基于肿瘤个体化治疗药物筛选的异种移植模型评价策略

肿瘤组织异种移植(PDX)模型在遗传学、病理学和生物学特性等方面与患者的原发肿瘤具有较好的相似性,药物临床反应一致性高,在肿瘤个体化治疗领域显示出良好的应用前景。利用PDX模型开展肿瘤靶向药物筛选可有效指导临床用药。本文针对用于化疗药物筛选的PDX模型评价策略进行综述,总结了建模标准和质量控制要求,提出组织形态学、测序分析、特异性标志物和STR检测四种模型溯源性评价方法,综合衡量药物毒性作用、肿瘤体积变化趋势和TGD数学模型结果进行疗效评价,为PDX模型指导肿瘤个体化治疗提供良好的应用策略。     

骨髓活检组织EBER原位杂交检测影响因素分析

目的探讨骨髓活检组织行EBV encoded RNA(EBER)原位杂交检测的影响因素,优化检测条件以期提高骨髓活检组织中EB 病毒的检出率。方法收集35 例EB 病毒相关疾病的骨髓活检标本,通过对比实验,比较不同脱钙方法、不同蛋白酶K 消化条件、不同抗体孵育温度下的骨髓活检组织行EBER 原位杂交检测的切片质量、脱片率及染色质量情况。结果脱钙以运用改良EDTA 脱钙液脱钙20~24h 后流水冲洗30min^1h 最佳;蛋白酶K 消化时间为9min 的组织切片脱片率低,杂交效果最好,阳性细胞着色深,定位准确;在37℃条件下进行抗体孵育杂交染色质量为佳。结论选取合适的脱钙方式,延长蛋白酶K 消化时间,选择最佳抗体孵育温度,可降低脱片率,显著提高骨髓活检组织行EBER 原位杂交检测的染色质量,为EBV 相关疾病的诊断和鉴别诊断提供重要依据。     

毒理学研究中的体外细胞毒性评价

体外细胞毒性评价作为传统动物模型毒性评价的替代方法正在得到越来越多的研究和应用,而其向高通量阶段的迈进则为新毒物和新药物的检测与目的物的筛选提供了更加快捷、高效的手段。将对体外细胞毒性评价常用细胞类型、体外细胞毒性评价的指标,及其检测技术方法的研究现状、进展和存在问题进行阐述,希望能为相关的研究提供一定的参考。     

缓解与未缓解髓系白血病干细胞表面抗原CD34~+CD123~+表达差异研究

目的:探索缓解与未缓解急性髓系白血病干细胞表面抗原表达差异,为判定化疗疗效及其预后提供依据。方法:按照急性白血病诊断标准,根据患者入院时骨髓白血病细胞数量多少分成临床缓解与未缓解两组,以流式细胞仪分别检测骨髓中白血病干细胞表面相关抗原表达情况,比较二者之间差异。其中经标准化疗方案治疗结束后,通过复查骨髓象判定疗效并比较化疗前后白血病干细胞表面相关抗原表达变化。结果:与缓解的急性髓系白血病患者骨髓白血病干细胞相关抗原表达值相比,未缓解的患者骨髓白血病干细胞表面相关抗原表达明显升高,差异具有统计学意义(P0.05,0.001);未缓解的患者经标准方案化疗后骨髓虽然已经获得完全缓解,但依然具有白血病干细胞表面抗原高表达,提示这部分患者依然有复发的可能性。结论:急性髓系白血病患者的白血病干细胞相关抗原表达值升高是急性白血病复发难治的根源之一。     

抗大口黑鲈弹状病毒发酵中草药的筛选及其效果评价

为筛选出能够对MSRV具有抑制效果的发酵中草药,研究通过MTT法检测8种发酵中草药(L1、L2、L3、L4、L5、X1、X2和ZHS)对EPC细胞的毒性作用,结果表明L1、L2、L3、X1、X2和ZHS未对EPC细胞造成明显的损伤和毒性, L4和L5在4 mg/mL时对细胞造成损害;通过荧光定量PCR法(qRT-PCR)筛选出X1药物(主要成分为板蓝根、鱼腥草、南芪和杏仁)对MSRV有良好的抗病毒效果;在细胞水平上通过qRT-PCR法发现X1药物能够抑制MSRV的感染;空斑实验结果表明X1药物可以降低约10倍MSRV的病毒滴度。在鱼体水平上设置空白对照组、药物对照组、攻毒组、预防组和治疗组,评价X1药物对MSRV的抗病毒效果,结果表明经X1药物处理后,治疗组可以将大口黑鲈的存活率提高20%;通过qRT-PCR法检测大口黑鲈组织中病毒载量,结果表明X1药物显著抑制MSRV在肝脏、脾脏组织中的复制;通过组织病理学观察发现X1药物可以抑制MSRV引起的在肝脏、脾脏组织病变。研究为发酵中草药在水产养殖中的应用提供新的思路,为开发抗MSRV药物提供参考依据。     

缓解与未缓解髓系白血病干细胞表面抗原CD34＋CD123＋表达差异研究

目的：探索缓解与未缓解急性髓系白血病干细胞表面抗原表达差异,为判定化疗疗效及其预后提供依据。方法：按照急性白血病诊断标准,根据患者入院时骨髓白血病细胞数量多少分成临床缓解与未缓解两组,以流式细胞仪分别检测骨髓中白血病干细胞表面相关抗原表达情况,比较二者之间差异。其中经标准化疗方案治疗结束后,通过复查骨髓象判定疗效并比较化疗前后白血病干细胞表面相关抗原表达变化。结果：与缓解的急性髓系白血病患者骨髓白血病干细胞相关抗原表达值相比,未缓解的患者骨髓白血病干细胞表面相关抗原表达明显升高,差异具有统计学意义（P〈0．05,0．001）;未缓解的患者经标准方案化疗后骨髓虽然已经获得完全缓解,但依然具有白血病干细胞表面抗原高表达,提示这部分患者依然有复发的可能性。结论：急性髓系白血病患者的白血病干细胞相关抗原表达值升高是急性白血病复发难治的根源之一。     

代谢组学及其在药物毒性评价中的应用

建立可靠的药物毒性评价方法是药物开发中的重要任务之一。近几年发展起来的代谢组学可以对生物体内的代谢产物变化进行定性定量分析,并对整体代谢轮廓进行模式识别,为药物毒性评价和毒性作用机制的研究提供了新的技术手段。本文就代谢组学、代谢组学研究方法及其在药物毒性评价中的应用进行了综述。     

通过时间基因表达谱分析探究异烟肼引起肝损伤的机制

抗结核药物异烟肼具有肝脏毒性,其发生机制需要进一步阐明。本研究使用异烟肼处理肝细胞,分析不同处理时间表达谱的差异。通过对差异表达基因进行聚类分析和功能富集分析,共得到6个与肝脏毒性相关的基因簇和一系列相关通路;进一步通过蛋白互作分析和时间序列差异分析方法,筛选出表达水平具有时间依赖性的13个关键基因。本研究结果为理解异烟肼引发肝脏毒性过程提供了思路,为今后药物性肝脏毒性的监测以及治疗提供了新的靶基因。     

石蜡包埋组织的DNA提取及其在免疫球蛋白基因重排分析中的应用

基因重排分析在淋巴瘤诊断中具有重要意义.文章应用改良DNA提取方法,从30例淋巴增生性病变石蜡包埋组织获得的DNA虽有不同程度的降解,但适于PCR扩增Ig重链基因重排分析;约1/3病例提出高分子量DNA,可用于DNA印迹杂交.因此,石蜡包埋组织同样可为某些疾患,如淋巴瘤疑难和罕见病例的回顾性分子病理学研究提供基因诊断的DNA来源.     

石蜡包时组织的DNA提取及其在免疫球蛋白基因重排…

基因重排分析在淋巴瘤诊断中具有重要意义。文章应用改良ＤＮＡ提取方法。从３０例淋巴生性病变石蜡包埋组织获得的ＤＮＡ虽有不同程度的降解,但适于ＰＣＲ扩增Ｉｇ重链箕因重排分析;约１／３病例提出高分子量ＤＮＡ,可用于ＤＮＡ印迹杂交。因此,石蜡包埋组织同样可为某些疾患,如淋巴瘤凝难和罕见病例的回顾性分子病理学研究提供基因诊断的ＤＮＡ来源。     

Granulocyte-dendritic cell unbalance in the non-obese diabetic mice

Several investigators, including ourselves, have reported lower yield of GM-CSF bone marrow-derived dendritic cells (DC) with altered MHC class II and co-stimulatory molecules expression in the non-obese diabetic (NOD) mice. However, whether this defect was intrinsic to the DC lineage and/or related to abnormal expansion of other cell types responding to GM-CSF remained an opened issue. We performed phenotypical and morphological analysis of cells from GM-CSF-supplemented-bone marrow-cultures and of freshly isolated bone marrow and blood cells from unmanipulated prediabetic NOD mice. The results show a heretofore undescribed bias towards generation of granulocytes in NOD mice, concomitant with quantitative and qualitative alterations of the DC lineage in both the bone marrow and the blood of this mouse strain. We propose that increased generation of granulocytes in NOD mice might contribute to autoimmunity. First, high numbers of granulocytes per se might favor inflammatory environment. Second, granulocytes, by interfering with DC development, might favor unbalanced antigen presenting cell function leading to T cell autoimmunity.     

Basic and 'special' stains for plastic sections in bone marrow histopathology, with special reference to May-Grünwald Giemsa and enzyme histochemistry

A battery of morphological, histochemical, and enzyme histochemical stains have been experimented on semithin sections of glycol-methacrylate-embedded bone marrow biopsies. We have been able to reproduce on sections the typical 'Romanowsky effect' which characterizes May-Grünwald Giemsa-stained smears of bone marrow or peripheral blood. This appears to be of critical importance for proper routine morphological evaluation of bone marrow biopsies. Conventional histochemical stains, and the enzyme histochemistry reactions that are most useful and widely used in the study of marrow aspiration smears have been successfully applied to plastic sections: in this way the evaluation of the cytochemical profiles of marrow diseases, especially leukemias, may be included in the histopathologist's diagnostic approach, with the additional advantage of preserving the architecture of the tissue and the relationship between haemapoietic cells and stromal components.     

Differential analysis of animal bone marrow by flow cytometry.

A simple procedure was developed for rapid analysis of animal bone marrow by flow cytometry using the lipophilic cationic dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)]. The batch process allows differentiation of bone marrow cells into lymphoid, erythroid, and myeloid populations and enables classification of erythroid and myeloid cells into proliferating and maturing subpopulations. From these data, myeloid:erythroid (M:E) ratios and maturation indices for erythroid and myeloid cells (EMI and MMI, respectively) can be derived. This procedure provides the opportunity to analyze bone marrow quantitatively and offers distinct advantages to current manual methods in terms of simplicity, throughput, and reproducibility. The method has been tested successfully using marrow from Wistar rats, B6C3F1 mice, beagle dogs, and cynomolgus monkeys. This technique facilitates the evaluation of bone marrow samples taken from preclinical safety studies or from animal colonies of large size.     

Novel technology to provide an enriched therapeutic cell concentrate from bone marrow aspirate

Current strategies to repair fractures rely on orthopaedic surgeons harvesting bone from one area of the body, typically pelvis and transferring it to the fracture site. The amount of tissue available is therefore limited, requiring a second surgical procedure and often causing the patient long term pain. An alternative approach is utilise therapeutic cells contained within bone marrow aspirate during the primary procedure. The number of therapeutic cells within a fresh aspirate is insufficient to provide clinically acceptable bone healing in a timescale that is satisfactory to the surgeon and the patient. Therefore methods to efficiently concentrate bone marrow in the clinical setting are required. Centrifugation is the current method of choice but has limitations in that it requires large capital equipment, servicing and there are potential issues of tissue contamination. We have developed a novel, acoustically‐assisted filtration device that addresses these limitations, delivering a concentrated bone marrow in a point of care, single use, fully disposable, compact device. An additional advantage is that the level of concentration required can be specified by the end user. The resulting bone marrow concentrate has been characterised in terms of cell number, viability and osteogenic potential using flow cytometry and alkaline phosphatase assay. When compared to recent clinical studies using bone marrow to repair non‐union fractures, the findings from our work suggest that the bone marrow concentrate is likely to be highly therapeutic and clinically efficacious as a bone fracture repair strategy. A product concept for use in the clinical setting is presented. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Comparison of flow cytometry- and microscopy-based methods for measuring micronucleated reticulocyte frequencies in rodents treated with nongenotoxic and genotoxic chemicals

The development of automated flow cytometric (FCM) methods for evaluating micronucleus (MN) frequencies in erythrocytes has great potential for improving the sensitivity, reproducibility, and throughput of the traditional in vivo rodent MN assay that uses microscopy-based methods for data collection. Although some validation studies of the FCM evaluation methods have been performed, a comprehensive comparison of these two data collection methods under routine testing conditions with a variety of compounds in multiple species has not been conducted. Therefore, to determine if FCM evaluation of MN frequencies in rodents was an acceptable alternative to traditional manual scoring methods in our laboratory, we conducted a comparative evaluation of MN-reticulocyte (MN-RET) frequencies determined by FCM- and microscopy-based scoring of peripheral blood and bone marrow samples from B6C3F1 mice and Fisher 344 rats. Four known inducers of MN (cyclophosphamide, ethyl methanesulfonate, vincristine sulfate, acrylamide) were assayed in bone marrow and peripheral blood of both mice and rats. In addition, MN-RET frequencies were measured in bone marrow (microscopy) and peripheral blood (FCM) of mice treated with five nongenotoxic chemicals (S-adenosylmethionine chloride, cefuroxime, diphenolic acid, 3-amino-6-methylphenol, pentabromodiphenyl oxide). No significant differences were observed between results obtained by the two methods in either species. These results support the use of FCM for determining MN-RET frequency in rodents after chemical exposure.     

参麦注射液对于白血病骨髓抑制期的治疗作用

目的：探讨参麦注射液对于白血病骨髓抑制期的治疗作用。方法：选取我院2011 年1 月至2012 年12 月收治的白血病的患者47 例,随机分为两组,观察组24 例化疗的同时给予参麦注射液,观察组患者23 例只给予化疗,比较两组患者化疗后的骨髓抑制情况和感染发生情况。结果：两组患者化疗效果显示总有效率分别为66.7%和65.2%,无显著差别（P〉0.05）。两组患者化疗骨髓抑制后WBC, Hb, PLT 的恢复时间分别为（6.32± 2.75）天,（9.32± 2.12）天和（7.31± 3.21）天,明显低于对照组（11.34± 4.34）天,（12.54± 3.21）天和（12.41± 4.32）天,差异显著有统计学意义（P〈0.05）;观察组患者中性粒细胞ANC〈0.5× 10^9/L持续的时间明显少于对照组,差异明显有统计学意义（P〈0.05）;观察组患者发生感染的比例明显低于对照组,差异显著有统计学意义（P〈0.05）。结论：参麦注射液可以明显减轻白血病化疗后的骨髓抑制现象,有效保护骨髓的造血功能,缩短骨髓受抑制时间,和降低感染率,明显提高患者生活质量。     

Allogeneic bone marrow supports human islet beta cell survival and function over six months

In this study, we have established a new strategy increasing human islet longevity utilizing allogeneic whole bone marrow (BM) co-cultured with human islets. The cultured islets' function and survival have been evaluated by analysis of insulin secretion in response to high-glucose-challenge, morphological evaluation of cell growth. Human islet only culture failed to reveal evidence of long term survival, growth or function in terms of insulin release or insulin response to glucose challenge. These results indicate that BM increases islet survival and function with the eventual formation of pancreatic endocrine tissue capable of sustaining beta cell fuction.     

Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation

Recent studies have suggested that bone marrow cells might possess a much broader differentiation potential than previously appreciated. In most cases, the reported efficiency of such plasticity has been rather low and, at least in some instances, is a consequence of cell fusion. After myocardial infarction, however, bone marrow cells have been suggested to extensively regenerate cardiomyocytes through transdifferentiation. Although bone marrow-derived cells are already being used in clinical trials, the exact identity, longevity and fate of these cells in infarcted myocardium have yet to be investigated in detail. Here we use various approaches to induce acute myocardial injury and deliver transgenically marked bone marrow cells to the injured myocardium. We show that unfractionated bone marrow cells and a purified population of hematopoietic stem and progenitor cells efficiently engraft within the infarcted myocardium. Engraftment was transient, however, and hematopoietic in nature. In contrast, bone marrow-derived cardiomyocytes were observed outside the infarcted myocardium at a low frequency and were derived exclusively through cell fusion.