血友病基因治疗和白血病免疫治疗基础研究(奚晓东)
发布日期:2010-04-16 浏览次数:363

(一)血栓与止血过程相关的信号转导和调控机制的研究
血栓与止血过程相关的信号转导和调控机制研究方向目前有两项国家自然科学基金课题(通过转基因小鼠模型的建立在体内研究整合素β3胞内段对血小板功能的调控作用30710103905和APL相关组织因子高表达的分子机制研究:PML/RAR融合蛋白的作用及调控方式30871107)及一项“863”项目子课题(血液恶性肿瘤相关抗体制备技术平台的建立和关键抗体的研制2006AA02A245)在研。
我们建立了稳定表达突变型整合素3的CHO细胞模型,分别为CHOIIb3E726Q、CHOIIb3E733Q、CHOIIb3E726QE733Q、CHOIIb3E726QE731QE733Q,并观察以上细胞模型在固相化的纤维蛋白原上的粘附伸展情况,研究了这些突变对整合素IIb3功能的影响,为下一步深入研究整合素3在信号转导中与胞内信号分子的相互作用奠定了基础。此外,整合素3缺陷小鼠的引进建立工作已经完成,目前已产出3(-/-)小鼠。同时,带有不同突变型整合素IIb3的逆转录病毒表达载体业已构建完成并达到高滴度的病毒感染效率。因此转基因研究的基础条件业已完备,下一步将实施转基因研究计划。
我们构建了原核表达载体pQE30-Kit4-5并获得高纯度的人c-Kit4-5重组蛋白免疫BALB/c小鼠,采用 B淋巴细胞杂交瘤技术进行细胞融合,成功制备了1株特异性的鼠抗人c-Kit mAb杂交瘤细胞。其所分泌的抗体能特异地识别人c-Kit分子,并且可能区分Kasumi白血病细胞表面表达的不同c-Kit分子。
在PML/RAR融合蛋白与组织因子启动子的相互作用及其分子机制的研究方面,我们证明了PML/RAR融合蛋白通过一种间接的、非DNA结合的机制与组织因子启动子相互作用从而上调其转录,同时我们还首次在组织因子启动子的调控区域内确定了含有GAGC核心区域的PML/RAR融合蛋白相互作用位点。
(二)凝血因子及遗传性出血病和血栓病的研究
凝血因子及遗传性出血病和血栓病研究方向目前在研有两项国家自然科学基金资助的科研项目,着重于凝血因子结构与功能的关系,分别是“凝血因子IX R327I 突变蛋白结构与功能的研究(30770904)”和“凝血因子VIIIHis99Arg突变蛋白的分子发病机制研究(30870942)”。
构建了FIX丙氨酸(R327A)、异亮氨酸(R327I)突变及FIXβ折叠结构域(Q324-329P)与FVII同源结构(M298-P303)互换的功能缺失性(FIXβ折叠FVII)和功能获得性(FVIIβ折叠FIX)表达质粒,体外瞬时表达显示R327I表达约为野生型的一半,而其他3种突变体的表达均与正常野生型相当,为稳定表达细胞株的建立及稳定表达蛋白纯化提供了相关依据。已成功构建突变PFⅧ His99Arg cDNA、PFⅧ His99Ala cDNA及野生型PFⅧcDNA表达质粒载体,并瞬时转染CHO和BHK细胞对突变蛋白进行了相关的活性及抗原检测。体外转染细胞后,表达突变蛋白FⅧ:C为野生型的3%,细胞上清液FⅧ:Ag为野生型的218%,细胞内FⅧ:Ag为野生型的119%。完成了突变蛋白的合成和分泌的相关检测。His99Arg和His99Ala突变型及野生型FⅧ蛋白的稳定表达与纯化尚在进行中。获得稳定表达的重组蛋白后将进行后续的实验。
对于血友病携带者和产前诊断工作,我们今年对诊断方法进行了改进。在已有的STRs位点上进行了进一步的优化筛选,采用了更接近于F8C的新的STRs组合:DXS1073、F8Civs13、Intron25、3_486、5_147、5_226,进一步提高诊断准确率。我们还对1435例血友病A进行了抑制物筛查,发生率为3.9%。
遗传性出血病方面,对21个少见的遗传性凝血因子缺陷症家系、2个血小板无力症家系、2个出血性毛细血管扩张症家系进行了相关基因与功能的研究。遗传性血栓病方面,对13个AT缺陷家系、16个PC缺陷家系、3个PS缺陷家系和2个PC/PS联合缺陷家系进行了相关基因诊断与功能的研究。
2009年共完成SCI论文2篇及国内核心期刊论文5篇,毕业硕士研究生4名。目前在读研究生16名(含博士研究生3名)。

Ⅸ. Study of hemophilia and immune biotherapy for hematological diseases (Xiao-Dong Xi)
1. Signaling and regulatory mechanisms involved in thrombosis and hemostasis processes
Major research projects of our team on signaling and regulatory mechanisms involved in thrombosis and hemosrasis processes are supported by the NSFC and the National High-tech “863” grants studying the in vivo effect of integrin β3 cytoplasmic domain on platelet function (30710103905), regulation of PML/RAR on tissue factor expression (30871107) and antibodies important in malignant blood neoplasms (2006AA02A245).
Stable CHO cell lines expressing different mutants of integrin 3 including CHOIIb3E726Q, CHOIIb3E733Q , CHOIIb3E726QE733Q and CHOIIb3E726QE731QE733Q have been established. By observing the adhesion and spreading of these cell lines on immobilized fibrinogen we studied the effect of these mutations on the function of integrin IIb3 and laid a solid basis for further analysis of the interaction of integrin 3 with cytoplasmic signaling molecules when transducing signals. In addition, we have obtained the integrin 3-deficient mice and the retroviral expression vectors encoding different integrin 3 mutants. These allowed us to perform the transgenic projects.
We have established a mouse monoclonal hybridoma cell line by fusing lymphocytes immunized with highly purified recombinant human c-Kit4-5 fragment. This hybridoma cell line stably secrets monoclonal antibody specifically recognizing human c-Kit. Most interestingly, this monoclonal antibody seems able to distinguish different c-Kit molecule populations expressed on the surface of leukemic Kasumi cells.
Our research project on the molecular mechanisms for the interaction between the PML/RAR fusion protein and the tissue factor promoter is developed steadily. We proved for the first time that the PML/RAR fusion protein interacts with and transactivates the tissue factor promoter through an indirect mechanism without direct DNA association and a GAGC-containing PML/RAR-reactive element in the tissue factor promoter is responsible for this regulation.
2. The coagulation factors and the inherited hemorrhagic and thrombotic diseases 
The main target of our team on the research of the coagulation factors and the inherited hemorrhagic and thrombotic diseases is the relationship between structure and function of the coagulation factors. Two NSFC-granted projects are currently conducted to study the function and structure of the factor IX with R327I mutation (30770904) and the pathological mechanism of factor VIII His99Arg mutation (30870942).
Five expression plasmids have been constructed, encoding mutants of FIX R327A, FIX R327I, FIX -fold domain (Q324-329P) and FVII homologous exchange gain-of-function (FIX -fold-FVII) or loss-of-function (FVII -fold-FIX) mutations, respectively. All constructs were in vitro transfected into CHO cells. Unlike the other three constructs that expressed an equal amount of protein as the wild type, transient transfection of R327I mutant resulted in a 50 % decrease of protein expression. Three expression constructs, i.e. FVIII His99Arg, FVIII His99Ala and the wild type FVIII have been constructed and transiently expressed in CHO and BHK cells. The results showed that the activity of expressed mutant proteins is only 3% of wild type. On the other hand, the antigen detection of the mutant proteins in cell lysates showed a level about 119% of wild type while 218% in supernatant. Stable expression cell lines are under establishment for further analysis of the effect of the mutants on the post-translational regulations and on the protein functions.
For the gene diagnosis of hemophilia, we optimized the diagnosis method by applying new STRs sites including DXS1073, F8Civs13, intron25, 3_486, 5_147, 5_226 to minimize the diagnosis errors and to improve the diagnostic accuracy. The FVIII inhibitor has been screened in 1435 hemophilia A patients revealing the prevalence about 3.9%. Gene diagnosis and functional study were conducted in different pedigrees of inherited hemorrhagic and thrombotic diseases including 21 of rare coagulation factor deficiencies, 2 of Glanzmann thrombasthenia, 2 of hereditary hemorrhagic telangiectasis, 13 of AT deficiency, 16 of PC deficiency, 3 of PS deficiency and 2 of combined PC/PS deficiency.
In 2009, we prepared 2 original papers for SCI journals and other 5 for domestic prestigious journals. 4 students were graduated and were awarded a master degree. There are 16 students who currently registered for our graduate programs including 3 for doctoral programs.

年度发表论文:
国家重点实验室第一作者单位文章:
1. Yan JS, Wang KK, Dong LM, Chen WQ, Liu HC, Zhao RH, Xi WD, Ding QL, Kieffer N, Wang ZY, Caen JP, Chen SJ, Chen Z, Xi XD. PML/RARα fusion protein transactivates the tissue factor promoter through a GAGC-containing element without direct DNA association. Proc Natl Acad Sci USA 2010 Feb 3. [Epub ahead of print] 
2. Yu T, Wang X, Ding Q, Fu Q, Dai J, Lu Y, Xi X, Wang H. Using a minigene approach to characterize a novel splice site mutation in human F7 gene causing inherited factor VII deficiency in a Chinese pedigree. Haemophilia 2009; 15: 1262.
合作文章:
1. Lu YL, Wang XF, Xie BS, Ding QL, Dai J, Xi XX, Wang MS, Wang HL. Occurrence of Haemophilia A and B in a Chinese family with mosaicism of the F9 gene mutation in HB index’s maternal grandfather. Thrombosis and Haemostasis. (In press)
2. Wang XF, Lu YL, Ding QL, Dai J, Xi XX, Wang HL. Haemophilia A in two unrelated females due to F8 gene inversions combined with skewed inactivation of X chromosome. Thromb Haemost 2009; 101: 775-778.