本文選題：雌二醇 + 丙酸睪酮　； 參考：《廣州醫(yī)學院》2011年碩士論文

【摘要】：目的 本課題擬通過觀察體外培養(yǎng)的兔淚腺上皮細胞經(jīng)雌二醇(Estradiol,E_2)及丙酸睪酮(Testosterone Propionate,TP)干預后細胞凋亡情況及基質(zhì)金屬蛋白酶(Matrix Metalloproteinase-9,MMP-9)蛋白表達情況,探討雌二醇及丙酸睪酮對兔淚腺上皮細胞的作用機制,為進一步研究雌激素及雄激素對干眼癥的影響奠定基礎(chǔ)。 方法 1.實驗分組 1)實驗分四組,分別為雌二醇組(E2組)、丙酸睪酮組(TP組)、凋亡對照組(AC組)、空白對照組(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, AC組只給予H_2O_2誘導凋亡;BC組即無藥物干預也無誘導凋亡。 2)實驗分為只H_2O_2誘導凋亡的AC組,1x10~(-5)mol/L雌二醇、丙酸睪酮處理的E組、T組,未加任何干預的BC組。 2.兔淚腺上皮細胞培養(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)淚腺細胞,純化細胞、倒置顯微鏡下觀察及拍照, HE染色觀察細胞形態(tài)。 3.淚腺上皮細胞鑒定 采用鼠抗廣譜細胞角蛋白單克隆抗體進行免疫細胞化學法鑒定細胞。 4. Hoechst 33342單染法 藥物干預后Hoechst 33342單染法定性檢測淚腺上皮細胞的凋亡 5. MTT法檢測淚腺上皮細胞活性 不同濃度的雌二醇及丙酸睪酮干預后,用MTT法檢測的OD值顯示不同組間淚腺細胞的活性。 6. Annexin V/PI雙染法檢測淚腺上皮細胞凋亡 藥物干預后,流式細胞術(shù)(Flow Cytometry, FCM)檢測分析細胞的凋亡,觀察雌二醇及丙酸睪酮對淚腺上皮細胞凋亡的影響。 7.免疫細胞化學法檢測細胞MMP-9蛋白表達 免疫細胞化學法檢測雌二醇及丙酸睪酮對H_2O_2誘導凋亡后MMP-9蛋白表達的影響。 結(jié)果 1.淚腺上皮細胞培養(yǎng)情況及HE染色觀察細胞形態(tài) 培養(yǎng)后細胞狀態(tài)良好,胞體透亮、胞膜清晰、遮光性較強,HE染色觀察淚腺上皮細胞呈不規(guī)則多邊形,細胞輪廓清晰,核染明顯,偶可見核仁。 2.淚腺上皮細胞鑒定結(jié)果 Pan-cytokeratin抗體免疫反應DAB顯色見棕色陽性顆粒均勻分布于細胞漿中。 3. Hoechst 33342染色觀察細胞凋亡情況 AC組中細胞呈現(xiàn)高強藍色熒光,不規(guī)則,且部分細胞凋亡脫落;空白對照組中細胞呈均勻的橢圓形染色,無明顯細胞脫落;E_2組及TP組有部分核染高熒光。 4. MTT檢測細胞活性結(jié)果 與空白對照組相比,加入1×10~(-4)mol/L H_2O_2誘導細胞凋亡后,淚腺上皮細胞活性明顯下降,OD值有統(tǒng)計學差異(P0.01);與AC組相比,同時加入不同濃度的雌二醇、丙酸睪酮后淚腺上皮細胞活性增強,總體而言E_2組與TP組細胞活性的OD值分別為0.4434±0.0981、0.3928±0.0518,雌二醇與丙酸睪酮對H_2O_2誘導的細胞凋亡具有抑制作用,其差異有統(tǒng)計學意義(P0.05);與丙酸睪酮組相比,雌二醇組抑制細胞凋亡作用強,有統(tǒng)計學意義(P0.05);其中,雌二醇對細胞的保護作用呈一定的劑量依賴性。 5.雌二醇及丙酸睪酮對淚腺上皮細胞凋亡的影響 不同濃度的雌二醇及丙酸睪酮對淚腺上皮細胞早晚期凋亡及壞死均有抑制作用,其凋亡率都較凋亡對照組低;且雌二醇組中,細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)計學意義(P0.01, P0.05)。 6.雌二醇及丙酸睪酮對淚腺細胞MMP-9蛋白表達的影響 經(jīng)H_2O_2誘導細胞凋亡后,淚腺細胞MMP-9蛋白表達累積光密度值在與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蛋白表達量降低;E組蛋白表達量比T組明顯降低;其差異均具有統(tǒng)計學意義(P0.01)。 7.淚腺上皮細胞凋亡率與MMP-9蛋白表達量的相關(guān)性分析 不同干預組間MMP-9蛋白表達量的變化與細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H_2O_2誘導的兔淚腺上皮細胞凋亡均有抑制作用。 2.雌二醇對淚腺上皮細胞凋亡的抑制作用呈一定的劑量依賴性。 3.雌二醇對淚腺上皮細胞的抑制作用強于丙酸睪酮。 4.雌二醇及丙酸睪酮對H_2O_2誘導的淚腺上皮細胞凋亡后MMP-9的表達有一定的抑制作用。 5.雌二醇及丙酸睪酮干預后MMP-9蛋白表達量的變化與細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淚腺上皮細胞凋亡抑制作用的機制可能與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 .

【學位授予單位】：廣州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363

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