维生素D_3的雌性性腺毒性及其机制的研究
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摘要
维生素D_3是一种脂溶性物质,主要储存在脂肪组织中,1,25—二羟维生素D_3是其最主要的活性形式。由于维生素D_3具有重要的生理作用,在临床上成为重要辅助用药和人们日常生活中的重要补充的营养素,其对健康,尤其对未成年儿童、老年妇女生殖健康问题更为突出。本项目研究以维生素D_3的生殖内分泌干扰作用为核心,从维生素D_3的雌激素样活性作用和维生素D_3的雌性性腺毒性、维生素D_3的雌性性腺毒性机制等方面展开研究,从而较系统、全面的研究维生素D_3的生殖内分泌干扰作用及其机制,为维生素D_3的危险性评价及防治提供科学依据,为维生素D_3的临床应用与合理补充提供指导。
一、维生素D_3的子宫增重效应及其机制
目的:通过未成年雌性小鼠的子宫营养试验,探讨维生素D_3的子宫增重效应及其分子机制
方法:清洁级昆明种小鼠66只,21天龄,按体重随机分为6组,每组11只,暴露剂量分别为(0.00926,0.0556,0.33,2.0mg/kg VitD_3),阳性对照组为30μg/kg雌二醇,溶剂对照为橄榄油。每天染毒一次,连续三天。观察小鼠子宫重量、脏器系数、子宫内膜腔上皮厚度、固有层腺体数量及PCNA、ER表达。
结果:
1.与阴性对照组比较,雌二醇组子宫湿重及脏器系数显著性升高(p＜0.05),维生素D_3的各剂量组子宫湿重较对照组无显著性差异(p＞0.05),但是有升高的趋势;0.0556mg/Kg、0.33mg/Kg和2.0mg/Kg VitD_3剂量组的子宫脏器系数较对照组显著性升高(p＜0.05);雌二醇组子宫湿重及脏器系数显著高于维生素D_3的各剂量组(p＜0.01);
2.子宫内膜腔上皮厚度:雌二醇组显著高于对照组及维生素D_3的各剂量组;子宫内膜腺体数量:维生素D_3的各剂量组随染毒剂量增高而增高,0.33mg/Kg和2.0 mg/Kg剂量组及阳性对照组均高于对照组,阳性对照组显著高于维生素D_3的各剂量组(p＜0.05或p＜0.01);
3.PCNA阳性率:VitD_3各剂量和雌二醇组均显著高于对照组,雌二醇组高于VitD_3各剂量组;ER阳性率:对照组低于0.00926 mg/Kg、0.0556 mg/Kg、0.33 mg/Kg剂量组,高于2.0 mg/Kg剂量组;雌二醇组显著高于对照组及0.0556 mg/Kg、0.33 mg/Kg、2.0 mg/Kg剂量组。
二、维生素D_3的雌性性腺毒性的研究
目的:研究亚慢性暴露VitD_3对雌性大鼠动情周期、血清性激素水平及子宫、卵巢组织病理学影响,探讨VitD_3的雌性性腺毒性作用。
方法:健康未成年雌性Wistar大鼠,清洁级,60只,体重80±5g,按体重随机分为5组,染毒剂量分别为0、0.0167、0.1、0.6、3.6mg/kg,溶剂为橄榄油。每天腹腔注射染毒1次,连续35天。实验期末,待动物进入动情期处死动物。观察指标包括体重、动情周期、子宫湿重及脏器系数、卵巢湿重及脏器系数、各级别卵泡数目、子宫内膜细胞的组织学观察、血清卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E_2)和孕酮(P_4)水平。
结果:
1.染毒结束时最高剂量的体重显著低于其它各组,与对照组比较差别有统计学意义(p＜0.01);
2.3.6mg/kg剂量组周期时间延长,其动情间期明显长于对照组,差别有统计学意义(p＜0.05);
3.3.6mg/kg染毒组的子宫湿重及脏器系数显著低于其余各组(p＜0.01),其卵巢湿重较对照组轻,差别有统计学意义(p＜0.01),但卵巢脏器系数与对照组比较差别均无统计学意义(p＞0.05)。
4.各剂量组闭锁卵泡数目与对照组相比,呈现上升的趋势,3.6mg/kg剂量组显著高于对照组,差别有统计学意义(p＜0.05)
5.3.6mg/kg剂量组血清FSH水平低于对照组(p＜0.05),各组LH水平和对照组相比,差别无统计学意义。各剂量组血清E_2水平和对照组相比,呈现上升的趋势,其中3.6mg/kg剂量组血清E_2显著高于对照组(p＜0.05);各剂量组血清P_4与对照组比较差别均无统计学意义(p＞0.05),但有下降趋势。
三、维生素D_3的雌性性腺毒性的机制(一)(对卵巢性激素分泌及卵巢、子宫ER表达的影响)
目的:研究VitD_3对卵巢分泌性激素和卵巢、子宫ER表达的影响,探讨VitD_3的雌性性腺毒性机制
方法:
1.采用正交表L16(4~5)设计正交实验,研究VitD_3(0、10~(-10)、10~(-7)、10~(-4)mol/L)和动情周期(动情前期、动情期、动情后期、动情间期)对卵巢切碎组织E_2和P_4的影响。
2.采用正交表L9(3~4)设计正交实验,研究VitD_3(0、10~(-7)、10~(-4)mol/L)和时间(4、8、24h)对卵巢颗粒细胞分泌E_2和P_4的影响。
3.健康未成年雌性Wistar大鼠,清洁级,50只,体重80±5g,实验动物模型同前。实验期末,待动物进入动情期处死动物。观察卵巢切碎组织体外分泌E_2和P_4水平、卵巢及子宫内膜ER表达。
结果:
1.体外染VitD_3可影响卵巢切碎组织体外分泌E_2(p＜0.01),但不会影响分泌P_4(p＞0.05),卵巢切碎组织体外分泌E_2和P_4与所处的动情周期有关(p＜0.01或p＜0.05);
2.不同浓度的VitD_3可影响颗粒细胞分泌E_2(p＜0.05),但不会影响颗粒细胞分泌P_4(p＞0.05);颗粒细胞分泌E_2与孵育时间无关(p＞0.05),颗粒细胞分泌P4与孵育时间有关(p＜0.05)。
3.3.6mg/kg VitD_3体内染毒:卵巢切碎组织P_4和E_2分泌明显降低,与对照组比较,差别有统计学意义(p＜0.05)
4.3.6mg/Kg剂量组子宫内膜下层ER表达阳性率显著高于对照组(p＜0.05);0.0167mg/kg、3.6mg/kg剂量组卵巢皮质ER表达阳性率低于对照组,而0.1mg/kg剂量组卵巢皮质ER表达阳性率高于对照组,差别均有统计学意义(p＜0.01)。
四、维生素D_3的雌性性腺毒性的机制(二)(诱导大鼠卵巢细胞凋亡及其可能机制)
目的:研究VitD_3对卵巢细胞凋亡的影响及其可能的机制,进一步探讨VitD_3的雌性性腺毒性机制
方法:
1.完体动物实验模型同前;采用透射电镜观察卵巢组织超微结构,Tunel法检测卵巢细胞凋亡,比色法检测血清及卵巢组织钙离子水平,Realtime RT-PCR法检测ERα、ERβ、Caspase-3、Caspase-8mRNA的表达;
2.卵巢颗粒细胞(GC)培养同前;采用FCM测定GC凋亡
结果:
1.3.6 mg/kg剂量组闭锁卵泡及黄体颗粒细胞凋亡率显著高于对照组(p＜0.01),而生长卵泡、成熟卵泡各剂量组比较,差别均无统计学意义。
2.对照组及各剂量组皆可见凋亡的细胞,与对照组组比较,3.6mg/kg剂量组异染色质增多,成块状,染色质边集,可见明显的凋亡小体,线粒体出现水肿,基质变稀,电子密度明显下降,甚至空泡状;
3.3.6mg/kg剂量组卵巢组织钙离子浓度高于对照组(p＜0.05)。
4.各染毒剂量组ERα、ERβmRNA表达及ERα/ERβmRNA比值与对照组比较差别无统计学意义(p＞0.05)。
5.0.0167 mg/kg剂量组Caspase3mRNA表达显著低于对照组(p＜0.05),随着染毒剂量增高,各染毒剂量组Caspase3、Caspase8mRNA表达有增加的趋势,而3.6 mg/kg剂量组Caspase3、Caspase8mRNA表达较对照组增高,其中,Caspase8mRNA表达显著高于对照组比较(p＜0.01)。
6.体外试验结果显示,10~(-7)和10~(-4) mol/L的维生素D_3可以增加GC的凋亡率,且随着培养时间的延长,凋亡率有增加的趋势。
结论:在本实验条件下,可得出如下结论:
1.维生素D_3具有子宫营养作用,表现为子宫细胞增殖及腺体数量增多,但其作用弱于雌二醇;
2.维生素D_3(亚慢性暴露)具有明显的雌性性腺毒性,可表现为动情间期延长,闭锁卵泡增多,血清性激素改变。
3.卵巢是维生素D_3的重要靶器官,维生素D_3可明显抑制卵巢性激素的分泌,还可以影响卵巢及子宫ER的表达,这可能是其雌性腺毒性的重要毒理学基础和可能毒性机制之一;
4.维生素D_3可诱导卵巢细胞凋亡:主要是通过导致细胞钙稳态失调;激活caspase家族途径,但具体机制有待进一步探讨。
Vitamin D3 is one fat soluble material,mainly stores up in the fatty tissue, 1,25- two hydroxy Vitamin D3 is its most main active form.Because Vitamin D_3 has the important physiological action,in clinical it becomes an important assistant medication and in the people daily life it becomes an important supplement nutrient,but to health,specially to the underage child and the old age woman,vitD_3 may reproduce the healthy question especially.This project takes the Vitamin D_3 reproduction and internal secretion interference effect as the core, including the Vitamin D3 estrogenic effects and the Vitamin D_3 female gonadal toxicity and its related mechanism,this systematic,comprehensive research focuses on Vitamin D3 reproduction and internal secretion interference effect and its related mechanism,then provides the scientific basis for the risk appraisal and preventing and controlling of the toxicities on Vitamin D3,and provides the instruction for the Vitamin D_3 clinical practice and the reasonable supplement.
Part 1 Study on the uterotrophic effect and its mechanism of VitD_3
Objectives:This experiment was designed to study the uterotrophic effect on young mice and its related mechanism of Vitamin D_3
Methods:66 mice,21 days of age,were randomly divided into 6 groups,exposure doses were respectively 0,0.00926,0.0556,0.33,2.0 mg / kg VitD_3,positive control group for 30μg/kg estradiol.The mice were exposed to VitD_3 once a day, for three consecutive days.Mice uterine weight,uterine organ coefficient, endometrial epithelial thickness,glands and PCNA,ER expression in endometrial lamina were observed.
Results:
1.The uterine wet weight and organ coefficient of estradiol group were significantly higher than those of the negative control group(p＜0.05),the uterine wet weight of all the vitamin D_3 groups were not significantly different from that of the negative control group(p＞0.05),but it had an increasing trends;the uterus coefficient of O.0556mg/Kg,0.33mg/Kg and 2.0mg/Kg doses of VitD_3 groups were significantly higher than the negative control group(p＜0.05);the uterine weight and organ coefficient of Estradiol group was significantly higher than those of all the vitamin D_3 groups(p＜0.01);
2.The endometrium epithelial thickness:the estradiol group was significantly higher than that of the control group and all the vitamin D3 dose groups;the number of endometrial glands:it increased with the increasing VitD_3 doses,that in 0.33 mg / Kg、2.0 mg / Kg doses of VitD3 groups and the positive control group were higher than the control group,the positive control group was significantly higher than that of all the vitamin D_3 dose group(p＜0.05 or p＜0.01);
3.The PCNA positive percentage of endometrial:that of all the VitD_3 groups and the estradiol group were significantly higher than that of the control group,and that of the estradiol group was higher than that of all the VitD_3 groups;ER-positive rate:that of the control group was lower than that of 0.00926 mg / Kg,0.0556 mg / Kg,0.33 mg/Kg groups,and higher than that of 2.0 mg / Kg dose group;that of the estradiol group was significantly higher than those of the control and 0.0556 mg / Kg,0.33 mg / Kg,2.0 mg / Kg dose groups.
PartⅡ:The study of female gonadal toxieities of VitD_3
Objectives:This experiment was designed to study on the effects of estrous cycle,serum hormone levels,uterine and ovarian pathology in female rats sub-chronicly exposure to VitD_3,and to explore the female gonadal toxicities of VitD_3.
Methods:60 healthy female Wistar rats,weight 80±5g,were randomly divided into 5 groups,the doeses were respectively 0,0.0167,0.1,0.6,3.6 mg / kg,and the solvent was olive oil.The rats were exposed to VitD_3 once a day and for 35 consecutive days.The exposure ended when the animals were in the estrus.The indicators such as weight,estrus stage,uterine wet weight and organ coefficient, ovarian wet weight and organ coefficient,the number of follicles,endometrial histology,serum follicle stimulating hormone(FSH),luteinizing hormone(LH), estradiol(E_2) and progesterone(P_4) levels were observed.
Results:
1.In the end of the experiment,the body weight of the rats exposed to the highest dose was significantly lower than other groups,and significantly lower than the control group(p＜0.01);
2.The period of estrus stage in rats of 3.6mg/kg vitD_3 was longer than that of the control group,and its metestrus period was significantly prolonged(p＜0.05);
3.The wet weight of the uterus and the its coefficient in 3.6mg/kg group were significantly lower than the other groups(p＜0.01),and the ovarian wet weight was lighter than the control group,the difference has statistical significance(p＜0.01), but ovarian organ coefficient had no statistically significant difference compared with the control group(p＞0.05).
4.Compared with the control group,the number of atretic follicles in all VitD_3 doses group showed an upward trend,and the number of atretic follicles in 3.6mg/kg dose group was significantly higher than that of the control group(p＜0.05).
5.The serum FSH level in 3.6mg/kg dose group was lower than the control group (p＜0.05),the level of LH in each group had no statistically significance compared with the control group,the levels of serum E_2 in all vitD_3 doses showed an upward trend,and the E_2 level of 3.6mg/kg dose group was significantly higher than that of control group(p＜0.05);there were no statistical difference of the serum P_4 levels in all doses group compared with the control group(p＞0.05),but there was a downward trend in serum P_4 level.
PartⅢStudy of the female gonadal toxicities mechanism(the hormone secretion of ovarian and ER expression in ovarian and uterine) of vitamin D3
Objectives:This experiment was designed to study the effects on ovarian sex hormone secretion,the ER expression in both ovarian and uterine,and to explore part of the mechanisms of female gonadal toxicities exposured to VitD_3
Methods:
1.Using orthogonal experimental design with orthogonal layout of L16(4~5),effects of VitD_3(0、10~(-10)、10~(-7)、10~(-4)mol/L )and estrous cycle(proestrous,estrous, postestrous,dioestrous) on steroidogenesis in the ovary were studied.
2.Using orthogonal experimental design with orthogonal layout of L9(3~4),effects of VitD_3(0、10~(-7)、10~(-4)mol/Land incubation time(4、8、24h) on steroidogenesis in granulosa cells were studied.
3.50 healthy female wistar rats,weight 80±5g,were randomly divided into 5 groups,the doeses were respectively 0,0.0167,0.1,0.6,3.6 mg / kg,and the solvent was olive oil.The rats were exposed to VitD_3 once a day and for 35 consecutive days.The exposure ended when the animals were in the estrus.Steroidogenesis in ovary in vitro,expression of ER in both ovary and uterus were observed.
Results:
1.In whole ovary culture,effects of VitD_3 on estradiol secretion were significant (p＜0.05),and effects of VitD_3 on progesterone secretion were not significant (p＞0.05).Effects of estrous cycle on estradiol and progesterone secretion in whole ovary culture were significant(p＜0.001 or p＜0.05);
2.Effects of VitD_3 on estradiol secretion in granulosa cells were significant (p＜0.01),and effects of VitD_3 on progesterone secretion were not significant (p＞0.05).Effects of incubation time on estradiol secretion in granulosa cells were not significant(p＞0.05),and effects of incubation time on progesterone secretion were significant(p＜0.05);
3.Estradiol and progesterone concentrations secreted in vitro by the ovaries of rats treated with 3.6mg/kg VitD_3 were significantly decreased(p＜0.05);
4.The expression of endometrial ER in 3.6mg/Kg group was significantly higher than that of the control group(p＜0.05);the expression of ER in ovarian cortex in 0.0167mg/kg,3.6mg/kg groups were lower than that of the control group,and that of 0.1mg / kg group was significantly higher than that of the control group(p＜0.01).
PartⅣStudy of the apoptosis in rat ovary and its possible mechanism in female gonadal toxicities of vitamin D_3
Objectives:This experiment was designed to study on ovarian cell apoptosis induced by VitD_3 s and its possible mechanism,and to explore the further mechanisms of female gonadal toxicities exposed to VitD_3
Methods:
1.50 healthy female wistar rats,weight 80±5g,were randomly divided into 5 groups,the doeses were respectively 0,0.0167,0.1,0.6,3.6 mg/ kg,and the solvent was olive oil.The rats were exposed to VitD_3 once a day and for 35 consecutive days.The exposure ended when the animals were in the estrus.The indicators such as the ultrastructure of ovarian tissue use of transmission electron microscopy, ovarian cell apoptosis by Tunel assay,the serum and ovarian tissue levels of calcium ions,ERα,ERβ,Caspase3,Caspase8 mRNA expression were observed.
2.Using orthogonal experimental design with orthogonal layout of L9(3~4),effects of VitD_3(0、10~(-7)、10~(-4)mol/L)and incubation time(4、8、24h) on apoptosis in granulosa cells were studied.The apoptosis of GC measured by FCM
Results:
1.The apoptosis in atretic follicles and luteal granulosa cell of 3.6 mg / kg dose group was significantly higher(p＜0.01),while the apoptosis in the growing follicles,mature follicles of all the VitD_3 groups,the differences were not statistically significant.
2.Apoptotic cells were visible in all groups;compared with the control group,an increased heterochromatin,chromatin margination,apoptotic bodies, mitochondrial edema,matrix-thinning,a decreased electron density,and even shaped vacuole were observed in 3.6mg/kg dose group;
3.The calcium concentration of ovarian tissue in 3.6mg/kg group was higer than that of the control group(p＜0.05).
4.The ERα,ERβmRNA expression and ERα/ ERβmRNA ratio in all vitD_3 groups had no significant difference compared with the control group(p＞0.05).
5.The expression of Caspase3mRNA of 0.0167 mg / kg dose group was significantly lower than that of the control group(p＜0.05),with the increasing VitD_3 dose,Caspase3,Caspase8mRNA expression in each VitD_3 group showed an increasing trend,and the Caspase3,Caspase8 mRNA expression of 3.6 mg / kg dose group were higher than the control group,but only Caspase8 mRNA expression was significantly higher than that of the control group(p＜0.01).
6.The results showed that in vitro,10~(-7) and 10~(-4) mol / L of vitamin D3 increased the apoptosis rate of GC,and with the culture time extended,the apoptosis rate of GC increased.
Conclusions:In this experimental condition,the following conclusions can be drawn:
1.Vitamin D3 has the uterotrophic effect in young mice,showing the effect of uterine cell proliferation and the number of glands increasing,but its effects are weaker than that of estradiol;
2.Vitamin D_3(sub-chronic exposure) has the obvious female gonadal toxicities: showing the effects of the prolonged metestrus period,the increased atretic follicles number,the changed serum sex hormones.
3.Ovarian is an important target organ of vitamin D_3,and vitamin D_3 can inhibit the sex hormones secretion of ovarian,and can affect the ER expression in both ovarian and uterine,which is the essential base of toxicological study and probably one of the mechanisms in its gonadal toxicities of uterotrophic effect vitamin D_3;
4.Vitamin D_3 can induce apoptosis in ovarian cells:The main mechenisms were calcium homeostasis disorders;activated caspases family,but the detailed mechanisms need to be further explored.
一、维生素D_3的子宫增重效应及其机制
目的:通过未成年雌性小鼠的子宫营养试验,探讨维生素D_3的子宫增重效应及其分子机制
方法:清洁级昆明种小鼠66只,21天龄,按体重随机分为6组,每组11只,暴露剂量分别为(0.00926,0.0556,0.33,2.0mg/kg VitD_3),阳性对照组为30μg/kg雌二醇,溶剂对照为橄榄油。每天染毒一次,连续三天。观察小鼠子宫重量、脏器系数、子宫内膜腔上皮厚度、固有层腺体数量及PCNA、ER表达。
结果:
1.与阴性对照组比较,雌二醇组子宫湿重及脏器系数显著性升高(p＜0.05),维生素D_3的各剂量组子宫湿重较对照组无显著性差异(p＞0.05),但是有升高的趋势;0.0556mg/Kg、0.33mg/Kg和2.0mg/Kg VitD_3剂量组的子宫脏器系数较对照组显著性升高(p＜0.05);雌二醇组子宫湿重及脏器系数显著高于维生素D_3的各剂量组(p＜0.01);
2.子宫内膜腔上皮厚度:雌二醇组显著高于对照组及维生素D_3的各剂量组;子宫内膜腺体数量:维生素D_3的各剂量组随染毒剂量增高而增高,0.33mg/Kg和2.0 mg/Kg剂量组及阳性对照组均高于对照组,阳性对照组显著高于维生素D_3的各剂量组(p＜0.05或p＜0.01);
3.PCNA阳性率:VitD_3各剂量和雌二醇组均显著高于对照组,雌二醇组高于VitD_3各剂量组;ER阳性率:对照组低于0.00926 mg/Kg、0.0556 mg/Kg、0.33 mg/Kg剂量组,高于2.0 mg/Kg剂量组;雌二醇组显著高于对照组及0.0556 mg/Kg、0.33 mg/Kg、2.0 mg/Kg剂量组。
二、维生素D_3的雌性性腺毒性的研究
目的:研究亚慢性暴露VitD_3对雌性大鼠动情周期、血清性激素水平及子宫、卵巢组织病理学影响,探讨VitD_3的雌性性腺毒性作用。
方法:健康未成年雌性Wistar大鼠,清洁级,60只,体重80±5g,按体重随机分为5组,染毒剂量分别为0、0.0167、0.1、0.6、3.6mg/kg,溶剂为橄榄油。每天腹腔注射染毒1次,连续35天。实验期末,待动物进入动情期处死动物。观察指标包括体重、动情周期、子宫湿重及脏器系数、卵巢湿重及脏器系数、各级别卵泡数目、子宫内膜细胞的组织学观察、血清卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E_2)和孕酮(P_4)水平。
结果:
1.染毒结束时最高剂量的体重显著低于其它各组,与对照组比较差别有统计学意义(p＜0.01);
2.3.6mg/kg剂量组周期时间延长,其动情间期明显长于对照组,差别有统计学意义(p＜0.05);
3.3.6mg/kg染毒组的子宫湿重及脏器系数显著低于其余各组(p＜0.01),其卵巢湿重较对照组轻,差别有统计学意义(p＜0.01),但卵巢脏器系数与对照组比较差别均无统计学意义(p＞0.05)。
4.各剂量组闭锁卵泡数目与对照组相比,呈现上升的趋势,3.6mg/kg剂量组显著高于对照组,差别有统计学意义(p＜0.05)
5.3.6mg/kg剂量组血清FSH水平低于对照组(p＜0.05),各组LH水平和对照组相比,差别无统计学意义。各剂量组血清E_2水平和对照组相比,呈现上升的趋势,其中3.6mg/kg剂量组血清E_2显著高于对照组(p＜0.05);各剂量组血清P_4与对照组比较差别均无统计学意义(p＞0.05),但有下降趋势。
三、维生素D_3的雌性性腺毒性的机制(一)(对卵巢性激素分泌及卵巢、子宫ER表达的影响)
目的:研究VitD_3对卵巢分泌性激素和卵巢、子宫ER表达的影响,探讨VitD_3的雌性性腺毒性机制
方法:
1.采用正交表L16(4~5)设计正交实验,研究VitD_3(0、10~(-10)、10~(-7)、10~(-4)mol/L)和动情周期(动情前期、动情期、动情后期、动情间期)对卵巢切碎组织E_2和P_4的影响。
2.采用正交表L9(3~4)设计正交实验,研究VitD_3(0、10~(-7)、10~(-4)mol/L)和时间(4、8、24h)对卵巢颗粒细胞分泌E_2和P_4的影响。
3.健康未成年雌性Wistar大鼠,清洁级,50只,体重80±5g,实验动物模型同前。实验期末,待动物进入动情期处死动物。观察卵巢切碎组织体外分泌E_2和P_4水平、卵巢及子宫内膜ER表达。
结果:
1.体外染VitD_3可影响卵巢切碎组织体外分泌E_2(p＜0.01),但不会影响分泌P_4(p＞0.05),卵巢切碎组织体外分泌E_2和P_4与所处的动情周期有关(p＜0.01或p＜0.05);
2.不同浓度的VitD_3可影响颗粒细胞分泌E_2(p＜0.05),但不会影响颗粒细胞分泌P_4(p＞0.05);颗粒细胞分泌E_2与孵育时间无关(p＞0.05),颗粒细胞分泌P4与孵育时间有关(p＜0.05)。
3.3.6mg/kg VitD_3体内染毒:卵巢切碎组织P_4和E_2分泌明显降低,与对照组比较,差别有统计学意义(p＜0.05)
4.3.6mg/Kg剂量组子宫内膜下层ER表达阳性率显著高于对照组(p＜0.05);0.0167mg/kg、3.6mg/kg剂量组卵巢皮质ER表达阳性率低于对照组,而0.1mg/kg剂量组卵巢皮质ER表达阳性率高于对照组,差别均有统计学意义(p＜0.01)。
四、维生素D_3的雌性性腺毒性的机制(二)(诱导大鼠卵巢细胞凋亡及其可能机制)
目的:研究VitD_3对卵巢细胞凋亡的影响及其可能的机制,进一步探讨VitD_3的雌性性腺毒性机制
方法:
1.完体动物实验模型同前;采用透射电镜观察卵巢组织超微结构,Tunel法检测卵巢细胞凋亡,比色法检测血清及卵巢组织钙离子水平,Realtime RT-PCR法检测ERα、ERβ、Caspase-3、Caspase-8mRNA的表达;
2.卵巢颗粒细胞(GC)培养同前;采用FCM测定GC凋亡
结果:
1.3.6 mg/kg剂量组闭锁卵泡及黄体颗粒细胞凋亡率显著高于对照组(p＜0.01),而生长卵泡、成熟卵泡各剂量组比较,差别均无统计学意义。
2.对照组及各剂量组皆可见凋亡的细胞,与对照组组比较,3.6mg/kg剂量组异染色质增多,成块状,染色质边集,可见明显的凋亡小体,线粒体出现水肿,基质变稀,电子密度明显下降,甚至空泡状;
3.3.6mg/kg剂量组卵巢组织钙离子浓度高于对照组(p＜0.05)。
4.各染毒剂量组ERα、ERβmRNA表达及ERα/ERβmRNA比值与对照组比较差别无统计学意义(p＞0.05)。
5.0.0167 mg/kg剂量组Caspase3mRNA表达显著低于对照组(p＜0.05),随着染毒剂量增高,各染毒剂量组Caspase3、Caspase8mRNA表达有增加的趋势,而3.6 mg/kg剂量组Caspase3、Caspase8mRNA表达较对照组增高,其中,Caspase8mRNA表达显著高于对照组比较(p＜0.01)。
6.体外试验结果显示,10~(-7)和10~(-4) mol/L的维生素D_3可以增加GC的凋亡率,且随着培养时间的延长,凋亡率有增加的趋势。
结论:在本实验条件下,可得出如下结论:
1.维生素D_3具有子宫营养作用,表现为子宫细胞增殖及腺体数量增多,但其作用弱于雌二醇;
2.维生素D_3(亚慢性暴露)具有明显的雌性性腺毒性,可表现为动情间期延长,闭锁卵泡增多,血清性激素改变。
3.卵巢是维生素D_3的重要靶器官,维生素D_3可明显抑制卵巢性激素的分泌,还可以影响卵巢及子宫ER的表达,这可能是其雌性腺毒性的重要毒理学基础和可能毒性机制之一;
4.维生素D_3可诱导卵巢细胞凋亡:主要是通过导致细胞钙稳态失调;激活caspase家族途径,但具体机制有待进一步探讨。
Vitamin D3 is one fat soluble material,mainly stores up in the fatty tissue, 1,25- two hydroxy Vitamin D3 is its most main active form.Because Vitamin D_3 has the important physiological action,in clinical it becomes an important assistant medication and in the people daily life it becomes an important supplement nutrient,but to health,specially to the underage child and the old age woman,vitD_3 may reproduce the healthy question especially.This project takes the Vitamin D_3 reproduction and internal secretion interference effect as the core, including the Vitamin D3 estrogenic effects and the Vitamin D_3 female gonadal toxicity and its related mechanism,this systematic,comprehensive research focuses on Vitamin D3 reproduction and internal secretion interference effect and its related mechanism,then provides the scientific basis for the risk appraisal and preventing and controlling of the toxicities on Vitamin D3,and provides the instruction for the Vitamin D_3 clinical practice and the reasonable supplement.
Part 1 Study on the uterotrophic effect and its mechanism of VitD_3
Objectives:This experiment was designed to study the uterotrophic effect on young mice and its related mechanism of Vitamin D_3
Methods:66 mice,21 days of age,were randomly divided into 6 groups,exposure doses were respectively 0,0.00926,0.0556,0.33,2.0 mg / kg VitD_3,positive control group for 30μg/kg estradiol.The mice were exposed to VitD_3 once a day, for three consecutive days.Mice uterine weight,uterine organ coefficient, endometrial epithelial thickness,glands and PCNA,ER expression in endometrial lamina were observed.
Results:
1.The uterine wet weight and organ coefficient of estradiol group were significantly higher than those of the negative control group(p＜0.05),the uterine wet weight of all the vitamin D_3 groups were not significantly different from that of the negative control group(p＞0.05),but it had an increasing trends;the uterus coefficient of O.0556mg/Kg,0.33mg/Kg and 2.0mg/Kg doses of VitD_3 groups were significantly higher than the negative control group(p＜0.05);the uterine weight and organ coefficient of Estradiol group was significantly higher than those of all the vitamin D_3 groups(p＜0.01);
2.The endometrium epithelial thickness:the estradiol group was significantly higher than that of the control group and all the vitamin D3 dose groups;the number of endometrial glands:it increased with the increasing VitD_3 doses,that in 0.33 mg / Kg、2.0 mg / Kg doses of VitD3 groups and the positive control group were higher than the control group,the positive control group was significantly higher than that of all the vitamin D_3 dose group(p＜0.05 or p＜0.01);
3.The PCNA positive percentage of endometrial:that of all the VitD_3 groups and the estradiol group were significantly higher than that of the control group,and that of the estradiol group was higher than that of all the VitD_3 groups;ER-positive rate:that of the control group was lower than that of 0.00926 mg / Kg,0.0556 mg / Kg,0.33 mg/Kg groups,and higher than that of 2.0 mg / Kg dose group;that of the estradiol group was significantly higher than those of the control and 0.0556 mg / Kg,0.33 mg / Kg,2.0 mg / Kg dose groups.
PartⅡ:The study of female gonadal toxieities of VitD_3
Objectives:This experiment was designed to study on the effects of estrous cycle,serum hormone levels,uterine and ovarian pathology in female rats sub-chronicly exposure to VitD_3,and to explore the female gonadal toxicities of VitD_3.
Methods:60 healthy female Wistar rats,weight 80±5g,were randomly divided into 5 groups,the doeses were respectively 0,0.0167,0.1,0.6,3.6 mg / kg,and the solvent was olive oil.The rats were exposed to VitD_3 once a day and for 35 consecutive days.The exposure ended when the animals were in the estrus.The indicators such as weight,estrus stage,uterine wet weight and organ coefficient, ovarian wet weight and organ coefficient,the number of follicles,endometrial histology,serum follicle stimulating hormone(FSH),luteinizing hormone(LH), estradiol(E_2) and progesterone(P_4) levels were observed.
Results:
1.In the end of the experiment,the body weight of the rats exposed to the highest dose was significantly lower than other groups,and significantly lower than the control group(p＜0.01);
2.The period of estrus stage in rats of 3.6mg/kg vitD_3 was longer than that of the control group,and its metestrus period was significantly prolonged(p＜0.05);
3.The wet weight of the uterus and the its coefficient in 3.6mg/kg group were significantly lower than the other groups(p＜0.01),and the ovarian wet weight was lighter than the control group,the difference has statistical significance(p＜0.01), but ovarian organ coefficient had no statistically significant difference compared with the control group(p＞0.05).
4.Compared with the control group,the number of atretic follicles in all VitD_3 doses group showed an upward trend,and the number of atretic follicles in 3.6mg/kg dose group was significantly higher than that of the control group(p＜0.05).
5.The serum FSH level in 3.6mg/kg dose group was lower than the control group (p＜0.05),the level of LH in each group had no statistically significance compared with the control group,the levels of serum E_2 in all vitD_3 doses showed an upward trend,and the E_2 level of 3.6mg/kg dose group was significantly higher than that of control group(p＜0.05);there were no statistical difference of the serum P_4 levels in all doses group compared with the control group(p＞0.05),but there was a downward trend in serum P_4 level.
PartⅢStudy of the female gonadal toxicities mechanism(the hormone secretion of ovarian and ER expression in ovarian and uterine) of vitamin D3
Objectives:This experiment was designed to study the effects on ovarian sex hormone secretion,the ER expression in both ovarian and uterine,and to explore part of the mechanisms of female gonadal toxicities exposured to VitD_3
Methods:
1.Using orthogonal experimental design with orthogonal layout of L16(4~5),effects of VitD_3(0、10~(-10)、10~(-7)、10~(-4)mol/L )and estrous cycle(proestrous,estrous, postestrous,dioestrous) on steroidogenesis in the ovary were studied.
2.Using orthogonal experimental design with orthogonal layout of L9(3~4),effects of VitD_3(0、10~(-7)、10~(-4)mol/Land incubation time(4、8、24h) on steroidogenesis in granulosa cells were studied.
3.50 healthy female wistar rats,weight 80±5g,were randomly divided into 5 groups,the doeses were respectively 0,0.0167,0.1,0.6,3.6 mg / kg,and the solvent was olive oil.The rats were exposed to VitD_3 once a day and for 35 consecutive days.The exposure ended when the animals were in the estrus.Steroidogenesis in ovary in vitro,expression of ER in both ovary and uterus were observed.
Results:
1.In whole ovary culture,effects of VitD_3 on estradiol secretion were significant (p＜0.05),and effects of VitD_3 on progesterone secretion were not significant (p＞0.05).Effects of estrous cycle on estradiol and progesterone secretion in whole ovary culture were significant(p＜0.001 or p＜0.05);
2.Effects of VitD_3 on estradiol secretion in granulosa cells were significant (p＜0.01),and effects of VitD_3 on progesterone secretion were not significant (p＞0.05).Effects of incubation time on estradiol secretion in granulosa cells were not significant(p＞0.05),and effects of incubation time on progesterone secretion were significant(p＜0.05);
3.Estradiol and progesterone concentrations secreted in vitro by the ovaries of rats treated with 3.6mg/kg VitD_3 were significantly decreased(p＜0.05);
4.The expression of endometrial ER in 3.6mg/Kg group was significantly higher than that of the control group(p＜0.05);the expression of ER in ovarian cortex in 0.0167mg/kg,3.6mg/kg groups were lower than that of the control group,and that of 0.1mg / kg group was significantly higher than that of the control group(p＜0.01).
PartⅣStudy of the apoptosis in rat ovary and its possible mechanism in female gonadal toxicities of vitamin D_3
Objectives:This experiment was designed to study on ovarian cell apoptosis induced by VitD_3 s and its possible mechanism,and to explore the further mechanisms of female gonadal toxicities exposed to VitD_3
Methods:
1.50 healthy female wistar rats,weight 80±5g,were randomly divided into 5 groups,the doeses were respectively 0,0.0167,0.1,0.6,3.6 mg/ kg,and the solvent was olive oil.The rats were exposed to VitD_3 once a day and for 35 consecutive days.The exposure ended when the animals were in the estrus.The indicators such as the ultrastructure of ovarian tissue use of transmission electron microscopy, ovarian cell apoptosis by Tunel assay,the serum and ovarian tissue levels of calcium ions,ERα,ERβ,Caspase3,Caspase8 mRNA expression were observed.
2.Using orthogonal experimental design with orthogonal layout of L9(3~4),effects of VitD_3(0、10~(-7)、10~(-4)mol/L)and incubation time(4、8、24h) on apoptosis in granulosa cells were studied.The apoptosis of GC measured by FCM
Results:
1.The apoptosis in atretic follicles and luteal granulosa cell of 3.6 mg / kg dose group was significantly higher(p＜0.01),while the apoptosis in the growing follicles,mature follicles of all the VitD_3 groups,the differences were not statistically significant.
2.Apoptotic cells were visible in all groups;compared with the control group,an increased heterochromatin,chromatin margination,apoptotic bodies, mitochondrial edema,matrix-thinning,a decreased electron density,and even shaped vacuole were observed in 3.6mg/kg dose group;
3.The calcium concentration of ovarian tissue in 3.6mg/kg group was higer than that of the control group(p＜0.05).
4.The ERα,ERβmRNA expression and ERα/ ERβmRNA ratio in all vitD_3 groups had no significant difference compared with the control group(p＞0.05).
5.The expression of Caspase3mRNA of 0.0167 mg / kg dose group was significantly lower than that of the control group(p＜0.05),with the increasing VitD_3 dose,Caspase3,Caspase8mRNA expression in each VitD_3 group showed an increasing trend,and the Caspase3,Caspase8 mRNA expression of 3.6 mg / kg dose group were higher than the control group,but only Caspase8 mRNA expression was significantly higher than that of the control group(p＜0.01).
6.The results showed that in vitro,10~(-7) and 10~(-4) mol / L of vitamin D3 increased the apoptosis rate of GC,and with the culture time extended,the apoptosis rate of GC increased.
Conclusions:In this experimental condition,the following conclusions can be drawn:
1.Vitamin D3 has the uterotrophic effect in young mice,showing the effect of uterine cell proliferation and the number of glands increasing,but its effects are weaker than that of estradiol;
2.Vitamin D_3(sub-chronic exposure) has the obvious female gonadal toxicities: showing the effects of the prolonged metestrus period,the increased atretic follicles number,the changed serum sex hormones.
3.Ovarian is an important target organ of vitamin D_3,and vitamin D_3 can inhibit the sex hormones secretion of ovarian,and can affect the ER expression in both ovarian and uterine,which is the essential base of toxicological study and probably one of the mechanisms in its gonadal toxicities of uterotrophic effect vitamin D_3;
4.Vitamin D_3 can induce apoptosis in ovarian cells:The main mechenisms were calcium homeostasis disorders;activated caspases family,but the detailed mechanisms need to be further explored.
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[27]张森,谭建华,大鼠发情周期中主要生殖激素的变化,动物医学进展,200526(12):1-6
[28]曹泽毅主编.激素受体及其临床应用[M].北京:北京医科大学中国协和医科大学联合出版社,1993:191
[29]史小林主编.人类生殖学[M].北京:科学出版社,2002:164
[30]王心如主编,毒理学基础[M],第五版,北京:人民卫生出版社,2007:93
[31]Zhivotovsky B.Caspases:the enzymes of death[J].Essays Biochem,2003,39:25-40
[32]Porter DA,Harman R M,Cowan R G,et al.Relationship of Fasligand expression and atresia during bovine follicle development[J]Reproduction,2001,121(4):561-566.
[33]Nilsson S,Gustafsson JA.Estrogen receptor transriptio and transactivation:basic aspects of estrogen action.Breast Cancer Res,2000,2:360-366
[34]杨世华,余四九.生殖激素控制卵泡细胞凋亡的研究进展[J]动物医学进展,2002,23(2):1-5.
[35]李育,王明艳,马红,卵巢颗粒细胞凋亡的调控机制,临床和实验医学杂志,2007年4月第6卷第4期,160-161
[36]刘忠华,刘海兰,岳奎忠,等.性未成熟小母猪注射PMSG对卵泡闭锁及颗粒细胞凋亡的影响[J].中国兽医学报,2002,22(4):384-386.
[37]Anthony J Z,Lynda L I,Steven G B.Developmentalexpression of Ca~(2+)/Mg~(2+)dependent endonuclease activity in rat granulose and luteal cells[J].Endocrinology,1989,125(4):2 216-2 222
[1]Holick MF.Vitamin D:A millenium perspective.J Cell Biochem.2003,88:296-307.
[2]Villena-Heinsen C,Meyberg R,Axt-FliednerR,etal.Immuno-histochemical analysis of 1,25-dihydroxyitamin-D3-receptors,estrogen and progesterone receptors and Ki-67 in ovarian carcinoma [J].Anticancer Res,2002,22(4):2261-2 2 67.
[3]Johnson JA,Grande JP,Roche PC,etal.Immuno-histochemical detection and distribution of the 1,25-dihydroxyvitamin D3 receptor in rat productive tissues.[J].Histo-chem Cell Biol,1996,105(1):7-15.
[4]Vigano P,Lattuada D,Mangioni S,etal.Cycling and early pregnant endometrium as a site of regulated expression of the vitamin D system [J].J Mol Endocrinol,2006,36(3):415-424.
[5]Reichrath J,Rafi L,Muller SM,etal.Immuno-histochemcal analysis of 1,25-dihydroxyvitaminD3 receptor in cervical carcinoma [J].Histchem J,1998,30 (8):561-567
[6]Carlberg C,Polly P.Gene-regulation by vitaminD3[J].Crit Rev Eukaryot Gene Exp,1998,8:19～42.
[7]Rastinejad F,Perlmann T,Evans RM,etal.Structural determinants of nuclear receptor assembly on DNA direct repeats [J].Nature,1995,375:203～211.
[8]Strugnell SA,Deluca HF.The vitaminD receptor2 structure and transcriptional activation [J].Proc Soc Exp Biol Med,1997,215:223～228
[9]Nakajima S,Hsieh JC,Jurutka P,etal.Examination of the potential functional role of conserved cysteine residues in the hormone-binding domain of the human1,25-dihydroxyvitamin-D3 receptor 1 [J].J Biol Chem,1996,271:5143-5149.
[10]Beer TM,Myrthue A.Calcitriol in cancer treatment:from the lab to clinic [J].Mol Cancer Ther,2004,3(3):373-381.
[11]Belkacemi L,Zuegel U,Steinmeyer A,et al.Calbindin-2D 8k (CaBP28k) identification and regulation by 1,25-dihydroxyvitaminD3 in human chorio-carcinoma cell line JEG-3 [J].Mol Cell Endocrinol,2005,236(1-2):31-41
[12]Norman AW,Ishizuka S,Okamura WH.Ligands for the vitamin D encocrine system:different shapes function as agonists and antiagonists for genomic and rapid response receptors or as a ligand for the plasma vitamin D binding protein.[J].J Steroid Biochem Mol Biol,2001,76(1-5):49-59
[13]Corhatt ST,Hill O,Nangia AK.Vitamin D receptor found in human sperm[J].Urology,2006,68(6):1345-1349
[14]Shao A,Wood RJ,Fleet JC.Increased vitamin D receptor level enhances 1,25-dihydroxyvitaminD3 -mediated gene expression and calcium transport in Caco-2cells[J].J Bone Miner Res,2001,16(4):615-624
[15]Marcinkowska E.A run for a membrane vitamin D receptor[J].Biol Signals Recept,2001,10(6):341-349.
[16]Norman AW.Minireview:vitamin D receptor:new assignments for an already busy receptor[J].Endocrinology 2006,14 7(12):5542-5548.
[17]向伟,丁宗一,郑维.维生素D及其受体与临床相关疾病的研究[J],中华儿科杂志,2004,42(7):54-544
[18]Tatsuya Yoshizawa,Yuki Handa,Yoshikatsu Uematsu etal,Mice lacking the vitamin D receptor exhibit impaired bone formation,uterine hypoplasia and growth retardation after weaning,Nature Genetics,1997,16,391 - 396
[19]Keiko Kinuta,Hiroyuki Tanaka,Tadashi Moriwake etal,Vitamin D Is an Important Factor in Estrogen Biosynthesis of Both Female and Male Gonads,Endocrinology 2000,141(4):1317-1324
[20]HPS Brain,G Bano,HD Mason,etal 1,25-dihydroxyvitaminD3 has a direct effect on steroid production by human granulose cells,Society for Endocrinology Annual Meeting 2001,Endocrine Abstracts 2:73
[21]Haussler MR,Whitfield GK,Hausshr CA.et al,The nuclear vitamin D receptor:biological and molecular regulatory properties revealed.J Bone Miner Res,1998,13:325.349。
[22]Haussler MR,Whitfield GK,Haussier CA.et al.The nuclear vitamin D receptor:biological and molecular regulatory properties revealed[J].J Bone Miner Res,1998,13:325.349.
[23]Delissalde F,Hernabdez MA,Barron A,et al.Vitamin D induces proliferation in rat endometirum cultured cells[J].Rev Invesl Clin,1998,50(2):113-118.
[24]Saunders DE,Chfistensen C,Lawrence WD,et al.Receptors for 1,25-dihydroxy-vitamin D_3 in gynecologic neoplasms[J].Gynecol Oncol,1992.44(2):131-136
[25]Srilatha Swami,Aruna V.Krishnan,et al,1α,25-Dihydroxyvitamin D_3Down-Regulates Estrogen Receptor Abundance and Suppresses Estrogen Actions in MCF-7 Human Breast Cancer Cells,Clinical Cancer Research 2000,6,3371-3379
[26]Horii I,Takizawa S,Fujii T.Effect of 1,25-dihydroxyvitamin D3 on the female reproductive system in rats.J Toxicol Sci.1992,17(3):91-105.
[27]陈炳卿主编,营养与食品卫生学[M],人民卫生出版社,2000:60
[28]邹纪平,钟琼莎,王耀光,对维生素D和钙的再认识:比较争鸣,中国临床康复,2004,29(8) 6478-6479
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[6]Johnson JA,Grande JP,Roche PC,etal.Immuno-histochemical detection and distribution of the 1,25-dihydroxyvitamin D3 receptor in rat productive tissues.[J].Histo-chem Cell Biol,1996,105(1):7-15.
[7]Vigano P,Lattuada D,Mangioni S,etal.Cycling and early pregnant endometrium as a site of regulated expression of the vitamin D system [J].J Mol Endocrinol,2006,36(3):415-424.
[8]Reichrath J,Rafi L,Muller SM,etal.Immuno-histochemcal analysis of 1,25-dihydroxyvitaminD3 receptor in cervical carcinoma [J].Histchem J,1998,30 (8):561-567
[9]Norman AW,Ishizuka S,Okamura WH.Ligands for the vitamin D encocrine system:different shapes function as agonists and antiagonists for genomic and rapid response receptors or as a ligand for the plasma vitamin D binding protein.[J].J Steroid Biochem Mol Biol,2001,76(l-5):49-59
[10]Corhatt ST,Hill O,Nangia AK.Vitamin D receptor found in human sperm [J].Urology,2006,68(6):1345-1349
[11]Horii I,Takizawa S,Fujii T.Effect of 1,25-dihydroxyvitamin D3 on the female reproductive system in rats.J Toxicol Sci.1992,17(3):91-105.
[12]1α,25-Dihydroxyvitamin D_3 Down-Regulates Estrogen Receptor Abundance and Suppresses Estrogen Actions in MCF-7 Human Breast Cancer Cells,Srilatha Swami,Aruna V.Krishnan,et al,Clinical Cancer Research 2000,August Vol.6,3371-3379
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[14]邹纪平,钟琼莎,王耀光,对维生素D和钙的再认识:比较争鸣,中国临床康复,2004,29(8)6478-6479
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[27]张森,谭建华,大鼠发情周期中主要生殖激素的变化,动物医学进展,200526(12):1-6
[28]曹泽毅主编.激素受体及其临床应用[M].北京:北京医科大学中国协和医科大学联合出版社,1993:191
[29]史小林主编.人类生殖学[M].北京:科学出版社,2002:164
[30]王心如主编,毒理学基础[M],第五版,北京:人民卫生出版社,2007:93
[31]Zhivotovsky B.Caspases:the enzymes of death[J].Essays Biochem,2003,39:25-40
[32]Porter DA,Harman R M,Cowan R G,et al.Relationship of Fasligand expression and atresia during bovine follicle development[J]Reproduction,2001,121(4):561-566.
[33]Nilsson S,Gustafsson JA.Estrogen receptor transriptio and transactivation:basic aspects of estrogen action.Breast Cancer Res,2000,2:360-366
[34]杨世华,余四九.生殖激素控制卵泡细胞凋亡的研究进展[J]动物医学进展,2002,23(2):1-5.
[35]李育,王明艳,马红,卵巢颗粒细胞凋亡的调控机制,临床和实验医学杂志,2007年4月第6卷第4期,160-161
[36]刘忠华,刘海兰,岳奎忠,等.性未成熟小母猪注射PMSG对卵泡闭锁及颗粒细胞凋亡的影响[J].中国兽医学报,2002,22(4):384-386.
[37]Anthony J Z,Lynda L I,Steven G B.Developmentalexpression of Ca~(2+)/Mg~(2+)dependent endonuclease activity in rat granulose and luteal cells[J].Endocrinology,1989,125(4):2 216-2 222
[1]Holick MF.Vitamin D:A millenium perspective.J Cell Biochem.2003,88:296-307.
[2]Villena-Heinsen C,Meyberg R,Axt-FliednerR,etal.Immuno-histochemical analysis of 1,25-dihydroxyitamin-D3-receptors,estrogen and progesterone receptors and Ki-67 in ovarian carcinoma [J].Anticancer Res,2002,22(4):2261-2 2 67.
[3]Johnson JA,Grande JP,Roche PC,etal.Immuno-histochemical detection and distribution of the 1,25-dihydroxyvitamin D3 receptor in rat productive tissues.[J].Histo-chem Cell Biol,1996,105(1):7-15.
[4]Vigano P,Lattuada D,Mangioni S,etal.Cycling and early pregnant endometrium as a site of regulated expression of the vitamin D system [J].J Mol Endocrinol,2006,36(3):415-424.
[5]Reichrath J,Rafi L,Muller SM,etal.Immuno-histochemcal analysis of 1,25-dihydroxyvitaminD3 receptor in cervical carcinoma [J].Histchem J,1998,30 (8):561-567
[6]Carlberg C,Polly P.Gene-regulation by vitaminD3[J].Crit Rev Eukaryot Gene Exp,1998,8:19～42.
[7]Rastinejad F,Perlmann T,Evans RM,etal.Structural determinants of nuclear receptor assembly on DNA direct repeats [J].Nature,1995,375:203～211.
[8]Strugnell SA,Deluca HF.The vitaminD receptor2 structure and transcriptional activation [J].Proc Soc Exp Biol Med,1997,215:223～228
[9]Nakajima S,Hsieh JC,Jurutka P,etal.Examination of the potential functional role of conserved cysteine residues in the hormone-binding domain of the human1,25-dihydroxyvitamin-D3 receptor 1 [J].J Biol Chem,1996,271:5143-5149.
[10]Beer TM,Myrthue A.Calcitriol in cancer treatment:from the lab to clinic [J].Mol Cancer Ther,2004,3(3):373-381.
[11]Belkacemi L,Zuegel U,Steinmeyer A,et al.Calbindin-2D 8k (CaBP28k) identification and regulation by 1,25-dihydroxyvitaminD3 in human chorio-carcinoma cell line JEG-3 [J].Mol Cell Endocrinol,2005,236(1-2):31-41
[12]Norman AW,Ishizuka S,Okamura WH.Ligands for the vitamin D encocrine system:different shapes function as agonists and antiagonists for genomic and rapid response receptors or as a ligand for the plasma vitamin D binding protein.[J].J Steroid Biochem Mol Biol,2001,76(1-5):49-59
[13]Corhatt ST,Hill O,Nangia AK.Vitamin D receptor found in human sperm[J].Urology,2006,68(6):1345-1349
[14]Shao A,Wood RJ,Fleet JC.Increased vitamin D receptor level enhances 1,25-dihydroxyvitaminD3 -mediated gene expression and calcium transport in Caco-2cells[J].J Bone Miner Res,2001,16(4):615-624
[15]Marcinkowska E.A run for a membrane vitamin D receptor[J].Biol Signals Recept,2001,10(6):341-349.
[16]Norman AW.Minireview:vitamin D receptor:new assignments for an already busy receptor[J].Endocrinology 2006,14 7(12):5542-5548.
[17]向伟,丁宗一,郑维.维生素D及其受体与临床相关疾病的研究[J],中华儿科杂志,2004,42(7):54-544
[18]Tatsuya Yoshizawa,Yuki Handa,Yoshikatsu Uematsu etal,Mice lacking the vitamin D receptor exhibit impaired bone formation,uterine hypoplasia and growth retardation after weaning,Nature Genetics,1997,16,391 - 396
[19]Keiko Kinuta,Hiroyuki Tanaka,Tadashi Moriwake etal,Vitamin D Is an Important Factor in Estrogen Biosynthesis of Both Female and Male Gonads,Endocrinology 2000,141(4):1317-1324
[20]HPS Brain,G Bano,HD Mason,etal 1,25-dihydroxyvitaminD3 has a direct effect on steroid production by human granulose cells,Society for Endocrinology Annual Meeting 2001,Endocrine Abstracts 2:73
[21]Haussler MR,Whitfield GK,Hausshr CA.et al,The nuclear vitamin D receptor:biological and molecular regulatory properties revealed.J Bone Miner Res,1998,13:325.349。
[22]Haussler MR,Whitfield GK,Haussier CA.et al.The nuclear vitamin D receptor:biological and molecular regulatory properties revealed[J].J Bone Miner Res,1998,13:325.349.
[23]Delissalde F,Hernabdez MA,Barron A,et al.Vitamin D induces proliferation in rat endometirum cultured cells[J].Rev Invesl Clin,1998,50(2):113-118.
[24]Saunders DE,Chfistensen C,Lawrence WD,et al.Receptors for 1,25-dihydroxy-vitamin D_3 in gynecologic neoplasms[J].Gynecol Oncol,1992.44(2):131-136
[25]Srilatha Swami,Aruna V.Krishnan,et al,1α,25-Dihydroxyvitamin D_3Down-Regulates Estrogen Receptor Abundance and Suppresses Estrogen Actions in MCF-7 Human Breast Cancer Cells,Clinical Cancer Research 2000,6,3371-3379
[26]Horii I,Takizawa S,Fujii T.Effect of 1,25-dihydroxyvitamin D3 on the female reproductive system in rats.J Toxicol Sci.1992,17(3):91-105.
[27]陈炳卿主编,营养与食品卫生学[M],人民卫生出版社,2000:60
[28]邹纪平,钟琼莎,王耀光,对维生素D和钙的再认识:比较争鸣,中国临床康复,2004,29(8) 6478-6479