Study on the effect of γδ T cells expanded in vitro to kill hepatocellular carcinoma cells.

Background: γδ T cells for tumor cell immunotherapy has recently become a hot topic. Objective: To investigate the stimulation of expanded γδ T cells in vitro to kill liver cancer cells and its mechanism, and in vivo validation. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated and amplified. The proportion of γδ T cells in T cells was determined using flow cytometry. γδ T cells were selected as effector cells, and HepG2 cells as target cells in the cytotoxicity experiment. NKG2D blocker was used to block effector cells from identifying target cells, and PD98059 was used to block intracellular signaling pathways. The nude mice tumor model was established in two batches, the tumor growth curve was drawn, and the tumor formation effect was tested using small animal imager to verify the killing effect of γδ T cells. Results: The γδ T cells in the three experimental groups exhibited a large amount of amplification (P < 0.01). In the killing experiment, the killing rate of γδ T cells stimulated by zoledronate (ZOL) in the experimental group was significantly higher than that in the HDMAPP group and the Mycobacterium tuberculosis H37Ra strain (Mtb-Hag) group (P < 0.05). The blocking effect of PD98059 is stronger than that of the NKG2D blocker (P < 0.05). Among them, in the HDMAPP group, when the target ratio was 40:1, the NKG2D blocker exhibited a significant blocking effect (P < 0.05). Alternatively, in the ZOL group, when the effect ratio was 10:1, the effector cells were blocked significantly after treatment using PD98059 (P < 0.05). In vivo experiments verified the killing effect of γδ T cells. According to the tumor growth curve, there was a difference between the experimental and control groups after cell treatment (P < 0.05). Conclusion: ZOL has high amplification efficiency and a positive effect on killing tumor cells.

Novel method for effectively amplifying human peripheral blood T cells in vitro.

The use of chimeric antigen receptor-modified T cells (CAR T cells) is an effective therapy for advanced cancer, especially hematological malignancies, and this method has attracted widespread attention in the last several years. The type, number and vitality of the effector cells clearly play important roles in this approach. In this study, to expand the possibility of curing cancer through adoptive cell therapy (ACT), we developed a novel method for effectively obtaining abundant T cells in vitro. The fusion proteins of three cytokines, SA-hIL-2, SA-hIL-7 and SA-hIL-21, were anchored onto biotin magnetic beads to increase the number of cytokines on the surface of the magnetic beads, which increased the local concentration of cytokines and thus promoted the binding of cytokines to T cells. Next, we examined the effects of these modified magnetic beads on the proliferation rate of T cells and CD19 CAR T cells. In this study, we report the expression and purification of the active bifunctional fusion proteins SA-hIL-2, SA-hIL-7 and SA-hIL-21, which were bound to biotin magnetic beads to develop a platform that was employed to increase the local concentration of cytokines. When the cells had been cultured for 14 days, the proliferation rate of the CD3+ T cells in the group that received cytokine-coupled biotin magnetic beads (Beads-SA-CK) was higher than that of the cells in the groups that received soluble cytokines (Soluble-SA-CK) and that of the cells in the standard group (Standard-CK). We speculate that this difference may be the result of the increased expression of Bcl-2 and the increased phosphorylation of Stat5. Moreover, our results preliminarily indicate that compared with the other two treatments, Soluble-SA-CK and Standard-CK, adding cytokine-coupled biotin magnetic beads more effectively increases the proliferation rate of CD19 CAR-T cells. As expected, the CD19 CAR-T cells stimulated by Beads-SA-CK had a stronger anticancer effect than the cells stimulated by the other two treatments. An effective method of preparing abundant T cells in vitro was developed, and it may provide a novel strategy for ACT.

Repairment of pancreas mesenchymal stem cells on injured pancreas tissues / 吉林大学学报(医学版)

Objective To study the repairment of pancreas mesenchymal stem cells(MSCs) on injured pancreas of rats,and find a new source of cells for treatment of diabetes. Methods ① Pancreases were taken out of three-day-old Wistar rats under bioclean condition,and cells were obtained by V-collagenase digestion. Generations were passed by conventional culture,cells were purified by adherence screening method,cell morphous was observed by Giemsa staining. The expressions of surface marker CD34 and CD44 were determined by FCM,and compared with bone marrow MSCs(BM-MSCs),the differences in character and function were observed. ②The experimental rats were divided into two groups randomly. Pancreas ischemic necrosis model was made by deligation,then the purified pancreas MSCs were marked with DAPI and then were transplantated partially. After two weeks,the survival rate was measured and histopathological detection was performed. Results ① The cells had concordant morphous gradually with vigorous generation. There was no significant difference in morphology and surface antigen compared with BM-MSCs.② The survival rate in experimental group was 75% ,the necrotic tissue had basi-rebounded. Blue fluorescent was observed in repaired pancreas tissues. The survival rate in control group was 20%. The survival rate in experimental group was higher than that in control group (P