中华临床医师杂志（电子版）
中華臨床醫師雜誌（電子版）
중화림상의사잡지（전자판）
CHINESE JOURNAL OF CLINICIANS(ELECTRONIC VERSION)
2013年
15期
7054-7058
,共5页
脐血干细胞移植%绿色荧光蛋白质类%嵌合体%小鼠%细胞分化
臍血榦細胞移植%綠色熒光蛋白質類%嵌閤體%小鼠%細胞分化
제혈간세포이식%록색형광단백질류%감합체%소서%세포분화
Cord blood stem cell transplantation%Green fluorescent proteins%Chimera%Mice%Cell differentiation
目的：本研究通过囊胚腔显微注射转增强型绿色荧光蛋白（EGFP）基因的脐血干细胞的方法制备嵌合体小鼠，以期为研究成体干细胞的体内分化提供一定的实验依据和理论基础。方法分离得到的表达绿色荧光的小鼠脐血干细胞用于囊胚腔显微注射。然后进行胚胎移植，出生的嵌合体小鼠首先进行毛色嵌合的观察，再对各组织进行基因组DNA和RNA水平的检测。流式细胞术检测阳性小鼠的组织中绿色荧光细胞的百分率。结果分离出的脐血干细胞，经流式检测表达绿色荧光的细胞所占的比例为80.25%。经显微注射和胚胎移植，共出生5只小鼠，均为白色，没有发生毛色嵌合。分别取心肌、肝、肺、皮肤、腿肌和脂肪共6种组织对EGFP基因进行PCR和RT-PCR检测。结果显示，有2只小鼠的腿肌和脂肪组织显示阳性，其他组织和其他3只小鼠的6种组织均为阴性。将这2只小鼠的腿肌和脂肪组织消化成单细胞悬液，进行流式细胞分析。结果显示2只嵌合小鼠腿肌和脂肪组织的平均嵌合率分别为9.87%和5.78%。结论来自成体的脐血干细胞在体内可以分化成腿部肌肉和脂肪组织的细胞。
目的：本研究通過囊胚腔顯微註射轉增彊型綠色熒光蛋白（EGFP）基因的臍血榦細胞的方法製備嵌閤體小鼠，以期為研究成體榦細胞的體內分化提供一定的實驗依據和理論基礎。方法分離得到的錶達綠色熒光的小鼠臍血榦細胞用于囊胚腔顯微註射。然後進行胚胎移植，齣生的嵌閤體小鼠首先進行毛色嵌閤的觀察，再對各組織進行基因組DNA和RNA水平的檢測。流式細胞術檢測暘性小鼠的組織中綠色熒光細胞的百分率。結果分離齣的臍血榦細胞，經流式檢測錶達綠色熒光的細胞所佔的比例為80.25%。經顯微註射和胚胎移植，共齣生5隻小鼠，均為白色，沒有髮生毛色嵌閤。分彆取心肌、肝、肺、皮膚、腿肌和脂肪共6種組織對EGFP基因進行PCR和RT-PCR檢測。結果顯示，有2隻小鼠的腿肌和脂肪組織顯示暘性，其他組織和其他3隻小鼠的6種組織均為陰性。將這2隻小鼠的腿肌和脂肪組織消化成單細胞懸液，進行流式細胞分析。結果顯示2隻嵌閤小鼠腿肌和脂肪組織的平均嵌閤率分彆為9.87%和5.78%。結論來自成體的臍血榦細胞在體內可以分化成腿部肌肉和脂肪組織的細胞。
목적：본연구통과낭배강현미주사전증강형록색형광단백（EGFP）기인적제혈간세포적방법제비감합체소서，이기위연구성체간세포적체내분화제공일정적실험의거화이론기출。방법분리득도적표체록색형광적소서제혈간세포용우낭배강현미주사。연후진행배태이식，출생적감합체소서수선진행모색감합적관찰，재대각조직진행기인조DNA화RNA수평적검측。류식세포술검측양성소서적조직중록색형광세포적백분솔。결과분리출적제혈간세포，경류식검측표체록색형광적세포소점적비례위80.25%。경현미주사화배태이식，공출생5지소서，균위백색，몰유발생모색감합。분별취심기、간、폐、피부、퇴기화지방공6충조직대EGFP기인진행PCR화RT-PCR검측。결과현시，유2지소서적퇴기화지방조직현시양성，기타조직화기타3지소서적6충조직균위음성。장저2지소서적퇴기화지방조직소화성단세포현액，진행류식세포분석。결과현시2지감합소서퇴기화지방조직적평균감합솔분별위9.87%화5.78%。결론래자성체적제혈간세포재체내가이분화성퇴부기육화지방조직적세포。
Objective The chimeric mice were prepared by microinjection of blastocyst cavity using umbilical cord blood stem cells(UCBSCs) of Enhanced Green Fluorescent Protein(EGFP)-transgenic mouse, which was expected to provide a theoretical and experimental basis for the study of in-vivo differentiation of adult stem cells. Methods Mouse UCBSCs expressing green fluorescence was microinjected into blastocyst cavity and several blastocysts were transferred into uterus of pseudo pregnant mouse. First of all, new-born candidate chimeric mouse were observed through feather color. Secondly, the genomic DNA and total RNA were extracted to analyze chimeric rate in several tissues. Finally, flow cytometry was used to detect percentage of green fluorescent cells mice in several tissues. Results The UCBSCs expressing green fluorescent protein were successfully isolated. After flow cytometry analysis, the proportion of cells expressing green fluorescence was 80.25%. Through microinjection and embryo transfer, we got five white new-born mice and no chimeric feather color was observed. The analyses of PCR and RT-PCR were carried out to detect EGFP gene using six tissues including, heart muscle, liver, lung, skin, leg muscle and adipose tissue. The results showed that the leg muscle and adipose tissue of two mice were positive and the other tissues and six tissues of the other 3 mice were all negative. The leg muscle and adipose tissue of two positive mice were digested into single-cells suspension and were carried out flow cytometry analysis. The Results showed that the average chimeric rates of leg muscle and adipose tissue of two positive mice were 9.87% and 5.78%, respectively. Conclusion The results demonstrated that adult UCBSCs could differentiate into leg muscle and adipose tissue in vivo.
