miR-21对视网膜母细胞瘤细胞增殖的影响及作用机制

目的　探讨MicroRNA-21（miR-21）对视网膜母细胞瘤(RB)细胞增殖的影响及作用机制。方法　采用Real-time PCR技术检测miR-21在正常视网膜组织和确诊RB组织中的表达情况；然后在转染的基础上运用MTT检查RB细胞存活率、流式细胞仪检测细胞凋亡率。Western blot法检测与细胞凋亡相关蛋白PDCD4、Bax、Bcl-2的表达。结果　与正常视网膜组织miR-21表达 （0.703±0.071）相比，RB组织miR-21为高表达（2.214±0.162），差异有统计学意义（P<0.01）。在Weri-Rb-1细胞中，与NC组(2.245±0.213)相比，miR-21抑制剂转染后明显降低了miR-21的表达水平，miR-21 inhibitor组为0.683±0.075,差异有统计学意义 （P<0.01）。两组细胞转染后24 h、48 h、72 h、96 h，MTT测定法检测细胞活力结果显示:两组24 h的A值比较，差异无统计学意义 （P>0.05），miR-21 inhibitor 组在 48 h、72 h、 96 h的A值均低于 NC 组，差异均有统计学意义 （均为P<0.01）。流式细胞术检测结果显示:NC组凋亡细胞在总细胞中百分比为（3.045±0.301）%和（4.832±0.493）%，miR-21 inhibitor组凋亡细胞在总细胞中百分比为（2.593±0.257）%和（40.167±4.014）%，miR-21 inhibitor组Weri-Rb-1细胞的凋亡率明显高于miR-21 NC组(P<0.01)。Western blot检测结果显示:NC组PDCD4表达（0.192±0.045）相比miR-21 inhibitor组（0.683±0.091）表达明显减少，NC组Bax的蛋白表达水平（0.143±0.036）相比miR-21 inhibitor组（1.192±0.054）也明显减少，差异均有统计学意义（P<0.01)，NC组Bcl-2蛋白表达（0.864±0.038）相比miR-21 inhibitor组（0.257±0.026）明显增多，差异有统计学意义（P<0.05)。结论　miR-21是RB的促癌基因，miR-21抑制剂可以通过降低miR-21表达抑制肿瘤细胞的增殖、促进细胞凋亡，这一过程与PDCD4、Bax、Bcl-2等凋亡相关蛋白有关。     

Responses of CDKs and p53 in Delayed Ischemic Neuronal Death

Stroke is a debilitating disease that affects millions each year.While in many cases cerebral ischemic in jury can be limited by effectivw resuscitation or thrombolytic treatment,the injured neurons wither in a process known as delayed neuronal death(DND).Mounting evidence indicates that DND is not simply necrosis played out in slow motion but apoptosis is triggered.Of particular interest are two groups of signal proteins that participate in apoptosis-cyclin dependent kinases(CDKs) and p53-among a myriad of signaling events after an ischemic insult.Recent investigations have shown that CDKs,a family of enzymes initially known for their role in cell cycle regulation,are activated in injured neurons in DND.As for p53,new reports suggest that its up-regulation may represent a failed attempt to rescue in jured neurons,although its up-regulation was previously considered an indication of apoptosis.These observations thus rekindle an old quest to identify new neuroprotective targets to minimize the stroke damage.In this review,the author will examine the evidence that indicates the participation of CDKs and p53 in DND and then introduce pre-clinical data to explore CDK inhibition as a potential neuroprotective target.Finally,using CDK inhibition as an example,this paper will discuss the pertinent criteria for a viable neuroprotective strategy for ischemic in jury.     

Rb1基因第16内含子内21个碱基缺失1例

目地研究双眼视网膜母细胞瘤患者Rb1基因杂合性突变的分子生物学特性。方法应用PCR—SSCP直接测序技术检测双眼视网膜母细胞瘤患者白细胞DNA中Rb1基因杂合性突变。结果50例证实有Rb1基因杂合性突变的病例中有1例发生于第16内含子中可以用3种定位方法解释、具有相同序列的21个碱基缺失。结论这种极为少见的Rb1基因突变方式可能是由于破坏了正常拼接位点的结构而激活了“隐蔽拼接位点”，导致异常的Rb1基因mRNA产生或由此影响整个拼接过程。     

Human Y-79 retinoblastoma cells express functional corticotropin-releasing hormone receptors

In human Y-79 retinoblastoma cells corticotropin-releasing hormone (CRH) produces a marked and rapid increase of adenylate cyclase activity. The concentration of the peptide producing half-maximal stimulation is 60 nM. The effect of CRH is significantly antagonized by the specific CRH receptor antagonist alpha-helical CHR 9-41 and is mimicked by sauvagine and urotensin I, two peptides displaying sequence homology with CRH. These results demonstrate the presence of functional CRH receptors in human Y-79 retinoblastoma cells and suggest that this cell line may be a suitable model in which to study the action of CRH on human retinal cell function.     

Rb基因对人视网膜母细胞瘤株SO-Rb50的影响

目的：探讨视网膜母细胞瘤（Rb）的发生与Rb基因（Rb1）缺失、失活等异常的关系。方法：用逆转录病毒载体pDOR与全长4．7kb野生型Rb1cDNA构建逆转录病毒表达载体pDOR－Rb1+。用脂质体介导法将pDOR－Rb1+转入CRIP包装细胞系。结果：实验产生了0．5X105Cfu具有一次感染能力的重组逆转病毒。利用该病毒感染SO-Rb50，经G418筛选，获得了抗G418细胞群体。运用PCR及Southern杂交技术对转染细胞进行检测，结果表明该抗G418细胞中有完整的外源Rb1存在。Northern杂交发现其Rb1mRNA表达水平有所提高。对细胞群体生长速率、软琼脂集落形成能力的测定表明，外源Rb1的表达使SO-Rb50在软琼脂中集落形成能力降低，而群体生长速率无明显影响。结论：外源Rb1对SO-Rb50恶性表型有一定影响。     

Second nonocular tumors in cured unilateral retinoblastoma patients

Summary According to recent data the incidence of second tumors in cured hereditary unilateral retinoblastoma patients is 20% within 10 years, 50% after 20 years and rises to 90% after 30 years. Nonhereditary unilateral retinoblastoma patients have not been regarded as susceptible for second nonocular tumors so far. A case is reported of such a patient having developed a second presacral retroperitoneal tumor after successful treatment of a nonhereditary unilateral retinoblastoma group V without intracranial extension or pulmonary metastases. Consequently all (hereditary and non-hereditary) retinoblastoma patients should receive at least quarterly intensive follow-up examinations after primary ophthalmologic treatment.     

视网膜母细胞瘤术后随访

复查69例视网膜母细胞瘤,56眼进入青光眼期。病理检查:未分化型占80%,对肿瘤细胞已浸润至视网膜、玻璃体及视神经断端者,术后深部X线或钴~(60)照射,很有价值,可提高存活率。本病易误诊为眼内炎、青光眼等。     

Deregulation of the G1/S-phase control in human testicular germ cell tumours

Deregulated cell cycle and defective genome-integrity checkpoints are among the hallmarks of cancer.Here we summarize our recent studies of key components of the GI/S machinery in normal human spermatogenesis, and their abnormalities in testicular germ cell tumours (TGCTs), with special emphasis on carcinoma in situ lesions (CIS). Our combined immunohistochemical and immunoblotting analyses of normal human adult and fetal testes, CIS, seminomas, embryonal carcinomas, and teratomas, revealed an 'unorthodox' spectrum of defects within the so-called RB pathway in TGCTs. The early aberrations included lack of expression of the retinoblastoma tumour suppressor (pRB) and the CDK inhibitor pl9ink4d, and overexpression of cyclin D2. Progression from CIS to invasive TGCTswas associated with loss of another two CDK inhibitors and tumour suppressors: pl6ink4a and pl8ink4c. We also found the lack of pRB and pl9ink4d in fetal gonocytes, the candidate target cell for all types of TGCTs. These findings, together with the status of the Chk2-p53 DNA-integrity checkpoint, are considered in relation to the origin, biology and pathogenesis of TGCTs, and potential implications of the GI/S defects for the curability of these tumours.     

视网膜母细胞瘤眼球的病理取材方法

目的：探讨视网膜母细胞瘤病理标本的不同取材方法对脉络膜浸润检出率的影响，寻求理想的常规取材方法。方法：复查297只原发摘除的视网膜母细胞瘤眼球的病理切片，比较五平面取材法（151只眼）和单平面取材法（146只眼）对脉络膜浸润的检出率。结果：五平面取材法对脉络膜浸润的检出率（31％）明显高于单平面取材法（17％），且主要是对玻璃膜在肿瘤细胞影响下发生变化的检出率高。脉络浸润的各期都主要分布在五平面取材法的各平面上。结论：五平面取材法能提供判断视网膜母细胞瘤患者预后的较大量病理信息，在日常医疗工作中从人力、物力、财力上能够承受，可作为常规取材方法应用于临床。连续切片取材只作为在特殊情况下对五平面取材法的补充。单平面取材法有可能丢失一些重要的病理信息而应由五平面取材法代替。