本文關(guān)鍵詞：Ad-HGF基因轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞移植治療兔肢體缺血的實(shí)驗(yàn)研究　出處：《山東大學(xué)》2012年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨髓間充質(zhì)干細(xì)胞 分離 擴(kuò)增 鑒定 血管新生 肝細(xì)胞生長因子 骨髓間充質(zhì)干細(xì)胞 移植 肢體缺血

【摘要】：研究背景 目前治療性血管新生主要有兩種方法,一是局部缺血區(qū)域注射外源性血管生長因子蛋白或基因,二是局部缺血組織移植具有多向分化潛能的干細(xì)胞,干細(xì)胞在缺血缺氧狀態(tài)下分化為血管內(nèi)皮細(xì)胞并產(chǎn)生促血管生長因子來進(jìn)一步促進(jìn)血管新生。 在基因工程范疇內(nèi)聯(lián)合基因治療和細(xì)胞治療,應(yīng)用基因修飾的干細(xì)胞促進(jìn)血管新生是一種極具前景的治療手段。HGF基因修飾的骨髓間充質(zhì)干細(xì)胞可以將治療性蛋白HGF轉(zhuǎn)入缺血的組織中以滿足基因治療的特定需要,安全性更高；它在體內(nèi)同時(shí)發(fā)揮治療性原材料和持續(xù)輸送促血管新生的生長因子HGF兩種作用,與單純的干細(xì)胞治療或基因治療相比更具優(yōu)越性。本課題分三部分,依次探討以下問題：(1)研究兔骨髓間充質(zhì)干細(xì)胞的分離、培養(yǎng)及鑒定方法。(2)利用腺病毒載體將肝細(xì)胞生長因子轉(zhuǎn)染兔骨髓間充質(zhì)干細(xì)胞,流式細(xì)胞儀檢測轉(zhuǎn)染效率及轉(zhuǎn)染對細(xì)胞生物學(xué)特性有無影響,ELISA及免疫組化檢測轉(zhuǎn)染后外源基因HGF的表達(dá)。(3)制備兔后肢缺血模型,HGF基因修飾的BMSCs移植與單純BMSCs移植或HGF基因治療在兔后肢缺血模型中的促血管新生作用及其機(jī)制。 第一部分：兔骨髓間充質(zhì)干細(xì)胞的體外培養(yǎng)及鑒定 目的：研究兔骨髓間充質(zhì)干細(xì)胞的分離、培養(yǎng)及鑒定方法,觀察其生物學(xué)特性。 方法：穿刺股骨采集兔骨髓血,應(yīng)用淋巴分離液密度梯度離心結(jié)合貼壁篩選培養(yǎng)骨髓間充質(zhì)干細(xì)胞。通過觀察細(xì)胞形態(tài)、流式細(xì)胞儀檢測細(xì)胞表面標(biāo)志物以及對其進(jìn)行誘導(dǎo)分化培養(yǎng),對培養(yǎng)的骨髓間充質(zhì)干細(xì)胞進(jìn)行鑒定。 結(jié)果：應(yīng)用淋巴分離液密度梯度離心結(jié)合貼壁篩選培養(yǎng)法得到純化的BMSCs。通過本方法培養(yǎng)得到的BMSCs貼壁生長,呈典型的長梭形,漩渦狀排列；流式細(xì)胞儀檢測細(xì)胞表面標(biāo)志物CD29、CD44表達(dá)陽性,CDllb、CD45表達(dá)陰性；細(xì)胞成骨誘導(dǎo)分化后I型膠原免疫細(xì)胞化學(xué)染色陽性,以上這些結(jié)果符合文獻(xiàn)報(bào)道的BMSCs特征。 結(jié)論：密度梯度離心結(jié)合貼壁篩選培養(yǎng)可以有效的體外擴(kuò)增骨髓間充質(zhì)干細(xì)胞,獲得高度純化的骨髓間充質(zhì)干細(xì)胞。 第二部分：Ad-HGF轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞及體外檢測 目的：利用重組腺病毒載體將肝細(xì)胞生長因子轉(zhuǎn)染兔骨髓間充質(zhì)干細(xì)胞,用流式細(xì)胞術(shù)檢測基因轉(zhuǎn)染效率,ELISA及免疫組化檢測轉(zhuǎn)染后外源基因HGF的表達(dá)。 方法：攜帶HGF基因和綠色熒光蛋白(green fluorescene protein, GFP)的重組腺病毒(adenovirus)載體Ad-HGF、Ad-GFP,均由蘭州軍區(qū)總醫(yī)院哈小琴教授提供。以不同MOI值(multiple of infection,感染復(fù)數(shù))的Ad-GFP病毒液轉(zhuǎn)染BMSCs,流式細(xì)胞檢測表達(dá)GFP陽性的細(xì)胞率。以MOI=150的Ad-HGF轉(zhuǎn)染BMSCs,ELISA檢測及免疫細(xì)胞化學(xué)檢測HGF表達(dá)；流式細(xì)胞儀檢測HGF-BMSC細(xì)胞表面標(biāo)志物以及誘導(dǎo)分化培養(yǎng),以確定基因轉(zhuǎn)染后細(xì)胞生物學(xué)特性有無變化；同時(shí)transwell檢測HGF介導(dǎo)BMSCs細(xì)胞遷移能力。 結(jié)果：Ad-GFP以不同的MOI轉(zhuǎn)染兔BMSCs48h后,表達(dá)GFP的陽性細(xì)胞率有顯著性差異(P0.05)；MOI=150時(shí)表達(dá)GFP陽性的細(xì)胞率達(dá)98%以上,MOI大于150以上時(shí),表達(dá)GFP陽性的細(xì)胞率無顯著性差異(P0.05)；因此選擇MOI=150轉(zhuǎn)染BMSCs。將Ad-HGF以MOI=150轉(zhuǎn)染兔BMSCs后,ELISA及免疫細(xì)胞化學(xué)檢測結(jié)果顯示：細(xì)胞培養(yǎng)上清及胞漿內(nèi)有豐富的HGF蛋白表達(dá),表明Ad-HGF可高效轉(zhuǎn)染BMSCs,BMSCs有HGF蛋白的高表達(dá)。流式細(xì)胞儀檢測及誘導(dǎo)分化培養(yǎng)結(jié)果顯示：腺病毒感染以及HGF基因修飾不改變BMSC表型和分化潛能。同時(shí)實(shí)驗(yàn)結(jié)果顯示HGF對BMSC有極強(qiáng)的趨化作用。 結(jié)論：Ad-GFP可成功轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞,以MOI=150轉(zhuǎn)染BMSCs,轉(zhuǎn)染效率最高,可達(dá)98%以上,轉(zhuǎn)染24h即可見綠色熒光,2-4天轉(zhuǎn)染率最高；表明重組腺病毒作為載體轉(zhuǎn)染BMSCs具有高轉(zhuǎn)染率與穩(wěn)定表達(dá)。肝細(xì)胞生長因子腺病毒表達(dá)載體Ad-HGF轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞不改變BMSCs的固有特性；經(jīng)ELISA及免疫組化檢測,BMSCs可高表達(dá)HGF蛋白。HGF對BMSC有明顯的趨化作用。 第三部分：肝細(xì)胞生長因子基因修飾的骨髓間充質(zhì)干細(xì)胞移植治療兔肢體缺血 目的：比較HGF基因修飾的BMSCs移植與單純BMSCs移植或HGF基因治療在兔后肢缺血模型中的促血管新生作用及其機(jī)制探討。 方法：后肢缺血的新西蘭大白兔模型隨機(jī)分為四組,每組10只,治療方法：a.空腺病毒對照組(Ad-GFP組)：缺血部位肌肉多點(diǎn)注射1×109Pfu Ad-GFP。b.骨髓間充質(zhì)干細(xì)胞(BMSC組)組：缺血部位肌肉多點(diǎn)注射1×107個骨髓間充質(zhì)干細(xì)胞。c.HGF基因治療組(Ad-HGF組)：缺血部位肌肉多點(diǎn)注射1×109Pfu Ad-HGF。d.轉(zhuǎn)染HGF的BMSC治療組(HGF-BMSC組)：缺血部位肌肉多點(diǎn)注射1×107個轉(zhuǎn)染HGF的BMSC。移植后28天行動脈造影及側(cè)枝血管計(jì)數(shù)；免疫組織化學(xué)檢測缺血區(qū)域毛細(xì)血管密度；免疫熒光檢測BMSCs在體內(nèi)的分布及分化；Western-blot檢測組織中HGF、C-met蛋白的表達(dá)；ELISA檢測移植前后兔血清中HGF的濃度。 結(jié)果：造影結(jié)果顯示骨髓干細(xì)胞組(BMSC組)、HGF基因治療組(Ad-HGF組)及HGF-BMSC組血管數(shù)明顯高于空腺病毒對照組(PO.05)；HGF-BMSC組的側(cè)支血管計(jì)數(shù)最高。移植后Ad-GFP標(biāo)記的骨髓間充質(zhì)干細(xì)胞散在分布于各肌纖維的間質(zhì)中,呈現(xiàn)明亮的綠色熒光。在細(xì)胞移植后3周,部分Ad-GFP標(biāo)記的骨髓間充質(zhì)干細(xì)胞表達(dá)內(nèi)皮細(xì)胞標(biāo)記物CD31。Western blot結(jié)果顯示,骨髓干細(xì)胞組(BMSC組)、HGF基因治療組(Ad-HGF組)及HGF-BMSC組HGF表達(dá)明顯高于空腺病毒對照組(P0.05),以HGF-BMSC組的HGF表達(dá)最高。ELISA結(jié)果顯示,HGF-BMSC移植前和移植后1周時(shí),兔血清HGF的濃度無明顯變化(P0.05) 結(jié)論：與單純的BMSC細(xì)胞移植或HGF基因治療相比,肝細(xì)胞生長因子基因修飾的骨髓間充質(zhì)干細(xì)胞可使HGF獲得穩(wěn)定而有效的表達(dá),具有更強(qiáng)的促進(jìn)血管新生能力,從而更加有效地改善肢體缺血。
[Abstract]:Research background
At present, therapeutic angiogenesis mainly has two kinds of methods, one is the local ischemia regional injection of exogenous vascular endothelial growth factor gene or protein, two ischemic tissue transplantation of pluripotent stem cells, stem cells in ischemia hypoxia differentiate into vascular endothelial cells and vascular growth factor to promote angiogenesis.
Genetic therapy and gene engineering in cells within the scope of application of gene modified stem cells to promote angiogenesis is the treatment of.HGF gene modified a promising bone marrow mesenchymal stem cells can be therapeutic protein HGF into ischemic tissue in order to meet the specific needs of gene therapy, it has higher safety; in vivo and play therapeutic raw materials and continuous delivery of angiogenic growth factor HGF two, and the pure stem cell therapy and gene therapy superior. The thesis is divided into three parts, in order to discuss the following issues: (1) study on rabbit bone marrow mesenchymal stem cells were separated and cultured. Identification methods. (2) using adenovirus vector HGF transfection of rabbit bone marrow mesenchymal stem cells, flow cytometry was used to detect the gene transfection efficiency and has no effect on the biological characteristics of cells, ELISA and free The expression of exogenous gene HGF was detected by histochemical examination. (3) rabbit hind limb ischemia model was prepared. The angiogenesis promoting effect and mechanism of HGF gene modified BMSCs transplantation combined with simple BMSCs transplantation or HGF gene therapy on hind limb ischemia model in rabbits were studied.
Part 1: in vitro culture and identification of rabbit bone marrow mesenchymal stem cells
Objective: To study the isolation, culture and identification of rabbit bone marrow mesenchymal stem cells (MSCs), and to observe their biological characteristics.
Methods: rabbit bone marrow puncture femoral blood collection, using lymphocyte separating medium density gradient centrifugation and adherent cultured bone marrow mesenchymal stem cells. Cell morphology was observed by flow cytometry, cell surface markers and differentiation culture on the identification of cultured bone marrow mesenchymal stem cells.
Results: the application of lymphocyte separating medium density gradient centrifugation and adherent culture method to obtain the purified BMSCs. cultured by the method to get the BMSCs adherent, spindle whorls; typically, detection of cell surface marker CD29 by flow cytometry, the expression of CD44, CDllb, CD45 negative expression cells; osteogenic differentiation of type I collagen after immunocytochemical staining, these results are consistent with the characteristics of BMSCs reported in the literature.
Conclusion: density gradient centrifugation combined with adherent screening and culture can effectively amplify bone marrow mesenchymal stem cells in vitro and obtain highly purified bone marrow mesenchymal stem cells.
The second part: Ad-HGF transfection of bone marrow mesenchymal stem cells and in vitro detection
Objective: to transfect rabbit bone marrow mesenchymal stem cells with recombinant adenovirus vector, and transfect rabbit bone marrow mesenchymal stem cells. The gene transfection efficiency was detected by flow cytometry, and the expression of HGF gene after transfection was detected by ELISA and immunohistochemistry.
Methods: carrying HGF gene and green fluorescent protein (green fluorescene, protein, GFP) of the recombinant adenovirus vector Ad-HGF (adenovirus), Ad-GFP, were provided by the General Hospital of Lanzhou military region ha xiao-qin2 professor. With different MOI values (multiple of infection Ad-GFP, MOI) virus was transfected by BMSCs, flow cytometry expression GFP positive cell rate. MOI=150 Ad-HGF was transfected into BMSCs, detect the expression of ELISA and immunocytochemistry HGF; flow cytometry HGF-BMSC cell surface markers and differentiation, gene transfer to determine cell biological characteristics after dyeing has no change; at the same time, Transwell detection of HGF mediated BMSCs cell migration.
Results: Ad-GFP with different MOI after BMSCs48h transfection, the expression of GFP positive cells rate has significant difference (P0.05); MOI=150 the expression of GFP positive cell rate of more than 98%, more than 150 MOI, the expression of GFP positive cells was no significant difference (P0.05); MOI=150 BMSCs. will therefore choose transfection Ad-HGF was transfected with MOI=150 after BMSCs, ELISA and immunocytochemical assay showed that the expression of HGF protein in cell culture supernatant and abundant cytoplasm, showed that Ad-HGF can efficiently transfected BMSCs, BMSCs have the high expression of HGF protein. Flow cytometry results showed: culture and induced differentiation of adenovirus infection and HGF gene modification does not change the BMSC phenotype and differentiation potential. The experimental results show that HGF has strong chemotactic effect on BMSC.
Conclusion: Ad-GFP can be successfully transfected into bone marrow mesenchymal stem cells transfected by MOI=150 BMSCs, the highest transfection efficiency can reach above 98%, 24h transfected with green fluorescence could be found in 2-4 days, the highest transfection rate; recombinant adenovirus as a vector transfected BMSCs with high transfection efficiency and stability. The expression of hepatocyte growth factor adenovirus expressing the inherent characteristics vector Ad-HGF was transfected into bone marrow mesenchymal stem cells did not alter BMSCs; by ELISA and immunohistochemistry, BMSCs high expression of HGF protein.HGF has obvious chemotactic effect on BMSC.
Third part: transplantation of bone marrow mesenchymal stem cells modified by hepatocyte growth factor gene in the treatment of limb ischemia in rabbits
Objective: To compare angiogenesis promoting effect of HGF gene modified BMSCs transplantation with BMSCs transplantation or HGF gene therapy in hind limb ischemia model of rabbits and explore its mechanism.
Methods: a model of New Zealand white rabbits were randomly divided into four groups of hind limb ischemia, 10 rats in each group, treatment methods: A. adenovirus control group (Ad-GFP group): ischemic muscle multi-point injection of 1 * 109Pfu Ad-GFP.b. of bone marrow mesenchymal stem cells group (BMSC group): ischemic muscle multi-point injection of 1 x 107 bone marrow mesenchymal stem cells,.C.HGF gene therapy group (Ad-HGF group): BMSC group ischemic muscle multi-point injection of 1 * 109Pfu Ad-HGF.d. transfected with HGF (HGF-BMSC group): BMSC. transplantation ischemic muscle multi-point injection of 1 * 107 HGF transfected 28 days after arterial angiography and collateral vessel count; immunohistochemical detection of ischemic area capillary density; immunofluorescence detection of BMSCs in distribution and differentiation in vivo; detection of Western-blot in HGF, the expression of C-met protein; concentration in rabbit serum HGF before and after transplantation of ELISA detection.
Results: the imaging results showed that bone marrow stem cells group (BMSC group), HGF gene therapy group (Ad-HGF group) and HGF-BMSC group was significantly higher than the number of vessels empty adenovirus control group (PO.05); HGF-BMSC group of collateral vessels after transplantation. The highest count of Ad-GFP labeled bone marrow mesenchymal stem cells scattered in the muscle fiber the interstitial, showed bright green fluorescence. In 3 weeks after transplantation, Ad-GFP labeled bone marrow mesenchymal stem cells expressed endothelial cell markers CD31.Western blot results showed that bone marrow stem cells group (BMSC group), HGF gene therapy group (Ad-HGF group) and HGF-BMSC group the expression of HGF was significantly higher than that of empty glands the virus control group (P0.05), in HGF-BMSC group the highest expression of HGF.ELISA showed that 1 weeks after HGF-BMSC transplantation, rabbit serum HGF concentration had no significant change (P0.05)
Conclusion: compared with simple BMSC cell transplantation or HGF gene therapy, hepatocyte growth factor gene modified bone marrow mesenchymal stem cells can achieve stable and effective expression of HGF, and have stronger angiogenesis promoting ability, thereby improving limb ischemia more effectively.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R329

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