Study On The Mechanism Of Pien Tze Huang On The Transplantation Of Osteosarcoma CSCs Cells In Nude Mice

Objective:Our group's preliminary experiments in vitro confirmed that Pien Tze Huang can inhibit the growth of osteosarcoma and reverse its drug resistance by inhibiting the expression of ABCB1 and ABCG2 in osteosarcoma cells,and further explore the in vivo studies of Pien Tze Huang on human osteosarcoma CSCs.the Transplantation of Osteosarcoma CSCs Cellsand the effect on the expression of ABCB1 and ABCG2 proteins.Method:1.Establishment of tumor stem cell transplantation tumor model of human osteosarcomaIn logarithmic phase,MG63/ADM cells were digested with trypsin to produce single cell suspension.The suspension culture was carried out on serum-free medium for 3 consecutive times.Tumor stem cells enriched in MG63/ADM cells.48 nude mice were inoculated subcutaneously with tumor stem cells to establish tumor transplantation model of human osteosarcoma.According to the random number table,they were divided into control group,adriamycin group,panzaihuang group,platyzaihuang combined with doxorubicin group.2.Measure tumor volume and weight,calculate tumor volume and weight inhibition rateThe nude mice were killed 24 hours after the last administration.The tumor volume and weight were measured and the tumor volume and weight inhibition rate were calculated.3.HE observation of tumor pathological conditionsHE observation of tumor pathology4.Immunohistochemistry(SP method)and western blot were used to detect the expression of ABCB1 and ABCG2 in tumors.Immunohistochemistry was used to detect the expression of ABCB1 and ABCG2 positive densities in different groups of tumors.Western blot was used to observe the expression of ABCB1 and ABCG2 proteins in tumors of different groups.Result:1.The results of measuring the tumor inhibition rate showed that the volume and weight of tumors in each drug group decreased compared with the control group(P<0.05).Compared with the adriamycin group,the tumor volume inhibition rate and weight inhibition rate of the Pien Tze Huang group were high(P<0.01).Compared with Pien Tze Huang group,the volume inhibition rate and weight inhibition rate of the combined group were significantly higher(P<0.01),indicating that the growth of transplanted tumor in nude mice was significantly inhibited.2.After HE staining,under the light microscope,the morphology of the cells in the control group was different,and the nucleoli size was different.There have some heterogeneous megakaryocyte,and the nucleus was heavily stained.The osteosarcoma cells in the dividing stage were seen.The size and shape of the tumor cells in the Pien Tze Huang group were similar,and a small amount of osteosarcoma cells showed apoptosis and nuclear decomposition.In the adriamycin group,the degree of differentiation of the tumor cells was low,the atypia was large,and the tumor tissue was incomplete.In the combined group,the cell morphology was small,the tissue necrosis was seen,the cell atypicality was small,some of the nuclei were shrinking and condensed,the chromatin was close to the nucleus,and the cytoplasm was dark and concentrated.3.The results of immunohistochemistry showed that In the control group,the positive target integrated optical density is the highest,and the positive target integrated optical density in the doxorubicin group is also expressed more,The Pien Tsai group has less expression and the combined group has the lowest,There was no significant difference in the positive target integral optical density between the adriamycin group and the control group.the positive target optical density was significantly decreased in the Pien Tze Huang group and the combined group compared with the control group(P<0.05).The optical density of the target integral was the lowest,and there was a statistically significant difference compared with the Pien Tze Huang group(P<0.05).4.Western blot results showed that compared with the control group,there was no significant difference in the protein expression of ABCB1 and ABCG2 in the adriamycin group;the expression of ABCB1 and ABCG2 protein in the Pien Tze Huang group and the combined group was decreased,and there was a statistical difference compared with the control group(P<0.01).This shows that Pien Tze Huang can reduce the expression of ABCB1 and ABCG2.Conclusion Pien Tze Huang can significantly inhibit the growth of human osteosarcoma tumor stem cells transplanted tumor in nude mice,and can enhance the role of doxorubicin,its mechanism of action and inhibition of membrane transport protein ABCB1,ABCG2 expression.