背景:骨髓间充质干细胞在骨髓中的含量非常少,而且与其他细胞特别是易与红细胞相混杂,因此在分离过程中需去除干扰,以获得尽可能多的高纯度的骨髓间充质干细胞.目的:采用红细胞裂解法分离培养大鼠骨髓间充质干细胞,同时进行生物学鉴定.设计:观察对比实验.单位:中国航天员科研训练中心航天细胞分子生物学实验室.材料:选用50只生后30 d的雄性SD大鼠,体质量100g,SPF级,购自北京实验动物中心(合格证:SCXK(京)2002-0003).DAB浓缩显色液(含过氧化氢,中杉公司),兔抗大鼠多克隆抗体(武汉博士德公司),FCS(PAA,Austria),LG-DMEM培养基(Sigma,USA).方法:实验于2004-09/2005-09在中国航天员科研训练中心航天细胞分子生物学实验室完成.将大鼠脱臼处死,暴露骨髓腔,收集细胞悬液,采用全骨髓培养法对骨髓间充质干细胞分离与原代培养.①红细胞裂解实验:取A、B、C、D 4管分别加入0.5 mL过滤并充分吹打均匀后的骨髓冲洗液,分别对应加入红细胞裂解液、氯化氨2 mL、磷酸盐缓冲生理盐水2 mL和体积分数为0.04的乙酸溶液0.5 mL,分别测量各组吸光度值及血红蛋白浓度,并观察细胞生长情况.②骨髓间充质干细胞生长曲线、倍增时间及表面标志分子表达情况的观察:根据公式:群体倍增时间(TD)=t[lg2/(lgNt-lgN0)](N0和Nt分别代表接种后和培养t小时的细胞数),计算出细胞的群体倍增时间并描绘并分析骨髓间充质干细胞第2、4、6代细胞的生长曲线;采用亚甲基兰染色测定骨髓间充质干细胞增殖情况;采用免疫细胞化学染色检测骨髓间充质干细胞表面标志分子表达情况.主要观察指标:①大鼠骨髓间充质干细胞分离和培养的观察结果.②不同处理方法对红细胞的裂解效果及其对骨髓间充质干细胞生长的影响.③第2、4、6代细胞的生长曲线及细胞倍增时间.④大鼠骨髓间充质干细胞表面标志分子表达情况.结果:①大鼠骨髓间充质干细胞的分离和培养结果:原代培养48 h后大部分细胞已贴壁,72 h时贴壁细胞已开始分裂增殖.7～8 d后可见有明显集落形成,之后集落迅速增多,不断扩大,互相融合,14～16 d时细胞克隆生长稠密.刚传代的细胞呈球形,很快沉降贴壁,少部分圆形细胞悬浮.贴壁细胞均匀分布,3～5 d增殖迅速.虽然传代后细胞形态未变,但增殖速度明显增快,约6 d长满培养瓶底.②不同处理方法对红细胞的裂解效果及其对骨髓间充质干细胞生长的影响:红细胞裂解液处理组,氯化氨处理组和4%乙酸处理组的血红蛋白浓度明显高于磷酸盐缓冲生理盐水处理组,差异有显著性(P＜0.01).③不同代骨髓间充质干细胞的生长曲线观察结果:第2,4,6代细胞生长曲线基本相同,经过一两天的潜伏适应期后进入对数生长期,第5天达顶点,以后进入平台期(在第5～7天).第6代骨髓间充质干细胞的潜伏期表现不明显骨髓间充质干细胞的群体倍增时间平均约为34 h.④不同代骨髓间充质干细胞的免疫表型鉴定:各代细胞CD44和CD106染色均呈阳性,表现为棕黄色颗粒沉淀,CD34染色呈阴性.结论:采用红细胞裂解液处理骨髓冲洗液后再行接种,能提高骨髓间充质干细胞的贴壁效率,同时不影响其贴壁后的生长,是一种可行的分离方法.细胞表面标记染色鉴定证明,本实验所分离细胞是骨髓间充质干细胞.
揹景:骨髓間充質榦細胞在骨髓中的含量非常少,而且與其他細胞特彆是易與紅細胞相混雜,因此在分離過程中需去除榦擾,以穫得儘可能多的高純度的骨髓間充質榦細胞.目的:採用紅細胞裂解法分離培養大鼠骨髓間充質榦細胞,同時進行生物學鑒定.設計:觀察對比實驗.單位:中國航天員科研訓練中心航天細胞分子生物學實驗室.材料:選用50隻生後30 d的雄性SD大鼠,體質量100g,SPF級,購自北京實驗動物中心(閤格證:SCXK(京)2002-0003).DAB濃縮顯色液(含過氧化氫,中杉公司),兔抗大鼠多剋隆抗體(武漢博士德公司),FCS(PAA,Austria),LG-DMEM培養基(Sigma,USA).方法:實驗于2004-09/2005-09在中國航天員科研訓練中心航天細胞分子生物學實驗室完成.將大鼠脫臼處死,暴露骨髓腔,收集細胞懸液,採用全骨髓培養法對骨髓間充質榦細胞分離與原代培養.①紅細胞裂解實驗:取A、B、C、D 4管分彆加入0.5 mL過濾併充分吹打均勻後的骨髓遲洗液,分彆對應加入紅細胞裂解液、氯化氨2 mL、燐痠鹽緩遲生理鹽水2 mL和體積分數為0.04的乙痠溶液0.5 mL,分彆測量各組吸光度值及血紅蛋白濃度,併觀察細胞生長情況.②骨髓間充質榦細胞生長麯線、倍增時間及錶麵標誌分子錶達情況的觀察:根據公式:群體倍增時間(TD)=t[lg2/(lgNt-lgN0)](N0和Nt分彆代錶接種後和培養t小時的細胞數),計算齣細胞的群體倍增時間併描繪併分析骨髓間充質榦細胞第2、4、6代細胞的生長麯線;採用亞甲基蘭染色測定骨髓間充質榦細胞增殖情況;採用免疫細胞化學染色檢測骨髓間充質榦細胞錶麵標誌分子錶達情況.主要觀察指標:①大鼠骨髓間充質榦細胞分離和培養的觀察結果.②不同處理方法對紅細胞的裂解效果及其對骨髓間充質榦細胞生長的影響.③第2、4、6代細胞的生長麯線及細胞倍增時間.④大鼠骨髓間充質榦細胞錶麵標誌分子錶達情況.結果:①大鼠骨髓間充質榦細胞的分離和培養結果:原代培養48 h後大部分細胞已貼壁,72 h時貼壁細胞已開始分裂增殖.7～8 d後可見有明顯集落形成,之後集落迅速增多,不斷擴大,互相融閤,14～16 d時細胞剋隆生長稠密.剛傳代的細胞呈毬形,很快沉降貼壁,少部分圓形細胞懸浮.貼壁細胞均勻分佈,3～5 d增殖迅速.雖然傳代後細胞形態未變,但增殖速度明顯增快,約6 d長滿培養瓶底.②不同處理方法對紅細胞的裂解效果及其對骨髓間充質榦細胞生長的影響:紅細胞裂解液處理組,氯化氨處理組和4%乙痠處理組的血紅蛋白濃度明顯高于燐痠鹽緩遲生理鹽水處理組,差異有顯著性(P＜0.01).③不同代骨髓間充質榦細胞的生長麯線觀察結果:第2,4,6代細胞生長麯線基本相同,經過一兩天的潛伏適應期後進入對數生長期,第5天達頂點,以後進入平檯期(在第5～7天).第6代骨髓間充質榦細胞的潛伏期錶現不明顯骨髓間充質榦細胞的群體倍增時間平均約為34 h.④不同代骨髓間充質榦細胞的免疫錶型鑒定:各代細胞CD44和CD106染色均呈暘性,錶現為棕黃色顆粒沉澱,CD34染色呈陰性.結論:採用紅細胞裂解液處理骨髓遲洗液後再行接種,能提高骨髓間充質榦細胞的貼壁效率,同時不影響其貼壁後的生長,是一種可行的分離方法.細胞錶麵標記染色鑒定證明,本實驗所分離細胞是骨髓間充質榦細胞.
배경:골수간충질간세포재골수중적함량비상소,이차여기타세포특별시역여홍세포상혼잡,인차재분리과정중수거제간우,이획득진가능다적고순도적골수간충질간세포.목적:채용홍세포렬해법분리배양대서골수간충질간세포,동시진행생물학감정.설계:관찰대비실험.단위:중국항천원과연훈련중심항천세포분자생물학실험실.재료:선용50지생후30 d적웅성SD대서,체질량100g,SPF급,구자북경실험동물중심(합격증:SCXK(경)2002-0003).DAB농축현색액(함과양화경,중삼공사),토항대서다극륭항체(무한박사덕공사),FCS(PAA,Austria),LG-DMEM배양기(Sigma,USA).방법:실험우2004-09/2005-09재중국항천원과연훈련중심항천세포분자생물학실험실완성.장대서탈구처사,폭로골수강,수집세포현액,채용전골수배양법대골수간충질간세포분리여원대배양.①홍세포렬해실험:취A、B、C、D 4관분별가입0.5 mL과려병충분취타균균후적골수충세액,분별대응가입홍세포렬해액、록화안2 mL、린산염완충생리염수2 mL화체적분수위0.04적을산용액0.5 mL,분별측량각조흡광도치급혈홍단백농도,병관찰세포생장정황.②골수간충질간세포생장곡선、배증시간급표면표지분자표체정황적관찰:근거공식:군체배증시간(TD)=t[lg2/(lgNt-lgN0)](N0화Nt분별대표접충후화배양t소시적세포수),계산출세포적군체배증시간병묘회병분석골수간충질간세포제2、4、6대세포적생장곡선;채용아갑기란염색측정골수간충질간세포증식정황;채용면역세포화학염색검측골수간충질간세포표면표지분자표체정황.주요관찰지표:①대서골수간충질간세포분리화배양적관찰결과.②불동처리방법대홍세포적렬해효과급기대골수간충질간세포생장적영향.③제2、4、6대세포적생장곡선급세포배증시간.④대서골수간충질간세포표면표지분자표체정황.결과:①대서골수간충질간세포적분리화배양결과:원대배양48 h후대부분세포이첩벽,72 h시첩벽세포이개시분렬증식.7～8 d후가견유명현집락형성,지후집락신속증다,불단확대,호상융합,14～16 d시세포극륭생장주밀.강전대적세포정구형,흔쾌침강첩벽,소부분원형세포현부.첩벽세포균균분포,3～5 d증식신속.수연전대후세포형태미변,단증식속도명현증쾌,약6 d장만배양병저.②불동처리방법대홍세포적렬해효과급기대골수간충질간세포생장적영향:홍세포렬해액처리조,록화안처리조화4%을산처리조적혈홍단백농도명현고우린산염완충생리염수처리조,차이유현저성(P＜0.01).③불동대골수간충질간세포적생장곡선관찰결과:제2,4,6대세포생장곡선기본상동,경과일량천적잠복괄응기후진입대수생장기,제5천체정점,이후진입평태기(재제5～7천).제6대골수간충질간세포적잠복기표현불명현골수간충질간세포적군체배증시간평균약위34 h.④불동대골수간충질간세포적면역표형감정:각대세포CD44화CD106염색균정양성,표현위종황색과립침정,CD34염색정음성.결론:채용홍세포렬해액처리골수충세액후재행접충,능제고골수간충질간세포적첩벽효솔,동시불영향기첩벽후적생장,시일충가행적분리방법.세포표면표기염색감정증명,본실험소분리세포시골수간충질간세포.
BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are few in bone marrow, and they are easily mingled with other cells, especially red blood cells. Therefore, intervention needs to be depleted in the process of isolation of red blood cells so as to obtain highlypurified BMSCs as many as possible.OBJECTIVE: To isolate and culture BMSCs of rats with red blood cell lysis method, and perform biological identification.DESIGN: Observation and controlled trial.SETTING: Laboratory of Aerospace Cell Molecular Biology, Scientific Research Training Center for Chinese Astronauts.MATERIALS: Fifty 30-day-old male SD rats, weighing about 100 g, of SPF degree, were purchased from Beijing Experimental Animal Center (License No. SCXK (Jing) 2002-0003) and involved in this trial. DAB condensed chromogen (hydrogen dioxide included, Zhongshan Company), rabbit anti-ret polyclonal antibody (Boster Co.,Ltd., Wuhan), fetal calf serum (PAA, Austria) and LG-DMEM medium (Sigma Company, USA) were used in this trial.METHODS: This trial was carried out in the Laboratory of Aerospace Cell Molecular Biology, Scientific Research Training Center for Chinese Astronauts during September 2004 to September 2005. The rats were sacrificed by dislocation to expose bone marrow cavity. Cell suspension was collected. BMSCs were isolated and cultured primarily by whole bonemarrow culture method. ① Red blood cells lysis test: A, B, C and D 4 tubes were chosen and filled with 0.5 mL bonemarrow rinse solution which was filtered and fully beat upon. Then, red blood cell lysis buffer of 2 mL, ammonium chloride of 2 mL, phosphate buffer normal saline of 2 mL and 0.04 volume fraction acetic acid of 0.5 mL were correspondingly added into the 4 tubes. In each tube, absorbance and hemoglobin concentration were measured and cell growth was observed. ② Observation of growth curve, doubling time and surface marker molecule expression of BMSCs: Based on the formula, population doubling time (TD) =t[lg2/(lgNt-lgN0)] (NO and Nt represented the cell number after inoculation and t hours after culture ,respectively), cell population doubling time was calculated and traced and growth curve of the 2nd, 4th and 6th generations of BMSCs were analyzed; The proliferation of BMSCs was measured by methylene blue staining method; Surface marker molecule expression of BMSCs was detected with immunocytochemical staining.MAIN OUTCOME MEASURES: ①Observation of isolation and culture of bone marrowmesenchymal stems of rats. ②Effect of different methods on the lysis of red blood cells and the growth of BMSCs. ③ The growth curve and cell doubling time of the 2nd, 4th and 6th generations of cells. ④Surface marker molecule expression of BMSCs of rats.RESULTS: ① Results of isolation and culture of BMSCs of rats: After 48-hour primary culture, most of the cells had adhered to the wall, and 72 hours later, division growth of the adherent cells presented. Seven to eight days later, cell colonies formed obviously, and then increased quickly, expanded incessantly and fused with each other. On 14 to 16 days, cell clones grew densely. Immediately generative cells presented ball-shape, subsided and adhered the wall verysoon. Some few round cells suspended. Adherent cells distributed evenly and proliferated quickly within 3 to 5 days. Although cell morphology of generative cells did not change after passage, cell proliferation was speeded up obviously and cells covered the bottom of the whole bottom on about 6 days. ② Effect of different methods on red blood cell lysis and the growth of BMSCs: hemoglobin concentration in the red blood cell lysate-treated group, ammonium chloride-treated group and 4％ acetic acid-treated group was significantly higher than that in the phosphate buffer normal saline-treated group, with significant difference (P ＜ 0.01). ③ Observation of growth curve of different generations of BMSCs: The growth curves of the 2nd, 4th and 6th generations of the cells were basically the same: latent period about 1 to 2 days, then logarithmic growth phase, peak on the 5th day and finally plateau phase (about on the 5th to 7th days). The latent period of the 6th generation of BMSCs was not obvious and the population doubling time of BMSCs was about 34 hours. ④ldentification of immunophenotype of different generations of BMSCs: Both CD44 and CD106 staining of each generation of cells were positive, presenting brown granule sediments, and CD34 staining was negative.CONCLUSION: Inoculation of red blood cells lysate-treated bone marrow rinse solution can boost the adherent rate of BMSCs, and does not influence its post-adherent growth, so it is a feasible separation method. Cell surface marker staining confirms that thecells isolated in this study are BMSCs.
