CAJ | 학술논문

干细胞%干细胞培养与分化%新生鼠睾丸%精原干细胞%Accutase酶%胰酶%透明质酸酶%磁珠分选%流式细胞分选%CD90.2%胶质细胞源性神经营养因子受体α1%国家自然科学基金%干细胞图片文章榦細胞%榦細胞培養與分化%新生鼠睪汍%精原榦細胞%Accutase酶%胰酶%透明質痠酶%磁珠分選%流式細胞分選%CD90.2%膠質細胞源性神經營養因子受體α1%國傢自然科學基金%榦細胞圖片文章
간세포%간세포배양여분화%신생서고환%정원간세포%Accutase매%이매%투명질산매%자주분선%류식세포분선%CD90.2%효질세포원성신경영양인자수체α1%국가자연과학기금%간세포도편문장

背景：有研究报道胰酶消化在一定程度上会破坏精原干细胞表面抗原并影响细胞活性，对后续的细胞分选及下游实验有一定影响。Accutae 酶具有蛋白酶和胶原酶的双重活性，在消化结束时无需终止和清洗，有保护细胞表面抗原的特殊作用，因而被应用于多种干细胞的培养及消化传代中。 　　目的：比较Accutase酶和胰酶在原代消化分离睾丸组织获取精原干细胞中的作用。 　　方法：新生5-7 d昆明雄鼠45只，取双侧睾丸胶原酶初步消化，混悬液定容后分为Accutase酶组、胰酶组、混合酶组(透明质酸酶+胰酶组)，不同组别小鼠睾丸组织分别于酶消化1，3，5 min后显微镜下观察并拍照，比较各组不同时间点的消化状态及形成单细胞所需的时间；获取单细胞悬液后分别计算单位体积内所得细胞总数及死亡率；通过差速贴壁方法去除间质细胞及支持细胞，经包被有干细胞标志分子 CD90.2的免疫磁珠进行分选，将所得 CD90+及 CD90-细胞用精原干细胞表面标志分子--胶质细胞源性神经营养因子受体α1标记，流式细胞仪检测不同组别CD90+及 CD90-细胞群中胶质细胞源性神经营养因子受体α1精原干细胞的阳性率。 　　结果与结论：不同类别的消化酶对小鼠睾丸的消化作用有明显差异，其中 Accutase 酶能更快获取单细胞悬液，其细胞团及破膜细胞明显少于胰酶组和混合酶组，其所得细胞总数高于其余2组，而细胞死亡率则低于其余2组。差速贴壁后细胞免疫磁珠分选结果显示Accutase酶组CD90+精原干细胞得率高，流式分选结果显示胶质细胞源性神经营养因子受体α1+/CD90+细胞为72.24%，高于胰酶组及混合酶组(51.16%，71.27%)；而胶质细胞源性神经营养因子受体α1+/CD90-细胞比率(15.03%)则低于胰酶组及混合酶组(18.8%，24.23%)。结果表明Accutase酶在分离和获取精原干细胞实验中效果优于胰酶。揹景：有研究報道胰酶消化在一定程度上會破壞精原榦細胞錶麵抗原併影響細胞活性，對後續的細胞分選及下遊實驗有一定影響。Accutae 酶具有蛋白酶和膠原酶的雙重活性，在消化結束時無需終止和清洗，有保護細胞錶麵抗原的特殊作用，因而被應用于多種榦細胞的培養及消化傳代中。 　　目的：比較Accutase酶和胰酶在原代消化分離睪汍組織穫取精原榦細胞中的作用。 　　方法：新生5-7 d昆明雄鼠45隻，取雙側睪汍膠原酶初步消化，混懸液定容後分為Accutase酶組、胰酶組、混閤酶組(透明質痠酶+胰酶組)，不同組彆小鼠睪汍組織分彆于酶消化1，3，5 min後顯微鏡下觀察併拍照，比較各組不同時間點的消化狀態及形成單細胞所需的時間；穫取單細胞懸液後分彆計算單位體積內所得細胞總數及死亡率；通過差速貼壁方法去除間質細胞及支持細胞，經包被有榦細胞標誌分子 CD90.2的免疫磁珠進行分選，將所得 CD90+及 CD90-細胞用精原榦細胞錶麵標誌分子--膠質細胞源性神經營養因子受體α1標記，流式細胞儀檢測不同組彆CD90+及 CD90-細胞群中膠質細胞源性神經營養因子受體α1精原榦細胞的暘性率。 　　結果與結論：不同類彆的消化酶對小鼠睪汍的消化作用有明顯差異，其中 Accutase 酶能更快穫取單細胞懸液，其細胞糰及破膜細胞明顯少于胰酶組和混閤酶組，其所得細胞總數高于其餘2組，而細胞死亡率則低于其餘2組。差速貼壁後細胞免疫磁珠分選結果顯示Accutase酶組CD90+精原榦細胞得率高，流式分選結果顯示膠質細胞源性神經營養因子受體α1+/CD90+細胞為72.24%，高于胰酶組及混閤酶組(51.16%，71.27%)；而膠質細胞源性神經營養因子受體α1+/CD90-細胞比率(15.03%)則低于胰酶組及混閤酶組(18.8%，24.23%)。結果錶明Accutase酶在分離和穫取精原榦細胞實驗中效果優于胰酶。
배경：유연구보도이매소화재일정정도상회파배정원간세포표면항원병영향세포활성，대후속적세포분선급하유실험유일정영향。Accutae 매구유단백매화효원매적쌍중활성，재소화결속시무수종지화청세，유보호세포표면항원적특수작용，인이피응용우다충간세포적배양급소화전대중。 　　목적：비교Accutase매화이매재원대소화분리고환조직획취정원간세포중적작용。 　　방법：신생5-7 d곤명웅서45지，취쌍측고환효원매초보소화，혼현액정용후분위Accutase매조、이매조、혼합매조(투명질산매+이매조)，불동조별소서고환조직분별우매소화1，3，5 min후현미경하관찰병박조，비교각조불동시간점적소화상태급형성단세포소수적시간；획취단세포현액후분별계산단위체적내소득세포총수급사망솔；통과차속첩벽방법거제간질세포급지지세포，경포피유간세포표지분자 CD90.2적면역자주진행분선，장소득 CD90+급 CD90-세포용정원간세포표면표지분자--효질세포원성신경영양인자수체α1표기，류식세포의검측불동조별CD90+급 CD90-세포군중효질세포원성신경영양인자수체α1정원간세포적양성솔。 　　결과여결론：불동유별적소화매대소서고환적소화작용유명현차이，기중 Accutase 매능경쾌획취단세포현액，기세포단급파막세포명현소우이매조화혼합매조，기소득세포총수고우기여2조，이세포사망솔칙저우기여2조。차속첩벽후세포면역자주분선결과현시Accutase매조CD90+정원간세포득솔고，류식분선결과현시효질세포원성신경영양인자수체α1+/CD90+세포위72.24%，고우이매조급혼합매조(51.16%，71.27%)；이효질세포원성신경영양인자수체α1+/CD90-세포비솔(15.03%)칙저우이매조급혼합매조(18.8%，24.23%)。결과표명Accutase매재분리화획취정원간세포실험중효과우우이매。
BACKGROUND:It is reported that the cellsurface antigen may be damaged by the trypsin enzyme in some extent and the cellactivity may also be influenced, as a result of which, the subsequent separation and fol ow-up-test wil be affected. Accutase enzymes possess the activities of protease and col agen enzyme and need no special termination or cleaning at the end of digestion. Another special function of accutase enzyme is to protect cellsurface antigen and thus it has been widely applied in the stem celldigestion and culture. OBJECTIVE:To compare the digestive effect of accutase enzymes and trypsin enzyme in the separation and original culture of spermatogonial stem cells. METHODS:The testes of 5-7 days male Kunming mice (n=45) were col ected and primarily digested with col agenas. The suspension of digested tissue was divided into three parts with the same volume named accutases enzyme group, trypsin enzyme group and mixed enzyme group (trypsin enzyme and hyaluronidase). For the comparison of testis digestive status and the time required for the formation of single cells, the micrographs were taken at 1, 3 and 5 minutes respectively after enzyme digestion. The total number and the mortality of the single cells were estimated and compared. The leydig cells and sertoli cells were removed by differential adherent method and the remained spermatogenic cells were then treated with CD90.2 immune magnetic beads. The selected spermatogonial stem cells were subsequently labeled by glial cellline-derived neurotrophic factor receptor alpha-1 and sorted with flow cytometry. The number of spermatogonial stem cells positive for glial cellline-derived neurotrophic factor receptor alpha-1 in CD90.2+and CD90.2-cells was compared within different groups. RESULTS AND CONCLUSION:The digestive capacities of different enzyme were different. Accutases enzyme obtained the single cellsuspension more quickly than trypsin enzyme and mixed enzyme, and had the least cellmasses and broken cellmembrane than the other two groups. After the differential attached treatment, the highest total number of CD90+spermatogonial stem cells and lowest cellmortality could be found in the accutases group, when compared with the other groups. The results of cellsorting by flow cytometry showed a higher rate (72.24%) of GFRα1+/CD90+cells in the accutases group than the trypsin group (51.16%) and mixed enzyme group (71.27%). The GFRα1+/CD90-number in the accutases group was lower (15.03%) than that of the trypsin group (18.8%) and mixed enzyme group (24.23%), respectively. The results indicate a better effect of accutases enzyme on the primary separation of spermatogonial stem cells than that of trypsin or mixed enzyme.