The Experimental Investigation Of Endothelin 1 On HEP 2 Cells In Vitro

Endothelin family is classified into three isotypes: endothelin 1, endothelin 2, endothelin 3, the amino acid sequences of three isotypes were highly homology. Endothelin 1 is a 21 amino acid peptides that originally isolated from the culture supernatent of porcine aortic endothelial cells. Endothelin 1 have many biological effects which include playing a role as the extremely potent vasoconstrictor as well as an regulator of pressure, fluid volume and ion balance.The diversity of actions of endothelin may be accounted for the existence of several types of endothelin receptor with different cellular function. At least two types of G protein-coupled receptors, type A and type B, can be distinguished according to their properties of intracellular signaling pathways they induced.ET-1 is overexpressed in many human cancers in vivo and vitro, and increasing evidence points to endothlin 1 as relevant mediators in tumor progressing in variety of malignancies. It has been proposed that endothelin 1 exerts autocrine or paracrine in malignant tumors, and exerts its function on stimulating cancer cells growth, acting as antiapoptoic factor, or decreasing gap junctional intercellular communication in tumor tissues.13.9ï¼…of maglinant tumors on head and neck are carcinoma of larynx, in some areas of China, the incidence of laryngeal carcinoma is 1.5-3.4/hundred thousands and laryngeal carcinoma harm the health of people severely. Comparing with other maglinant tumors, there are seldomly studies about the effect of endothelin 1 on the progression of laryngeal carcinoma, Shichiri founded that endothelin 1 can stimulate laryngeal carcinoma cell's proliferation, but the progression of maglinant tumors have connect not only with the proliferation of tumor cells, but also with apoptosis and movement of cells. whether laryngeal carcinoma cell exist endothelin 1 and it's receptors, what is the effect of endothelin 1 on the progression of laryngeal carcinoma need indepth study. Our study is aimed to approach the expression of endothelin 1 and it's receptor in HEP 2 cells and explore the effect of endothelin 1 on the growth, movement and apopotosis of HEP 2 cells.Objects1. To study the expression of gene and-protein of endothelin 1, endothelin receptor A, and endothelin receptor B in HEP 2 cells.2. To investigate whether endothelin 1 can stimulate the proliferation of HEP 2 cells.3. To observe the effects of endothelin 1 on the movement of HEP 2 cells in vitro.4. To assess effect of endothelin 1 on the apoptosis of HEP 2 cells.Methods1. Detection of endothelin 1, endothelin receptor A, and endothelin receptor B by RT PCR in HEP 2 cells, and investigate the expression of endothelin 1, endothelin receptor A, and endothelin receptor B in HEP 2 cells by immunohistochemistry ABC method.2. HEP 2 cells were seeded in 96 wells plates at density of 2×10³ cells/well. Endothelin 1 were added at increasing concentrations (1×10^-7mol/L, 1×10^-8mol/L, 1×10^-9mol/L, 1×10^-10mol/L, 1×10^-11mol/L) to the cultures, MTT methods were used to explore the effect of endothelin 1 on the proliferation of HEP 2 cells. After being cultured for 24h, 48h, 72h, 96h, the optical density were detected.3. HEP 2 cells were plated into 24 wells plates at density of 300 cells/well and grown for 48 hours in increasing concentrations(1×10^-7mol/L, 1×10^-9 mol/L, 1×10^-11mol/L) endothelin 1, then the wells were washed by PBS and in cubated in serum with fully supplemented medium for 10 days, colony formation test were used to detect the effect of drug on cells in vitro.4. HEP 2 cells were plated into 6 wells plates at density of 3×10⁵cells/well, then endothelin 1 at concentrations of 1×10^-7mol/L, 1×10^-9mol/L, 1×10^-11mol/L were added into the wells for 48h, the cell movement was determined with the use of wound assay.5. Cells were incubated in endothelin 1 at concentrations of 1×10^-7mol/L, 1×10^-9mol/L, 1×10^-11mol/L for 24 and 48h, the apoptosis index(AI) were analysed by flow cytometry respectively.Results1. Endothelin 1, endothelin receptor A, and endothelin receptor B mRNAs were detected by RT PCR in HEP 2 cells. We detected the PCR products of 129bp for endothelin 1, 175 bp for endothelin receptor A, 130 bp for endothelin receptor B. To ensure the quality of RNAs, amplification reations were performed with pairs of primers specific for human glyceraldehyde phosphate dehydrogenage(Gapdh), We detectde 141bp for Gapdh.2. Endothelin 1, endothelin receptor A and endothelin receptor B like activity were founded in HEP 2 cells. The positive reactions of endothelin 1 were mainly demonstrated in cytoplasm; the positive reactions of endothelin receptor A and endothelin receptor B were mainly demonstrated in cell membrane.3. Addition of endothelin 1 resulted in a dose dependent increase in cell numbers which was significant at 1×10^-7mol/L for HEP 2 cells, endothelin 1 with concentration of 1×10^-11mol/L～1×10^-8mol/L has no effect on the proliferation of HEP 2 cells.4. After 48 hours, the HEP 2 cells migratting from the injure margin in the control group were 8.00±1.00; in the endothelin 1 group at concentrations of 1×10^-7mol/L, 1×10^-9mol/L and 1×10^-11mol/L, the cells migrating from the injure margin were 21.6±1.57, 17.3±1.52, 15.6±2.08 respectively. Compared with control group, the cells migrating of all endothelin 1 groups were statistically higher.5. Volume of conony treated by endothelin 1(1×10^-7mol/L)were bigger than that of control group. The colony number of control group was 31.0±1.00, and the colony number were 59.0±2.00, 32.06±1.15 and 30.6±2.08 respectively following the treatment with endothelin 1 at concentrations of 1×10^-7mol/L, 1×10^-9 mol/L and 1×10^-11mol/L. Compared with control group, the colony number of endothelin 1(1×10^-7mol/L) group were statistically higher.6. After treated with endothelin 1 for 24 and 48 hours, the AI among the different endothelin 1 treated groups and control group has no statistically difference.Conclusions1. Endothelin 1, endothelin receptor A, and endothelin receptor B mRNAs and protein expresse in cultured HEP 2 cells.2. Endothelin 1 can stimulate the proliferation of HEP 2 cells; this function of stimulating are dose dependent.3. Endothelin 1 promotes the migration and invasion abilities of HEP 2 cells.4. Endothelin 1 has no effect on the apoptosis of HEP 2 cells.