| Objective: To construct a lentivirus-mediated BMSCs that overexpresses IL-12 gene,and to observe the targeted therapeutic effect of IL-12 gene transfected BMSCs on gastric cancer xenograft model mice,and to explore the effect of Banxia Xiexin Decoction on IL-12 gene transfer.Whether BMSCs are targeted for the treatment of gastric cancer have synergistic effects.Methods: 1.Under the sterile conditions,the fetus and tibia of male SD rats of 2 months old were taken,the bone marrow cavity was fully washed,and an appropriate amount of low-dose DMEM containing 10% fetal bovine serum was added,centrifuged for 5-10 min,the supernatant was discarded,and complete medium culture was added..When the cells meet more than 90%,0.25% trypsin-EDTA is digested and passaged.Take well-mixed P3 generation BMSCs,digest,centrifuge,discard the upper layer of liquid,add primary antibody,secondary antibody,incubate,resuspend the cells and test on FCM.2.Transfected BMSC cells with LV5 null-borne lentivirus and IL-12 overexpressing LV5 lentivirus.After 48 h and 72 hours,the transfection of BMSCs was observed by inverted fluorescence microscopy.The efficiency of lentivirus transfection of BMSCs was calculated..The cells were screened by puromycin and the expression of IL-12 in BMSCs was detected by real-time PCR.BMSCs cells stably overexpressing IL-12 gene were established.3.SPF-grade healthy BALB/C nude mice were inoculated subcutaneously into human gastric cancer BCG-823 cells in nude mice,and a mouse model of human gastric cancer xenograft was established.4.Human gastric cancer xenograft mice were randomly divided into tumor formation group(model group),LV5 empty lentivirus transfected BMSCs group(empty group),LV5-IL-12 overexpression lentivirus transfected BMSCs group(transfer)Dyeing group),Banxia Xiexin Decoction intervention group(drug group),Banxia Xiexin Decoction combined with LV5-IL-12 overexpression lentivirus transfection BMSCs group(combined group).The model group was given normal saline to the mice.In the empty group,BMSCs transfected with lentivirus were administered to the tail vein of the mice.The transfected group transfected the IL-12 gene into the BMSCs and was injected into the tail vein.The drug group was administered with Banxia Xiexin Decoction by intragastric administration.In the combined group,BMSCs were transfected with IL-12 overexpression in the tail vein,and Banxia Xiexin Decoction was administered intragastrically for 13 days.5.After intervention,the mice were observed for general conditions.The tumor volume,body weight and growth of the mice in each group were calculated every other day,and the tumor inhibition rate was calculated.6.The tissues of gastric cancer xenografts were embedded,sliced and patched.The distribution of BMSCs in tumor tissues was observed by immunofluorescence staining,and the cell count was performed to calculate the BMSCs in tumor tissues.distributed.7.The tissues of gastric cancer xenografts were embedded and sectioned,and the TUNEL reaction mixture was prepared.The nuclear staining solution was added,stained with hematoxylin,sealed,and the apoptosis of tumor cells was observed under light microscope.The apoptotic index was calculated.AI).8.Embed and slice the tumor tissues of gastric cancer xenografts in each group,add primary antibody,secondary antibody,incubate,and seal with neutral gum.The expression of Bax and Bcl-2 protein in tumor tissues was detected by immunohistochemistry,and the average optical density value was determined.Results: 1.After 7 days of BMSCs culture,the cells were fused at 70%-80%,and the cells were typical long spindle-shaped and colony growth.After passage,the cells proliferated vigorously,and the cell morphology was relatively uniform,long fusiform and polygonal cells,and other forms of cells were rare,reaching the standard of passage.The positive expression of CD44 in the 3rd generation BMSCs was 96.68%,while the positive expression of CD34 was extremely low,only 1.25%.2.After BMSCs transfected for 48 h,the morphology of the cells was normal under normal light microscope,and the growth of BMSCs and IL-12 gene transfected BMSCs under fluorescent microscope increased.After 72 h,the cell fusion phenomenon was obvious,the green fluorescence intensity reached the highest,and the transfection efficiency reached 90%.3.The results of Q-PCR showed that IL-12 mRNA was stably expressed in BMSC after lentivirus transfection.The expression of IL-12 mRNA was significantly higher than that of normal BMSCs and empty lentivirus transfected BMSCs group.The difference was statistically significant.(P<0.05),there was no significant difference in the expression of IL-12 mRNA between normal BMSCs and vacant lentivirus-transfected BMSCs group(P>0.05).4.The tumor formation of nude mice showed that the lumps gradually appeared in the underarms of the mice after the 3rd day,and gradually increased.There was no significant change in the 5~7d masses.After the 9th day,the masses increased significantly.On the 13 th day,the tumors of the nude mice increased significantly.Compared with the model group and the empty group,the tumor block of the transfection group,the drug group and the combined group had a small increase,which was statistically significant(P<0.05).Compared with the transfection group and the drug group,the tumor group had the smallest increase,which was statistically significant(P<0.05).Compared with the model group and the empty group,the tumor inhibition rates of the transfection group,the drug group and the combined group were 36.9%,42.3%,and 60.5%,respectively,and the difference was statistically significant(P<0.05).5.Laser confocal results were shown by immunofluorescence staining.Blue fluorescence was observed in the tumor tissues of the empty group,the transfection group and the combined group,and the distribution was dense.Compared with the empty group,the distribution of BMSCs in the transfection group and the combined group was significantly increased,and the difference was statistically significant(P<0.05).Compared with the transfection group,the combined group increased significantly,and the difference was statistically significant(P < 0.05).6.TUNEL results showed that a large number of apoptotic bodies(brown granules)appeared in the tumor tissues of nude mice infected with gastric cancer,the drug group and the combined group.Only a small number of apoptotic bodies appeared in the model group and the empty group.Compared with the model group and the empty group,the apoptotic index of the transfection group,the drug group and the combined group were significantly increased,and the difference was statistically significant(P<0.05).Compared with the transfection group and the drug group,the apoptotic bodies of the combined group increased significantly,and the apoptotic index increased significantly,the difference was statistically significant(P<0.05).7.Immunocytochemistry results showed that there were a small number of Bax positive cells in the model group and the empty group,and the cytoplasm showed a light yellow-brown staining,and the cell morphology was round.The Bax positive cells in the transfection group,the drug group and the combined group were significantly increased,and the Bax positive cells were observed,and the cytoplasm was yellowish brown.Compared with the model group and the empty group,the expression of Bax protein increased significantly,and the difference was statistically significant(P<0.05).The expression of Bax protein in the combined group was significantly higher than that in the transfection group and the drug group,and the difference was statistically significant(P<0.05).There were a large number of Bcl-2 positive cells in the model group and the empty group.The positive cells were expressed in the granules,and the cytoplasm showed a light yellow-brown staining.The cell morphology was round.The Bcl-2 positive cells in the transfection group,the drug group and the combined group were significantly reduced.Compared with the model group and the empty group,the expression of Bcl-2 protein was significantly decreased,and the difference was statistically significant(P<0.05).The expression of Bcl-2 protein in the combined group was significantly lower than that in the transfection group and the drug group.Academic significance(P<0.05).Conclusion: 1.This experiment successfully constructed a lentivirus-mediated overexpression of IL-12 gene transfected BMSCS.2.LMSCs with lentiviral empty load and BMSCs overexpressing IL-12 gene can be targeted to the tumor tissues of gastric cancer xenografts in nude mice.3,BMSCs overexpressing IL-12 gene and Banxia Xiexin Decoction have a certain inhibitory effect on gastric cancer xenografts in nude mice.The combined intervention has obvious synergistic effect,and its inhibition and lowering of Bcl-2 Gene expression,increased Bax gene expression,and induction of apoptosis in gastric cancer cells. |
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