中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2007年
15期
2971-2975
,共5页
嗅球%嗅鞘细胞%纯化%纯度%细胞活力
嗅毬%嗅鞘細胞%純化%純度%細胞活力
후구%후초세포%순화%순도%세포활력
背景:嗅鞘细胞的纯化方法以及嗅鞘细胞纯度的不同被认为与嗅鞘细胞移植后的效果有关.因此,开发高效、便于统一的纯化方法对嗅鞘细胞移植研究的标准化非常重要.目的:为嗅鞘细胞移植研究的标准化提供一个高效、便于统一的纯化方法.设计:随机对照实验.单位:东南大学临床医学院附属中大医院骨科,东南大学临床医学院中心实验室,东南大学临床医学院实验动物中心.材料:实验于2006-02/08在东南大学临床医学院中心实验室完成.选用28只成年雌性SD大鼠,体质量200～250 g;DMEM/F-12(GIBCO);2.5 g/L胰蛋白酶(GIBCO);左旋多聚赖氨酸(SlGMA);牛垂体萃取液(SIGMA);胎牛血清(杭州四季青生物试剂公司);兔抗p75抗体(SIGMA);生物素化羊抗兔IgG(武汉博士德生物技术公司);MTT试剂盒(SlGMA).方法:将SD大鼠嗅球中分离出嗅鞘细胞的原代培养物,体外培养8 d,将培养物分为4组:差速贴壁组、免疫吸附组、改良方法组、对照组.①改良方法组细胞悬液接种到未经包被的培养瓶在37℃、体积分数为0.05CO2的环境中孵育1 h,将上清液接种到培养瓶中(底面用1 mg/L的兔抗p75抗体润湿后在37℃下烘干,再用DMEM/F-12洗涤1次).上清液在这种已包被兔抗p75抗体的培养瓶中37℃、体积分数为0.05 CO2下孵育45min,DMEM/F-12洗涤5遍以清除未贴壁的细胞,用细胞刮刀收获贴壁细胞、离心、重新悬浮在含20 mg/L牛垂体萃取液和10万U/L青链霉素的D/F-10S中;Nash差速贴壁组细胞按Nash的方法进行操作;抗p75抗体免疫吸附组细胞按照Ramo'n-Cueto的方法进行操作;上述纯化方法获得的3组细胞悬液分别接种到包被左旋多聚赖氨酸的24孔细胞培养板中,在37℃、体积分数为0.05 CO2的环境中培养14 d.对照组未经纯化的细胞悬液同样重新悬浮在含20 mg/L牛垂体萃取液和10万U/L青链霉素的D/F-10S中并接种到包被左旋多聚赖氨酸的24孔细胞培养板中,培养条件同其他组.②在各组处理结束后2,5,8,10,12,14 d进行嗅鞘细胞纯度比较,每组每个时间点都随机选择15个视野计算嗅鞘细胞比例,这15个数值的平均值代表嗅鞘细胞纯度,从而评估这种改良方法的纯化效率.③分别用MTT法检测处理后第14天各组细胞活力.主要观察指标:各组嗅鞘细胞纯度、处理后14 d细胞活力检测结果.结果:①改良方法组每个时间点的嗅鞘细胞纯度都高于其他3组(P＜0.05～0.01),各组嗅鞘细胞纯度都随培养时间延长而降低,但改良方法组的纯度变化最小,改良方法组最后1个时间点的纯度仍然很高(92.1±1.2)%,而其他组最高只有(85.2±2.2)%.②处理结束后第14天,改良方法组嗅鞘细胞细胞活力与其他组细胞活力差异无显著性(P=0.895).结论:本组纯化成年大鼠嗅球嗅鞘细胞的改良方法是高效的,而且对嗅鞘细胞的活力无额外损害,将有益于嗅鞘细胞研究的标准化.
揹景:嗅鞘細胞的純化方法以及嗅鞘細胞純度的不同被認為與嗅鞘細胞移植後的效果有關.因此,開髮高效、便于統一的純化方法對嗅鞘細胞移植研究的標準化非常重要.目的:為嗅鞘細胞移植研究的標準化提供一箇高效、便于統一的純化方法.設計:隨機對照實驗.單位:東南大學臨床醫學院附屬中大醫院骨科,東南大學臨床醫學院中心實驗室,東南大學臨床醫學院實驗動物中心.材料:實驗于2006-02/08在東南大學臨床醫學院中心實驗室完成.選用28隻成年雌性SD大鼠,體質量200～250 g;DMEM/F-12(GIBCO);2.5 g/L胰蛋白酶(GIBCO);左鏇多聚賴氨痠(SlGMA);牛垂體萃取液(SIGMA);胎牛血清(杭州四季青生物試劑公司);兔抗p75抗體(SIGMA);生物素化羊抗兔IgG(武漢博士德生物技術公司);MTT試劑盒(SlGMA).方法:將SD大鼠嗅毬中分離齣嗅鞘細胞的原代培養物,體外培養8 d,將培養物分為4組:差速貼壁組、免疫吸附組、改良方法組、對照組.①改良方法組細胞懸液接種到未經包被的培養瓶在37℃、體積分數為0.05CO2的環境中孵育1 h,將上清液接種到培養瓶中(底麵用1 mg/L的兔抗p75抗體潤濕後在37℃下烘榦,再用DMEM/F-12洗滌1次).上清液在這種已包被兔抗p75抗體的培養瓶中37℃、體積分數為0.05 CO2下孵育45min,DMEM/F-12洗滌5遍以清除未貼壁的細胞,用細胞颳刀收穫貼壁細胞、離心、重新懸浮在含20 mg/L牛垂體萃取液和10萬U/L青鏈黴素的D/F-10S中;Nash差速貼壁組細胞按Nash的方法進行操作;抗p75抗體免疫吸附組細胞按照Ramo'n-Cueto的方法進行操作;上述純化方法穫得的3組細胞懸液分彆接種到包被左鏇多聚賴氨痠的24孔細胞培養闆中,在37℃、體積分數為0.05 CO2的環境中培養14 d.對照組未經純化的細胞懸液同樣重新懸浮在含20 mg/L牛垂體萃取液和10萬U/L青鏈黴素的D/F-10S中併接種到包被左鏇多聚賴氨痠的24孔細胞培養闆中,培養條件同其他組.②在各組處理結束後2,5,8,10,12,14 d進行嗅鞘細胞純度比較,每組每箇時間點都隨機選擇15箇視野計算嗅鞘細胞比例,這15箇數值的平均值代錶嗅鞘細胞純度,從而評估這種改良方法的純化效率.③分彆用MTT法檢測處理後第14天各組細胞活力.主要觀察指標:各組嗅鞘細胞純度、處理後14 d細胞活力檢測結果.結果:①改良方法組每箇時間點的嗅鞘細胞純度都高于其他3組(P＜0.05～0.01),各組嗅鞘細胞純度都隨培養時間延長而降低,但改良方法組的純度變化最小,改良方法組最後1箇時間點的純度仍然很高(92.1±1.2)%,而其他組最高隻有(85.2±2.2)%.②處理結束後第14天,改良方法組嗅鞘細胞細胞活力與其他組細胞活力差異無顯著性(P=0.895).結論:本組純化成年大鼠嗅毬嗅鞘細胞的改良方法是高效的,而且對嗅鞘細胞的活力無額外損害,將有益于嗅鞘細胞研究的標準化.
배경:후초세포적순화방법이급후초세포순도적불동피인위여후초세포이식후적효과유관.인차,개발고효、편우통일적순화방법대후초세포이식연구적표준화비상중요.목적:위후초세포이식연구적표준화제공일개고효、편우통일적순화방법.설계:수궤대조실험.단위:동남대학림상의학원부속중대의원골과,동남대학림상의학원중심실험실,동남대학림상의학원실험동물중심.재료:실험우2006-02/08재동남대학림상의학원중심실험실완성.선용28지성년자성SD대서,체질량200～250 g;DMEM/F-12(GIBCO);2.5 g/L이단백매(GIBCO);좌선다취뢰안산(SlGMA);우수체췌취액(SIGMA);태우혈청(항주사계청생물시제공사);토항p75항체(SIGMA);생물소화양항토IgG(무한박사덕생물기술공사);MTT시제합(SlGMA).방법:장SD대서후구중분리출후초세포적원대배양물,체외배양8 d,장배양물분위4조:차속첩벽조、면역흡부조、개량방법조、대조조.①개량방법조세포현액접충도미경포피적배양병재37℃、체적분수위0.05CO2적배경중부육1 h,장상청액접충도배양병중(저면용1 mg/L적토항p75항체윤습후재37℃하홍간,재용DMEM/F-12세조1차).상청액재저충이포피토항p75항체적배양병중37℃、체적분수위0.05 CO2하부육45min,DMEM/F-12세조5편이청제미첩벽적세포,용세포괄도수획첩벽세포、리심、중신현부재함20 mg/L우수체췌취액화10만U/L청련매소적D/F-10S중;Nash차속첩벽조세포안Nash적방법진행조작;항p75항체면역흡부조세포안조Ramo'n-Cueto적방법진행조작;상술순화방법획득적3조세포현액분별접충도포피좌선다취뢰안산적24공세포배양판중,재37℃、체적분수위0.05 CO2적배경중배양14 d.대조조미경순화적세포현액동양중신현부재함20 mg/L우수체췌취액화10만U/L청련매소적D/F-10S중병접충도포피좌선다취뢰안산적24공세포배양판중,배양조건동기타조.②재각조처리결속후2,5,8,10,12,14 d진행후초세포순도비교,매조매개시간점도수궤선택15개시야계산후초세포비례,저15개수치적평균치대표후초세포순도,종이평고저충개량방법적순화효솔.③분별용MTT법검측처리후제14천각조세포활력.주요관찰지표:각조후초세포순도、처리후14 d세포활력검측결과.결과:①개량방법조매개시간점적후초세포순도도고우기타3조(P＜0.05～0.01),각조후초세포순도도수배양시간연장이강저,단개량방법조적순도변화최소,개량방법조최후1개시간점적순도잉연흔고(92.1±1.2)%,이기타조최고지유(85.2±2.2)%.②처리결속후제14천,개량방법조후초세포세포활력여기타조세포활력차이무현저성(P=0.895).결론:본조순화성년대서후구후초세포적개량방법시고효적,이차대후초세포적활력무액외손해,장유익우후초세포연구적표준화.
BACKGROUND: The diversity of purification procedures resulting in various purities of olfactory ensheathing cells (OECs) used for grafting is considered to be relevant in the effectiveness of OECs transplant. It is important to develop a well-defined method which produces OECs of great purity and is easy to unify for the future standardization of research involving OECs.OBJECTIVE: To establish a method being easy to unify for purifying OECs to acquire highly and uniformly enriched population of OECs for standardized studies on cell transplantation.DESIGN: Randomized and controlled experiment.SETTING: Department of Orthopaedics, Affiliated Zhongda Hospital of Southeast University School of Clinical Medicine;Central Laboratory of Southeast University School of Clinical Medicine; Experimental Animal Center of Southeast University School of Clinical Medicine.MATERIALS: This experiment was carried out in the Central Laboratory of Southeast University School of Clinical Medicine from February to August 2006. Twenty-eight adult female SD rats weighing 200-250 g were selected in this study. The main reagents were detailed as follows: DMEM/F-12 (GIBCO); 2.5 g/L trypsin (GIBCO); poly-L-lysine (SIGMA); bovine pituitary extract (BPE, SIGMA); fetal bovine serum (FBS, Sijiqing Biological Agent Co., Ltd., Hangzhou);rabbit anti-low-affinity nerve growth factor receptor (anti-P75, SIGMA); biotinylated goat anti-rabbit IgG (Boster Bioengineering Co., Ltd., Wuhan); methyl thiazolyl tetrazolium (MTT) kit (SIGMA).METHODS: Primary cultures of OECs were separated from adult SD rats olfactory bulbs. At day 8 in vitro, the primary cultures were divided randomly into 4 groups, namely differential adhesion method group, immunoadsorption method group,the modified method group,and control group.①The cell suspension in the modified method group was seeded into uncoated flasks and incubated at 37 ℃ in 0.05 volume fraction of CO2 for 1 hours. The supematants were seeded into flasks that had been prepared as follows. The bottoms of these flasks were moistened with anti-P7s (1 mg/L) and were made to dry at 37 ℃, and then they were washed one time with DMEM/F-12. The supernatants were incubated on the anti-p75-treated flasks for 45 minutes at 37 ℃, 0.05 volume fraction of CO2. For removing unbound cells, the flasks were washed five times with DMEM/F-12. The bound cells were detached from the flasks with a cell scraper, centrifuged,and resuspended in D/F-10S with 105 U/L penicillin/streptomycin and 20 mg/L BPE. The cell suspension in differential anchoring method group or immunoadsorption method group was purified as previously described by Nash or Ramo'n-Cueto respectively. Three groups of cell suspensions resulted from the above three methods were seeded respectively onto poly-L-lysine-coated 24-well cell culture chambers and incubated for 14 days at 37 ℃ in 0.05 volume fraction of CO2. Without purification, the cell suspension in control group was also resuspended in D/F-10S with 105 U/L penicillin/streptomycin and 20 mg/L BPE and seeded onto poly-L-lysine-coated 24-well cell culture chambers and incubated under the same culture condition as the other groups.②Purity comparisons for the four groups were made at 2, 5, 8, 10, 12 and 14 days after the end of their respective manipulation to evaluate the effectiveness of the modified method. At per time point in each of the four groups, fifteen visual fields of cultures were selected randomly to count the At the day of 14, viabilities of OECs in the four groups were assessed by MTT assays.MAIN OUTCOME MEASURES: OECs purities at per time point and viabilities of OECs at the day of 14 in each of the four groups after the end of their respective manipulation.RESULTS:① The purities of OECs in the modified method group at each time point were greater (P＜0.05-0.01) than counterparts in the other three groups. OECs purities decreased with culture prolongation in all groups, but the changes of purities over the whole period of observation in the modified method group were the least. The last purities of OECs yielded from the modified method were still extremely great (92.1±1.2)%, whereas the parallels in the others were no more than (85.2±2.2)%.② There was no significant difference in viabilities of OECs between the modified method group and any of the others at the day of 14 (P=0.895).CONCLUSION: The modified method for purifying OECs from the adult rat olfactory bulb is highly effective without extra impairment on the viability of OECs and will be beneficial to the future standardization of research involving OECs.
