中华小儿外科杂志
中華小兒外科雜誌
중화소인외과잡지
Chinese Journal of Pediatric Surgery
2015年
8期
626-631
,共6页
刘淼清%胡书奇%王永飚%黄晓忠%朱利斌%夏立广%李仲荣
劉淼清%鬍書奇%王永飚%黃曉忠%硃利斌%夏立廣%李仲榮
류묘청%호서기%왕영표%황효충%주리빈%하립엄%리중영
干细胞%细胞增殖%细胞分化%磷脂酰肌醇3-激酶
榦細胞%細胞增殖%細胞分化%燐脂酰肌醇3-激酶
간세포%세포증식%세포분화%린지선기순3-격매
Stem cells%Cell proliferation%Cell Differentiation%Phosphatidylinositol 3-Kinase
目的 通过抑制PI3K/AKT信号通路,探讨该信号通路在一氧化氮对体外培养的大鼠肠神经干细胞增殖、分化的调控情况.方法 从孕15d胚鼠肠道提取肠神经干细胞,传代后的肠神经干细胞分为LY294002(25 μmol/L)组、LY294002(50μmol/L)组及空白对照组,培养48 h后,形态学观察分析各组的神经球大小情况,流式细胞仪检测各组肠神经干细胞nestin、BrdU标记的增殖细胞、子代细胞神经元Tuj-1染色阳性细胞百分比;传代后的肠神经干细胞分为50 μmol/l DETA/NO+ 20 ng/ml EGF组、100 μmol/L L-NAME+ 20 ng/ml EGF组及空白对照组(20 ng/ml EGF),培养1 h后,利用Western Blot法检测及gel-pro图像软件分析各组P-AKT磷酸化水平.结果 形态学观察发现LY294002(50 μmol/L)组神经球较小、部分细胞有退化现象、LY294002 (25 μmol/L)组次之、空白对照组神经球较大;流式检测发现LY294002 (25 μmol/L)组与空白对照组相比,肠神经干细胞nestin百分比显著降低[(18.42±1.22)％比(27.44±1.08)％,(P＜0.05)],BrdU标记的增殖细胞百分比显著降低[(2.20±0.30)％比(12.27±0.59)％,(P＜0.05)],神经元Tuj-1百分比显著增加[(17.23±0.78)％比(13.98±0.53)％,(P＜0.05)];而LY294002(50 μmol/L)组与空白对照组相比,肠神经干细胞nestin百分比[(8.77±0.90)％比(27.44±1.08)]、BrdU标记的增殖细胞百分比[(0.62±0.03)％比(12.27±0.59)]及神经元百分比[(9.88±1.22)％比(13.98±0.53)％]差异均有统计学意义(P＜0.05).Western Blot法检测发现100 μmol/L L-NAME+ 20 ng/ml EGF组pAKT磷酸化的程度显著增强[(15.71±1.09)％比(39.42±1.02)％,P＜0.05],50 μmol/l DETA/NO+ 20 ng/ml EGF组显著降低[(58.44±1.73)％比(39.42±1.02)％,P＜0.05].结论 一氧化氮能抑制肠神经干细胞增殖、促进其向神经元分化,其作用机制与抑制PI3K/AKT信号通路有关.
目的 通過抑製PI3K/AKT信號通路,探討該信號通路在一氧化氮對體外培養的大鼠腸神經榦細胞增殖、分化的調控情況.方法 從孕15d胚鼠腸道提取腸神經榦細胞,傳代後的腸神經榦細胞分為LY294002(25 μmol/L)組、LY294002(50μmol/L)組及空白對照組,培養48 h後,形態學觀察分析各組的神經毬大小情況,流式細胞儀檢測各組腸神經榦細胞nestin、BrdU標記的增殖細胞、子代細胞神經元Tuj-1染色暘性細胞百分比;傳代後的腸神經榦細胞分為50 μmol/l DETA/NO+ 20 ng/ml EGF組、100 μmol/L L-NAME+ 20 ng/ml EGF組及空白對照組(20 ng/ml EGF),培養1 h後,利用Western Blot法檢測及gel-pro圖像軟件分析各組P-AKT燐痠化水平.結果 形態學觀察髮現LY294002(50 μmol/L)組神經毬較小、部分細胞有退化現象、LY294002 (25 μmol/L)組次之、空白對照組神經毬較大;流式檢測髮現LY294002 (25 μmol/L)組與空白對照組相比,腸神經榦細胞nestin百分比顯著降低[(18.42±1.22)％比(27.44±1.08)％,(P＜0.05)],BrdU標記的增殖細胞百分比顯著降低[(2.20±0.30)％比(12.27±0.59)％,(P＜0.05)],神經元Tuj-1百分比顯著增加[(17.23±0.78)％比(13.98±0.53)％,(P＜0.05)];而LY294002(50 μmol/L)組與空白對照組相比,腸神經榦細胞nestin百分比[(8.77±0.90)％比(27.44±1.08)]、BrdU標記的增殖細胞百分比[(0.62±0.03)％比(12.27±0.59)]及神經元百分比[(9.88±1.22)％比(13.98±0.53)％]差異均有統計學意義(P＜0.05).Western Blot法檢測髮現100 μmol/L L-NAME+ 20 ng/ml EGF組pAKT燐痠化的程度顯著增彊[(15.71±1.09)％比(39.42±1.02)％,P＜0.05],50 μmol/l DETA/NO+ 20 ng/ml EGF組顯著降低[(58.44±1.73)％比(39.42±1.02)％,P＜0.05].結論 一氧化氮能抑製腸神經榦細胞增殖、促進其嚮神經元分化,其作用機製與抑製PI3K/AKT信號通路有關.
목적 통과억제PI3K/AKT신호통로,탐토해신호통로재일양화담대체외배양적대서장신경간세포증식、분화적조공정황.방법 종잉15d배서장도제취장신경간세포,전대후적장신경간세포분위LY294002(25 μmol/L)조、LY294002(50μmol/L)조급공백대조조,배양48 h후,형태학관찰분석각조적신경구대소정황,류식세포의검측각조장신경간세포nestin、BrdU표기적증식세포、자대세포신경원Tuj-1염색양성세포백분비;전대후적장신경간세포분위50 μmol/l DETA/NO+ 20 ng/ml EGF조、100 μmol/L L-NAME+ 20 ng/ml EGF조급공백대조조(20 ng/ml EGF),배양1 h후,이용Western Blot법검측급gel-pro도상연건분석각조P-AKT린산화수평.결과 형태학관찰발현LY294002(50 μmol/L)조신경구교소、부분세포유퇴화현상、LY294002 (25 μmol/L)조차지、공백대조조신경구교대;류식검측발현LY294002 (25 μmol/L)조여공백대조조상비,장신경간세포nestin백분비현저강저[(18.42±1.22)％비(27.44±1.08)％,(P＜0.05)],BrdU표기적증식세포백분비현저강저[(2.20±0.30)％비(12.27±0.59)％,(P＜0.05)],신경원Tuj-1백분비현저증가[(17.23±0.78)％비(13.98±0.53)％,(P＜0.05)];이LY294002(50 μmol/L)조여공백대조조상비,장신경간세포nestin백분비[(8.77±0.90)％비(27.44±1.08)]、BrdU표기적증식세포백분비[(0.62±0.03)％비(12.27±0.59)]급신경원백분비[(9.88±1.22)％비(13.98±0.53)％]차이균유통계학의의(P＜0.05).Western Blot법검측발현100 μmol/L L-NAME+ 20 ng/ml EGF조pAKT린산화적정도현저증강[(15.71±1.09)％비(39.42±1.02)％,P＜0.05],50 μmol/l DETA/NO+ 20 ng/ml EGF조현저강저[(58.44±1.73)％비(39.42±1.02)％,P＜0.05].결론 일양화담능억제장신경간세포증식、촉진기향신경원분화,기작용궤제여억제PI3K/AKT신호통로유관.
Objective To explore the role of phosphoinositide-3-kinase (PI3K)/AKT signaling pathway in nitric oxide on the in vitro proliferation and differentiation of enteric neural stem cells (ENSCs).Methods ENSCs were isolated from gut of 15-day-old rat embryos and cultured into 3 groups of LY294002 25 μmol/L (PB-K inhibitor) intervention,LY294002 50 μmol/L intervention and control.After 48h culture,neurospheric sizes in each group were observed.The percentages of positive cells for nestin,5-bromo-2-deoxyuridine (BrdU) and Tuj-1 were measured by flow cytometry.And ENSCs were also cultured into 3 groups of DETA/NO 50μmol/L + 20 ng/ml EGF intervention,L-NAME100 μmol/L+ 20 ng/ml EGF intervention and control (20 ng/ml EGF).After 1h culture,quantitative Western blot and gel-pro imaging system were used to detect the levels of phosphorylatedAKT level.Results Neurospheric sizes were the smallest,some cells had a phenomenon of degradation in LY294002(50μmol/L) group and the largest in control and LY294002 (25 μmol/L)groups.Flow cytometry showed that positive rates for nestin decreased significantly [(18.42 ±1.22) ％ vs (27.44 ± 1.08) ％,P＜0.05],BrdU decreased significantly [2.20 ± 0.30) ％ vs (12.27 ±0.59),P＜0.05] and Tuj-1 increased significantly [17.23 ± 0.78) ％ vs (13.98 ± 0.53) ％,P＜0.05] in LY294002 (25 μmol/L) group.There were all significant decreases (P＜0.05) for nestin,BrdU and Tui-1 in LY294002 (50 μmol/L) group (8.77 ± 0.90) ％ vs (27.44 ± 1.08);(0.62 ± 0.03) ％ vs (12.27 ± 0.59);(9.88 ± 1.22) ％ vs (13.98 ± 0.53) ％.Western blot quantitative analysis showed that the expression level of P-AKT increased significantly (P＜0.05) in L-NAME (100 μmol/L) + 20 ng/ml EGF (15.71 ± 1.09) ％ vs (39.42 ± 1.02) and decreased significantly in DETA/NO (50μmol/L) + 20 ng/ml EGF[(15.71 ± 1.09)％ vs (39.42 ± 1.02),P＜0.05].Conclusions Nitric oxide decreases the proliferation of ENSCs and promotes its differentiation into neurons through an inhibition of PBK/AKT pathway.
