CAJ | 학술논문

目的：比较来源于正常与退变髓核的髓核间充质干细胞（NPMSCs）的细胞代谢活性及干性基因表达情况。方法：收集6例非退变患者髓核组织（正常组）与6例腰椎间盘突出症患者的退变髓核组织（退变组），采用酶消化法分离细胞，应用标准间充质干细胞培养基（standard MSC culture medium）进行细胞培养并观察细胞形态。两组内各取1例分离得到的细胞进行流式细胞仪检测间充质干细胞表面蛋白分子标记CD90、CD105、CD73、CD45、CD34及人类白细胞抗原（HLA）-DR表达情况；并进行成骨、成脂及成软骨诱导分化，诱导28d后分别应用茜素红染色鉴定细胞成骨能力、油红O染色鉴定细胞成脂能力、甲苯胺蓝染色鉴定细胞成软骨分化能力，并按照国际干细胞治疗协会（ISCT）提出的间充质干细胞的判定标准，对分离得到的细胞进行综合评估鉴定。采用CCK-8检测两组P2代NPMSCs的代谢活力。提取两组每例P2代细胞总RNA，行RT-PCR检测P2代细胞“干性维持”相关基因Oct4及Nanog表达情况。结果：两组P0代细胞均贴壁生长，形态学方面两组并无明显差异。免疫表型鉴定显示正常组和退变组间充质干细胞表面分子标记CD90、CD105、CD73表达比例分别高达96％、98％、95%以上，两组均低表达造血细胞标志物CD45、CD34、HLA-DR（均低于4%）。茜素红染色、油红O染色及甲苯胺蓝染色分别证实正常组与退变组细胞均可向骨、脂肪及软骨细胞三系诱导分化。上述结果证实分离得到的细胞即NPMSCs。细胞代谢活性测定示P2代细胞在培养后5d、7d、9d、11d、13d正常组细胞活性均强于退变组，两组细胞活性有统计学差异（P<0.05）。正常组“干性维持”相关基因Oct4及Nanog表达量分别为退变组的4.63±1.17、7.36±1.19倍，正常组均明显高于退变组（P<0.05）。结论：正常与退变髓核组织内均存在NPMSCs，但正常椎间盘来源的NPMSCs具有较强的细胞代谢活性，较”强的“干性维持”基因表达。目的：比較來源于正常與退變髓覈的髓覈間充質榦細胞（NPMSCs）的細胞代謝活性及榦性基因錶達情況。方法：收集6例非退變患者髓覈組織（正常組）與6例腰椎間盤突齣癥患者的退變髓覈組織（退變組），採用酶消化法分離細胞，應用標準間充質榦細胞培養基（standard MSC culture medium）進行細胞培養併觀察細胞形態。兩組內各取1例分離得到的細胞進行流式細胞儀檢測間充質榦細胞錶麵蛋白分子標記CD90、CD105、CD73、CD45、CD34及人類白細胞抗原（HLA）-DR錶達情況；併進行成骨、成脂及成軟骨誘導分化，誘導28d後分彆應用茜素紅染色鑒定細胞成骨能力、油紅O染色鑒定細胞成脂能力、甲苯胺藍染色鑒定細胞成軟骨分化能力，併按照國際榦細胞治療協會（ISCT）提齣的間充質榦細胞的判定標準，對分離得到的細胞進行綜閤評估鑒定。採用CCK-8檢測兩組P2代NPMSCs的代謝活力。提取兩組每例P2代細胞總RNA，行RT-PCR檢測P2代細胞“榦性維持”相關基因Oct4及Nanog錶達情況。結果：兩組P0代細胞均貼壁生長，形態學方麵兩組併無明顯差異。免疫錶型鑒定顯示正常組和退變組間充質榦細胞錶麵分子標記CD90、CD105、CD73錶達比例分彆高達96％、98％、95%以上，兩組均低錶達造血細胞標誌物CD45、CD34、HLA-DR（均低于4%）。茜素紅染色、油紅O染色及甲苯胺藍染色分彆證實正常組與退變組細胞均可嚮骨、脂肪及軟骨細胞三繫誘導分化。上述結果證實分離得到的細胞即NPMSCs。細胞代謝活性測定示P2代細胞在培養後5d、7d、9d、11d、13d正常組細胞活性均彊于退變組，兩組細胞活性有統計學差異（P<0.05）。正常組“榦性維持”相關基因Oct4及Nanog錶達量分彆為退變組的4.63±1.17、7.36±1.19倍，正常組均明顯高于退變組（P<0.05）。結論：正常與退變髓覈組織內均存在NPMSCs，但正常椎間盤來源的NPMSCs具有較彊的細胞代謝活性，較”彊的“榦性維持”基因錶達。
목적：비교래원우정상여퇴변수핵적수핵간충질간세포（NPMSCs）적세포대사활성급간성기인표체정황。방법：수집6례비퇴변환자수핵조직（정상조）여6례요추간반돌출증환자적퇴변수핵조직（퇴변조），채용매소화법분리세포，응용표준간충질간세포배양기（standard MSC culture medium）진행세포배양병관찰세포형태。량조내각취1례분리득도적세포진행류식세포의검측간충질간세포표면단백분자표기CD90、CD105、CD73、CD45、CD34급인류백세포항원（HLA）-DR표체정황；병진행성골、성지급성연골유도분화，유도28d후분별응용천소홍염색감정세포성골능력、유홍O염색감정세포성지능력、갑분알람염색감정세포성연골분화능력，병안조국제간세포치료협회（ISCT）제출적간충질간세포적판정표준，대분리득도적세포진행종합평고감정。채용CCK-8검측량조P2대NPMSCs적대사활력。제취량조매례P2대세포총RNA，행RT-PCR검측P2대세포“간성유지”상관기인Oct4급Nanog표체정황。결과：량조P0대세포균첩벽생장，형태학방면량조병무명현차이。면역표형감정현시정상조화퇴변조간충질간세포표면분자표기CD90、CD105、CD73표체비례분별고체96％、98％、95%이상，량조균저표체조혈세포표지물CD45、CD34、HLA-DR（균저우4%）。천소홍염색、유홍O염색급갑분알람염색분별증실정상조여퇴변조세포균가향골、지방급연골세포삼계유도분화。상술결과증실분리득도적세포즉NPMSCs。세포대사활성측정시P2대세포재배양후5d、7d、9d、11d、13d정상조세포활성균강우퇴변조，량조세포활성유통계학차이（P<0.05）。정상조“간성유지”상관기인Oct4급Nanog표체량분별위퇴변조적4.63±1.17、7.36±1.19배，정상조균명현고우퇴변조（P<0.05）。결론：정상여퇴변수핵조직내균존재NPMSCs，단정상추간반래원적NPMSCs구유교강적세포대사활성，교”강적“간성유지”기인표체。
Objectives: To compare the cell activity and stem cell-related genes expression of mesenchymal stem cells(MSCs) in nucleus pulposus(NP) from non-degenerative and degenerative disc . Methods: The human nucleus pulposus mesenchymal stem cells (NPMSCs) were isolated by using standard MSC culture medium as suitable for MSCs culture by Cyagen from 6 non-degenerative patients of either scoliosis or burst fracture and 6 patients with degenerative intervertebral disc separately . One case of cells was selected from each group, respectively, and immunophenotype(CD90/CD105/CD73/CD45/CD34 and HLA-DR) and multilineage(Osteogenic, chondrogenic and adipogenic) differentiation potential were analyzed under the criteria to define MSCs, which was stated by the International Society for Cellular Therapry (ISCT). The cell activity of second passage from each group was analyzed by using cell counting kit-8(CCK-8). The total RNA of both non-degenerative and degenerative group were extracted, then the Real-Time PCR was used to detect the relative expressions of stem cell-related genes Oct4 and Nanog. Results: The original cells from both non-degenerative and degener-ative group showed adherent growth, cell morphology of two groups showed no significant difference. The im-mune phenotype showed both normal and degenerative cells highly expressed the mesenchymal stem cell sur-face markers CD90, CD105, CD73 (expression ratio as high as 96%, 98%, 95%, respectively). And hematopoietic markers CD45, CD34, HLA-DR were lower expressions(all less than 4%). Multilineage differen-tiation results showed cells obtained from both non-degenerative and degenerative NP developed red stained calcium salts, and adipocyte-like cells which were stained red by Oil red O, and chondrocyte-like cells stained blue by toluidine blue, after Osteogenic, chondrogenic and adipogenic differentiation for 28 days. To-gether with the immunophenotype, cells obtained from both non-degenerative and degenerative NP fulfilled the criteria to define MSCs stated by ISCT. Cell viability assay by CCK-8 showed that cell activity in normal group was stronger than that in the degeneration group at 5, 7, 9, 11, 13 days, there was significant differ-ence between two groups(P<0.05). The Real-Time PCR results showed that Stemness maintenance related gene Oct4 and Nanog expression in normal group was 4.63 ±1.17, 7.36 ±1.19 times respectively than degeneration group. The Stemness maintenance related gene expression in normal group was significantly higher than that in the degeneration group (P<0.05). Conclusions: Both the non-degenerative and degenerative NP contain NPMSCs. Moreover, the non-degenerative NPMSCs show stronger ability of cell metabolic activity and stem cell-related genes expression.