Exploration On The Phenotype Of Gene Therapy

ANP(atrial natriuretic peptide) is a polypeptide hormone with potent natriuretic, diuretic and vasorelaxant effects which is also involved in the homeostatic regulation of fluid and electrolyte balance. These properties permit efficacious applications of ANP in a variety of clinical therapeutic uses including essential hypertension, congestive heart failure and renal failure, et al. Most of ANP used in clinical research were chemically synthesized by solid-phase method, which was costly; the half life of ANP is very short(2.5 minutes), it must be intravenous injected continuously in clinical uses, these limit its application. The concept of Phenotypic Gene Therapy as our proposal can overcome these limitations and results in the reduction of systemic blood pressure and distinct diuresis.The Phenotypic Gene Therapy treats the symptoms of diseases directly and it is not necessary to know the genetic backgrounds of diseases in detail. In order to treat and cure the disease of patients individually, to inject one or several gene(s) which product(s) should be able to eliminate the symptom and restore the normal physiological phenotype well. The gene(s) can be expressed continuously and steadily in vivo so as to cure the disease surely. It is just like a minimal pharmaceutical factory is established in vivo in the body of patients and continuously provides medicine for treatment.We chose ANP gene as the functional gene to treat essential hypertension and nephrotic syndrome. The animal models are spontaneously hypertensive rat (SHR) and nephrotic syndrome (NS) rat which is induced by adriamycin (ADR) respectively.First at all, we cloned rat ANP (rANP) cDNA into retrovirus vector pLXSN to construct two recombinant plasmids pLHY2 and pLHY4. By using the standard liposome-mediated protocal, the pLHY2 and pLHY4 were transfected respectively into PA317 packaging cell line. The transfected cell line was screened with G418 to get G418-resistant colonies. The target cells-SHR skin fibroblast (SHRFT) were infected with the viruses getting from PA317/pLHY2 and PA317/pLHY4 cells respectively. The infected SHRFT were screened out with G418 to get G418-resistant colonies. The radioimmunoassay (RIA) was performed by using commercially available kit to detect the rANP in the medium of the infected SHRFT and the rANP expression level of SHRFT/pLHY2 cells was highest. The SHRFT/pLHY2 cells was cultivated in large amounts. The SHRFT/pLHY2 cells were transplanted into 6-week-old SHR by means of the collagen or the capsule respectively. The significant decrease in blood pressure which lasted 7 weeks and a little diuresis was shown. The means of the capsule may make gene therapy to be a reversible process.