| BackgroundHuman hypertension is a complex systemic disorder which is considered to be one of the most common and the most important cardiovascular diseases in clinical currently and is the characteristic of sustaining blood pressure elevation. Tremendous achievements have been made in pathogenesis and treatment of hypertension at present, but hypertension and its concomitant risk factors remain uncontrolled. Indicating that there are some unknown factors involved in the pathophysiological progress of hypertension.Recent studies have found the autoantibodies (α1-AA) against the second extracellular loop (α1-AR-ECII) ofα1-adrenergic receptors in patients with malignant, essential and refractory hypertension. And the autoantibodies were demonstrated to exert agonist-like effect, such as a positive chronotropic effect on isolated neonatal rat cardiomyocytes, and stimulating their activities via activation of L-type calcium channels. All these indicated thatα1-AA may be closely associated with the development of hypertension. However, the current studies mainly focus onα1A-AA. While theα1-AR is divided into three subtypes, they areα1A,α1B andα1C-It has been found that the main subtype distributed in human and rat aorta isα1D-AR, which has roles in artery and small artery contration and involved in development of hypertension. Therefore, whether there isα1D-AA in hypertensive patients and its relationship with disease should be concerned. But there are many basic research need to be done to solve the above problems. First, the existing kit forα1-AA detection is used for detecting autoantibody againstα1A-AR. So, it's the necessary to establish a kit for detecting autoantibody againstα1D-AA. Second, the pathophysiological significance ofα1D-AA is to be confirmed. The former study of our team have showed that although immunize nomal rats with synthesizedα1D-AR-ECⅡpeptide did not lead to hypertension, the endothelium of immunized rats were damaged. Howerer, it is not unclear that the endothelial dysfuction is due to the antigen or theα1D-AA. In addition, the immune system were activated in hypertension without clear reason. It has been known that T lymphocytes play an essential role in the start-up phase of immune respones and the proliferation of T lymphocytes is an important stage of immune respones to antigen. Some studies hsve shown that there isα1A-AR in the membrane of T lymphocytes, so whetherα1D-AA can affect the immune regulation function in addition to cardiovascular discovered previously by binding the corresponding membrane receptor thereby.In conclusion, we designed this research project as follows:(1) According to the principle of ELISA method, design the antibody detection kit ofα1D-AA for the hypertensives, prepared in the future research, and contribute to the clinical diagnosis of hypertension. (2) With the passive immunity byα1D-AA in normal rats, observe if the endothelial dysfunction was induced byα1D-AA.; (3) In hypertension model, test the immune system and observe the direct effect ofα1D-AA on T lymphocytes.Objectives1. To establish a special method for detecting serum autoantibodies against humanα1D adrenoceptor.2. The blood pressure of passive immunized rats withα1-AA slightly elevated, but can not lead to hypertension. But for a long time, it would damage the vascular relaxation and contraction and lead to vascular endothelium dysfunction.3. The production ofα1D-AA may be related to immune system accentuation, and the producedα1D-AA can promote the proliferation of T lymphocytes.Methods1. A synthetic peptide corresponding to the amino acid sequence 192-218 of the second extracellular loop of humanα1D adrenoceptor was used as an antigen to develop a new assay named indirect Streptavidin-Enzyme-Linked immunoabsorbent assay (SA-ELISA), which was used in detection of the autoantibodies against humanα1D adrenoceptor in serum collected from 69 patients with primary hypertension and 100 healthy subjects.2. Withα1D-AA passive immunity normal rats, observed the changes of blood pressure; Use the blood pipe ring tensity determination technology to observe the constriction and relaxation of vessels; Take the endothelin to observe the injury ofα1D-AA on vessels.3. Made the 2K1C hypertension mouse model, and examined in the blood serumα1D-AA and antibody formation ability; in cultured lymphocytes from mesenteric lymph nodes, we observed the effects ofα1D-AA positive IgGs on T lymphocytes proliferation induced by Con A by CCK-8 Detection Kit. Results1. Theα1D-AA kit and its preliminary application.The optimal working concentration of antigen is 2.5mg/L, of biotinylated goat anti-human IgG is 1:1500 and of SA-HRP is 1:750 in this experimental method.To measureα1D-AA in positive and negative sera for 20 times, the coefficients of variation (CV) within were 5.8% and 4.2%, respectively, and the inter-assay CV were 12.7% and 9.8% during continuous 20 days' measurement. Indicate that this method is of good repeatability and stability. After absorption with specialα1D-AR antigen, OD value significantly decreased, indicate the strong specificity of this method.. The positive frequencies of autoantibody againstα1D adrenoceptor among patients with primary hypertension was 23.2%(16/69), which was higher than that of healthy subjects 9%(9/100), (P<0.05, Table 1-2).2.Theα1D-AA titer of the passive immunized rats.Theα1D-AA titer of the passive immunized rats increased rapidly, then maintained at a stable level. There was no statistical difference among theα1D-AA titer of passive immunized group after immunizing for 1,2,3 and 4 months. However, there was significant difference when compared with control group (P<0.05). Theα1D-AA titer of the passive immunized rats maintain at 0.43±0.13 to 0.49±0.11 in the whole process of immunizing. Indicate that theα1D-AA passive immunized model was sucdessful (Figure 2-1).3. The blood pressure of the passive immunized rats.The blood pressure of immunization group and control group rats were 105±5.4mmHg, and 104±4.4mmHg, respectively (P>0.05). Two weeks later, the blood pressure of passive immunized rats began to significantly rise on the 2nd week after passive immunity and peaked at the 4th week (125±6.9mmHg), kept the high level until the 6th week (125±7.3mmHg), then gradually decreased to baseline. However, the pressure of control group had no significant fluctuation at all the time (Figure 2-2).4. The ET-1 of the passive immunized ratsThe results showed that the ET-1 of immunization group started to increase after passive immunized 4 weeks(12.0±1.4), and peaked at the 12th week (21.0±1.6), which was significantly higher than the control group(11.0±1.1), (P<0.05), and kept the level until the 16th week (16.3±0.6), (P<0.05, vs. control group, Figure 2-3). Indicate thatα1D-AA may damaged the endothelium continuously.5. The vaesculars reactivity in passive immunized rats. As showed in figure 2-4, the contraction of isolated rat thoracic aorta rings gradually increased with the increase of the concentrations of PE. When the concentrations of PE were 10-7 mol/L and 10-6 mol/L, the contraction amplitude of immunization group were higher than that of control group (P<0.05, P<0.05, Figure 2-4). While the endothelium-dependent vasodilation of immunization group was weaker than that of the control group. When the concentrations of PE were 10-6 mol/L and 10-5 mol/L, the vasodilation of two group have significant differerce (P<0.05, P<0.05, Figure 2-5).6. The blood pressure (BP) of rats after 2K1C surgery.The BP of surgery group and sham group were 114.8±3.9mmHg and 114.3±3.3mmHg before 2K1C surgery. Two weeks later, the BP of surgery group(134.3±8.7mmHg)was higher than sham group (116.2±3.9mmHg)(P<0.05). The BP peaked at the 4th week (141.5±10.5mmHg), (P<0.01, vs. sham) and at 16th week still maitained a high level (137.6±7.4mmHg, P<0.01, vs. sham, Figure 3-1).7. The production ofα1D-AA in rats after 2K1C surgery.Theα1D-AA of surgery group increaced at 2th week after 2K1C (0.19±0.02), which was higher than that of sham group (0.15±0.01, P<0.01); at 4th week,α1D-AA reached the peak (0.21±0.01, P<0.01, vs. sham); and maintain at a high level till 12th week (0.21±0.01, P<0.01, vs. sham). Then, theα1D-AA begain to fall down and till to baseline at 16th week (0.16±0.01, Figure 3-2). Indicate that,α1D-AA is produced regularly during the formation of Hypertension.8. The antibody-producing capacity after 2K1C.The number of Direct Plaque Forming Cells increased significantly (2.5±0.01, P<0.01, vs. sham, Figure 3-3). Indicate that, the antibody-producing capacity increased after 2K1C.9. The effect ofα1D-AA on the proliferation of T lymphocytes.The results detected by CCK-8 kit showed that three concentrations ofα1D-AA (0.01,0.1, 1μmol/L) promoted the proliferation of T lymphocytes induced by Con A in a dose-dependent manner (OD values were 0.49±0.05,0.69±0.08,0.82±0.07), which was similar to the role ofα1D-AR agonist phenylephrine. In addition, the effects ofα1-AA could be neutralized by theα1D-AR-specific blockers Bunazosin or the antigen peptides corresponding to the second extracellular loop ofα1D-AR, but simple Bunazosin orα1D-AR antigen peptides had no significant effect on the proliferation of T lymphocytes. Conclusions1. This method is stable, and can be used for the measure ofα1D-AA by SA-ELISA repeatly.2. Theα1D-AA does not change the blood pressure of normal rats, but damage the constriction and relaxation of vessels and endothelial cells.3. The generation ofα1D-AA may associate with abnormal immune system, andα1D-AA can promote the proliferation of T lymphocytes by activating ofα1D-AR directly. |
