Umbilical DCs Tranfected With CD137L Can Regulate The Function Of Nks From Peripheral Blood Of The Patients With Colorectal Cancer-An Experimental Study

Part one An experimental study of different treatment of DCs in affecting the function of volunteers`s PBMCs in vitroObjective: To observe the DCs with different treatments about their proliferation ability in vitro, cell phenotypic variations and differences of the ratio of the tumor target cells’ lethality, after getting PBMCs through the method of density gradient centrifugation.Methods:1 Density gradient centrifugation was used to obtain the PBMCs from volunteers`s peripheral blood and MNCs from umbilical cord blood. DCs with different treatments were obtained purified and independently cultured.2 Human colon cancer cell lines SW-1116 cells were used as target cells to extract tumor specific antigen which can be in took by DCs and be used to activate PBMCs which came from the peripheral blood and umbilical cord blood source.3 The DCs were cultured with PBMCs together after different treatments and then they were proliferated. The classification of lymphocytes, the cells phenotypic variations, the secretion of cytokines and the abilities of killing targeted cells of proliferation PBMCs were observed by flowing cytometry, enzyme-linked immunoassay and cell counting kit-8.Results: 1 The difference of cell phenotypic variations and antigen presenting ability between different treatment group DCs: The expression of CD83, CD86 in the group of DCs with loading tumor special antigen were increased obviously compared with the group of DCs previously. It was significant statistical difference in the group of DCs with loading TSA which had more mature DCs and stronger antigen presenting ability. The expression of CD83, CD86 in the group of DCs without loading tumor special antigen were increased not obviously compared with the group of DCs previously, especially that of CD83. It was significant statistical difference in the group of DCs without loading TSA which had less mature DCs and weaker antigen presenting ability. The expression of CD83, CD86 in the group of human umbilical cord DCs with loading tumor special antigen were increased compared with the group of DCs previously. But it was no significant statistical difference in the group of DCs with loading TSA which had a little difference mature DCs and antigen presenting ability. The expression of CD83, CD86 in the group of human umbilical cord DCs without loading tumor special antigen were not increased compared with the group of DCs previously. It was no significant statistical difference in the group of DCs without loading TSA which had less difference mature DCs and weaker antigen presenting ability. 2 The ex vivo expansion difference of PBMCs cultured with different treatment group DCs: The group of DCs with loading tumor special antigen had stimulated proliferation of PBMCs obviously which including more CD3+CD8+T cells and a little CD3+CD4+Tcells, CD3-CD56+NK cells. The group of DCs without loading tumor special antigen had promoted proliferation of PBMCs a little which including the CD3+CD8+T cells, CD3+CD4+T cells and CD3-CD56+NK cells. The number of CD3+CD8+T cells had decreased compared with the group of DCs with loading tumor special antigen. It was significant statistical difference. The group of PBMCs without DCs could propagated which contained CD3+CD4+T cells, CD3+CD8+T cells and CD3-CD56+NK cells. The number of CD3+CD4+T cells had increased compared with other treatment groups. It was significant statistical difference. The group of human umbilical cord DCs with loading tumor special antigen had a role in promoted proliferation of PBMCs which including the CD3-CD56+NK, CD3+CD4+T cells and CD3+CD8+T cells. The number of CD3-CD56+NK cells had increased compared with other treatment groups. It was significant statistical difference. The group of human umbilical cord DCs without loading tumor special antigen could stimulate proliferation of PBMCs which contained the CD3-CD56+NK, CD3+CD4+T cells and CD3+CD8+T cells. The number of CD3-CD56+NK cells had no difference with the group of human umbilical cord DCs with loading TSA. It was no significant statistical difference. 3 The IFN-gamma secretion difference of PBMCs culture supernatant with different treatment group DCs: The group of DCs with loading tumor special antigen had high level of IFN-gamma secretion which would more enhance as time goes on. The group of DCs without loading tumor special antigen had poor level of IFN-gamma secretion compared with the group of DCs with loading tumor special antigen. It was significant statistical difference that the level of IFN-gamma secretion had no change alone with time. The group of PBMC without DCs had low level of IFN-gamma secretion which would no change as time goes on. The group of human umbilical cord DCs with loading tumor special antigen had medium level of IFN-gamma secretion which would increase alone with time. But it was no significant statistical difference between with other groups. The group of human umbilical cord DCs without loading tumor special antigen had same level of IFN-gamma secretion with the group of DCs with laoding TSA. The level of that would increase as time goes on. But it was no significant statistical difference between with other groups. 4 The difference of cytotoxic cells killing ability on target human colon tumor SW-1116 cells with different treatment group DCs: The group culture a week with the DCs which laoding tumor special antigen had more ability of killing target human colon tumor SW-1116 cells. It was significant statistical difference that ability of killing target cells was selectivity compared with other groups. The group culture a week with the DCs which without loading tumor special antigen could kill target human colon tumor SW-1116 cells. It was significant statistical difference that ability of killing target cells was less selectivity compared with the groups of DCs with loading TSA. The group culture a week with no DCs had little ability of killing target human colon tumor SW-1116 cells. It was significant statistical difference that ability of killing target cells was poorer and no selectivity compared with the groups of DCs with laoding TSA. The group culture a week with the human umbilical cord DCs with loading tumor special antigen had strong capability of killing target human colon tumor SW-1116 cells. It was significant statistical difference that ability of killing target cells was no selectivity compared with the groups of DCs which laoding TSA. The group culture a week with the human umbilical cord DCs which without loading tumor special antigen had same capability of kiling target human colon tumor SW-1116 cells with the groups of human umbilical cord DCs with laoding TSA. Part two An experimental study of umbilical DCs tranfected with CD137L/p EGFP-N1 in affecting the function of volunteers`s PBMCs in vitro Objective: To observe the in vitro proliferation ability, cell phenotypic variations and tumor target cells killing efficiency of PBMCs separated by density gradient centrifugation which cultured with human umbilical cord DCs transfected with constructed CD137L/pEGFP-N1 plasmid.Methods:1 The core gene sequence of CD137 L was acquired through gene bank. The primer which contained the enzyme sites of xho1 and EcoR1 was designed accord with the CDS regions of CD137 L gene.2 PCR amplification was used to get purpose gene by the design primer as template. The amplification products were detected by agarose gel electrophoresis and obtained to purify according to the instructions of DNA gel extraction kit.3 The PCR amplification products were digested by restriction enzymes sites of xho1 and EcoR1 and recombined with pEGFP-N1 plasmid. The CD137L/pEGFP-N1 plasmid was confirmed to be constructed as expectation by DH5 alpha cells transformation, positive plasmid extraction and sequence reaction.4 The CD137L/pEGFP-N1 plasmid was confirmed to transfect human umbilical cord DCs using Lipofectamine Reagent PoloDeliver 3000 by fluorescence microscope and flow cytometry.5 After cultured with umbilical DCs tranfected with CD137L/pEGFP-N1, the classification of lymphocytes, the cells phenotypic variations, the secretion of cytokines and the abilities of killing target cells in proliferation PBMCs were detected by flow cytometry, enzyme-linked immunoassay and cell counting kit-8.Results: 1 The CD137L/pEGFP-N1 plasmid was constructed and transfect human umbilical cord DCs which could express CD137 L successfully(31.65±0.23% was detected by FCM). 2 The expression of CD83, CD86 in human umbilical cord DCs were increased after they had been transfected. It was significant statistical difference that high levels of CD83, CD86 compared with other groups. 3 The umbilical DCs tranfected with CD137L/pEGFP-N1 had more promoted proliferation of PBMCs which including the CD3-CD56+NK cells, CD3+CD4+T cells and CD3+CD8+T cells. It was significant statistical difference that the increased number of CD3-CD56+NK cells compared with other groups. 4 The group of the umbilical DCs tranfected with CD137L/pEGFP-N1 had high level of IFN-gamma secretion which would more enhance as time goes on. It was significant statistical difference that the level of IFN-gamma secretion compared with other groups. 5 The cells of the group(most CD3-CD56+NK cells) culture a week with the umbilical DCs tranfected with CD137L/pEGFP-N1 could kill target human colon tumor SW-1116 cells. It was no difference that ability of killing target cells compared with the group of the umbilical DCs with laoding the TSA. It was significant statistical difference that ability of killing target cells compared with the group of umbilical DCs tranfected with pEGFP-N1 and the group of umbilical DCs without loading the TSA. The cells of the group(most CD3-CD56+NK cells) culture a week with the umbilical DCs tranfected with CD137L/pEGFP-N1 could kill target human colon tumor LS-174-T cells too. It was significant statistical difference that ability of killing target cells compared with the groups of umbilical DCs tranfected with pEGFP-N1 and the groups of umbilical DCs with or without loading the TSA. It was significant statistical difference that killing target cells ability on SW-1116 and LS-174-T cells of the umbilical DCs with loading the TSA groups. But it was no significant statistical difference that killing target cells ability on SW-1116 and LS-174-T cells of the umbilical DCs without loading the TSA group, umbilical DCs tranfected with pEGFP-N1 group and the umbilical DCs tranfected with CD137L/pEGFP-N1 group. Compared with the group of umbilical DCs with loading the TSA, the cells of the umbilical DCs tranfected with CD137L/pEGFP-N1 group was no special killing target cells ability on SW-1116 and LS-174-T cells. Part three An experimental study of umbilical DCs tranfected with CD137 L in affecting the function of colorectal carcinoma patient’s PBMCs in vitroObjective: To observe the immune ability of colorectal carcinoma patients PBMCs separated by density gradient centrifugation and the in vitro proliferation ability, cell phenotypic variations and tumor target cells killing efficiency of those which cultured with the umbilical DCs tranfected with CD137L/pEGFP-N1.Methods:1 Density gradient centrifugation was used to obtain the PBMCs from colorectal carcinoma patient’ s peripheral blood. The immune abilities of those cells were detected by flow cytometry.2 Lipofectamine Reagent PoloDeliver 3000 was used to transfect human umbilical cord DCs by constructed CD137L/pEGFP-N1 plasmid. After cultured with these DCs, the PBMCs were to proliferate. The classification of lymphocytes, the cells phenotypic variations, the secretion of cytokines and the abilities of killing target cells in proliferation PBMCs were detected by flow cytometry, enzyme-linked immunoassay and cell counting kit-8.Results: 1 The immune function of PBMCs from colorectal carcinoma patients’ peripheral blood were in low level. 2 The umbilical DCs tranfected with CD137L/pEGFP-N1 had more promoted proliferation of colorectal carcinoma patients PBMCs which including the CD3-CD56+NK cells, CD3+CD4+T cells and CD3+CD8+T cells. It was significant statistical difference that the increased number of CD3-CD56+NK cells compared with other groups. 3 The group of the umbilical DCs tranfected with CD137L/pEGFP-N1 had high level of IFN-gamma secretion which would more enhance as time goes on. It was no difference that the level of IFN-gamma secretion compared with the group of umbilical DCs which loading the TSA and the groups of the umbilical DCs tranfected withpEGFP-N1 which without loading the TSA. 4 The cells of the group(most CD3-CD56+NK cells) culture a week with the umbilical DCs tranfected with CD137L/pEGFP-N1 could kill target human colon tumor SW-1116 and LS-174-T cells. It was significant statistical difference that ability of killing target human colon tumor SW-1116 and LS-174-T cells compared with the group of umbilical DCs tranfected with pEGFP-N1 which without loading the TSA. It was no difference that the special killing target human colon tumor SW-1116 cells ability of umbilical DCs tranfected with CD137L/pEGFP-N1 group compared with the group of umbilical DCs which loading TSA. It was significant statistical difference that the nonspecial killing target human colon tumor LS-174-T cells ability of umbilical DCs tranfected with CD137L/pEGFP-N1 group compared with the group of umbilical DCs which loading the TSA. Compared with the group of umbilical DCs which loading the TSA,the cells of the umbilical DCs tranfected with CD137L/pEGFP-N1 group was no special killing target cells ability on SW-1116 and LS-174-T cells. Part four An experimental study of umbilical DCs tranfected with CD137 L in antineoplastic effect on SCID/Beige nude mice ex vivoObjective: Human colon tumor SW-1116 cells were inoculated in SCID/Beige nude mice to build human colon tumor model simulation and the tumor microenvironment ex vivo state. Humanized immune environment system was constructed in SCID/Beige nude mice by injecting of human immune cells through rat caudal vein. To observe the changes in lifestyle, survival time and tumor size of the tumor-burdened SCID/Beige nude mice by injecting of PBMCs which activated by the transfected CD137L/pEGFP-N1 plasmid human umbilical cord DCs through rat caudal vein.Methods:1 Human colon cancer cell lines SW-1116 cells were used as target cells by develop amplification and purification.2 5×105/0.5ml human colon cancer cell lines SW-1116 were choose to inoculate in SCID/Beige nude mice to build human colon tumor model simulation. 1×106/0.5ml human immune cells were selected to construct humanized immune environment system in SCID/Beige nude mice through rat caudal vein. The difference of tumor form time and size were observed between different treatment groups.3 The change of tumor size was observed in SCID/Beige nude mice who burdened tumor to assess tumor inhibition ratios between different immune effectors cells in experimental group.Results: 1 The tumor formed in 3 to 5 days after inoculate human colon cancer cell lines SW-1116 cells. The tumors were clearly and stability in 7 to 10 days. 2 The tumor volume and weight of the humanized immune SCID/Beige nude mice were lower than that of the control group. It was significant statistical difference that volume and weight of tumor compared with the control group. The humanized immune environment system in SCID/Beige nude mice was useful to inhibit the growth of tumor. 3 The cells of the group(most CD3-CD56+NK cells) culture a week with the umbilical DCs tranfected with CD137L/pEGFP-N1 could reduce the tumor volume and weight of the SCID/Beige nude mice which burdened tumor. It was significant statistical difference that ability of reducing tumor volume and weight compared with the humanized immune mice group and the control mice group. But it was no significant statistical difference that reducing tumor volume and weight compared with the umbilical DCs loaded the special Ag group.Conclusions:1 Human umbilical cord DCs can promote PBMCs amplification in vitro under circumstances of existence both IL-2 and IL-15. The amplification PBMCs can be induced to convert in the direction of CD3-CD56+NK cells.2 Lipofectamine Reagent PoloDeliver 3000 can transfect human umbilical cord DCs with CD137L/pEGFP-N1 plasmid. Transfected human umbilical cord DCs can express CD137 L on their membrane.3 Human umbilical cord DCs transfected with CD137L/pEGFP-N1 plasmid can strong promote proliferation of PBMCs and induce them convert to a lot of CD3-CD56+NK cells.4 Human umbilical cord DCs transfected with CD137L/pEGFP-N1 plasmid can strong up-regulate the immune ability and reduce the inhibiting of colorectal carcinoma patients PBMCs. They also can promote high ability of killing tumor cells of colorectal carcinoma patients CD3-CD56+NK cells.5 Human umbilical cord DCs transfected with CD137L/pEGFP-N1 plasmid will provide a new technology to amplify and purify the CD3-CD56+NK cells in vitro and ex vivo. The amplification CD3-CD56+NK cells have a wide variety of tumor cell killing effect. They will be an important clinical immunotherapy cells to provide a new strategy for clinical tumor immunotherapy in cancer prevention and prevention of postoperative recurrence.