本文選題：豹皮樟總黃酮 + 縫隙連接　； 參考：《蚌埠醫(yī)學院》2014年碩士論文

【摘要】：目的： 1.觀察豹皮樟總黃酮（Total Flavonoids of Litsea Coreana，TFLC）對睪丸間質(zhì)正常/腫瘤細胞縫隙連接（Gap Junction，GJ）功能和連接蛋白43（connexin43，Cx43）表達的影響。 2.分別觀察TFLC在睪丸間質(zhì)腫瘤細胞（I-10）和睪丸間質(zhì)正常細胞（TM_3）中對奧沙利鉑（oxalipatin，OHP）細胞毒性的影響及其這種影響與GJ的關系。 3.探討TFLC影響奧沙利鉑抗增殖及誘導凋亡作用的可能機制。 方法： 1.TFLC對睪丸間質(zhì)細胞中GJ功能的影響 1.1細胞接種熒光示蹤法檢測不同濃度TFLC對睪丸間質(zhì)細胞GJ功能的影響。 1.2Western blotting和細胞免疫熒光法檢測不同濃度的TFLC（0、5、10、20μg/ml）分別作用于睪丸間質(zhì)腫瘤細胞（I-10）和睪丸間質(zhì)正常細胞（TM3）24h，對細胞Cx43總蛋白和膜表面Cx43蛋白表達的影響。 2. TFLC對OHP細胞毒性的影響 2.1用接種不同密度細胞的方法，獲得生長融合細胞（高密度，有條件形成GJ）與生長未融合細胞（低密度，無條件形成GJ），在上述不同接種密度的細胞中，采用MTT法分別觀察OHP對I-10和TM3細胞的毒性。 2.2為了進一步驗證TFLC對OHP細胞毒性的影響與GJ有關，我們采用siRNA法成功干擾掉兩株細胞中Cx43蛋白后，檢測TFLC對OHP細胞毒性的影響。 3. TFLC對OHP誘導細胞凋亡作用的影響 3.1在有GJ形成的條件下，先用20μg/ml TFLC預處理I-10和TM_3細胞24h，再用30μM OHP處理對應細胞8h，然后采用Annexin V/PI雙染法觀察TFLC對OHP誘導細胞凋亡率的變化。 3.2在有GJ形成的條件下，采用Hoechst33258熒光染色法檢測20μg/ml TFLC作用細胞24h后，對OHP誘導細胞晚期凋亡情況的影響。 4. TFLC對OHP作用后凋亡相關蛋白Bax、Bcl-2的表達和caspase3、caspase9的影響 先用20μg/ml TFLC處理I-10和TM3細胞24h后，再用10μM OHP處理24h，然后采用Western blotting法檢測凋亡相關蛋白Bax、Bcl-2的表達和caspase3、caspase9的剪切情況。 結(jié)果： 1. TFLC增強睪丸間質(zhì)細胞（I-10和TM3）GJ功能 1.1細胞接種熒光示蹤結(jié)果顯示TFLC（0、5、10、20μg/ml）分別處理I-10和TM3細胞24h，可以顯著增強GJ介導的細胞間熒光傳遞。在I-10細胞中，與對照組相比，TFLC處理后細胞的熒光傳遞功能分別增加了20.12%±20.06%（P0.05）、50.00%±7.30%（P0.05）和155.00%±13.40%（P0.01）；在TM3細胞中，與對照組相比，TFLC處理后細胞的熒光傳遞功能分別增加了36.50%±2.90%（P0.05）、85.00%±2.12%（P0.05）和180.00%±1.41%（P0.01）。結(jié)果表明，在I-10和TM3細胞中TFLC均可以顯著增強GJ功能。 1.2采用Western blotting檢測TFLC對兩株睪丸細胞中Cx43蛋白的表達，結(jié)果顯示：在睪丸腫瘤細胞I-10中，隨著TFLC濃度的增加Cx43表達水平相應升高；同樣在睪丸正常細胞TM3中，隨著TFLC濃度的增加Cx43表達水平也相應升高。 1.3細胞免疫熒光的結(jié)果與Western blotting實驗結(jié)果一致：在I-10細胞中，隨著TFLC濃度的增加，膜表面Cx43表達水平相應升高；同樣在TM3細胞中，隨著TFLC濃度的增加，膜表面Cx43表達水平也相應升高。 2. TFLC對OHP細胞毒性的影響 2.1MTT法檢測結(jié)果顯示在高密度接種（有GJ形成）條件下，用20μg/ml TFLC增強GJ功能后，10μM OHP處理I-10和TM3細胞24h。在I-10細胞中，聯(lián)合用藥組細胞存活率顯著低于OHP單用組（P0.01）；而在低密度接種（無GJ形成）條件下，聯(lián)合用藥組的細胞存活率與OHP單用組無顯著差別（P0.05）。同時在TM3細胞中，聯(lián)合用藥組細胞存活率顯著高于OHP單用組（P0.01）；而在低密度接種（無GJ形成）條件下，聯(lián)合用藥組的細胞存活率與OHP單用組無明顯差異（P0.05）。 2.2siRNA法結(jié)果顯示：在I-10細胞中，抑制Cx43后，TFLC和OHP的聯(lián)合用藥組的細胞存活率與OHP單用組無顯著差異（P0.05），而在未轉(zhuǎn)染時聯(lián)合用藥組細胞存活率顯著低于OHP單用組（P0.05）；在TM3細胞中，抑制Cx43后，聯(lián)合用藥組的細胞存活率與OHP單用組無顯著差異（P0.05），而在未轉(zhuǎn)染時聯(lián)合用藥組細胞存活率明顯高于OHP單用組（P0.05）。以上結(jié)果均表明，在I-10細胞中TFLC可以通過增強GJ功能而顯著增強OHP的細胞毒性；而在TM3細胞中TFLC增強GJ功能反而會顯著抑制OHP的 細胞毒性。 3. TFLC對OHP誘導細胞凋亡的影響 3.1Annexin V/PI雙染結(jié)果顯示在高密度接種（有GJ形成）條件下，在I-10細胞中，用20μg/ml TFLC增強GJ功能后，30μM OHP處理細胞8h，細胞凋亡率顯著高于OHP單用組（P0.05）；而在TM3細胞中，用20μg/ml TFLC增強GJ功能后，30μM OHP處理細胞8h，細胞凋亡率顯著低于OHP單用組（P0.01）。 3.2Hoechst33258熒光染色結(jié)果也顯示有GJ形成的條件下，在I-10細胞中，用20μg/ml TFLC增強GJ功能后，20μM OHP處理細胞24h，細胞晚期凋亡率顯著高于OHP單用組（P0.05）；而在TM3細胞中，用20μg/ml TFLC增強GJ功能后，20μM OHP處理細胞24h，細胞晚期凋亡率顯著低于OHP單用組（P0.01），單用TFLC組與對照組的細胞凋亡率無顯著差異。結(jié)果表明，在I-10細胞中TFLC可以顯著增強OHP誘導的細胞凋亡作用，而在TM3細胞中TFLC明顯減輕OHP誘導的細胞凋亡作用。 4. TFLC對OHP誘導細胞凋亡相關蛋白的影響 Western blotting結(jié)果顯示：在I-10細胞中，20μg/ml TFLC預處理細胞24h后，與單用OHP組相比可以顯著增強促凋亡蛋白Bax的表達，而抑制抑凋亡蛋白Bcl-2的表達。而在TM3細胞中，同樣的處理，與單用OHP組相比，預處理可以明顯降低促凋亡蛋白Bax的表達，而增強抑凋亡蛋白Bcl-2的表達。線粒體凋亡途徑是細胞凋亡過程中的重要環(huán)節(jié)，，本實驗還檢測了caspase3、caspase9的表達。Western blotting結(jié)果顯示：在I-10細胞中，與單用OHP組相比，聯(lián)合用藥組的caspase3和caspase9的剪切均明顯增多；而在TM3細胞中，與單用OHP組相比，聯(lián)合用藥組的caspase3和caspase9的剪切均明顯減少。 綜合上述結(jié)果表明，在睪丸腫瘤細胞I-10中TFLC可以通過增強caspase3、caspase9的剪切和上調(diào)Bax/Bcl-2的表達而增強OHP的細胞毒性；而在睪丸正常細胞TM3中TFLC卻減少caspase3、caspase9的剪切和下調(diào)Bax/Bcl-2的表達而減輕OHP的細胞毒性。 結(jié)論： 1. TFLC增強I-10和TM3細胞中GJ功能和Cx43蛋白的表達。 2.在睪丸腫瘤細胞I-10中TFLC增強OHP的細胞毒性，而在睪丸正常細胞TM3中TFLC卻減輕OHP的細胞毒性。 3.在睪丸腫瘤細胞I-10中TFLC增強OHP誘導的細胞凋亡，而在睪丸正常細胞TM3中TFLC卻減輕OHP誘導的細胞凋亡。 4. TFLC對睪丸細胞中奧沙利鉑細胞毒性的影響可能與Bcl-2/Bax、 caspase3/9有關。
[Abstract]:Purpose :

1 . To observe the effects of total flavonoids of Litsea Coreana ( TFLC ) on the expression of Gap Junction and Cx43 in normal / tumor cells of the testis .

2 . The effects of TFLC on the cytotoxicity of oxaliplatin ( oxalipatin , OHP ) and the relationship between the effects of TFLC on the cytotoxicity of oxaliplatin ( oxalipatin , OHP ) were observed .

3 . To investigate the possible mechanism of TFLC on the anti - proliferation and apoptosis of oxaliplatin .

Method :

1 . Effect of TFLC on the function of bone marrow cells in rat testis

1 . The effects of different concentrations of TFLC were detected by cell inoculation fluorescence tracing method .

1.2 Western blotting and immunofluorescence assay were used to detect the effects of different concentrations of TFLC ( 0 , 5 , 10 , 20 渭g / ml ) on the expression of Cx43 protein and Cx43 protein in human testis stromal tumor cells ( I - 10 ) and normal testis cells ( TM3 ) .

2 . Effect of TFLC on OHP cytotoxicity

2.1 Using the method of inoculating different density cells , the growth - fused cells ( high - density , conditional - forming ) and unfused cells ( low - density and unconditional ) were obtained . In the above - mentioned cells with different densities , the toxicity of OHP on I - 10 and TM3 cells was observed by MTT method .

2.2 In order to further verify the effect of TFLC on OHP cytotoxicity , we successfully interfered with the effect of TFLC on OHP cytotoxicity by interfering with the expression of Cx43 protein in two cells successfully .

3 . Effect of TFLC on OHP - induced Apoptosis

3.1 After pretreatment with 20 渭g / ml TFLC , I - 10 and TM _ 3 cells were pretreated with 20 渭g / ml TFLC for 24 h , then treated with 30 渭M OHP for 8 h .

3.2 After 24 h of 24 h of TFLC , the effect of 20 渭g / ml TFLC cells on the apoptosis of OHP - induced cells was studied .

4 . Effects of TFLC on the Expression of Bax , Bcl - 2 and caspase3 and caspase9 after OHP

After 24 hours of treatment I - 10 and TM3 cells with 20 渭g / ml TFLC , the expression of Bax , Bcl - 2 and caspase3 and caspase9 were detected by Western blotting .

Results :

1 . TFLC enhances the functions of testicular interstitial cells ( I - 10 and TM3 )

1 . The results showed that TFLC ( 0 , 5 , 10 , 20 渭g / ml ) treated with TFLC ( 0 , 5 , 10 , 20 渭g / ml ) could significantly enhance inter - cell fluorescence transfer . In I - 10 cells , the fluorescence transfer function of TFLC cells increased by 20.12 % 鹵 20.06 % ( P0.05 ) , 50.00 % 鹵 7.30 % ( P0.05 ) and 155.00 % 鹵 13.40 % ( P0.01 ) .
In TM3 cells , the fluorescence transfer function of TFLC cells increased by 36.50 % 鹵 2.90 % ( P0.05 ) , 85.00 % 鹵 2.12 % ( P0.05 ) and 180.00 % 鹵 1.41 % ( P0.01 ) .

1.2 Expression of Cx43 protein in testicular cells was detected by Western blotting . The results showed that the level of Cx43 expression increased with the increase of TFLC concentration in tumor cells I - 10 .
Similarly , in TM3 , the level of Cx43 expression increased with the increase of TFLC concentration .

1 . The results of cell immunofluorescence were consistent with that of Western blotting : in I - 10 cells , the level of Cx43 expression increased correspondingly with the increase of TFLC concentration .
Also in TM3 cells , the level of Cx43 expression increased correspondingly with the increase of TFLC concentration .

2 . Effect of TFLC on OHP cytotoxicity

2 . The results of MTT assay showed that 10 渭M OHP treated I - 10 and TM3 cells for 24 h with 20 渭g / ml TFLC in high density inoculation . The survival rate of cells in combination therapy group was significantly lower than that in OHP alone group ( P0.01 ) .
At the same time , in TM3 cells , the survival rate of combined group cells was significantly higher than that in OHP alone group ( P0.01 ) .
However , there was no significant difference between the cell survival rate and OHP alone group ( P0.05 ) .

2 . The results of siRNA showed that in I - 10 cells , the cell survival rate of TFLC and OHP was significantly lower than that in OHP alone group ( P0.05 ) , but the survival rate of combined group cells was significantly lower than that in OHP alone group ( P0.05 ) .
In TM3 cells , there was no significant difference between the survival rate of OHP alone and OHP alone ( P0.05 ) . The results showed that TFLC could significantly enhance the cytotoxicity of OHP in I - 10 cells .
In TM3 cells , TFLC could significantly inhibit OHP .

Cytotoxicity .

3 . Effect of TFLC on OHP - induced Apoptosis

3 . The results showed that under the condition of high density inoculation , the cell apoptosis rate was significantly higher than that of OHP alone group ( P0.05 ) .
In TM3 cells , the cell apoptosis rate was significantly lower than that of OHP alone group ( P0.01 ) .

3 . The results of the fluorescent staining showed that the apoptotic rate of the cells was significantly higher than that in OHP alone group ( P0.05 ) .
The results showed that TFLC could significantly enhance the apoptosis of OHP - induced apoptosis in TM3 cells , while TFLC significantly reduced OHP - induced apoptosis in TM3 cells .

4 . Effects of TFLC on Apoptosis - related Proteins in OHP - induced Apoptosis

Western blotting showed that the expression of Bax and caspase - 3 and caspase - 9 could be significantly decreased compared with the single OHP group after 24 h of pretreatment with 20 渭g / ml TFLC , but the expression of Bcl - 2 was enhanced in TM3 cells .
鑰屽湪TM3緇嗚優(yōu)涓

本文編號：2087337