BMP-4及其拮抗剂对PRL腺瘤细胞分泌、增殖和凋亡的影响

Effects of BMP-4 and its antagonism Noggin on secreting prolatin,and cell proliferation and apoptosis in human prolactinomas

目的研究骨形态生成蛋白4(BMP-4)及其拮抗剂Noggin对PRL腺瘤细胞分泌、增殖和凋亡的影响。方法用不同浓度的BMP-4和Noggin对原代培养的PRL腺瘤细胞进行干预,观察不同时间的细胞形态,测量PRL的浓度。以免疫细胞化学、Western blot及RT-PCR检测BMP-4蛋白和BMP-4mRNA在PRL腺瘤细胞的表达,甲基噻唑基四唑(MTT)法计算Noggin对PRL腺瘤细胞生长的半最大抑制浓度(IC50)值,透射电镜,流式细胞仪观察检测Noggin处理后的细胞形态和周期改变,计算凋亡率。结果 5ng/ml的BMP-4培养基可促进PRL腺瘤细胞分泌,最大效应浓度为20ng/m·l MTT法测定IC50值为18μg/m·l 加入Noggin后;免疫细胞化学,Western blot显示,与对照组相比,作用后24hPRL腺瘤细胞表达BMP-4无明显差异,但作用48h和72h的表达显著降低(P<0.05),RT-PCR示Noggin作用24h,48h和72h后BMP-4mRNA基因扩增条带亮度逐渐减弱,电镜观察显示。Noggin作用24h、48h后PRL腺瘤细胞形态无明显改变,72h时可见典型的凋亡细胞。结论 BMP-4在一定浓度范围内呈剂量依赖性地促进PRL腺瘤细胞生长,增殖与分泌。

Objective To explore the effects of bone morphogenetic protein-4（BMP-4） and its antagonism Noggin on secreting prolactin（PRL）,and cell proliferation and apoptosis in human prolactinomas.Methods The prolactinoma cells after the primary culture were treated by the different concentration of BMP-4 and Noggin.The cell configuration was observed and PRL concentration in the culture medium was measured by radioimmunoassay at defferemt time after the treatment.The expressions of BMP-4 protein and BMP-4 mRNA were determined respectively by the cell immunocytochemical technique,Western blot and RT-PCR.The half maximal inhibitory concentration（IC 50） of Noggin for prolactinoma cell growth was determined by methyl thiazolyl tetrazolium（MTT） method.The cell apoptosis was determined by flow cytometry and electron microscope.Results The secreting of PRL by prolactinoma cells could be accelerated in the medium containing 5 ng BMP-4 per milliliter and the maximal effect concentration of BMP-4 was 20 ng/ml.IC 50 of Noggin was 18 μg/ml.The apoptosis of prolactinoma cells induced by Noggin was proven by the cytometry and electronmicroscope examinations.Conclusion The promotive effect of BMP-4 on prolactinoma cell secreting and proliferation is dose-dependent within a certain concentration.Noggin can inhibit prolactinoma cells proliferation and induce apoptosis of prolactinoma cells in a time-dependent manner.