牛黄体细胞与C6胶质瘤细胞共培养及凋亡相关基因在大鼠黄体中的表达
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摘要
为了探讨黄体细胞分泌物抑制肿瘤细胞增殖的可能性及机制,本研究建立黄体细胞培养模型,通过黄体细胞与C6胶质瘤细胞共培养以及给培养的C6胶质瘤细胞中分别添加OT、PG-Cl和5-HT的方法,观察黄体细胞及黄体分泌物对C6胶质瘤细胞增殖的影响;为了探讨黄体细胞凋亡相关基因表达变化,进一步研究黄体细胞的凋亡机理,本研究采用RT-PCR方法对SD大鼠不同期黄体p21、p53、K-ras和c-Myc mRNA的表达进行检测。主要结果如下:
1.成功建立黑白花奶牛黄体细胞培养模型。牛黄体细胞组织块法培养效果较好,贴壁细胞数较多,消化法细胞贴壁数不多。
2.成功建立SD大鼠黄体细胞培养模型。SD大鼠黄体细胞培养消化法效果较好,组织块法培养速度较慢。
3. C6胶质瘤细胞与黑白花奶牛黄体细胞共培养后出现空泡化现象,存活率与时间和黄体细胞浓度呈负相关,提示C6胶质瘤细胞空泡化现象与黄体分泌物有关。
4. OT可以抑制C6胶质瘤细胞增殖并引起C6胶质瘤细胞空泡化。PGF2α类似物PG-Cl可以抑制C6胶质瘤细胞增殖,但不引起C6胶质瘤细胞空泡化。5-HT对C6胶质瘤细胞增殖几乎没有影响。
5. p21、p53、K-ras和c-Myc基因mRNA在SD大鼠黄体妊娠第5 d、15 d及分娩后第3 d(即第20 d)均有表达,不同时间表达量不同,p21从妊娠第5 d到分娩后第3 d先降后增,c-Myc略有降低;p53先增后降,且分娩后第3d下降幅度异常大,结果提示存在其他基因或机制抑制p53表达;K-ras表达一直升高,分娩后第3d增加幅度异常大。
结论:牛黄体细胞分泌物对C6胶质瘤细胞增殖具有一定程度的抑制作用;OT、PGF2α的类似物PG-Cl可以抑制C6胶质瘤细胞增殖且OT能够引起C6胶质瘤细胞空泡化;p21和c-Myc mRNA在妊娠第5 d、15 d及分娩后第3 d(第20 d)变化幅度不大;p53先增后降,分娩后第3 d下降幅度异常大,提示有其他基因或机制抑制p53表达;K-ras表达一直升高,分娩后第3 d上调幅度异常大,提示K-ras在黄体退化中可能发挥重要作用。
In order to study the inhibitory effect of luteal cell secretions on proliferation of cultured C6 glioma cells, the holstein dairy cattle’s luteal cells were cocultured with C6 glioma cells. The C6 glioma cells were treated with OT, PG-Cl and 5-HT and the inhibitory rate were measured to investigate the inhibited mechanism of luteal cell secretions. What’s more, the mRNA expression of p21, p53, c-Myc, and K-ras were investigated to explore the change of apoptosis related genes in corpus luteum by RT-PCR method. The main results as follows:
1. The model of luteal cells of holstein dairy cattle was established and the result showed that tissue block method was better than enzyme digestion method.
2. The model of luteal cell of SD rat was established and the result showed that enzyme digestion method was better than tissue block method.
3. The C6 rat glioma cells were vacuolizated by cocultured method with holstein dairy cattle luteal cells and the survival rate of C6 glioma cells was negatively correlated with time and luteal cells’concentration. The results showed that vacuolization of C6 Glioma cells were related with the luteal cell secretions.
4. The proliferation of C6 glioma cells could be inhibited by OT and PG-Cl, but not by 5-HT. The vacuolization of C6 glioma cells were found in OT treated group.
5. p21, p53, K-ras and c-Myc mRNA were expressed in the 5 d, 15 d, and 3 d after delivery. The expression of P21 was increased from 5 d to 15 d but decreased in 3 d after delivery and the expression of c-Myc was decreased lightly from 5 d to 3 d after delivery. The K-ras’s expression was increased significantly from 5 d to 3 d after delivery and the p53’s expression was increased lightly from 5 d to 15 d but decreased obviously in 3d after delivery.
Conclusion: The C6 glioma cells’proliferation was inhibited by bovine’s luteal cell secretions. The OT and PG-Cl could inhibit C6 glioma cells’proliferation in different concentration and the inhitied rate of C6 glioma cell were positively correlated with time under suitable concentration of OT and PG-Cl. p21, p53, K-ras and c-Myc mRNA were expressed in the 5 d, 15 d, and 3 d after delivery. The expression of P21 was increased from 5 d to 15 d but decreased in 3 d after delivery and the expression of c-Myc was decreased lightly from 5 d to 3 d after delivery. The K-ras’s expression was increased significantly from 5 d to 3 d after delivery and the p53’s expression was increased lightly from 5 d to 15 d but decreased obviously in 3 d after delivery.
1.成功建立黑白花奶牛黄体细胞培养模型。牛黄体细胞组织块法培养效果较好,贴壁细胞数较多,消化法细胞贴壁数不多。
2.成功建立SD大鼠黄体细胞培养模型。SD大鼠黄体细胞培养消化法效果较好,组织块法培养速度较慢。
3. C6胶质瘤细胞与黑白花奶牛黄体细胞共培养后出现空泡化现象,存活率与时间和黄体细胞浓度呈负相关,提示C6胶质瘤细胞空泡化现象与黄体分泌物有关。
4. OT可以抑制C6胶质瘤细胞增殖并引起C6胶质瘤细胞空泡化。PGF2α类似物PG-Cl可以抑制C6胶质瘤细胞增殖,但不引起C6胶质瘤细胞空泡化。5-HT对C6胶质瘤细胞增殖几乎没有影响。
5. p21、p53、K-ras和c-Myc基因mRNA在SD大鼠黄体妊娠第5 d、15 d及分娩后第3 d(即第20 d)均有表达,不同时间表达量不同,p21从妊娠第5 d到分娩后第3 d先降后增,c-Myc略有降低;p53先增后降,且分娩后第3d下降幅度异常大,结果提示存在其他基因或机制抑制p53表达;K-ras表达一直升高,分娩后第3d增加幅度异常大。
结论:牛黄体细胞分泌物对C6胶质瘤细胞增殖具有一定程度的抑制作用;OT、PGF2α的类似物PG-Cl可以抑制C6胶质瘤细胞增殖且OT能够引起C6胶质瘤细胞空泡化;p21和c-Myc mRNA在妊娠第5 d、15 d及分娩后第3 d(第20 d)变化幅度不大;p53先增后降,分娩后第3 d下降幅度异常大,提示有其他基因或机制抑制p53表达;K-ras表达一直升高,分娩后第3 d上调幅度异常大,提示K-ras在黄体退化中可能发挥重要作用。
In order to study the inhibitory effect of luteal cell secretions on proliferation of cultured C6 glioma cells, the holstein dairy cattle’s luteal cells were cocultured with C6 glioma cells. The C6 glioma cells were treated with OT, PG-Cl and 5-HT and the inhibitory rate were measured to investigate the inhibited mechanism of luteal cell secretions. What’s more, the mRNA expression of p21, p53, c-Myc, and K-ras were investigated to explore the change of apoptosis related genes in corpus luteum by RT-PCR method. The main results as follows:
1. The model of luteal cells of holstein dairy cattle was established and the result showed that tissue block method was better than enzyme digestion method.
2. The model of luteal cell of SD rat was established and the result showed that enzyme digestion method was better than tissue block method.
3. The C6 rat glioma cells were vacuolizated by cocultured method with holstein dairy cattle luteal cells and the survival rate of C6 glioma cells was negatively correlated with time and luteal cells’concentration. The results showed that vacuolization of C6 Glioma cells were related with the luteal cell secretions.
4. The proliferation of C6 glioma cells could be inhibited by OT and PG-Cl, but not by 5-HT. The vacuolization of C6 glioma cells were found in OT treated group.
5. p21, p53, K-ras and c-Myc mRNA were expressed in the 5 d, 15 d, and 3 d after delivery. The expression of P21 was increased from 5 d to 15 d but decreased in 3 d after delivery and the expression of c-Myc was decreased lightly from 5 d to 3 d after delivery. The K-ras’s expression was increased significantly from 5 d to 3 d after delivery and the p53’s expression was increased lightly from 5 d to 15 d but decreased obviously in 3d after delivery.
Conclusion: The C6 glioma cells’proliferation was inhibited by bovine’s luteal cell secretions. The OT and PG-Cl could inhibit C6 glioma cells’proliferation in different concentration and the inhitied rate of C6 glioma cell were positively correlated with time under suitable concentration of OT and PG-Cl. p21, p53, K-ras and c-Myc mRNA were expressed in the 5 d, 15 d, and 3 d after delivery. The expression of P21 was increased from 5 d to 15 d but decreased in 3 d after delivery and the expression of c-Myc was decreased lightly from 5 d to 3 d after delivery. The K-ras’s expression was increased significantly from 5 d to 3 d after delivery and the p53’s expression was increased lightly from 5 d to 15 d but decreased obviously in 3 d after delivery.
引文
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