| ?Objective? Study the killing effect for BCSC enriched by microsphere culture method through co-culturing DC pulsed by pcDNA3.1~+-CD44(s1-s5) and homologous CIK cell, laying a foundation for later development of the vivo animal experiment and clinical promotion of adoptive immunotherapy.?Methods? 1?Routinely culture breast cancer cell lines MCF-7?SK-BR-3?MDA-MB-231?T-47D?ZR-75-1?BT-549, then detect the CD44 content of each cell by RT-PCR. 2?Put the c DNA reversely transcribed from RNA as a template for PCR, then ligate and transform JM109 after digested by double enzymes with the vector pcDNA3.1~+ respectively. Identify the recombinant by digesting and gene sequencing. 3?The plasmid is extracted in a large amount after proliferated massively of the correct bacterial colony, and transfect 293 T cells with p EGFP-C3 respectively, then detect the transfection efficiency and protein expression. 4 ? Cut the target fragment from pcDNA3.1~+-CD44(s1-s5) and construct the p ET32a-CD44(s1-s5) with subcloning method, then transform BL21 quickly and amplificate in vitro. 5?Inoculate according to the proportion of 1:100 of bacteria and medium. Collect some bacterium liquid used as protein expression contrast before inducing when OD600 value is 0.6, then induce for 4h by adding IPTG. Collect the bacterium suspension next day and boil 30 minutes after adding Loading Buffer to detect CD44 expression with Western blot. 6?Inoculate the MDA-MB-231 cells according to 5×103/m L in serum-free medium containing EGF?b FGF?B27. Routinely culture and replace medium every 2 to 3 days. Observe and record morphologic change of the cell before and after microsphere culturing. 7?Induce separated mononuclear cells through adding proper cytokines to form DC and CIK respectively, then co-culture CIK and DC. Kill the above enriched BCSC when they are mature. 8?Detect LDH release value in 492 nm wavelength of experimental group and control group in a different effector-target ratio, then calculate the cytotoxicity percentage to distinguish the killing effect.?Results? 1?The content of CD44 in MDA-MB-231 cells is the highest, and it can be used as the cell origin of gene clone and microsphere culturing. 2?The construction is successful which is confirmed by enzyme digesting and gene sequencing. However, the protein size is inconsistent with the predicted size. 3?The expression of CD44 protein after induced by IPTG is good. However, its molecular weight is inconsistent with the eukaryotic and the predicted size. 4?The MDA-MB-231 cells turn into sphere significantly in day 7. Thereinto the sphere is compact and at its best in day 10, fitting to collect and standby apply. 5?The cell morphology of DCs are tending to mature and the number of CIK cells increase significantly in 7 days. 6?The killing activity of experimental group is higher than the control group at any effector-target ratio(P<0.05).?Conclusions? 1?Plasmid pcDNA3.1~+-CD44(s1-s5) and p ET32a-CD44(s1-s5) can be constructed successfully with gene recombination technology. 2?The molecular weight of eukaryotic expressed protein is not consistent slightly with the predicted size. This prompt that CD44 protein may exist posttranslational modification just as glycosylation. 3?The kill effect of CD44-DC-CIK for BCSC is better than carrier vector-DC-CIK, and this lays a good experimental basis for the adoptive immunotherapy applied in the treatment of tumor. |
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