负载结肠癌干细胞热休克蛋白96的树突状细胞与细胞因子诱导的杀伤细胞对同源肿瘤干细胞的杀伤研究

The study on the effect of dendritic cells combined with cytokine-induced killer cells pulsed hot shock protein 96 from colon cancer stem cell on homologous cancer stem cells

目的研究结肠癌干细胞来源的gp96冲击树突细胞(Dendritic cell,DC),与细胞因子诱导的杀伤细胞(Cytokine killer cell,CIK)共培养,探讨其对结肠癌干细胞的杀伤效果。方法用无血清悬浮培养法从结肠癌细胞HT29中富集结肠癌干细胞,肿瘤干细胞可以通过其表面的特异性标记分子进行鉴定,结肠癌肿瘤通常选用人类白细胞分化抗原44(CD44)、人类白细胞分化抗原133(CD133)、乙醛脱氢酶-1(ALDH1)等分子。本研究选取CD133对HT29肿瘤细胞球进行肿瘤干细胞的鉴定。然后,将从结肠癌细胞HT29微球中提取的总蛋白通过硫酸铵分级盐析、Con A亲和层析、DEAE离子交换层析纯化出热休克蛋白96(gp96),所得蛋白经10%聚丙烯酰胺凝胶电泳(10%SDS-PAGE)和蛋白质印记(Western Blot)进行分子量及纯度的鉴定,并用二喹啉甲酸(BCA)法测其蛋白浓度。然后用gp96-肽复合物冲击DC,与CIK共培养,采用乳酸脱氢酶(LDH)法检测gp96冲击DC-CIK细胞对结肠癌干细胞(CSCs)的杀伤活性。结果流式细胞术检测第5代左右HT29微球中CD133+阳性的细胞比例为(73.92±2.76)%,明显高于HT29细胞(30.45±4.50)%。每克(湿重)肿瘤细胞可纯化出30μg左右的gp96。负载结肠癌干细胞gp96的DC-CIK对结肠癌干细胞的杀伤率高于DC-CIK组的杀伤率,差异有统计学意义(P<0.05)。结论负载结肠癌干细胞gp96的DC-CIK可以更有效地杀伤结肠癌干细胞,临床应用的可能性尚需深入研究。

Objective To investigate the tumor destructive effect of cytokine-induced killer cells(CIK) co-cultured with dendritic cells(DC) pulsed gp96 on HT29 colon cancer stem cells(CSCs). Methods Used serum-free suspension culture enrichment colon cancer stem cells from the HT29 colon cancer cells.The stem cells could be identified by their specific marker molecules.In colon cancer tumor,chose cluster of differentiation 44( CD44), Cluster of differentiation 133(CD133),Acetaldehyde dehydrogenase 1(ALDH1) and other molecules.In this study,CD133 was used to identify the tumor stem cells of HT29 tumor cell spheres.After that,gp96 was purified from colon cancer HT29 sphere cells by ammonium sulfate precipitation,Con A-Sepharose affinity Chromatography and DEME anion exchange Chromatography.The protein was indentified as gp96 by SDS-PAGE and Western Blotting.The concentration was measured by BCA.Gp96 was used to impact DC,after co-cultured with CIK,used the LDH method to detect the killing activity of colon cancer stem cells. Results The percentage of the fifth generation CD133+of HT29 sphere cells expressed higher levels of stem cell markers was(73.92 ±2.76)% which was higher than CD133+of HT29 cells(30.45±4.50)%. The quatity of gp96 obtained from 1g HT29 sphere cells was about 30μg. DC-CIK which pulsed gp96 had higher killing activity than DC-CIK group(P<0.05). Conclusion CIK co-cultured with DC which pulsed gp96 from colon cancer stem cell has higher killing activity.More researches are needed to evaluate the clinical applications.