Particulate endocytosis mediates biological responses of human mesenchymal stem cells to titanium wear debris.

Continual loading and articulation cycles undergone by metallic (e.g., titanium) alloy arthroplasty prostheses lead to liberation of a large number of metallic debris particulates, which have long been implicated as a primary cause of periprosthetic osteolysis and postarthroplasty aseptic implant loosening. Long-term stability of total joint replacement prostheses relies on proper integration between implant biomaterial and osseous tissue, and factors that interfere with this integration are likely to cause osteolysis. Because multipotent mesenchymal stem cells (MSCs) located adjacent to the implant have an osteoprogenitor function and are critical contributors to osseous tissue integrity, when their functions or activities are compromised, osteolysis will most likely occur. To date, it is not certain or sufficiently confirmed whether MSCs endocytose titanium particles, and if so, whether particulate endocytosis has any effect on cellular responses to wear debris. This study seeks to clarify the phenomenon of titanium endocytosis by human MSCs (hMSCs), and investigates the influence of endocytosis on their activities. hMSCs incubated with commercially pure titanium particles exhibited internalized particles, as observed by scanning electron microscopy and confocal laser scanning microscopy, with time-dependent reduction in the number of extracellular particles. Particulate endocytosis was associated with reduced rates of cellular proliferation and cell-substrate adhesion, suppressed osteogenic differentiation, and increased rate of apoptosis. These cellular effects of exposure to titanium particles were reduced when endocytosis was inhibited by treatment with cytochalasin D, and no significant effect was seen when hMSCs were treated only with conditioned medium obtained from particulate-treated cells. These findings strongly suggest that the biological responses of hMSCs to wear debris are triggered primarily by the direct endocytosis of titanium particulates, and not mediated by secreted soluble factors. In this manner, therapeutical approaches that suppress particle endocytosis could reduce the bioreactivity of hMSCs to particulates, and enhance long-term orthopedic implant prognosis by minimizing wear-debris periprosthethic osteolysis.     

直肠癌切除前后腹腔冲洗液脱落细胞学对比研究

目的研究直肠癌术中腹腔多次冲洗，对于降低患者腹腔内脱落癌细胞阳性率的临床意义。方法对63例直肠癌患者术中腹腔冲洗分6次进行，肿瘤切除前3次，切除后3次；将6次腹腔冲洗液行常规病理学涂片细胞学检查结果进行对比。结果肿瘤切除前，全组患者腹腔冲洗液脱落癌细胞均呈阳性表达。肿瘤切除后第1次腹腔冲洗液脱落癌细胞阳性40例，第2次阳性33例，第3次阳性13例。肿瘤切除后第1次冲洗液的脱落癌细胞阳性结果与切除后第2次冲洗液阳性结果比较，P〉0.05，差异无统计学意义；但第3次冲洗液的脱落癌细胞阳性结果与第2次比较，P〈0.01，差异有统计学意义。结论对直肠癌患者进行术中多次腹腔冲洗可降低腹腔内脱落癌细胞的阳性率。     

Gastrointestinal stromal tumours: a regular origin in the muscularis propria, but an extremely diverse gross presentation

Background Gastrointestinal stromal tumours (GISTs) are thought to arise from the interstitial cells of Cajal (ICCs). ICCs form a network surrounding the myenteric plexus and between-muscle fibres of the muscularis propria of the tubular GI tract. The cell of origin of so-called extra-gastrointestinal stromal tumours (EGISTs) is not known.Aim and methods To study the diversity of gross presentation of GISTs and to critically assess the incidence of EGISTs and their relationship to mural GISTs, a total of 200 neoplasms with typical morphologic and immunohistochemical features of GISTs were reviewed, looking for any degree of association with the muscularis propria of the gut wall.Results There were 130 gastric (65%), 9 duodenal (4.5%), 48 small intestinal (24%), 9 colorectal (4.5%), 1 appendiceal (0.5%) and 3 unclassifiable GISTs (1.5%). Fourteen cases (7%) were initially submitted as EGISTs (four mesenteric, four omental, one pararectal/prostatic, one pelvic/Douglas, one perivesical, one located between root of mesentery and tail of pancreas, one involving the mesentery, omentum and abdominal wall extensively and one located between liver and stomach). After critical re-evaluation of surgical reports and remote clinical history and a careful search for residual muscular tissue from the gut wall in the tumour pseudocapsule (in some cases supported by desmin immunoreactivity), it was possible to reclassify most of these cases (11/14) as either GISTs with extensive extramural growth resulting in loss of contact to the external muscle coat of the gut (8/14) or as metastases from an inoperable GIST (2/14) or from a previously resected deceptively benign tumour (1/14).Conclusion EGISTs are probably rarer than previously reported (1.5% or less in this study). We concluded that most so-called EGISTs represent apparent EGISTs that should have arisen from the outermost muscle coat, but have lost their contact to the point of origin due to extensive extramural growth pattern. From a surgical point of view, it is crucial to document and mark any focal attachment or adhesions to the gut wall noticed during surgery for an apparent EGIST. In contrast to most other neoplasms, GISTs should be defined by virtue of any degree of association with the muscularis propria (no matter how minimal), but not by localisation of the bulk of the tumour.     

MBP对PBMC产生IFN-γ和NO的影响

目的探讨T细胞非特异性活化在CNS脱髓鞘性疾病中的作用。方法分离实验性自身免疫性脑脊髓炎(EAE)易感性BALB/c小鼠外周血单个核细胞(PBMC),采用体外细胞培养方法在体外与碱性髓鞘蛋白(MBP)共培养,测定培养上清液中IFN-γ、NO水平。结果经MBP刺激的PBMC产生IFN-γ[(43.83±6.06)pg/mL]和NO[(180.76±20.75)μmol/L]明显增加,与对照组产生的IFN-γ[(28.52±2.18)pg/mL]和NO[(95.61±13.09)μmol/L]相比差异有统计学意义(P<0.01)。结论在CNS脱髓鞘性疾病发病过程中,活化的T细胞、单核细胞等分泌致炎细胞因子和其他有害物质增多。     

人胶质瘤干细胞内在自我更新能力

目的 了解胶质瘤干细胞内在的自我更新和增殖能力。方法 观察原代胶质瘤细胞在单纯改良Eagle/F12培养液（DMEM/F12）中胶质瘤干细胞球的形成,并检测其CD133、胶质纤维酸性蛋白（GFAP）、微管相关蛋白（MAP2）、髓磷脂碱性蛋白（MBP）的表达。通过二代球体形成、细胞增殖测定、分化实验分析其自我更新、增殖、多能分化能力。通过裸鼠移植瘤实验观察所分离细胞球细胞与原代培养胶质瘤细胞成瘤能力的差异。结果 在单纯DMEM/F12培养液中形成的胶质瘤细胞球细胞表达神经干细胞标记CD133,不表达分化标志GFAP、MAP2,少数细胞表达MBP。分离出的胶质瘤细胞球细胞可在单纯DMEM/F12培养基中增殖,并能形成CD133阳性的二代细胞球,可分化为GFAP、MAP2、MBP阳性表达的肿瘤细胞。裸鼠成瘤实验显示其成瘤能力显著高于原代胶质瘤细胞。结论 胶质瘤干细胞能在无血清、无外源性细胞因子培养基中形成肿瘤干细胞球,胶质瘤干细胞的自我更新和增殖不依赖于外源性生长因子,它可能拥有自我更新的自身活化机制。     

制作角膜缘干细胞完全缺乏动物模型的新方法

目的提出一种新的角膜缘干细胞完全缺乏模型制作方法,为干细胞缺乏疾病的治疗性研究提供可靠的模型。方法利用9mm环钻划界,板层切除角膜组织,通过临床评分、印迹细胞学、组织病理及免疫组织化学验证模型的成功。结果板层切除术后30~45d,模型眼角膜混浊、上皮缺损、大量新生血管长入,印迹细胞学可见PAS( )的杯状细胞,组织病理学检查上皮符合结膜细胞表型,免疫荧光染色可见结膜杯状细胞特异性标志MUC5AC阳性表达。结论环钻划界加板层切除法为一种可靠、有效的角膜缘干细胞完全缺乏模型制作方法。     

巯基化合物对内毒素诱导大鼠枯否细胞NF—kB活性和肿瘤坏死因子释放的影响

目的 观察巯基化合物对内毒素诱导大鼠枯否细胞（KC）NF-kB活性和肿瘤坏死因子-a（TNF-a）释放的影响。方法采用胶原酶灌注和percoll密度梯度离心法分离得到高纯度大鼠KC，在过夜培养后冲洗两遍（不含血清），加入内毒素（LPS）10ng／ml刺激原代培养的KC，观察不同浓度谷胱甘肽乙基酯（GSHEE）及N-乙酰半胱氨酸（NAC）对大鼠KCTNF-a释放和KC内谷胱甘肽（GSH）及NF-kB活性的影响。并观察使用谷胱甘肽合成抑制剂BSO后，对NAC抑制炎症因子的影响，采用凝胶滞留电泳法测NF-kB活性，采用高效液相色谱分析法测GSH水平。结果 LPS诱导KC中GSH水平无变化；GSHEE和NAC可提高KC中GSH水平（P〈0．05），降低NF-kB活性和TNF-a释放（P〈0．05）。NAC与BSO合用时KC中GSH水平无变化，TNF-a释放降低（P〈0．05）。结论 NAC通过抑制KC中NF-kB的激活和TNF-a的释放调节KC功能，但这种抑制作用并非通过增加GSH介导。     

Diagnostic accuracy of the Retinal Thickness Analyser: differentiation between normal eyes and eyes with glaucoma or macular pathologies

Background The Retinal Thickness Analyser (RTA) is intended to detect glaucomatous changes as well as macular pathologies at the posterior pole. We determined the diagnostic accuracy for eyes with manifest glaucoma or macular diseases.Methods We examined 71 eyes with long-term, established eye conditions. Included were 28 eyes with glaucoma, 21 with different macular diseases and 22 normal eyes. All examinations were evaluated in a blind-test by RTA experts without any clinical information on the patients. After comparison of the RTA interpretation with the clinical diagnosis, we determined sensitivity, specificity, positive and negative predictive values.Results Of 71 examinations, 15 (21%) were not interpretable. If these results are excluded, the following diagnostic accuracy values were calculated for glaucoma and macular disorders respectively: sensitivity 75 and 59%, specificity 55 and 97%, positive predictive value 48 and 90% and negative predictive value 80 and 84%. These values were not significantly different when both eyes of each patient were included in the final analysis (n=133).Conclusion The diagnostic values of the RTA determined in this case control study were not satisfactory. However, no clinical information was used in the assessment. The extent to which additional clinical information increases the diagnostic value remains to be determined.     

Endometrial cancer patients have a significant risk of harboring isolated tumor cells in histologically negative lymph nodes

In this study, we examine the prevalence of finding isolated tumor cells (ITCs) in negative lymph nodes of endometrial cancer patients using immunohistochemistry. Seventy-six endometrial cancer patients with lymph nodes histologically negative for metastatic disease were examined. Nodal tissue sections were stained with anticytokeratin antibodies AE-1 and CAM 5.2. Nodes with single or groups of cells (two to four cells) < or =0.2 mm and showing cytokeratin reactivity were positive for ITCs. Findings were compared to features of the primary tumor and patient outcome. ITCs were present in 31 of 1712 lymph nodes. Fifteen (19.7%) patients had ITC-positive nodes. ITCs involved only pelvic nodes in nine cases, only para-aortic nodes in five cases, and pelvic and para-aortic in one case. Tumor in adnexa was the only pathologic feature associated with nodal ITCs (P= 0.0485). All 15 patients with nodal ITCs were alive at follow-up. One (6.7%) patient suffered recurrent disease but was alive at last encounter. Disease recurred in 5 (8.8%) of 57 patients without nodal ITCs. Two are alive without disease, two alive with disease, and one died from her cancer. In summary, a significant proportion of endometrial cancer patients have ITCs detected by immunohistochemistry in histologically negative regional lymph nodes.