抗PTD-bcr/abl多克隆抗体的制备及鉴定

目的 制备多克隆抗体并初步用于PTD－bcr/abl融合的研究，为慢性粒细胞白血病的免疫治疗提供实验依据。方法 在大肠杆菌中表达PTD－bcr/abl融合蛋白，用所获得的蛋白免疫家兔得到多克隆抗体，并用免疫组化染色，Western-blot等方法进行此抗体的鉴定。结果 （1）表达PTD－bcr/abl融合蛋白。（2）制备了抗PTD－bcr/abl融合蛋白多克隆抗体。（3）并用多克隆抗体以多种方法，检测PTD－bcr/abl融合蛋白，证实此抗体效价高，特异性强。结论 此抗体可用于PTD－bcr/abl融合蛋白的进一步研究。     

TAT蛋白转导域介导BCR/ABL融合蛋白通过小鼠血脑屏障的作用

目的　探讨HIV - 1反式激活蛋白 (TAT)的蛋白转导结构域 (PTD)介导的BCR/ABL(TATPTD -BCR/ABL)融合蛋白通过血脑屏障的作用。方法　在大肠杆菌中表达PTD -BCR/ABL融合蛋白。将经Ni-NTA树脂亲和层析纯化的融合蛋白 ,经尾静脉注射小鼠体内 ,用免疫组织化学染色法 ,对小鼠脑组织细胞内的PTD -BCR/ABL融合蛋白进行检测。结果　①表达和纯化了相对分子质量 (Mr)为 2 3× 10 3 的PTD -BCR/ABL融合蛋白 ;②将其经静脉注射到小鼠体内 ,在小鼠脑组织细胞中可检测到PTD -BCR/ABL融合蛋白。结论　PTD可在体内介导较大Mr的融合蛋白通过血脑屏障 ,有望为治疗性的药物通过血脑屏障进入中枢神经系统提供了一种新的方法     

蛋白转导域介导BCR/ABL抗原对CML患者T细胞的活化作用

目的：研究蛋白转导域(PTD)介导的BCR／ABL抗原对慢性髓细胞白血病(CML)患者T细胞的特异性活化作用。方法：利用基因工程技术，将PTD基因与CML b3a2 bcr／abl基因融合并原核表达。将纯化的PTD—BCR／ABL融合蛋白与CML患者外周血单个核细胞(PBMC)体外共孵育，用流式细胞仪分别检测CD4^ 、CD8^ T细胞上活化抗原CD25的表达。结果：终浓度为100mg／L的PTD—BCR／ABL抗原体外刺激4d后，10例CML患者中，5例表现为CD8^ T细胞活化，2例表现为CD4^ T细胞活化，其中有1例CD8^ 和CD4^ T细胞同时活化；而作为对照的BCR／ABL抗原刺激组无一例表现为CD8^ 或CD4^ T细胞活化。结论：PTD能将外源性BCR／ABL抗原转导入抗原呈递细胞内，加工呈递后激活抗原特异性CD8^ 及CD4^ T细胞，为CML特异性CD8^ 、CD4^ T细胞的体外活化及细胞免疫治疗开辟一条新的途径。     

骨髓增殖性疾病Bcr／Abl融合基因的表达及其意义

目的：为探讨几种骨髓增殖性疾病bcr／abl 融合基因出现的频率及临床意义．方法：采用逆转录多聚酶键反应（RT－PCR）技术,研究了慢性粒细胞白血病（CML）,真性红细胞增多症（PV）,原发性血小板增多症（ET）,原发性骨髓纤维化（MF）bcr／abl基因的变化．结果：48例慢性期CML,43例呈现bcr／abl基因阳性,占90％,14例加速,急变期CML,6例出现bcr／abl基因,阳性率为43％,与慢性CML相比P＜0.01;10例PV出现1例bcr／abl基因阳性,占10％,6例ET中未发出bcr／abl基因．2例MF中发现1例bcr／abl基因阳性．结论：bcr／abl基因检测对于CML的临床分型有重要意义,bcr／abl阴性的CML预后差,而bcr／abl基因的阳性有助于初诊时的CML急变与急性非淋巴细胞白血病（AML）M2的鉴别和CML与慢性粒单细地白血病（CMML）的鉴别．在其它三种骨髓增殖性疾病中MF可能与CML关系最为密切,而ET关系较远,少数PV可能转化为CML．     

慢性粒细胞白血病和原发性骨髓纤维化患者bcr/abl融合基因表达及临床意义

目的探讨bcr/abl融合基因的表达水平在慢性粒细胞白血病(CML)和原发性骨髓纤维化(MF)患者的诊断、疗效及预后观察中的意义.方法采用荧光定量逆转录多聚酶链反应(RT-PCR)技术,对18例初、复治CML、7例MF患者的骨髓或外周血单个核细胞进行了bcr/abl融合基因检测,并对其中2例行异基因骨髓移植(allo-BMT)患者进行了短期随防.结果 13例慢性期(CP)CML,12例不同程度表达bcr/abl融合基因,占92.3%,3例加速期(AP),急变期(BC)CML,2例表达bcr/abl融合基因,占66.7%,2例患者allo-BMT后一年内未检出 bcr/abl融合基因,三者之间有非常显著差异P＜0.01;8例初诊患者WBC数及bcr/abl融合基因表达水平明显高于常规治疗组;7例MF患者有1例表达bcr/abl基因.结论 bcr/abl基因是CML发病的分子基础,定量检测bcr/abl融合基因对于CML的诊断、临床分型、疗效观察及预后判断有重要意义;MF患者可能与CML关系密切,对MF患者应进行跟踪观察.     

蛋白转导在基因治疗中的应用

蛋白转导是近几年生命科学领域发现的一种独特现象,具有蛋白转导功能的蛋白通过几个短的碱性小肽组成的蛋白转导域(proteintransductiondomain,PTD)的介导,能将与其共价连接的DNA、多肽或蛋白质以及其他大分子物质通过非经典途径穿过细胞膜甚至血脑屏障,并在细胞间自由传递。PTD这种能携带融合蛋白自由进入细胞的独特功能为人类一些疾病的基因治疗提供了一种新的运载工具,在基因治疗中有着美妙的应用前景。     

温热处理对K562细胞系bcr／abl融合基因mRNA转录和Caspase-3表达的影响

目的观察不同温度刺激对K562细胞bcr／abl融合基因mRNA转录和Caspase-3蛋白表达的影响。方法将培养获得的K562细胞分别进行40℃、43℃、46℃恒温刺激30min,以37％作为对照;RT-PCR技术检测bcr／abl融合基因mRNA的转录,Westernbloting技术检测Caspase-3的表达。结果K562细胞热刺激30min后,bcr／abl融合基因mRNA的转录随温度升高而下调,Caspase-3的表达随温度升高而上调。结论温热刺激可降低K562细胞内bcr／abl融合基因mRNA的转录,提高Caspase-3的表达。     

25例慢性粒细胞白血病患者bcr/abl融合基因结果分析

目的探讨逆转录聚合酶链反应(RT—PCR)在检测慢性粒细胞白血病(CML)bcr/abl融合基因方面的应用价值。方法对25例慢性粒细胞白血病患儿血标本提取细胞RNA,用RT—PCR方法检测bcr/abl融合基因。结果 25例慢性粒细胞白血病bcr/abl融合基因分析,阳性24例(96%);阴性1例(4%)。结论 RT—PCR检测bcr/abl融合基因敏感性好,稳定性高,对临床治疗和预后判断具有指导意义。     

细胞穿透肽PEP-1介导增强型绿色荧光蛋白在小鼠体内跨膜转导

目的 研究细胞穿透肽PEP-1介导的大分子物质在小鼠体内的跨膜转导能力.方法 用基因工程的方法制备并纯化增强型绿色荧光蛋白EGFP和PEP-1-EGFP融合蛋白,分别将500 μg的EGFP蛋白和PEP-1-EGFP融合蛋白通过尾静脉注射入昆明小鼠体内,2 h后麻醉小鼠,PBS充分灌流并取心、脑、肝、脾、肾快速冷冻切片后立即置荧光显微镜下观察.结果 2 h后PEP-1-EGFP融合蛋白处理的小鼠大脑、心肌、肝、脾和肾组织里出现均一的明亮绿色荧光,而EGFP蛋白处理的小鼠各脏器内均未见到绿色荧光.结论 细胞穿透肽PEP-1能携带增强型绿色荧光蛋白穿透小鼠细胞膜并分布于心、脑、肝、脾、肾组织内,为将来用PEP-1介导各种大分子药物跨膜转导进行各种疾病的蛋白治疗提供实验依据.     

66例血液病患者bcr/abl融合基因、Ph染色体结果分析

CML特异性费城染色体（Philadephia,Ph）是由t（9：22）（q34：11）易位形成。9号染色体原癌基因abl（Abelson protooncogene）易位至22号染色体的断裂点簇集区（breakpoint cluster region,bcr）,发生重排,产生bcr／abl融合基因,引起CML的始动突变。融合基因bcr／abl几乎见于所有的慢性粒细胞性自血病、25％-50％的急性B系淋巴细胞性白血病（ALL）和约5％的急性粒细胞性白血病中,本文统计了进行染色体和ber／abl融合基因检查的初诊血液病66例,对其骨髓细胞染色体核型及bcr／abl融合基因进行分析。     

慢性粒细胞白血病特异性单、双及三单位核酶载体的构建

本文拟针对在慢性粒细胞白血病发病中起关键作用的ｂｃｒａｂｌ 融合基因, 构建特异性的系列核酶载体。为此,我们针对ｂｃｒａｂｌ 融合位点设计、合成了３ 个相邻的锤头状核酶。通过基因重组将３ 个单核酶按顺序定向克隆入改建的ｐＧＥＭ３ｚｆ 载体中,构建了ｂｃｒａｂｌ 单核酶、双核酶及三核酶体外转录载体, 并在此基础上将三单位核酶克隆入ｐ ＤｏＲｎｅｏ 载体中构建三核酶逆转录病毒载体。此外, 通过ＰＣＲ 方法, 扩增了ｂｃｒａｂｌ 融合位点附近约３７６ｂｐ 的序列,成功地构建了ｂｃｒａｂｌ 融合基因体外转录载体。本文构建的ｂｃｒａｂｌ 特异性系列载体,为今后遴选特异、高效的核酶打下了基础,并为将核酶用于自体骨髓移植的体外骨髓净化创造了条件。     

Short-term culturing influences the number of bcr/abl-fused cells detected by fluorescence in situ hybridisation in bone marrow aspirates of CML patients.

Fluorescence in situ hybridisation (FISH) has been proven as a helpful tool in diagnosis and monitoring of bcr/abl fusion in chronic myelogeneous leukaemia (CML). Since long-term cultures are known to decrease the number of bcr/abl-fused cells, it is questionable whether similar effects are detectable in short-term cultures, a technique often preceding FISH analysis. Therefore, we evaluated bone marrow aspirates of 10 CML patients at biopsy and after culturing for between 24 and 144 h by FISH. The percentage of bcr/abl-fused cells in FISH varied between 15 and 70% at biopsy. In samples with 15 and 30% of aberrant cells at biopsy, an increase of about 20% per day was seen within the first 48 h. In longer lasting cultures, the percentage of leukaemic cells then asymptotically approached a value of 60-70%. In patients with 38 and 50% of bcr/abl-fused cells at biopsy, an increase of about 20% could be detected in the first 24 h. Then 65-70% of the cells already bore the bcr/abl fusion, and the percentage of leukaemic cells was almost constant for longer lasting cultures up to 144 h. In patients with a percentage of about 70% before culturing, no increase in positive cells was detected. These results emphasize the impact of short-term culturing on the number of bcr/abl-fused cells. In particular, the importance for monitoring CML patients is obvious. Therefore the effect described should be taken into account in order to avoid misinterpretation and incorrect therapy decisions in CML patients.     

Megakaryocytes carry the fused bcr-abl gene in chronic myeloid leukaemia: a fluorescence in situ hybridization analysis from bone marrow biopsies

Histological examination of bone marrow biopsies shows that about one-third of chronic myeloid leukaemia (CML) patients exhibit an increase of megakaryocytes. The megakaryocytic predominance may be so striking that differentiation from other chronic myeloproliferative disorders (CMPD) may be difficult in some CML patients. Megakaryocytes in CML are clonal as demonstrated by loss of glucose-6-phosphate dehydrogenase isoenzymes. The Ph translocation, fusing the abl and bcr genes on chromosomes 9 and 22, however, obviously occurs as a second step in tumour development. So far, the Ph translocation has not been assigned explicitly to megakaryocytes. The question is whether the megakaryocytic cell lineage could harbour the bcr/abl fusion in those CML cases with striking proliferation of megakaryocytes but lack this genetic defect in cases with normal or decreased megakaryocyte counts. We therefore performed triple-colour fluorescence in situ hybridization (FISH) for portions of the bcr and abl genes flanking the breakpoint in CML in paraffin sections of CML cases with normal and with increased numbers of megakaryocytes. This method allows identification of the bcr/abl fusion in single, morphologically intact cells, whereas conventional cytogenetics requires lysis and thus destruction of the cell. Among the 21 CML patients examined by FISH, 10 were informative for bcr and abl genes and displayed distinct hybridization signals within nuclei of bone marrow cells. Besides the granulopoietic cells, megakaryocytes of all those patients (4 without and 6 with varying grades of megakaryocytic increase) displayed bcr/abl fusion signals indiciative of a Ph translocation. The lack of hybridization signals in the remaining 11 cases indicates that this technique is not of value diagnostically and should be reserved for scientific questions. Positive controls consisted of conventional chromosome preparations from bone marrow aspirates demonstrating the Ph chromosome in all patients examined, and negative controls of paraffin sections of bone marrow biopsies from non-CML patients. These showed no fusion signals in bone marrow cells, including megakaryocytes, using FISH. Our results demonstrate clearly that not only the transforming event but also the Ph translocation leading to the bcr/abl fusion happens prior to the differentiation of the pluripotent stem cell into different myeloid lineages. The megakaryocytic proliferation evident in some CML cases is probably a consequence of the disease progress.     

荧光原位杂交检测慢性粒细胞白血病的染色体畸变

目的 检测费城染色体（Ｐｈｉｌａｄｅｌｐｈｉａ ｃｈｒｏｍｏｓｏｍｅ，Ｐｈ染色体）阴性，变异型Ｐｈ染色体及伴有其它染色体异常的慢性粒细胞白血病（ｃｈｒｏｎｉｃ ｍｙｅｌｏｉｄ ｌｅｕｋｅｍｉａ，ＣＭＬ）的ｂｃｒ／ａｂｌ融合基因。方法 双色荧光原位杂交技术，检测伴有８种不同的骨髓细胞染色体畸变ＣＭＬ患者的ｂｃｌ／ａｂｌ融合基因。结果 ３例变异型Ｐｈ和７例有标准型Ｐｈ的ＣＭＬ患者均为ｂｃｒ／ａｂｌ融合     

PTD4-GFP-VP3融合蛋白在人肝癌细胞HepG2中的定位观察及凋亡诱导效应的实验研究

目的观测PTD4-GFP-VP3融合蛋白诱导人肝癌细胞HepG_2的凋亡效应及其在肿瘤细胞中的定位与其凋亡效应关系的研究。方法构建PTD4-GFP-VP3融合蛋白的原核表达载体,利用人源肝癌细胞株HepG_2,经细胞透膜实验在激光共聚焦显微镜下观察PTD4介导的融合蛋白透膜效应,DAPI染色观测该蛋白在细胞中的定位;利用TUNEL法观察PTIM-GFP-VP3融合蛋白诱导肿瘤细胞的凋亡效应,并利用流式细胞仪检测其凋亡率。结果PTD4-GFP-VP3融合蛋白在孵育细胞0.5h后即可透过细胞膜进入到细胞质中,12h后定位于细胞核并诱导肿瘤细胞凋亡,48h达到最高凋亡效应。结论PTD4能携带GFP-VP3融合蛋白穿透细胞膜,在肿瘤细胞中具有核定位效应,并能诱导肿瘤细胞凋亡,为后期进一步研究VP3的凋亡机制及其抗肿瘤治疗奠定了基础。