Study Of The Effect Of β-elemene On Differentiation Of Endothelial Progenitor Cell Derived From Rat Bone Marrow And Notch Signal Pathway In The Process Of Differentiation

IntroductionEndothelial progenitor cell (EPC) is the precursor of endothelial cells, which can differentiate into endothelial cell (EC) and have not express the mature EC phenotype. The main source for the isolation of postnatal EPC is the bone marrow, thouroug along with the research, more and more evidence proved that EPC pays an important role in the process of tumor angiogenesis. And EPC differentiates to EC is the key step to educe function.At present, our mainly research about the differentiation of EPC from tow directions which are the shape and the cell surface marker. We could not distinguish EPC from other cell, so we use molecule marker to distinguish it. CD133 is the latest stem cell marker which dose not express in mature EC, and it is considered the most important marker to distinguish vessel EPC and EC. vWF exists in Weibel-Palade body of EC, vWF in blood plasma is secreted from EC, it is the molecule marker of blood vessel endothelium. The study result indicated that purified CD133+cell could differentiate to EC in vitro. In the process of differentiation, EPC misses CD133 obviously, and beginning to express the typical surface marker of mature EC, such as vWF. Notch signal pathway is the conservative signal pathway in the process of evolutionary, it pays an important role in cell fate decision and modeling. In the process of embryonic development, Notch signal pathway is the key signal of nervous system, cardiovascular system, immune system, liver and kidney. To be grown up, notch signal pathway involves in tissue regeneration and stem cell educes function.β-elemene, it is the anticancer active ingredient from the rhizome of Zingiber offioinale (Curcuma aromatica Salisb), Curcuma aromatica Salisb has function of trade spirit, removing qi stagnation,relieve pain,anticancer in traditional Chinese medicine, it pays important role in induce tumor cell apoptosis, differentiation anti-tumor metastasis, reversal rumor cell drug resistance, depress tumor microvessel density and many other directions. At present,β-elemene is used in chemotherapeutic of malignant serous cavity fluidify,lung cancer, alimentary canal tumor, cerebroma and other shallow tumor in clinically. Then it has some curative effect in leukemia, malignant lymphoma, esophageal carcinoma, stomach cancer and breast cancer. But the influence ofβ-elemene about the differentiation of EPC needs deep study.Methods1.The marrow originates EPC separation, culture and identificationThe application density gradient centrifuge process separates the single nuclear cell from the rat marrow, vaccinates in the endothelial cell nutrient fluid, after the blood vessel bast growth factor induction,carries on cell specificity designated object CD133,vWF related antigen examination, appraises it for the bast ancestor cell.2. Activation of Notch signal pathway in the differentiation process of EPCWe use RT-PCR to test the target gene Hey-2 and Hes-1 mRNA of Notch signal pathway in EPC which was cultured with different time(0天,4天,7天,10天,14天).3.the Effect ofβ-elemene on differentiation of endothelial progenitor cell and Notch signal pathway in the process of differentiationAdd different colorβ-elemene (0μg/ml,5μg/ml,10μg/ml,20μg/ml) at cultured day 4,and 7 day. After affect 24h, test the mRNA expression of CD 133 and vWF by RT-PCR.Add different colorβ-elemene (0μg/ml,5μg/ml,10μg/ml,20μg/ml)at cultured day 7. After affect 24h, test the mRNA expression of Hey-2 and Hes-1 by RT-PCR. Results一,Cultivation and identification of EPCAfter 24h original generation cultured, the cells are small and circular majority; after 4 days, some cells extend to spindle-shaped; at the 7th day, there are cell colonies, the spindle-shaped cells continue to adhere and grow well; at 14 day, the cells fuse to the monolayer like endothelial cell as the paving-stone shape. The cells that CD133 and vWF are both positive are the differeftiating EPC.二,The activation of Notch signal pathway in the differentiation process of EPCWe use RT-PCR to test the target gene Hey-2 and Hes-1 mRNA of Notch signal pathway in EPC which was cultured with different time(0天,4天,7天,10天,14天).The expression of Hey-2 and Hes-1 are none. At 4 day, the expression of Hey-2 and Hes-1 are weak. The expression of Hey-2 and Hes-1 strengthen gradually at 7th 10th 14th day. It is p＜0.05, compares day 0.三,Effect ofβ-elemene on the differentiation of EPC and Notch signal pathway in the process of differentiation(一) Effect ofβ-elemene on the differentiation of EPCAdd different colorβ-elemene (0μg/ml,5μg/ml,10μg/ml,20μg/ml) at cultured day 4,and 7 day. After affect 24h, test the mRNA expression of CD133 and vWF by RT-PCR. The result reveal that the mRNA expression level of CD133 is up-regulation and vWF is down-regulation with the color increase.It is p＜0.01, compared with control group.(二) Effect ofβ-elemene on Notch signal pathway in the process of differentiation of EPC.Add different colorβ-elemene (0μg/ml,5μg/ml,10μg/ml,20μg/ml)at cultured day 7. After affect 24h, test the mRNA expression of Hey-2 and Hes-1 by RT-PCR. The result reveals that the mRNA expression level of Hey-2 and Hes-1 are down-regulation with the color decrease. It is p＜0.05,compared with control group. ConclutionIn the differentiation process of EPC, Notch signal pathway may participate in this process.(3-elemene can inhibit the differentiation of EPC.(3-elemene can inhibit the Notch signal pathway in the differentiation process of EPC.