深低温冷冻灭活技术在骨肉瘤保肢治疗中的应用及进展

骨肉瘤保肢治疗已成为骨肿瘤外科主要发展方向,骨肉瘤生物重建逐渐成为保肢治疗的首选方法.深低温冷冻灭活回植技术以其手术操作简单、成本低廉、保留了骨诱导生成能力等诸多优点而成为一种有广泛应用前景的生物型保肢技术.本文就深低温冷冻灭活技术在骨肉瘤保肢治疗中的应用及研究进展作一综述.     

碱性成纤维细胞生长因子复合天然型无机骨修复兔下颌骨缺损的形态计量研究

观察了碱性成纤维细胞生长因子(bFGF)复合天然型无机骨(N.N.B)与单纯天然型无机骨对骨生长的促进作用.在人工兔下颌骨缺损部位置放3组不同的骨组织,17实验单位置入N.N.B,17实验单位置入bFGF+N.N.B,9实验单位置入自体骨,分别于3周、6周、12周观察不同组别单位面积内的成骨面积、血管密度(MVD)、间质细胞增殖指数(PCNA增殖指数).结果发现bFGF+N.N.B组成骨时间早,成骨面积多,血管增生明显,PCNA增殖指数高,与N.N.B组有明显差异(p<0.001),与自体骨组基本接近(p>0.05).可见,bFGF可促进新骨在无机骨中的生长,可作为临床的一种较为理想的复合型人工植骨材料.     

羟基磷灰石、红骨髓、骨形态发生蛋白及纤维蛋白复合物异位诱导成骨的实验研究

目的 探讨骨形态发生蛋白、自体红骨髓、羟基磷灰石及纤维蛋白复合物的制备方法及其诱导成骨活性。方法 在大白鼠股后肌群分别植入(1)红骨髓(RBM)、(2)纤维蛋白-红骨髓复合物(FS-RBM)、(3)纤维蛋白-羟基磷灰石-红骨髓复合物(FS-HA-RBM)、(4)纤维蛋白-羟基磷灰石复合物(FS-HA)、(5)纤维蛋白-羟基磷灰石-骨形态发生蛋白复合物(FS-HA-BMP)、(6)纤维蛋白-羟基磷灰石-骨形态发生蛋白-红骨髓复合物(FS-HA-BMP-RBM),按1,2,4周三个时间点进行大体标本、组织学观察及ALP检测,比较各组间诱导成骨活性。结果 FS-HA-BMP-RBM组复合物有较多的新生软骨和骨形成。各组ALP活性比较显示,HA-FS-BMP-RBM组最强,且于第2周最高。结论 FS-HA-BMP-RBM复合物具有良好的诱导成骨能力。     

藻酸钙凝胶复合骨形态发生蛋白、红骨髓异位诱导成骨的实验研究

目的探讨骨形态发生蛋白、自体红骨髓及藻酸钙凝胶复合物的制备方法及其诱导成骨活性。方法在大白鼠股后肌群分别注入CaAG-BMP-RBM、CaAG-BMP、CaAG,按1、2、4周三个时间点进行大体标本、组织病理学观察及AKP检测,比较各组间诱导成骨活性。结果CaAG-BMP-RBM组凝胶复合物有较多的新生软骨和骨形成,AKP活性各组比较显示CaAG-BMP-RBM组最强,且于第2周最高。结论CaAG-BMP-RBM复合物具有良好的诱导成骨能力。     

耐药性小鼠乳腺癌细胞的超微结构及形态计量研究

目的：探讨小鼠乳腺癌经化疗后存活的耐药性癌细胞的超微 形态计量特征以及这类癌细胞的种类。方法：对TAⅡ小鼠乳腺癌联合化疗，进行光镜、电镜观察，用图像分析系统进行细胞及细胞器的形态计量分析。结果：坏死癌组织的碎屑中有残留的癌细胞存在。从超微结构上分析，这些癌细胞仍存活。残留癌细胞体积小，具有低分化细胞的形态特征。结论：化疗后残留的小鼠乳腺癌细胞可能是具有特殊的超微结构及形态计量特征的异质性癌细胞群，对多种化疗药物具有强大的抵抗力，它们可能是肿瘤局部复发的潜在因素。     

脑星形细胞瘤形态计量病理分级诊断的研究

目的:探讨星形细胞瘤形态定量病理分级诊断方法。方法:应用图像分析定量技术,对136例人星形细胞瘤和3例正常对照的细胞核的面积等8项形态参数、肿瘤4项分布密度参数和细胞核分维值进行量化。结果:经图像分析定量,说明细胞核的面积、周长、直径、短轴和长轴、细胞核数、巨细胞面数密度、血管交点面数密度和细胞核面数密度等9项指标能够较好地反映星形细胞瘤4级间的差异,具有较好的鉴别诊断价值。结论:应用形态计量方法进行星形细胞瘤的病理分级诊断更加客观可靠。     

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。     

异体DBM骨粒复合rhBMP-2及骨水泥材料修复骨缺损的实验研究

目的探讨异体脱钙骨基质（DBM）骨粒、骨水泥（BC）及重组人骨形态发生蛋白-2（rhBMP-2）复合材料修复实验性骨缺损的能力。方法复合材料植入兔实验性尺骨骨缺损部位（A组），B组植入纯骨水泥棒材作为对照。不同时间行组织学、印度墨汁动脉血管灌注及材料自然断面扫描电镜观察。结果材料植入第2周表面纤维组织样完整包膜形成，材料外周及两端血管形成密集，并向材料中长入；第4、8、12周新生血管明显增多，且深入至材料中央，新生间充质组织继续大量长入；包绕DBM骨粒的间充质组织部分4周转化为软骨。第8、12周软骨趋于成熟，可见含有造血骨髓的新生骨组织形成，其间可见较多的哈佛氏系统；同时，可见明显DBM骨粒被新生组织爬行吸收现象。第4周原自然裂隙部分已被结构紊乱的胶原纤维束充填。第8至12周，可见大量排列紊乱的矿化骨结构沿多方向长入，新生骨组织附于脱钙骨基质骨粒及骨水泥表面，原自然裂隙几近消失。植入B组材料者除外被纤维膜外余无变化。结论脱钙骨基质骨粒、骨形态发生蛋白、骨水泥复合材料具有良好的骨诱导及骨引导生成作用，是良好的骨缺损修复材料。     

骨肉瘤细胞(MG-63)条件培养基中骨形态蛋白(BMP)的实验研究--BMP的分离、纯化、鉴定

目的 探索从骨肉瘤细胞条件培养基中提取骨形态蛋白(BMP)的方法，并测定其生物学活性。方法 收集骨肉瘤细胞(MG-63)条件培养基，通过浓缩、透析，SephcrylS—100凝胶层析纯化，BMP单克隆抗体鉴定所需洗脱峰，SDS—PAGE测定分子量，小鼠肌袋实验检测其骨诱导活性。结果 BMP单抗鉴定所提蛋白为BMP，SDS—PAGE显示分子量在21kD，能够在小鼠肌肉内产生异位骨化。结论 骨肉瘤细胞条件培养基中含有BMP，分离后具有良好的生物学活性，而骨肉瘤细胞可以在体外长期培养生长，为BMP的大量提取、临床应用提供一个有益的方法。     

The effects of permanent I-125 interstitial implantation on cortical bone

From 1973 to 1980, 1,427 cancer patients were treated by the Brachytherapy Service, Radiation Oncology Department of Memorial Sloan-Kettering Cancer Center (MSKCC) with I-125 permanent interstitial implantation. Concern has been expressed with regard to the possible toxic effects of the low average photon energy (28 kev) of I-125 on bone, secondary to its increased absorption in these tissues because of the photoelectric effect. In an attempt to address this concern with respect to cortical bone, we reviewed pertinent data on those patients whose site of implant was at close proximity to osseous tissue in the pelvic, chest wall and head and neck regions. The review included 74 sites in 58 patients having no prior history or clinical evidence of any pathologic or metabolic bony disorder. Long term (greater than 1 year) follow-up was available in over 50% of the cases, with 10 patients followed more than 4 years. Local control rate for the 74 implanted sites was 93%. The implanted activity ranged from 1.6 to 48.8 mCi and the implanted volume from 0.5 to 152 cm³. At this follow-up range, there appears to be no excessive osseous toxicity either clinically or radiologically with I-125 permanent implantation.     

神经导航辅助微骨窗入路在脑深部肿瘤手术中的应用

目的探讨神经导航辅助下微骨窗入路显微手术切除脑内深部肿瘤的方法和疗效。方法收集140例脑深部肿瘤患者,分为对照组(54例)和试验组(86例)。对照组患者采用传统手术治疗,试验组患者采用神经导航辅助的显微手术切除肿瘤。结果试验组患者中,全切除78例(90.7%),近全切除5例(5.8%),大部分切除2例(2.3%),活检1例(1.2%)。对照组患者中,全切除40例(74.1%),近全切除9例(16.7%),大部分切除5例(9.3%)。两组患者的骨窗大小、手术时间和全切率比较,差异有统计学意义(P<0.05)。结论神经导航辅助微骨窗入路可显著提高脑深部肿瘤患者的治疗效果,提高全切率,缩短手术时间,并减少手术创伤。     

尾部悬吊大鼠骨质疏松模型骨的微观结构及力学性能变化的研究

目的 观察模拟失重尾部悬吊大鼠模型松质骨骨密度、骨小梁结构及力学性能变化.方法 3个月龄雄性SD大鼠24只,随机分为3组,尾部悬吊14d、28d和空白对照组.到期处死动物,测定L4椎体、股骨髁部骨密度,并进行显微CT及生物力学测试.结果 尾部悬吊组较对照组椎体、股骨髁部骨密度均显著下降,且尾部悬吊大鼠骨质疏松随时间加重,松质骨的骨体积分数（BV/TV）、骨小梁厚度（Tb. Th）、骨小梁数目（Tb. N）较对照组显著降低（P＜0.05）,股骨髁部表面积体积比（BS/BV）、椎体骨小梁间隙（Tb. Sp）则较对照显著增高.生物力学测试表明,尾部悬吊14d、28d组腰椎的最大压缩应力分别较空白对照组下降11.8%、26.3%,股骨抗弯曲载荷较空白对照组下降13.9%,24.6%,力学强度显著下降.结论 尾部悬吊大鼠从14d即表现出明显的松质骨骨密度下降,骨小梁三维微结构破坏,椎体力学强度也显著下降,并随时间发展不断加重,理论上骨折风险性增加.     

555例乳腺病变冷冻切片病理诊断分析

目的:探讨乳腺病变冷冻切片诊断的准确性,进一步提高临床与病理对冷冻切片的认识,从而降低误诊率。方法:回顾分析我院2005年-2009年555例乳腺病变冷冻切片与石蜡切片对比诊断结果。结果:555例冷冻切片报告中,确诊546例,确诊率为98.38%,7例延迟诊断,2例假阴性误诊,假阴性率为0.59%。结论:术中冷冻切片诊断技术是乳腺病变的重要诊断手段之一,全面深入了解其病变特点及局限性,才能减少误诊。     

BRMS1 expression alters the ultrastructural,biomechanical and biochemical properties of MDA-MB-435 human breast carcinoma cells: An AFM and Raman microspectroscopy study

Restoring BReast cancer Metastasis Suppressor 1 (BRMS1) expression suppresses metastasis in MDA-MB-435 human breast carcinoma cells at ectopic sites without affecting tumor formation at orthotopic site in the body. BRMS1 expression induces many phenotypic alterations in 435 cells such as cell adhesion, cytoskeleton rearrangement, and the down regulation of epidermal growth factor receptor (EGFR) expression. In order to better understand the role of cellular biomechanics in breast cancer metastasis, the qualitative and quantitative detection of cellular biomechanics and biochemical composition is urgently needed. In the present work, using atomic force microscopy (AFM) and fluorescent microscopy we revealed that BRMS1 expression in 435 cells induced reorganization of F-actin and caused alteration in cytoarchitectures (cell topography and ultrastructure). Results from AFM observed increase in biomechanical properties which include cell adhesion, cellular spring constant, and Young’s modulus in 435/BRMS1 cells. Raman microspectroscopy showed weaker vibrational spectroscopic bands in 435/BRMS1 cells, implying decrease in concentration of cellular biochemical components in these cells. This was despite the similar spectral patterns observed between 435 and 435/BRMS1 cells. This work demonstrated the feasibility of applying AFM and Raman techniques for in situ measurements of the cellular biomechanics and biochemical components of breast carcinoma cells. It provides vital clues in understanding of the role of cellular biomechanics in cancer metastasis, and further the development of new techniques for early diagnosis of breast cancer.     

Intraoperative frozen section analysis of sentinel lymph nodes in breast cancer patients

BACKGROUND:

Accurate intraoperative pathologic examination of sentinel lymph nodes (SLNs) has been an important tool that can reduce the need for reoperations in patients with SLN‐positive breast cancer. The objective of the current study was to determine the accuracy of intraoperative frozen section (IFS) of SLNs during breast cancer surgery.

METHODS:

The authors retrospectively reviewed the records of 326 patients with breast cancer who underwent IF analysis of SLNs at a single institution. Then, they conducted a meta‐analysis that included 47 published studies of IFS of SLNs in patients with breast cancer.

RESULTS:

Hematoxylin and eosin (H&E) staining revealed metastasis in SLNs in 99 patients (30.4%), including 61 patients with macrometastasis (MAM) (>2 mm) (the MAM group) and 38 patients with micrometastasis (Mi) or isolated tumor cell (ITC) deposits (the Mi/ITC group). The overall sensitivity of the institutional series was 60.6% (60 of 99 patients), and overall specificity was 100% (227 of 227 true negatives). The sensitivity of IFS was significantly lower in the Mi/ITC group (28.9%) than in the MAM group (80.3%; P < .0001). According to the meta‐analysis of published studies and data from the author's institution (47 studies, for a total of 13,062 patients who underwent SLN dissection with IFS of SLNs), the mean sensitivity was 73%, and the mean specificity was 100%. The mean sensitivity was 94% for the MAM group and 40% for the Mi/ITC group.

CONCLUSIONS:

IFS of SLNs was more reliable for detecting MAM than for detecting Mi/ITC deposits. It lacked sufficient accuracy to rule out Mi/ITC deposits. Cancer 2011. © 2010 American Cancer Society.     

粒细胞集落刺激因子减轻混合骨髓移植后移植物抗宿主病的实验研究

Objective: How to reduce the incidence and severity of acute graft-versus-host disease (aGVHD) is a crucial step to improve the overall survival of allogeneic bone marrow transplantation (allo-BMT). The low incidence of severe aGVHD observed in allogeneic peripheral blood stem cell transplantation (allo-PBSCT), which may be related to modulating immune function of T lymphocytes by granulocyte colony-stimulating factor (G-CSF) primed donors. The study aimed to explore whether aGVHD could be alleviated by syngeneic bone marrow mixed with G-CSF-mobilized H-2 haploidentical marrow grafting. Methods: Female BALB/c mice and neonatal BALB/c mice were recipients and male (BALB/c × C57BL/6)F1(BCF1) mice were donor mice respectively. Donor mice were injected subcutaneously with G-CSF daily at 0.01 μg/g body weight or saline for 6 days, and splenocytes were harvested on day 6. Spleen index (SI) represented GVHD in neonatal mice after the intraperitoneal injection of mixed spleen cells. Lethally irradiated (60Co, 8.5 Gy) adult mice were transplanted with a mixture of syngeneic plus G-CSF-mobilized (control diluents) H-2 haploidentical marrow cells. Survival time and survival rate of the recipients were observed after mixed marrow transplantation (MBMT). GVHD was assessed by observing signs of weight loss, ruffled fur, diarrhea and histological change of skin, liver and small intestines. Enzyme-linked immunosorbent assay (ELISA) method was used to detect cytokines (IL-2, IL-4 and INF-γ). Fluorescence-activated cell sorting (FACS) analysis was used to detect T cells phenotype. Results: (1) The neonatal mice subject to injection of 2:1 and 1:1 mixed spleen cells and H-2 haploidentical spleen cells all suffered from aGVHD. The severity of aGVHD in recipient mice receiving G-CSF-mobilized splenocytes was dramatically reduced. (2) The aGVHD signs and histological change were observed in most mice of 2:1 and 1:1 MBMT groups. However, the survival time of G-CSF-mobilized MBMT was longer than in control groups and these mice had signs of moderate GVHD. (3) L3T4 cells and relative ratio in both subsets was significantly reduced in G-CSF-treated donor mice. The total number of Thy1.2 and lyt2 cells was increased after G-CSF pretreatment of donors, but no statistical difference. (4) The supernatants from a primary MLR were collected at 48 h for cytokine measurement. The results showed an increased production of IL-4 and a decreased production of IL-2 and INF-γ after stimulating with concanavalin A for 48 h. Conclusion: The GVHD could be reduced using syngeneic bone marrow mixed with H-2 haploidentical marrow cells. The severity of aGVHD in recipient mice receiving G-CSF-mobilized splenocytes or marrow cells could be further moderated, which is associated with increased IL-4 production and decreased IL-2 and INF-γ production.     

逆转录病毒载体介导的双耐药基因转染小鼠骨髓细胞的实验研究

目的:将耐药基因以逆转录病毒为载体转染骨髓细胞以解决大剂量化疗所造成的骨髓抑制。方法:以逆转录病毒为载体,将双突变的二氢叶酸还原酶基因(DH-FR)和胞苷脱氨基酶基因(CD)转染小鼠骨髓细胞,观察该细胞耐MTX及Ara-C的CFU-GM生成情况;RT-PCR检测转基因的表达情况。结果:转基因的小鼠骨髓细胞有耐药克隆形成(14%);基因转染后小鼠骨髓细胞对MTX和Ara-C的耐受明显增加,P<0·005;RT-PCR显示转基因细胞有转染的基因条带。结论:双耐药基因通过逆转录病毒转染小鼠骨髓细胞并且获得表达,提高了骨髓细胞对MTX和Aar-C的耐药性。     

锶在兔体内 部分组织器官分布 与富集规律的 模拟研究

目的：研究锶在兔体内不同器官中的分布和富集规律。方法：将日本大耳白兔分6组,1个空白对照组和5个实验组,每组4个平行样。空白组（第1组）白兔喂去离子水,5个实验组（第2~6组）白兔饮用添加了六水氯化锶（浓度0.002 g/mL）的去离子水,隔天灌喂1次,第2~6组饮用水体积依次递增分别为1、2、3、4、5 mL。30 d后取兔血液、肝脏和后肢胫骨,样品经烘干、粉碎、硝解后送检。利用电感耦合等离子体发射光谱仪（ICP-AES）检测样品的锶含量。结果：白兔血液锶含量随着摄入锶总量的增加而增加,呈正相关关系,线性拟合R2值为0.82。肝脏中的锶含量与摄入锶总量之间无明显相关关系。后肢胫骨中锶含量随着摄入锶总量的增加而增加,呈正相关关系,线性拟合R²值为0.98。血液和胫骨中的锶含量呈正相关关系,线性拟合R²值为0.76。结论：锶进入生物体后经过血液循环最后富集在骨骼中。