Mechanism Of The Effect Of Xiaozheng Huayu Method On The Proliferation Of Lung Cancer Stem Cells By Tregs Cells

Objective:By analyzing the effect of Huazheng Huisheng Dan on the proliferation and differentiation of lung cancer stem cells?LCSC?and regulatory T cells?Tregs?in peripheral blood,the mechanism and relationship were discussed.Improve the theoretical basis of the treatment of lung cancer by eliminating phlegm and dissipating phlegm,and enrich the theoretical system of TCM treatment of lung cancer.Method:1.Using the flow cytometric sorting technique to obtain the side population cells?SP cells?in the lung cancer cell line Lewis,and verify the stem cell characteristics through relevant in vitro culture experiments;using flow cytometry to obtain the cell lines SP cells and non-side cells?non-SP cells?,and whether SP cells,non-SP cells,unsorted cells?Total cells?have tumor stem cells by detecting SP cell-related functions and in vitro and in vivo cloning experiments characteristic.2.The selected SP cells were cultured in vitro,and the disease was changed by using the Huazheng Huisheng Dan.The SP cells were injected into the skin of nude mice,and the anti-tumor rate was observed by using the disease.3.60 C57BL/6 mice were randomly divided into 4 groups.Three of them were randomly selected and inoculated with lung cancer cells to obtain a mouse model of lung cancer.The remaining group was a blank group.After successful modeling,two groups were randomly selected.The patients were treated with Huazheng Huisheng Dan and cisplatin,and the remaining group was used as the blank model group and the blank control group.The rats were intragastrically administered with the same amount of normal saline for 4 weeks.After 4 weeks,the peripheral blood of the mice was taken and the flow cells were used.Screening was performed to obtain CD4⁺CD25⁺,CD4⁺and CD4⁺CD25⁺Foxp3⁺,and the ratio of the two pairs was calculated.Result:1.The results of cell flow fluorescence sorting showed that the average ratio of SP cells in Lewis and the percentage of reduction after blocking were?1.79±0.12?%and?0.43±0.08?%respectively.In vitro cloning experiments showed:SP cells,The number of clones of non-SP cells and Total cells was?403±17?,?106±11?and?154±19?respectively.The differentiation ability of SP cells showed that the ratio of SP cells was?1.49.±0.05?%and?0.16±0.04?%?P<0.05?;in vitro proliferation experiments showed that from the third day,the proliferation curve of SP cells increased rapidly,followed by Total cells,while non-SP cells The cell cycle results showed that the ratio of cells in the G₀/G₁ phase was?60.03±4.52?%in non-SP cells,and the cell ratio in SP cells was?78.41±5.39?%.The experiment showed that by the 28th day,the average tumor volume of the SP cell group,the non-SP cell group and the Total cell group were?821±19?mm3,?179±14?mm3 and?452±17?mm3,respectively..2.Effect of drugs on SP cells:Compared with the blank group,the use of high and medium doses of Huazheng Huisheng Dan and cisplatin can increase the number of cells in G₁ phase,and decrease the number of cells in S phase and G₂ phase.Both were statistically significant?P<0.05?;in vivo tumor formation results:compared with the blank group,high and medium doses of Huazheng Huisheng Dan and cisplatin were used,the tumor weight was reduced?P<0.05?,tumor inhibition The rate was significantly increased?P<0.05?,and the tumor weight was reduced after the low-dose regenerative gynecological use,but it was not statistically significant.3.The percentages of CD4⁺CD25⁺/CD4⁺,CD4⁺CD25⁺Foxp3⁺/CD4⁺and CD4⁺CD25⁺Foxp3⁺/CD4⁺CD25⁺expression in the peripheral blood of the model group were?8.47±0.56?%,?2.99±0.10?%and?28.53?.±2.01)%,after treatment with Huazheng Huisheng Dan and cisplatin,the percentages of expression in the peripheral blood of mice with rejuvenation group were?5.95±0.37?%,?1.02±0.04?%and?17.21±1.39?%,the difference was statistically significant.The cisplatin group CD4⁺CD25⁺/CD4⁺,CD4⁺CD25⁺Foxp3⁺/CD4⁺and CD4⁺CD25⁺Foxp3+/CD4⁺CD25⁺were?9.02±0.63?%,?4.15±0.22?%and?29.44±1.87?%respectively.Compared with the model group,the proportion of expression in the peripheral blood increased.Conclusion:SP cells in the lung cancer cell line Lewis have cancer stem cell characteristics.The disease-reducing phlegm and blood stasis method can inhibit the growth and development of lung cancer stem cells,which is characterized by the cancer stem cells staying in the G₁ phase,preventing the transformation of cells into the S phase;effectively inhibiting the growth of tumors in nude mice,It can increase the tumor inhibition rate;it can also inhibit the growth of lung cancer by regulating the proliferation of Tregs cells in the tumor microenvironment.The mechanism may be that Tregs cells affect the growth of lung cancer stem cells.