大鼠骨髓間充質(zhì)干細(xì)胞的分離、培養(yǎng)、鑒定、基因轉(zhuǎn)導(dǎo)及眼內(nèi)移植
發(fā)布時(shí)間：2018-09-03 20:38
【摘要】： 隨著細(xì)胞和基因治療技術(shù)和方法的發(fā)展,載體細(xì)胞或種子細(xì)胞逐漸成為影響細(xì)胞和基因治療成功與否的關(guān)鍵環(huán)節(jié)之一。干細(xì)胞因其具有高度的自我更新能力和多向分化潛能,已成為組織工程中首選的種子細(xì)胞。骨髓間充質(zhì)干細(xì)胞(rMSCs)來(lái)源充足、取材容易、移植后排斥反應(yīng)弱,因此,成為細(xì)胞和基因治療等方面研究的熱點(diǎn)。為了使骨髓間充質(zhì)干細(xì)胞更好地發(fā)揮治療效果,有時(shí)需要對(duì)其進(jìn)行修飾和誘導(dǎo)。病毒載體是最常用的基因傳遞載體,不同類型的病毒載體具有各自的優(yōu)缺點(diǎn),為了篩選能夠高效轉(zhuǎn)導(dǎo)MSCs的載體,本研究對(duì)重組腺病毒Ad5、Ad5F35,腺相關(guān)病毒AAV1和AAV2,以及慢病毒載體對(duì)體外培養(yǎng)的MSCs的轉(zhuǎn)導(dǎo)效率和外源基因表達(dá)水平進(jìn)行了比較,為以后利用MSCs作為種子或載體細(xì)胞進(jìn)行細(xì)胞和基因治療提供了實(shí)驗(yàn)依據(jù)。 遺傳性視網(wǎng)膜變性,包括視網(wǎng)膜光感受器細(xì)胞的丟失,一直是年輕人群致盲的最主要原因。目前還沒有有效的治療方法,盡管過(guò)去有研究表明不同來(lái)源的細(xì)胞包括胎兒視網(wǎng)膜細(xì)胞、胚胎干細(xì)胞、基因修飾過(guò)的RPE細(xì)胞、IPE細(xì)胞,以及MSCs等移植有助于保存視網(wǎng)膜光感受器,有的顯示移植的細(xì)胞可表達(dá)視網(wǎng)膜細(xì)胞的某些標(biāo)記等,利用移植細(xì)胞替代丟失的光感受器細(xì)胞或延緩光感受器細(xì)胞變性凋亡,為治療這種疾病提供了很好的前景,同時(shí)也是對(duì)現(xiàn)今治療方法和技術(shù)的挑戰(zhàn)。在移植細(xì)胞的選擇、受體對(duì)移植細(xì)胞的反應(yīng)、在不同的受體中移植細(xì)胞存活和分化途徑等等,還有大量的科學(xué)問(wèn)題沒有解決。在本研究中,我們主要想解決移植細(xì)胞在正常和視網(wǎng)膜變性大鼠視網(wǎng)膜下的命運(yùn)如何?究竟有多少細(xì)胞能存活、分布怎樣、能存活多久,與受體視網(wǎng)膜間的關(guān)系如何等問(wèn)題。為此,我們利用gfp標(biāo)記大鼠MSCs,熒光顯微鏡直接觀察并記錄移植到正常大鼠視網(wǎng)膜下的MSCs在活體視網(wǎng)膜內(nèi)的存活和分布情況,并且利用遺傳性視網(wǎng)膜變性RCS大鼠動(dòng)物模型研究移植MSCs細(xì)胞在減緩視網(wǎng)膜變性和光感受丟失方面的治療效果。 第一部分不同的重組病毒載體在大鼠骨髓間充質(zhì)干細(xì)胞中的轉(zhuǎn)導(dǎo)效率及基因表達(dá)水平 [目的]:探討不同的重組病毒載體對(duì)體外培養(yǎng)的SD大鼠骨髓間充質(zhì)干細(xì)胞(rMSCs)的感染效率和外源基因表達(dá)水平,為利用骨髓間充質(zhì)干細(xì)胞作細(xì)胞和基因治療提供實(shí)驗(yàn)依據(jù)。 [方法]:采用淋巴細(xì)胞分離液密度梯度離心及體外培養(yǎng)方法分離rMSCs,流式細(xì)胞儀檢測(cè)細(xì)胞表面CD11b、CD45和CD90的表達(dá)鑒定細(xì)胞類型。進(jìn)一步用攜帶綠色熒光蛋白(EGFP)報(bào)告基因的重組Ad5-EGFP、Ad5F35-EGFP、rAAV1/2-EGFP、rAAV2-EGFP及Lentivirus- EGFP感染體外培養(yǎng)的rMSCs,熒光顯微鏡觀察、流式細(xì)胞儀檢測(cè)EGFP陽(yáng)性率及熒光強(qiáng)度。 [結(jié)果]: rMSCs細(xì)胞表面CD11b、CD45和CD90陽(yáng)性率分別為(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天后流式細(xì)胞儀檢測(cè),EGFP陽(yáng)性率分別為(33.6±2.7)%、(88.6±1.0)%及(99.9±0.1)%,熒光平均強(qiáng)度為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天后陽(yáng)性率分別為(96.9±0.4)%、(99.9±0.1)%及(99.7±0.1)%,熒光平均強(qiáng)度為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/細(xì)胞)感染rMSCs, 6天后陽(yáng)性率分別為(0.94±0.31)%及(1.30±0.36)%,和(2.16±0.38)%及(3.90±0.33)%;LV-EGFP按30 (TU/細(xì)胞)感染rMSCs,6天后陽(yáng)性率為(60.5±3.2)%,平均熒光強(qiáng)度為27.0±3.6。 [結(jié)論]: Ad5、Ad5F35及LV能夠有效感染體外培養(yǎng)的rMSCs并表達(dá)外源基因。感染效率與病毒的用量間存在量效關(guān)系。 第二部分骨髓間充質(zhì)干細(xì)胞視網(wǎng)膜下移植后存活狀態(tài)及對(duì)視網(wǎng)膜變性治療作用的觀察 [目的]:研究穩(wěn)定表達(dá)綠色熒光蛋白(GFP)的大鼠骨髓間充質(zhì)干細(xì)胞(rMSCs)移植到正常SD大鼠視網(wǎng)膜下間隙后的存活和分布情況,同時(shí)以RCS大鼠作模型,研究rMSCs視網(wǎng)膜下間隙移植對(duì)光感受器變性的保護(hù)作用。 [方法]:體外培養(yǎng)rMSCs,用攜帶報(bào)告基因EGFP的Lentivirus感染rMSCs,建立穩(wěn)定表達(dá)GFP的rMSCs細(xì)胞(gfp-MSCs),將1×105個(gè)gfp-MSCs移植到成年SD大鼠視網(wǎng)膜下間隙,熒光體視鏡觀察眼底并拍照記錄,分別于移植后2和9周取眼球作冰凍切片,DAPI染色后共聚焦顯微鏡觀察gfp-MSCs細(xì)胞的存活和分布,以及與受體視網(wǎng)膜細(xì)胞的關(guān)系。同時(shí)以RCS大鼠作模型,將1×105個(gè)rMSCs移植到3周齡RCS大鼠左眼視網(wǎng)膜下間隙,右眼注射PBS作為對(duì)照。術(shù)后5周處死動(dòng)物,取眼球做石蠟切片,HE染色,顯微鏡觀察視網(wǎng)膜外核層的厚度及殘留的光感受器的數(shù)目,并拍照記錄。 [結(jié)果]:熒光體視鏡觀察眼底,術(shù)后2周移植的gfp-MSCs在SD大鼠視網(wǎng)膜下間隙已擴(kuò)散開,但隨時(shí)間延長(zhǎng)移植的細(xì)胞逐漸減少,術(shù)后9周僅殘留少量移植細(xì)胞,主要分布在注射位點(diǎn)附近;移植的gfp-MSCs細(xì)胞一直保留在SD大鼠視網(wǎng)膜下間隙,沒有觀察到移植細(xì)胞在神經(jīng)視網(wǎng)膜內(nèi)遷移情況。RCS大鼠眼球石蠟切片顯微鏡觀察顯示細(xì)胞移植眼保留的光感受器數(shù)量明顯較對(duì)側(cè)僅注射PBS的對(duì)照眼多,凋亡細(xì)胞則較對(duì)照眼少。 [結(jié)論]:移植的rMSCs在大鼠視網(wǎng)膜下可存在較長(zhǎng)時(shí)間,rMSCs的移植可延緩RCS大鼠視網(wǎng)膜光感受器變性,為治療視網(wǎng)膜變性提供了新的途徑。
[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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329;R779.6
本文編號(hào)：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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329;R779.6
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
1 侯玲玲,鄭敏,王冬梅,袁紅豐,李海民,陳琳,白慈賢,張涌,裴雪濤;人骨髓間充質(zhì)干細(xì)胞在成年大鼠腦內(nèi)的遷移及分化[J];生理學(xué)報(bào);2003年02期
2 王運(yùn)濤,鄭啟新,郭曉東,劉勇,李忠玉,吳永超;大鼠骨髓間充質(zhì)干細(xì)胞的優(yōu)化獲取及生物學(xué)鑒定[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2003年05期
3 吳繼紅;張圣海;劉焰;陳霞芳;徐萍;吳小兵;黃倩;;1型及2型腺相關(guān)病毒對(duì)視網(wǎng)膜細(xì)胞轉(zhuǎn)導(dǎo)效率的比較研究[J];中華醫(yī)學(xué)雜志;2006年40期
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