【摘要】 本试验旨在建立猪颗粒细胞、胎儿成纤维细胞和新生巴马小型猪不同组织来源成纤维细胞的分离及传代培养技术体系和冷冻保存方法,并比较猪卵丘/颗粒细胞和新生巴马小型猪肌肉成纤维细胞HDAC1基因的mRNA表达的差异,从分子水平上鉴定新生巴马小型猪肌肉成纤维作为供体细胞的可行性。运用组织块贴壁培养方法分离不同来源的组织细胞进行培养,并比较了常规冷冻保存和微量冷冻保存对细胞复苏率的影响,同时,运用实时定量PCR的方法检测猪卵丘/颗粒细胞和新生巴马小型猪肌肉成纤维细胞HDAC1基因mRNA表达的动态变化。研究结果表明:(1)猪颗粒细胞单层细胞的生长需要5～6d;用组织块法获得猪胎儿成纤维细胞单层的生长时间为10～11d;用组织块法可以成功地对新生巴马小型猪的肌肉、肺、肾脏、心、睾丸组织的成纤维细胞进行分离和传代培养,肾和睾丸组织的成纤维细胞贴壁生长后,长满形成单层细胞需要12～13d,而肺、肌肉和心脏组织的则需要15～16d。(2)细胞的微量冷冻保存效果优于常规冷冻保存(P<0.05)。(3)猪卵丘/颗粒细胞与新生巴马小型猪肌肉成纤维细胞HDACl mRNA的相对表达量为1.000比0.985,两者差异不显著(P>0.05)。新生广西巴马小型猪不同组织来源的成纤维细胞的分离及传代培养和微量冷冻法,丰富了这种猪体细胞的分离培养种类,为其体细胞核移植研究提供了丰富的供体细胞,并从表观遗传的角度分析,鉴定了新生巴马小型猪胎儿成纤维细胞与猪卵丘/颗粒细胞一样适合作供体,为核移植前的供体细胞鉴定提供了新的方法。更多还原

【Abstract】 This experiment was conducted to establish the culture sysytem for the donor cells,and to optimize cryopreservation method;and the expression level of mRNA for HDAC was analyzed between new born bama mini pig fibroblasts and granulosa cells(GC)/cumulus cells(CC) by using the technics of real-time PCR in order to investigate the feasibility of bama mini pig muscle fibroblast cell for donor cell at a molecular level.The new-born bama min-pig different tissue fibroblasts were prepared by the tissue explant culture.the donor cells,and percentage of living cells after thawing was compared between micro-cryopreservation and conventional cryopreservation.Real-time PCR method was used for quantitative analysis of HDAC1 mRNA level in porcine GC/ CC in the 2nd generation and the 5th generation of newborn bama mini pig muscle fibroblast cells.The results were as follows:(1) the time required for the formation of monolayers for culture of cells derived from GC,porcine fetal fibroblasts,kidney and testis,lung,muscle and heart was 5-6,10-11,12-13 and 15-16 d,respectively.(2) The percentage of living cells subjected to micro-cryopreservation was significantly higher than that of conventional cryopreservation(P<0.05).(3) There was no significant difference in expression level of mRNA for HDAC between new born bama minipig fibroblasts and GC/CC(P>0.05).Different tissue fibroblasts of bama min-pig were isolated and cultured and micro-cryopreservation method,which enriched for somatic cell nuclear transplantation as donor cells.Expression level of mRNA for HDAC between new born Bama mini pig fibroblasts and GC/CC which indicates that the two kinds of cells are suitable for dornor cells of SCNT with regard to HDAC1 expression.更多还原