中华小儿外科杂志
中華小兒外科雜誌
중화소인외과잡지
CHINESE JOURNAL OF PEDIATRIC SURGERY
2010年
3期
216-220
,共5页
陈青江%马燕%贾慧敏%张世伟%张树成%白玉作%袁正伟%王维林
陳青江%馬燕%賈慧敏%張世偉%張樹成%白玉作%袁正偉%王維林
진청강%마연%가혜민%장세위%장수성%백옥작%원정위%왕유림
直肠,畸形%细胞凋亡%胚胎发育
直腸,畸形%細胞凋亡%胚胎髮育
직장,기형%세포조망%배태발육
Rectum,abnormalities%Apoptosis%Embryonic development
目的 研究肛门直肠畸形鼠盆底肌胚胎发育过程中的凋亡现象, 探索凋亡在盆底肌异常发育中的可能致病作用.方法 体重250～300 g的Wistar大白鼠74只随机分组,雌雄午夜合笼后次晨雌鼠阴道涂片,显微镜下见到精子即确认已交配,此日计妊娠第0天.ETU组:52只大鼠于妊娠第10天经胃管注入125 mg/kg的乙烯硫脲.正常对照组:22只大鼠于妊娠第10天经胃管注入等量的生理盐水.妊娠第16、17、19、21天刮宫取胎, 经连续石蜡切片及提取盆底横纹肌复合体, 应用TUNEL染色及DNA Ladder方法检测盆底肌的凋亡, 免疫组织化学及RT-PCR方法分析Bcl-2/Bax在盆底肌中的表达.结果 ①正常对照组共孕雄性胎鼠108只,未见畸形发生.ETU致畸组共得雄性胎鼠247只,肛门直肠畸形173只, 其中伴发脊柱裂或脊膜膨出21只(12.1%:21/173),3.5%(6/173)伴有肢体畸形(肢体缺如或马蹄内翻足);②在正常组中.孕17 d后出现TUNEL阳性染色,散在分布于横纹肌复合体与球海绵体肌交界区及直肠后两侧横纹肌复合体汇合处;畸形组, 孕16 d已有TUNEL阳性染色,大量的阳性染色细胞主要分布在横纹肌复合体的背侧;两组DNA电泳时出现均出现DNA梯度,以畸形组明显;③在两组中,孕17d时开始小现Bax及Bcl-2的表达,孕19d及孕21 d表达增强.与正常组相比,Bcl-2表达下凋(分别为0.44±0.06比0.49±0.07,0.69土0.09比0.90±0.14,0.51±0.07比0.71±0.11),孕19d及21 d时差异有统汁学意义.而Bax基因在畸形组中表达上调(分别为0.39±0.05比0.31±0.05, 0.47±0.06比0.37±0.06,0.40±0.05比0.37±0.05),孕17d及19d时差异有统汁学意义.RT-PCR的结果提示畸形组Bax表达显著增强而Bcl-2表达减弱,差异有统计学意义(P<0.05).结论 肛门直肠畸形鼠盆底肌存在异常凋亡及Bax/Bcl-2表达紊乱, 过早、过度及异位的凋亡可能是肛门直肠畸形时盆底肌发育异常机制之一;Bax/Bcl-2在盆底肌中的时空表达, 提示其在盆底肌凋广过程中的重要调节作用.
目的 研究肛門直腸畸形鼠盆底肌胚胎髮育過程中的凋亡現象, 探索凋亡在盆底肌異常髮育中的可能緻病作用.方法 體重250～300 g的Wistar大白鼠74隻隨機分組,雌雄午夜閤籠後次晨雌鼠陰道塗片,顯微鏡下見到精子即確認已交配,此日計妊娠第0天.ETU組:52隻大鼠于妊娠第10天經胃管註入125 mg/kg的乙烯硫脲.正常對照組:22隻大鼠于妊娠第10天經胃管註入等量的生理鹽水.妊娠第16、17、19、21天颳宮取胎, 經連續石蠟切片及提取盆底橫紋肌複閤體, 應用TUNEL染色及DNA Ladder方法檢測盆底肌的凋亡, 免疫組織化學及RT-PCR方法分析Bcl-2/Bax在盆底肌中的錶達.結果 ①正常對照組共孕雄性胎鼠108隻,未見畸形髮生.ETU緻畸組共得雄性胎鼠247隻,肛門直腸畸形173隻, 其中伴髮脊柱裂或脊膜膨齣21隻(12.1%:21/173),3.5%(6/173)伴有肢體畸形(肢體缺如或馬蹄內翻足);②在正常組中.孕17 d後齣現TUNEL暘性染色,散在分佈于橫紋肌複閤體與毬海綿體肌交界區及直腸後兩側橫紋肌複閤體彙閤處;畸形組, 孕16 d已有TUNEL暘性染色,大量的暘性染色細胞主要分佈在橫紋肌複閤體的揹側;兩組DNA電泳時齣現均齣現DNA梯度,以畸形組明顯;③在兩組中,孕17d時開始小現Bax及Bcl-2的錶達,孕19d及孕21 d錶達增彊.與正常組相比,Bcl-2錶達下凋(分彆為0.44±0.06比0.49±0.07,0.69土0.09比0.90±0.14,0.51±0.07比0.71±0.11),孕19d及21 d時差異有統汁學意義.而Bax基因在畸形組中錶達上調(分彆為0.39±0.05比0.31±0.05, 0.47±0.06比0.37±0.06,0.40±0.05比0.37±0.05),孕17d及19d時差異有統汁學意義.RT-PCR的結果提示畸形組Bax錶達顯著增彊而Bcl-2錶達減弱,差異有統計學意義(P<0.05).結論 肛門直腸畸形鼠盆底肌存在異常凋亡及Bax/Bcl-2錶達紊亂, 過早、過度及異位的凋亡可能是肛門直腸畸形時盆底肌髮育異常機製之一;Bax/Bcl-2在盆底肌中的時空錶達, 提示其在盆底肌凋廣過程中的重要調節作用.
목적 연구항문직장기형서분저기배태발육과정중적조망현상, 탐색조망재분저기이상발육중적가능치병작용.방법 체중250～300 g적Wistar대백서74지수궤분조,자웅오야합롱후차신자서음도도편,현미경하견도정자즉학인이교배,차일계임신제0천.ETU조:52지대서우임신제10천경위관주입125 mg/kg적을희류뇨.정상대조조:22지대서우임신제10천경위관주입등량적생리염수.임신제16、17、19、21천괄궁취태, 경련속석사절편급제취분저횡문기복합체, 응용TUNEL염색급DNA Ladder방법검측분저기적조망, 면역조직화학급RT-PCR방법분석Bcl-2/Bax재분저기중적표체.결과 ①정상대조조공잉웅성태서108지,미견기형발생.ETU치기조공득웅성태서247지,항문직장기형173지, 기중반발척주렬혹척막팽출21지(12.1%:21/173),3.5%(6/173)반유지체기형(지체결여혹마제내번족);②재정상조중.잉17 d후출현TUNEL양성염색,산재분포우횡문기복합체여구해면체기교계구급직장후량측횡문기복합체회합처;기형조, 잉16 d이유TUNEL양성염색,대량적양성염색세포주요분포재횡문기복합체적배측;량조DNA전영시출현균출현DNA제도,이기형조명현;③재량조중,잉17d시개시소현Bax급Bcl-2적표체,잉19d급잉21 d표체증강.여정상조상비,Bcl-2표체하조(분별위0.44±0.06비0.49±0.07,0.69토0.09비0.90±0.14,0.51±0.07비0.71±0.11),잉19d급21 d시차이유통즙학의의.이Bax기인재기형조중표체상조(분별위0.39±0.05비0.31±0.05, 0.47±0.06비0.37±0.06,0.40±0.05비0.37±0.05),잉17d급19d시차이유통즙학의의.RT-PCR적결과제시기형조Bax표체현저증강이Bcl-2표체감약,차이유통계학의의(P<0.05).결론 항문직장기형서분저기존재이상조망급Bax/Bcl-2표체문란, 과조、과도급이위적조망가능시항문직장기형시분저기발육이상궤제지일;Bax/Bcl-2재분저기중적시공표체, 제시기재분저기조엄과정중적중요조절작용.
Objective To investigate the role of apoptosis during the emhryogenesis of pelvic floor muscle (PFM) in ARM rat.Methods ARM rat embryos were induced by injecting the E10 pregnant rats with ethyle-nethiourea (ETU).Normal and ARM rat embryos from E16 to E21 were serial-sectioned transversely or sag-ittally.and striated muscle compleres (SMCs) were dissected out and snap frozen.TUNEL staining and DNA Ladder analysis were performed to identify the apoptosis.expression of Bax/Bcl-2 were confirmed with im-munohistochemical staining and RT-PCR analysis.Results Hypoplastic and disordered SMC sling shifted significantly cephalad, ventrally, and converged inferior to the rectourethral fistula, infiltrated with connective tissue in ARM embryos.In normal group, TUNEL-positive cells became evident on El7, sporadic positive staining mainly localized in two areas, the junction area between SMC and M bulbocarvemosus, and posteriot to the rectum where bilateral SMC converged In ARM group, massive positive staining nuclei were observed from El6 to E21 , and were mainly distributed in the dorsal part of the SMC.Electrophoresis of UNA samples yielded a "ladder" pattern of migration both in normal and in ARM group from E17 to E21 (the ladders were stronger in ARM group.In both groups, the expressioas of Bax /Bcl-2 were detectable on E17, the immuno-reactivity increased on E19 and E21.Compared with normal group, the expression of Bax were increased, while Bcl-2 were declined in ARM group.Significant up-regulation of Rax mRNA levels and down-regulation of Bcl-2 mRNA levels were observed in ARM group.Conclusions In the current study.abnormal apoptosis and disturbed expression of Bax/Bcl-2 were identified during SMC development in ARM rats.It suggests that excessive and dislocated apoptosis might contribute to the malformation of SMC in ARM rats.The temporo-spatial expressioas of Bax/Bcl-2 indicated their important role in the regulation of apoptosis of SMC.
