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CELL AND GENE THERAPY IN A MOUSE MODEL OF HUMAN OSTEOGEN

人类成骨小鼠模型中的细胞和基因治疗

基本信息

批准号：
6054661
负责人：
CHRISTOPHER NIYIBIZI
金额：
$ 3.59万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-02-01 至 2000-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6054661
关键词：
bone marrow transplantation collagen disease /disorder model gene expression gene therapy genetic transcription immunofluorescence technique laboratory mouse nonhuman therapy evaluation osteogenesis osteogenesis imperfecta polymerase chain reaction transfection /expression vector

项目摘要

Osteogenesis imperfecta (OI) is a group of heritable disorders of connective tissue whose common feature is bone fragility. Most forms of OI are the result of mutations in the genes that encode proalpha1 and proalpha2 polypeptide chains of type I collagen the major protein of bone. The long-term objective of the present proposal is to test the feasibility of using cell therapy or gene therapy for some forms of OI and other bone related diseases. The major focus of the proposal is to utilize a mouse model of human OI type III that has defective synthesis of proalpha2(I) chains to evaluate the feasibility of reversing OI defects by bone marrow stromal cell transplantation or by use of bone marrow stromal cells to deliver therapeutic genes to bone. The aims are: (1) to evaluate the potential of bone marrow stromal cells from normal donor mice transplanted into syngeneic OI mice to synthesize and deposit normal type I collagen in bone matrix of the recipient mice and (2), to test the feasibility of gene therapy by demonstrating that bone marrow stromal cells transduced with collagen genes will home to bone and express the genes in bone. As a prelude to this, bone marrow stromal cells will be established from the normal mice by flushing the marrow from femurs and tibias. The established bone marrow stromal cells will be transduced with a retroviral vector containing LacZ and neo genes (BAG-LacZ Neo) prior to transplanting them to the recipient mice to aid in cell tracking. The bone marrow stromal cells established from normal mice will be injected in the femurs of the irradiated or non-irradiated OI mice and the expression of the proalpha2(I) chains will be determined by immunofluorescence localization using a mouse alpha2(I) anti-serum and cyanogen bromide digestion of the bone collagen of the recipient mice. To test for the gene expression of collagen genes in bone by bone marrow stromal cells, bone marrow stromal cells transduced with the mouse proalpha2(I) cDNA will be injected in the femurs of the oim mice. The presence of the transduced cells in the recipient mice will be assessed by PCR and gene expression will be determined by immunofluorescence localization of the alpha2(I) chain in bone matrix. Future plans will involve determining the amount of collagen made by the transplanted cells in the bones of the recipient mice and the assessment of the bone quality by radiographic, histological and biomechanical analysis of the bones of the recipient mice.

成骨不全（OI）是一组遗传性结缔组织疾病，其共同特征是骨脆性。大多数形式的OI是编码I型胶原蛋白（骨的主要蛋白质）的前α 1和前α 2多肽链的基因突变的结果。本提案的长期目标是测试使用细胞疗法或基因疗法治疗某些形式的OI和其他骨相关疾病的可行性。该提案的主要重点是利用具有proalpha 2（I）链合成缺陷的人III型OI的小鼠模型来评估通过骨髓基质细胞移植或通过使用骨髓基质细胞将治疗基因递送至骨来逆转OI缺陷的可行性。其目标是：（1）评估移植到同基因OI小鼠中的来自正常供体小鼠的骨髓基质细胞合成并存款受体小鼠骨基质中的正常I型胶原的潜力，和（2）通过证明用胶原基因转导的骨髓基质细胞将归巢到骨并在骨中表达基因来测试基因治疗的可行性。作为其前奏，将通过从股骨和胫骨冲洗骨髓从正常小鼠中建立骨髓基质细胞。将建立的骨髓基质细胞用含有LacZ和neo基因（BAG-LacZ Neo）的逆转录病毒载体转导，然后将其移植到受体小鼠中，以帮助细胞追踪。将从正常小鼠建立的骨髓基质细胞注射到经辐射或未经辐射的OI小鼠的股骨中，并使用小鼠α 2（I）抗血清和受体小鼠骨胶原的溴化氰消化，通过免疫荧光定位来测定前α 2（I）链的表达。为了测试骨髓基质细胞在骨中胶原基因的基因表达，将用小鼠proalpha 2（I）cDNA转导的骨髓基质细胞注射到oim小鼠的股骨中。通过PCR评估受体小鼠中转导细胞的存在，并通过骨基质中α 2（I）链的免疫荧光定位确定基因表达。未来的计划将包括确定受体小鼠骨骼中移植细胞产生的胶原蛋白量，以及通过对受体小鼠骨骼的放射学、组织学和生物力学分析来评估骨骼质量。