CAJ | 학술논문

目的：探讨 TNF-α活化的人源骨髓间充质干细胞（MSC）对结肠癌细胞株 HT29生长、转移的影响。方法制备、收集 MSC 条件培养液（CM）及 TNF-α活化的 MSC（T-MSC）条件培养液（TCM）。采用 MTT 比色法检测在α-最低基础培养液（α-MEM）、CM、TCM 中培养2、4、6 d 的 HT29细胞增殖率。制备下层不接种细胞、接种 MSC 或接种 T-MSC，上层均含有 HT29细胞的双层软琼脂糖共培养体系，表面加入α-MEM 后培养30 d，检测单细胞肿瘤集落形成能力。利用悬滴法制备的 HT29肿瘤球进行3D 基质胶侵袭实验，检测α-MEM、CM、TCM 中 HT29细胞的侵袭能力。实时聚合酶链反应检测在α-MEM、CM、TCM 中培养的 HT29细胞中上皮钙黏素 mRNA 表达变化。两组数据比较采用成组 t 检验，多组数据比较采用单因素方差分析。结果培养4～6 d 后，CM 或 TCM 组 HT29细胞增殖率显著高于α-MEM 组（第4、6天各组吸光度值分别为0．57±0．06、0．51±0．02、0．22±0．01和0．45±0．01、0．73±0．04、0．15±0．02），差异均有统计学意义（tCM ＝8．090、21．140，tTCM ＝21．190、17．950；P 均＜0．05）。软琼脂糖实验培养后30 d，T-MSC 共培养的 HT29单细胞集落数目明显多于其他两组。3D侵袭实验中 TCM 刺激后 HT29向周围侵袭能力强于α-MEM 组[侵袭率分别为（82±2）％和（65±8）％]，差异有统计学意义（t＝2．848，P ＜0．05）。TCM 刺激后 HT29细胞中膜上皮钙黏素 mRNA 水平较α-MEM 组显著下降（0．32±0．02比1．00±0．00），差异有统计学意义（t ＝35．190，P ＜0．05）。结论TNF-α活化的人源 MSC 可促进结肠癌细胞生长及侵袭。
목적：탐토 TNF-α활화적인원골수간충질간세포（MSC）대결장암세포주 HT29생장、전이적영향。방법제비、수집 MSC 조건배양액（CM）급 TNF-α활화적 MSC（T-MSC）조건배양액（TCM）。채용 MTT 비색법검측재α-최저기출배양액（α-MEM）、CM、TCM 중배양2、4、6 d 적 HT29세포증식솔。제비하층불접충세포、접충 MSC 혹접충 T-MSC，상층균함유 HT29세포적쌍층연경지당공배양체계，표면가입α-MEM 후배양30 d，검측단세포종류집락형성능력。이용현적법제비적 HT29종류구진행3D 기질효침습실험，검측α-MEM、CM、TCM 중 HT29세포적침습능력。실시취합매련반응검측재α-MEM、CM、TCM 중배양적 HT29세포중상피개점소 mRNA 표체변화。량조수거비교채용성조 t 검험，다조수거비교채용단인소방차분석。결과배양4～6 d 후，CM 혹 TCM 조 HT29세포증식솔현저고우α-MEM 조（제4、6천각조흡광도치분별위0．57±0．06、0．51±0．02、0．22±0．01화0．45±0．01、0．73±0．04、0．15±0．02），차이균유통계학의의（tCM ＝8．090、21．140，tTCM ＝21．190、17．950；P 균＜0．05）。연경지당실험배양후30 d，T-MSC 공배양적 HT29단세포집락수목명현다우기타량조。3D침습실험중 TCM 자격후 HT29향주위침습능력강우α-MEM 조[침습솔분별위（82±2）％화（65±8）％]，차이유통계학의의（t＝2．848，P ＜0．05）。TCM 자격후 HT29세포중막상피개점소 mRNA 수평교α-MEM 조현저하강（0．32±0．02비1．00±0．00），차이유통계학의의（t ＝35．190，P ＜0．05）。결론TNF-α활화적인원 MSC 가촉진결장암세포생장급침습。
Objective To explore the effects of tumor necrosis factor (TNF)-α activated human bone marrow mesenchymal stem cell (MSC)on proliferation and invasion of colorectal cancer (CRC)cell line HT29.Methods The conditional medium of MSC (CM)and TNF-α activated MSC (T-MSC) (TCM)were collected.The proliferation rates of HT29 cells in α-minimum essential medium (α-MEM), CM and TCM for two,four and six days were detected by methyl thiazol tetrazolium (MTT)method. Based agar were maken in plates without seeding or seeded with MSC or T-MSC and HT29 cells were seeded on the top agarose.The colony forming ability was tested after culturing with freshα-MEM for 30 days.Tumor sphere of HT29 cells was formed by suspension method.And three-dimensional matrix gel was immplemented for invasion examination.The invasion ability of HT29 cells inα-MEM,CM and TCM was measured.The expression of E-cadherin of HT29 cells inα-MEM,CM and TCM at mRNA level was determined by real-time polymerase chain reaction (PCR).The t test was used for two groups comparison and single factor analysis of variance was performed for multiple groups comparison.Results After cultured for four to six days,the proliferation rates of HT29 cells in CM or TCM were higher than that inα-MEM (the absorption value of each group at four days:0.57 ±0.06 and 0.51 ±0.02,0.22 ±0.01 ;at six days 0.45 ±0.01 and 0.73 ±0.04,0.15 ±0.02 ),and the differences were statistically significant (t CM =8.090 and 21 .140,t TCM =21 .190 and 17.950,all P <0.05).After cultured in soft agarose medium for 30 days,the colony number HT29 in T-MSC was more than other two groups.And in three-dimensional matrix gel invasion examination,the invasion ability of HT29 in T-MSC was significantly higher than that in α-MEM ((82 ± 2 )% and (65 ± 8 )%,t = 2.848,P < 0.05 ).The expression of E-cadherin of HT29 cells in TCM at mRNA level was lower than that inα-MEM (0.32±0.02 vs 1 .00± 0.00),and the difference was statistically significant (t = 35 .190,P < 0.05 ).Conclusion TNF-αactivated human bone marrow MSC may promote proliferation and invasion of CRC cells.