自体成体干细胞移植治疗兔眼外肌机械性损伤的实验研究
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摘要
第一章肌卫星细胞的体外分离、原代培养和免疫荧光特性
目的:研究眼外肌卫星细胞的体外生长特点;比较其与骨骼肌卫星细胞免疫荧光特性的异同。
方法:采用肌块组织酶原消化法体外分离并原代培养眼外肌卫星细胞和骨骼肌卫星细胞,光镜下动态观察两种细胞的生长变化。免疫荧光法检测肌管及新生纤维标记物(新生型肌球蛋白重链)、肌源性标记物(结蛋白、肌生成素)、肌纤维母细胞标记物(α-平滑肌肌动蛋白)、肌细胞标记物(肌球蛋白轻链)、眼外肌特异性肌球蛋白等5类6种抗体在细胞的染色情况,比较两种细胞免疫荧光特性的异同。
结果:眼外肌卫星细胞与骨骼肌卫星细胞在体外生长和增殖的过程中形态学变化一致:肌纤维种植后第1天起大量析出,1周内培养皿内呈小梭形细胞样贴壁、迅速增殖并呈相互融合趋势,2周后肌纤维旁赘生大细胞团和细胞团外放射状的肌管形成(表现为新生型肌球蛋白重链的染色阳性),并形成三维的网状结构(表现为α-平滑肌肌动蛋白染色阳性)。两种细胞的免疫荧光染色:结蛋白、肌生成素α-平滑肌肌动蛋白、肌球蛋白轻链均表达阳性;眼外肌特异性肌球蛋白抗体的染色结果,在眼外肌卫星细胞表达阳性而骨骼肌卫星细胞表达阴性。
结论:眼外肌卫星细胞可用酶消化法成功提取,其细胞生长的形态学特点与免疫荧光特性与骨骼肌卫星细胞一致。这两种肌源性干细胞都具有体外增殖并向成肌方向定向分化的能力。
第二章机械性损伤兔眼外肌动物模型的建立和自体成体干细胞移植对模型动物的治疗
目的:建立机械性损伤大白兔眼外肌的动物模型,观察眼外肌自然修复规律;用自体成体干细胞移植治疗模型动物,观察治疗后眼外肌的修复规律。
方法:用割裂法建立眼外肌机械性损伤模型,取肌肉标本行冰冻切片后给予Masson染色、影像学定量(ImageProPlus软件),观察切片中肌肉组织含量(muscle,M)、胶原含量(collagen,C)、非胶原含量(non-collagen,NC)及细胞核数量(nuclei,N)等指标的变化。体外分离扩增骨髓间充质干细胞(简称骨髓干细胞)。将自体骨髓干细胞和肌卫星细胞用CM-DiI(长效细胞示踪剂)标记并移植到受伤区域,观察外源性供体细胞注入眼外肌组织后的转归及组织相容性。随后用同样两种细胞移植于受伤1周的模型动物,设立移植组2组(两种干细胞移植)和对照组3组(阳性对照组、阴性对照1组和阴性对照2组)等5组,分析各组目标肌肉的组织学变化,对M、C、NC、N等指标进行统计学分析。
结果:眼外肌机械性受损后初期以胶原组织增生为特点,1周后纤维化程度逐渐加重:2周时广泛纤维化和疤痕形成,细胞核数量也达到高峰,纤维化进行性加重。CM-DiI标记的移植细胞在体内存留至少14天:第1天即与受体组织相融合,第14天注射区域大片肌纤维着染。各时间点各组肌肉组织含量相对恒定。移植组与阳性对照组相比,后者胶原和非胶原组织含量明显增加,有统计学差异P＜0.05。移植组与阴性对照组间没有统计学差异P＞0.05。5组术后两周胶原与非胶原组织的增生与前期比均达到高峰,有明显统计学差异P＜0.05。细胞核数量表现波动,在移植后第7天为低值,第3和第14天均为高值。骨髓干细胞移植和肌卫星细胞移植对眼外肌修复过程的影响没有统计学差异P＞0.05。
结论:机械性眼外肌损伤导致肌肉及周围结缔组织的退行性变、纤维化和疤痕形成。骨髓间充质干细胞和骨骼肌卫星细胞移植到眼外肌微环境后均有很好的局部组织相容性,且能明显减轻机械性损伤导致的肌肉退形性变,促进再生和抗纤维化的修复过程。
Chapter 1:Isolation,primary culture,and characterizing the immunofluorescence of muscle satellite cells
Objective:To identify the morphological changes of extraocular muscle satellite cells(ESC)in vitro.To compare the expression of immunofluorescene of ESC with that of skeletal muscle satellite cells (SSC).
Methods:To isolate and culture ESC in vitro by muscle block enzyme digestion.Light microscopy was used to monitor the morphological changes of the cells during the period of their growth.And to compare immunofluorescene characters of ESC with that of SSC, which are the marker of myotube(Mosin heavy chain-neonatal,MyHC-n), myogenic markers(Desmin and Myogenin,F5D),marker of myoblast (α-smooth muscle actin,α-SMA),marker of striated muscle(light meromyosin,MF20),and marker of extraocular muscle(myosin heavy chain-extraocular specific,N2.261).
Results:ESC and SSC had the same appearance on the morphological changes while growing in vitro.They came out from first day after seeding the muscle fibers.The cells adhered to the matrix in 1 week,expanded,and fused to each other.Cell mass also found after 2 weeks,which located nearby one of the residual independent myofibril and myotube formed radially on it(MyHC-n were stained positive). 3-dimensional meshwork formed shortly after myotube merged,which showedα-SMA positive.Both ESC and SSC stained as positive results in those antibodies,such as Desmin,Myogenin,α-SMA,and MF20.It was only differed in N2.261 staining between ESC and SSC,while ESC+ and SSC-.
Conclusion:ESC has been isolated and cultured in vitro successfully. The morphological and immuno-fluorescene characters are intact features of ESC just as SSC are.Both of the two myogenic cells are able to be self-expanded and differentiated to form myofibril,they have same features of regeneration and myogenic potential in vitro.
Chapter 2:Establishing laceration model of extraocular muscle in rabbit and treating those rabbits by transplanting autologous adult stem cells
Objective:To establish laceration model of extraocular muscle in rabbits and find its nature of wound healing.To evaluate the effect of auto-transplantation by adult stem cell on the process of wound healing in this model.
Methods:Established the model of wound healing in eye by lacerating the extraocular muscle.The muscle samples were harvested, frozen,sliced and stained by Masson's trichrome staining.Imaging processing(using software of ImageProPlus,IPP)was used to quantify the process of natural healing.4 targets were analyzed.They were: muscle(M,collagen(C),non-collagen(NC)and nuclei(N).Bone marrow mesenchymal stem cells(MSC)were isolated,cultured and expanded in vitro by a putative method.ESC and SSC were labeled by Cell tracker CM-DiI,and transplanted and traced the destiny of transplanted cells in the traumatic area.Both ESC and SSC transplantation were performed to the wound area 1 week after trauma.5 groups were set up as two treatment groups(MSC and SSC transplantation)and three control groups,which were:negative control group 1 and 2,positive control group.The morphological changes were recorded by Masson's staining and analyzed by IPP.
Results:Laceration accelerated the formation of collagen in early stage of wound healing process.Apparent fibrosis showed up just one week after being suffered laceration.Diffused fibrosis and scar emerged dramatically after two weeks.Number of nuclei reached its peak at day 14.Inflammatory cells and fibrosis developed heavier than ever.CM-DiI labeled cells could fuse to adjacent muscles from first day on and stayed in site at least for 14 days.Positive stained muscle fibers were found abounded in the wound area at day 14.Cell transplantation resulted of consistent muscle area at different time points of all groups.In comparison of treatment groups and positive control groups,the later got significant higher amount of non-muscle connective tissue(including collagen and non-collagen part)statistically(P＜0.05),while they showed the same level of increasing amount of non-muscle tissue(P＞0.05). Non-muscle tissue proliferated dramatically after 2 weeks of transplantation while compared with earlier stages(P＜0.05)in all groups. Number of nuclei fluctuated within these 2 weeks,which showed low level at day 7 and high level at day 3 or 14.SSC and MSC played similar way in benefiting the mechanical trauma of extraocular muscle.
Conclusions:Laceration could lead to degeneration of both muscle and the surrounding connective tissue,which act as fibrosis and scar formation.Both MSC and SSC has good consistent with traumatic muscle in ocular environment.And they could slow down the development of fibrosis and scar formation,and also benefit the process of wound healing of extraocular muscle in the way of upgrading the regeneration and downgrading the fibrosis.
目的:研究眼外肌卫星细胞的体外生长特点;比较其与骨骼肌卫星细胞免疫荧光特性的异同。
方法:采用肌块组织酶原消化法体外分离并原代培养眼外肌卫星细胞和骨骼肌卫星细胞,光镜下动态观察两种细胞的生长变化。免疫荧光法检测肌管及新生纤维标记物(新生型肌球蛋白重链)、肌源性标记物(结蛋白、肌生成素)、肌纤维母细胞标记物(α-平滑肌肌动蛋白)、肌细胞标记物(肌球蛋白轻链)、眼外肌特异性肌球蛋白等5类6种抗体在细胞的染色情况,比较两种细胞免疫荧光特性的异同。
结果:眼外肌卫星细胞与骨骼肌卫星细胞在体外生长和增殖的过程中形态学变化一致:肌纤维种植后第1天起大量析出,1周内培养皿内呈小梭形细胞样贴壁、迅速增殖并呈相互融合趋势,2周后肌纤维旁赘生大细胞团和细胞团外放射状的肌管形成(表现为新生型肌球蛋白重链的染色阳性),并形成三维的网状结构(表现为α-平滑肌肌动蛋白染色阳性)。两种细胞的免疫荧光染色:结蛋白、肌生成素α-平滑肌肌动蛋白、肌球蛋白轻链均表达阳性;眼外肌特异性肌球蛋白抗体的染色结果,在眼外肌卫星细胞表达阳性而骨骼肌卫星细胞表达阴性。
结论:眼外肌卫星细胞可用酶消化法成功提取,其细胞生长的形态学特点与免疫荧光特性与骨骼肌卫星细胞一致。这两种肌源性干细胞都具有体外增殖并向成肌方向定向分化的能力。
第二章机械性损伤兔眼外肌动物模型的建立和自体成体干细胞移植对模型动物的治疗
目的:建立机械性损伤大白兔眼外肌的动物模型,观察眼外肌自然修复规律;用自体成体干细胞移植治疗模型动物,观察治疗后眼外肌的修复规律。
方法:用割裂法建立眼外肌机械性损伤模型,取肌肉标本行冰冻切片后给予Masson染色、影像学定量(ImageProPlus软件),观察切片中肌肉组织含量(muscle,M)、胶原含量(collagen,C)、非胶原含量(non-collagen,NC)及细胞核数量(nuclei,N)等指标的变化。体外分离扩增骨髓间充质干细胞(简称骨髓干细胞)。将自体骨髓干细胞和肌卫星细胞用CM-DiI(长效细胞示踪剂)标记并移植到受伤区域,观察外源性供体细胞注入眼外肌组织后的转归及组织相容性。随后用同样两种细胞移植于受伤1周的模型动物,设立移植组2组(两种干细胞移植)和对照组3组(阳性对照组、阴性对照1组和阴性对照2组)等5组,分析各组目标肌肉的组织学变化,对M、C、NC、N等指标进行统计学分析。
结果:眼外肌机械性受损后初期以胶原组织增生为特点,1周后纤维化程度逐渐加重:2周时广泛纤维化和疤痕形成,细胞核数量也达到高峰,纤维化进行性加重。CM-DiI标记的移植细胞在体内存留至少14天:第1天即与受体组织相融合,第14天注射区域大片肌纤维着染。各时间点各组肌肉组织含量相对恒定。移植组与阳性对照组相比,后者胶原和非胶原组织含量明显增加,有统计学差异P＜0.05。移植组与阴性对照组间没有统计学差异P＞0.05。5组术后两周胶原与非胶原组织的增生与前期比均达到高峰,有明显统计学差异P＜0.05。细胞核数量表现波动,在移植后第7天为低值,第3和第14天均为高值。骨髓干细胞移植和肌卫星细胞移植对眼外肌修复过程的影响没有统计学差异P＞0.05。
结论:机械性眼外肌损伤导致肌肉及周围结缔组织的退行性变、纤维化和疤痕形成。骨髓间充质干细胞和骨骼肌卫星细胞移植到眼外肌微环境后均有很好的局部组织相容性,且能明显减轻机械性损伤导致的肌肉退形性变,促进再生和抗纤维化的修复过程。
Chapter 1:Isolation,primary culture,and characterizing the immunofluorescence of muscle satellite cells
Objective:To identify the morphological changes of extraocular muscle satellite cells(ESC)in vitro.To compare the expression of immunofluorescene of ESC with that of skeletal muscle satellite cells (SSC).
Methods:To isolate and culture ESC in vitro by muscle block enzyme digestion.Light microscopy was used to monitor the morphological changes of the cells during the period of their growth.And to compare immunofluorescene characters of ESC with that of SSC, which are the marker of myotube(Mosin heavy chain-neonatal,MyHC-n), myogenic markers(Desmin and Myogenin,F5D),marker of myoblast (α-smooth muscle actin,α-SMA),marker of striated muscle(light meromyosin,MF20),and marker of extraocular muscle(myosin heavy chain-extraocular specific,N2.261).
Results:ESC and SSC had the same appearance on the morphological changes while growing in vitro.They came out from first day after seeding the muscle fibers.The cells adhered to the matrix in 1 week,expanded,and fused to each other.Cell mass also found after 2 weeks,which located nearby one of the residual independent myofibril and myotube formed radially on it(MyHC-n were stained positive). 3-dimensional meshwork formed shortly after myotube merged,which showedα-SMA positive.Both ESC and SSC stained as positive results in those antibodies,such as Desmin,Myogenin,α-SMA,and MF20.It was only differed in N2.261 staining between ESC and SSC,while ESC+ and SSC-.
Conclusion:ESC has been isolated and cultured in vitro successfully. The morphological and immuno-fluorescene characters are intact features of ESC just as SSC are.Both of the two myogenic cells are able to be self-expanded and differentiated to form myofibril,they have same features of regeneration and myogenic potential in vitro.
Chapter 2:Establishing laceration model of extraocular muscle in rabbit and treating those rabbits by transplanting autologous adult stem cells
Objective:To establish laceration model of extraocular muscle in rabbits and find its nature of wound healing.To evaluate the effect of auto-transplantation by adult stem cell on the process of wound healing in this model.
Methods:Established the model of wound healing in eye by lacerating the extraocular muscle.The muscle samples were harvested, frozen,sliced and stained by Masson's trichrome staining.Imaging processing(using software of ImageProPlus,IPP)was used to quantify the process of natural healing.4 targets were analyzed.They were: muscle(M,collagen(C),non-collagen(NC)and nuclei(N).Bone marrow mesenchymal stem cells(MSC)were isolated,cultured and expanded in vitro by a putative method.ESC and SSC were labeled by Cell tracker CM-DiI,and transplanted and traced the destiny of transplanted cells in the traumatic area.Both ESC and SSC transplantation were performed to the wound area 1 week after trauma.5 groups were set up as two treatment groups(MSC and SSC transplantation)and three control groups,which were:negative control group 1 and 2,positive control group.The morphological changes were recorded by Masson's staining and analyzed by IPP.
Results:Laceration accelerated the formation of collagen in early stage of wound healing process.Apparent fibrosis showed up just one week after being suffered laceration.Diffused fibrosis and scar emerged dramatically after two weeks.Number of nuclei reached its peak at day 14.Inflammatory cells and fibrosis developed heavier than ever.CM-DiI labeled cells could fuse to adjacent muscles from first day on and stayed in site at least for 14 days.Positive stained muscle fibers were found abounded in the wound area at day 14.Cell transplantation resulted of consistent muscle area at different time points of all groups.In comparison of treatment groups and positive control groups,the later got significant higher amount of non-muscle connective tissue(including collagen and non-collagen part)statistically(P＜0.05),while they showed the same level of increasing amount of non-muscle tissue(P＞0.05). Non-muscle tissue proliferated dramatically after 2 weeks of transplantation while compared with earlier stages(P＜0.05)in all groups. Number of nuclei fluctuated within these 2 weeks,which showed low level at day 7 and high level at day 3 or 14.SSC and MSC played similar way in benefiting the mechanical trauma of extraocular muscle.
Conclusions:Laceration could lead to degeneration of both muscle and the surrounding connective tissue,which act as fibrosis and scar formation.Both MSC and SSC has good consistent with traumatic muscle in ocular environment.And they could slow down the development of fibrosis and scar formation,and also benefit the process of wound healing of extraocular muscle in the way of upgrading the regeneration and downgrading the fibrosis.
引文
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