| NHE gene family encodes a series of exchangers locating at the membranes of eukaryocytes. Among them, NHE-1 isoform is ubiquitous and exchanges extracellular Na+ with intracellular H+ at the ratio of 1:1. It is known that NHE gene plays a key role in regulating the intracellular pH and cells' volume. Moreover, the activation of NHE gene is an early event during such processes as cells' proliferation, differentiation, apoptosis and so on. Studies show us that NHE gene is an essential factor involving in the development of many kinds of disease, especially of tumour, diabetes and heart disease.Recently, a new chemotherapic strategy is proposed, which is based on the regulation of tumour cells' pHi. Therefore, NHE-1 has been recognized as a potential target. NHE-1 gene is highly expressed in many kinds of malignant cells, including lung cancer cells, breast carcinoma cells, hepatoma cells and melanoma cells. In addition to this, level of NHE-1 mRNA in such cells is more than ten times of the normal cells'. Thus, it's evident that the density and activity of NHE-1 has been significantly raised. Although the glycolysis in tumour cells is markedly enhanced even under aerobic conditions and produces large amount of H+, tumour cells can strengthen Na+/H+ exchange to discharge excessive H+ in the cells and prevent intracellular acidification through up-regulation of NHE-1 expression. As a result, the high expression of NHE-1 gene may be important fortheir special property of anti-apoptosis. Based on these points, mis study aims to clarify the significance of NHE-1 gene in regulation of the proliferation/apoptosis of HepG2 cell lines. NHE-1 gene may be a new target of gene therapy for hepatoma. This study is divided into two parts:In the first part, asODN, which is complementary to NHE-1 gene, was employed to inhibit NHE-1 gene expression of HepG2 cells to study the inductive effects of intracellular acidification on cells' apoptosis. HepG2 cells were incubated with asODN of varied concentration for certain time or with some concentration for different time course. The morphological and apoptotic changes of cells were observed by light-, electro-microscope and in situ cell death detection. In the second part, we construct an antisense expression vector of NHE-1, then select the positive clones to investigate the potential inductive of hepatoma in Balb/c nude mice by HepG2 cells which has been successfully transfected.The main results are as follows:1. We have established the standard curve for determining pHi of HepG2 cells and found that pHi correlated with fluorescence intensity in a line manner.2. According to the standard curve, pHi of HepG2 cells is determined as 7.12 0.04.3. We confirmed the acidic effect of asODN on HepG2 cells. The effect has shown a dose- and time-dependent manner. In the first 24 hours of incubation, asODN of lumol/L didn't have any acidic effect, while asODN of 20 mol/L effected significantly on the cells'acidification. So 20 mol/L was an appreciable concentration and at different time points its acidic effect was varied, especially after 48 hours' incubation.4. We chose the cells, which had been incubated with 20 mol/L asODN for 24 hours, for in situ cell death detection. DAB staining showed many brown positive cells in the treatment, while onlybackground staining in the control.5. With electron microscope, we observed the apoptotic changes in the cells which had been incubated for 72 hours.6. We cloned the sense and antisense oligonucleotides of NHE-1 gene and constructed its sense and antisense expression vectors.7. After it transfected HepG2 cells, the antisense expression vector inhibited the cells' proliferation(inhibition rates=32.6%), while the sense expression vector and the vacant one had no effects.8. Transfected HepG2 cells could induce the tumour formation in Balb/c nude mice, but the formation time was prolonged compared to the control(inhibition rates=30.2%).Conclusion: The over expression of NHE-1 gene correlates with the prol...
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