雌二醇对大鼠生长板软骨细胞C型利钠肽及胰岛素样生长因子1表达的影响

Effect of estrodial on C type natriuretic peptide and insulin like growth factor 1 expression in rat growth plate chondrocytes

目的·探讨不同浓度的17β雌二醇(17βE2)对大鼠生长板软骨细胞C型利钠肽(CNP)、利钠肽受体B(NPR-B)及胰岛素样生长因子1(IGF1)的调控作用,及其对软骨细胞增殖的影响。方法·Wistar大鼠8只,于出生后14 d处死,分离胫骨上段骺软骨板,取得软骨细胞并培养48 h后,于培养液中加入17βE2,并使其终浓度分别为10^(-4)、10^(-6)、10^(-8)、10^(-10)和10^(-12) mol/L,同时设空白对照组。继续培养48 h。之后利用CCK8法测定软骨细胞增殖能力,应用ELISA法测定培养液中CNP和IGF1的浓度,并利用实时定量PCR检测软骨细胞CNP、NPR-B及IGF1 mRNA的相对水平。结果·不同浓度的17βE2对生长板软骨细胞增殖有显著影响。当17βE2的浓度为10^(-8) mol/L时,其对软骨细胞的促增殖效应最强;当浓度进一步增至10^(-4) mol/L时,则抑制其增殖。随着17βE2浓度的增加,细胞培养液中CNP的浓度以及软骨细胞中CNP mRNA相对水平均出现明显差异,以10^(-8) mol/L组CNP的浓度和mRNA水平最高,10^(-4) mol/L组为最低。IGF1同样在10^(-8) mol/L组的浓度和mRNA表达水平达到最高,但最低值分别出现在10^(-10) mol/L组和10^(-12) mol/L组。软骨细胞增殖水平与CNP的mRNA及蛋白浓度均呈正相关(均P=0.000),但与IGF1的mRNA及蛋白浓度水平无明显相关性(均P>0.05)。结论·17βE2对大鼠生长板软骨细胞增殖调控具有剂量-效应关系,呈现出高浓度抑制、一定浓度范围内(10^(-10)~10^(-8) mol/L)促进的作用;该作用与其调节生长板软骨细胞表达CNP水平呈正相关。

Objective · To observe effect of 17β estrodial （17β E2） with different concentrations on C type natriuretic peptide （CNP）, insulin like growth factor 1 （IGF1）, and natriuretic peptide receptor B （NPR-B） expression and proliferation of growth plate chondrocytes of rats in vitro. Methods · Eight Wistar rats were sacrificed and their epiphyseal cartilages of the upper tibias were separated to obtain chondrocytes on the 14th day after birth. Then chondrocytes were cultured with 17β E2 in different concentrations （10-4、10-6、10-8、10-10 and 10-12 mol/L） for 48 h, while control group was cultured without 17β E2. CCK8 method, ELISA and qRT-PCR were used to analyze the proliferation of chondrocytes, the levels of CNP and IGF1 in culture medium and mRNA levels of CNP, NPR-B and IGF1, respectively. Results · 17β E2 in different concentrations affected the proliferation of growth plate chondrocytes significantly. When the concentration of 17β E2 was 10-8 mol/L, it had the strongest effect on the cell proliferation. When the concentration increased to 10-4 mol/L, the proliferation of chondrocytes was inhibited. With the increasement of 17β E2 concentration, the levels of CNP in the culture medium and the mRNA levels of CNP in the chondrocytes were significantly different. The highest levels of CNP protein and mRNA both appeared in 10-8 mol/L group, while the lowest levels both appeared in 10-4 mol/L group. IGF1 and its mRNA also reached the highest levels in 10-8 mol/L group, but the lowest concentration and mRNA level were in 10-10 mol/L group and 10-12 mol/L, respectively. Both CNP mRNA and protein levels were positive correlated with the proliferation of chondrocytes （P=0.000）. Nevertheless, there was no significant correlation between the proliferation of chondrocytes and IGF1 mRNA or protein levels （P〉0.05）. Conclusion · 17β E2 modulates proliferation of rat growth plate chondrocytes in a dose-effect manner. It enhances proliferation at relatively low concentrations （10-10-10-8 mol/L） and inhibits proliferation at high concentration. This effect is positively related to CNP expression in chondrocytes.