FN-TPO基因修饰的间充质干细胞对造血细胞扩增和造血重建的影响

发布时间：2017-12-31 01:00

  本文关键词：FN-TPO基因修饰的间充质干细胞对造血细胞扩增和造血重建的影响　出处：《重庆医科大学》2007年硕士论文　论文类型：学位论文

  更多相关文章： 间充质干细胞 纤维连接蛋白 血小板生成素 基因修饰 造血重建

【摘要】： 背景:造血干/祖细胞(hematopoietic stem/progenitor cells, HSPC)移植是治疗多种严重危害人类生命和健康的疾病的有效手段,而HSPC数量不足和骨髓造血诱导微环境( hematopoietic inductive micro-environment,HIM)功能不全或遭到严重破坏是影响HSPC移植的主要因素。间充质干细胞(mesenchymal stem cells, MSCs)是骨髓中除HSPC外的另一种干细胞,它是构成骨髓HIM的基质细胞的主要来源。MSCs能够效的支持造血细胞体外扩增,MSCs与HSPC共移植后可以恢复HIM,有利于促进造血重建;血小板生成素(thrombopoietin, TPO)是一个有效的早期造血促进因子,纤维连接蛋白(fibronectin,FN)是介导HSPC与HIM间相互作用的主要细胞外基质成分之一,在骨髓中两者均主要由MSCs发育而来的基质细胞分泌。大量研究表明,通过基因修饰的方法可以改善MSCs的生物学性状。能否通过基因修饰的方法增强MSCs的造血支持能力,已经逐渐成为研究的热点。 目的:建立体外逆转录病毒载体介导的骨髓MSCs纤维连接蛋白-血小板生成素(FN-TPO)基因修饰体系;观察FN-TPO基因修饰的MSCs对造血细胞扩增和造血重建的影响。 方法:构建携带FN-TPO基因的重组逆转录病毒载体,该载体经包装细胞包装后产生具有感染能力的逆转录病毒,以其对骨髓MSCs进行基因修饰;RT-PCR检测基因修饰后MSCs对FN-TPO基因的表达,台盼兰拒染法检测基因修饰后骨髓MSCs的体外增殖能力,MTT法检测基因修饰后MSCs黏附造血细胞能力,ELISA法检测基因修饰后MSCs分泌TPO能力。将免疫磁珠分选的脐血CD34+细胞按照2.0×10~4/ml的细胞密度接种到基因修饰后骨髓MSCs形成的滋养层上并添加必要的细胞因子(SCF、FLT3-L、IL-11)扩增7 d,流式细胞仪检测CD34+细胞扩增倍数,半固体培养基集落培养检测扩增后细胞集落形成能力。FN-TPO基因修饰后骨髓MSCs联合脐血单个核细胞(mononuclear cells, MNCs)移植亚致死剂量放射线照射的严重联合免疫缺陷(severe combined immunodeficiency disease,SCID)小鼠,观察动物的一般情况、不同时间点外周血常规情况、移植4周后存活动物骨髓及外周血中人CD45+细胞含量和人beta-actin基因嵌合情况,判断FN-TPO基因修饰后骨髓MSCs的造血重建能力。 结果:成功构建携Fn-TPO基因的重组逆转录病毒载体且以该逆转录病毒载体对骨髓MSCs进行体外基因修饰;RT-PCR法检测到FN-TPO基因在骨髓MSCs内能够正常转录;基因修饰后的骨髓MSCs较未修饰细胞体外增殖能力无明显改变[(6.92±0.77)×10~4/ml Vs(7.18±0.89)×10~4/ml,P0.05];黏附造血细胞能力增强(0.188±0.018 Vs 0.167±0.017, P0.01),分泌TPO能力增强(7.46±0.59 ng/ml Vs 5.58±0.37 ng/ml,P0.01)且不随培养时间延长而显著减弱(7.46±0.59 ng/ml Vs 7.15±0.19 ng/ml,P0.05),但受细胞生长状态影响。2.0×10~4脐血CD34+细胞经基因修饰后骨髓MSCs联合必要细胞因子体外扩增7 d,有核细胞数、CD34+细胞比例、BFU-E、CFU-GM及CFU-GEMM分别为(29.9±2.7)×104、(33.3±2.8)%、109.3±4.1、163.7±7.1、13.3±1.5,较对照组明显增加(P0.01)。FN-TPO基因修饰后MSCs联合脐血MNCs共移植能有效重建亚致死剂量放射线照射后的SCID小鼠造血,提高小鼠存活率。 结论: 1.成功建立了携带FN-TPO基因的逆转录病毒载体基因修饰系统,并且成功对骨髓MSCs进行基因修饰; 2. FN-TPO基因修饰后MSCs黏附造血细胞、分泌TPO能力增强; 3. FN-TPO基因修饰后MSCs,能够更有效的扩增脐血CD34+细胞,并有利于维持扩增后细胞集落形成能力; 4. FN-TPO基因修饰后骨髓MSCs联合脐血MNCs共移植有利于经经亚致死剂量放射线破坏后的SCID小鼠造血恢复并提高SCID小鼠的存活率。
[Abstract]:Background: hematopoietic stem / progenitor cells (hematopoietic stem/progenitor cells, HSPC) transplantation is an effective therapy for a variety of serious harm to human life and health of the disease, while HSPC insufficiency and bone marrow hematopoietic microenvironment (hematopoietic inductive, micro-environment, HIM) dysfunction or damage are the main factors influencing HSPC mesenchymal transplantation. Mesenchymal stem cells (mesenchymal stem cells, MSCs) in the bone marrow except HSPC another kind of stem cells, it is amplified in support of hematopoietic cells in vitro.MSCs constitutes a major source of bone marrow stromal cells from HIM to MSCs and HSPC were effective, after transplantation can restore HIM, is conducive to the promotion of hematopoietic reconstruction; thrombopoietin (thrombopoietin TPO) is an effective early hematopoietic growth factors, fibronectin (fibronectin, FN) is mediated mainly by HSPC cell interaction between HIM and matrix into One, the secretion of stromal cells in bone marrow in both development and is mainly composed of MSCs. A large number of studies show that biological traits through genetic modification method can improve the MSCs method. The gene modified MSCs enhanced hematopoietic support capabilities, has gradually become a hot research topic.
Objective: to establish an in vitro retroviral vector mediated bone marrow MSCs fibronectin thrombopoietin (FN-TPO) - modified system; influence and hematopoietic reconstruction observation of FN-TPO gene modified MSCs on proliferation of hematopoietic cells.
Methods: to construct recombinant retroviral vector carrying FN-TPO gene, the vector by packaging cell line with retroviral infection ability, the bone marrow of MSCs gene modification on the expression of MSCs FN-TPO gene; RT-PCR detection of genetically modified, the proliferation ability of bone marrow trypan blue exclusion staining detection of MSCs gene modified the detection method, MTT gene modified MSCs adhesion of hematopoietic cells, the TPO secretion ability of MSCs for detection of ELISA gene modified method. The immunomagnetic separation of umbilical cord blood CD34+ cells according to the cell density of 2 * 10~4/ml were inoculated into the gene modified bone marrow MSCs formation of trophoblast and add necessary cytokines (SCF, FLT3-L, IL-11) 7 d amplification, amplification multiples of CD34+ cells were detected by flow cytometry and semi solid culture medium detected by colony culture after the cell colony formation ability of.FN-TPO gene modified bone marrow MSCs Umbilical cord blood mononuclear cells (mononuclear, cells, MNCs) severe combined immunodeficiency transplantation sublethal dose irradiation (severe combined immunodeficiency disease, SCID) mice, observe the general condition of animal, routine blood at different time points of peripheral transplant survival of animal bone marrow and peripheral blood in human CD45+ cells and the content of human beta-actin chimeric gene after 4 weeks, judge MSCs FN-TPO of bone marrow hematopoietic reconstitution ability after gene modification.
Results: the successful construction of recombinant retroviral vector carrying Fn-TPO gene and in vitro gene modification of the bone marrow MSCs in the retroviral vector; RT-PCR method to detect FN-TPO gene transcription in MSCs in normal bone marrow; gene modified bone marrow MSCs compared with unmodified cell proliferation in vitro had no obvious change in [(6.92 + 0.77) * 10~4/ml Vs (7.18 + 0.89) * 10~4/ml, P0.05]; enhance the ability of hematopoietic cell adhesion (0.188 + 0.018 Vs 0.167 + 0.017, P0.01), enhance the ability of secretion of TPO (7.46 + 0.59 + 0.37 ng/ml Vs 5.58 ng/ml, P0.01) and not with the prolonged culture time was decreased significantly (7.46 + 0.59 ng/ml Vs + 0.19 ng/ml 7.15, P0.05), but by the impact of.2.0 * 10~4 cells of umbilical cord blood CD34+ cells by gene modified bone marrow in vitro MSCs combined with cytokines necessary amplification of the 7 d, the number of nucleated cells, the proportion of CD34+ cells BFU-E, CFU-GM and CFU-GEMM respectively (29.9 + 2 .7) x 104, (33.3 + 2.8)%, 109.3 + 4.1163.7 + 7.1,13.3 + 1.5, significantly increased compared with the control group (P0.01). After.FN-TPO gene modified MSCs combined with cord blood MNCs co transplantation, the hematopoietic rate of SCID mice after sublethal dose irradiation can be effectively reconstructed, and the survival rate of mice can be increased.
Conclusion:
1. the gene modification system of retroviral vector carrying FN-TPO gene was successfully established, and the gene modification of bone marrow MSCs was successfully carried out.
After 2. FN-TPO gene modification, MSCs adhered to the hematopoietic cells, and the ability to secrete TPO was enhanced.
3. FN-TPO gene modified MSCs can more effectively amplify CD34+ cells in umbilical cord blood and help to maintain the colony forming ability after amplification.
4. FN-TPO gene modified bone marrow MSCs combined with cord blood MNCs co transplantation is beneficial to hematopoietic recovery of SCID mice after sublethal dose irradiation and to improve the survival rate of SCID mice.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

【参考文献】

相关期刊论文 前1条

1 周光飚,董颖,王月英,印彤,亢卉,陈竺,陈赛娟;白血病致病基因产物靶向治疗:从急性早幼粒到其他类型白血病[J];中国科学C辑:生命科学;2004年06期

，

本文编号：1357344