Compound Sophora Injection Intervention Bmscs Interaction Mechanism To Breast Cancer Cells In Vitro Experimental Study

In China, the morbidity of breast cancer increase3%every year, which is the maximum growth around the world. According to the first Large-scale breast cancer epidemics research project in China, the age of Chinese female breast cancer spans from20to more than70years old, of which the age range of40to49is the peak period. The median age is48years old which is10years early than the western country. Also the relapse and metastasis of breast cancer is the one of the most challenge for the cancer treatment. And the problem is its reasons and mechanisms are still not clear. Based on this, we need pull more focus on this area in the future to explore new way for the better breast cancer treatment. During the past25years, tumor genetics, especially cancer gene and tumor-suppressor genes function have been over stated, which lead to the cancer cell focused concept. In the other hand, the tumor cells and immune cell tumor matrix have been ignored, which cause the partial understanding of the biological character of tumor cell. After more than ten years debate and argument, cancer researchers gradually agree with one point that the tumor mesenchymal cells as the main body building and the tumor inflammatory microenvironment may trigger and promote the origin and development of the tumor and inflammation of the tumor microenvironment also accelerate the progress of the tumor growth.Bone mesenchymal stem cells (BMSCs)are the multi functional stem cells from the bone marrow. It can be drivedby the chemotactic agent to the local inflammatory when the body happen wounds and inflammation, the cells actively participate in the repair of body. Tumor is always called as the ceaselessinflammation, this type of cell in recent years is proved to be one of the important mesenchymal cells, it can secrete chemotactic agent through the paracrine way and combine with the receptor on the surface of tumor, and simultaneously activate downstream cancer genetic pathways, causing tumors malign hyperplasia, hit and the increase of the transfer capability, eventually leading to the malignant tumor recurrence and metastasis.When the recurrence and metastasis of breast cancer happened, the warm hot is one of the usual symptom, which simultaneously is the external performance of the internal inflammation microenvironment of the tumor. This symptom can be treated by qingrejiedu method. Kushen injection is one of the common Chinese medicine injection with the QinReJieDu function. We adopt it as a means of intervention, with tumor microenvironment inflammation of the important bone marrow stromal cells mesenchymal stem cells for research object, to control the active situation of Axis chemotactic agent and downstream genes within the interaction of the breast cancer cells, observe how theKushen injectionintervene the malignant behavior by the bone marrow stromal to breast cancer stem cells. The study mainly includes the following parts.1In vitro purification, identification and pluripotency comparison withindifferent sources of BMSCsBMSCs can be divided to variety types of tissue cell, including the osteoblast cells, cartilage cells and fat cells, it can be induced in vitro and further growth in vivo by transplantation, this method is already widely appliedin tissue engineering. In this study, MBSCs were obtained from the morrow of SD rats andBalb/c mice.According to the cell growth curve, induces osteogenesis, adipogenic differentiation and flow cytometry (FLM) identification technique, the MBSCs of SD rats is identified as the one with better cell activity and ’pluripotency, and finally choosed as the MBSCs sources for thefollowing experiment.2The intervention of Kushen injectionto the phenotypic differentiation of BMSC in tumor microenvironmentStem cell research can cure nearly80%of clinical diseases, but the treatment of tumor and infectious diseases is still the blind area, and its safety application in tissue engineering(mutation for tumor cells) is one of the important barrier in the present research, so the study of the relationship between adult stem cells and tumor is currently one of the key area. Recently the MBSCs have been found play a crucial role in the breast cancer occurrence, recurrence and metastasis, but this point is still controversial. Research front consider cancer as the ceaselessinflammation, and have similarperformance with inflammatory response of the body after damage repair. MBSCs will be drived into chemotactic stroma in tumor by inflammatory chemotactic agent gradient, and then differentiated into fiber mother cell phenotype cells in tumor micro environment. After differentiation, it keep secreting SDF-1etc chemotactic agent, and combine with CXCR4receptor on the surface of the tumor,then activate downstream cancer genes through the paracrine way, which consequently promote the tumors growth, malignant proliferation and angiogenesis, and will eventually lead to the tumor recurrence and metastasis. Through simulating the tumorlnflammatory micro environmental in the tumor growth condition medium, we observe the phenotype change of MBSCsin microenvironment to verify the above pathological changes occurrence.3The in vitro study on the effect of Kushen injectionto the chemotactic factors axis between BMSCs and breast cancer cellsKushen injection,which is clinically substantiated with good inhibition to inflammatory response, was chosen as the intervention means. The observe target was set at the chemotactic factors SDF-1/CXCR4axis between the interaction ofBMSCs and breast cancer cells4T1,single intervention and TRANSWELL co-culture system in vitro were applied to observe the SDF-1/CXCR4dynamic change when the system interacted.4The in vitro study on how Kushen injection affect promotion behavior of the BMSC-sto breast cancerThe effects of BMSCs caused to breast cancer cell growth were observed in TRANSWELL migration Chamber under TRANSWELL co-culture system. The migration situation of both BMSCs and4T1cell were observed in RANSWELL Boyden Chamber under trained and co-culture medium. The change of attack abilities of4T1breast cancer cell were detected by mRNA array gene sequence under co-culture condition.Conclusion:1The study successfully established the platform of BMSCs separation, purification and identification in the lab, and compares the characters of different sources of Bone marrow stromal,the experiment will provide the good cells sources. 2The study separatelyobserved the difference between BMSCs and breast cancer cells4Tlon the inhibition rate, apoptosis induction and cell cycle effects. The result indicate that, under same concentration level at which the cancer cell is inhibited and induced to apoptosis, Kushen injection didn’t show obvious effect on the normal BMSCs, which implied that Kushen injectionis ideal long-term intervention and chemotherapy drugs. Comparing with the DDP, the results suggest that Kushen injectionmay not be completely through the direct suppresses to tumor to resist the relapse and metastasis of cancer cell under the low concentration level intervention condition.3In the tumor microenvironment, the phenotype of BMSCschanged, the results show that the specific amount of protein expression and strength increased along time. After intervention of Kushen injection, it found that the change of phenotype of related fibroblasts from BMSCs inhibited.4With the intervention of different concentration of Kushen injection, the CXCR4receptor expression on the surface of breast cancer cells4T1cellswas decreased under both normoxic and hypoxic conditions. Under thypoxic condition, BMSCs have stronger cell proliferation capacity to the4T1breast cancer than it’s under normoxic condition. It also more likely migrate to breast cancer cell4T1under thypoxic condition than under normixic condition.In the co-culture conditions, the migration and attack capability of4T1breast cancer cells didn’t change too much from normoxic condition to hypoxic condition. The results also indicate that Kushen injection obviously decreased the SDF-1concentration in the supernatant of cell co-culture solution and affect the migration between each other (P>0.05) after48h thypoxic co-culture cultivation.5The results indicate that the malignant behavior of breast cancer cell increased after co-culture cultivation. The proliferation, attackcapability and migration ability of BMSCs all enhanced under the thypoxic condition. After mRNAarray gene detection, the gene of relative malignant behavior raise under thypoxic co-culture condition.