中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2009年
3期
234-238
,共5页
井莹莹%杨吉成%盛伟华%胡志清%郁心%单云波%刘铁连%韩亚丽%包婉蓉%张日%朱南康%缪竞诚
井瑩瑩%楊吉成%盛偉華%鬍誌清%鬱心%單雲波%劉鐵連%韓亞麗%包婉蓉%張日%硃南康%繆競誠
정형형%양길성%성위화%호지청%욱심%단운파%류철련%한아려%포완용%장일%주남강%무경성
hLIF%腺病毒载体%造血干/祖细胞%SCID小鼠
hLIF%腺病毒載體%造血榦/祖細胞%SCID小鼠
hLIF%선병독재체%조혈간/조세포%SCID소서
hLIF%Recombinant adenovirus vector%Hematopoietic stem/progenitor cell%SCID mice
目的 用腺病毒载体介导的人白血病抑制因子基因(Ad-hLIF)感染WI-38人胚肺成纤维细胞制备饲养层细胞,体外观察转基因细胞对CD34+造血干/柑细胞增殖和分化的影响,体内研究对辐射损伤SCID小鼠造血功能恢复的效果.方法 用RT-PCR和ELISA法鉴定Ad-hLIF转基因饲养层细胞目的 基因的表达后,将经免疫磁珠法分离和流式细胞术检测后的CD34+细胞在饲养层和/或细胞因子培养体系中扩增28 d,检测不同时间点的单个核细胞(MNC)数量及CD34+细胞阳性率;扩增后的MNC经CFDA SE荧光标记后移植入辐射损伤SCID小鼠体内,RT-PCR和细胞荧光标记法检测小鼠内含Alu基因人源细胞的门巢情况.结果 感染50MOI(multiplicity of infection)Ad-hLIF的饲养层细胞均有绿色荧光,RT-PCR和ELISA法结果 显示hLIF目的 基因能在WI-38饲养层细胞中表达,免疫磁珠法分离的CD34+造血干/祖细胞经流式细胞术检测其纯度可达95.60%±2.58%,MNC在Ad-hLIF转基因饲养层培养体系持续扩增,最高可达356.95±0.87倍,其中CD34+细胞仪在0~14 d能维持较高水平,最高可扩增52.11±1.13倍,以后逐渐降低.将其移植辐射损伤SCID小鼠后,可明显提高小鼠存活率,4周内小鼠骨髓中不仅可观察到CFDA SE荧光标记的细胞,而且经RT-PCR法搭定后.还可检测到表达Alu人源基因的人脐血造血归巢细胞.结论 成功建立的Ad-hLIF转基因饲养层细胞不仅体外可以有效地扩增CD34+造血干/祖细胞,并延缓其分化.扩增的CD34+细胞对辐射损伤SCID小鼠具有造血功能恢复的功能.
目的 用腺病毒載體介導的人白血病抑製因子基因(Ad-hLIF)感染WI-38人胚肺成纖維細胞製備飼養層細胞,體外觀察轉基因細胞對CD34+造血榦/柑細胞增殖和分化的影響,體內研究對輻射損傷SCID小鼠造血功能恢複的效果.方法 用RT-PCR和ELISA法鑒定Ad-hLIF轉基因飼養層細胞目的 基因的錶達後,將經免疫磁珠法分離和流式細胞術檢測後的CD34+細胞在飼養層和/或細胞因子培養體繫中擴增28 d,檢測不同時間點的單箇覈細胞(MNC)數量及CD34+細胞暘性率;擴增後的MNC經CFDA SE熒光標記後移植入輻射損傷SCID小鼠體內,RT-PCR和細胞熒光標記法檢測小鼠內含Alu基因人源細胞的門巢情況.結果 感染50MOI(multiplicity of infection)Ad-hLIF的飼養層細胞均有綠色熒光,RT-PCR和ELISA法結果 顯示hLIF目的 基因能在WI-38飼養層細胞中錶達,免疫磁珠法分離的CD34+造血榦/祖細胞經流式細胞術檢測其純度可達95.60%±2.58%,MNC在Ad-hLIF轉基因飼養層培養體繫持續擴增,最高可達356.95±0.87倍,其中CD34+細胞儀在0~14 d能維持較高水平,最高可擴增52.11±1.13倍,以後逐漸降低.將其移植輻射損傷SCID小鼠後,可明顯提高小鼠存活率,4週內小鼠骨髓中不僅可觀察到CFDA SE熒光標記的細胞,而且經RT-PCR法搭定後.還可檢測到錶達Alu人源基因的人臍血造血歸巢細胞.結論 成功建立的Ad-hLIF轉基因飼養層細胞不僅體外可以有效地擴增CD34+造血榦/祖細胞,併延緩其分化.擴增的CD34+細胞對輻射損傷SCID小鼠具有造血功能恢複的功能.
목적 용선병독재체개도적인백혈병억제인자기인(Ad-hLIF)감염WI-38인배폐성섬유세포제비사양층세포,체외관찰전기인세포대CD34+조혈간/감세포증식화분화적영향,체내연구대복사손상SCID소서조혈공능회복적효과.방법 용RT-PCR화ELISA법감정Ad-hLIF전기인사양층세포목적 기인적표체후,장경면역자주법분리화류식세포술검측후적CD34+세포재사양층화/혹세포인자배양체계중확증28 d,검측불동시간점적단개핵세포(MNC)수량급CD34+세포양성솔;확증후적MNC경CFDA SE형광표기후이식입복사손상SCID소서체내,RT-PCR화세포형광표기법검측소서내함Alu기인인원세포적문소정황.결과 감염50MOI(multiplicity of infection)Ad-hLIF적사양층세포균유록색형광,RT-PCR화ELISA법결과 현시hLIF목적 기인능재WI-38사양층세포중표체,면역자주법분리적CD34+조혈간/조세포경류식세포술검측기순도가체95.60%±2.58%,MNC재Ad-hLIF전기인사양층배양체계지속확증,최고가체356.95±0.87배,기중CD34+세포의재0~14 d능유지교고수평,최고가확증52.11±1.13배,이후축점강저.장기이식복사손상SCID소서후,가명현제고소서존활솔,4주내소서골수중불부가관찰도CFDA SE형광표기적세포,이차경RT-PCR법탑정후.환가검측도표체Alu인원기인적인제혈조혈귀소세포.결론 성공건립적Ad-hLIF전기인사양층세포불부체외가이유효지확증CD34+조혈간/조세포,병연완기분화.확증적CD34+세포대복사손상SCID소서구유조혈공능회복적공능.
Objective To establish Ad-hLIF transgenic feeder cells for the expansion of umbilical cord blood CD34+ HSPC in vitro and study the SCID mice model of hematopoietic stem/progenitor cell (HSPC) transplantation. Methods The expression of objective gene in Ad-hLIF transgenic feeder cells was detected by RT-PCR and ELISA. The purity of umbilical cord blood CD34+ HSPC separated by magnetic-activated cell sorting(MACS) was detected by the flow cytometry. After expanded with various combinant of cytokines and transgenic feeder layer cells for 28 d, the quantity of mono-nuclear cell (MNC) and CD34+ cells rate was detected in different time. MNC after expansion stained by CFDA SE was injected to the sublethally irradiated SCID mice. Humanize gene Alu was detected by RT-PCR and fluorescence microscope. Results The green fluorescence was observed in the transgenic cells infected with 50MOI( multiplicity of infection) Ad-hLIF, and the objective gene was confirmed by RT-PCR and ELISA. The purity of umbilical cord blood CD34+ HSPC separated by MACS could reach 95.60% ±2.58%, Ad-hLIF transgenic feeder cells and various cytokines system increased MNC by 356.95±0.87 fold, and maximal expansion of CD34+ cells was observed during 0-14 d of culture, then down-expansion gradually. Four weeks after transplanted in SCID mice, fluorescently-labeled humanize cells still can be observed. The existence of the humanized gene Alu was confirmed by RT-PCR. Conclusion Ad-hLIF transgenic feeder cells can effectively proliferate umbilical cord blood CD34+ HSPC in vitro and delay it differentiate, what's more, it has high transplant efficacy and haematogenesis activity.