破骨细胞体外分离培养及其骨吸收活动的观察

目的 建立大鼠破骨细胞体外分离培养方法，为体外研究破骨细胞骨吸收机理奠定基础。方法 采用出生24h内的Wistar大鼠，从其四肢长骨中分离出破骨细胞，与盖玻片、骨磨片共同培养，观察破骨细胞的形态结构及体外骨吸收活性。结果 相差显微镜及光镜观察到分离的细胞含多个核，能够移动，胞浆有伪足样突起，这些细胞用目前公认的鉴定破骨细胞的标志—抗酒石酸酸性磷酸酶染色呈阳性反应，扫描电镜观察到这些细胞能在骨片上形成典型的骨吸收陷窝。结论 用此方法分离培养的细胞为具有骨吸收活性的破骨细胞。     

骨疏康对去卵巢雌鼠骨质疏松症防治作用的研究

目的 为验证骨疏康颗粒防治原发性骨质疏松症(POP)的疗效和探讨其治疗机理。方法 以切除卵巢雌性大鼠复制绝经后骨质疏松症模型,观察该药对去卵巢大鼠抗失骨作用。实验分为模型组、骨疏康组、尼尔雌醇组和正常对照组。术后1周开始灌胃治疗,持续12周。结果 实验性骨质疏松症大鼠一般状态、器官指数、腰椎骨矿含量、胫骨骨组织形态计量学、股骨生物力学性能等指标明显异常,病理光镜和计算机图像分析显示POP病变典型。骨疏康颗粒对上述改变有明显缓解作用,综合疗效优于对照西药。结论 骨疏康颗粒可通过体内多方位调节作用达到预防和抑制骨质疏松症的发生和发展,具有较好的应用前景。     

肺癌A549细胞诱导RAW264.7破骨细胞分化中AMPK信号通路的作用

目的:探讨AMPK信号通路在体外肺癌细胞诱导破骨细胞分化中的作用。方法:将肺癌A549细胞与RAW264.7细胞共培养后随机分为阴性对照组、阳性对照组、AICAR组,药物干预后行TRAP染色观察破骨细胞的分化,流式细胞仪观察破骨细胞细胞凋亡情况,qPCR检测破骨细胞AMPK、mTOR和CTSK mRNA的表达,Western blot检测破骨细胞AMPK、p-AMPK、mTOR和p-mTOR蛋白的表达。结果:与阴性对照组比较,AICAR组破骨细胞数明显减少,差异具有统计学意义（P＜0.05）;对诱导破骨细胞凋亡的作用无差异;上调AMPK的mRNA和蛋白表达,下调mTOR的mRNA和蛋白表达,差异具有统计学意义（P＜0.05）。结论:AICAR可以抑制肺癌细胞诱导破骨细胞分化,而AMPK/mTOR信号通路可能参与了肺癌细胞诱导破骨细胞分化过程。     

17β-雌二醇对破骨细胞骨架和骨吸收功能影响的实验研究

目的　研究17β-雌二醇对体外培养破骨细胞细胞骨架的影响,以及这种影响与骨吸收功能之间的关系。方法　在破骨细胞培养液中加入不同浓度的17β-雌二醇,用F-actin特异性抗体进行免疫荧光染色,激光共聚焦显微镜观察破骨细胞细胞骨架的变化情况,同时用扫描电镜观察破骨细胞在骨片形成骨吸收陷窝数目及面积的变化。结果　随着17β-雌二醇浓度的升高,实验组破骨细胞F-actin相对荧光强度从(89 6±7 5)%下降至(34 7±5 4)% (与对照组荧光强度相比),细胞内微丝收缩变短,排列紊乱,细胞波状缘消失,破骨细胞伸展面积从(1289±53)μm2 /细胞核下降至(406±42)μm2 /细胞核,同时,骨吸收陷窝的数目和面积从(131 5 ±11 7)个/片和(2157±51)μm2 减少到(16 8±4 0 )个/片和(965±75)μm2。生理浓度(10-7mol/l)及其以上17β-雌二醇处理组与对照组比较,差异有显著性(P<0 01 )。结论　17β-雌二醇影响破骨细胞内细胞骨架的结构,下调F-actin的表达,降低破骨细胞的活动能力,从而抑制破骨细胞的骨吸收功能。     

人参皂苷Rb2在体外调控破骨细胞的作用

目的探究人参皂苷Rb2在体外对破骨细胞的作用及机制。方法通过培养RAW264.7破骨前体细胞,加入不同浓度Rb2溶液(0.1μM,1μM,10μM),采用CCK-8检测Rb2药物毒性,利用TRAP染色观察破骨细胞形态并计数,使用RT-PCR方法检测TRAP、NFATc1破骨活动特异性基因m RNA的表达,通过Western blot技术检测破骨细胞中LC3 I、LC3 II、m TOR的蛋白表达水平。结果 TRAP阳性计数,与空白对照组相比(168.2±22.6),0.1μM组(131.0±16.3),P=0.018;1μM组(98.8±17.9),P0.01;10μM组(85.8±17.3),P0.01,破骨细胞数量明显减少。对照组TRAP、NFATc1破骨分化特异性基因m RNA表达明显下降,与对照组相比各组P0.01。LC3 II/LC3 I的比值显著增高,m TOR量下降,与对照组相比各组P0.01。结论 Rb2可能通过自噬途径影响破骨基因的表达,从而减少破骨细胞的形成,m TOR通路在此过程中起重要作用。     

骨疏康冲剂与钙剂联合应用治疗绝经后骨质疏松症的临床研究

目的 观察骨疏康冲剂与钙剂联合应用治疗绝经后骨质疏松症的效果。方法 对年龄为50～75岁、绝经5～30年确诊为骨质疏松症的妇女62例,随机分为骨疏康冲剂加钙剂联合用药组和单用骨疏康冲剂组和单用钙剂组。分别于治疗前、治疗后3个月和6个月观察患者临床症状,用DEXA监测髋部骨密度,测定血清碱性磷酸酶(ALP)、血钙、血磷。结果 骨疏康冲剂加钙剂联合用药组服药后6个月,临床症状改善明显优于单用骨疏康冲剂组(P<0.01),其骨密度也高于单用骨疏康冲剂组(P<0.05),但骨代谢指标各组间无显著差异(P>0.05)。结论 骨疏康冲剂与钙剂联合应用治疗绝经后骨质疏松症较单用骨疏康冲剂有着较好的疗效。     

同种异体骨移植后骨吸收和破骨细胞的形态学特点

目的探讨骨移植后骨吸收和破骨细胞的型态学特点。方法利用扫描电镜,结合光镜,观察8例临床植骨后因严重骨吸收被取出的标本,5例人类正常骨标本和20例大鼠肌内埋入的骨标本,分析严重骨吸收过程中破骨细胞和破骨区表面的形态学特征,以及破骨细胞的形态学演变。结果与正常对照骨标本不同,骨吸收部位有大量幼稚的单核破骨细胞聚集,伴有表面结构变异退变,内部纤维裸露和红细胞吞噬破骨区表现为局灶性切割而不是弥漫性骨质疏松。结论破骨的特点是出现单核破骨细胞和局灶性切割。     

骨疏康对氢化可的松引起的大鼠骨丢失的影响

目的 通过氢化可的松造成实验性骨质疏松，观察中成药骨疏康对防止骨质丢失的效应。结果 模型组与给药组大鼠骨钙、骨磷和羟脯氨酸含量有显著差异。骨疏康有效量可防止氢化可的松引起的大鼠骨矿物质及有机质的丢失。结论 骨疏康能有效预防皮质类固醇诱导的骨质疏松症。     

胫骨内接种建立乳腺癌骨转移大鼠模型的特点

目的研究胫骨内接种MRMT-1细胞制作的乳腺癌骨转移大鼠模型在行为学、影像学、核医学、病理学和分子生物学等方面的特点。方法使用雌性SD大鼠,随机分为假手术组和模型组,使用胫骨内注射法制成乳腺癌骨转移模型。造模后第19天时进行疼痛测定;第21天取材,测定肿瘤体积,通过影像技术评估骨质缺损程度,核医学测定骨矿物质含量(BMC)和骨密度(BMD),HE染色观察形态,抗酒石酸酸性磷酸酶(TRAP)染色并计数破骨细胞,免疫组化法测定增殖细胞核抗原(PCNA)、护骨素(OPG)和核因子kB受体活化因子配体(RANKL),荧光实时定量RT-PCR测定甲状旁腺激素相关蛋白(PTHrP)。结果模型组在造模后第19天已出现机械痛觉超敏、机械痛觉过敏和热痛觉过敏(P0.01)。第21天取材后胫骨影像评分升高(P0.01),BMD下降(P0.05);肉眼观察肿瘤生长明显(P0.01),镜下可见溶骨病变为主的混合性骨质破坏;破骨细胞数量和活性增加(P0.01),PTHrP、OPG水平与OPG/RANKL比值均下降(P0.05、P0.01),而RANKL无明显变化。结论乳腺癌骨转移大鼠模型具有疼痛和骨质破坏的表现,但未表现出PTHrP和RANKL升高,其损伤途径是通过抑制OPG破坏了OPG-RANKL-RANK系统的平衡,引起破骨细胞过度激活,造成骨吸收作用亢进。     

ANGPTL4在调控骨巨细胞瘤细胞增殖、血管生成和破骨分化作用的实验研究

目的探讨人血管生成素样蛋白4(ANGPTL4)基因沉默对骨巨细胞瘤单核基质细胞(GCTSC)增殖、血管生成以及破骨分化的作用。方法体外培养GCTSC细胞系,采用ANGPTL4 shRNA、Scrambled shRNA转染作为ANGPTL4 shRNA组和阴性对照组,另设置无处理的空白对照组。采用实时荧光定量PCR(QPCR)和Western blotting法检测ANGPTL4 mRNA和蛋白表达。MTT法和流式细胞术检测细胞增殖和凋亡。将GCTSC细胞分别与人脐静脉内皮细胞(HUVECs)、小鼠骨髓单核细胞(BMM)共培养,观察HUVECs成管能力和BMM破骨分化能力。结果与空白对照组和阴性对照组比较,ANGPTL4 shRNA组ANGPTL4 mRNA(0.174±0.045)和蛋白表达量(0.098±0.020)均明显降低;而ANGPTL4 shRNA组GCTSC细胞增殖活性降低,凋亡率升高(P<0.05)。与HUVECs共培养后,ANGPTL4 shRNA组闭合管数量[(57.35±17.24)%]减少(P<0.05);与BMM共培养后,ANGPTL4 shRNA组TRAP染色阳性的破骨细胞样多核巨细胞数量[(48.36±21.79)%]减少(P<0.05)。结论沉默骨巨细胞瘤GCTSC细胞ANGPTL4基因表达后,细胞增殖活性降低,凋亡率增加,并且对肿瘤血管生成和多核巨细胞破骨分化有一定的抑制作用。     

Oncogénétique et psycho-oncologie, une collaboration recommandée...

Résumé: La possibilité depuis 1994, de connaître la probabilité individuelle de développer certains cancers a permis de proposer de nouvelles modalités de prévention, de traitements et contribué au développement actuel de loncogénétique. Une meilleure connaissance des répercussions psychologiques tant pour les patients que pour les apparentés est désormais possible et limplication des psycho-oncologues dans ce cadre de la réalisation des tests prédictifs, recommandée. La mission de «messager» qui incombe au «cas-index» doit faire lobjet dune attention particulière. La complexité de linformation et la dimension paradoxale que peut avoir parfois la communication à propos des choix, rend difficile lévaluation de la qualité du consentement. La situation particulièrement délicate dune aide à la décision à légard de la chirurgie prophylactique, exige une collaboration étroite des généticiens et des psycho-oncologues.Les soins de support en oncologie     

The role of papillomaviruses in human cancers

This review comprehensively evaluates the influence of gene-gene, gene-environment and multiple interactions on the risk of colorectal cancer (CRC). Methods of studying these interactions and their limitations have been discussed herein. There is a need to develop biomarkers of exposure and of risk that are sensitive, specific, present in the pathway of the disease, and that have been clinically tested for routine use. The influence of inherited variation (polymorphism) in several genes has been discussed in this review; however, due to study limitations and confounders, it is difficult to conclude which ones are associated with the highest risk (either individually or in combination with environmental factors) to CRC. The majority of the sporadic cancer is believed to be due to modification of mutation risk by other genetic and/or environmental factors. Micronutrient deficiency may explain the association between low consumption of fruit/vegetables and CRC in human studies. Mitochondrial modulation by dietary factors influences the balance between cell renewal and death critical in colon mucosal homeostasis. Both genetic and epigenetic interactions are intricately dependent on each other, and collectively influence the process of colorectal tumorigenesis. The genetic and environmental interactions present a good prospect and a challenge for prevention strategies for CRC because they support the view that this highly prevalent cancer is preventable.     

Antifungal combination therapy in localized candidosis

A mouse model of localized candidosis in air-filled subcutaneous cysts imitating thrush has been developed. We have now tested various antifungal combinations in this animal model. Flucytosine (5-FC) + amphotericin B (Amph B) showed the highest efficacy, a clear additive or even synergistic effect was seen. The combination of 5-FC + imidazole or triazole derivative was less efficacious, an additive effect was rare. The combination of 5-FC + Amph B was also tested against Candida albicans strains showing various degrees of 5-FC-resistance. A significant reduction in 5-FC-resistant mutants was seen after the treatment with the combination.     

Les génériques en cancérologie: à molécule identique, médicaments identiques? Les biosimilaires, des super-génériques?

Résumé: Les biosimilaires vont bientôt voir leur apparition en Europe. Comment un laboratoire peut-il aborder le développement de son dossier dAMM? Quelles sont les bases légales et les recommandations officielles? Comment la similarité et/ou le caractère générique peuvent-ils être démontrés? Les règles sont-elles identiques à celles des produits chimiques conventionnels pour lesquels, notamment en cancérologie, il existe des médicaments génériques? Comment faire pour que la sécurité et lefficacité des médicaments biosimilaires soient assurées pour les patients?     

Cancer immunotherapy

Unprecedented progress has seen made in the last decade in the field of cancer immunotherapy. The recent approval of nivolumab （Opdivo）, the first anti-programmed cell death-1 （PD-1） antibody, for metastatic melanoma in Japan, marked a milestone in the rapidly advancing field of cancer immunotherapy. Nivolumab together with ipilimumab （Yervoy）, the anti-cytotoxic T-lymphocyte-associated antigen-4 （CTLA-4） antibody, are the first 2 drugs in the class of ＂immune checkpoint inhibitors＂ that have delivered impressive responses in patients with metastatic melanoma and renal cell cancer （RCC） as well as a variety of solid tumors.     

Effect of tumor therapeutic irradiation on the mechanical properties of teeth tissue

Tumor irradiation of the head-neck area is accompanied by the development of a so-called radiation caries in the treated patients. In spite of conservative therapeutic measures, the process results in tooth destruction. The present study investigated the effects of irradiation on the demineralization and remineralization of the dental tissue. For this purpose, retained third molars were prepared and assigned either to a test group, which was exposed to fractional irradiation up to 60 Gy, or to a non-irradiated control group. Irradiated and non-irradiated teeth were then demineralized using acidic hydroxyl-cellulose gel; afterwards the teeth were remineralized using either Bifluorid12 or elmex gelee. The nanoindentation technique was used to measure the mechanical properties, hardness and elasticity, of the teeth in each of the conditions. The values were compared to the non-irradiated control group. Irradiation decreased dramatically the mechanical parameters of enamel and dentine. In nonirradiated teeth, demineralization had nearly the same effects of irradiation on the mechanical properties. In irradiated teeth, the effects of demineralization were negligible in comparison to non-irradiated teeth. Remineralization with Bifluorid12 or elmex gelee led to a partial improvement of the mechanical properties of the teeth. The enamel was more positively affected by remineralization than the dentine.     

Consultation multidisciplinaire pour les patients atteints d’une pathologie tumorale (carcinome infiltrant ou lésion précancéreuse) liée à HPV : la consultation multidisciplinaire papillomavirus

Given the recent increase in the number of human papillomavirus (HPV)-induced cancers in other locations than gynaecological, the number of patients with two cancers at distinct sites, and because of the lack of exhaustive data, we decided to create a multidisciplinary network around an HPV consultation at the Georges-Pompidou European Hospital (HEGP). This network aims to set up the best tools for detecting HPV-associated “multisite” precancerous lesions in order to determine the possible impact of dedicated care for this at-risk population. This monthly consultation was created at the HEGP in June 2014. It is currently organized around five consultations: gynaecological, ENT, urological, digestive and immunological. Every patient who has been diagnosed with HPV-related cancer and whose care is provided at the HEGP is offered this particular follow-up: systematically, once the initial lesion has been treated, the patient is convened annually for a day during which it benefits from the consultations mentioned above. A consultation with a psychologist is systematically proposed. Local samples are taken at each site: a cytological examination, the analysis of known predictive and prognostic virological markers are carried out. This study fits more broadly in a theme of clinical and fundamental research around cancers related to HPV.     

Differentiation state and invasiveness of human breast cancer cell lines

Summary Eighteen breast cancer cell lines were examined for expression of markers of epithelial and fibroblastic differentiation: E-cadherin, desmoplakins, ZO-1, vimentin, keratin and ₁ and ₄ integrins. The cell lines were distributed along a spectrum of differentiation from epithelial to fibroblastic phenotypes. The most well-differentiated, epithelioid cell lines contained proteins characteristic of desmosomal, adherens and tight junctions, were adherent to one another on plastic and in the basement membrane matrix Matrigel and were keratin-positive and vimentin-negative. These cell lines were all weakly invasive in anin vitro chemoinvasion assay. The most poorly-differentiated, fibroblastic cell lines were E-cadherin-, desmoplakin- and ZO-1-negative and formed branching structures in Matrigel. They were vimentin-positive, contained only low levels of keratins and were highly invasive in thein vitro chemoinvasion assay. Of all of the markers analyzed, vimentin expression correlated best within vitro invasive ability and fibroblastic differentiation. In a cell line with unstable expression of vimentin, T47D_CO, the cells that were invasive were of the fibroblastic type. The differentiation markers described here may be useful for analysis of clinical specimens and could potentially provide a more precise measure of differentiation grade yielding more power for predicting prognosis.