大鼠骨髓间充质干细胞的分离、培养、鉴定、基因转导及眼内移植
发布时间：2018-09-03 20:38
【摘要】： 随着细胞和基因治疗技术和方法的发展,载体细胞或种子细胞逐渐成为影响细胞和基因治疗成功与否的关键环节之一。干细胞因其具有高度的自我更新能力和多向分化潜能,已成为组织工程中首选的种子细胞。骨髓间充质干细胞(rMSCs)来源充足、取材容易、移植后排斥反应弱,因此,成为细胞和基因治疗等方面研究的热点。为了使骨髓间充质干细胞更好地发挥治疗效果,有时需要对其进行修饰和诱导。病毒载体是最常用的基因传递载体,不同类型的病毒载体具有各自的优缺点,为了筛选能够高效转导MSCs的载体,本研究对重组腺病毒Ad5、Ad5F35,腺相关病毒AAV1和AAV2,以及慢病毒载体对体外培养的MSCs的转导效率和外源基因表达水平进行了比较,为以后利用MSCs作为种子或载体细胞进行细胞和基因治疗提供了实验依据。 遗传性视网膜变性,包括视网膜光感受器细胞的丢失,一直是年轻人群致盲的最主要原因。目前还没有有效的治疗方法,尽管过去有研究表明不同来源的细胞包括胎儿视网膜细胞、胚胎干细胞、基因修饰过的RPE细胞、IPE细胞,以及MSCs等移植有助于保存视网膜光感受器,有的显示移植的细胞可表达视网膜细胞的某些标记等,利用移植细胞替代丢失的光感受器细胞或延缓光感受器细胞变性凋亡,为治疗这种疾病提供了很好的前景,同时也是对现今治疗方法和技术的挑战。在移植细胞的选择、受体对移植细胞的反应、在不同的受体中移植细胞存活和分化途径等等,还有大量的科学问题没有解决。在本研究中,我们主要想解决移植细胞在正常和视网膜变性大鼠视网膜下的命运如何?究竟有多少细胞能存活、分布怎样、能存活多久,与受体视网膜间的关系如何等问题。为此,我们利用gfp标记大鼠MSCs,荧光显微镜直接观察并记录移植到正常大鼠视网膜下的MSCs在活体视网膜内的存活和分布情况,并且利用遗传性视网膜变性RCS大鼠动物模型研究移植MSCs细胞在减缓视网膜变性和光感受丢失方面的治疗效果。 第一部分不同的重组病毒载体在大鼠骨髓间充质干细胞中的转导效率及基因表达水平 [目的]:探讨不同的重组病毒载体对体外培养的SD大鼠骨髓间充质干细胞(rMSCs)的感染效率和外源基因表达水平,为利用骨髓间充质干细胞作细胞和基因治疗提供实验依据。 [方法]:采用淋巴细胞分离液密度梯度离心及体外培养方法分离rMSCs,流式细胞仪检测细胞表面CD11b、CD45和CD90的表达鉴定细胞类型。进一步用携带绿色荧光蛋白(EGFP)报告基因的重组Ad5-EGFP、Ad5F35-EGFP、rAAV1/2-EGFP、rAAV2-EGFP及Lentivirus- EGFP感染体外培养的rMSCs,荧光显微镜观察、流式细胞仪检测EGFP阳性率及荧光强度。 [结果]: rMSCs细胞表面CD11b、CD45和CD90阳性率分别为(14.1±3.3)%,(1.1±0.4)%和(82.3±5.7)%。Ad5-EGFP按10、100和1000 MOI感染rMSCs,2天后流式细胞仪检测,EGFP阳性率分别为(33.6±2.7)%、(88.6±1.0)%及(99.9±0.1)%,荧光平均强度为4.4±0.3、39.8±1.5及811.4±3.9 ; Ad5F35-EGFP按10、100和1000 MOI感染rMSCs,2天后阳性率分别为(96.9±0.4)%、(99.9±0.1)%及(99.7±0.1)%,荧光平均强度为369.3±14.8、895.4±7.5及703.2±38.4;rAAV1/2-EGFP及rAAV2-EGFP按1×104和1×105 (vg/细胞)感染rMSCs, 6天后阳性率分别为(0.94±0.31)%及(1.30±0.36)%,和(2.16±0.38)%及(3.90±0.33)%;LV-EGFP按30 (TU/细胞)感染rMSCs,6天后阳性率为(60.5±3.2)%,平均荧光强度为27.0±3.6。 [结论]: Ad5、Ad5F35及LV能够有效感染体外培养的rMSCs并表达外源基因。感染效率与病毒的用量间存在量效关系。 第二部分骨髓间充质干细胞视网膜下移植后存活状态及对视网膜变性治疗作用的观察 [目的]:研究稳定表达绿色荧光蛋白(GFP)的大鼠骨髓间充质干细胞(rMSCs)移植到正常SD大鼠视网膜下间隙后的存活和分布情况,同时以RCS大鼠作模型,研究rMSCs视网膜下间隙移植对光感受器变性的保护作用。 [方法]:体外培养rMSCs,用携带报告基因EGFP的Lentivirus感染rMSCs,建立稳定表达GFP的rMSCs细胞(gfp-MSCs),将1×105个gfp-MSCs移植到成年SD大鼠视网膜下间隙,荧光体视镜观察眼底并拍照记录,分别于移植后2和9周取眼球作冰冻切片,DAPI染色后共聚焦显微镜观察gfp-MSCs细胞的存活和分布,以及与受体视网膜细胞的关系。同时以RCS大鼠作模型,将1×105个rMSCs移植到3周龄RCS大鼠左眼视网膜下间隙,右眼注射PBS作为对照。术后5周处死动物,取眼球做石蜡切片,HE染色,显微镜观察视网膜外核层的厚度及残留的光感受器的数目,并拍照记录。 [结果]:荧光体视镜观察眼底,术后2周移植的gfp-MSCs在SD大鼠视网膜下间隙已扩散开,但随时间延长移植的细胞逐渐减少,术后9周仅残留少量移植细胞,主要分布在注射位点附近;移植的gfp-MSCs细胞一直保留在SD大鼠视网膜下间隙,没有观察到移植细胞在神经视网膜内迁移情况。RCS大鼠眼球石蜡切片显微镜观察显示细胞移植眼保留的光感受器数量明显较对侧仅注射PBS的对照眼多,凋亡细胞则较对照眼少。 [结论]:移植的rMSCs在大鼠视网膜下可存在较长时间,rMSCs的移植可延缓RCS大鼠视网膜光感受器变性,为治疗视网膜变性提供了新的途径。
[Abstract]:With the development of cell and gene therapy technology and methods, carrier cells or seed cells have become one of the key factors that affect the success of cell and gene therapy. Stem cells have become the preferred seed cells in tissue engineering because of their high self-renewal ability and multi-directional differentiation potential. Bone marrow mesenchymal stem cells (rMSCs) In order to make bone marrow mesenchymal stem cells play a better therapeutic effect, it is sometimes necessary to modify and induce them. Viral vectors are the most commonly used gene delivery vectors, and different types of viral vectors have their own characteristics. In order to screen the vector that can transduce MSCs efficiently, we compared the transduction efficiency of recombinant adenovirus Ad5, Ad5F35, adeno-associated virus AAV1 and AAV2, and lentiviral vector to MSCs cultured in vitro and the expression level of exogenous genes, so as to provide cell and gene therapy with MSCs as seed or carrier cells in the future. Experimental basis.
Hereditary retinal degeneration, including loss of retinal photoreceptor cells, has been the leading cause of blindness in young people. There is no effective treatment, although previous studies have shown that cells from different sources include fetal retinal cells, embryonic stem cells, genetically modified RPE cells, IPE cells, and MSCs. Transplantation helps to preserve the photoreceptors of the retina. Some cells show that transplanted cells can express some markers of retinal cells. The use of transplanted cells to replace lost photoreceptor cells or delay the degeneration and apoptosis of photoreceptor cells provides a good prospect for the treatment of this disease, but also a challenge to current treatment methods and techniques. In this study, we mainly want to solve the fate of transplanted cells under the retina of normal and retinal degeneration rats, and how many cells can survive. To solve these problems, we used GFP to label rat MSCs, observed and recorded the survival and distribution of MSCs transplanted into normal rat retina in vivo by fluorescence microscopy, and studied the animal model of hereditary retinal degeneration in RCS rats. Transplantation of MSCs cells in the treatment of retinal degeneration and photoreceptor loss.
Part I Transduction efficiency and gene expression level of different recombinant viral vectors in rat bone marrow mesenchymal stem cells
[Objective] To investigate the infection efficiency and exogenous gene expression level of different recombinant viral vectors in vitro cultured bone marrow mesenchymal stem cells (rMSCs) of SD rats, and to provide experimental basis for the use of bone marrow mesenchymal stem cells as cells and gene therapy.
[Methods] rMSCs were isolated by density gradient centrifugation of lymphocyte isolates and cultured in vitro. The expression of CD11b, CD45 and CD90 on the cell surface was detected by flow cytometry to identify the cell type. Further, the recombinant Ad5-EGFP, Ad5F35-EGFP, rAAV1/2-EGFP, rAAV2-EGFP and Lentivirus-EGFP were used to infect the cells. RMSCs was cultured in vitro and observed by fluorescence microscopy. The positive rate and intensity of EGFP were detected by flow cytometry.
[Results]: The positive rates of CD11b, CD45 and CD90 on the surface of rMSCs cells were (14.1 ((14.1 +3.3)%, (1.1 (1.1 +0.4)% and (82.3 ((82.3 +5.7)%%% (14.1 ((1.1 +0.4)%)%, (1.1 ((1.1 +0.4.4 (0.4.4 +0.3,3.3.3.3,39.8.1.8.8.1.5,39.8.1.8.5 and 1 000 MOI infected with Ad5,100,100 and 1000MOI, respectively, respectively. The positive rates of EGFPwere (33.6 ((88.6 35-EGF The positive rates of rMSCs infected with 10,100 and 1000MOI at 10,100 and 1000MOI at 2 days were (96.9 [(96.9 [0.4)%%,(99.9 [(99.9 [0.1)%%,(99.7 [(99.7 [0.1)%)%,(99.7 [(99.7 [(99.7 [) 0.1 69.3 [14.8, 895.4 [7.4] 7.5 and 703.2.2.2 [(38.4)%,(69.3 [(69.3 [14.3], 895.5.4 [7.4 [7.5]] 7.5 and 703.2.16 + 0.38)% and LV-EGFP infected rMSCs at 30 (TU/cell) and the positive rate was (60.5+3.2)% 6 days later, with an average fluorescence intensity of 27.0+3.6.
[Conclusion] Ad5, Ad5F35 and LV can effectively infect rMSCs cultured in vitro and express foreign genes.
Part 2 Survival status of bone marrow mesenchymal stem cells after subretinal transplantation and its therapeutic effect on retinal degeneration
[Objective] To study the survival and distribution of rat bone marrow mesenchymal stem cells (rMSCs) stably expressing green fluorescent protein (GFP) after transplantation into the subretinal space of normal SD rats, and to study the protective effect of rMSCs transplantation in the subretinal space of RCS rats on photoreceptor degeneration.
[Methods] rMSCs were cultured in vitro and infected with entivirus carrying EGFP to establish stable GFP-expressing rMSCs. One hundred and fifty-five gfp-MSCs were transplanted into the subretinal space of adult SD rats. The fundus was observed by fluorescence stereoscopy and photographed. The eyeballs were taken for frozen section 2 and 9 weeks after transplantation, and then copolymerized with DAPI staining. The survival and distribution of gfp-MSCs and the relationship between them and the retinal cells of the recipients were observed by focal microscopy. At the same time, 1 105 rMSCs were transplanted into the subretinal space of the left eye of RCS rats aged 3 weeks, and PBS was injected into the right eye as control. The thickness of nuclear layer and the number of residual photoreceptors were recorded.
[Results] After 2 weeks, the transplanted gfp-MSCs had diffused into the subretinal space of SD rats, but the number of transplanted cells decreased gradually with time. Only a few transplanted cells remained near the injection site at 9 weeks after operation. The transplanted gfp-MSCs cells remained in the subretinal space of SD rats without any observation. The migration of transplanted cells in the retina was observed. The number of photoreceptors in the transplanted eyes of RCS rats was significantly more than that of the control eyes only injected PBS, while the number of apoptotic cells was less than that of the control eyes.
[Conclusion] The transplanted rMSCs can survive for a long time under the retina of rats. The transplantation of rMSCs can delay the degeneration of retinal photoreceptors in RCS rats and provide a new way for the treatment of retinal degeneration.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329;R779.6
本文编号：2221102
[Abstract]:With the development of cell and gene therapy technology and methods, carrier cells or seed cells have become one of the key factors that affect the success of cell and gene therapy. Stem cells have become the preferred seed cells in tissue engineering because of their high self-renewal ability and multi-directional differentiation potential. Bone marrow mesenchymal stem cells (rMSCs) In order to make bone marrow mesenchymal stem cells play a better therapeutic effect, it is sometimes necessary to modify and induce them. Viral vectors are the most commonly used gene delivery vectors, and different types of viral vectors have their own characteristics. In order to screen the vector that can transduce MSCs efficiently, we compared the transduction efficiency of recombinant adenovirus Ad5, Ad5F35, adeno-associated virus AAV1 and AAV2, and lentiviral vector to MSCs cultured in vitro and the expression level of exogenous genes, so as to provide cell and gene therapy with MSCs as seed or carrier cells in the future. Experimental basis.
Hereditary retinal degeneration, including loss of retinal photoreceptor cells, has been the leading cause of blindness in young people. There is no effective treatment, although previous studies have shown that cells from different sources include fetal retinal cells, embryonic stem cells, genetically modified RPE cells, IPE cells, and MSCs. Transplantation helps to preserve the photoreceptors of the retina. Some cells show that transplanted cells can express some markers of retinal cells. The use of transplanted cells to replace lost photoreceptor cells or delay the degeneration and apoptosis of photoreceptor cells provides a good prospect for the treatment of this disease, but also a challenge to current treatment methods and techniques. In this study, we mainly want to solve the fate of transplanted cells under the retina of normal and retinal degeneration rats, and how many cells can survive. To solve these problems, we used GFP to label rat MSCs, observed and recorded the survival and distribution of MSCs transplanted into normal rat retina in vivo by fluorescence microscopy, and studied the animal model of hereditary retinal degeneration in RCS rats. Transplantation of MSCs cells in the treatment of retinal degeneration and photoreceptor loss.
Part I Transduction efficiency and gene expression level of different recombinant viral vectors in rat bone marrow mesenchymal stem cells
[Objective] To investigate the infection efficiency and exogenous gene expression level of different recombinant viral vectors in vitro cultured bone marrow mesenchymal stem cells (rMSCs) of SD rats, and to provide experimental basis for the use of bone marrow mesenchymal stem cells as cells and gene therapy.
[Methods] rMSCs were isolated by density gradient centrifugation of lymphocyte isolates and cultured in vitro. The expression of CD11b, CD45 and CD90 on the cell surface was detected by flow cytometry to identify the cell type. Further, the recombinant Ad5-EGFP, Ad5F35-EGFP, rAAV1/2-EGFP, rAAV2-EGFP and Lentivirus-EGFP were used to infect the cells. RMSCs was cultured in vitro and observed by fluorescence microscopy. The positive rate and intensity of EGFP were detected by flow cytometry.
[Results]: The positive rates of CD11b, CD45 and CD90 on the surface of rMSCs cells were (14.1 ((14.1 +3.3)%, (1.1 (1.1 +0.4)% and (82.3 ((82.3 +5.7)%%% (14.1 ((1.1 +0.4)%)%, (1.1 ((1.1 +0.4.4 (0.4.4 +0.3,3.3.3.3,39.8.1.8.8.1.5,39.8.1.8.5 and 1 000 MOI infected with Ad5,100,100 and 1000MOI, respectively, respectively. The positive rates of EGFPwere (33.6 ((88.6 35-EGF The positive rates of rMSCs infected with 10,100 and 1000MOI at 10,100 and 1000MOI at 2 days were (96.9 [(96.9 [0.4)%%,(99.9 [(99.9 [0.1)%%,(99.7 [(99.7 [0.1)%)%,(99.7 [(99.7 [(99.7 [) 0.1 69.3 [14.8, 895.4 [7.4] 7.5 and 703.2.2.2 [(38.4)%,(69.3 [(69.3 [14.3], 895.5.4 [7.4 [7.5]] 7.5 and 703.2.16 + 0.38)% and LV-EGFP infected rMSCs at 30 (TU/cell) and the positive rate was (60.5+3.2)% 6 days later, with an average fluorescence intensity of 27.0+3.6.
[Conclusion] Ad5, Ad5F35 and LV can effectively infect rMSCs cultured in vitro and express foreign genes.
Part 2 Survival status of bone marrow mesenchymal stem cells after subretinal transplantation and its therapeutic effect on retinal degeneration
[Objective] To study the survival and distribution of rat bone marrow mesenchymal stem cells (rMSCs) stably expressing green fluorescent protein (GFP) after transplantation into the subretinal space of normal SD rats, and to study the protective effect of rMSCs transplantation in the subretinal space of RCS rats on photoreceptor degeneration.
[Methods] rMSCs were cultured in vitro and infected with entivirus carrying EGFP to establish stable GFP-expressing rMSCs. One hundred and fifty-five gfp-MSCs were transplanted into the subretinal space of adult SD rats. The fundus was observed by fluorescence stereoscopy and photographed. The eyeballs were taken for frozen section 2 and 9 weeks after transplantation, and then copolymerized with DAPI staining. The survival and distribution of gfp-MSCs and the relationship between them and the retinal cells of the recipients were observed by focal microscopy. At the same time, 1 105 rMSCs were transplanted into the subretinal space of the left eye of RCS rats aged 3 weeks, and PBS was injected into the right eye as control. The thickness of nuclear layer and the number of residual photoreceptors were recorded.
[Results] After 2 weeks, the transplanted gfp-MSCs had diffused into the subretinal space of SD rats, but the number of transplanted cells decreased gradually with time. Only a few transplanted cells remained near the injection site at 9 weeks after operation. The transplanted gfp-MSCs cells remained in the subretinal space of SD rats without any observation. The migration of transplanted cells in the retina was observed. The number of photoreceptors in the transplanted eyes of RCS rats was significantly more than that of the control eyes only injected PBS, while the number of apoptotic cells was less than that of the control eyes.
[Conclusion] The transplanted rMSCs can survive for a long time under the retina of rats. The transplantation of rMSCs can delay the degeneration of retinal photoreceptors in RCS rats and provide a new way for the treatment of retinal degeneration.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329;R779.6
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2 王运涛,郑启新,郭晓东,刘勇,李忠玉,吴永超;大鼠骨髓间充质干细胞的优化获取及生物学鉴定[J];华中科技大学学报(医学版);2003年05期
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