破骨样细胞中骨保护素和NF-κB配体受体的表达及其意义

目的探讨破骨样细胞中骨保护素(OPG)和NF-κB配体受体(RANKL)表达情况。方法单独培养骨髓单核细胞前体,用巨噬细胞集落刺激因子(M-CSF)和维生素D诱导其转化为破骨样细胞,在0、5、10、15 d用光镜观察和TRAP染色(抗酒石酸染色)评估破骨样细胞的转化程度,用RT-PCR检测OPG和RANKL的表达情况;然后构建主动脉中膜平滑肌细胞(SMC)与骨髓单核细胞前体共培养的模型,用维生素C和β-磷酸甘油诱导SMC转化为成骨样细胞,并在0、5、10、15 d用同样方法再次检测共培养中破骨样细胞转化的情况,以及两种细胞中OPG和RANKL的表达情况。结果不管是单独培养、还是共培养,破骨样细胞中始终没有OPG和RANKL的表达。结论破骨样细胞的转化可能主要受成骨样细胞分泌的OPG和RANKL调控,本身并没有分泌OPG和RANKL进行自身调节的机制存在。     

两种大鼠骨髓源破骨样细胞诱导方法的比较

目的 比较两种不同的大鼠骨髓源破骨样细胞(Osteoclast-like cells,OLC)体外分离培养的方法.方法 收集5周龄SD大鼠骨髓细胞悬液,加入M-CSF(10 ng/ml)培养24 h后置入不同的诱导培养体系中,即A组:巨噬细胞集落刺激因子(M-CSF)+破骨细胞分化因子(RANKL),B组:M-CSF+1,25二羟基维生素D3[1,25(OH)2D3]+地塞米松(Dexamethasone),分别于培养3、5、7、9、12 d利用抗酒石酸碱性磷酸酶(TRAP)染色、骨吸收陷窝检测等对获得的OLC进行形态学和功能观察,并进行计数比较.结果 两种方法 均可诱导出TRAP染色阳性的多核细胞.两组细胞数量均于第7天达到最高峰,B组于7、9、12 d获得的细胞数量较A组多(P<0.05);B组骨吸收陷窝数于9、12 d时多于A组(P<0.05).结论 两种方法 均诱导出破骨样细胞,B组诱导OLC的细胞活性和细胞数量高于A组.     

慢病毒介导大鼠肾脏基因转染的实验研究

目的 研究活体大鼠肾脏中慢病毒载体介导的基因转染及表达情况。方法 以水泡性口炎病毒包膜蛋白(VSV-G)为包膜、携带以磷酸甘油酸激酶(PGK)启动子启动的LacZ报告基因的慢病毒载体注射Lewis大鼠右侧肾脏实质，分别于注射后1、2、3、7天处死大鼠(每个时间点实验组n=3，对照组n=2)，通过β-半乳糖苷酶(β-Gal)组织化学染色检测报告基因的表达。结果 实验组大鼠右肾可见β-Gal染色阳性细胞，而对侧肾脏及其他脏器均未见β-Gal表达；β-Gal的表达在。肾实质注射2天后达到峰值，到第7天仍能维持较高水平。β-Gal阳性细胞周围未见明显炎症细胞浸润。病毒载体注射的肾脏外观及组织形态学未见异常。结论 慢病毒载体能有效而稳定的将外源基因导入活体大鼠。肾脏而无明显毒副作用。     

RANKL和M-CSF诱导THP-1细胞向破骨细胞样细胞分化的研究

目的探讨建立有效诱导THP-1细胞分化为破骨细胞样细胞的方法。方法 THP-1细胞首先在TPA的刺激下分化为贴壁细胞,然后在破骨细胞生成因子（RANKL）和巨噬细胞集落刺激因子（M-CSF）的联合诱导下向破骨细胞样细胞分化。通过抗酒石酸酸性磷酸酶（TRAP）染色检测分化来的细胞是否为破骨细胞样细胞。结果 THP-1在RANKL和M-CSF的作用下分化为TRAP阳性的破骨细胞样细胞。结论 RANKL和M-CSF可联合诱导THP-1细胞分化为破骨细胞样细胞。     

蛇床子素/壳聚糖衍生物胶束对破骨细胞分化及其特异性分子表达的影响

目的观察蛇床子素(osthale,OST)/壳聚糖衍生物胶束(osthole-loaded N-octyl-O-sulfonyl chitosan micelles,NSC-OST)对破骨细胞分化及其特异性分子表达的影响,初步探讨其抗骨质疏松的分子机制。方法通过使用大鼠骨髓单核细胞体外建立破骨细胞诱导模型;将细胞分为空白组、对照组、OST组、NSC-OST组,以巨噬细胞集落刺激因子(macrophage colony-stimulating factor,M-CSF)和核因子κB受体活化因子配基(receptor activator of nuclear factor kappa-Βligand,RANKL)诱导细胞分化,药物组以相同的有效浓度干预细胞;采用CCK-8法观察NSC-OST对细胞活性的影响;通过抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色法测定阳性细胞数;制备骨磨片并将其与细胞共培养,通过甲苯胺蓝染色测定各组骨吸收陷窝面积;通过扫描电镜观察骨吸收陷窝的微观结构;使用荧光素酶报告基因法观察NSC-OST对细胞中活化T细胞核因子(nuclear factor of activated T cells,NFAT)转录表达的影响;应用Western blot法检测NSC-OST对细胞中NFATc1等相关破骨特异分子蛋白表达的影响。结果 CCK-8法发现OST和NSC-OST对骨髓前体细胞均没有细胞毒性;对照组、OST组、NSC-OST组TRAP染色阳性细胞数分别为148.8±12.5、107.6±9.7、75.0±7.4 (个),OST和NSC-OST均可抑制破骨细胞的生成(P<0.05),但NSC-OST的抑制效果更明显(P<0.05);该3组诱导的破骨细胞生成的骨陷窝吸收面积分别为:1 344 942±164 150、824 080±37 484、597 716±14 659 (μm2/片),两药物组的骨吸收活性均受到明显抑制(P<0.05),且NSC-OST组的抑制效果更明显(P<0.05);对照组、Cs A组、OST组和NSC-OST组的荧光素酶报告基因表达量分别为29 174±1 540、9 073±824、21 661±1 430、13 434±879,两药物组均可明显抑制NFAT的转录表达,且NSC-OST组的抑制效果更好;与对照组相比,药物组均可抑制破骨特异分子NFATc1、Fos蛋白(cellular oncogene fos,c-Fos)、组织蛋白酶K (cathepsin K,CTSK)、抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)的蛋白表达(P<0.05),且NSC-OST组的效果更显著(P<0.05)。结论NSC-OST可以抑制破骨细胞的分化以及NFATc1等相关破骨特异分子的表达,且其效果优于OST。     

活化T细胞核因子转录因子家族在T细胞中的作用及与哮喘的关系

活化T细胞核因子（NFAT）是一类具有多向调节功能的转录因子,在活化T细胞中,NFAT能与许多其它信号转导系统的转录因子在核内相互作用,完成不同信号间整合,激活特异性的基因表达。NFAT可能在Th0细胞的分化中起重要作用,并能调节Th2细胞因子的产生,在变态反应性疾病中具有重要意义。NFAT与哮喘也有很密切的关系。     

人骨诱导因子和绿色荧光蛋白基因共表达慢病毒载体的构建和鉴定及在HT-29细胞中的表达

目的构建和鉴定人骨诱导因子基因(hOGN)和绿色荧光蛋白(GFP)共表达慢病毒载体,并检测其在人大肠癌细胞株HT-29中的表达水平。方法采用PCR方法克隆hOGN基因;将hOGN基因连接到带有GFP的慢病毒表达载体pEZ-Lv201中构建慢病毒载体;将构建的慢病毒载体质粒pEZ-hOGN-SV40-eGFP-IRES-Puro(pEZ-hOGN)与慢病毒包装质粒混合物Lenti-PacHIVmix共转染293T细胞,收集慢病毒悬液;重组病毒转染H1299细胞,定量PCR法检测重组病毒滴度;将构建的pEZ-hOGN感染HT-29细胞,荧光显微镜观察和Western印迹检测感染后HT-29细胞中GFP表达情况和目的基因hOGN的表达情况。结果基因测序分析证实克隆的hOGN基因与GenBank提供的序列完全一致;构建的重组慢病毒载体经双酶切、琼脂糖凝胶电泳鉴定证实OGN正确插入pEZ-Lv201;定量PCR测定慢病毒滴度为0.1×10~9~1×10~9TU/ml。在HT-29细胞中重组病毒感染96h后在荧光显微镜下可检测到较强的绿色荧光蛋白表达,Western印迹检测到在HT-29细胞中hOGN蛋白过表达。结论成功构建携带hOGN基因的重组慢病毒载体,在HT-29细胞中有效表达。     

携带Foxp3基因慢病毒载体的构建

目的构建携带叉头样转录因子p3（Foxp3）基因的重组慢病毒载体PWPXL-MOD-Foxp3,并包装成慢病毒,为体外获得CD4＋CD2＋5调节性T细胞（CD4＋CD2＋5 Tregs）奠定基础。方法采用双酶切法构建慢病毒表达载体PWPXL-MOD-Foxp3,用磷酸钙沉淀法将包装慢病毒所需的四质粒系统共转染人胚肾细胞293T,慢病毒系统转染48h后收集病毒上清液,纯化、浓缩后采用流式细胞术测定慢病毒滴度。结果重组的慢病毒载体滴度为3.3×108IU/ml。结论成功构建了含有Foxp3基因的高滴度的慢病毒载体,为体外获得CD 4＋CD 2＋5 Tregs奠定基础。     

破骨细胞分化发育中的信号转导及转录因子研究进展

破骨细胞是骨组织中特有的一种多核细胞,在骨吸收过程中起重要作用。破骨细胞分化过程中,巨噬细胞集落刺激因子（M-CSF）与细胞核因子κB受体活化因子配基（RANKL）结合于细胞表面受体上,提供破骨细胞存活、增殖的信号并激活相应的信号转导通路,使分化中的破骨细胞表达特异性基因,使成熟的破骨细胞执行骨吸收功能。在此过程中,转录因子（PU．1）、核转录因子κB（NF-κB）、活化T细胞核因子c1（NFATc1）、     

17β-雌二醇对新生大鼠成骨细胞护骨素和破骨细胞分化因子表达的调节

体外培养成骨细胞，用不同浓度（10^-11～10^-6mol／L）17β-雌二醇干预，RT—PCR测定护骨素、破骨细胞分化因子（ODF）mRNA的表达水平。雌二醇作用后，成骨细胞护骨素表达上调（P〈0．05），以10^-8mol／L最显著；ODF表达无明显变化。雌激素治疗骨质疏松症的作用可能与其在生理浓度时促进成骨细胞护骨素表达有关。     

Gastric carcinoma with osteoclast-like giant cells

Extraskeletal neoplasms with osteoclast-like giant cells are uncommon. These tumors are most frequently reported in the breast and pancreas, and are relatively rare in other sites. We report a case of primary gastric adenocarcinoma with an infiltrate of osteoclast-like giant cells. The patient is a 64-yr-old black woman who presented with epigastric pain and was found to have a mass in the gastric antrum. Histological examination showed a poorly differentiated adenocarcinoma with an infiltrate of osteoclast-like giant cells. The giant cells were present both in the primary gastric adenocarcinoma and in the lymph node metastases. Immunohistochemical stains demonstrated that the giant cells were of monocytic/histiocytic origin and probably represent a distinctive host response to the tumor. The patient is alive and well 12 months after resection. This is the second published report of gastric carcinoma with osteoclast-like giant cells. Based on this limited experience, gastric carcinoma with osteoclast-like giant cells may represent a distinct clinicopathological entity with a more favorable prognosis.     

Gallbladder carcinoma with osteoclast-like giant cells

Extraskeletal tumors containing multinucleated, osteoclast-like giant cells (OGCs) are uncommon. These neoplasms are most frequently reported in the breast and pancreas. Recently, some authors have suggested that carcinomas containing OGCs may represent a distinct clinicopathological entity with a more favorable prognosis. Occurrence in the gallbladder is extremely rare, with only one previous case. We report here on an additional case of gallbladder carcinoma with an infiltrate of OGCs. A 72-year-old woman presented with postprandial abdominal pain and was found to have a mass in the body of the gallbladder with direct liver invasion. Histological examination showed an adenosquamous carcinoma with an infiltrate of benign OGCs. Immunohistochemical analysis demonstrated that the giant cells were of histiocytic origin. The patient survived for 6 years without evidence of recurrence. This case adds to a small body of literature on gallbladder carcinoma with OGCs. Further studies are required to clearly define the prognostic significance of these giant cells in gallbladder cancer and the differences between adenosquamous carcinoma with OGCs and other gallbladder carcinomas (such as adenocarcinoma and squamous cell carcinoma) with those cells.     

Undifferentiated carcinoma with osteoclast-like giant cells of the pancreas

Pure undifferentiated carcinoma with osteoclast-like giant cells of the pancreas is very rare. Its prognosis is grim. The clinicopathologic findings of a case of this unusual tumor are presented. Following resection, the patient at 9 mo follow-up developed local recurrence.     

Pleomorphic carcinoma of the pancreas with osteoclast-like giant cells

Summary We studied two cases of pleomorphic carcinoma of the pancreas with osteoclast-like giant cells. The cut surface of both tumors was firm and whitish-tan in color, with extensive hemorrhage and necrosis. Microscopically, these tumors were composed mainly of pleomorphic mononuclear cells and numerous bizarre giant cells, with a spindle-cell sarcomatoid appearance and adenocarcinomatous elements exhibiting varying degrees of differentiation. Multinucleated giant cells resembling osteoclasts were frequently located around sites of necrotic hemorrhage. Immunohistochemically, most tumor cells of sarcomatous areas and some anaplastic giant cells were positive for vimentin and cytokeratin. Both carcinoembryonic antigen and CA 19-9 were detected in tumor cells in one case, forming ducts or glands. These findings suggest that the tumors in these two cases originated from pancreatic-duct cells with mesenchymal differentiation. In contrast, osteoclast-like giant cells in both cases showed strong immunoreactivity with vimentin and with KP1 and PG-M1 (CD68), which are monoclonal antibodies that react with a histiocyte-macrophage-associated antigen; however, there was no reaction with any epithelial markers. Thus, osteoclast-like giant cells are not epithelial in nature, suggesting that their origin of histiocyte-macrophage lineage is possibly induced as a paraneoplastic product.     

Osteoinductive factor inhibits formation of human osteoclast-like cells.

Osteoinductive factor (OIF) is a glycoprotein in bone that induces ectopic bone formation. Implantation of OIF plus transforming growth factor beta (TGF-beta) type 1 or 2 into subcutaneous tissues of rats induces formation of bone at the implantation site. Since TGF-beta is also present in bone matrix and inhibits formation of multinucleated cells that express an osteoclast phenotype in long-term human marrow cultures, we tested the effects of OIF on formation of these osteoclast-like cells to determine the effects of OIF on cells in the osteoclast lineage. We found that OIF inhibited total multinucleated cell (MNC) formation in a dose-dependent fashion and preferentially inhibited formation of MNCs that react with monoclonal antibody 23c6 (23c6-positive MNCs), an antibody that identifies osteoclasts. In addition, low concentrations of OIF in combination with low concentrations of TGF-beta acted synergistically to inhibit 23c6-positive MNC formation. The inhibition of 23c6-positive MNC formation by OIF was not mediated by prostaglandin synthesis. These data suggest that regulatory growth factors, such as OIF or TGF-beta, that are stored within the bone matrix and released when bone is resorbed can serve as natural inhibitors of osteoclast activity by inhibiting osteoclast formation.     

Undifferentiated carcinoma with osteoclast-like giant cells of the pancreas]

Undifferentiated carcinoma with osteoclast-like giant cells is a rare neoplasm of exocrine pancreas. Till recently, some cases have been reported, however histogenesis of the tumors are controversial and their characteristic findings have not been described yet. Thirty five-year-old men and 75-year-old men were presented with upper abdominal pain and a palpable mass. On computed tomography, one case showed a well enhancing solid tumor with low density and the other was showed a mainly cystic tumor with peripheral enhancement in the body and tail of the pancreas. One case accompanied multiple metastatic liver masses with subhepatic lymph node enlargement. Tumor staining was seen on angiography. Biopsy and pancreatectomy were performed. Pathological findings revealed tumors composed of neoplastic spindle shaped or pleomorphic large cells with scattered non-neoplastic osteoclast-like giant cells. In one case, there were small foci of adenocarcinoma components in the periphery of the tumor. On immunohistochemical stain, neoplastic cells showed focal positivity for epithelial membrane antigen and vimentin. Tumors were diagnosed as undifferentiated carcinoma with osteoclast-like giant cells. We report these rare cases with a review of literature.     

Huge undifferentiated carcinoma of the pancreas with osteoclast-like giant cells

Undifferentiated carcinoma of the pancreas with osteoclast-like giant cells(OGCs)is very rare,less than1%of all pancreatic malignancies,and shows worse prognosis than that of invasive ductal adenocarcinoma of the pancreas.We present a case of en bloc resection for a huge undifferentiated carcinoma with OGCs that invaded the stomach and transverse mesocolon.A 67-year female was admitted for left upper quadrant pain and computed tomography demonstrated a mass occupying the lesser sac and abutting the stomach and pancreas.There were no distant metastases and the patient underwent subtotal pancreatectomy with splenectomy,total gastrectomy,and segmental resection of the transverse colon.Histopathological examination confirmed an 11 cm-sized undifferentiated carcinoma of the pancreas with OGCs.Immunohistochemical staining revealed reactivity with pan-cytokeratin in adenocarcinoma component,with vimentin in neoplastic multinucleated cells,with CD45/CD68 in OGCs,and with p53 in tumor cells,respectively.The patient had suffered from multiple bone metastases and survived9 mo after surgery.This case supports the ductal epithelial origin of undifferentiated carcinoma with OGCs and early diagnosis could result in favorable surgical outcomes.Investigations on the surgical role and prog-nostic factors need to be warranted in this tumor.     

伴有破骨细胞样巨细胞的胆管肉瘤样癌1例报告

胆管癌是起源于胆管上皮细胞具有高度侵袭性的胆道恶性肿瘤,其发病率和死亡率在全球范围内呈上升趋势。目前具有特殊的组织学表现的胆管肿瘤并不多见,其中上皮细胞肿瘤的肉瘤样变化可见于肝脏、胆囊、胰腺和壶腹部[1]。肉瘤样胆管癌是由上皮细胞和间质细胞组成的罕见肿瘤,大多数肉瘤样胆管癌具有肉瘤组分,表现为梭形细胞或多形性细胞分化。而伴有破骨细胞样巨细胞的胆管肉瘤样癌是一种非常罕见的恶性肿瘤,国内外文献少有报道,现就笔者所在医院诊断的1例伴破骨细胞样巨细胞的胆管肉瘤样癌,结合文献对其临床特点、病理学特征、诊治和预后进行初步探讨。