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
2008年
38期
7566-7572
,共7页
汤锋武%修波%范存刚%崔志强%萧凯%韩中朝
湯鋒武%脩波%範存剛%崔誌彊%蕭凱%韓中朝
탕봉무%수파%범존강%최지강%소개%한중조
脐带血%干细胞%AC133 神经分化
臍帶血%榦細胞%AC133 神經分化
제대혈%간세포%AC133 신경분화
背景:近年来,在啮齿类动物中发现,造血干细胞具有向神经分化的塑型性,而在人类,这种造血的塑型性仍然具有争议.目的:检测人脐血来源的AC133+造血干细胞是否具有向神经分化的潜能.设计、时间及地点:本实验为成组对照设计实验,于2005-08/2007-12在天津血液学研究所和清华大学玉泉医院神经中心实验室完成.材料:人脐带取自健康足月新生儿.胎脑滋养层细胞来源于22周流产胎儿脑组织.方法:应用免疫磁珠分选人脐血AC133+细胞,流式细胞仪检测其纯度为99%以上.同时以机械分离及酶消化法制备胎脑滋养层细胞.选用4种培养条件对脐血来源AC133+造血干细胞进行诱导分化:①生长培养液DMEM/F12加上皮生长因子和碱性成纤维生长因子.②DMEM/F12 加上皮生长因子和碱性成纤维生长因子并联合使用脑源性生长因子,其间用全反式维甲酸处理3 d.③非细胞接触的共培养体系,先将制备的胎脑滋养层细胞生长在培养板内插件的半透膜上,与培养板内的脐血来源的AC133+细胞进行共培养.④细胞直接接触共培养,将预先用BrdU标记的脐血来源的AC133+细胞直接种在胎脑滋养层细胞上进行共培养.主要观察指标: 1,2,4周收集诱导分化的脐血细胞,以RT-PCT检测是否有巢蛋白、骨形态生成蛋白2及神经细胞黏附分子的表达,免疫细胞化学方法检测细胞分型特异性抗原.结果:部分脐血来源的AC133+细胞,在体外存在生长必需的因子上皮生长因子和碱性成纤维生长因子时,能表达一些与神经细胞分化有关的基因,如巢蛋白、骨形态生成蛋白,条件不同时,这些基因出现下调和关闭.在DMEM/F12 加上皮生长因子和碱性成纤维生长因子联合使用脑源性生长因子诱导培养液中,上述基因表达在2周时可检测到,同时可检测到神经发育过程中出现的另一分子神经细胞黏附分子,这一分子在造血过程中并不出现,在使用内插件的胎脑滋养层细胞共培养的脐血细胞中也检测到相同结果.在优化的非细胞接触条件中主要表达胶质酸性蛋白,在细胞直接接触的共培养体系中出现神经元分化的标志物Ⅲβ-tubulin蛋白的表达.这种基因表达变化提示,在特定的培养环境下,脐血来源的AC133+细胞内可能出现了基因重排,使造血潜能的干细胞表达神经细胞发育分子.结论:脐血来源的AC133+细胞包含部分具有向神经分化潜能的多能干细胞,在适合条件下,表达神经分化相关抗原.
揹景:近年來,在齧齒類動物中髮現,造血榦細胞具有嚮神經分化的塑型性,而在人類,這種造血的塑型性仍然具有爭議.目的:檢測人臍血來源的AC133+造血榦細胞是否具有嚮神經分化的潛能.設計、時間及地點:本實驗為成組對照設計實驗,于2005-08/2007-12在天津血液學研究所和清華大學玉泉醫院神經中心實驗室完成.材料:人臍帶取自健康足月新生兒.胎腦滋養層細胞來源于22週流產胎兒腦組織.方法:應用免疫磁珠分選人臍血AC133+細胞,流式細胞儀檢測其純度為99%以上.同時以機械分離及酶消化法製備胎腦滋養層細胞.選用4種培養條件對臍血來源AC133+造血榦細胞進行誘導分化:①生長培養液DMEM/F12加上皮生長因子和堿性成纖維生長因子.②DMEM/F12 加上皮生長因子和堿性成纖維生長因子併聯閤使用腦源性生長因子,其間用全反式維甲痠處理3 d.③非細胞接觸的共培養體繫,先將製備的胎腦滋養層細胞生長在培養闆內插件的半透膜上,與培養闆內的臍血來源的AC133+細胞進行共培養.④細胞直接接觸共培養,將預先用BrdU標記的臍血來源的AC133+細胞直接種在胎腦滋養層細胞上進行共培養.主要觀察指標: 1,2,4週收集誘導分化的臍血細胞,以RT-PCT檢測是否有巢蛋白、骨形態生成蛋白2及神經細胞黏附分子的錶達,免疫細胞化學方法檢測細胞分型特異性抗原.結果:部分臍血來源的AC133+細胞,在體外存在生長必需的因子上皮生長因子和堿性成纖維生長因子時,能錶達一些與神經細胞分化有關的基因,如巢蛋白、骨形態生成蛋白,條件不同時,這些基因齣現下調和關閉.在DMEM/F12 加上皮生長因子和堿性成纖維生長因子聯閤使用腦源性生長因子誘導培養液中,上述基因錶達在2週時可檢測到,同時可檢測到神經髮育過程中齣現的另一分子神經細胞黏附分子,這一分子在造血過程中併不齣現,在使用內插件的胎腦滋養層細胞共培養的臍血細胞中也檢測到相同結果.在優化的非細胞接觸條件中主要錶達膠質痠性蛋白,在細胞直接接觸的共培養體繫中齣現神經元分化的標誌物Ⅲβ-tubulin蛋白的錶達.這種基因錶達變化提示,在特定的培養環境下,臍血來源的AC133+細胞內可能齣現瞭基因重排,使造血潛能的榦細胞錶達神經細胞髮育分子.結論:臍血來源的AC133+細胞包含部分具有嚮神經分化潛能的多能榦細胞,在適閤條件下,錶達神經分化相關抗原.
배경:근년래,재교치류동물중발현,조혈간세포구유향신경분화적소형성,이재인류,저충조혈적소형성잉연구유쟁의.목적:검측인제혈래원적AC133+조혈간세포시부구유향신경분화적잠능.설계、시간급지점:본실험위성조대조설계실험,우2005-08/2007-12재천진혈액학연구소화청화대학옥천의원신경중심실험실완성.재료:인제대취자건강족월신생인.태뇌자양층세포래원우22주유산태인뇌조직.방법:응용면역자주분선인제혈AC133+세포,류식세포의검측기순도위99%이상.동시이궤계분리급매소화법제비태뇌자양층세포.선용4충배양조건대제혈래원AC133+조혈간세포진행유도분화:①생장배양액DMEM/F12가상피생장인자화감성성섬유생장인자.②DMEM/F12 가상피생장인자화감성성섬유생장인자병연합사용뇌원성생장인자,기간용전반식유갑산처리3 d.③비세포접촉적공배양체계,선장제비적태뇌자양층세포생장재배양판내삽건적반투막상,여배양판내적제혈래원적AC133+세포진행공배양.④세포직접접촉공배양,장예선용BrdU표기적제혈래원적AC133+세포직접충재태뇌자양층세포상진행공배양.주요관찰지표: 1,2,4주수집유도분화적제혈세포,이RT-PCT검측시부유소단백、골형태생성단백2급신경세포점부분자적표체,면역세포화학방법검측세포분형특이성항원.결과:부분제혈래원적AC133+세포,재체외존재생장필수적인자상피생장인자화감성성섬유생장인자시,능표체일사여신경세포분화유관적기인,여소단백、골형태생성단백,조건불동시,저사기인출현하조화관폐.재DMEM/F12 가상피생장인자화감성성섬유생장인자연합사용뇌원성생장인자유도배양액중,상술기인표체재2주시가검측도,동시가검측도신경발육과정중출현적령일분자신경세포점부분자,저일분자재조혈과정중병불출현,재사용내삽건적태뇌자양층세포공배양적제혈세포중야검측도상동결과.재우화적비세포접촉조건중주요표체효질산성단백,재세포직접접촉적공배양체계중출현신경원분화적표지물Ⅲβ-tubulin단백적표체.저충기인표체변화제시,재특정적배양배경하,제혈래원적AC133+세포내가능출현료기인중배,사조혈잠능적간세포표체신경세포발육분자.결론:제혈래원적AC133+세포포함부분구유향신경분화잠능적다능간세포,재괄합조건하,표체신경분화상관항원.
BACKGROUND: At present, hemopoietic stem cells have been proved to differentiate into nerves in rodents animals. As for the human, this topic is in debate.OBJECTIVE: To investigate the neural differentiation potential of human umbilical cord blood-derived AC133+ cells. DESIGN, TIME AND SETTING: Control experiments by grouping were performed in the Hematology Institute of Tianjin Hematology Hospital and Central Laboratory of Neurosurgery in Yuquan Hospital of Tsinghua University from August 2005 to December 2007.MATERIALS: Human umbilical cord blood was sampled from full-term newborn infant. Fetal brain-derived trophic support cells were harvested from aborted fetus of 22 weeks old.MAIN OUTCOME MEASURES: After the induction, human cord blood cells were collected at weeks 1, 2 and 4. RT-PCR was used to detect the expression of nestin, bone morphogenetic protein-2 and neural cell adhesion molecule. Immunocytochemistry method was applied to detect the cytotype-specific antigen. RESULTS: In the culture medium containing epidermal growth factor and basic fibroblast growth factor, human cord blood AC133+ cells could express nestin and bone morphogenetic protein-2, which were down-regulated even closed up in suboptimal condition. In the DMEM/F12 medium supplemented with epidermal growth factor, basic fibroblast growth factor and brain-derived neurotrophic factor, the gene expression of bone morphogenetic protein-2 and nestin continued in optimal condition at 2 weeks. Moreover neural cell adhesion molecule, another gene of neural cells, also expressed in this condition. AC133+ cells co-cultured with fetal brain-derived trophic support cells exhibited similar expressions. In the optimal non-cell-cell contact co-culture system, glial fibrillary acidic protein-positive cells were found by immuocytochemistry, while neuronal marker β-tubulin Ⅲwas expressed in the cell-cell direct contact system. These outcomes indicated that human cord blood isolated AC133+ cells may have an effect through gene rearrangement on inducing stem cells to express nerve cell development factors.CONCLUSION: The human umbilical cord blood-derived AC133+ cells contain some multipotential stem cells with differentiation potential, neural differentiation-related antigen when exposed to a suitable microenvironment.
