本文關(guān)鍵詞：FN-TPO基因修飾的間充質(zhì)干細(xì)胞對造血細(xì)胞擴(kuò)增和造血重建的影響　出處：《重慶醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 間充質(zhì)干細(xì)胞 纖維連接蛋白 血小板生成素 基因修飾 造血重建

【摘要】： 背景:造血干/祖細(xì)胞(hematopoietic stem/progenitor cells, HSPC)移植是治療多種嚴(yán)重危害人類生命和健康的疾病的有效手段,而HSPC數(shù)量不足和骨髓造血誘導(dǎo)微環(huán)境( hematopoietic inductive micro-environment,HIM)功能不全或遭到嚴(yán)重破壞是影響HSPC移植的主要因素。間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)是骨髓中除HSPC外的另一種干細(xì)胞,它是構(gòu)成骨髓HIM的基質(zhì)細(xì)胞的主要來源。MSCs能夠效的支持造血細(xì)胞體外擴(kuò)增,MSCs與HSPC共移植后可以恢復(fù)HIM,有利于促進(jìn)造血重建;血小板生成素(thrombopoietin, TPO)是一個(gè)有效的早期造血促進(jìn)因子,纖維連接蛋白(fibronectin,FN)是介導(dǎo)HSPC與HIM間相互作用的主要細(xì)胞外基質(zhì)成分之一,在骨髓中兩者均主要由MSCs發(fā)育而來的基質(zhì)細(xì)胞分泌。大量研究表明,通過基因修飾的方法可以改善MSCs的生物學(xué)性狀。能否通過基因修飾的方法增強(qiáng)MSCs的造血支持能力,已經(jīng)逐漸成為研究的熱點(diǎn)。 目的:建立體外逆轉(zhuǎn)錄病毒載體介導(dǎo)的骨髓MSCs纖維連接蛋白-血小板生成素(FN-TPO)基因修飾體系;觀察FN-TPO基因修飾的MSCs對造血細(xì)胞擴(kuò)增和造血重建的影響。 方法:構(gòu)建攜帶FN-TPO基因的重組逆轉(zhuǎn)錄病毒載體,該載體經(jīng)包裝細(xì)胞包裝后產(chǎn)生具有感染能力的逆轉(zhuǎn)錄病毒,以其對骨髓MSCs進(jìn)行基因修飾;RT-PCR檢測基因修飾后MSCs對FN-TPO基因的表達(dá),臺盼蘭拒染法檢測基因修飾后骨髓MSCs的體外增殖能力,MTT法檢測基因修飾后MSCs黏附造血細(xì)胞能力,ELISA法檢測基因修飾后MSCs分泌TPO能力。將免疫磁珠分選的臍血CD34+細(xì)胞按照2.0×10~4/ml的細(xì)胞密度接種到基因修飾后骨髓MSCs形成的滋養(yǎng)層上并添加必要的細(xì)胞因子(SCF、FLT3-L、IL-11)擴(kuò)增7 d,流式細(xì)胞儀檢測CD34+細(xì)胞擴(kuò)增倍數(shù),半固體培養(yǎng)基集落培養(yǎng)檢測擴(kuò)增后細(xì)胞集落形成能力。FN-TPO基因修飾后骨髓MSCs聯(lián)合臍血單個(gè)核細(xì)胞(mononuclear cells, MNCs)移植亞致死劑量放射線照射的嚴(yán)重聯(lián)合免疫缺陷(severe combined immunodeficiency disease,SCID)小鼠,觀察動物的一般情況、不同時(shí)間點(diǎn)外周血常規(guī)情況、移植4周后存活動物骨髓及外周血中人CD45+細(xì)胞含量和人beta-actin基因嵌合情況,判斷FN-TPO基因修飾后骨髓MSCs的造血重建能力。 結(jié)果:成功構(gòu)建攜Fn-TPO基因的重組逆轉(zhuǎn)錄病毒載體且以該逆轉(zhuǎn)錄病毒載體對骨髓MSCs進(jìn)行體外基因修飾;RT-PCR法檢測到FN-TPO基因在骨髓MSCs內(nèi)能夠正常轉(zhuǎn)錄;基因修飾后的骨髓MSCs較未修飾細(xì)胞體外增殖能力無明顯改變[(6.92±0.77)×10~4/ml Vs(7.18±0.89)×10~4/ml,P0.05];黏附造血細(xì)胞能力增強(qiáng)(0.188±0.018 Vs 0.167±0.017, P0.01),分泌TPO能力增強(qiáng)(7.46±0.59 ng/ml Vs 5.58±0.37 ng/ml,P0.01)且不隨培養(yǎng)時(shí)間延長而顯著減弱(7.46±0.59 ng/ml Vs 7.15±0.19 ng/ml,P0.05),但受細(xì)胞生長狀態(tài)影響。2.0×10~4臍血CD34+細(xì)胞經(jīng)基因修飾后骨髓MSCs聯(lián)合必要細(xì)胞因子體外擴(kuò)增7 d,有核細(xì)胞數(shù)、CD34+細(xì)胞比例、BFU-E、CFU-GM及CFU-GEMM分別為(29.9±2.7)×104、(33.3±2.8)%、109.3±4.1、163.7±7.1、13.3±1.5,較對照組明顯增加(P0.01)。FN-TPO基因修飾后MSCs聯(lián)合臍血MNCs共移植能有效重建亞致死劑量放射線照射后的SCID小鼠造血,提高小鼠存活率。 結(jié)論: 1.成功建立了攜帶FN-TPO基因的逆轉(zhuǎn)錄病毒載體基因修飾系統(tǒng),并且成功對骨髓MSCs進(jìn)行基因修飾; 2. FN-TPO基因修飾后MSCs黏附造血細(xì)胞、分泌TPO能力增強(qiáng); 3. FN-TPO基因修飾后MSCs,能夠更有效的擴(kuò)增臍血CD34+細(xì)胞,并有利于維持?jǐn)U增后細(xì)胞集落形成能力; 4. FN-TPO基因修飾后骨髓MSCs聯(lián)合臍血MNCs共移植有利于經(jīng)經(jīng)亞致死劑量放射線破壞后的SCID小鼠造血恢復(fù)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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329

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相關(guān)期刊論文 前1條

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