| Essential hypertension is a familiar chronic disease associated with many genetic disorders and environmental factors, which can not only cause vascular complications of the heart and brain, but also result in a group of serious pathological disorders such as abnormal changes of metabolic in lipolysis, insulin resistance, or diabetes. Though it is so harmful to mankind health and its incidence and mortality rate rank top among all diseases, there isn't a very efficient cure method to prevent this disease.Though traditional druggery has efficiency on blood pressure control, yet the final outcome may not be of satisfaction. Many of them are on the purpose of promoting vasodilatation and reducing resistance of peripheral vascular. Their effective period relatively is all short. They are lack of better specific functions and must be taken intermittently year after year, which lead to side effects on patients and decrease the response on reducing blood pressure because of the patients'bad compliancy. Some synthesized polypeptides such as ANP, are proofed that they can improve vasodilatation which depends on endothelium, suppress the growth of vascular smooth muscle, and reduce the peripheral resistance so as to control the hypertension. Most of the druggery, however, has disadvantages such as a short half-lifetime in plasma, high costs in production, resulting in the restricted usage in clinical practice. Therefore, it comes to the necessity to find a treatment integrated with more special functions, less side effects, lasting long term on blood pressure control. Following the development and progress of molecular cardio-vascular study, the continuous expression of active polypeptides can successfully make up for the shortcoming which the half-lifetime is so short, which make gene therapy become a new promising method for hypertension control and take on exciting prospect on clinic application.During the current two decades, about one hundred genes to be considered to participate in blood pressure control have been cloned. Significant progress has achieved and adjustment actions are generally identified in gene therapy by measures of experiments on the demo animals, targeting at proofing some of their functions in pressure adjusting. According to the different mechanisms of hypertension, different gene is introduced to make control, such as to promote expression of dilation related gene or suppression of constriction related gene, and finally to catch the aim to control or heal the hypertension.Atrial natriuretic peptide, which is simply called ANP and firstly found by de.Bold from rat atrium, was studied widely in its functions in the recent several years. Associated study shows that besides its strong functions in vascular dilation, promotion of sodium excretion and urine output, ANP can also suppress the rennin angiotensin aldehyde system (RAAS), or suppress the activity of sympathetic system, which inturn lead to lowering of cardiac output, or suppressing the growth of smooth muscle. For all of the advantages, it has become a very important target gene on hypertension therapy in recent years.Recent study discovered that mice with ANP gene transferred may have continuous hypotension, however, if they who are lack of this gene may develop hypertension. Lots of fundamental and clinical data demonstrated that intravenous injection of ANP gene can treat ESH and some secondary hypertension. Some domestic scholars also have constructed several plasmids to transfer ANP gene into different hypertensive rat models, and confirmed the efficacy of continuously expression of pro-ANP by intramuscular method, which finally lead to reduction of blood pressure. All these data cited above clearly confirmed the important role of ANP gene expression in hypertension control among all related factors.Accompanying the progressing of hypertension, lots of unbeneficial changes of both functional and structural inc. reconstitution of left ventricle or vascular remodeling, may develop. And it finally leads to the malignant circle of hypertension. Since the mid term of the 90th, Mr. Chao and his mates had started to observe the effects of some vascular-dilating gene, such as ANP gene, on blood pressure control via gene delivery. The results indicated that this method can also reverse the end-organ damages besides the effects of pressure reduction. Therefore we realized that expression of pro-ANP not only can lower blood pressure, but also can reduce the damage of end-organ, which make it becomes one of the best target genes of hypertension therapy.Within the couple of years, scientists were very interested in gene therapy with plasmid adopted as vector. Based on its such advantages as easy to be constructed, no infectivity, no carcinoma-inducing, low-immunogenic happening, long term of expression, no serious side-effect, no integration with host cell gene, etc. they benefits for the application and extension of gene therapy in clinical practice in the future. In the study project, we choose plasmid pcDNA3.1+ as vector for gene therapy. The reason is mainly because it has a powerful promoter CMV from cytomegalovirus, which has the highest transcription activity, and has a polyA tail from bovine growth hormone, which ensures the stability of mRNA after transcription. There is no related report of using this vector to carry ANP gene in hypertension therapy yet, though it has been so widely used in transgenetic study.The study aims to construct a recombinant plasmid of hANP by means of adopting pcDNA3.1+ as vector, which has a CMV promoter. Through intramuscular or intravenous injection to inspect its expression in spontaneously hypertensive rats after delivering of the vector, we carefully observed the effects by monitoring physical parameters such as blood pressure and urine sodium excretion, together with inspection of the changes in SHR's end-organ damage, for example, the reconstruction of the heart or the kidney, and discussed some issues associated with safety of gene therapy.In order to do so, we choose 64 healthy male SHRs featuring in 4-week-old, weight 50~100 grams, and divided them into two big groups (im&IV), eight small groups (four in each big group) randomly. Then we began our transgenetic experiment when the rat was 5 or 9 week old for each big group. The dosage of pcDNA3.1+/hANP or pcDNA3.1+ plasmids injected is 200ug/0.2ml for 5w-group and 500ug/0.5ml for 9w-group respectively.We observed the physical parameters, such as body weight, blood pressure, heart rate, urine volume and sodium excretion, body temperature, et al, of the SHRs periodically, and collected the plasma of associated groups, according to the efficacy of pressure control, to detect the ANP levels. We would kill two rats at some time point, according to the comparison result of the experiment and its control group, to detect the expression of the plasmid. When the rats were 20-week-old, all would be killed for the purpose of collecting samples, such as the heart and kidney, to check the pathological and histological changes.As for the security problem of gene therapy, we mainly monitored the body temperatures and other physical conditions, such as activity, food and water intake, urine volume, et al, checked five biochemical parameters (GOT,GPT,LDH,ALP,WBC), and detected the antibodies of hANP which may existed in SHRs'plasma. The items would be regarded as main discussion contents.Results of PCR, enzyme digestion, and sequencing assay proved that we successfully harvested the hANP cDNA fragment and inserted it into the vector of pcDNA3.1+ plasmid, which set foundation for further study of gene therapy in animals. Monitoring results of gene expression and other physical parameters after gene delivery demonstrated that, the recombinant plasmid pcDNA3.1+/hANP could express effectively the mRNA of hANP and its pre-protein, which can be released into blood circulation and rise the plasma ANP level, and finally cause reduction of blood pressure to a maximal extent of 30 mmHg. At the same time, the therapy also caused changes of water intake and urine excretion for a short period, yet no significant changes in heart rate, body weight and food consumption. We did not observed the same efficacy of pressure reduction in the intravenous injection groups as in the intramuscular injection ones, which suggested intravenous therapy not an ideal selection and its failure reason needs further study.Pathological and histological findings show that, together with the reduction of blood pressure, the end-organ damages, such as the heart and the kidney, also were effectively attenuated or reversed in the im-groups. The ratio (LVMI &HBR), which we used to evaluate the heart reconstruction, was significantly decreased in the 5wimexp group as compared with that of its control group. Further micro photographic observations demonstrated that, when we ranked the scores of the glomerular, the tubular and the micro vascular damage and compared with Mann-Whitney U test in the two 5wim-groups; there is a significant reduction of the scores in the 5wimexp group than that of its control group. These results proved that early administration of gene therapy before formation of hypertension, with pcDNA3.1+/hANP plasmid by way of intramuscular injection, can effectively reduce or prevent the end-organ damage that succeeded hypertension, both from macro and micro histological structure.Nowadays, most of the gene therapy methods of hypertension are still in the phase of transferring from animal experiment to clinical practice, and it is far away from the real practice. As is known that the safety problem of gene therapy is the core of obstacles to decide whether it can be put into clinical practice. Wherefore, we also collected some important physical, biochemical, and immunological parameters associated with security evaluation of our study. The final analysis demonstrated that there were no significant differences in body weight, heart rate, and food consumption between the experimented and its control groups. No abnormal symptoms such as vomiting, diarrhea, and irritation, etc, which were normally considered side effects associated with gene therapy, appeared among the SHR after gene transfer. During the first eight days after gene transfer, the comparison of daily average body temperature among the groups did not show any obvious difference. And the comparison of the five biochemical parameters between the experimental and its control groups caught the same result as well. We neither found detectable immunoreactive hANP antibodies in the SHRs'serum. All of the data listed above proves that it might be a safe way to deliver the recombinant plasmid vector pcDNA3.1+/hANP into SHR by intramuscular or intravenous injection method.Essential hypertension is a congenital disease that is proved to be associated with multiple genetic disorders; however, its etiology is still not clarified till now. Though some gene therapy methods were proved to have efficacy in pressure control, yet most is at the stage of making experiments on animal models, which means there's still a long distance to go before clinical practice. So is our aim of this study mainly to explore some new methods and experience for gene therapy of hypertension. It is believed that people will find more and more excellent target genes, or construct even fitter vectors, or explore smarter deliver methods, to make our dream of eradicating this disease come true.
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