目的 建立新生大鼠坏死性小肠结肠炎(NEC)模型,探讨糖巨肽对NEC新生大鼠肠道的保护作用.方法 36只SD新生大鼠采用抽签法随机分为NEC模型组、糖巨肰干预组和正常对照组,每组12只.大鼠出生后第1～3天均为母乳喂养；在第4～6天,正常对照组继续母乳喂养,而NEC模型组和糖巨肽干预组则通过人工喂养+缺氧复氧冷刺激的方法进行造模.第6天喂养结束后三组大鼠均置于保育箱中空腹24h,然后用颈椎脱臼法处死大鼠,取回盲部近段回肠组织进行固定、切片,行病理检查、TUNEL凋亡检测和电镜检查；同时取回盲部附近肠段制备组织匀浆,检测肠组织肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)及白细胞介素1β(Interleukin-1β,IL-1β)的含量,并采用荧光定量PCR的方法检测PAF mRNA的表达.结果 (1)病理评分:NEC模型组为(2.17±0.83)分,糖巨肽干预组(0.92±0.79)分,正常对照组(0.17±0.39)分,糖巨肽干预组与NEC模型组比较,差异有统计学意义(H=8.819,P=0.003)；(2)肠组织TNF-α水平:模型组(41.94±13.51)pg/ml,干预组(31.69±11.68) pg/ml,正常组(17.42±7.18)pg/ml,干预组与模型组相比,差异有统计学意义(F=3.959,P=0.030)；(3)肠组织IL-1β水平:模型组(150.33±36.41) pg/ml,干预组(118.36±33.00)pg/ml,正常组(28.44±15.04) pg/ml,干预组与模型组相比,差异有统计学意义(F =5.080,P=0.013)；(4)肠组织PAF mRNA表达水平(2- △△C1值):模型组为(3.01±0.96),干预组为(1.56±0.29),正常组为(1.01±0.13),干预组与模型组比较,差异有统计学意义(F=25.251,P=0.000)；(5)电镜观察:正常对照组可见其细胞核占细胞体积的大部分,结构清晰,有核膜包被,提示正常细胞相；NEC模型组可见凋亡小体,提示凋亡晚期相；糖巨肽干预组可见染色质靠核膜边集,核孔扩大,提示凋亡早期相；(6)TUNEL检测肠上皮细胞凋亡率:NEC模型组为(38.79±9.79),糖巨肽干预组为( 29.54±7.30),正常对照组相为(6.37±1.96),糖巨肽干预组肠上皮细胞凋亡率低于NEC模型组,两组比较,差异具有统计学意义(F =6.888,P=0.003).结论 在人工喂养条件下,缺氧、复氧和冷刺激方法的NEC造模成功率为75％ (9/12).糖巨肽对NEC新生大鼠肠道具有保护作用.
目的 建立新生大鼠壞死性小腸結腸炎(NEC)模型,探討糖巨肽對NEC新生大鼠腸道的保護作用.方法 36隻SD新生大鼠採用抽籤法隨機分為NEC模型組、糖巨肰榦預組和正常對照組,每組12隻.大鼠齣生後第1～3天均為母乳餵養；在第4～6天,正常對照組繼續母乳餵養,而NEC模型組和糖巨肽榦預組則通過人工餵養+缺氧複氧冷刺激的方法進行造模.第6天餵養結束後三組大鼠均置于保育箱中空腹24h,然後用頸椎脫臼法處死大鼠,取迴盲部近段迴腸組織進行固定、切片,行病理檢查、TUNEL凋亡檢測和電鏡檢查；同時取迴盲部附近腸段製備組織勻漿,檢測腸組織腫瘤壞死因子α(tumor necrosis factor-α,TNF-α)及白細胞介素1β(Interleukin-1β,IL-1β)的含量,併採用熒光定量PCR的方法檢測PAF mRNA的錶達.結果 (1)病理評分:NEC模型組為(2.17±0.83)分,糖巨肽榦預組(0.92±0.79)分,正常對照組(0.17±0.39)分,糖巨肽榦預組與NEC模型組比較,差異有統計學意義(H=8.819,P=0.003)；(2)腸組織TNF-α水平:模型組(41.94±13.51)pg/ml,榦預組(31.69±11.68) pg/ml,正常組(17.42±7.18)pg/ml,榦預組與模型組相比,差異有統計學意義(F=3.959,P=0.030)；(3)腸組織IL-1β水平:模型組(150.33±36.41) pg/ml,榦預組(118.36±33.00)pg/ml,正常組(28.44±15.04) pg/ml,榦預組與模型組相比,差異有統計學意義(F =5.080,P=0.013)；(4)腸組織PAF mRNA錶達水平(2- △△C1值):模型組為(3.01±0.96),榦預組為(1.56±0.29),正常組為(1.01±0.13),榦預組與模型組比較,差異有統計學意義(F=25.251,P=0.000)；(5)電鏡觀察:正常對照組可見其細胞覈佔細胞體積的大部分,結構清晰,有覈膜包被,提示正常細胞相；NEC模型組可見凋亡小體,提示凋亡晚期相；糖巨肽榦預組可見染色質靠覈膜邊集,覈孔擴大,提示凋亡早期相；(6)TUNEL檢測腸上皮細胞凋亡率:NEC模型組為(38.79±9.79),糖巨肽榦預組為( 29.54±7.30),正常對照組相為(6.37±1.96),糖巨肽榦預組腸上皮細胞凋亡率低于NEC模型組,兩組比較,差異具有統計學意義(F =6.888,P=0.003).結論 在人工餵養條件下,缺氧、複氧和冷刺激方法的NEC造模成功率為75％ (9/12).糖巨肽對NEC新生大鼠腸道具有保護作用.
목적 건립신생대서배사성소장결장염(NEC)모형,탐토당거태대NEC신생대서장도적보호작용.방법 36지SD신생대서채용추첨법수궤분위NEC모형조、당거연간예조화정상대조조,매조12지.대서출생후제1～3천균위모유위양；재제4～6천,정상대조조계속모유위양,이NEC모형조화당거태간예조칙통과인공위양+결양복양랭자격적방법진행조모.제6천위양결속후삼조대서균치우보육상중공복24h,연후용경추탈구법처사대서,취회맹부근단회장조직진행고정、절편,행병리검사、TUNEL조망검측화전경검사；동시취회맹부부근장단제비조직균장,검측장조직종류배사인자α(tumor necrosis factor-α,TNF-α)급백세포개소1β(Interleukin-1β,IL-1β)적함량,병채용형광정량PCR적방법검측PAF mRNA적표체.결과 (1)병리평분:NEC모형조위(2.17±0.83)분,당거태간예조(0.92±0.79)분,정상대조조(0.17±0.39)분,당거태간예조여NEC모형조비교,차이유통계학의의(H=8.819,P=0.003)；(2)장조직TNF-α수평:모형조(41.94±13.51)pg/ml,간예조(31.69±11.68) pg/ml,정상조(17.42±7.18)pg/ml,간예조여모형조상비,차이유통계학의의(F=3.959,P=0.030)；(3)장조직IL-1β수평:모형조(150.33±36.41) pg/ml,간예조(118.36±33.00)pg/ml,정상조(28.44±15.04) pg/ml,간예조여모형조상비,차이유통계학의의(F =5.080,P=0.013)；(4)장조직PAF mRNA표체수평(2- △△C1치):모형조위(3.01±0.96),간예조위(1.56±0.29),정상조위(1.01±0.13),간예조여모형조비교,차이유통계학의의(F=25.251,P=0.000)；(5)전경관찰:정상대조조가견기세포핵점세포체적적대부분,결구청석,유핵막포피,제시정상세포상；NEC모형조가견조망소체,제시조망만기상；당거태간예조가견염색질고핵막변집,핵공확대,제시조망조기상；(6)TUNEL검측장상피세포조망솔:NEC모형조위(38.79±9.79),당거태간예조위( 29.54±7.30),정상대조조상위(6.37±1.96),당거태간예조장상피세포조망솔저우NEC모형조,량조비교,차이구유통계학의의(F =6.888,P=0.003).결론 재인공위양조건하,결양、복양화랭자격방법적NEC조모성공솔위75％ (9/12).당거태대NEC신생대서장도구유보호작용.
Objective To establish an appropriate neonatal rat model of necrotizing enterocolitis (NEC) and to investigate the protective effects of glycomacropeptide (GMP) on the gut from injury in neonatal rats with NEC.Method A total of 36 neonatal SD rats were randomly divided into 3 groups:NEC model group( Group M ),NEC + GMP group( Group G) and normal control group( Group N),each group had 12 rats.All the neonatal rats were fed with breast milk in the first 3 days after birth.During the second 3 days after birth,the rats of Group N were still maternal breast-fed,but the rats of Group M and Group G were separated from their mothers and lived in incubator and began to be formula fed,and were subjected to cold exposure shortly after hypoxic-reoxygenation treatment.After being fed in such means for 6 days,all the neonatal rats were placed into the incubator and fasted for 24 hours.Then all the rats were sacrificed by cervical dislocation. Intestinal tissue located at the boundary of ileum and cecum was obtained for:① histological examination after HE staining,② TUNEL detection,③electron microscopic observation; and the tissue homogenate was obtained for checking TNF-α and IL-1β levels by ELISA and platelet activating factor (PAF) mRNA expression by quantitative fluorescence ( QF )-PCR.Result ( 1 ) The pathological scores of the 3 groups were 2.17 ± 0.83 ( Group M ),0.92 ± 0.79 ( Group G) and 0.17 ± 0.39 ( Group N) separately.There was significant difference between Group M and Group G (H=8.819,P=0.003).(2) TNF-α levels of 3 groups were (41.94 ± 13.51 ) pg/ml ( Group M ),(31.69 ± 11.68 ) pg/ml ( Group G) and ( 17.42 ± 7.18 ) pg/ml ( Group N) separately,and TNF-α level in Group G was significantly lower than that of Group M ( F =3.959,P =0.030). ( 3 ) 1L-1β levels of 3 groups were ( 150.33 ± 36.41 ) pg/ml ( Group M ),( 118.36 ± 33.00) pg/ml ( Group G ) and ( 28.44 ± 15.04 ) pg/ml ( Group N ) separately,and IL-1β level in Group G was lower than that of Group M (F =5.080,P =0.013 ).(4)Expression levels of intestinal PAF mRNA (2-△△C1 value):3.01 ±0.96 (Group M),1.56 ±0.29 (Group G),1.01 ±0.13 (Group N),the level of Group G was significantly lower than that of Group M (F=25.251,P =0.000).(5) Electron microscopy:Group N showed that its cell volume was mostly occupied by the nucleus,the structure was clear,nuclear membrane existed,suggesting the normal phase of cell; Group M showed that apoptotic body existed,suggestiug that the advanced stage phase of apoptosis; Group G showed that condensed chromatin marginated around the nuclear envelope,nuclear pores expanded,suggesting the early phase of apoptosis.(6)The apoptosis rate of intestinal epithelial cells by TUNEL detection:38.79 ±9.79 ( Group M),29.54 ± 7.30 ( Group G),6.37 ± 1.96 ( Group N ) ; the apoptosis rate of intestinal epithelial cells of Group G was significantly lower than that of Group M ( F =6.888,P =0.003 ).Conclusion GMP has protective effects on guts of neonatal rats with NEC,which may probably work by reducing TNF-α,IL-1 β and PAF expression,inhibiting the apoptosis of intestinal epithelial cells and reducing intestinal tissue injury.
