建立科研使用FFPE肿瘤组织库的几点建议

福尔马林固定石蜡包埋( formalin fixed paraffn embed-ded, FFPE)是病理科普遍采用的组织处理和保存方法。组织标本经取材、固定、脱水、透明、浸蜡以及包埋等程序处理之后,可长时间保存[1]。《临床技术操作规范—病理学分册》(以下简称《操作规范》)要求, FFPE 组织需保存15年[2]。该操作由于流程简单,易于掌握,可以实现高度自动化处理,且代价低廉,适合大多数医院病理科开展。目前, FFPE组织已广泛应用于病理科的常规病理工作中。     

组织固定处理及包埋常见问题与对策

对于石蜡切片而言,组织的固定是组织随后脱水、透明、浸蜡等处理的第一步,也是最重要的一步.包埋操作是否规范对后续组织切片同样具有重要的决定意义.本文就组织固定、处理及包埋常见的问题与对策,进行分析与讨论,旨在与技术人员一起学习交流,以提高组织固定处理及包埋的质量,为组织切片和染色以及病理诊断提供有力的保证.     

石蜡包埋对心、肺和脾组织的收缩影响

在石蜡包埋过程中,酒精、二甲苯和石蜡等都对组织产生明显的收缩影响,严重地影响了显微镜下对细胞和组织的观察及测量结果.1998年我们曾对石蜡切片和恒冷切片进行比较,认为石蜡切片距新鲜组织结构的距离相差甚远[1].后来我们又系统地研究了石蜡包埋过程对肝、肾组织的收缩影响,并对其收缩系数进行了推算[2],为显微镜下研究细胞和组织的大小提供了数据.但不同的组织由于其细胞密度和细胞间质的多少不同,表现的收缩影响程度各不相同,因此,有必要对全身其它组织作进一步的调查,为显微镜下准确地判断和测量各种组织的大小提供参考依据.     

学校组织公平与教师组织公民行为的关系分析

目的:本研究旨在对学校组织公平与教师组织公民行为的关系进行分析.方法:采用自编的量表对随机抽取重庆市21所学校856教师进行调查.考察了学校组织公平感与教师组织公民行为的关系.结果:学校组织公平感与教师组织公民行为有显著的正相关.经过逐步回归分析进一步表明:程序公平与教师组织公民行为回归十分显著.结论:学校组织公平感越高,教师组织公民行为水平越高.程序公平是教师组织公民行为的正向预测因子.     

脂肪组织工程研究进展

组织工程为临床疑难问题的解决提供了新的思路，而工程化脂肪组织具有组织重建与填充作用，广泛应用于外科领域。本就脂肪组织工程涉及的前脂肪细胞生长分化、分子调控、聚合物支架及细胞外基质、微环境等方面进行综述。     

脂肪组织工程研究进展

组织工程为临床疑难问题的解决提供了新的思路 ,而工程化脂肪组织具有组织重建与填充作用 ,广泛应用于外科领域。本文就脂肪组织工程涉及的前脂肪细胞生长分化、分子调控、聚合物支架及细胞外基质、微环境等方面进行综述     

3D-SC人工皮肤材料组织相容性的实验研究

目的观察实验材料3D-SC人工皮肤的组织相容性。方法将阴性对照材料DERMMATRIXTM和实验材料3D-SC组织工程材料分别植入SD大鼠背部皮下组织中,分别于1、4、12周在光镜下观察组织学反应。结果植入1周,实验组和对照组的纤维包膜厚度相比差异有显著统计学意义（P〈0.01）;阴性对照材料和3D-SC组织工程材料炎细胞浸润以淋巴细胞和中性粒细胞为主,个别对照材料发现少量异物巨细胞,实验材料未见异物巨细胞;两种材料的淋巴细胞、中性粒细胞数量相当（P〉0.05）。植入4周,对照组的纤维厚度增长显著（P〈0.01）,与对照组包膜厚度相当（P〉0.05）;实验组与对照组中淋巴细胞、中性粒细胞及异物巨细胞数量相当（P〉0.05）;与植入1周相比,实验组的淋巴细胞数量减少明显（P〈0.01）,而对照组材料变化不明显（P〉0.05）;对照组的中性粒细胞数量减少明显（P〈0.05）,实验组的中性粒细胞数量变化不大（P〉0.05）;两种材料均出现少量异物巨细胞,差别不大（P〉0.05）。植入12周,对照组和实验组的包膜厚度相当（P〉0.05）,各种炎细胞浸润显著减少,表现相当（P〉0.05）;两种材料未见异物巨细胞;与植入1周相比,实验组的中性粒细胞数量减少显著（P〈0.05）。结论3D-SC组织工程材料具有较好的组织相容性。     

组织芯片技术在检测正常组织和肿瘤组织抗原表达中的应用

组织芯片 (ｔｉｓｓｕｅｃｈｉｐ) [1] 技术具有高产出 (ｈｉｇｈ ｔｈｒｏｕｇｈｐｕｔ) ,实验误差小和省时、省力、节约经费的优点 ,并能应用于科研、教学、生物试剂测试、质量监控及标准化等领域[2 ,3 ] 。我们采用了组织芯片这项技术并选用了 5种常用抗体 (ＡＥ1/ 3、波形蛋白、ＣＤ45、结蛋白和Ｓ 10 0蛋白 )对 5 4例正常组织及 6 0例肿瘤标本进行了抗原表达的检测 ,以了解该技术在这方面的使用价值。一、材料和方法5 4例正常组织和 6 0例肿瘤标本选自内蒙古中蒙医院病理科和内蒙古乌兰察布盟医院病理科外检标本 (1988～ 2 0 0 0年 )。…     

带植入物病理组织标本切磨片制作技术探讨

硬组织病理切磨技术主要是针对骨组织、带植入物的骨组织、埋置有坚硬植入物的其他组织标本,或在动物试验阶段进行了亲骨荧光素标记的不能进行脱钙的骨组织,通过脱水、浸润、包埋处理,由硬组织切磨系统完成的组织病理切片制作的技术。有文献报道骨组织及埋置坚硬植入物的组织进行病理组织切片制作,通常利用EXAKT硬组织切磨系统[1-3]。硬组织病理切磨技术最显著的特点是不破坏组织中的植入物,且保持了组织与植入物之间原有的组织结构形态,这对研究组织内植入物的长入情况具有重要意义。本文结合本实验室日常硬组织制片工作经验,分析硬组织病理切片制作的每个处理环节,以期能为其他技术人员提供参考。     

肌腱构建与组织工程技术研究新进展

本综述组织工程化肌腱领域中取得的多项成果，如肌腱组织替代物，种子细胞，组织工程用生物材料及转基因技术应用研究等。论述建立生长，增殖可调的肌腱种子细胞系可行性。研制有利于细胞生长与粘附的生物材料及转基因等亲技术在组织工程学中的应用必将推动组织工程化 肌腱的研究。     

不同来源骨髓间充质干细胞的体内成软骨

背景：骨髓间充质干细胞经体外诱导后可修复软骨缺损,但目前采用的种子细胞多来源于自体或同种异体。 目的：观察同种异体及异种来源的骨髓间充质干细胞诱导成软骨后修复喉软骨缺损的效果。 方法：分别取人胚胎骨髓间充质干细胞和刚出生兔骨髓间充质干细胞的第3代细胞种植于聚乳酸-羟基乙酸共聚物生物支架上,并加入转化生长因子β1和软骨形态发生蛋白诱导成软骨细胞。将两种细胞体系植入新西兰白兔体内,并于植入后4,8周取材行大体、组织学观察。 结果与结论：植入后4,8周人胚胎骨髓间充质干细胞和兔骨髓间充质干细胞均有新生组织填充,经组织学观察大部分为软骨细胞,分泌软骨细胞基质糖胺聚糖和Ⅱ型胶原,且两种细胞支架复合物所生成的软骨细胞数大致相同,并无明显的免疫排斥反应。提示异种来源的骨髓间充质干细胞复合聚乳酸-羟基乙酸共聚物在转化生长因子β1和软骨形态发生蛋白联合诱导下所得的组织工程化软骨,与同种来源的骨髓间充质干细胞所获得的组织工程化软骨修复喉软骨缺损具有可比性。     

以转染CDMP1基因的BMSCs修复软骨缺损

 目的 探讨CDMP1基因转染的骨髓间充质干细胞（BMSCs）负载于聚乳酸-羟基乙酸（PLGA）支架上修复喉软骨缺损的能力,并对其修复效果做出初步评估。方法 用反转录聚合酶链式反应（RT-PCR）和免疫印迹法(Western blot)检测hCDMP1mRNA和蛋白的表达;用免疫组织化学方法检测Ⅱ型胶原蛋白(ColⅡ)以及糖胺聚糖(GAG)的表达;将转染前后的细胞支架培养体系移植入兔甲状软骨全层缺损处,从大体、组织学方面观察其对软骨缺损的修复作用。结果 腺病毒感染方法可以将外源hCDMP1基因成功转入BMSCs,并使其获得稳定表达;和对照组比较,转染hCDMP1基因的BMSCs分泌ColⅡ、GAG等软骨特异性基质的能力增强,有促进软骨分化趋势;转染细胞支架复合物可更加有效地修复喉软骨缺损。结论 转染hCDMP1基因的BMSCs/PLGA三维生物支架复合物移植动物体内可修复喉软骨缺损。     

脱钙骨基质与骨髓间充质干细胞共培养关节腔内的成软骨活性

背景：将骨髓间充质干细胞附着到支架材料上再植入关节软骨缺损处,细胞不但不消失,而且可形成新的软骨。 目的：观察同种异体脱钙骨基质与骨髓间充质干细胞共培养在关节内的成软骨活性。 方法：在54只青紫蓝兔单侧膝关节制作关节软骨全层缺损模型,随机分组：实验组在缺损处植入自体骨髓间充质干细胞与同种异体脱钙骨基质复合物,对照组缺损处仅植入同种异体脱钙骨基质,空白对照组未植入任何物质。 结果与结论：植入后12周,实验组缺损处修复组织呈软骨样,表面光滑平坦,与周围软骨整合的软骨细胞更为成熟,修复组织与软骨下骨结合牢固;修复组织的细胞为透明软骨样细胞,柱状排列,Ⅱ型胶原染色阳性,与周围软骨及软骨下骨整合良好,且实验组组织学评分优于对照组和空白对照组 (P < 0.01)。对照组缺损处修复组织呈纤维样,与周围软骨未结合,空白对照组缺损区无修复组织,两组均无Ⅱ型胶原染色阳性表达。表明同种异体脱钙骨基质与骨髓间充质干细胞共培养后植入膝关节可形成软骨样组织,有效修复关节软骨缺损。     

自体脂肪间充质干细胞复合人脐带Wharton胶支架修复兔膝关节软骨缺损****◇

背景：脐带Wharton胶富含透明质酸,糖胺多糖及胶原等,成分与天然软骨细胞外基质类似,因此由人脐带提取的Wharton胶很可能是一种较为理想的软骨组织工程支架材料。 目的：评价自体脂肪间充质干细胞复合人脐带Wharton胶支架修复兔膝关节软骨缺损的效果。 方法：将终浓度为1010 L -1、成软骨方向诱导后的兔自体脂肪间充质干细胞与人脐带Wharton胶支架复合,继续培养1周构建组织工程软骨,对兔膝关节全层软骨缺损进行修复(实验组),并与单纯支架修复的对照组及空白组进行比较。术后3个月对修复组织行大体观察、组织学检测、糖胺多糖、总胶原定量检测及生物力学测定。 结果与结论：实验组的缺损多为透明软骨修复,对照组以纤维组织修复为主,空白组无明显组织修复。提示脂肪间充质干细胞作为软骨组织工程种子细胞具有可行性;实验构建的组织工程软骨能有效的修复关节软骨缺损,人脐带Wharton胶可作为软骨组织工程良好的支架材料。     

羟基丁酸-羟基辛酸共聚体/胶原骨软骨一体化支架修复膝关节软骨损伤

BACKGROUND: Due to the complex physiological characteristics of the osteochondral tissue, the clinical repair of knee cartilage injury often has dissatisfied outcomes. Tissue engineering methods and tools provide a new idea for osteochondral repair. OBJECTIVE: To observe the effect of poly(hydroxybutyrate-co-hydroxyoctanoate/collagen) osteochondral tissue-engineered scaffold on the repair of articular cartilage injury in a rabbit. METHODS: The poly(hydroxybutyrate-co-hydroxyoctanoate/collagen) osteochondral tissue-engineered scaffold was prepared by solvent casting/particle leaching method. Then, seed cells were isolated and cultured on the scaffold. Twenty-four healthy New Zealand white rabbits, 4 weeks of age, were used for the study. Under balanced anesthesia, an articular cartilage defect (4.5 mm in diameter, 5 mm in depth) was created on the rabbit’s femoral condyle using a bone drill. After modeling, rabbits were randomized into three groups and given direct suture in blank group, pure scaffold implantation in control group and implantation of the scaffold-cell complex in experimental group. Femoral condyle of each rabbit was taken out for gross and histological observations at 8, 20 weeks after surgery. RESULTS AND CONCLUSION: At 8 weeks after surgery, transparent film-covered defects and small/irregular cells were found in the experimental group; the defects were filled with fibrous tissues in the control group; while there was no repair in the blank group. Until the 20th week, the defects were covered with hyaline cartilage-like tissues, accompanied by regular cell arrangement in the experimental group; in the control group, the defects were covered with white membranous tissues, and many chondrocytes were found at the basement and edge; in the blank group, some newborn tissues were visible at the defect region. These findings suggest that the poly (hydroxybutyrate-co- hydroxyoctanoate/collagen) osteochondral tissue-engineered scaffold carrying seed cells contributes to articular cartilage repair.     

多孔丝素蛋白/羟基磷灰石复合脂肪间充质干细胞修复兔关节软骨及软骨下骨缺损

背景：丝素蛋白/羟基磷灰石是细胞立体培养的良好支架,是临床常用的骨缺损修复材料,具有良好的生物相容性。脂肪干细胞具有向骨及软骨细胞分化的潜能,适合骨软骨缺损修复。 目的：观察转化生长因子β1和胰岛素样生长因子1联合成软骨诱导脂肪干细胞与丝素蛋白/羟基磷灰石复合后修复兔关节软骨及软骨下骨缺损的效果。 方法：取新西兰大白兔56只,2只用于传代培养脂肪间充质干细胞,以3×109 L-1浓度接种到丝素蛋白/羟基磷灰石。其余54只新西兰大白兔,在股骨髁间制备软骨缺损模型,随机分为细胞复合材料组、单纯材料组和空白对照组,细胞复合材料组植入复合脂肪间充质干细胞的丝素蛋白/羟基磷灰石;单纯材料组植入丝素蛋白/羟基磷灰石;空白对照组不作任何植入。从大体、影像学、组织学观察比较缺损的修复情况。 结果与结论：12周时大体观察、CT、磁共振和组织学检查细胞材料复合组软骨及软骨下骨缺损区完全被软骨组织修复,修复组织与周围软骨色泽相近,支架材料基本吸收,未见明显退变和白细胞浸润,所有标本均未见丝素蛋白残留。单纯材料组缺损区缩小、部分修复,且呈纤维软骨样修复。空白对照组缺损无明显修复。提示复合脂肪间充质干细胞的丝素蛋白/羟基磷灰石修复兔关节软骨及软骨下骨缺损能力优于单纯丝素蛋白/羟基磷灰石材料。丝素蛋白/羟基磷灰石复合脂肪间充质干细胞可形成透明软骨修复动物膝关节全层软骨缺损,重建关节的解剖结构和功能,可作为新型骨软骨组织工程支架。     

凝胶包埋骨髓基质干细胞复合脱钙骨基质修复关节软骨缺损简

目的探索Ⅱ型胶原凝胶包埋的自体骨髓基质干细胞（BMSCs）接于同种异体脱钙骨基质（DBM）材料修复兔关节软骨缺损的效果。方法15只健康成年新西兰大白兔,雌雄不限,体质量约3．0kg,兔龄6。9个月;以Urist方法制作同种异体DBM材料。以Ⅱ型胶原蛋白配制水凝胶．以水凝胶包埋兔BMSCs并接种于同种异体DBM材料,构建组织工程复合物。在新西兰大白兔股骨髁关节面制造软骨缺损。分组进行修复。将健康成年新西兰大白兔27只（雌雄不限,体质量约2．5kg．兔龄3～4个月）共54侧膝关节随机分为Ⅱ型胶原／DBM／BMSCs修复组（实验组）、Ⅱ型胶原／DBM修复组（实验对照组）及空白对照组。于术后4周、8周及12周各处死9只动物,取材对修复组织进行大体及组织学观察,根据Wakitani法对修复组织进行评分．数据输入SPSS11．5软件进行统计学分析,比较各组的评分差异是否具有统计学意义。结果实验组Ⅱ型胶原／DBM／BMSCs植入后形成透明软骨样修复．表面光滑平坦,与周围软骨及软骨下骨结合良好;实验对照组Ⅱ型胶原／DBM植入后有部分软骨样修复：而空白对照组仅有少量纤维性修复。根据组织学评分标准,实验组组织学评分为（20．25±1．64）分,高于实验对照组[（7．46±1．29）分]及空白对照组[（6．00±2．09）分]。结论Ⅱ型胶原自体BMSCs复合同种异体DBM支架材料修复全层关节软骨缺损的效果良好,是一种修复软骨缺损的行之有效的方法。     

The use of poly(lactic-co-glycolic acid) microspheres as injectable cell carriers for cartilage regeneration in rabbit knees

The use of injectable scaffolding materials for in vivo tissue regeneration has raised great interest because it allows cell implantation through minimally invasive surgical procedures. Previously, we showed that poly(lactic-co-glycolic acid) (PLGA) microspheres can be used as an injectable scaffold to engineer cartilage in the subcutaneous space of athymic mice. The purpose of this study was to determine whether PLGA microspheres can be used as an injectable scaffold to regenerate hyaline cartilage in the osteochondral defects of rabbit knees. A full-thickness wound to the patellar groove of the articular cartilage was made in the knees of rabbits. Rabbit chondrocytes were mixed with PLGA microspheres and injected immediately into these osteochondral wounds. Both chondrocyte transplantations without PLGA microspheres and culture medium injections without chondrocytes served as controls. Sixteen weeks after implantation, chondrocytes implanted using the PLGA microspheres formed white cartilaginous tissues. Histological scores indicating the extent of the cartilaginous tissue repair and the absence of degenerative changes were significantly higher in the experimental group than in the control groups (P < 0.05). Histological analysis by a hematoxylin and eosin stain of the group transplanted with microspheres showed thicker and better-formed cartilage compared to the control groups. Alcian blue staining and Masson's trichrome staining indicated a higher content of the major extracellular matrices of cartilage, sulfated glycosaminoglycans and collagen in the group transplanted with microspheres than in the control groups. In addition, immunohistochemical analysis showed a higher content of collagen type II, the major collagen type in cartilage, in the microsphere transplanted group compared to the control groups. In the group transplanted without microspheres, the wounds were repaired with fibro-cartilaginous tissues. This study demonstrates the feasibility of using PLGA microspheres as an injectable scaffold for cartilage regeneration in a rabbit model of osteochondral wound repair.     

The use of poly(lactic-co-glycolic acid) microspheres as injectable cell carriers for cartilage regeneration in rabbit knees

The use of injectable scaffolding materials for in vivo tissue regeneration has raised great interest because it allows cell implantation through minimally invasive surgical procedures. Previously, we showed that poly(lactic-co-glycolic acid) (PLGA) microspheres can be used as an injectable scaffold to engineer cartilage in the subcutaneous space of athymic mice. The purpose of this study was to determine whether PLGA microspheres can be used as an injectable scaffold to regenerate hyaline cartilage in the osteochondral defects of rabbit knees. A full-thickness wound to the patellar groove of the articular cartilage was made in the knees of rabbits. Rabbit chondrocytes were mixed with PLGA microspheres and injected immediately into these osteochondral wounds. Both chondrocyte transplantations without PLGA microspheres and culture medium injections without chondrocytes served as controls. Sixteen weeks after implantation, chondrocytes implanted using the PLGA microspheres formed white cartilaginous tissues. Histological scores indicating the extent of the cartilaginous tissue repair and the absence of degenerative changes were significantly higher in the experimental group than in the control groups (P < 0.05). Histological analysis by a hematoxylin and eosin stain of the group transplanted with microspheres showed thicker and better-formed cartilage compared to the control groups. Alcian blue staining and Masson's trichrome staining indicated a higher content of the major extracellular matrices of cartilage, sulfated glycosaminoglycans and collagen in the group transplanted with microspheres than in the control groups. In addition, immunohistochemical analysis showed a higher content of collagen type II, the major collagen type in cartilage, in the microsphere transplanted group compared to the control groups. In the group transplanted without microspheres, the wounds were repaired with fibro-cartilaginous tissues. This study demonstrates the feasibility of using PLGA microspheres as an injectable scaffold for cartilage regeneration in a rabbit model of osteochondral wound repair.     

同种异体软骨细胞-聚羟基乙酸支架复合物修复甲状软骨缺损

BACKGROUND: Tissue-engineered bone can be obtained by the combination of chondrocytes and polyglycolic acid scaffold. OBJECTIVE: To investigate the effect of allogeneic chondrocytes/polyglycolic acid scaffold compound in the repair of thyroid cartilage defects in rabbits. METHODS: Twenty New Zealand adult rabbits were randomly divided into experimental group with implantation of allogeneic chondrocytes/polyglycolic acid scaffold compound and control group with implantation of polyglycolic acid scaffold. Gross and histological observations were done at 4 and 8 weeks after implantation.   RESULTS AND CONCLUSION: (1) Gross observation results: 4 weeks after surgery, cartilage defects in the experimental group were repaired certainly, and no necrosis appeared in the repair area; in the control group, the defects were filled with muscle and connective tissues. At 8 weeks after implantation, cartilage defects in the experimental group were further repaired, with unclear repair boundaries, and in the control group, cartilage defects were no repaired and showed a notable boundary with the surrounding normal cartilage tissues. (2) Immunohistochemical staining results: the expression of type II collagen in the experimental group was higher than that in the control group (P < 0.05) at 4 and 8 weeks after implantation. These findings indicate that the allogeneic chondrocytes/polyglycolic acid scaffold compound can promote the repair of thyroid cartilage defects in rabbits.