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1、 Estrogen progesterone ET1 induced cardiomyocyte hypertrophy reaction Abstract Objective To observe the different concentrations of 17 estradiol (E2 and progesterone (P separate and joint use of endothelin-1 (ET 1) induced cardiomyocyte hypertrophy reaction. ET 1 stimulate the in vitro culture of ne
2、wborn rat cardiomyocytes, model cardiac mast cells and cardiac myocytes 3H leucine (3H leu incorporation as indicators of myocardial hypertrophy results E2 the ET induced myocardial cells could significantly inhibit the incorporation of 3H leu increased P this no significant effect, the joint use of
3、 E2 / P, P of E2 to reduce ET 1 induced myocardial cells 3H leu incorporation to increase the role with a certain degree of inhibition. conclusion E2 inhibited ET 1 induced myocardial hypertrophy the joint use of the reaction, E2 / P, P can be a certain degree of inhibition of E2 on myocardial prote
4、ction effect should be applied in appropriate low concentrations. Keywords: 17 estradiol progesterone cardiomyocyte hypertrophy reaction Abstract Objective To investigate the effects of 17 estradiol (E2) or progesterone (P) alone and combined E2 / P treatment on cardiomyocyte hypertrophy induced by
5、endothelin 1 (ET 1.Methods Myocardial cells from neonatal rats were cultured in vitro and cardiomyocyte hypertrophy model was established with ET 1. Cardiomyocyte hypertrophic response was assayed by 3H leucine (3H leu incorporation.Results Pretreatment with E2 could inhibit the increase of 3H leu i
6、ncorporation induced by ET 1, while P had no significant effect on this increase.With combined E2 / P Treatment, P could weaken the inhibitive effect of E2 on the increase of 3H leu incorporation induced by ET 1 to some extent.Conclusion E2 can inhibit cardiomyocyte hypertrophy induced by ET 1.With
7、combined E2 / P treatment, P can attenuate the protective effect of E2 on myocardium to some extent, so it is better for us to use the lower concentration of E2 and P. Keywords: 17 estradiol, progesterone, cardiocyte, hypertrophic response A large number of basic and clinical pilot study confirmed t
8、hat estrogen has a protective effect on the cardiovascular system 1, estrogen replacement therapy (estrogen replacement therapy, ERT) can significantly improve the female menopause triggered by a series of symptoms, reduce the incidence of cardiovascular events improve the quality of life of older w
9、omen, but the long-term simple application the estrogen increased female incidence of endometrial cancer 2, the clinical trials with a small dose progesterone can inhibit the carcinogenic side effects of estrogen, effectively reduce the womb the danger of the film cancer 3, but whether progesterone
10、interfere with the protective effect of estrogen on the cardiovascular system, yet controversial 4,5 This topic is intended to neonatal SD rat cardiomyocytes cultured to myocardial cells 3H leucine (3H leucine, 3H leu to incorporation as cardiomyocyte hypertrophy response indicators observe 17-estra
11、diol (17 estradiol, E2 and dextral pregnant 4-en-3, 20 dione (progesterone, P used alone E2 / P joint use of endothelin-1 (endothelin of ET 1) induced cardiomyocyte hypertrophy response impact. aims to explore the impact of female hormones on the cardiovascular system function, and postmenopausal wo
12、men with safe and effective application of hormone replacement Treatment provided new experimental reference and basis. 1 Materials and methods 1 3 d the 1.1 experimental animals born Spragure Dawley germline rats, male or female, provided by the Experimental Animal Center of Sun Yat-sen University,
13、 animals Certificate of Conformity: Cantonese and Certificate word 2004A089. The 1.2 Main reagents E2, P ET 1,5 bromo-deoxyuridine nucleoside (5 bromodeoxyuridine 5 Brdu, trypsin (trypsin purchased from Sigma, DMEM medium was purchased from Gibco Company HEPES were purchased from Sangon (Shanghai co
14、mpany, newborn calf serum Hyclone products, 3H leu products for the China Institute of Atomic Energy, and the rest were of analytical grade. 1.3 major high-speed experimental instrument Eppendorf centrifuge (centrifuge5417, Germany, 78HW type 1 temperature magnetic stirrer (Hangzhou Electric Instrum
15、ent Factory, Galaxy carbon dioxide incubator (Stephen Clark Fabrications Ltd, a United Kingdom, XSZ D2 inverted microscope (Chongqing Optical Instrument Factory, LS 3801 liquid scintillation counter (Beckman, Germany, IBAS image analysis system (Kontron Elektronik, Germany. 1.4 cultured cardiac myoc
16、ytes cultured neonatal rat cardiomyocytes by Simpson et al 6 described remove the SD rat heart Health 1 3 d, 0.08% sterile trypsin digestion and separation ventricular myocytes, differential adherence Purification of myocardial cells and in the culture for 48 h before adding 0.1 mmol / L of 5 Brdu o
17、f myocardial cells in culture medium to inhibit the growth of non-myocardial cells. 1.5 cardiomyocytes 3H leu incorporation was measured differential adherence myocardial cells to 3 105 cells / ml density were seeded in 24-well culture plates .48 h for serum-free phenol red-free DMEM medium incubate
18、d for 24 h , with or without addition of different concentrations (10-10 10-6mol / L), E2, P pretreatment 24 h, and then added a 10-8mol / L ET 1 for 24 h, added to each well of 37 kBq / ml 3H Leu incubated for 16 h, remove culture medium, with 10% trichloroacetic acid, 0.5 ml of 4 C fixed 30 min, D
19、-Hanks solution and then precooled washing twice, and finally with 1% SDS-containing 0.2 mol / L, 0.5 ml of NaOH solution collected after cell lysis at 37 C for 12 h incubation lysis buffer containing 8 ml of scintillation liquid measuring cup, shake placed in a liquid scintillation determination of
20、 radioactivity. 1.6 statistical data are presented as mean + - standard deviation (x + - s, t-test and ANOVA analysis for statistical analysis, P <0.05 was considered statistically significant. 2 Results 2.1 ET 1 myocardial cell morphology digested with trypsin isolated neonatal SD rat ventricula
21、r myocytes cultured for 72 h after adding ET 1 (10-8mol / L, 24 h after the myocardial cells showed obvious hypertrophy (Figure 1). a normal cardiomyocytes the b. hypertrophy of cardiomyocytes Figure 1 ET 1 induced hypertrophy of myocardial cells ( 200) The 2.2 E2 pretreatment ET 1 induced myocardia
22、l cells 3H leu incorporation to increase the impact compared to the control group ,10-8 mol / L ET myocardial cells can increase 3H leu incorporation (88.67 + -15.05% (n = 6 , P <0.01, this effect may be after 24 h E2 pretreatment significantly inhibited ,10-10 10-6mol / L E2 respectively ET 1 pr
23、ocessing cardiomyocytes 3H leu incorporation decreased as shown in Table 1, wherein 10 -8mol / L E2 effect is most obvious, given alone E2 3H leu incorporation of myocardial cells had no significant effect (n = 6, P> 0.05, Table 1, different concentrations of E2 the ET induced myocardial cells 3H
24、 leu Note: blank control group * P <0.01, ET 1 group # P <0.01 ET 1-induced myocardial cells 3H leu incorporation 2.3 P pretreatment increased compared with control group ,10-8 mol / L ET myocardial cells can 3H leu incorporation to increase (94.48 + -27.18% ( n = 6, P <0.01,10-10 10-6mol/L
25、P pretreatment 24 h increased 3H leu incorporation ET 1 induced myocardial cells no significant impact, see Table 2 Table 2 different concentrations of P ET 1-induced cardiac myocyte 3H leu incorporation to increase the impact of Note: compared with the blank control group, * P <0.01 2.4 E2 and P
26、 use increased incorporation of 3H leu ET 1 induced myocardial cells combined in order to investigate the E2 / P cardiac hypertrophic response, we further 10-10,10-8,10-6 mol / L E2 decreased ET induced myocardial cells 3H leu incorporation to increase as a control, were observed 10-10,10-8,10-6 mol
27、 / L E2 and 10-10,10-8,10-6 mol / L of P combined use of cardiac myocytes 3H leu incorporation found that, compared with control group ,10-8 mol / L ET myocardial cells can 3H leu incorporation to increase (76.92 + -23.30% (n = 6 , P <0.01; The role of the 24 h after E2 pretreatment significantly
28、 inhibited ,10-10 ,10-8 ,10-6mol / L E2, respectively, of the myocardial cells 3H leu incorporation decreased (21.41 + -1.66%, (31.30 + -3.10%, (25.90 + -1.75% (n = 6, P <0.01, when E2 / P combination ,10-6mol / L P 10-6,10-8 mol / L of E2 reduced ET myocardial cells induced 3H Leu incorporation
29、increased significantly inhibited (N = 6, P <0.05), this inhibition is not present a significant dose effect related, and when the concentration of E2 was the 10-10mol / L , the same 10-6mol / L P of E2 is no significant impact (n = 6, P> 0.05, joint use of other concentrations of E2 and P, P
30、reduced the E2 ET 1 induced myocardial cells 3H leu doped into increased role no significant inhibition (n = 6, P> 0.05, see Table 3 Table 3 E2 / the P joint use of the ET 1 induced myocardial cells 3H leu incorporation of the impact of increased NOTE: the blank control group Compare P <0.01,
31、ET 1 group comparison * P <0.01, and E2 (10-8mmol / L + ET group # P <0.05, E2 (10-6 mol / L) + the ET 1 group P <0.01. 3 Discussion The myocardial cells ET 1 specific receptor 7, the ET 1 can be used as an important cause hypertrophy factor involved in myocardial hypertrophy 8, the present
32、 study demonstrated that adding ET 1 after myocardial cells showed obvious hypertrophy. Myocardial hypertrophy as high blood pressure common complication of the disease, has now been listed as an independent risk factor for cardiovascular disease morbidity and mortality 9, and the prevention and tre
33、atment of cardiac hypertrophy has a very important significance. study found that estrogen has a cardiovascular protective effect 1, hormone replacement therapy can significantly reduce postmenopausal women the risk of left ventricular hypertrophy 10, suggesting that estrogen may inhibit cardiac hyp
34、ertrophy we observed in experiments, in ET 1 stimulation of state, 10 -10 10-6mol / L E2 significantly inhibited the ET 1 induced cardiomyocyte hypertrophy response, manifested as ET 1 induced myocardial cells 3H leu incorporation increased significantly reduced, while the basic state, E2 3H leu inc
35、orporation normal myocardial cells itself had no significant impact, suggesting that ET 1 induced myocardial hypertrophy at the cellular level E2 inhibit. Estrogen hormone protective effect on the cardiovascular system has been a hot research over the years, and the pregnancy hormone is a relatively
36、 small impact on cardiac and vascular function, different conclusions. Vedernikov, etc. 11 found that progesterone alone on potassium chloride induced aortic ring contraction and acetylcholine-induced aortic ring relaxation effect of no significant impact. study found the progesterone by the diastol
37、ic pulmonary vascular play certain vascular protective effects 12. in our experiments ET 1-induced cardiac myocyte hypertrophy reaction observed 10-10 10-6mol / L, P alone had no significant effect. Posted in the free papers Download Center http:/prolonged solitary use the ERT can increase the incid
38、ence of endometrial cancer danger, to avoid danger, often combined use of estrogen and progestin, but the joint use of estrogen and progesterone will weaken the protective effect of estrogen on the cardiovascular system? Our experimental results show that, when used in combination of E2 / P, when E2
39、 is the low concentration (10-10mol / L, even if a high concentration of P (10-6mol / L not affect E2 on the inhibition of ET 1 induced cardiac hypertrophic response, but when the concentration of E2 10-6,10-8 mol / L, the same concentration of P to produce significant inhibition of E2 anti-myocardi
40、al hypertrophy. This indicates that the P in a certain concentration range can antagonize the inhibitory effect of E2 on cardiac hypertrophy, and this antagonistic effect is not only concerned with the concentration of P, but also with the concentration of E2. When P and E2 are at low concentration,
41、 P so as not to affect the inhibitory effect of E2 on cardiac hypertrophic response. Wassmann et al 13 in rat vascular smooth muscle cells have been similar to the results of our experiments. suggesting that part of the joint use of progesterone may offset estrogen protective effect on the cardiovas
42、cular system. early termination in July 2002, the WHI estrogen and progestin continuous combined treatment group (daily conjugated estrogen 0.625 mg + 2.5 mg of medroxyprogesterone clinical study, the interim summary report found that hormone replacement therapy can increase the risk of cardiovascul
43、ar disease, the reason may also be associated with the joint use of progesterone Therefore, the need for further in-depth study of hormone replacement therapy plus progestin on the function of the cardiovascular system, in order to clarify its mechanism experimental reference and basis for the safe
44、and effective hormone replacement therapy in postmenopausal women. In summary, our results show that E2 can be used alone significantly inhibited ET 1 induced cardiomyocyte hypertrophy response in cell experiments, and P is no significant effect. E2 / P joint use, high concentration of P can be the
45、effect of a certain degree of inhibition of E2 on myocardial protection, when P and E2 are low concentration, P so as not to weaken the inhibitory effect of E2 on cardiac hypertrophic response, but this reserved P on E2-induced endometrial excessive concentration the proliferative inhibition unclear
46、, need to continue to explore in the next step of the experiment and the experiment will help to deepen our understanding of the role of the cardiovascular system of sex hormones in the application the appropriate compatibility issues of estrogen and progesterone in hormone replacement therapy for p
47、ostmenopausal women some experimental Reference. References 1 Mikkola TS, Clarkson TB. Estrogen replacement therapy, atherosclerosis, and vascular function J. Cardiovasc Res, 2002,53 (3): 605 619. 2 Bernstein L. The risk of breast, endometrial and ovarian cancer in users of hormonal preparations J.
48、Basic Clin Pharmacol Toxicol, 2006,98 (3): 288 296. 3 Pentti K, Honkanen R, Tuppurainen MT, et al.Hormone replacement therapy and mortality in 52 to 70 year old women: the kuopio osteoporosis risk factor and prevention study J. Eur J Endocrinol, 2006,154 (1 ): 101 107. 4 Lamon Fava S, Postfai B, Diffenderfer M, et al. Role of the estrogen and progestin in hormonal replacement therapy on apolipoprotein AI kinetics in postmenopausal women J. Arterioscler Thromb Vasc Biol, 2006,26 (2): 385 391. 5 Shultz JM, Zhu XD, Knopp RH, et al. Norgestimate and medroxyprogesterone acetate do not attenu