本文選題：雌二醇 + 丙酸睪酮�。� 參考：《廣州醫(yī)學(xué)院》2011年碩士論文

【摘要】：目的 本課題擬通過觀察體外培養(yǎng)的兔淚腺上皮細(xì)胞經(jīng)雌二醇(Estradiol,E_2)及丙酸睪酮(Testosterone Propionate,TP)干預(yù)后細(xì)胞凋亡情況及基質(zhì)金屬蛋白酶(Matrix Metalloproteinase-9,MMP-9)蛋白表達(dá)情況,探討雌二醇及丙酸睪酮對(duì)兔淚腺上皮細(xì)胞的作用機(jī)制,為進(jìn)一步研究雌激素及雄激素對(duì)干眼癥的影響奠定基礎(chǔ)。 方法 1.實(shí)驗(yàn)分組 1)實(shí)驗(yàn)分四組,分別為雌二醇組(E2組)、丙酸睪酮組(TP組)、凋亡對(duì)照組(AC組)、空白對(duì)照組(BC組)。E2組、TP組分別給予1x10~(-5)、1x10~(-6)、1x10~(-7)、1x10~(-8)mol/L雌二醇、丙酸睪酮干預(yù), AC組只給予H_2O_2誘導(dǎo)凋亡;BC組即無藥物干預(yù)也無誘導(dǎo)凋亡。 2)實(shí)驗(yàn)分為只H_2O_2誘導(dǎo)凋亡的AC組,1x10~(-5)mol/L雌二醇、丙酸睪酮處理的E組、T組,未加任何干預(yù)的BC組。 2.兔淚腺上皮細(xì)胞培養(yǎng)及HE染色 無菌摘取兔淚腺組織,用組織塊培養(yǎng)法及混合消化法培養(yǎng)淚腺細(xì)胞,純化細(xì)胞、倒置顯微鏡下觀察及拍照, HE染色觀察細(xì)胞形態(tài)。 3.淚腺上皮細(xì)胞鑒定 采用鼠抗廣譜細(xì)胞角蛋白單克隆抗體進(jìn)行免疫細(xì)胞化學(xué)法鑒定細(xì)胞。 4. Hoechst 33342單染法 藥物干預(yù)后Hoechst 33342單染法定性檢測(cè)淚腺上皮細(xì)胞的凋亡 5. MTT法檢測(cè)淚腺上皮細(xì)胞活性 不同濃度的雌二醇及丙酸睪酮干預(yù)后,用MTT法檢測(cè)的OD值顯示不同組間淚腺細(xì)胞的活性。 6. Annexin V/PI雙染法檢測(cè)淚腺上皮細(xì)胞凋亡 藥物干預(yù)后,流式細(xì)胞術(shù)(Flow Cytometry, FCM)檢測(cè)分析細(xì)胞的凋亡,觀察雌二醇及丙酸睪酮對(duì)淚腺上皮細(xì)胞凋亡的影響。 7.免疫細(xì)胞化學(xué)法檢測(cè)細(xì)胞MMP-9蛋白表達(dá) 免疫細(xì)胞化學(xué)法檢測(cè)雌二醇及丙酸睪酮對(duì)H_2O_2誘導(dǎo)凋亡后MMP-9蛋白表達(dá)的影響。 結(jié)果 1.淚腺上皮細(xì)胞培養(yǎng)情況及HE染色觀察細(xì)胞形態(tài) 培養(yǎng)后細(xì)胞狀態(tài)良好,胞體透亮、胞膜清晰、遮光性較強(qiáng),HE染色觀察淚腺上皮細(xì)胞呈不規(guī)則多邊形,細(xì)胞輪廓清晰,核染明顯,偶可見核仁。 2.淚腺上皮細(xì)胞鑒定結(jié)果 Pan-cytokeratin抗體免疫反應(yīng)DAB顯色見棕色陽(yáng)性顆粒均勻分布于細(xì)胞漿中。 3. Hoechst 33342染色觀察細(xì)胞凋亡情況 AC組中細(xì)胞呈現(xiàn)高強(qiáng)藍(lán)色熒光,不規(guī)則,且部分細(xì)胞凋亡脫落;空白對(duì)照組中細(xì)胞呈均勻的橢圓形染色,無明顯細(xì)胞脫落;E_2組及TP組有部分核染高熒光。 4. MTT檢測(cè)細(xì)胞活性結(jié)果 與空白對(duì)照組相比,加入1×10~(-4)mol/L H_2O_2誘導(dǎo)細(xì)胞凋亡后,淚腺上皮細(xì)胞活性明顯下降,OD值有統(tǒng)計(jì)學(xué)差異(P0.01);與AC組相比,同時(shí)加入不同濃度的雌二醇、丙酸睪酮后淚腺上皮細(xì)胞活性增強(qiáng),總體而言E_2組與TP組細(xì)胞活性的OD值分別為0.4434±0.0981、0.3928±0.0518,雌二醇與丙酸睪酮對(duì)H_2O_2誘導(dǎo)的細(xì)胞凋亡具有抑制作用,其差異有統(tǒng)計(jì)學(xué)意義(P0.05);與丙酸睪酮組相比,雌二醇組抑制細(xì)胞凋亡作用強(qiáng),有統(tǒng)計(jì)學(xué)意義(P0.05);其中,雌二醇對(duì)細(xì)胞的保護(hù)作用呈一定的劑量依賴性。 5.雌二醇及丙酸睪酮對(duì)淚腺上皮細(xì)胞凋亡的影響 不同濃度的雌二醇及丙酸睪酮對(duì)淚腺上皮細(xì)胞早晚期凋亡及壞死均有抑制作用,其凋亡率都較凋亡對(duì)照組低;且雌二醇組中,細(xì)胞的凋亡率與濃度呈一定的劑量依賴性,濃度越高,凋亡率越小;E2組凋亡率較TP組低;差異均具有統(tǒng)計(jì)學(xué)意義(P0.01, P0.05)。 6.雌二醇及丙酸睪酮對(duì)淚腺細(xì)胞MMP-9蛋白表達(dá)的影響 經(jīng)H_2O_2誘導(dǎo)細(xì)胞凋亡后,淚腺細(xì)胞MMP-9蛋白表達(dá)累積光密度值在與BC組相比,AC組、E組、T組均明顯增加;與AC組相比,E組與T組MMP-9蛋白表達(dá)量降低;E組蛋白表達(dá)量比T組明顯降低;其差異均具有統(tǒng)計(jì)學(xué)意義(P0.01)。 7.淚腺上皮細(xì)胞凋亡率與MMP-9蛋白表達(dá)量的相關(guān)性分析 不同干預(yù)組間MMP-9蛋白表達(dá)量的變化與細(xì)胞凋亡率之間存在正相關(guān)關(guān)系(r=0.943, p0.01)。 結(jié)論 1.雌二醇及丙酸睪酮對(duì)H_2O_2誘導(dǎo)的兔淚腺上皮細(xì)胞凋亡均有抑制作用。 2.雌二醇對(duì)淚腺上皮細(xì)胞凋亡的抑制作用呈一定的劑量依賴性。 3.雌二醇對(duì)淚腺上皮細(xì)胞的抑制作用強(qiáng)于丙酸睪酮。 4.雌二醇及丙酸睪酮對(duì)H_2O_2誘導(dǎo)的淚腺上皮細(xì)胞凋亡后MMP-9的表達(dá)有一定的抑制作用。 5.雌二醇及丙酸睪酮干預(yù)后MMP-9蛋白表達(dá)量的變化與細(xì)胞凋亡率之間存在正相關(guān)關(guān)系。 6.雌二醇及丙酸睪酮對(duì)淚腺上皮細(xì)胞凋亡抑制作用的機(jī)制可能與MMP-9有關(guān)。
[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 .

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

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本文編號(hào)：1896502