목적: 다운증후군의 신경계 결함에 관련된 양수유래 줄기세포의 표지자를 정상과 비교 분석하였다. 연구 방법: 임신 중기에 산전 진단을 위해 시행한 양수천자를 통해 채취된 4개 표본의 양수세포는 염색체검사에서 2개는 21번 삼염색체로 확인되었고 다른 2개는 정상이었다. 두 단계로 이루어진 양수유래 줄기세포 배양을 통해 얻어진 줄기세포들을 분석하였다. 형태학 관찰, 염색체검사, 역전사효소 중합반응 및 Western blot 분석을 시행하였다. 결과: 양수유래 줄기세포는 계대배양에서 높은 증식력을 보였으며 대부분 섬유아세포와 같은 방추상의 단핵세포로 관찰되었으며 드물게 원형의 단핵세포들도 있었다. 줄기세포의 염색체검사에서는 양수의 염색체검사와 동일하게 다운증후군 두 개의 표본은 21 삼염색체증 (47,XX,+21)이었고 정상 임신의 양수유래 줄기세포도 염색체검사 결과는 정상 (46,XX)이었다. 다운증후군과 관련된 것으로 알려진 단백 분석에서는 S100β는 다운증후군에서 정상보다 증가되어 나타났으며 COL6A1은 정상에서는 발현되었으나 다운증후군에서는 발현되지 않았다. Insulin like growth factor binding protein-1의 발현은 정상과 다운증후군 모두에서 차이가 없었다. 줄기세포 표지자 유전자인 Oct4, nanog, SOX2는 모두 발현되었다. 반면에, c-Kit의 발현은 정상에서는 나타났으나 다운증후군에서는 검출되지 않았다. 신경세포 표지자인 NSE는 모두 발현되었으나, microtubule associated protein 2와 glial fibrillary acidic protein는 다운증후군에서 발현되지 않았다. 세포자멸사 (apoptosis)와 관련된 Bcl-2 gene family 단백에 대한 분석에서는 Bcl-XL은 다운증후군에서 정상보다 증가되어 발현되었으며 Bcl-2, Bid 단백 발현은 정상과 다운증후군에서 차이가 없었다. Bax는 다운증후군에서 감소되었다. 결론: 양수유래 줄기세포는 조직 공학과 세포치료에 탁월한 재료가 될 것이다. 다운증후군의 양수유래 줄기세포의 분자생물학적 특성에 대한 분석은 앞으로의 연구를 위한 기초를 제공할 것이다.

Objective: To assess molecular markers of amniotic fluid derived stem cells (AFSCs) in aspects of increased neurological deficit in Down syndrome. Methods: Amniotic fluid samples through amniocentesis for prenatal diagnosis from four mid trimester pregnancies; by routine chromosomal analysis, two of them were trisomy 21 (Down syndrome) and others were normal, were selected after informed consent. Cells from two- stage culture protocol were assayed; morphology through phase contrast microscopy, chromosomal analysis, reverse transcriptase- polymerase chain reaction and Western blot analysis. Results: AFSCs were highly proliferative in subcultures and most of them were mononuclear, fibroblast-like, fusiform cells. There were also a few ovoid cells. The chromosomal analysis of amniotic fluid stem cells was identical to that of amniotic fluid cells. Two of four samples were 47,XX,+21, others were 46,XX. Of the proteins related to Down syndrome, the expression of S100β were increased in AFSCs of Down syndrome, COL6A1 (Collagen IV, alpha 1) was down-regulated in them and insulin like growth factor binding protein-1 was expressed in all AFSCs. Stem cell markers were expressed heterogeneously. Oct4 (POU5F1), nanog, and SOX2 (sex determining region Y) were expressed in both groups. But c-Kit was not expressed in AFSCs of Down syndrome. The neural cell marker, neuron specific enolase was detected in both groups. Other neural cell markers, microtubule associated protein 2, glial fibrillary acidic protein were undetectable in ASFCs of Down syndrome. Bcl-2 gene family proteins related with apoptosis were assayed. The expression of Bcl-XL was increased in Down syndrome more than in normal pregnancy. Bcl-2 and BID were expressed in all AFSCs and Bax was down-regulated in Down syndrome. Conclusion: AFSCs are an excellent choice for many future tissue engineering strategies and cell based therapies. Analysis of molecular features of AFSCs from normal and Down syndrome will provide the basis of further experimental study.

Objective: To assess molecular markers of amniotic fluid derived stem cells (AFSCs) in aspects of increased neurological deficit in Down syndrome. Methods: Amniotic fluid samples through amniocentesis for prenatal diagnosis from four mid trimester pregnancies; by routine chromosomal analysis, two of them were trisomy 21 (Down syndrome) and others were normal, were selected after informed consent. Cells from two- stage culture protocol were assayed; morphology through phase contrast microscopy, chromosomal analysis, reverse transcriptase- polymerase chain reaction and Western blot analysis. Results: AFSCs were highly proliferative in subcultures and most of them were mononuclear, fibroblast-like, fusiform cells. There were also a few ovoid cells. The chromosomal analysis of amniotic fluid stem cells was identical to that of amniotic fluid cells. Two of four samples were 47,XX,+21, others were 46,XX. Of the proteins related to Down syndrome, the expression of S100β were increased in AFSCs of Down syndrome, COL6A1 (Collagen IV, alpha 1) was down-regulated in them and insulin like growth factor binding protein-1 was expressed in all AFSCs. Stem cell markers were expressed heterogeneously. Oct4 (POU5F1), nanog, and SOX2 (sex determining region Y) were expressed in both groups. But c-Kit was not expressed in AFSCs of Down syndrome. The neural cell marker, neuron specific enolase was detected in both groups. Other neural cell markers, microtubule associated protein 2, glial fibrillary acidic protein were undetectable in ASFCs of Down syndrome. Bcl-2 gene family proteins related with apoptosis were assayed. The expression of Bcl-XL was increased in Down syndrome more than in normal pregnancy. Bcl-2 and BID were expressed in all AFSCs and Bax was down-regulated in Down syndrome. Conclusion: AFSCs are an excellent choice for many future tissue engineering strategies and cell based therapies. Analysis of molecular features of AFSCs from normal and Down syndrome will provide the basis of further experimental study.

Amniotic fluid derived stem cell, Down syndrome