薯蓣皂苷元对IGF-1诱导的人甲状腺原代细胞增殖的影响
摘要
研究背景：
Graves病(简称GD)是一种常见的自身免疫性甲状腺疾病,弥漫性甲状腺肿是其特征性表现之一。患者甲状腺肿越明显,病情越难控制,药物治疗的疗程越长。因此探讨甲状腺肿大的机制,研发有效的治疗方法十分必要。
在甲状腺肿大的发生发展过程中,生长因子的作用日益受到重视,其中以胰岛素样生长因子-1(IGF-1)的作用较为突出。我们前期研究结果显示,GD患者血清和甲状腺组织中的IGF-1水平与甲状腺大小呈正相关；IGF-1通过影响PI3K和核因子NF-κB,上调抗凋亡蛋白FLIP和细胞周期蛋白cyclin D1的表达水平；同时该信号通路还参与细胞周期的调节；提示IGF-1是一种重要的致肿大因子,参与甲状腺肿大的发生和发展过程。
薯蓣皂苷(dioscin)系从薯蓣科薯蓣属植物穿龙薯蓣、盾叶薯蓣或穿龙薯蓣、黄山药等植物根茎中提取的一类甾体皂苷,其水解产物为薯蓣皂苷元(diosgenin, Dio)。薯蓣皂苷元作为合成多种甾体激素和甾体避孕药比较理想的前体,其应用前景非常广阔。现代药理研究表明,薯蓣皂苷及其衍生物具有免疫调节、降血脂、抗衰老、抗氧化、抗关节炎、保护胃黏膜、祛痰、溶血、脱敏、灭钉螺、抗肿瘤、抗艾滋病、抗血小板聚集、增强心脏收缩力、减慢心率、抗动脉硬化、改善微循环等多种药理活性。近年来免疫相关性疾病的发病率渐高,尤其是Graves病的发病率增高并呈现发病年龄降低的趋势,薯蓣皂苷既有调节免疫作用,又有抗增殖和诱导凋亡作用,其治疗Graves病的可能性值得我们探讨。
细胞周期蛋白在增殖性甲状腺疾病中的作用也受到很大关注。细胞周期蛋白D1(cyclin D1)是一种重要的细胞周期蛋白,在细胞增殖中起正性调控作用, cyclinD1与CDK4和CDK6形成复合体,使Rb蛋白磷酸化,促进G1/S期转移。当cyclin D1蛋白高表达时,就有可能导致细胞异常增殖甚至癌变。细胞周期蛋白B1 (cyclinB1)是典型的G2/M期周期蛋白,G2/M期转换的关键调控分子是cyclin B1/p34cdc2复合物,cyclin B1在S期开始合成并在G2期定位于细胞质,与p34cdc2结合形成有丝分裂促进因子(MPF),促进细胞从G2期进入M期,从而完成有丝分裂。但是在甲状腺细胞内,IGF-1是否通过影响cylinD1、cyclin B1的表达及细胞周期的变化从而参与甲状腺肿大发生发展的过程尚不明确。
基于上述研究现状,为了更深入的认识IGF-1在GD甲状腺肿大中的作用机制和薯蓣皂苷元对人甲状腺细胞增殖的影响,以便为临床上探索治疗GD提供实验依据,本研究确定研究的目的与方法如下：
目的：
1.观察不同浓度IGF-1对体外培养的人甲状腺原代细胞增殖的影响。
2.观察薯蓣皂苷元对IGF-1诱导的人甲状腺原代细胞增殖的影响
3.观察IGF-1和薯蓣皂苷元是否通过调控cyclin D1、cyclin B1蛋白表达水平影响人甲状腺原代细胞的细胞周期,探讨可能的机制。
研究方法：
1.人甲状腺原代细胞的培养：外科甲状腺手术中取正常甲状腺组织1-3g,剪成lmm3左右的小块；用加有100 U/ml青霉素和100μg/ml链霉素的不含钙镁的HankS液反复漂洗,然后500 g离心5分钟；用适量的消化液(0.2%I型胶原酶,0.125%胰酶,溶于D-HankS液)8-10ml,在磁力搅拌器上37℃水浴消化,消化时间一般为40-60分钟；消化后加5ml含血清的培养液终止消化,用200目不锈钢筛网过滤,500 g离心5分钟。DMEM/F12培养基洗涤沉淀2-3次,于加有10% NBS、2 mIU/ml的TSH、100U/ml青霉素和100ug/ml链霉素、2mmol/L谷氨酰胺的DMEM/F12培养基中培养;过夜后弃上清,PBS冲洗去除未贴壁细胞,用DMEM/F12培养(10% NBS、2mIU/ml的TSH、100U/ml青霉素和100ug/ml链霉素、2mmol/L谷氨酰胺)1到2天后,用含0.2%NBS的DMEM/F12培养基培养(饥饿-同步化)24 h,之后进入实验干预阶段-即用不用浓度的IGF-1在有或无薯蓣皂苷元的情况下培养。
2.人甲状腺原代细胞的鉴定：人甲状腺原代细胞提取后在培养皿中进行细胞爬片,用加有10% NBS、2mIU/ml的TSH的DMEM/F12培养基培养2-3天,然后采用免疫荧光方法进行细胞鉴定,采用DAPI染核,抗-TSH受体抗体染胞膜。
3.细胞活力的测定：细胞在皿中培养至密度至70-80%时,消化后转至96孔板,用加有10% NBS、2mIU/ml的TSH的DMEM/F12培养基培养2-3天,用含0.2%NBS的DMEM/F12培养基培养(同步化)24 h,之后进入实验干预阶段；然后采用MTT方法检测细胞活力。
4.细胞增殖的检测：人甲状腺原代细胞在培养皿中干预结束后,采用EdU方法检测细胞增殖,荧光显微镜下所有的细胞核显示蓝色(Hoechst染色),增殖中的细胞核显示红色(EdU染色)。
5.流式细胞仪检测细胞周期：人甲状腺原代细胞在培养皿中干预结束后,消化后用PBS洗后离心,采用流式细胞仪检测细胞周期,
6. cyclin D1、cyclin B1蛋白表达水平检测：采用Western blot检测cyclin D1、cyclinB1蛋白表达水平。
结果：
1、人甲状腺原代细胞的鉴定
免疫荧光结果显示,细胞核被DAPI染成蓝色,细胞膜被抗-TSH受体抗体染成绿色,荧光显微镜下人甲状腺细胞呈现单层、不规则形状并聚集成岛状,抗-TSH受体抗体阳性细胞超过95%。
2、MTT结果：MTT结果显示100ng/ml IGF-1能显著增加人甲状腺原代细胞的细胞活力,IGF-1对人甲状腺原代细胞的细胞活力的作用呈现没有量效和时效的影响；20μmol/L薯蓣皂苷元开始能明显抑制人甲状腺原代细胞的细胞活力,而且呈现量效和时效的影响；不同浓度的薯蓣皂苷元和100 ng/ml IGF-1作用时,细胞活力明显下降,薯蓣皂苷元的作用仍呈现量效和时效的影响。
3、EdU结果：EdU阳性细胞数(即增殖期细胞)在用IGF-1处理后明显增加,而在薯蓣皂苷元处理后EdU阳性细胞数明显减少；同时用IGF-1和薯蓣皂苷元处理后,EdU阳性细胞数明显减少。
4、流式细胞仪检测细胞周期：IGF-1促使更多的细胞进入S期,Go/G1期细胞减少；薯蓣皂苷元阻滞细胞在Go/G1期,S期细胞减少；当用25μmol/L薯蓣皂苷元和100ng/ml IGF-1共同作用时,Go/G1期细胞增加而S期细胞减少,G2/M期细胞没有明显变化。
5、Western blot检测结果：100 ng/ml IGF-1作用6 h和12 h能增加cyclin D1蛋白表达,作用24 h后cyclin D1蛋白水平开始减少,作用48 h后cyclin D1蛋白水平明显减少；25μmol/L的薯蓣皂苷元作用6 h对cyclin D1蛋白水平无明显影响,作用12 h和48 h后能降低cyclin D1蛋白的水平;当用25μmol/L薯蓣皂苷元和100 ng/mlIGF-1分别作用12 h和48 h时,明显降低cyclin D1蛋白水平；在以上所有的实验中,cyclin B1蛋白水平无明显变化。
结论：
IGF-1能促进人甲状腺原代细胞的增殖并促使更多细胞进入S期；薯蓣皂苷元能抑制人甲状腺原代细胞的增殖并阻滞细胞在Go/G1期；而当两者共同作用时,其效果更接近于薯蓣皂苷元单独作用时。
Background:
Graves' disease (GD) is a kind of common autoimmune thyroid disease. Diffuse thyroid goiter is one of the characteristic features of GD. The bigger the thyroid volumes are, the longer the period of medical treatment for GD is. So it is very important to study the mechanism of thyroid goiter formation to find effective therapeutic targets for this disease.
Previous studies also showed that growth factors, especially insulin-like growth factor-1 (IGF-1), plays important roles in thyroid cell growth and human thyroid diseases. IGF-1 is an important hypertrophic factor for thyroid cells. It regulates cell proliferation and a vast variety of differentiated cell functions. Our previous research showed that in patients with untreated GD, there was a significant increase in serum IGF-1 levels, and localized IGF-1 expression in thyroid tissues was higher in those patients compared with healthy subjects. Meanwhile, thyroid volumes were positively correlated with IGF-1 levels in untreated patients. In addition, IGF-1 promoted cell cycle progression and up-regulated cyclin Dl expression by stimulating the PI3K/ NF-κB pathway in thyroid cells. These findings indicated that IGF-1 plays a key role in the formation of thyroid goiters.
Diosgenin is a steroidal sapogenin belonging to the group of triterpenes. It is found in several plants including Dioscorea species (yams), fenugreek and Costus speciosus. So far the benefits of diosgenin were limited to preclinical studies demonstrating efficacy against skin aging, hyperglycemia, hypercholesterolemia, and hypertriacylglycerolemia. A recent study has demonstrated that diosgenin controlled hypercholesterolemia in rats fed a high-cholesterol supplemented diet by improving the lipid profile as well as by modulating oxidative stress. An interesting biological activity of diosgenin was demonstrated by Turchan-Cholewo who concluded that diosgenin might have potential therapeutic effect against an increased risk of developing dementia in opiate abusers with HIV infection. All these studies showed that diosgenin had various biologic activities, but the effects of diosgenin on human primary thyrocytes have never been reported.
The role of cyclins in proliferative thyroid diseases also get great attention. Cyclin D1 is a G1 phase cell cycle protein. Accumulation of this kind of protein by stimulation of extracellular signals promotes cell proliferation. Cyclin D1 can be combined with CDK4 and CDK6, which can make Rb phosphorylated and progress G1 phase to S phase. It has been reported that in thyroid papillary carcinomas, the expression of cyclin D1 was elevated, which was thought to be a contributory factor to thyroid tumorigenesis. While cyclin B1 regulates a G2 checkpoint and promotes cell entry into M phase. Cyclin B1 is produced in S phase and located in cytoplasma in G2 phase. Cyclin B1 can be combined with p34cdc2 to Mitosis-Promoting Factor(MPF), which promotes cell entry into M phase. But in normal thyroid cells, informations regarding the relationship between cyclin D1、cyclin B1 induced by IGF-1 and cell proliferation are uncertain.
In addition, IGF-1 may play a important role in Graves'disease. The aim of the present study was to evaluate the possible effects of diosgenin on cell proliferation induced by IGF-1 in primary human thyroid cells. And this makes a possible alternative therapy for goiter patients.
Objectives:
1. The effects of IGF-1 in different concentration on cell proliferation in primary human thyroid cells.
2. The effects of diosgenin on cell proliferation induced by IGF-1 in primary human thyroid cells.
3. IGF-1 and diosgenin possibly control the cyclin D1 and cyclin B1 protein expression to affect the cell progress.
Methods:
1. Cell culture:The specimens were obtained from normal thyroid tissue of euthyroid patients operated on for benign follicular nodules. The tissue was incubated with Ca2+- and Mg2+-free Hanks' salt solution containing type I collagenase, trypsin (0.25%),0.75 mg/ml heat-inactivated dialyzed chicken serum, in a shaking water bath at 37℃for 40-60 minutes. The supernatants were collected, combined, and washed in DMEM/F12 culture medium for 2-3 times, then centrifuged. Thyrocytes were suspended in DMEM/F12 culture medium containing 10% newborn calf serum (NBS), TSH (Thyroid Stimulating Hormone,2 mU/ml), penicillin-streptomycin (100 U/ml), fungizone (2μg/ml). Cells were plated in Falcon 25-cm2 tissue culture flasks for primary culture with DMEM/F12 medium and incubated at 37℃in humidified air atmosphere containing 5% CO2. After 24 h the supernatant containing no adherent cells was removed. The primary thyroid cells formed a confluent monolayer within 3-5 d; and the cells were treated when they reached 70-80% confluence.
2. Identification of primary huaman thyroid cells:Primary human thyrocytes were seeded on a glass slide. The cells were assessed by immunofluorescence. Nuclei were stained by DAPI, and membranes were specially stained by anti-TSHR antibody.
3. Cell proliferation assay:The effects of IGF-1 and diosgenin on cell proliferation were measured by 3-(4,5-dimefhylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and 5-ethynyl-20-deoxyuridine (EdU) incorporation assay using a MTT cell proliferation assay kit and EdU assay kit, respectively.
4. Cell cycle analysis was performed by using FACS:Briefly, after starvation overnight, IGF-1 was added and treated for 24h,then diosgenin was added and treated for another 24h. Cells harvested by trypsinization were washed with PBS and stained with propidium iodide (PI) using Coulter DNA PREPTM Reagents kit. Samples were analyzed by FACS on a Coulter Elite ESP using standard filter sets. Results were expressed graphically and quantified as the fraction of cells in Go/G1, S, or G2/M phase using Coulter cytologic software.
5. The protein expression of cyclin D1 and cyclin B1 was determined by Western blot analysis:After treatment cells were harvested, centrifuged, washed with PBS and lysed for 20 min on ice in RIPA buffer containing 1×PBS,1% NP-40,0.1% SDS,5 mM EDTA,0.5% sodium dexyccholate,1 mM sodium orthovanadate, 1mM PMSF. Cells were then centrifugated at 12000 rpm for 10 min at 4℃. The resulting supernatants (whole cell lysates) were collected and frozen at-80℃or used immediately. Protein concentrations were determined by BCA protein assay (Pierce, USA).40μg of each sample was heated for 30 min at 60℃, then analyzed by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes. Membranes were block-ed in 5% nonfat milk for 1 h and then incubated with the specific primary antibody overnight at 4℃, washed and incubated with appropriate horseradish peroxidase conjugated secondary antibody. After that, immune complexes were detected using the ECL method, and immunoreactive bands were quantified using Alphalmager 2200. Values were corrected with the absorbency of the internal control (β-actin).
Results:
1. Immunofluorescence
Primary human thyrocytes were identified by expression of TSH receptor using immunofluorescence. Nuclei were stained by DAPI, and membranes were specially stained by anti-TSHR antibody. The immunofluorescent photograph confirmed that the monolayer, irregular phenotype cells which aggregated to islands were human thyrocytes.
2. Effects of diosgenin and IGF-1 on cell viability (MTT)
We found that 100 ng/ml IGF-1 significantly increased cell viability (p< 0.001). However, there was no dose-dependent or time-dependent effect of IGF-1 on cell viability in primary human thyrocytes.20μmol/L diosgenin began to markedly inhibit cell viability (p< 0.01). And there were dose-dependent and time-dependent effects of diosgenin on cell viability. Importantly, when human thyrocytes were exposed to diosgenin in the present of IGF-1, the inhibition effect of diosgenin on the cell viability was significant (p< 0.01) and presented a dose-and time-dependent effect.
3. Effects of diosgenin and IGF-1 on cell proliferation (EdU)
We found that the number of EdU+ cells (EdU-labeled replicating cells) was increased after treated with IGF-1, while the number was significantly reduced in diosgenin treatment group. More importantly, the number of EdU+ cells was also significantly decreased after treated with both diosgenin and IGF-1, compared with that of the control.
4. Effects of diosgenin and IGF-1 on cell cycle (FACS);
Compared with the control group, the proportion of cells treated by IGF-1 in G0/G1 phase was decreased, and the proportion in the S phase was increased. However, the proportion in G2/M phase was not changed. When the cells were exposed to 25μmol/L diosgenin and IGF-1100 ng/ml together, the proportion of the cells in G0/G1 phase was increased, while S phase decreased. And the proportion in G2/M phase was still not changed.
5. Effects of diosgenin and IGF-1 on cyclin D1 and cyclin B1 expression
The results showed that IGF-1 treatment for 6 h and 12 h increased the cyclin D1 protein level. However, after 24 h treatment with IGF-1, the cyclin D1 protein level began to decrease. Consequently, the cyclin D1 protein level was markedly decreased after 48 h treatment compared with the control. On the other hand, Diosgenin treatment for 6 h alone had no apparent effects on cyclin D1 protein expression, whereas diosgenin treatment for 12 h and 48 h decreased the cyclin D1 protein expression. Interestingly, diosgenin treatment in the present of IGF-1 could dramatically decrease the cyclin Dl expression. However, the cyclin B1 protein level was not significantly changed in each group.
Conclusions:
In summary, this study showed that IGF-1 enhanced proliferation and promoted cell cycle progression to S, whereas diosgenin reduced cell proliferation induced by IGF-1 and rendered G0/G1 arrest by decreasing cyclin D1 expression in primary human thyrocytes. The effect of diosgenin on cell proliferation in present of IGF-1 in primary human thyrocytes was much more similar to that of diosgenin alone, and this implicated that diosgenin and IGF-1 might have a same signal pathway on proliferation and cell cycle in primary human thyrocytes.
Graves病(简称GD)是一种常见的自身免疫性甲状腺疾病,弥漫性甲状腺肿是其特征性表现之一。患者甲状腺肿越明显,病情越难控制,药物治疗的疗程越长。因此探讨甲状腺肿大的机制,研发有效的治疗方法十分必要。
在甲状腺肿大的发生发展过程中,生长因子的作用日益受到重视,其中以胰岛素样生长因子-1(IGF-1)的作用较为突出。我们前期研究结果显示,GD患者血清和甲状腺组织中的IGF-1水平与甲状腺大小呈正相关；IGF-1通过影响PI3K和核因子NF-κB,上调抗凋亡蛋白FLIP和细胞周期蛋白cyclin D1的表达水平；同时该信号通路还参与细胞周期的调节；提示IGF-1是一种重要的致肿大因子,参与甲状腺肿大的发生和发展过程。
薯蓣皂苷(dioscin)系从薯蓣科薯蓣属植物穿龙薯蓣、盾叶薯蓣或穿龙薯蓣、黄山药等植物根茎中提取的一类甾体皂苷,其水解产物为薯蓣皂苷元(diosgenin, Dio)。薯蓣皂苷元作为合成多种甾体激素和甾体避孕药比较理想的前体,其应用前景非常广阔。现代药理研究表明,薯蓣皂苷及其衍生物具有免疫调节、降血脂、抗衰老、抗氧化、抗关节炎、保护胃黏膜、祛痰、溶血、脱敏、灭钉螺、抗肿瘤、抗艾滋病、抗血小板聚集、增强心脏收缩力、减慢心率、抗动脉硬化、改善微循环等多种药理活性。近年来免疫相关性疾病的发病率渐高,尤其是Graves病的发病率增高并呈现发病年龄降低的趋势,薯蓣皂苷既有调节免疫作用,又有抗增殖和诱导凋亡作用,其治疗Graves病的可能性值得我们探讨。
细胞周期蛋白在增殖性甲状腺疾病中的作用也受到很大关注。细胞周期蛋白D1(cyclin D1)是一种重要的细胞周期蛋白,在细胞增殖中起正性调控作用, cyclinD1与CDK4和CDK6形成复合体,使Rb蛋白磷酸化,促进G1/S期转移。当cyclin D1蛋白高表达时,就有可能导致细胞异常增殖甚至癌变。细胞周期蛋白B1 (cyclinB1)是典型的G2/M期周期蛋白,G2/M期转换的关键调控分子是cyclin B1/p34cdc2复合物,cyclin B1在S期开始合成并在G2期定位于细胞质,与p34cdc2结合形成有丝分裂促进因子(MPF),促进细胞从G2期进入M期,从而完成有丝分裂。但是在甲状腺细胞内,IGF-1是否通过影响cylinD1、cyclin B1的表达及细胞周期的变化从而参与甲状腺肿大发生发展的过程尚不明确。
基于上述研究现状,为了更深入的认识IGF-1在GD甲状腺肿大中的作用机制和薯蓣皂苷元对人甲状腺细胞增殖的影响,以便为临床上探索治疗GD提供实验依据,本研究确定研究的目的与方法如下：
目的：
1.观察不同浓度IGF-1对体外培养的人甲状腺原代细胞增殖的影响。
2.观察薯蓣皂苷元对IGF-1诱导的人甲状腺原代细胞增殖的影响
3.观察IGF-1和薯蓣皂苷元是否通过调控cyclin D1、cyclin B1蛋白表达水平影响人甲状腺原代细胞的细胞周期,探讨可能的机制。
研究方法：
1.人甲状腺原代细胞的培养：外科甲状腺手术中取正常甲状腺组织1-3g,剪成lmm3左右的小块；用加有100 U/ml青霉素和100μg/ml链霉素的不含钙镁的HankS液反复漂洗,然后500 g离心5分钟；用适量的消化液(0.2%I型胶原酶,0.125%胰酶,溶于D-HankS液)8-10ml,在磁力搅拌器上37℃水浴消化,消化时间一般为40-60分钟；消化后加5ml含血清的培养液终止消化,用200目不锈钢筛网过滤,500 g离心5分钟。DMEM/F12培养基洗涤沉淀2-3次,于加有10% NBS、2 mIU/ml的TSH、100U/ml青霉素和100ug/ml链霉素、2mmol/L谷氨酰胺的DMEM/F12培养基中培养;过夜后弃上清,PBS冲洗去除未贴壁细胞,用DMEM/F12培养(10% NBS、2mIU/ml的TSH、100U/ml青霉素和100ug/ml链霉素、2mmol/L谷氨酰胺)1到2天后,用含0.2%NBS的DMEM/F12培养基培养(饥饿-同步化)24 h,之后进入实验干预阶段-即用不用浓度的IGF-1在有或无薯蓣皂苷元的情况下培养。
2.人甲状腺原代细胞的鉴定：人甲状腺原代细胞提取后在培养皿中进行细胞爬片,用加有10% NBS、2mIU/ml的TSH的DMEM/F12培养基培养2-3天,然后采用免疫荧光方法进行细胞鉴定,采用DAPI染核,抗-TSH受体抗体染胞膜。
3.细胞活力的测定：细胞在皿中培养至密度至70-80%时,消化后转至96孔板,用加有10% NBS、2mIU/ml的TSH的DMEM/F12培养基培养2-3天,用含0.2%NBS的DMEM/F12培养基培养(同步化)24 h,之后进入实验干预阶段；然后采用MTT方法检测细胞活力。
4.细胞增殖的检测：人甲状腺原代细胞在培养皿中干预结束后,采用EdU方法检测细胞增殖,荧光显微镜下所有的细胞核显示蓝色(Hoechst染色),增殖中的细胞核显示红色(EdU染色)。
5.流式细胞仪检测细胞周期：人甲状腺原代细胞在培养皿中干预结束后,消化后用PBS洗后离心,采用流式细胞仪检测细胞周期,
6. cyclin D1、cyclin B1蛋白表达水平检测：采用Western blot检测cyclin D1、cyclinB1蛋白表达水平。
结果：
1、人甲状腺原代细胞的鉴定
免疫荧光结果显示,细胞核被DAPI染成蓝色,细胞膜被抗-TSH受体抗体染成绿色,荧光显微镜下人甲状腺细胞呈现单层、不规则形状并聚集成岛状,抗-TSH受体抗体阳性细胞超过95%。
2、MTT结果：MTT结果显示100ng/ml IGF-1能显著增加人甲状腺原代细胞的细胞活力,IGF-1对人甲状腺原代细胞的细胞活力的作用呈现没有量效和时效的影响；20μmol/L薯蓣皂苷元开始能明显抑制人甲状腺原代细胞的细胞活力,而且呈现量效和时效的影响；不同浓度的薯蓣皂苷元和100 ng/ml IGF-1作用时,细胞活力明显下降,薯蓣皂苷元的作用仍呈现量效和时效的影响。
3、EdU结果：EdU阳性细胞数(即增殖期细胞)在用IGF-1处理后明显增加,而在薯蓣皂苷元处理后EdU阳性细胞数明显减少；同时用IGF-1和薯蓣皂苷元处理后,EdU阳性细胞数明显减少。
4、流式细胞仪检测细胞周期：IGF-1促使更多的细胞进入S期,Go/G1期细胞减少；薯蓣皂苷元阻滞细胞在Go/G1期,S期细胞减少；当用25μmol/L薯蓣皂苷元和100ng/ml IGF-1共同作用时,Go/G1期细胞增加而S期细胞减少,G2/M期细胞没有明显变化。
5、Western blot检测结果：100 ng/ml IGF-1作用6 h和12 h能增加cyclin D1蛋白表达,作用24 h后cyclin D1蛋白水平开始减少,作用48 h后cyclin D1蛋白水平明显减少；25μmol/L的薯蓣皂苷元作用6 h对cyclin D1蛋白水平无明显影响,作用12 h和48 h后能降低cyclin D1蛋白的水平;当用25μmol/L薯蓣皂苷元和100 ng/mlIGF-1分别作用12 h和48 h时,明显降低cyclin D1蛋白水平；在以上所有的实验中,cyclin B1蛋白水平无明显变化。
结论：
IGF-1能促进人甲状腺原代细胞的增殖并促使更多细胞进入S期；薯蓣皂苷元能抑制人甲状腺原代细胞的增殖并阻滞细胞在Go/G1期；而当两者共同作用时,其效果更接近于薯蓣皂苷元单独作用时。
Background:
Graves' disease (GD) is a kind of common autoimmune thyroid disease. Diffuse thyroid goiter is one of the characteristic features of GD. The bigger the thyroid volumes are, the longer the period of medical treatment for GD is. So it is very important to study the mechanism of thyroid goiter formation to find effective therapeutic targets for this disease.
Previous studies also showed that growth factors, especially insulin-like growth factor-1 (IGF-1), plays important roles in thyroid cell growth and human thyroid diseases. IGF-1 is an important hypertrophic factor for thyroid cells. It regulates cell proliferation and a vast variety of differentiated cell functions. Our previous research showed that in patients with untreated GD, there was a significant increase in serum IGF-1 levels, and localized IGF-1 expression in thyroid tissues was higher in those patients compared with healthy subjects. Meanwhile, thyroid volumes were positively correlated with IGF-1 levels in untreated patients. In addition, IGF-1 promoted cell cycle progression and up-regulated cyclin Dl expression by stimulating the PI3K/ NF-κB pathway in thyroid cells. These findings indicated that IGF-1 plays a key role in the formation of thyroid goiters.
Diosgenin is a steroidal sapogenin belonging to the group of triterpenes. It is found in several plants including Dioscorea species (yams), fenugreek and Costus speciosus. So far the benefits of diosgenin were limited to preclinical studies demonstrating efficacy against skin aging, hyperglycemia, hypercholesterolemia, and hypertriacylglycerolemia. A recent study has demonstrated that diosgenin controlled hypercholesterolemia in rats fed a high-cholesterol supplemented diet by improving the lipid profile as well as by modulating oxidative stress. An interesting biological activity of diosgenin was demonstrated by Turchan-Cholewo who concluded that diosgenin might have potential therapeutic effect against an increased risk of developing dementia in opiate abusers with HIV infection. All these studies showed that diosgenin had various biologic activities, but the effects of diosgenin on human primary thyrocytes have never been reported.
The role of cyclins in proliferative thyroid diseases also get great attention. Cyclin D1 is a G1 phase cell cycle protein. Accumulation of this kind of protein by stimulation of extracellular signals promotes cell proliferation. Cyclin D1 can be combined with CDK4 and CDK6, which can make Rb phosphorylated and progress G1 phase to S phase. It has been reported that in thyroid papillary carcinomas, the expression of cyclin D1 was elevated, which was thought to be a contributory factor to thyroid tumorigenesis. While cyclin B1 regulates a G2 checkpoint and promotes cell entry into M phase. Cyclin B1 is produced in S phase and located in cytoplasma in G2 phase. Cyclin B1 can be combined with p34cdc2 to Mitosis-Promoting Factor(MPF), which promotes cell entry into M phase. But in normal thyroid cells, informations regarding the relationship between cyclin D1、cyclin B1 induced by IGF-1 and cell proliferation are uncertain.
In addition, IGF-1 may play a important role in Graves'disease. The aim of the present study was to evaluate the possible effects of diosgenin on cell proliferation induced by IGF-1 in primary human thyroid cells. And this makes a possible alternative therapy for goiter patients.
Objectives:
1. The effects of IGF-1 in different concentration on cell proliferation in primary human thyroid cells.
2. The effects of diosgenin on cell proliferation induced by IGF-1 in primary human thyroid cells.
3. IGF-1 and diosgenin possibly control the cyclin D1 and cyclin B1 protein expression to affect the cell progress.
Methods:
1. Cell culture:The specimens were obtained from normal thyroid tissue of euthyroid patients operated on for benign follicular nodules. The tissue was incubated with Ca2+- and Mg2+-free Hanks' salt solution containing type I collagenase, trypsin (0.25%),0.75 mg/ml heat-inactivated dialyzed chicken serum, in a shaking water bath at 37℃for 40-60 minutes. The supernatants were collected, combined, and washed in DMEM/F12 culture medium for 2-3 times, then centrifuged. Thyrocytes were suspended in DMEM/F12 culture medium containing 10% newborn calf serum (NBS), TSH (Thyroid Stimulating Hormone,2 mU/ml), penicillin-streptomycin (100 U/ml), fungizone (2μg/ml). Cells were plated in Falcon 25-cm2 tissue culture flasks for primary culture with DMEM/F12 medium and incubated at 37℃in humidified air atmosphere containing 5% CO2. After 24 h the supernatant containing no adherent cells was removed. The primary thyroid cells formed a confluent monolayer within 3-5 d; and the cells were treated when they reached 70-80% confluence.
2. Identification of primary huaman thyroid cells:Primary human thyrocytes were seeded on a glass slide. The cells were assessed by immunofluorescence. Nuclei were stained by DAPI, and membranes were specially stained by anti-TSHR antibody.
3. Cell proliferation assay:The effects of IGF-1 and diosgenin on cell proliferation were measured by 3-(4,5-dimefhylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and 5-ethynyl-20-deoxyuridine (EdU) incorporation assay using a MTT cell proliferation assay kit and EdU assay kit, respectively.
4. Cell cycle analysis was performed by using FACS:Briefly, after starvation overnight, IGF-1 was added and treated for 24h,then diosgenin was added and treated for another 24h. Cells harvested by trypsinization were washed with PBS and stained with propidium iodide (PI) using Coulter DNA PREPTM Reagents kit. Samples were analyzed by FACS on a Coulter Elite ESP using standard filter sets. Results were expressed graphically and quantified as the fraction of cells in Go/G1, S, or G2/M phase using Coulter cytologic software.
5. The protein expression of cyclin D1 and cyclin B1 was determined by Western blot analysis:After treatment cells were harvested, centrifuged, washed with PBS and lysed for 20 min on ice in RIPA buffer containing 1×PBS,1% NP-40,0.1% SDS,5 mM EDTA,0.5% sodium dexyccholate,1 mM sodium orthovanadate, 1mM PMSF. Cells were then centrifugated at 12000 rpm for 10 min at 4℃. The resulting supernatants (whole cell lysates) were collected and frozen at-80℃or used immediately. Protein concentrations were determined by BCA protein assay (Pierce, USA).40μg of each sample was heated for 30 min at 60℃, then analyzed by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes. Membranes were block-ed in 5% nonfat milk for 1 h and then incubated with the specific primary antibody overnight at 4℃, washed and incubated with appropriate horseradish peroxidase conjugated secondary antibody. After that, immune complexes were detected using the ECL method, and immunoreactive bands were quantified using Alphalmager 2200. Values were corrected with the absorbency of the internal control (β-actin).
Results:
1. Immunofluorescence
Primary human thyrocytes were identified by expression of TSH receptor using immunofluorescence. Nuclei were stained by DAPI, and membranes were specially stained by anti-TSHR antibody. The immunofluorescent photograph confirmed that the monolayer, irregular phenotype cells which aggregated to islands were human thyrocytes.
2. Effects of diosgenin and IGF-1 on cell viability (MTT)
We found that 100 ng/ml IGF-1 significantly increased cell viability (p< 0.001). However, there was no dose-dependent or time-dependent effect of IGF-1 on cell viability in primary human thyrocytes.20μmol/L diosgenin began to markedly inhibit cell viability (p< 0.01). And there were dose-dependent and time-dependent effects of diosgenin on cell viability. Importantly, when human thyrocytes were exposed to diosgenin in the present of IGF-1, the inhibition effect of diosgenin on the cell viability was significant (p< 0.01) and presented a dose-and time-dependent effect.
3. Effects of diosgenin and IGF-1 on cell proliferation (EdU)
We found that the number of EdU+ cells (EdU-labeled replicating cells) was increased after treated with IGF-1, while the number was significantly reduced in diosgenin treatment group. More importantly, the number of EdU+ cells was also significantly decreased after treated with both diosgenin and IGF-1, compared with that of the control.
4. Effects of diosgenin and IGF-1 on cell cycle (FACS);
Compared with the control group, the proportion of cells treated by IGF-1 in G0/G1 phase was decreased, and the proportion in the S phase was increased. However, the proportion in G2/M phase was not changed. When the cells were exposed to 25μmol/L diosgenin and IGF-1100 ng/ml together, the proportion of the cells in G0/G1 phase was increased, while S phase decreased. And the proportion in G2/M phase was still not changed.
5. Effects of diosgenin and IGF-1 on cyclin D1 and cyclin B1 expression
The results showed that IGF-1 treatment for 6 h and 12 h increased the cyclin D1 protein level. However, after 24 h treatment with IGF-1, the cyclin D1 protein level began to decrease. Consequently, the cyclin D1 protein level was markedly decreased after 48 h treatment compared with the control. On the other hand, Diosgenin treatment for 6 h alone had no apparent effects on cyclin D1 protein expression, whereas diosgenin treatment for 12 h and 48 h decreased the cyclin D1 protein expression. Interestingly, diosgenin treatment in the present of IGF-1 could dramatically decrease the cyclin Dl expression. However, the cyclin B1 protein level was not significantly changed in each group.
Conclusions:
In summary, this study showed that IGF-1 enhanced proliferation and promoted cell cycle progression to S, whereas diosgenin reduced cell proliferation induced by IGF-1 and rendered G0/G1 arrest by decreasing cyclin D1 expression in primary human thyrocytes. The effect of diosgenin on cell proliferation in present of IGF-1 in primary human thyrocytes was much more similar to that of diosgenin alone, and this implicated that diosgenin and IGF-1 might have a same signal pathway on proliferation and cell cycle in primary human thyrocytes.
引文
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