摘要
在已经证实外源性hIGF-1和携hIGF-1基因成肌细胞可以促进损伤骨骼肌再生和一定程度抑制损伤骨骼肌纤维化的研究基础上,本课题重点研究hIFNγ对损伤骨骼肌发生纤维化的影响。本研究第一部分首先证实外源性hIFNγ可以明显抑制损伤骨骼肌纤维化,与外源性hIGF-1联合应用能够促进损伤骨骼肌修复。第二部分采用基因治疗的手段重组hIFNγ基因,并转染至成肌细胞,经体外实验证实该基因编码的产物能在胞内稳定表达并具有生物活性。第三部分将转hIFNγ基因的成肌细胞和转hIGF-1基因的成肌细胞联合移植到损伤骨骼肌,观察其在损伤骨骼肌微环境下的存活,以及两种基因是否能在骨骼肌内分别表达,第四部分探讨了联合移植携hIGF-1基因和携hIFNγ成肌细胞对损伤骨骼肌的修复。
第一部分联合应用外源性hIFNγ和hIGF-1治疗小鼠骨骼肌急性钝挫伤
目的:观察小鼠骨骼肌急性钝挫伤后,单独或联合注射外源性hIFNγ和hIGF-1对骨骼肌修复过程中再生和纤维化的影响。
方法:64只7～12周雄性C3H小鼠,制作右侧腓肠肌钝挫伤动物模型,随机分成4组,即A组(注射hIFNγ)、B组(注射hIGF-1)、C组(联合注射hIFNγ和hIGF-1)、D组(注射生理盐水)。挫伤后第10d,各组小鼠在腓肠肌损伤处分别注射不同药物进行干预。分别于干预前(伤后7d)和干预后4d、18d、32d(伤后14d、28d、42d)从各组中随机抽取4只小鼠取腓肠肌损伤部位肌肉,用荧光定量PCR和免疫荧光化学检测不同时间点Ⅱb型肌球蛋白重链(Myosin heavychain-Ⅱb,MHC-Ⅱb)及波形蛋白(Vimentin)的表达。统计学分析使用SPSS 10.0统计软件,采取One-Way ANOVA方法进行检验,p＜0.05具有统计学意义。
结果:(1)干预后各时间点,B、C组小鼠损伤骨骼肌MHC-Ⅱb表达水平(包括mRNA与蛋白)较A、D组明显升高;(2)干预后各时间点A、B、C组小鼠损伤骨骼肌Vimentin表达(包括mRNA与蛋白)较D组降低,其中以A组和B组降低更明显。
结论:(1)在骨骼肌急性损伤后,局部注射hIGF-1有明显促进骨骼肌再生和一定程度抑制纤维化的作用;(2)局部注射hIFNγ仅表现出抑制纤维化的作用,其作用远比hIGF-1明显;(3)联合应用hIGF-1和hIFNγ,能够同时促进骨骼肌再生和抑制纤维化,有效地促进了损伤骨骼肌的愈合。
第二部分
hIFNγ真核表达质粒的构建及转染后在成肌细胞内的表达与活性鉴定
目的:构建携带hIFNγ目的基因的真核表达重组质粒pEGFP-C2-hIFNγ,将其转染至小鼠C2C12成肌细胞后,观察hIFNγ基因在细胞内的表达,以及该基因编码产物hIFNγ蛋白的生物活性。
方法:(1)应用基因重组技术,将hIFNγ基因克隆到pEGFP-C2真核表达载体中,以酶切和测序方法鉴定重组质粒pEGFP-C2-hIFNγ的正确性;(2)经脂质体介导将pEGFP-C2-hIFNγ转染至C2C12成肌细胞,24h后,在荧光倒置显微镜下观察增强绿色荧光蛋白(enhance green fluorescentp rotein,EGFP)瞬时表达情况;(3)通过G418进行梯度筛选,获得能够稳定表达hIFNγ的细胞克隆,用RT-PCR和Western Blot法检测转染细胞hIFNγ的表达;(4)以TGFβ-1处理WI-38成纤维细胞,再与转hIFNγ基因的成肌细胞或空白成肌细胞共培养,以荧光定量PCR和免疫荧光化学方法检测α-SMA的表达。
结果:(1)酶切和测序结果均证实正确构建了hIFNγ真核表达质粒pEGFP-C2-hIFNγ;(2)细胞转染pEGFP-C2-hIFNγ后24h,荧光显微镜下可观察到EGFP的表达,40%以上细胞发出绿色荧光;(3)RT-PCR和Western Blot检测到转染细胞中有明显的hIFNγmRNA和蛋白表达;(4)转hIFNγ基因的成肌细胞能够抑制经TGFβ-1处理的WI-38细胞表达α-SMA。
结论:(1)成功制备了含hIFNγ基因的真核表达质粒,并成功转染至小鼠C2C12成肌细胞;(2)转hIFNγ基因的C2C12成肌细胞能够稳定表达有活性的hIFNγ。
第三部分联合移植携hIFNγ基因和携hIGF-1基因的成肌细胞在小鼠损伤骨骼肌的存活及基因产物的表达
目的:观察联合移植携hIFNγ基因和携hIGF-1基因的成肌细胞,在小鼠损伤骨骼肌的存活以及重组MFNγ基因和重组hIGF-1基因产物的表达
方法:64只7～12周雄性C3H小鼠,除4只小鼠作正常对照外,60只小鼠被制作右侧腓肠肌钝挫伤动物模型,随机分成5组,即A组(注射转hIFNγ基因的C2C12细胞),B组(注射转MGF-1基因的C2C12细胞),C组(联合注射转hIFNγ基因的和转hIGF-1基因的C2C12细胞),D组(注射空白C2C12成肌细胞),E组(注射生理盐水)。挫伤后第10d,各组小鼠于腓肠肌损伤局部注射不同药物。伤后14d、28d、42d,于各组中随机抽取4只小鼠取右侧腓肠肌损伤部位肌肉,行BrdU免疫荧光化学染色,检测外源细胞在体内的存活情况;行荧光定量PCR和免疫荧光化学检测hIFNβ和hIGF-1在损伤骨骼肌局部的表达。实验结果使用SPSS 10.0统计软件进行统计学分析,以P＜0.05为有统计学意义。
结果:(1)细胞移植后4d-32d,A、B、C、D小鼠均有BrdU免疫组化阳性染色,四组间无明显差异;(2)只有A、C两组出现hIFNγmRNA和蛋白表达,两组间无明显差异;(3)只有B、C两组出现hIGF-1 mRNA和蛋白表达,两组间无明显差异。
结论:(1)转基因成肌细胞和空白成肌细胞移植后,在损伤骨骼肌局部均能存活一定时间,重组基因对细胞的存活能力没有影响;(2)携hIFNγ基因的成肌细胞和携hIGF-1基因的成肌细胞移植后,在损伤骨骼肌局部可以分别表达hIFNγ和hIGF-1;(3)联合注射携hIFNγ基因和携hIGF-1基因的成肌细胞,在存活能力和产物表达上,相互之间无明显影响。
第四部分联合应用携hIFNγ基因和携hIGF-1基因的成肌细胞治疗小鼠骨骼肌急性钝挫伤
目的:观察损伤骨骼肌局部联合注射携hIFNγ基因和携hIGF-1的成肌细胞,对小鼠急性钝挫伤骨骼肌修复的影响。
方法:80只7～12周雄性C3H小鼠,制作右侧腓肠肌钝挫伤动物模型,随机分成5组,即A组(注射转hIFNγ基因的C2C12细胞),B组(注射转hIGF-1基因的C2C12细胞),C组(联合注射转hIFNγ基因的和转hIGF-1基因的C2C12细胞),D组(注射空白C2C12成肌细胞),E组(注射生理盐水)。挫伤后第10d,各组小鼠于腓肠肌损伤局部注射不同药物。伤后7d、14d、28d、42d,于各组中随机抽取4只小鼠取右侧损伤腓肠肌,以荧光定量PCR和免疫荧光化学检测不同时间点波形蛋白(Vimentin)、α-SMA及MHC-Ⅱb的表达。
结果:(1)干预后各时间点,A组、C组小鼠损伤骨骼肌局部的Vimentin表达较B、D、E各组降低;伤后28d、42d,B组损伤骨骼肌Vimentin表达低于D、E组;A、C组间或D、E组间Vimentin表达无明显差异;(2)干预后各时间点,A组、C组小鼠损伤骨骼肌局部的α-SMA表达较B、D、E各组降低;伤后28d、42d,B组损伤骨骼肌α-SMA表达低于D、E组;A、C组间或D、E组间α-SMA表达无明显差异;(3)干预后各时间点,B组、C组小鼠损伤骨骼肌局部的MHC-Ⅱb表达较B、D、E各组增高;B、C组间或A、D、E组间MHC-Ⅱb表达无明显差异。
结论:(1)注射转hIFNγ基因的C2C12成肌细胞,可以抑制损伤骨骼肌Vimentin和α-SMA的表达,提示有抑制损伤骨骼肌纤维化的作用;(2)注射转hIGF-1基因的C2C12成肌细胞,可以使损伤骨骼肌MHC-Ⅱb表达增高,但Vimentin和α-SMA的表达轻度下降,具有促进损伤骨骼肌再生和一定程度的抑制损伤骨骼肌纤维化的作用;(3)联合注射转hIFNγ基因的C2C12成肌细胞和转hIGF-1基因成肌细胞可以使损伤骨骼肌MHC-Ⅱb表达增高和Vimentin和α-SMA的表达下降,具有同时促进骨骼肌再生和抑制损伤骨骼肌纤维化的作用。
On the basis of the research that exogenous hIGF-1 and myoblasts with hIGF-1 gene could promote muscle regeneration and mildly inhibit muscle fibrosis,we would emphasize about the effects of hIFNγon muscle fibrosis.After confirming the anti-fibrosis for skeletal muscle by exogenous hIFNγ,we constructed a recombinant plasmid pEGFP-C2-hIFNγand transfected C2C12 myoblasts.Following analyzing the expression and activity of hIFNγin transfected cells in vitro,the myoblasts with hIFNγgene or with hIGF-1 gene were combined to be transplanted into injured skeletal muscle.Then the survival potential and expression of different transfected cells were observed,and their effects for repair of injured skeletal muscle were explored.
PartⅠCombined Injection with Exogenous hIFNγand hIGF-1 for Acute Skeletal Muscle Contusion in the Mouse
Objective To observe the effects of hIFNγor/and hIGF-1 on regeneration and fibrosis of skeletal muscle after acute contusion.
Methods A standard contusion model was reproduced at the right gastrocnemius in 64 male mice of 7-12 weeks.All the mice were randomly divided into 4 groups,such as group A(injection with hIFNγ),group B(injection with hIGF-1),group C(injection with hIGF-1 and hIFNγ),and group D(injection with physiological saline).All injections were introduced on day 10 after injury at local injured gastrocnemius with different interventions.On day 7,14,28,42(eg,the day before intervention,and 4d, 18d,32d and intervention) following contusion,the local injured gastrocnemius were harvested of 4 mice from each group.Then the expression of MHC-Ⅱb and vimentin was detected by fluorescent quantitation PCR and immunofluorescence cytochemistry technology.The data were analyzed by one-way ANOVA using a statistical software package program(SPSS10.0),and were tested.The results were considered to be significant at p values＜0.05.
Results(1)At the time following intervention,the expression of MHC-Ⅱb mRNA and protein in local injured muscle of group B and group C were significantly higher than those of group A and D;(2)After intervention,the expression of vimentin mRNA and protein in local injured muscle of group A,group B,and group C were more inhibited than those of group D.It was more significant for the inhibition of vimentin expression in group A and group C.
Conclusions(1) It was indicated that hIGF-1 could enhance muscle regeneration,and inhibit fibrosis to some extent,by injection into the injured skeletal muscle following acute contusion;(2) It was identified that hIFNγinjected into injured muscle had the effect of anti-fibrosis,what is more significant than that of hIGF-1;(3)Combined injection with hIGF-1 and hIFNγcould improve muscle regeneration and inhibit fibrosis simultaneously,and promot the injured muscle healing.
PartⅡExpression and Activity Detection of hIFNγProtein Following Construction and Transfection of pEGFP-C2-hIFNγinto C2C12 Myoblasts
Objective To construct and transfect recombinant plasmid of pEGFP-C2-hIFNγinto the C2C12 myoblasts,and to observe the expression of hIFN gene and the activity of hIFNγprotein in the C2C12 myoblasts.
Methods(1) with the technology of gene rearrangement,hIFNγgene was cloned into pEGFP-C2 plasmid;then its correctness was evaluated by the means of restriction enzyme analysis and sequencing;(2) pEGFP-C2-hIFNγwas transfected into myoblasts C2C12 with liposome mediated transfection;(3) After 24h,the transient expression of EGFP in the transfected C2C12 cells was observed under fluorescence microscope;(3) After transfection,the positive cell clones selected with G418 were culturing for 4 weeks;the hIFNγgene expression of transfected C2C12 cells were tested by RT-PCR and Western Blot analysis;(4) The WI-38 fibroblasts treated with TGFβ-1 were cultured with myoblasts with or without hIFNγgene,then theα-SMA expression of WI-38 cells were analyzed by fluorescent quantitation PCR and immunofluorescence cytochemistry technology.
Results(1) Correct construction of pEGFP-C2-hIFNγwas identified by methods of restriction enzyme analysis and nucleotide;(2) Green fluorescence was emitted from transfected cells under fluorescent microscope after 24h;(3) the hIFNγexpression in myoblasts C2C12 transfected with pEGFP-C2-hγIFN was detected by RT-PCR and western blot analysis;(4) theα-SMA expression of WI-38 cells treated with TGFβ-1 were inhibited by transfected myoblasts with hIFNγgene.
Conclusions(1) The pEGFP-C2-hIFNγ,a eukaryotic expression plasmid of hIFNγ, gene,has been constructed.And the mouse myoblasts C2C12 could be transfected with the recombinant plasmid with hIFNγ;(2) The transfected C2C12 myoblasts could express hIFNγwith activity stablely.
PartⅢSurvival and Expression of Myoblasts with hIFNγGene or hIGF-1 Gene Following Transplantation into Injured Skeletal Muscle in the Mouse
Objective To observe the survival and expression of transfected myoblasts with hIFNγgene or with hIGF-1 gene following combined transplantation into injured skeletal muscle.
Methods Acute skeletal muscle contusion models were produced on right gastrocnemius in 60 male mice of 7-12 weeks,and another 4 normal mice as control. The 60 mice were divided into 5 groups randomly as group A(injection with C2C12 cells transfected hIFNγgene),group B(injection with C2C 12 cells transfected hIGF-1 gene),group C(combined injection with C2C12 cells transfected hIFNγgene and C2C12 cells transfected hIGF-1 gene),group D(injection with blank C2C12 cells), group E(injection with physiological saline).The intervention was introduced on the 10~(th) day following injury by different injection into the injured muscle.The injured right gastrocnemius were harvested from 4 mice in every group on day 4,18,and 32 following intervention,which were tested by BrdU staining to evaluate cells surviving, and analyzed the expression of hIFNγand hIGF-1 by fluorescent quantitation PCR and immunofluorescence cytochemistry technology.
Results(1)The BrdU staining in group A,B,D,and D were positive,and no difference was found among groups;(2) hIFNγexpression were found only in the group A and C without difference;(3) hIGF-1 expression were found only in the group B and C without difference.
Conclusions(1) The transfected myoblasts and the blank myoblasts could survive for a period in the injured muscle following transplantation.It indicated that transfection had no influence on cells survival potential;(2)The Myoblasts carried with hIFNγgene or hIGF-1 gene could express hIFNγor hIGF-1 respectively in the injured muscle;(3) There were no difference about cells survival and expression following combined transplantation of transfected cells with hIFNγor hIGF-1 gene.
PartⅣCombined Transplantaion of Myoblasts with hIFNγGene and Myoblasts with hIGF-1 Gene for Acute Skeletal Muscle Contusion in the Mouse
Objective To observe the effects of combined transplantation of myoblasts with hIFNγgene and myoblasts with hIGF-1 gene on acute skeletal muscle contusion in the mouse.
Methods Acute skeletal muscle contusion models were produced on right gastrocnemius in 80 male mice of 7-12 weeks.The 80 mice were divided into 5 groups randomLy as group A(injection with C2C12 cells transfected with hIFNγgene),group B(injection with C2C12 cells transfected with hIGF-1 gene),group C(combined injection with C2C12 cells transfected with hIFNγgene and C2C12 cells transfected with hIGF-1 gene),group D(injection with blank C2C12 cells),group E(injection with physiological saline).The intervention was introduced on the 10~(th) day following injury by different injection into the injured muscle.The injured right gastrocnemius were harvested from 4 mice in every group on day 7,14,28,and 42 following injury(eg,the day before intervention,and 4d,18d,32d after intervention),which were tested about expression of MHC-Ⅱb,α-SMA and vimentin by fluorescent quantitation PCR and immunofluorescence cytochemistry technology.
Results(1) After intervention,the expression of vimentin in the group A and group C was significantly lower than that of group B,D,and E.the vimentin expression of group B was lower than that of group D,and E on the day 28 and day 42.It was similar of vimentin expression between group A and C,or group D and E.(2) After intervention,the expression ofα-SMA in the group A and group C was significantly lower than that of group B,D,and E.theα-SMA expression of group B was lower than that of group D,and E on the day 28 and day 42.It was similar ofα-SMA expression between group A and C,or group D and E.(3) Following intervention,the expression of MHC-Ⅱb in the group B and group C was significantly higher than that of group A, D,and E.It was similar of MHC-Ⅱb expression between group B and C,or among group A,D and E.
Conclusion(1) Local injection of C2C 12 myoblasts with hIFNγgene could inhibit the expression of vimentin andα-SMA significantly,what indicated its function of anti-fibrosis;(2) Local injection of C2C 12 myoblasts with hIGF-1 gene could promote skeletal muscle regeneration and inhibit fibrosis moderately,evidenced by its effects of significant enhancement of the MHC-Ⅱb expression and mild inhibition of the expression of vimentin andα-SMA.(3) It was indicated that combined injection of myoblasts with hIFNγgene or hIGF-1 gene would promote skeletal muscle repair by stimulation of muscle regeneration and anti-fibrosis.
引文
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