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学科主题: 内科学
题名:
粒细胞集落刺激因子对急性损伤人胎肝细胞保护作用的实验研究
作者: 孙子健
答辩日期: 2016-05-24
导师: 胡瑾华
专业: 内科学
授予单位: 北京大学
授予地点: 北京大学解放军302医院教学医院
学位: 硕士
关键词: 粒细胞集落刺激因子 ; D-半乳糖胺 ; 人胎肝细胞 ; 细胞凋亡
其他题名: Protective Effect of Granulocyte Colony-stimulating Factor on Acute Injured Human Fetal Hepatocytes
分类号: R575.3
摘要:

目的:运用D-半乳糖胺(D-GalN)构建人胎肝细胞急性损伤模型,观察粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)对人胎肝细胞急性损伤是否具有保护作用,并研究G-CSF保护人胎肝细胞可能的分子机制。

方法 :

1.体外培养人胎肝细胞,分别用梯度浓度的D-GalN和不同的D-GalN作用时长处理人胎肝细胞,用MTT法检测细胞活性,以确定人胎肝细胞急性损伤模型的最佳构建条件。

2.细胞急性损伤造模前和造模同时均给予相同剂量的G-CSF处理。设置不同剂量G-CSF以及不同G-CSF给药时间间隔处理急性损伤人胎肝细胞模型,MTT法检测细胞活性,选择适宜的G-CSF给药方案。实验设置空白对照组、药物(G-CSF)对照组、损伤模型对照组和G-CSF治疗组,采用MTT法检测各组细胞存活状态,同时采用LDH法检测各组细胞死亡情况,观察G-CSF对急性损伤模型是否具有保护作用。

3.Western Blot方法检测人胎肝细胞G-CSF受体的表达以及G-CSF处理正常人胎肝细胞5、10、15、30、60min后p-Akt、p-ERK1/2表达水平及变化规律,G-CSF处理正常人胎肝细胞12h后Bcl-2表达水平;用qPCR和Western Blot检测G-CSF治疗急性损伤模型3h后,损伤模型中存活胎肝细胞内Bcl-2的mRNA和蛋白水平。

结果 :

1. D-GalN对人胎肝细胞具有毒性作用,随浓度增加和作用时间的延长杀伤作用增强,当10mg/ml D-GalN作用12h,可使细胞活性减少约90%,以此作为急性损伤模型建模条件。

2. G-CSF剂量为10μg/ml时对急性损伤模型保护作用显著(P=0.029),并且G-CSF给药时间间隔12h组细胞活性高于其他各组,建立G-CSF给药方案为:G-CSF10μg/ml 急性损伤造模前和造模同时两次给药处理,间隔12h。结果显示:G-CSF治疗组存活细胞多于损伤模型对照组(MTT分别为0.221±0.033和0.164±0.032(t=3.029,P=0.013));G-CSF治疗组坏死细胞少于损伤模型对照组(LDH水平分别为4.0±3.8 U/L和20.7±4.7 U/L(t=6.779,P=0.000)),即G-CSF对急性损伤人胎肝细胞具有显著的保护作用。而药物对照组与空白对照细胞相比,细胞活性没有显示出明显差异,说明G-CSF对细胞无毒性作用,观察期内未显示明显的增殖作用。

3.人胎肝细胞G-CSF受体表达丰富。G-CSF处理正常人胎肝细胞5 min时,Akt和ERK1/2分子的磷酸化水平迅速升高,并持续至30 min逐渐恢复基础水平。经G-CSF处理12 h的正常人胎肝细胞,Bcl-2蛋白表达水平显著增加(P=0.020)。G-CSF间隔12小时两次给药治疗急性损伤人胎肝模型3h时,qPCR和Western Blot结果均提示G-CSF有使损伤细胞Bcl-2表达增加的趋势。

结论:

1.应用10 mg/mL D-GalN体外处理人胎肝细胞12 h可以成功构建D-GalN致人胎肝细胞急性损伤模型,此模型可以达到杀伤约90%人胎肝细胞的效果,并且可以为G-CSF治疗预留足够的药物反应窗口期。

2. G-CSF对D-GalN所致人胎肝细胞急性损伤具有显著保护作用。实验表明此保护作用可能的机制是G-CSF与人胎肝细胞G-CSF受体结合,通过激活Akt和ERK1/2,进一步促进其下游抗凋亡蛋白Bcl-2表达上调,从而抑制细胞凋亡。

 

英文摘要:

Objects: To establish the acute injury model of human fetal hepatocytes caused by D-galactosamine (D-GalN) and observe the protective effect of granulocyte colony-stimulating factor (G-CSF) on injuried cells and explore its mechanism.

Methods:

1. Human fetal hepatocytes were incubated by D-GalN of different concentrations and  administration durations. The cell survival rate was determined by MTT method, which is used to optimize parameters for establishing the acute injured human fetal hepatocyte model.

2. Different concentrations and time intervals between the first and second treatment of G-CSF are used for the treatment of injured human fetal hepatocytes. The cell viability was tested by MTT method, which helped to optimize G-CSF administration protocal.    Human fetal hepatocytes were cultured and divided into four groups, including blank control group, G-CSF control group, injured model group and G-CSF treatment group. The cell survival rates were tested by MTT and LDH method, which is used to observe the protective effect of G-CSF on the acute injuried cell model.

3. The expressions of G-CSF receptor (G-CSFR), the levels of p-ERK1/2 and p-Akt at 5, 10, 15, 30 and 60 min after G-CSF treatment and Bcl-2 levels 12 hours after G-CSF administration in the normal human fetal hepatocytes were tested by Western Blot. The mRNA and protein levels of Bcl-2 3 hours after G-CSF treatment to the injured human fetal hepatocytes were tested by qPCR and Western Blot, respectively.

Results:

1. D-GalN was toxic to human fetal hepatocytes. The toxicity increased in a concentration and time depended manner. The cell viability decreased by about 90% when the concentration 10 mg/mL and incubation time 12 h of D-GalN was used, which was adopted as the condition of the acute injury model.

2. G-CSF at a dose of 10 μg/mL significantly protected the cells from D-GalN-induced injury (P=0.029), and time interval of 12h between the two times of G-CSF treatment leads to the highest cell viability. Thus the G-CSF administration protocol included two times of 10 μg/mL G-CSF treatment, one at 12h before and the other at the same time of D-GalN administration. The absorbance of MTT was 0.221±0.033 in the G-CSF treatment group, which was significantly increased than those in the injured model group (0.164±0.032, P=0.013). Meanwhile, the levels of LDH were significantly decreased in the G-CSF treatment group than those in the injured model group (4.0±3.8 U/L vs. 20.7±4.7 U/L, P=0.000). The results showed that G-CSF treatment increased cell viability and decreased cell death, indicating that G-CSF had a protective effect on acute injured human fetal hepatocytes. Compared to that of the blank control group, the cell viability of the G-CSF control group showed no significant difference, which suggested that G-CSF had no toxicity or proliferation effect on the cells.

3. The results from Western Blot showed that human fetal hepatocytes expressed G-CSFR. p-ERK1/2 and p-Akt levels elevated at 5 min after G-CSF administration on the normal cells, and gradually decrease to initial level at 30 min after the G-CSF treatment. The level of Bcl-2 protein in the human fetal hepatocytes was significantly increased at 12 h after incubation with G-CSF (P=0.020). The results of qPCR and Western Blot showed that at 3h after the human fetal hepatocytes subjected to D-GalN and G-CSF administration, G-CSF had a trend to increase Bcl-2 expression in the injured cells.

Conclusion:

1. The D-GalN-induced acute injured human fetal hepatocyte model is successfully established by using the concentration 10 mg/mL and incubation time 12 h of D-GalN. About 90% cells is killed in this model, and the incubation time is enough for the G-CSF action.

2. G-CSF has a significant protective effect on the acute injured human fetal hepatocytes induced by D-GalN. The mechanism may be that G-CSF binds to the G-CSFR of human fetal hepatocytes and then rapidly increases the levels of p-Akt and p-ERK1/2, which promotes the expression of Bcl-2 and inhibits cell apoptosis.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/125288
Appears in Collections:北京大学解放军302医院教学医院_学位论文

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作者单位: 北京大学解放军302医院教学医院

Recommended Citation:
孙子健. 粒细胞集落刺激因子对急性损伤人胎肝细胞保护作用的实验研究[D]. 北京大学解放军302医院教学医院. 北京大学. 2016.
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