豹皮樟总黄酮对睾丸间质细胞中奥沙利铂作用的影响及其可能机制

发布时间：2018-07-01 09:32

  本文选题：豹皮樟总黄酮 + 缝隙连接　； 参考：《蚌埠医学院》2014年硕士论文

【摘要】：目的： 1.观察豹皮樟总黄酮（Total Flavonoids of Litsea Coreana，TFLC）对睾丸间质正常/肿瘤细胞缝隙连接（Gap Junction，GJ）功能和连接蛋白43（connexin43，Cx43）表达的影响。 2.分别观察TFLC在睾丸间质肿瘤细胞（I-10）和睾丸间质正常细胞（TM_3）中对奥沙利铂（oxalipatin，OHP）细胞毒性的影响及其这种影响与GJ的关系。 3.探讨TFLC影响奥沙利铂抗增殖及诱导凋亡作用的可能机制。 方法： 1.TFLC对睾丸间质细胞中GJ功能的影响 1.1细胞接种荧光示踪法检测不同浓度TFLC对睾丸间质细胞GJ功能的影响。 1.2Western blotting和细胞免疫荧光法检测不同浓度的TFLC（0、5、10、20μg/ml）分别作用于睾丸间质肿瘤细胞（I-10）和睾丸间质正常细胞（TM3）24h，对细胞Cx43总蛋白和膜表面Cx43蛋白表达的影响。 2. TFLC对OHP细胞毒性的影响 2.1用接种不同密度细胞的方法，获得生长融合细胞（高密度，有条件形成GJ）与生长未融合细胞（低密度，无条件形成GJ），在上述不同接种密度的细胞中，采用MTT法分别观察OHP对I-10和TM3细胞的毒性。 2.2为了进一步验证TFLC对OHP细胞毒性的影响与GJ有关，我们采用siRNA法成功干扰掉两株细胞中Cx43蛋白后，检测TFLC对OHP细胞毒性的影响。 3. TFLC对OHP诱导细胞凋亡作用的影响 3.1在有GJ形成的条件下，先用20μg/ml TFLC预处理I-10和TM_3细胞24h，再用30μM OHP处理对应细胞8h，然后采用Annexin V/PI双染法观察TFLC对OHP诱导细胞凋亡率的变化。 3.2在有GJ形成的条件下，采用Hoechst33258荧光染色法检测20μg/ml TFLC作用细胞24h后，对OHP诱导细胞晚期凋亡情况的影响。 4. TFLC对OHP作用后凋亡相关蛋白Bax、Bcl-2的表达和caspase3、caspase9的影响 先用20μg/ml TFLC处理I-10和TM3细胞24h后，再用10μM OHP处理24h，然后采用Western blotting法检测凋亡相关蛋白Bax、Bcl-2的表达和caspase3、caspase9的剪切情况。 结果： 1. TFLC增强睾丸间质细胞（I-10和TM3）GJ功能 1.1细胞接种荧光示踪结果显示TFLC（0、5、10、20μg/ml）分别处理I-10和TM3细胞24h，可以显著增强GJ介导的细胞间荧光传递。在I-10细胞中，与对照组相比，TFLC处理后细胞的荧光传递功能分别增加了20.12%±20.06%（P0.05）、50.00%±7.30%（P0.05）和155.00%±13.40%（P0.01）；在TM3细胞中，与对照组相比，TFLC处理后细胞的荧光传递功能分别增加了36.50%±2.90%（P0.05）、85.00%±2.12%（P0.05）和180.00%±1.41%（P0.01）。结果表明，在I-10和TM3细胞中TFLC均可以显著增强GJ功能。 1.2采用Western blotting检测TFLC对两株睾丸细胞中Cx43蛋白的表达，结果显示：在睾丸肿瘤细胞I-10中，随着TFLC浓度的增加Cx43表达水平相应升高；同样在睾丸正常细胞TM3中，随着TFLC浓度的增加Cx43表达水平也相应升高。 1.3细胞免疫荧光的结果与Western blotting实验结果一致：在I-10细胞中，随着TFLC浓度的增加，膜表面Cx43表达水平相应升高；同样在TM3细胞中，随着TFLC浓度的增加，膜表面Cx43表达水平也相应升高。 2. TFLC对OHP细胞毒性的影响 2.1MTT法检测结果显示在高密度接种（有GJ形成）条件下，用20μg/ml TFLC增强GJ功能后，10μM OHP处理I-10和TM3细胞24h。在I-10细胞中，联合用药组细胞存活率显著低于OHP单用组（P0.01）；而在低密度接种（无GJ形成）条件下，联合用药组的细胞存活率与OHP单用组无显著差别（P0.05）。同时在TM3细胞中，联合用药组细胞存活率显著高于OHP单用组（P0.01）；而在低密度接种（无GJ形成）条件下，联合用药组的细胞存活率与OHP单用组无明显差异（P0.05）。 2.2siRNA法结果显示：在I-10细胞中，抑制Cx43后，TFLC和OHP的联合用药组的细胞存活率与OHP单用组无显著差异（P0.05），而在未转染时联合用药组细胞存活率显著低于OHP单用组（P0.05）；在TM3细胞中，抑制Cx43后，联合用药组的细胞存活率与OHP单用组无显著差异（P0.05），而在未转染时联合用药组细胞存活率明显高于OHP单用组（P0.05）。以上结果均表明，在I-10细胞中TFLC可以通过增强GJ功能而显著增强OHP的细胞毒性；而在TM3细胞中TFLC增强GJ功能反而会显著抑制OHP的 细胞毒性。 3. TFLC对OHP诱导细胞凋亡的影响 3.1Annexin V/PI双染结果显示在高密度接种（有GJ形成）条件下，在I-10细胞中，用20μg/ml TFLC增强GJ功能后，30μM OHP处理细胞8h，细胞凋亡率显著高于OHP单用组（P0.05）；而在TM3细胞中，用20μg/ml TFLC增强GJ功能后，30μM OHP处理细胞8h，细胞凋亡率显著低于OHP单用组（P0.01）。 3.2Hoechst33258荧光染色结果也显示有GJ形成的条件下，在I-10细胞中，用20μg/ml TFLC增强GJ功能后，20μM OHP处理细胞24h，细胞晚期凋亡率显著高于OHP单用组（P0.05）；而在TM3细胞中，用20μg/ml TFLC增强GJ功能后，20μM OHP处理细胞24h，细胞晚期凋亡率显著低于OHP单用组（P0.01），单用TFLC组与对照组的细胞凋亡率无显著差异。结果表明，在I-10细胞中TFLC可以显著增强OHP诱导的细胞凋亡作用，而在TM3细胞中TFLC明显减轻OHP诱导的细胞凋亡作用。 4. TFLC对OHP诱导细胞凋亡相关蛋白的影响 Western blotting结果显示：在I-10细胞中，20μg/ml TFLC预处理细胞24h后，与单用OHP组相比可以显著增强促凋亡蛋白Bax的表达，而抑制抑凋亡蛋白Bcl-2的表达。而在TM3细胞中，同样的处理，与单用OHP组相比，预处理可以明显降低促凋亡蛋白Bax的表达，而增强抑凋亡蛋白Bcl-2的表达。线粒体凋亡途径是细胞凋亡过程中的重要环节，，本实验还检测了caspase3、caspase9的表达。Western blotting结果显示：在I-10细胞中，与单用OHP组相比，联合用药组的caspase3和caspase9的剪切均明显增多；而在TM3细胞中，与单用OHP组相比，联合用药组的caspase3和caspase9的剪切均明显减少。 综合上述结果表明，在睾丸肿瘤细胞I-10中TFLC可以通过增强caspase3、caspase9的剪切和上调Bax/Bcl-2的表达而增强OHP的细胞毒性；而在睾丸正常细胞TM3中TFLC却减少caspase3、caspase9的剪切和下调Bax/Bcl-2的表达而减轻OHP的细胞毒性。 结论： 1. TFLC增强I-10和TM3细胞中GJ功能和Cx43蛋白的表达。 2.在睾丸肿瘤细胞I-10中TFLC增强OHP的细胞毒性，而在睾丸正常细胞TM3中TFLC却减轻OHP的细胞毒性。 3.在睾丸肿瘤细胞I-10中TFLC增强OHP诱导的细胞凋亡，而在睾丸正常细胞TM3中TFLC却减轻OHP诱导的细胞凋亡。 4. TFLC对睾丸细胞中奥沙利铂细胞毒性的影响可能与Bcl-2/Bax、 caspase3/9有关。
[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 % 卤 20.06 % ( P0.05 ) , 50.00 % 卤 7.30 % ( P0.05 ) and 155.00 % 卤 13.40 % ( P0.01 ) .
In TM3 cells , the fluorescence transfer function of TFLC cells increased by 36.50 % 卤 2.90 % ( P0.05 ) , 85.00 % 卤 2.12 % ( P0.05 ) and 180.00 % 卤 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缁嗚優涓

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