| Apoptin,an apoptotic protein expressed by the VP3 gene of chicken anemia virus,is not cytotoxic in normal cells,but can specifically induce apoptosis in tumor cells.The recombinant adenovirus Ad-VT and Ad-VP3 expressing chicken anemia virus VP3 gene can specifically express Apoptin protein in a variety of tumor cells to induce tumor cell apoptosis,and Ad-VT can specifically replicate in tumor cells to have an oncolytic effect.Cancer is one of the main causes of human death.In 2020,breast cancer was surpassing lung cancer to become the most common cancer;and breast cancer mortality ranked first among female cancer deaths.The current treatments for breast cancer have large side effects and poor curative effect on patients with metastases,and patients still have the risk of recurrence after treatment.Therefore,there is still a need to continue to find more effective treatments.There are a part of the cells inside the cancer called cancer initiating cells or cancer stem cells,which play a key role in cancer initiation,treatment resistance,metastasis and recurrence.This means that targeting breast cancer stem cells may be a promising treatment strategy to eradicate breast cancer.So far,there are no specific drugs for cancer stem cells.Therefore,inducing cancer stem cells to differentiate and targeting them to kill cancer stem cells may help to develop new treatment strategies against cancer,which may have clinical advantages in the treatment of cancer.The purpose of our research is to enrich and sort breast cancer stem cells with CD44~+CD24~-phenotype and analyze their stem cell characteristics.Using the recombinant adenoviruses Ad-VT and Ad-VP3 expressing chicken anemia virus VP3 gene constructed in the previous study to act on breast cancer stem cells,analyze the killing effect of recombinant adenovirus on breast cancer stem cells,inhibit the stemness of breast cancer stem cells and increase the sensitivity of breast cancer stem cells to drugs.By targeting cancer stem cells,inducing their differentiation,and inhibiting their resistance to treatment,we are looking for new strategies for clinical cancer treatment.The first is the use of serum-free suspension culture and magnetic bead sorting methods to enrich and sort breast cancer stem cells,and analyze their self-renewal,differentiation,drug resistance,etc.Secondly,use recombinant adenovirus to act on breast cancer stem cells to prevent breast cancer.The proportion of stem cells in the stem cells,cell proliferation ability,induced cell apoptosis level,cell migration and invasion ability were tested and analyzed.Then the recombinant adenovirus's effect on breast cancer stem cells'in vivo tumor formation ability and in vivo tumor killing effect were detected.Finally,Ad-VT and paclitaxel are combined with breast cancer stem cells to detect the proportion of stem cells in breast cancer stem cells,cell proliferation ability,induced apoptosis level,cell migration and invasion ability,etc,to analyze whether Ad-VT can enhance the sensitivity of breast cancer stem cells to chemotherapy drugs,thereby inhibiting the treatment resistance of cancer stem cells.The main results of this research are as follows:1.To detect and analyze the stem characteristics of breast cancer stem cells.Under serum-free culture conditions,breast cancer stem cells can form breast cancer cell spheres.Serum induction experiments show that breast cancer stem cells have the ability to differentiate and have strong self-renewal ability.Up-regulation of stem cell regulatory factors and multiple genes related to human cancer stem cells are overexpressed,they are also resistant to the chemotherapy drug paclitaxel.2.To detect the inhibitory effect of recombinant adenovirus on breast cancer stem cells in vitro,the results showed that the proportion of CD44~+CD24~-cell population decreased,the expression of stem cell regulatory factors was down-regulated,and the stemness of breast cancer stem cells was inhibited.And it was found that Ad-VT and Ad-VP3 can inhibit cell proliferation.Detection of cell mitochondrial membrane potential and apoptosis protein expression showed that Ad-VT and Ad-VP3 can induce breast cancer stem cell mitochondrial pathway apoptosis.The cell migration and invasion experiment results also show that the migration and invasion ability of breast cancer stem cells is inhibited.3.To detect the in vivo inhibitory effect of recombinant adenovirus on breast cancer stem cells.On the one hand,the tumorigenesis ability of the cells was tested by the mouse in vivo tumorigenesis experiment.The results showed that the stemness of breast cancer stem cells after the action of Ad-VT and Ad-VP3was inhibited,and the ability to form tumors in the body was lost.On the other hand,the breast cancer stem cells in good condition were injected subcutaneously into tumor-bearing cells to restructure the glands.The virus was injected into tumor-bearing mice,and the results showed that Ad-VT and Ad-VP3 have killing effects on breast cancer stem cell tumor-bearing models in vivo,and can inhibit tumor growth in mice.4.The combined effect of Ad-VT and paclitaxel can inhibit the drug resistance of breast cancer stem cells.The combined use of Ad-VT and paclitaxel can inhibit the stemness of breast cancer stem cells,induce the level of apoptosis,inhibit cell proliferation,and inhibit migration and invasion.It is significantly stronger than Ad-VT single treatment group and paclitaxel single treatment group,and the combined application enhances the inhibition of breast cancer stem cells'in vivo tumor formation ability.In summary,the enriched and sorted breast cancer stem cells have the characteristics of self-renewal,differentiation,and treatment resistance.Recombinant adenovirus Ad-VT and Ad-VP3 have certain inhibitory effects on breast cancer stem cells in vivo and in vitro.Inhibiting the stemness of breast cancer stem cells,Ad-VT can enhance the sensitivity of breast cancer stem cells to drugs,inhibit the treatment resistance of breast cancer stem cells,and enhance the inhibitory effect of paclitaxel on breast cancer stem cells. |
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