p16INK4a与Fas对椎间盘细胞功能影响的比较

目的分析p16^INK4a及Fas在椎间盘细胞中的功能作用，探讨椎间盘退变的机制。方法利用p16^INK4a及Fas特异性短链干扰RNA（siRNA）转染体外培养的内破裂（IDD）及突出（LIDP）椎间盘细胞。观察p16^INK4a及Fas表达的沉默情况。分析视网膜母细胞瘤（Rb）蛋白磷酸化状态、β-半乳糖苷酶（β-gal）染色阳性率、细胞形态和增殖的变化；放射性同位素标记培养细胞，观察细胞合成氨基葡聚糖（GAG）及核心蛋白的变化。结果p16^INK4a。及Fas特异性siRNA分别有效地沉默了椎间盘细胞内p16^INK4a及Fas表达。p16^INK4a表达沉默使退变椎间盘细胞衰老表型及合成能力得以明显改善；Fas特异性siRNA转染的LIDP椎间盘细胞的生理功能有所恢复，但未及正常水平，而转染的IDD椎间盘细胞的功能无明显变化。结论p16^INK4a。基因介导的细胞衰老是椎间盘退变的一个关键启动因子。     

不同病理类型椎间盘突出组织的超微结构观察

目的：从细胞水平了解不同病理类型的突出椎间盘细胞结构的病变程度。方法：将4例凸起型、9例破裂型、6例游离型椎间盘突出髓核组织分别切成小块，经组织学处理后行透射电镜观察。结果：(1)凸起型椎问盘突出髓核组织中观察到部分退变细胞、严重退变细胞及坏死细胞；(2)破裂型椎间盘突出髓核组织中观察到少部分退变细胞，大部分为严重退变细胞及坏死细胞；(3)游离型椎间盘突出髓核组织中观察到严重退变细胞及坏死细胞。结论：不同临床病理类型的椎间盘突出组织，其细胞退变严重程度不同，以凸起型为轻，破裂型较重．游离型最为严重．     

退变椎间盘聚集蛋白聚糖中硫酸软骨素链变化的实验研究

目的：研究内破裂及突出椎间盘细胞合成的聚集蛋白聚糖中硫酸软骨素链（CS）在长度及数量分布上的变化.方法：将正常椎间盘、内破裂（IDD）及突出椎间盘（LIDP）髓核或纤维环的组织20mg培养于24孔板中,用放射性同位素35S-硫酸盐和3H-丝氨酸标记新合成的蛋白聚糖分子.将聚集蛋白聚糖单体从培养的组织片中提取,用四氢硼酸钠或木瓜蛋白酶消化后,凝胶包谱分析硫酸软骨链的变化特征.结果：IDD椎间盘组织内细胞合成的聚集蛋白聚糖内CS链的数量明显减少,但长度保持相对正常;突出椎间盘组织中CS的数量和长度有明显下降和缩短,CS链数量的减少较IDD组织中更为严重.结论：IDD组织合成CS的减少主要是由于生成的CS链数量减少所致,而数量的进一步减少以及CS链长度的缩短是LIDP组织中CS合成量下降的主要原因.     

退变腰椎间盘组织中碱性成纤维细胞生长因子的表达研究

目的 探讨碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF)在正常和退变椎间盘组织中的表达情况。方法 交30例来源于腰椎间盘突出症患者手术中所取得的椎间盘组织（观察组，男11例，女19例；年龄25－78岁，平均48岁；病程3个月－30年，平均9年11个月）与6例来源于脊柱侧凸患者前路松解术所取得的椎间盘组织（对照组，男女各3例，年龄10－17岁，平均14.2岁）进行对比，首先经病理组织学检查证实为退变椎间盘组织和政党椎间盘组织，然后将两组椎间盘组织分别通过免疫组织化学方法和原位杂交方法，检测各自椎间盘组织中的bFGF及其mRNA的表达。观察组30例均为退变椎间盘组织，免疫组化阳性率为90％（27／30），原位杂交阳性率为20％（6／30）；对照组6例均为正常椎间盘组织，其免疫组及原位杂交均为阴性，两组间免疫组化方法检测阳性率在统计学上差异有非常显著性意义。结论 bFGF在正常和退变椎间盘组织中表达的差异有显著性意义。提示 bFGF可能作为增生刺激因子促进椎间盘组织中的软骨细胞增生和细胞外基质合成，进而加速椎间盘退变。     

Fas配体mRNA在正常睾丸组织及精原细胞瘤中的表达

为明确Fas配体mRNA在正常睾丸组织及精原细胞瘤中的表达情况，采用原位杂交，反转录PCR（RT－PCR）法检测正常睾丸组织8例Fas配体mRNA的表达，采用原位杂交法检测33例精原细胞瘤组织中的Fas配体mRNA的表达。结果RT－PCR方法证明正常人睾丸组织中有Fas配体mRNA的表达；原位杂交法证明8例正常睾丸组织中均有Fas配体mRNA的表达，且表达局限于睾丸Sertoli细胞，正常生殖细胞中无Fas配体mRNA的表达；原位杂交方法在33例精原细胞瘤组织中检测到Fas配体mRNA表达阳性11例，阳性率为33．33％，多呈现小片状或点状表达。提示Fas配体mRNA仅表达于人类睾丸的Sertoli细胞中，Fas配体mRNA在精原细胞瘤组织中的出现可能是精原细胞瘤形成及进展的原因。     

胆囊癌组织细菌L型的检测与p21及p53基因突变的关系

目的 探讨胆囊癌的发生与细菌L型感染和p21及p53基因突变的关系。方法 采用免疫组化SP法和革兰氏染色技术，对40例胆囊癌及40例慢性胆囊炎组织中的细菌L型和突变型p53蛋白及p21蛋白进行检测，并对胆囊癌组织中的细菌L型阳性伴p21及p53蛋白阳性表达结果和细菌L型阴性伴p21及p53蛋白阳性表达结果进行对比分析。结果 胆囊癌组织中革兰氏染色细菌L型检出率为77．5％(31／40)，明显高于胆囊炎的57．5％(23／40)，P＜0．05，且与免疫组化细菌L型抗原表达阳性率[80．0％(32／40)]具有一致性；胆囊癌组织中的p21及p53蛋白表达阳性率分别为62．5％(25／40)和65．0％(26／40)，明显高于胆囊炎组织中的p21及p53蛋白表达阳性率[2．5％(1／40)和5.0％(2／40)]，P＜0．05；胆囊癌组织中的细菌L型阳性者其p21及p53蛋白表达阳性率分别为75．0％(24／32)和78．1％(25／32)，明显高于细菌L型阴性者的p21及p53蛋白的表达阳性串率[12.5％(1／8)，12．5％(1／8)]，P＜0。05。结论 细菌L型参与了p21及p53基因的突变，并在胆囊癌的发生中可能起协同作用。     

微创针刺旋切制备兔椎间盘退变模型

目的探讨微创针刺旋切制备兔椎间盘退变(intervertebral disc degeneration,IDD)模型的可行性。方法取40只新西兰大白兔,雌雄不限,体质量(2.9±0.3)kg;随机分为对照组和实验组(n=20)。对照组不予处理;实验组采用18G穿刺针在C臂X线机引导下经皮侧后方穿刺进入L4、5、L5、6椎间盘内,旋切髓核组织以促进椎间盘的退变。术后4、8、12、16周行大体观察、MRI观察并根据Pfirrmann分级法评价椎间盘退变情况,然后处死动物取材行Masson染色和番红O染色观察。结果实验组髓核组织颜色较对照组暗,弹性降低。对照组MRI T2加权像椎间盘信号强度早期未见明显改变,后期略减弱;实验组椎间盘信号强度随时间延长呈减弱趋势。根据Pfirrmann分级法评价椎间盘退变程度,两组随时间延长椎间盘退变程度均逐渐加重(P0.05);两组间比较除术后4周差异无统计学意义(P0.05)外,其余术后各时间点实验组椎间盘退变程度较对照组严重(P0.05)。Masson染色示随时间延长,对照组纤维环出现排列不规整,但结构仍完整;实验组纤维环排列紊乱,甚至出现断裂现象。番红O染色示对照组髓核细胞未见明显减少,实验组髓核细胞明显减少。结论微创针刺旋切法可成功制备兔IDD模型。     

结缔组织生长因子在椎间盘纤维化和退变中的表达和作用

目的研究疼痛椎间盘组织中结缔组织生长因子（connective tissue growth factor, CTGF）的表达及其在椎间盘纤维化和退变中的作用。方法收集腰椎后路融合过程中切除的43个疼痛的病理椎间盘，来自于28例行腰椎后路椎体间融合手术的严重椎间盘源性下腰痛患者；同时收集16个在MRIT2加权像信号强度明显减弱的无腰痛症状的退变椎间盘，取自于6例腰椎管狭窄症和8例多节段腰椎后路融合的患者（年龄44～75岁，平均53．5岁，男女比例为8：6）和8个正常对照椎间盘，来自于4具新鲜尸体标本（22～39岁，平均28岁）的L。和蛉．椎间盘。均行组织学检查并用免疫组化方法检测CTGF在不同椎间盘组织的表达。结果组织学检查发现，疼痛椎间盘组织显示不同程度的慢性血管化炎症反应。纤维环组织失去正常的胶原纤维板层结构，板层结构断裂、紊乱或相互交叉融合，正常的成纤维细胞被软骨细胞替代。髓核显示明显纤维化、血管浸润或形成炎性肉芽组织，软骨细胞被成纤维细胞所替代。免疫组化染色显示CTGF在疼痛椎间盘大量表达，无腰痛症状的退变椎间盘有少量表达，正常对照椎间盘没有表达。结论疼痛的退变椎间盘在组织学上明显不同于无腰痛症状的退变椎间盘。CTGF在疼痛椎间盘的大量表达可能与椎间盘纤维化和退变过程密切相关。     

"穿刺抽取髓核"诱导腰椎间盘退变的时间相关性评估

目的对穿刺纤维环抽取髓核诱导的腰椎间盘退变模型，进行时间相关的放射学和组织学评估，明确椎间盘退变程度的时间相关性。方法1岁山羊12只，以粗针穿刺纤维环抽取髓核（L1，2-15，6）建立腰椎间盘退变模型。术后第2、4、8、16周分别行放射学观察、髓核蛋白多糖（GAG）定量、组织学及微观结构评估。结果影像学示术后16周椎间隙高度显著降低（P〈0．01），但各椎间盘间差异无统计学意义（P〉0．05）。GAG定量示所有节段髓核内GAG含量随时间持续下降（P〈0．01），但与椎间盘序列间无相关性。大体标本及组织学观察显示，椎间盘退变组织学表现与抽取髓核后时间显著相关：术后2周组织学未见明显异常；4周始出现退变表现；16周时髓核已近完全纤维化。电镜观察示术后2—16周，髓核细胞从基本正常至大量凋亡，髓核基质逐渐纤维化。结论针刺抽取髓核法是较理想的腰椎间盘退变模型诱导方法。本研究观察16周，椎间盘退变未见缓解及自行修复，诱发的退变严重度与术后时间因素显著相关，而与椎间盘节段序列间无相关性。该模型在术后2周尚未出现明显组织学改变，或许是进行干预的良好时机。     

颈椎间盘纤维环及髓核的超微结构观察

目的：探讨在颈椎间盘纤维环及髓核退变中组织形态的变化。方法：对正常人、单纯颈椎间盘突出症、脊髓型颈椎病三组椎间盘纤维环及髓核进行电镜观察。结果：三组椎间盘胶原纤维无明显变化,单纯颈椎间盘突出症与脊髓型颈椎病人的退变椎间盘细胞较正常人有明显变化,表现为严重退变或细胞坏死。结论：单纯颈椎间盘突出症患者椎间盘以退变细胞为主,为退变早期阶段功能代偿期,脊髓型颈椎病椎间盘以坏死细胞为主,为退变晚期阶段,为不可逆期;颈腰椎间盘退变的组织形态学不完全相同。     

Prognostic implications of aberrations in p16/pRb pathway in urothelial bladder carcinomas: a multivariate analysis including p53 expression and proliferation markers

OBJECTIVE: To assess the prognostic value of the expression of two negative regulators of the cell cycle, namely CDKN2/INK4a gene product (p16) and retinoblastoma gene product (pRb), in urinary bladder cancer in relation to clinicopathological parameters, proliferative fraction and p53 protein accumulation. METHODS: Paraffin sections from 139 patients with urothelial carcinomas were stained immunohistochemically with antibodies to p16 (F12), pRb (PMG3-245), p53 (DO1), PCNA (PC10) and Ki-67 (MIB-1). RESULTS: Diminished p16 and pRb expression occurred in 29 and 74% of cases, respectively, being associated with advanced stage but not with histological grade, papillary status or proliferation rate. In most cases (53%) with some fault in the p16/pRb pathway, only one gene was affected. A double-negative p16/pRb phenotype was comparatively uncommon (25%) and was usually seen in T3-T4 tumours. In survival analysis (either univariate or multivariate) aberrant p16 expression was an adverse prognostic parameter only in T3-T4 tumours. In contrast, the abnormal p16/pRb and p53/p16 phenotypes were linked to a diminished overall and disease-free survival (univariate analysis); p53/p16 abnormal expression was also found to be an independent predictor of reduced survival in muscle-invasive tumours, while proliferation markers were the only parameters with independent significance in superficial (Ta-T1) tumours. CONCLUSION: Our results suggest that lack of p16 immunoexpression, when combined with p53 accumulation, plays an important role in determining the clinical outcome in muscle-invasive urothelial carcinomas.     

FasL在家兔退变髓核组织中的表达及其与白细胞介素-6和肿瘤坏死因子-α的相关性

目的 观察生理解剖学屏障与FasL的分子生物学效应之间的关系,探讨椎间盘退变的发病机制.方法 采用18规格的皮肤穿刺针穿刺家兔纤维环,在术后的3、6、10周收集正常及穿刺后的椎间盘组织,免疫组织化学染色观察FasL及白细胞介素(IL)-6和肿瘤坏死因子(TNF)-α等炎性因子的表达.结果 正常髓核组织中可见少许髓核细胞质中FasL呈弱阳性染色(8.7%),实验组髓核细胞质则呈强阳性染色(41.6%、48.8%、46.4%).FasL阳性细胞百分比在正常组与各实验组之间的两两比较,差异有统计学意义(P<0.01).实验组髓核细胞FasL阳性表达百分比与IL-6及TNF-α阳性表达百分比之间显著相关(r=0.424,P<0.05及r=0.527,P<0.01).结论 FasL与理解剖学屏障的共同作用,可能是使髓核组织产生免免效应的重要因素.当生理解剖学屏障受到损伤后(如穿刺纤维环),FasL、IL-6及TNF-α的表达程度增强,FasL所介导的免疫炎性反应是引起椎间盘退变的重要机制之一.     

The expression of key cell cycle markers and presence of human papillomavirus in squamous cell carcinoma of the tonsil

BACKGROUND: Chemical carcinogens induce squamous cell carcinoma (SCC) of the head and neck by targeting the p53 and the retinoblastoma (pRb) pathways. Human papillomavirus (HPV) might have an etiologic role in these cancers at particular sites. Few studies have compared cell cycle protein expression in HPV-positive and HPV-negative tumors in this region. METHODS: Fifty tonsil SCCs were analyzed for HPV by PCR and for expression of cell cycle proteins (p53, pRb, p16(INK4A), p21(CIP1/WAF1), p27(KIP1), and cyclinD1) by immunohistochemistry. RESULTS: HPV was present in 42%; almost all were type 16. There were statistical associations between HPV positivity and reduced expression of pRb and cyclinD1, overexpression of p16, and younger patient age. Tumor with down-regulated p27 tended to have down-regulated pRb and p21. CONCLUSIONS: HPV-positive tonsil SCCs have distinct molecular pathways. Their association with younger patient age suggests that they are biologically distinct from HPV-negative tumors.     

肝硬化组织中p16INK4a和Rb的基因甲基化状态和蛋白表达

目的：探讨肝硬化组织中p16INK4a和视网膜母细胞瘤易感基因（Rb基因）启动子区域的甲基化状态与其蛋白的表达的关系。方法：分别采用PCR和免疫组织化学方法测定65例肝硬化患者和20例正常人肝组织中p16INK4a和Rb启动子区域甲基化状况和蛋白的表达。结果：20例正常肝组织（对照组肝组织）中均未检测到p16INK4a和Rb甲基化异常,肝硬化组肝组织中p16INK4a和Rb基因甲基化率分别为40.0%（26/65）和36.9%（24/65）;p16INK4a和Rb蛋白表达的积分光密度（IOD）在正常肝组织为225.7±27.4和254.7±34.8,在肝硬化组肝组织中为32.4±7.5和45.2±6.4,差异均有统计学意义（均P<0.05）。结论：肝硬化组织中存在p16INK4a和Rb异常甲基化和p16INK4a和Rb蛋白的表达降低,由此导致的细胞周期失控可能参与了肝硬化的发生发展过程。     

p16INK4a expression and clinicopathologic parameters in renal cell carcinoma

OBJECTIVES: The tumour suppressor gene p16INK4a is a cyclin-dependent kinase inhibitor, for which inactivation attributable to promoter hypermethylation or homozygous deletion has been described in malignancies. Little is known about p16INK4a protein levels in renal cell carcinoma (RCC) and its association with clinicopathologic parameters or disease progression. METHODS: The expression of the p16INK4a gene was analysed with the use of immunohistochemistry and tissue microarrays (TMA). Tissue cores were obtained from the primary tumour itself, the tumoural invasion front, and histologically benign peritumoural tissue of 397 nephrectomies. For statistical analysis, sections were classified into four groups according to the relative amount of positively stained cells: negative (0%), low (1-10%), intermediate (11-50%), and high positivity (>50%). Follow-up data were analyzed for 198 patients (follow-up period: 2-240 mo; median: 138 mo). RESULTS: Absent or low expression of p16INK4a was observed in 82% of tumour samples. No statistically significant association was found between protein levels detected in tumour, invasion front, or normal renal tissues and any of the clinicopathologic variables. Survival analysis by Kaplan-Meier revealed a significant association between high expression (>50%) of p16INK4a in tumours and better disease-specific survival (p=0.03, log-rank test). Cox regression analysis showed that p16INK4a expression is an independent covariate in disease-specific survival (p<0.01). CONCLUSIONS: The absence of p16INK4a expression in most tumour cells indicates that p16INK4a could be involved in the tumourigenesis of RCC. Immunohistochemically detected positivity for p16INK4a is a positive prognosticator for specific survival in both uni- and multivariate analyses.     

Accelerated expression of senescence associated cell cycle inhibitor p16INK4A in kidneys with glomerular disease

The cell cycle inhibitor p16(INK4A) (also known as cyclin-dependent kinase inhibitor 2A) is expressed in vivo in many tissues with age. The exposure of certain chronic stresses can trigger p16(INK4A) expression and a senescence-like phenotype. We studied whether p16(INK4A) expression is induced in glomerular disease (GD). We performed p16(INK4A) immunostaining on 35 biopsies with GD, 12 tubulointerstitial nephritis (TIN), and 19 normal live donor kidneys at transplantation. Based on values for 42 normal kidneys, we calculated expected nuclear p16(INK4A) expression for age and compared the observed values in diseased kidneys to those expected for age. In GD, p16(INK4A) expression was strikingly increased in glomerular and interstitial cell nuclei compared to normals and TIN, and could not be attributed to age (P<0.05). By multivariate analyses, GD was independently associated with increased nuclear p16(INK4a) expression in glomeruli (P<0.001) and interstitium (P=0.01). The p16(INK4A) expression in glomerular and interstitial cell nuclei, and tubular cytoplasm was higher in kidneys with proteinuria and with atrophy/fibrosis (P<0.05). Older age was associated with increased nuclear p16(INK4a) expression in tubules (P=0.01), and interstitial inflammation was associated with increased nuclear p16(INK4a) expression in interstitial cells (P=0.001). The p16(INK4a) staining in tubular cytoplasm was increased in both GD and TIN compared to normals (P<0.001), and was not related to age (P>0.05). Thus, kidneys with GD display increased expression of senescence marker p16(INK4A) in glomerular and interstitial cell nuclei compared to kidneys with normal aging or TIN. The findings suggest a role for somatic cell senescence mechanisms in progression of GD.     

CDKN2／p16^INK4a基因克隆,探针制备及其在肺癌基因诊断中的 …

OBJECTIVES: To clone CDKN2/p16(INK4a) gene, prepare its probe, and to study the change of CDKN2/p16(INK4a) gene in lung cancers. METHODS: Total RNA of normal lung tissue was extracted, CDKN2/p16(INK4a) gene cDNA synthesized, and CDKN2/p16(INK4a) gene recombinant vector, constructed. Southern blot was used to study CDKN2/p16(INK4a) gene in 46 cases of lung cancers, 3 cases of normal lung tissues, 6 cases of lung tissues near cancer, and 3 cases of lymph nodes with lung cancer metastasis. RESULTS: Cloned CDKN2/p16(INK4a) cDNA was proved by enzyme digestion and sequencing. Southern blot showed 4.3 kb band in normal lung tissues and lung tissues near cancers, and deletion of CDKN2/p16(INK4a) gene in cancer tissues and lymph nodes with lung cancer metastasis, with a deletion rate of 17.4% (8/46). CONCLUSION: CDKN2/p16(INK4a) gene may play a role to some extent in progression of lung cancers.     

Micro-RNA与椎间盘退行性变的研究进展

下腰疼痛正在成为目前影响人们生活质量的重要因素,其发病的年龄越来越趋于年轻化,每年因下腰痛带来的社会经济损失巨大。椎间盘退变（intervertebral disc degeneration,IDD）是引起下腰疼的重要原因,在多重因素作用下,椎间盘组织出现生物力学和结构的变化,发生纤维环破裂、髓核组织突出,使脊髓和神经...     

Differential Expression of p16INK4A and Cyclin D1 in Benign and Malignant Salivary Gland Tumors: A Study of 44 Cases

Salivary gland tumors (SGT) are a heterogeneous group of lesions. There is conflicting data concerning the molecular events involving the tumour suppressor retinoblastoma protein (pRb) pathway in these tumors. Few studies examined the alterations in components of the Rb pathway by immunohistochemical (IHC) methods in benign and malignant SGTs. Furthermore, recent evidence implicates human papillomavirus (HPV) in mucoepidermoid carcinoma (MEC) carcinogenesis. The purpose of our study is to examine p16^INK4A and cyclin D1 expression in a variety of benign and malignant salivary gland tumors, and to investigate p16^INK4A expression as a surrogate marker for HPV infection in MEC. Our series includes 30 malignant tumors [14 MEC, 6 acinic cell carcinomas (ACC), 5 polymorphous low grade adenocarcinomas (PLGA), 5 (AdCC)] and 14 benign tumors (4 benign cysts, 5 Warthin tumors and 5 pleomorphic adenomas (PA). All cases were tested by IHC for p16^INK4A and cyclin D1. Testing for HPV wide spectrum (HPV-WS) was performed by in situ hybridization in all MEC cases. Staining intensity was recorded semi quantitatively (on a scale from 0 to 4+). Fisher’s exact test and Pearson X² test with a p < 0.05 were used. Cyclin D1 and p16^INK4A are expressed similarly in malignant and benign tumors (p = 0.146 and p = 0.543, respectively). None of the MEC cases showed nuclear reactivity for HPV-WS. Statistical analysis showed positive correlation between cyclin D1 and p16^INK4A expression. Our findings suggest that p16^INK4A overexpression is likely secondary to cyclin D1 gene upregulation or amplification. Further molecular studies are warranted.