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雌二醇及丙酸睾酮对兔泪腺细胞凋亡的影响及其作用机制的研究

发布时间:2018-05-16 10:38

  本文选题:雌二醇 + 丙酸睾酮 ; 参考:《广州医学院》2011年硕士论文


【摘要】:目的 本课题拟通过观察体外培养的兔泪腺上皮细胞经雌二醇(Estradiol,E_2)及丙酸睾酮(Testosterone Propionate,TP)干预后细胞凋亡情况及基质金属蛋白酶(Matrix Metalloproteinase-9,MMP-9)蛋白表达情况,探讨雌二醇及丙酸睾酮对兔泪腺上皮细胞的作用机制,为进一步研究雌激素及雄激素对干眼症的影响奠定基础。 方法 1.实验分组 1)实验分四组,分别为雌二醇组(E2组)、丙酸睾酮组(TP组)、凋亡对照组(AC组)、空白对照组(BC组)。E2组、TP组分别给予1x10~(-5)、1x10~(-6)、1x10~(-7)、1x10~(-8)mol/L雌二醇、丙酸睾酮干预, AC组只给予H_2O_2诱导凋亡;BC组即无药物干预也无诱导凋亡。 2)实验分为只H_2O_2诱导凋亡的AC组,1x10~(-5)mol/L雌二醇、丙酸睾酮处理的E组、T组,未加任何干预的BC组。 2.兔泪腺上皮细胞培养及HE染色 无菌摘取兔泪腺组织,用组织块培养法及混合消化法培养泪腺细胞,纯化细胞、倒置显微镜下观察及拍照, HE染色观察细胞形态。 3.泪腺上皮细胞鉴定 采用鼠抗广谱细胞角蛋白单克隆抗体进行免疫细胞化学法鉴定细胞。 4. Hoechst 33342单染法 药物干预后Hoechst 33342单染法定性检测泪腺上皮细胞的凋亡 5. MTT法检测泪腺上皮细胞活性 不同浓度的雌二醇及丙酸睾酮干预后,用MTT法检测的OD值显示不同组间泪腺细胞的活性。 6. Annexin V/PI双染法检测泪腺上皮细胞凋亡 药物干预后,流式细胞术(Flow Cytometry, FCM)检测分析细胞的凋亡,观察雌二醇及丙酸睾酮对泪腺上皮细胞凋亡的影响。 7.免疫细胞化学法检测细胞MMP-9蛋白表达 免疫细胞化学法检测雌二醇及丙酸睾酮对H_2O_2诱导凋亡后MMP-9蛋白表达的影响。 结果 1.泪腺上皮细胞培养情况及HE染色观察细胞形态 培养后细胞状态良好,胞体透亮、胞膜清晰、遮光性较强,HE染色观察泪腺上皮细胞呈不规则多边形,细胞轮廓清晰,核染明显,偶可见核仁。 2.泪腺上皮细胞鉴定结果 Pan-cytokeratin抗体免疫反应DAB显色见棕色阳性颗粒均匀分布于细胞浆中。 3. Hoechst 33342染色观察细胞凋亡情况 AC组中细胞呈现高强蓝色荧光,不规则,且部分细胞凋亡脱落;空白对照组中细胞呈均匀的椭圆形染色,无明显细胞脱落;E_2组及TP组有部分核染高荧光。 4. MTT检测细胞活性结果 与空白对照组相比,加入1×10~(-4)mol/L H_2O_2诱导细胞凋亡后,泪腺上皮细胞活性明显下降,OD值有统计学差异(P0.01);与AC组相比,同时加入不同浓度的雌二醇、丙酸睾酮后泪腺上皮细胞活性增强,总体而言E_2组与TP组细胞活性的OD值分别为0.4434±0.0981、0.3928±0.0518,雌二醇与丙酸睾酮对H_2O_2诱导的细胞凋亡具有抑制作用,其差异有统计学意义(P0.05);与丙酸睾酮组相比,雌二醇组抑制细胞凋亡作用强,有统计学意义(P0.05);其中,雌二醇对细胞的保护作用呈一定的剂量依赖性。 5.雌二醇及丙酸睾酮对泪腺上皮细胞凋亡的影响 不同浓度的雌二醇及丙酸睾酮对泪腺上皮细胞早晚期凋亡及坏死均有抑制作用,其凋亡率都较凋亡对照组低;且雌二醇组中,细胞的凋亡率与浓度呈一定的剂量依赖性,浓度越高,凋亡率越小;E2组凋亡率较TP组低;差异均具有统计学意义(P0.01, P0.05)。 6.雌二醇及丙酸睾酮对泪腺细胞MMP-9蛋白表达的影响 经H_2O_2诱导细胞凋亡后,泪腺细胞MMP-9蛋白表达累积光密度值在与BC组相比,AC组、E组、T组均明显增加;与AC组相比,E组与T组MMP-9蛋白表达量降低;E组蛋白表达量比T组明显降低;其差异均具有统计学意义(P0.01)。 7.泪腺上皮细胞凋亡率与MMP-9蛋白表达量的相关性分析 不同干预组间MMP-9蛋白表达量的变化与细胞凋亡率之间存在正相关关系(r=0.943, p0.01)。 结论 1.雌二醇及丙酸睾酮对H_2O_2诱导的兔泪腺上皮细胞凋亡均有抑制作用。 2.雌二醇对泪腺上皮细胞凋亡的抑制作用呈一定的剂量依赖性。 3.雌二醇对泪腺上皮细胞的抑制作用强于丙酸睾酮。 4.雌二醇及丙酸睾酮对H_2O_2诱导的泪腺上皮细胞凋亡后MMP-9的表达有一定的抑制作用。 5.雌二醇及丙酸睾酮干预后MMP-9蛋白表达量的变化与细胞凋亡率之间存在正相关关系。 6.雌二醇及丙酸睾酮对泪腺上皮细胞凋亡抑制作用的机制可能与MMP-9有关。
[Abstract]:Purpose

The mechanism of estradiol and testosterone on lacrimal gland epithelial cells was investigated by observing the expression of estradiol ( E2 ) and testosterone propionate ( TP ) in rabbit lacrimal gland epithelial cells cultured in vitro .

method

1 . Experimental grouping

1 ) The experimental groups were divided into four groups : estradiol group ( E2 group ) , testosterone propionate group ( TP group ) , apoptosis control group ( AC group ) , blank control group ( BC group ) .

2 ) The experiment was divided into the group of AC , 1x10 ~ ( -5 ) mol / L estradiol and testosterone treated with H _ 2O _ 2 - induced apoptosis .

2 . Rabbit lacrimal gland epithelial cell culture and HE staining

The cells of lacrimal gland were cultured in sterile rabbit lacrimal gland , the cells were cultured by tissue block culture and mixed digestion method , the cells were purified , the cells were observed and photographed under reversed microscope , and the morphology of cells was observed by HE staining .

3 . Identification of lacrimal gland epithelial cells

Immunocytochemistry was used to identify cells by using mouse anti - wide - spectrum cytokeratin monoclonal antibody .

4 . Single - staining method of hoechen 33342

The apoptosis of lacrimal gland epithelial cells was detected by single - staining method after drug intervention

5 . MTT assay for the detection of lacrimal gland epithelial cell activity

After intervention of testosterone and testosterone propionate at different concentrations , the activity of lacrimal gland cells was measured by MTT assay .

6 . Apoptosis of lacrimal gland epithelial cells was detected by annexin V / PI double staining method .

Flow Cytometry ( FCM ) was used to detect the apoptosis of cells and observe the effect of estradiol and testosterone on the apoptosis of lacrimal gland epithelial cells .

7 . Detection of MMP - 9 protein expression by immunohistochemical method

The effect of testosterone and testosterone propionate on the expression of MMP - 9 after induction of apoptosis in H _ 2O _ 2 induced by H _ 2O _ 2 was detected by immunohistochemical method .

Results

1 . Cell morphology of lacrimal gland epithelial cells and HE staining

After the culture , the cells were in good condition , the cell body was bright , the membrane was clear , the light - shielding property was stronger , HE staining observed that the epithelial cells of lacrimal gland were irregular polygon , the outline of the cell was clear , the nuclear staining was obvious , even the nucleolus is visible .

2 . Result of identification of lacrimal gland epithelial cells

The immunoreaction DAB of Pan - cytokeratin antibody showed that the brown positive particles were homogeneously distributed in the cytoplasm .

3 . Apoptosis of the cells was observed by the staining method in 33342 stain .

In the AC group , the cells presented high - intensity blue fluorescence , irregular and partial apoptosis . The cells in the blank control group had uniform oval staining and no obvious cell shedding ; the E _ 2 group and TP group had partial nuclear staining and high fluorescence .

4 . MTT assay for cell activity results

Compared with the control group , the activity of the lacrimal gland epithelial cells decreased significantly after the addition of 1 脳 10 ~ ( -4 ) mol / L H _ 2O _ 2 - induced apoptosis .

Effect of testosterone and testosterone on apoptosis of lacrimal gland epithelial cells

In the estradiol group , the apoptosis rate and the apoptosis rate of the cells were dose dependent , the higher the concentration and the lower the apoptosis rate , the apoptosis rate of the E2 group was lower than that in the TP group , and the difference was statistically significant ( P0.01 , P0.05 ) .

Effects of estradiol and testosterone on the expression of MMP - 9 protein in lacrimal gland cells

Compared with the AC group , the expression of MMP - 9 protein decreased significantly in both group E and group E , and the expression of E group was significantly lower than that in group T , and the difference was statistically significant ( P0.01 ) .

7 . Correlation between apoptosis rate of lacrimal gland epithelial cells and expression of MMP - 9 protein

There was a positive correlation between the expression of MMP - 9 and the rate of apoptosis in different intervention groups ( r = 0.943 , p0.01 ) .

Conclusion

1 . Both estradiol and testosterone inhibited the apoptosis of rabbit lacrimal gland epithelial cells induced by H _ 2O _ 2 .

2 . The inhibitory effect of estradiol on the apoptosis of lacrimal gland epithelial cells was dose - dependent .

3 . The inhibitory effect of estradiol on lacrimal gland epithelial cells was stronger than that of testosterone propionate .

4 . The expression of MMP - 9 induced by H _ 2O _ 2 induced by estradiol and testosterone inhibited the expression of MMP - 9 .

5 . There was a positive correlation between the changes of MMP - 9 protein expression and apoptosis rate after the intervention of estradiol and testosterone propionate .

6 . The mechanism of estradiol and testosterone inhibiting the apoptosis of lacrimal gland epithelial cells may be related to MMP - 9 .

【学位授予单位】:广州医学院
【学位级别】:硕士
【学位授予年份】:2011
【分类号】:R363

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