由Cx43组成的缝隙连接在耐顺铂睾丸癌细胞侵袭转移中的作用
发布时间：2019-01-18 16:56
【摘要】：目的:观察由Cx43组成的缝隙连接(gap junction,GJ)在耐顺铂睾丸癌细胞侵袭转移中的作用。方法:利用浓度递增法逐步诱导耐药细胞株I-10/DDP,用不同浓度的顺铂(0,2,4,8,16,32,64,128μM)分别作用小鼠睾丸癌敏感株(I-10)和小鼠睾丸癌耐顺铂耐药株(I-10/DDP)24 h后采用MTT法测定细胞存活率,计算耐药指数。1.采用Western blotting法检测I-10和I-10/DDP中耐药相关蛋白:多药耐药蛋白1(MDR1)及P-糖蛋白(P-gp)的表达。2.免疫荧光法检测I-10和I-10/DDP细胞膜Cx43的表达。3.荧光示踪法(Parachute)检测细胞间荧光传递功能(即GJ功能),以及工具药改变GJ功能后对GJ功能的调控作用。4.采用si RNA干扰的方法下调Cx43的表达;用过表达的方法上调Cx43的表达。5.采用Transwell法检测I-10和I-10/DDP的侵袭转移能力,以及工具药直接调控改变GJ功能与干扰和过表达Cx43间接改变GJ功能后细胞的侵袭转移能力。结果:1.耐药株的建立I-10/DDP的IC50为84.171μM,I-10的IC50为21.451μM。耐药指数RI为3.924;耐药细胞中耐药相关蛋白MDR1及P-gp表达显著高于正常I-10细胞。结合以上两个结果表明:I-10/DDP对顺铂产生了获得性耐药。2.细胞膜Cx43的表达与I-10相比,I-10/DDP细胞膜Cx43的表达量显著降低。3.GJ功能与I-10相比,I-10/DDP细胞间的GJ功能明显降低(*P0.05)。4.侵袭转移能力与I-10相比,I-10/DDP细胞的侵袭转移能力明显增强(**P0.01)。5.调控GJ功能对侵袭转移的影响10μM RA增强GJ功能,25μM Oleamide降低GJ功能。RA抑制细胞的侵袭转移能力(*P0.05;**P0.01),Oleamide增强细胞的侵袭转移能力(*P0.05)。6.分子生物学方法调控Cx43表达对侵袭转移的影响沉默Cx43可明显增强细胞的侵袭转移能力(*P0.05)。过表达Cx43可明显抑制细胞的侵袭转移能力(*P0.05)。结论:1.Cx43表达量降低可能与睾丸癌I-10对顺铂获得性耐药的产生有关;2.Cx43组成的缝隙连接可抑制睾丸癌耐药株I-10/DDP细胞的侵袭转移能力。
[Abstract]:Aim: to investigate the role of gap junction (gap junction,GJ) composed of Cx43 in the invasion and metastasis of cisplatin resistant testicular cancer cells. Methods: drug resistant cell line I-10 / DDP was induced by increasing concentration, and different concentrations of cisplatin were used to induce the drug resistance cell line I-10 / DDP, and the drug resistance cell line I-10 / DDP was treated with different concentrations of cisplatin. Mice testicular carcinoma sensitive strain (I-10) and mouse testicular carcinoma resistant Cisplatin resistant strain (I-10/DDP) were treated with 1632Ti64128 渭 M for 24 h. The cell survival rate was measured by MTT assay, and the drug resistance index was calculated. 1. The expression of multidrug resistance protein 1 (MDR1) and P- glycoprotein (P-gp) in I-10 and I-10/DDP were detected by Western blotting assay. 2. Immunofluorescence assay was used to detect the expression of Cx43 on cell membrane of I-10 and I-10/DDP. 3. 3. Fluorescence tracer (Parachute) was used to detect the intercellular fluorescence transfer function (GJ function) and the regulatory effect of the tool drug on the GJ function after changing the GJ function. 4. Si RNA interference was used to down-regulate the expression of Cx43, and overexpression was used to up-regulate the expression of Cx43. The invasiveness and metastasis of I-10 and I-10/DDP were detected by Transwell assay, and the ability of invasion and metastasis of the cells after direct regulation of GJ and indirect alteration of GJ function by interfering and overexpression of Cx43 was detected. Results: 1. The IC50 of I-10/DDP was 84.171 渭 m ~ (-1) and the IC50 of 84.171 渭 M ~ (-1) I-10 was 21.451 渭 m ~ (-1). The drug resistance index (RI) was 3.924, and the expression of MDR1 and P-gp in resistant cells was significantly higher than that in normal I-10 cells. Combined with the above two results, I-10/DDP produced acquired resistance to cisplatin. 2. 2. Compared with I-10, the expression of Cx43 in I-10/DDP cell membrane was significantly lower than that of I-10. Compared with I-10, the GJ function of I-10/DDP cells was significantly lower than that of I-10 (* P0.05). Compared with I-10, the invasion and metastasis ability of I-10/DDP cells was significantly increased (* P0.01). The effect of GJ on invasion and Metastasis: 10 渭 M RA enhanced the function of GJ, 25 渭 M Oleamide decreased the function of GJ. RA inhibited the ability of invasion and metastasis of the cells (* P0.05P0.01), Oleamide enhanced the ability of invasion and metastasis (* P0.05). Effects of Cx43 expression on invasion and Metastasis by Molecular Biological methods silencing Cx43 could significantly enhance the ability of cell invasion and metastasis (* P0.05). Overexpression of Cx43 significantly inhibited the ability of cell invasion and metastasis (* P0.05). Conclusion: the decrease of 1.Cx43 expression may be related to the production of cisplatin acquired resistance to cisplatin in testicular carcinoma I-10, and the gap junction of 2.Cx43 can inhibit the invasion and metastasis of I-10/DDP cell line.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.21
本文编号：2410920
[Abstract]:Aim: to investigate the role of gap junction (gap junction,GJ) composed of Cx43 in the invasion and metastasis of cisplatin resistant testicular cancer cells. Methods: drug resistant cell line I-10 / DDP was induced by increasing concentration, and different concentrations of cisplatin were used to induce the drug resistance cell line I-10 / DDP, and the drug resistance cell line I-10 / DDP was treated with different concentrations of cisplatin. Mice testicular carcinoma sensitive strain (I-10) and mouse testicular carcinoma resistant Cisplatin resistant strain (I-10/DDP) were treated with 1632Ti64128 渭 M for 24 h. The cell survival rate was measured by MTT assay, and the drug resistance index was calculated. 1. The expression of multidrug resistance protein 1 (MDR1) and P- glycoprotein (P-gp) in I-10 and I-10/DDP were detected by Western blotting assay. 2. Immunofluorescence assay was used to detect the expression of Cx43 on cell membrane of I-10 and I-10/DDP. 3. 3. Fluorescence tracer (Parachute) was used to detect the intercellular fluorescence transfer function (GJ function) and the regulatory effect of the tool drug on the GJ function after changing the GJ function. 4. Si RNA interference was used to down-regulate the expression of Cx43, and overexpression was used to up-regulate the expression of Cx43. The invasiveness and metastasis of I-10 and I-10/DDP were detected by Transwell assay, and the ability of invasion and metastasis of the cells after direct regulation of GJ and indirect alteration of GJ function by interfering and overexpression of Cx43 was detected. Results: 1. The IC50 of I-10/DDP was 84.171 渭 m ~ (-1) and the IC50 of 84.171 渭 M ~ (-1) I-10 was 21.451 渭 m ~ (-1). The drug resistance index (RI) was 3.924, and the expression of MDR1 and P-gp in resistant cells was significantly higher than that in normal I-10 cells. Combined with the above two results, I-10/DDP produced acquired resistance to cisplatin. 2. 2. Compared with I-10, the expression of Cx43 in I-10/DDP cell membrane was significantly lower than that of I-10. Compared with I-10, the GJ function of I-10/DDP cells was significantly lower than that of I-10 (* P0.05). Compared with I-10, the invasion and metastasis ability of I-10/DDP cells was significantly increased (* P0.01). The effect of GJ on invasion and Metastasis: 10 渭 M RA enhanced the function of GJ, 25 渭 M Oleamide decreased the function of GJ. RA inhibited the ability of invasion and metastasis of the cells (* P0.05P0.01), Oleamide enhanced the ability of invasion and metastasis (* P0.05). Effects of Cx43 expression on invasion and Metastasis by Molecular Biological methods silencing Cx43 could significantly enhance the ability of cell invasion and metastasis (* P0.05). Overexpression of Cx43 significantly inhibited the ability of cell invasion and metastasis (* P0.05). Conclusion: the decrease of 1.Cx43 expression may be related to the production of cisplatin acquired resistance to cisplatin in testicular carcinoma I-10, and the gap junction of 2.Cx43 can inhibit the invasion and metastasis of I-10/DDP cell line.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.21
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