摘要
下丘脑-垂体-性腺轴(Hypothalamic-Pituitary-gonad Axial, HPG轴)是机体重要的神经生殖调控轴。种类繁多、作用机制复杂的细胞因子、神经递质、激素及其受体之间的平衡是维持动物HPG轴神经免疫内分泌机能平衡的物质基础,其中任何物质表达失衡,都会打破HPG轴的平衡,引起一系列组织结构和功能的改变,进而引发细胞的衰老凋亡。为了探讨激素和细胞因子对HPG轴因雌激素缺乏所引起的组织功能改变和细胞衰老凋亡的调节作用,本试验拟通过建立去卵巢(OVX)大鼠模型,人为破坏HPG轴中神经-免疫-内分泌网络间的平衡状态,用免疫组织化学SP法来研究在这种病理状态下下丘脑和垂体中凋亡相关蛋白Bcl-2和Bax的表达情况以及体外补充雌激素(Estrogen,E)和细胞因子γ-干扰素(Interferon-γ, IFN-γ)后对其表达的影响。主要结果如下:
1.大鼠下丘脑中Bcl-2和Bax的表达:大鼠下丘脑视上核、室旁核、弓状核、下丘脑腹内侧核及乳头体内侧核等35个核团中均有Bcl-2和Bax免疫反应阳性产物的表达,主要在细胞膜、细胞质和核膜分布。去卵巢后,Bcl-2的相对表达量随时程逐渐减少,4周后最少,6周后又逐渐上升,并且维持在一定的水平;Bax则逐渐增加,4周后达峰值,与假手术(SHAM)组差异均显著(P<0.05)。给予外源性雌激素后,Bcl-2的相对表达量增加,与OVX组差异显著(P<0.05);Bax的相对表达量在不同的核团出现不同程度的变化,在视上核、弓状核等核团Bax的相对表达量显著减少,与OVX差异显著(P<0.05),而在室旁核、下丘脑腹内侧核等核团Bax的变化则不显著,与OVX相比P>0.05。雌激素联合200 IUγ-干扰素注射,各核团内Bcl-2的相对表达量较OVX组差异极显著(P<0.01),与OVX+E2组差异显著(P<0.05),与SHAM组相比,P>0.05;Bax的含量总体变化趋势与单独注射雌激素后一致,表达量减少的核团与OVX组相比,差异也极显著(P<0.01);雌激素联合50 IU和800 IU的γ-干扰素,Bcl-2和Bax的相对表达量与OVX+E2差异不显著(P>0.05)。
2.大鼠垂体内Bcl-2和Bax的表达:腺垂体和神经垂体内均有Bcl-2和Bax阳性细胞的表达,主要在细胞膜、细胞质着色,腺垂体内阳性产物明显较神经垂体内多,着色也深。去卵巢后,Bcl-2的表达减少,Bax增加,与SHAM组差异显著(P<0.05)。补充外源性雌激素后,Bcl-2较OVX组显著增加(P<0.05),Bax显著减少(P<0.05);雌激素联200IUγ-干扰素注射后,凋亡情况较单独注射雌激素有所改善,Bcl-2的表达较OVX组极显著增加(P<0.01),Bax极显著减少(P<0.01),两者与SHAM组差异均不显著(P>0.05)。50 IU/800 IU的γ-干扰素联合雌激素注射后凋亡情况与单独注射雌激素后差异不显著(P>0.05)。
以上结果表明,雌激素缺乏状态下,下丘脑和垂体中的细胞对凋亡的敏感性增强,而外源性的生理剂量的雌激素与适当剂量的γ-干扰素对下丘脑神经元和垂体在一定时期内有重要的保护作用,可以调节Bcl-2和Bax蛋白的表达和两者的比例。
Hypothalamic-Pituitary-gonad (HPG) axis is important to regulation mechanisms of neuroreproduction. The balance of a great variety cytokines, neurotransmitters and hormones and their receptors with complex coordination mechanisms is the basic to matain the balance of immune-nero-endocrine network. If the expressions of the matters above-mentioned lose balance, the structure and effunction of the tissue may vary, and then the cells may ageing and apoptosis. In order to study the regulation mechanisms of hormones and cytokines to this variation for estrogen deficiency, ovariectomizedrat (OVX) modelwas made and sensitive immunohistochemical SP method was used to study the expressions of the apoptosis associated protein Bcl-2 and Bax in this pathologic state and in the state of treated with 17-βestrogen and Interferon-γ( IFN-γ). The main rusults were as followed:
1. The expressions of Bcl-2 and Bax in hypothalami of rat: In 35 nucleuses in hypothalamus of the rats, include supraoptic nucleus, periventricular nucleus, arcuatus nucleus, hypothalamic ventromedialis nucleus and medial mamillary nucleus, et al, Bcl-2 and Bax immunohistochemical positive substances were located in these nucleuses and main located in cellular membrane, cytoplasm and caryotheca. After ovariectomized, relative expresions of Bcl-2 positive productions(μ2) tapered with the cours and to be the minumum after 4 weeks, concurrently, theμ2 of Bax increased, and to be the maximum after 4 weeks, which had significant difference with the SHAM group(P<0.05). After treated with 17-βestrogen, theμ2 of Bcl-2 increased and had remarkable difference with OVX group(P<0.05), but theμ2 of Bax had different in different nucleus, it decreased significantly in supraoptic nucleus, arcuate nucleus,et al, which had remarkable difference with OVX group(P<0.05); and it had no significant variation in periventricular nucleus, hypothalamic nucleus,et al. After treated with 17-βestrogen and 200 IU IFN-γtogether, theμ2 of Bcl-2 had very significant difference with OVX group(P<0.01), and had significant difference with OVX+E2 group(P<0.05),but had no remarkble difference with SHAM group(P>0.05). The total tendency of theμ2 of Bax was the to OVX+E2 group, it also had very significant difference with OVX group(P<0.01) in the nucleus which theμ2 of Bax decreased. Compared with OVX +E2 group, theμ2 of Bcl-2 and Bax of the grouop treated with 17-βestrogen and 50 IU/800 IU IFN-γtogether had no significant difference (P>0.05).
2. The expressions of Bcl-2 and Bax in pituitary of rat: Bcl-2 and Bax immunohistochemical positive substances were located in both and neurohypophysis in cellular membrane, cytoplasm.The number of Bcl-2 and Bax positive products in neurohypophysis is obviously less than that in adenohypophysis . After ovariectomized, relative expresions of Bcl-2 positive productions(μ2) tapered with the cours and to be the minumum after 4 weeks, concurrently, theμ2 of Bax increased, and to be the maximum after 4 weeks, which had significant difference with the SHAM group(P<0.05). After treated with 17-βestrogen, theμ2 of Bcl-2 increased and had remarkable difference with OVX group(P<0.05), and theμ2 of Bax decreased and had significant difference with OVX group(P<0.05) too. After treated with 17-βestrogen and 200 IU IFN-γtogether, theμ2 of Bcl-2 had very significant difference with OVX group(P<0.01), and had significant difference with OVX+E2 group(P<0.05),but had no remarkble difference with SHAM group(P>0.05). The total tendency of theμ2 of Bax was the to OVX+E2 group, it also had very significant difference with OVX group(P<0.01) in the nucleus which theμ2 of Bax decreased. Compared with OVX +E2 group, theμ2 of Bcl-2 and Bax of the grouop treated with 17-βestrogen and 50 IU/800 IU IFN-γtogether had no significant difference (P>0.05).
The result aboved indicate that the cells of hypothalami and pituitary were more sensitive to the apoptosis in the state of lacking estrogen. Ectogenic physiological dose estrogen and proper does IFN-γhave important protected effect on the hypothalami neuron and pituitary in a certain period, which can regulating the express and the ratio of the contents of Bcl-2 and Bax.
引文
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