TUNEL）检测细胞凋亡。将两组结果进行比较。结果AG治疗组创伤区及周边NO含量、iNOSmRNA和iNOS阳性细胞表达量较NS组明显降低,凋亡细胞数量也相应明显减少。结论AG能选择性地抑制iNOS表达,减少NO过量产生,从而减少细胞凋亡,发挥神经保护作用。     

大鼠创伤性脑损伤后海马细胞凋亡及bcl-2、NGF-R阳性细胞的变化

目的：探讨大鼠创伤性脑损伤后不同时相海马细胞凋亡及。bcl-2、NGF—R阳性细胞的变化规律。方法：采用大鼠自由落体脑损伤模型，伤后1、2、3、4、5、7、10d取脑切片，作：Nissl染色，用TUNEL法检测细胞凋亡，免疫组化染色观察bcl-2、NGF-R阳性细胞。结果：Nissl染色可见损伤侧海马CA2、CA3区锥体细胞层细胞消失或排列紊乱。损伤侧海马凋亡细胞伤后5d达到高峰，正常侧海马各时相点均未见到凋亡细胞。损伤侧海马bcl-2阳性细胞数量下降，伤后3d至最低点；NGF—R阳性细胞数量增加，伤后5d达到高峰。结论：大鼠创伤性脑损伤后损伤侧海马细胞凋亡数量的变化与伤后时程有关。伤后细胞bcl-2表达下降、NGF-R表达增加是导致细胞凋亡的因素。     

何首乌饮对衰老大鼠卵巢增殖与凋亡的影响

目的探讨中药方剂何首乌饮对衰老大鼠卵巢增殖与凋亡的影响。方法 D-半乳糖连续腹腔注射致亚急性衰老动物模型,造模后灌胃何首乌饮连续60天后,大鼠断头取卵巢。采用免疫组织化学分析方法和原位末端标记法（TUNEL）检测大鼠卵巢组织PCNA,细胞凋亡情况。结果模型组大鼠卵巢PCNA阳性细胞数低于正常对照组大鼠（P〈0.05）。何首乌饮各剂量组PCNA阳性细胞数高于模型组（P〈0.05）。模型组大鼠卵巢组织TUNEL阳性细胞数高于正常对照组大鼠（P〈0.05）。何首乌饮各剂量组TUNEL阳性细胞数均低于模型组（P〈0.05）。并呈现一定的剂量依赖性。差异有统计学意义（P〈0.05）。结论何首乌饮可明显提高衰老大鼠卵巢的增殖能力并抑制其凋亡,具有一定的延缓衰老作用。     

外阴部、阴囊疣状黄瘤临床病理特点及文献复习

目的 探讨外阴部、阴囊疣状黄瘤（verruciform xanthoma,VX）的临床病理学特征、诊断、鉴别诊断及发病机制。方法 对1例阴囊VX、2例外阴VX进行光镜、免疫组化、特殊染色、HPV原位杂交观察并结合文献分析。结果 VX表皮疣状增生,棘层增厚,钉突延长位于真皮同一水平;表面过度角化伴角化不全,内有中性粒细胞浸润;真皮乳头层内上皮钉突间见黄色瘤样细胞（泡沫细胞）浸润。免疫组化标记黄色瘤样细胞CD68、α1-AT、Mac387阳性,CK（AE1/AE3）弱阳性,S-100、Ki-67、HPV阴性。PAS阳性。原位杂交HPV6/11呈阴性。结论 外阴部、阴囊VX与发生于口腔黏膜的VX临床病理学特征相似,黄色瘤样细胞来源于单核/巨噬细胞。     

Ultrastructural visualization of human papillomavirus DNA in verrucous and precancerous squamous lesions.

Human papillomavirus (HPV) DNA was ultrastructurally localized by the non-isotopic in situ hybridization technique in formalin-fixed, paraffin-embedded specimens of verruca vulgaris of the skin, condyloma acuminatum of the penis and severe dysplasia of the uterine cervix. Biotinylated DNA probe cocktails were employed for the visualization of HPV-DNA, types 6 and 11 (HPV 6/11) and types 16 and 18 (HPV 16/18). The papillomavirus genus-specific antigen was also visualized by pre-embedding immunoelectron microscopy using rabbit antiserum. In verruca vulgaris, HPV antigen-positive 50-60 nm-particles of mature viral size were observed in the nuclei of the granular cells and parakeratotic cells with perinuclear haloes, whereas HPV 6/11 and HPV 16/18 DNA were negative. In condyloma acuminatum, the nuclei were positive for the HPV antigen and HPV 6/11 DNA, but were negative for HPV 16/18 DNA. More cells were labeled for the viral DNA than for the viral antigen. The ultrastructural observation indicated the presence of the naked (plasmid) viral DNA as fine particles sized 15-20 nm. In the dysplastic cervical mucosa, dot-like positivity of HPV 16/18 DNA was recognized. The HPV antigen and HPV 6/11 DNA were undetectable. HPV 16/18 DNA was localized in part of the nuclear chromatin. This pattern of localization may suggest integration of the viral DNA into the host cell DNA.     

Ultrastructural Visualization of Human Papillomavirus DNA in Verrucous and Precancerous Squamous Lesions

Human papillomavirus (HPV) DNA was ultrastructurally localized by the non-isotopic in situ hybridization technique in formalin-fixed, paraffin-embedded specimens of verruca vulgaris of the skin, condyloma acuminatum of the penis and severe dysplasia of the uterine cervix. Biotinylated DNA probe cocktails were employed for the visualization of HPV-DNA, types 6 and 11 (HPV 6/11) and types 16 and 18 (HPV 16/18). The papillomavirus genus-specific antigen was also visualized by pre-embedding immunoelectron microscopy using rabbit antiserum. In verruca vulgaris, HPV antigen-positive 50-60 nm-particles of mature viral size were observed in the nuclei of the granular cells and parakeratotic cells with perinuclear haloes, whereas HPV 6/11 and HPV 16/18 DNA were negative. In condyloma acuminatum, the nuclei were positive for the HPV antigen and HPV 6/11 DNA, but were negative for HPV 16/18 DNA. More cells were labeled for the viral DNA than for the viral antigen. The ultrastructural observation indicated the presence of the naked (plasmid) viral DNA as fine particles sized 15-20 nm. In the dysplastic cervical mucosa, dotlike positivity of HPV 16/18 DNA was recognized. The HPV antigen and HPV 6/11 DNA were undetectable. HPV 16/18 DNA was localized in part of the nuclear chromatin. This pattern of localization may suggest integration of the viral DNA into the host cell DNA. Acta Pathol Jpn 41: 757-762, 1991.     

Warty Carcinoma Arising in Condyloma Acuminatum of Urinary Bladder: A Case Report

We describe the case of a 62-year-old man with chronic irritation of the urinary bladder resulting in dysuria and hypogastric pain. Three neoplasms measuring 0.5, 1, and 1.5 cm, respectively, were observed by cystoscopy and removed by transurethral resection (TUR). Histologic examination showed a complex folding of squamous hyperplastic epithelium around a connective tissue core. The superficial epithelium contained numerous koilocytes. The double polymerase chain reaction (PCR) detected DNA of type 11 human papillomavirus (HPV). The diagnosis was condyloma acuminatum of bladder. Three months later the patient presented with fever, and a new cytoscopy demonstrated an ulcerated, exophytic 4.5 cm mass. Histopathology showed a squamous carcinoma with papillomatous structure, pronounced viral koilocytosis, and irregular invasive deep margin. HPV type 11 was found with double PCR. The diagnosis was warty carcinoma arising in condyloma acuminatum. To the best of our knowledge, this is the first case of warty carcinoma of the urinary bladder described in the literature. We discuss the relationship between the infection by HPV and the development of condyloma acuminatum, its evolution toward a well-differentiated squamous carcinoma, and its distinction from verrucous carcinoma. Int J Surg Pathol 8(3):253-259, 2000     

Penile verrucous carcinoma: a clinicopathologic, human papillomavirus typing and flow cytometric analysis.

The relationship of various verruciform squamous cell proliferations of the penis such as verrucous carcinoma, with or without anaplasia and giant condyloma, is uncertain. We conducted clinicopathologic, flow cytometric, and HPV typing studies on 15 cases of penile verrucous carcinoma to investigate its place in the spectrum of genital squamous proliferations. The results show a high degree of morphologic uniformity with respect to Ackerman's original diagnostic criteria, as well as to several other histopathologic features evaluated. The latter include polygonal squamous cells with glassy cytoplasm, centrally located vesicular nuclei, intercellular edema, well-formed cellular bridges, and absence or paucity of koilocytes, true fibrovascular cores, and keratohyalin granules. Intraepithelial abscesses and crust-formation were present in many cases. Four cases contained microscopic foci of cellular anaplasia. These hybrid verrucous-squamous carcinomas presented and behaved similarly to the pure verrucous carcinomas. Tumor recurrence was correlated with extent of initial surgical management. DNA ploidy analysis by flow cytometry performed on eight pure and two hybrid tumors showed uniform diploid populations with similar G1/G2 fractions in both groups. Eight pure and two hybrid tumors evaluated for HPV by isotopic in situ hybridization were uniformly negative for HPV types 6, 11, 16, 18, and 31. The results show that penile verrucous carcinoma demonstrates characteristic and uniform morphologic features and does not contain the HPV types typically associated with condyloma acuminatum, giant condyloma of Buschke-L?wenstein, and condylomatous carcinoma.     

尖锐湿疣角质形成细胞凋亡及Ki-67的表达

目的：研究尖锐湿疣（ＣＡ）角质形成细胞的凋亡及Ｋｉ－６７的表达及其相关性。方法：对４８例ＣＡ采用ＴＵＮＥＬ技术原位检测其细胞的凋亡,用免疫组化方法,观察Ｋｉ－６７阳性的表达。结果：表皮中见到典型的凋亡细胞,表皮各层中有较多Ｋｉ－６７阳性细胞。病理严重度与凋亡指数无显著差异性（Ｐ〉０．０５）;与Ｋｉ－６７阳性细胞指数比较则有显著差异性（Ｐ〈０．００５）。凋亡细胞与Ｋｉ－６７阳性细胞之间呈负相关（ｒ＝     

肛管和肛周尖锐湿疣中人乳头状瘤病毒的检测及意义

将３２例肛管和肛周尖锐湿疣进行Ａｐｇａｒ评分和凹空细胞分型，应用聚合酶链反应和免疫组化技术检测ＨＰＶ抗原和ＤＮＡ，并观察ＨＰＶ在尖锐湿疣组织中的分布状况。结果发现Ａ型１４例，Ｂ型１８例，Ａ型与高积分尖锐湿疣的关系较Ｂ型密切。ＰＣＲ和免疫组化检测阳性率分别为２５％（８／３２）和２２％（７／３２）。我们发现在尖锐湿疣组织中，不论是否是凹空细胞或凹空细胞分型如何及Ａｐｇａｒ积分高低，均有ＨＰＶ分布，但阳性率不同。这表明在正确诊断尖锐湿疣时，凹空细胞核形态、组织学改变和特殊检测三者各起作用并彼此相关。本文还探讨了浆膜型抗原发生机制。     

Absence of human papillomavirus DNA in nongenital seborrheic keratosis

Seborrheic keratosis (SK) is a benign epidermal tumor of unknown etiology. Because of its wart-like morphology, human papillomavirus (HPV) has been suggested as a possible causative agent. Viral involvement, however, has not been confirmed yet despite extensive research. The aim of this study was to evaluate the presence of HPV 6/11, 31, 33 DNA in nongenital SK. We analyzed 40 biopsy specimens taken from patients with nongenital SK using in situ polymerase chain reaction (PCR) and PCR with tissue extracts. The SK specimens (n=40), analyzed by in situ PCR, were negative for all HPV probes tested (types 6/11, 31, 33). Control slides (condyloma acuminatum, n=3) were positive for type 6/11, 31, and 33 HPV probes tested. Melasma samples (n=4), the negative controls, were consistently negative. No HPV DNA band was detected by PCR with the tissue extracts from paraffin-embedded SK samples, while condyloma acuminatum, the positive controls, showed DNA bands of the correct molecular weights. Our results show that HPV type 6/11, 31, and 33 cannot be recognized as causative agents for nongenital SK, which is in contrast to the previous studies. Further studies are required to reveal the presence of other types (more than 90) of HPV DNA.     

Detection of five different human papillomavirus types in cervical lesions by in situ hybridization. A study of 110 cases using sulfonated probes

Several findings suggest an etiologic relationship between genital tract squamous cell carcinoma and certain types of human papillomavirus (HPV). Detection of these HPV types in cervical lesions considered as preneoplastic states (ie, cervical intraepithelial neoplasia or CIN) is extremely important but difficult because the morphology of these states is highly heterogeneous and clinical course is rarely predictable. In situ hybridization (ISH) is the only technique allowing correlation between HPV type and tissue or cell morphology. In this report, 110 biopsy specimens from uterine cervix lesions were studied: 66 CIN, 10 invasive carcinoma, 28 metaplasia, and six condyloma acuminata. A new ISH technique based on direct modification of DNA probes by sulfonation was used. The hybridized DNA was revealed first by a specific monoclonal antibody against sulfonated DNA, and then by an alkaline phosphatase system. In order to determine the sensitivity level of this method, 14 biopsy specimens were also submitted to Southern blot hybridization. Five probes were used separately (HPV 6, 11, 16, 18, and 33) for each biopsy specimen. Results of ISH were correlated with morphologic criteria such as number of koilocytes and mitoses. Oncogenic HPV was found exclusively in CIN. The number of labeled cells varied with CIN grade. These data suggest that, whatever the grade, CIN represents a unique preneoplastic process, and that HPV replication depends on the squamous maturation of the pathologic epithelium.     

Detection of human papillomavirus DNA in anogenital exophytic lesions by in situ hybridization to paraffin sections

In situ hybridization was used to detect human papillomavirus (HPV) nucleic acids (type 6b, 11, 16, 18, 31, and 33) and immunohistochemistry was done to detect papillomavirus common antigen in paraffin sections of biopsy specimens. Two patients suffering from condyloma acuminatum contained HPV-6b and HPV-11. Both cases showed small foci of the antigen-positive cells. One patient having condyloma acuminatum with dysplastic features contained a small quantity of HPV-16 without any antigen-positive cells. One case of verrucous carcinoma showed neither HPV-DNA nor antigen. In situ hybridization is a powerful tool in the analysis of the pathogenesis of HPV-associated neoplasms.     

Histochemical investigation into the molecular mechanisms of malignant transformation in a benign glomus tumour

A glomangiosarcoma arose in a benign glomus tumour. The histological and immunohistochemical characteristics of the tumour were investigated. Apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end labelling (TUNEL). The proportion of apoptotic cells was found to be low and TUNEL positive nuclei were present in the benign part of the tumour. Bcl-2 protein, an inhibitor of apoptosis, was strongly expressed in the glomangiosarcoma with only weak staining in the benign area. The proliferation index of the glomangiosarcoma was almost 10-fold higher than that of the benign glomus tumour. Numerous nuclei in the glomangiosarcoma were intensely stained for the tumour suppressor protein p53. The results of the this study may contribute to an understanding of the molecular basis of malignant transformation in benign glomus tumours.