CAJ | 학술논문

目的研究肛门直肠畸形胎鼠盆底肌卫星细胞及其微环境的胚胎发育,从干细胞水平探索盆底肌发育不良的可能发生机制.方法乙烯硫脲致畸建立肛门直肠畸形动物模型,正常组及给药组孕16～2 d取胎,经连续石蜡切片及提取盆底横纹肌复合体,应用免疫荧光化学染色、透射电镜、细胞培养技术及实时定量PCR方法研究盆底肌卫星细胞的胚胎发育.结果正常组:孕16 d时盆底肌已经开始形成,未见Pax7阳性表达细胞,孕17 d后盆底肌出现Pax7弱阳性染色细胞,孕19、21 d Pax7表达增强,孕21 d时阳性细胞比例略下降,占肌细胞核的35.07%.畸形组:孕16 d时即见Pax7较强阳性染色细胞,孕17～21 d阳性染色细胞比例增加,以孕19 d最明显,孕21 d时占肌细胞核的44.16%.Real-time PCR结果提示,与正常组比较,畸形组Pax7表达增强,而Myogenin表达减弱,各组间差异有统计学意义.透射电镜可见肛门直肠畸形组盆底肌卫星细胞胞核不规则,异染色质明显增多,胞膜与肌膜间间隙增大,基底膜明显增厚,周围肌纤维排列紊乱,横纹不明显.细胞分离培养后畸形组卫星细胞分化障碍,培养72 h后仍有较高比例的MyoD阳性表达细胞.畸形组盆底肌vWF及Neurofilament表达减弱而Vimentin表达明显增强,存在微环境发育缺陷.结论肛门直肠畸形时盆底肌卫星细胞提前形成使得分化成肌的前体细胞减少,卫星细胞的超微结构异常及分化功能障碍,同时存在卫星细胞微环境发育缺陷,使得卫星细胞不能向骨骼肌分化,可能是导致盆底肌发育不良的机制之一.
목적 연구항문직장기형태서분저기위성세포급기미배경적배태발육,종간세포수평탐색분저기발육불량적가능발생궤제.방법 을희류뇨치기건립항문직장기형동물모형,정상조급급약조잉16～2 d취태,경련속석사절편급제취분저횡문기복합체,응용면역형광화학염색、투사전경、세포배양기술급실시정량PCR방법연구분저기위성세포적배태발육.결과 정상조:잉16 d시분저기이경개시형성,미견Pax7양성표체세포,잉17 d후분저기출현Pax7약양성염색세포,잉19、21 d Pax7표체증강,잉21 d시양성세포비례략하강,점기세포핵적35.07%.기형조:잉16 d시즉견Pax7교강양성염색세포,잉17～21 d양성염색세포비례증가,이잉19 d최명현,잉21 d시점기세포핵적44.16%.Real-time PCR결과제시,여정상조비교,기형조Pax7표체증강,이Myogenin표체감약,각조간차이유통계학의의.투사전경가견항문직장기형조분저기위성세포포핵불규칙,이염색질명현증다,포막여기막간간극증대,기저막명현증후,주위기섬유배렬문란,횡문불명현.세포분리배양후기형조위성세포분화장애,배양72 h후잉유교고비례적MyoD양성표체세포.기형조분저기vWF급Neurofilament표체감약이Vimentin표체명현증강,존재미배경발육결함.결론 항문직장기형시분저기위성세포제전형성사득분화성기적전체세포감소,위성세포적초미결구이상급분화공능장애,동시존재위성세포미배경발육결함,사득위성세포불능향골격기분화,가능시도치분저기발육불량적궤제지일.
Objective To investigate the embryonic development of pelvic floor muscle (PFM)satellite cell niche in ARM rats. Methods Embryonic development of satellite cell niche and the myogenic potential of satellite cell from striated muscle complex (SMC) were studied with immunohistochemistry, transmission electron microscopy (TEM), cell culture technique and real-time quantitative PCR analysis in rat embryos with ethylenethiourea (ETU) induced ARM. Results No Pax7+ cells were identified on embryonic day 16 (E16) in normal rats. Weak Pax7 expression was detected on E17, and the expression increased on E19 and E21. Strong Pax7-expression were evident on E16 in ARM rats, the positive rate increased with gestational age. Significant up-regulation of Pax7 mRNA levels and down-regulation of Myogenin mRNA levels were observed in ARM group, as demonstrated by real-time PCR. Abnormal differentiation of satellite cells was convincing in ARM embryos. Irregularly shaped satellite cell with increased nuclear heterochromatin, thickened basal lamina, widened gap between satellite cell and myofiber, disarrangement of muscle fibers were confirmed under TEM in the ARM group. The expression of vWF and Neurofilament in ARM group decreased, while the expression of Vimentin increased, indicated an unfavorable satellite cell niche. Conclusions Our results suggest that premature development of satellite cells result in depletion of myogenic precursors and poor muscle growth. Intrinsic satellitecell ultrastructure change and extrinsic impairment of microenvironment compromised the myogenic potential of satellite cell and might be associated with the pathogenesis of ARM. Further investigations are needed to decipher the underlying mechanisms mediating premature specification and dysdifferentiation of satellite cell.