肿瘤坏死因子-α活化的人源骨髓间充质干细胞对结肠癌细胞HT29生长及侵袭的影响

Impact of tumor necrosis factor-a activated human hone marrow mesenchymal stem cells on proliferation and invasion of colon cancer cell HT29

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、6d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30d，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4～6d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30d，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。

Objective To explore the effects of tumor necrosis factor （TNF）-α activated human bone marrow mesenehymal 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 fresh α-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 in α-MEM, CM and TCM was measured. The expression of E-cadherin of HT29 cells in α-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 in α-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 （tCM =8. 090 and 21. 140, tTCM =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 （654-8）%, t=2. 848, P 〈0. 05）. The expression of E-cadherin of HT29 cells in TCM at mRNA level was lower than that in α-MEM （0.32±0.02 vs 1.00±0.00）, and the difference was statistically significant （t = 35. 190, P 〈 0. 05）. Conclusion TNF-α activated human bone marrow MSC may promote proliferation and invasion of CRC cells.