中华神经医学杂志
中華神經醫學雜誌
중화신경의학잡지
CHINESE JOURNAL OF NEUROMEDICINE
2011年
5期
456-460
,共5页
兰艳纤%李金恒%王克万%王勇
蘭豔纖%李金恆%王剋萬%王勇
란염섬%리금항%왕극만%왕용
神经干细胞%海马%胰酶%细胞增殖%细胞凋亡
神經榦細胞%海馬%胰酶%細胞增殖%細胞凋亡
신경간세포%해마%이매%세포증식%세포조망
Neural stem cell%Hippocampus%Trypsinization%Proliferation%Apoptosis
目的 探讨低浓度胰酶不同消化分离时间对体外培养新生大鼠海马NSCs增殖与凋亡的影响.方法 取出生24 h内SD大鼠海马组织,以1.25 g/L胰酶37℃分别消化5、10、15、20和25min(依次为A～E组),获得单细胞悬液后进行培养.通过台盼蓝染色计数、细胞形态观察和神经球数目比较不同消化时间对NSCs活力和生长的影响;用5-溴-2脱氧尿嘧啶核苷(BrdU)标记法检测NSCs的增殖能力;用免疫荧光细胞化学法检测BrdU、nestin的表达:用Annexin V-FITC/PI染色和流式细胞仪检测细胞凋亡率.结果 原代和传代培养的NSCs都能快速增殖并形成神经球;免疫荧光染色结果显示神经球细胞均表达NSCs特异性标志物nestin;所获得的细胞能将BrdU结合到细胞核中;各组培养3、5、7 d后,C组(消化15 min)NSCs成球数最多,BrdU标记克隆率最高,细胞凋亡率最低,与其他组比较差异有统计学意义(P<0.05).结论 体外分离培养的新生大鼠海马NSCs具有增殖能力,1.25 g/L胰酶在不同消化时间对NSCs增殖能力和凋亡率的影响有所不同,消化时间过长或过短都会抑制NSCs增殖,诱导NSCs凋亡,且消化时间越长NSCs的凋亡率越高.
目的 探討低濃度胰酶不同消化分離時間對體外培養新生大鼠海馬NSCs增殖與凋亡的影響.方法 取齣生24 h內SD大鼠海馬組織,以1.25 g/L胰酶37℃分彆消化5、10、15、20和25min(依次為A～E組),穫得單細胞懸液後進行培養.通過檯盼藍染色計數、細胞形態觀察和神經毬數目比較不同消化時間對NSCs活力和生長的影響;用5-溴-2脫氧尿嘧啶覈苷(BrdU)標記法檢測NSCs的增殖能力;用免疫熒光細胞化學法檢測BrdU、nestin的錶達:用Annexin V-FITC/PI染色和流式細胞儀檢測細胞凋亡率.結果 原代和傳代培養的NSCs都能快速增殖併形成神經毬;免疫熒光染色結果顯示神經毬細胞均錶達NSCs特異性標誌物nestin;所穫得的細胞能將BrdU結閤到細胞覈中;各組培養3、5、7 d後,C組(消化15 min)NSCs成毬數最多,BrdU標記剋隆率最高,細胞凋亡率最低,與其他組比較差異有統計學意義(P<0.05).結論 體外分離培養的新生大鼠海馬NSCs具有增殖能力,1.25 g/L胰酶在不同消化時間對NSCs增殖能力和凋亡率的影響有所不同,消化時間過長或過短都會抑製NSCs增殖,誘導NSCs凋亡,且消化時間越長NSCs的凋亡率越高.
목적 탐토저농도이매불동소화분리시간대체외배양신생대서해마NSCs증식여조망적영향.방법 취출생24 h내SD대서해마조직,이1.25 g/L이매37℃분별소화5、10、15、20화25min(의차위A～E조),획득단세포현액후진행배양.통과태반람염색계수、세포형태관찰화신경구수목비교불동소화시간대NSCs활력화생장적영향;용5-추-2탈양뇨밀정핵감(BrdU)표기법검측NSCs적증식능력;용면역형광세포화학법검측BrdU、nestin적표체:용Annexin V-FITC/PI염색화류식세포의검측세포조망솔.결과 원대화전대배양적NSCs도능쾌속증식병형성신경구;면역형광염색결과현시신경구세포균표체NSCs특이성표지물nestin;소획득적세포능장BrdU결합도세포핵중;각조배양3、5、7 d후,C조(소화15 min)NSCs성구수최다,BrdU표기극륭솔최고,세포조망솔최저,여기타조비교차이유통계학의의(P<0.05).결론 체외분리배양적신생대서해마NSCs구유증식능력,1.25 g/L이매재불동소화시간대NSCs증식능력화조망솔적영향유소불동,소화시간과장혹과단도회억제NSCs증식,유도NSCs조망,차소화시간월장NSCs적조망솔월고.
Objective To study the influence of digestion times of low concentration trypsin on the proliferation and apoptosis of neural stem cells (NSCs) in the hippocampus of neonate rats.Methods Hippocampus of neonatal rats (within 24 h) were taken out, and treated with trypsin at 1.25g/L concentration and 37 ℃ for 5, 10, 15, 20 and 25 minutes; unicellular suspension was then successfully got and primary culture and subculture were performed. Effects of trypsinization on cell viability and growth of NSCs were compared by observing the cell morphology and Trypan blue staining.The 5-bromodeoxyuridine labeling was performed to assess the self-renewing and proliferative activities of NSCs. Fluorescence immunocytochemistry was carried out to examine the expressions of BrdU and nestin. Apoptosis was measured by Annexin V-FITC/PI assay and flow cytometry. Results Primary and passage culture of NSCs enjoyed rapid proliferation and formation of neurospheres. The neurosphere cells expressed NSCs specific marker nestin by immunofluorescence; all the neurosphere cells could incorporate BrdU into the nucleus; of the neurospheres obtained from the 3rd, 5th and 7th d, those digested for 15 rain enjoyed the highest level of NSCs neurospheres, the highest BrdU labeled clone and the lowest cell apoptosis as compared with those digested for 5, 10, 20 and 25 min (P<0.05). Conclusion The NSCs isolated from the hippocampus of neonatal rats have the ability of proliferation in vitro. And 1.25 g/L concentration of trypsin with digestion times could positively change the proliferative and apoptosis capacity of NSCs: too short or long digestion times can inhibit the proliferation of NSCs and induce the apoptosis of NSCs; the longer the digestion time, the higher the apoptosis of NSCs.
