【摘要】：目的:觀察由Cx43組成的縫隙連接(gap junction,GJ)在耐順鉑睪丸癌細(xì)胞侵襲轉(zhuǎn)移中的作用。方法:利用濃度遞增法逐步誘導(dǎo)耐藥細(xì)胞株I-10/DDP,用不同濃度的順鉑(0,2,4,8,16,32,64,128μM)分別作用小鼠睪丸癌敏感株(I-10)和小鼠睪丸癌耐順鉑耐藥株(I-10/DDP)24 h后采用MTT法測定細(xì)胞存活率,計算耐藥指數(shù)。1.采用Western blotting法檢測I-10和I-10/DDP中耐藥相關(guān)蛋白:多藥耐藥蛋白1(MDR1)及P-糖蛋白(P-gp)的表達(dá)。2.免疫熒光法檢測I-10和I-10/DDP細(xì)胞膜Cx43的表達(dá)。3.熒光示蹤法(Parachute)檢測細(xì)胞間熒光傳遞功能(即GJ功能),以及工具藥改變GJ功能后對GJ功能的調(diào)控作用。4.采用si RNA干擾的方法下調(diào)Cx43的表達(dá);用過表達(dá)的方法上調(diào)Cx43的表達(dá)。5.采用Transwell法檢測I-10和I-10/DDP的侵襲轉(zhuǎn)移能力,以及工具藥直接調(diào)控改變GJ功能與干擾和過表達(dá)Cx43間接改變GJ功能后細(xì)胞的侵襲轉(zhuǎn)移能力。結(jié)果:1.耐藥株的建立I-10/DDP的IC50為84.171μM,I-10的IC50為21.451μM。耐藥指數(shù)RI為3.924;耐藥細(xì)胞中耐藥相關(guān)蛋白MDR1及P-gp表達(dá)顯著高于正常I-10細(xì)胞。結(jié)合以上兩個結(jié)果表明:I-10/DDP對順鉑產(chǎn)生了獲得性耐藥。2.細(xì)胞膜Cx43的表達(dá)與I-10相比,I-10/DDP細(xì)胞膜Cx43的表達(dá)量顯著降低。3.GJ功能與I-10相比,I-10/DDP細(xì)胞間的GJ功能明顯降低(*P0.05)。4.侵襲轉(zhuǎn)移能力與I-10相比,I-10/DDP細(xì)胞的侵襲轉(zhuǎn)移能力明顯增強(qiáng)(**P0.01)。5.調(diào)控GJ功能對侵襲轉(zhuǎn)移的影響10μM RA增強(qiáng)GJ功能,25μM Oleamide降低GJ功能。RA抑制細(xì)胞的侵襲轉(zhuǎn)移能力(*P0.05;**P0.01),Oleamide增強(qiáng)細(xì)胞的侵襲轉(zhuǎn)移能力(*P0.05)。6.分子生物學(xué)方法調(diào)控Cx43表達(dá)對侵襲轉(zhuǎn)移的影響沉默Cx43可明顯增強(qiáng)細(xì)胞的侵襲轉(zhuǎn)移能力(*P0.05)。過表達(dá)Cx43可明顯抑制細(xì)胞的侵襲轉(zhuǎn)移能力(*P0.05)。結(jié)論:1.Cx43表達(dá)量降低可能與睪丸癌I-10對順鉑獲得性耐藥的產(chǎn)生有關(guān);2.Cx43組成的縫隙連接可抑制睪丸癌耐藥株I-10/DDP細(xì)胞的侵襲轉(zhuǎn)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.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R737.21

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