| Part I : The Expression of Estrogen Receptors and VascularSmooth Muscle Phynotype Protein inHuman Intracranial AneurysmsObjective: To investigate the expression of estrogen receptors (ERs) on intracranial aneurysmal vascular smooth muscle cell (VSMC). To investigate the VSMC phynotype protein a-smooth muscle actin (α-SMA) and desmin on intracranial aneurismal VSMC. To investigate the correlation between the expression of ERs and VSMC phynotype protein on aneurysms. Methods: The external carotid artery fragment and intracranial aneursyms wall were resected from 8 non-aneurysm patients and 18 aneurysm patients respectively. The vascular tissue was fixed by 4% paraformaldehyde, then embedded in OCT. 5μm sections dyeing with HE or PI were prepared to observation under light microscope. Immunohistochemical method was used to study the expression of ERs, a-SMA and Desmin on VSMC of vascular and aneursyms. The section optical density was measured by Image-Pro-Plus 6.0 software and analysed by SPSS 13.0 for Windows.Results: VSMC aligned disorder in aneursyms wall, display heteromorphosis and disappeared in some hyalinosis aneurysms. The expression of ERa and ERβ on normal VSMC in women were higher than that in men. The OD value of ERa and ERp expression on aneurysms were 0.2800±0.0958 and 0.3512±0.0848 which were more than those expressed on normal artery VSMC (0.1800±0.0258 and 0.2299±0.0336). This difference were significant (T=3.174, P=0.004; T=3.877, P=0.001). The OD value of a-SMA and Desmin expression on aneurysms were0.1371 ±0.0469 and 0.0961 ±0.0368, which were less than either expression on normal artery VSMC (0.3780±0.0427 和 0.3134±0.0581). This difference were significant (T=12.393, P<0.001; T=11.604, P<0 001). The expression of ERs on VSMC was correlated with the expression of a-SMA and Desmin. Conclusion: 1. The level of ERs expression on VSMC of normal artery were higher in women than those in men. 2. Inverse correlation is between the elevated expression of ERs and down-regulation of VSMC phenotype protein in aneurysms VSMC, which correspond to the formation of aneurysms.Part II: The Expression of Estrogen Receptors andVascular Smooth Muscle Phynotype Protein inRat Intracranial Aneurysms ModelObjective: To investigate the expression of ERs and VSMC phynotype protein on rat intracranial aneurysm model and the relationship each other. Methods: Sprague-Dawley rats were randomly divided into two groups: operation group and control group. The operation group rats were ligated right common carotid artery (CCA) and bilateral ramus posterior arteriae renalis (RPAR) feeding 1% saline 1 week later which group had 15 female rats and 15 male rats. The control group no operation were performed with normal feeding. The rats were sacrificed 6 months after operation, perfused with 4% parafonnaldehyde. The ACA-OA bifurcation were get under microscope, embedded in OCT, get successive sections, dyeing with HE or PI. Immunohistochemical method was used to study the expression of ERs, α-SMA and Desmin on VSMC of ACA-OA in both groups. The section optical density was measured by Image-Pro-Plus 6.0 software and analysed by SPSS 13.0 for Windows. Results: 5 cases of injury of blood vessel and 10 cases of aneurismal changes happen to hypertension rats' left ACA-OA bifurcation, neither to right ACA-OA bifurcation nor to control rats. The expression of ERα and ERβ in normal femalewere higher than that in male rats (T=3.757, P=0.004; T=2.552, P=0.029). The expression of ERα and ERβ were higher in male rats left aneurismal ACA-OA bifurcation of hypertension group than that in male of control group, which difference were significant (T=2.462, P=0.030; T=2.389, P=0.034). So did in female rats between hypertension group and control group (T=2.441, P=0.033; T=2.292, P=0.043). The expression of α-SMA and Desmin were lower in male rats left aneurismal ACA-OA bifurcation of hypertension group than in male of control group, which difference were significant (T=3.314, P=0.006; T=5.351; P<0.001). So did in female rats between hypertension group and control group (T=2.800, P=0.017; T=4.958, P<0.001). The expression of ERα and ERβ were no changes on left ACA-OA bifurcation between vascular injury of hypertension group and control group (T=0.098, P=0.925; T=0.443, P=0.673). So did between the two groups in female rats(T=0.225, P=0.829; T=0.299, P=0.773). The expression of ERs on VSMC were correlated that of VSMC phynotype protein. Conclusion: 1. The intracranial aneursyms developing procedure can be studied by the hypertension rat model. 2. The phynotype of VSMC is changed from contraction type to synthesis type in the formation of rat aneursyms, which is correlated with elevated ERs expression. 3. The down-regulation of Desmin is perhaps the early indication of artery injury, which could prefigure the aneursyms formation.Part III: The Relationship between ERα and ERβ Gene Polymorphism and Intracranial Aneurysm FormationObjective: To investigate the relationship between gene restriction fragment length polymorphism (RFLP) of ERs and intracranial aneurysm. Methods: 55 aneurysm patients and 66 non-aneurysm patients were chosen in during February, 2006 to November, 2006 hospitalized by PLA General hospital neurosurgery department. The ages were 46.62±17.62 and 47.85±12.96respectively. Venous blood was harvested with 5ml, which was used to extracting the genome DNA. The fragment of the first intron of ERa, the 5th extron and 8th extron of ERβ were augmented by PCR. The augment fragments were incise by enzyme: the fragments of the first intron of ERα incised by Pvu II and Xba I ; the fragments of 5th extron of ERβ incised by Rsa I ; the fragments of 8th extron of ERβ incised by Alu I . The fragments of post incised were electrophoretic analysed. Statistic analysis was by SPSS 13.0 for Windows. Results: the PCR augment fragment length were as follows: ERα 1st 1.3kbp, ERβ 5th 156bp and ERβ 8th 307bp. The RFLP of control group Pvu II was PP 12.1%, Pp 50.0% and pp 37.9%; The RFLP of aneurysm group Pvu II was PP 21.8%, Pp 60.0% and pp 18.2%. There was significant difference between aneurysm group and control group (x2=6.280, P<0.05) . The RFLP of control group Xba I was XX 1.5%, Xx 36.4% and xx 62.1%; The RFLP of aneurysm group Xba I was XX 1.8%, Xx 40.0% and xx 58.2%. There was no significant difference between aneurysm group and control group (x2=0.198, P>0.05) .The RFLP of control group Rsa I was RR 53.0%, Rr 36.4% and rr 40.9%; The RFLP of aneurysm group Rsa I was RR 32.7%, Rr 63.6% and rr 3.7%. There was significant difference between aneurysm group and control group (x2=6.203, P<0.05). The RFLP of control group Alu I was AA 54.5 %, Aa 39.4% and aa 6.1 %; The RFLP of aneurysm group Alu I was AA 45.5%, Aa 49.0% and aa 5.5%. There was no significant difference between aneurysm group and control group (x2=1.155,P>0.05).Conclusion: 1. The RFLP of ERα 1st intron Pvu II and ERβ 5th extron Rsa I gene polymorphism are related to aneurysm formation. 2. The polymorphism of ERα and ERβ are corresponded to the expression ability of ERs and structure of ERs, which are affected the formation of aneurysm.Part IV: The Effect of Estrognen and Tamoxifen on theExpression of Estrogen Receptor and VSMC PhynotypeProtein on Rat Artery VSMC invitroObjective: To investigate the affection of 17β-estrodiol and Tamoxifen (TAM) on the expression of ERs and phynotype protein of rat aorta thoracalis VSMC invitro. Methods: The rat aorta thoracalis VSMC was cultured invitro. VSMC was disposed with 10-9M, 10-8M and 10-7M 17β-estrodiol; 10-8M, 10-7M and 10-6M TAM; 10-6M TAM and 10-7M 17β-estrodiol for 24h. The total mRNA was extracted for further experiment. The ERα , ERβ , α-SMA and OPN relative mRNA fragment concentration was analysis by RT-PCR.Results: 10-9M, 10-8M and 10-7M 17β-estrodiol could elevated the expression of ERα and ERβ (F=29.973, P<0.001; F=66.186, P<0.001). TAM did not affect the expression of ERα and ERβ , but which could interrupt the effect of 17β-estrodiol. 10-9M, 10-8M and 10-7M 17β-estrodiol could elevated the expression of α-SMA (F=173.836, P<0.001) and suppress expression of OPN(F=10.243, P=0.004). TAM did not affect the expression of both α-SMA and OPN, but which could interrupt the effect of 17β-estrodiol.Conclusion: 1. Estrongen can elevated the expression of ERα and ERβ on VSMC and convert VSMC from synthesis type to construction type. 2. The elevated expression of ERs and changes of VSMC phynotype are depended on estrogen, which can be interrupted by TAM. 3. The elevated expression of ERs promotes the effect of estrogen. |
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