乳源抗菌—免疫调节融合肽对人卵巢癌细胞的影响

发布时间：2018-03-10 04:34

本文选题：乳源抗菌--免疫调节融合肽　切入点：卵巢癌　出处：《安徽医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的探讨乳源抗菌-免疫调节融合肽（antibacterial-immunomodulating fusionpeptide，AIFP）对卵巢癌细胞增殖、凋亡和周期的影响，探究其可能的分子机制，并初步研究其抗菌活性。方法免疫荧光染色鉴定原代培养细胞的纯度；MTT法检测AIFP对卵巢癌细胞增殖的影响；流式细胞技术检测AIFP对卵巢癌细胞凋亡和周期的影响；RT-PCR和Western Blot检测AIFP对卵巢癌细胞Akt、CDC25C、cyclinB1、bax和Bcl-xl基因和相关蛋白表达水平影响；细菌生长曲线的绘制和纸片琼脂糖扩散法检测AIFP的抗菌活性。结果免疫荧光鉴定原代卵巢癌细胞细胞纯度的鉴定结果显示，原代卵巢癌细胞的纯度可达84.61%，可以进行下一步实验。MTT法显示，AIFP作用于SKOV3细胞24、48和72h的ID50分为2.75×10-4mg/ml、3.96×10-5mg/ml和3.96×10-6mg/ml，与对阴性照组相比较，具有时间和剂量依赖性，流式细胞技术结果显示：AIFP的浓度为5×10-3mg/ml，作用于原代卵巢癌细胞24、48和72h的凋亡率分别可达6.10%、19.31%和40.63%，与阴性对照组相比较差异用统计学意义（p＜0.05）。AIFP的浓度为×10-3mg/ml，作用于SKOV3细胞24、48和72h的凋亡率分别可为2.82%、7.82%和29.81%，与阴性对照组比较差异有统计学意义（P＜0.05）。用5×10-3mg/mlAIFP作用于SKOV3细胞24、48和72h后G2/M期延长（分别为7.15%、8.81%、12.00%）与空白对照组（2.32%、2.93%、3.02%）比较差异有统计学意义，用5×10-6mg/ml AIFP作用于SKOV3细胞24、48和72h后，G2/M期延长（分别为5.25%、6.90%、8.14%），与空白对照组（2.32%、2.93%、3.02%）比较差异有统计学意义。PCR实验显示，AIFP作用于卵巢癌细胞后，Akt、CDC25C、cyclinB1、BcL-xl基因的表达水平下调， Bax的表达水平上调，与空白对照组比较差异有统计学意义（P＜0.05）。Western Blot实验结果显示，Akt、CDC25C、cyclinB1和BcL-xl蛋白表达水平上升，Bax蛋白表达水平下降的，与空白对照组比较差异有统计学意义（P＜0.05）。AIFP组大肠杆菌生长曲线的稳定区段位于对照组的下方，表明细菌生长受到抑制。纸片琼脂糖扩散实验中，AIFP组出现抑菌环，抑菌环的直径小于氨苄青霉素组，但明显大于空白对照组，且差异有统计学意义（P＜0.05）。结论1、成功培养了原代卵巢癌细胞，经鉴定其纯度可以进行下一步实验。 2、AIFP能够抑制原代卵巢癌细胞和SKOV3细胞株的增殖。 3、AIFP能够促进卵巢癌细胞的凋亡。 4、AIFP对卵巢癌细胞周期的影响表现在其可以将卵巢癌细胞周期阻止在G2/M期 5、AIFP可以下调卵巢癌细胞Akt、CDC25C、cyclinB1、BcL-xl基因及蛋白的表达水平，，上调Bax基因及蛋白的表达水平。 6、AIFP影响大肠杆菌的生长曲线，对大肠杆菌有一定的抗菌作用。
[Abstract]:Objective to investigate the effects of anti-bacterial and immunomodulating fusion peptide (AIFP) from milk source on proliferation, apoptosis and cell cycle of ovarian cancer cells, to explore its possible molecular mechanism, and to study its antibacterial activity. Methods the effects of AIFP on the proliferation of ovarian cancer cells were detected by immunofluorescence staining. The effect of AIFP on apoptosis and cell cycle of ovarian cancer cells was detected by flow cytometry. RT-PCR and Western Blot were used to detect the effect of AIFP on the expression of gene and Bcl-xl genes and related proteins in AkttCDC25Cncyclin B1. The bacterial growth curve and disk agarose diffusion method were used to detect the antibacterial activity of AIFP. Results the purity of primary ovarian cancer cells was identified by immunofluorescence assay. The purity of primary ovarian cancer cells was up to 84.61. The further experiment. MTT method showed that the ID50 of SKOV3 cells treated with AIFP for 24 h and 72 h were 2.75 脳 10-4 mg / ml and 3.96 脳 10-5 mg / ml and 3.96 脳 10-6 mg / ml, respectively, which were in a time-and dose-dependent manner as compared with the negative group. Flow cytometry showed that the concentration of 5 脳 10 ~ (-3) mg / ml of AIFP was 5 脳 10 ~ (-3) mg / ml, and the apoptotic rates of primary ovarian cancer cell line 24448 and 72 h were 6.1010 ~ 19.31% and 40.63%, respectively. Compared with the negative control group, the concentration of AIFP was less than 0.05 mg / ml, the concentration of AIFP was 脳 10 ~ (-3) mg / ml, and the effect on SKOV3 cells was 244.48 mg / ml. The apoptotic rates were 2.82% and 29.81%, respectively, and there was significant difference between the two groups (P < 0.05). After treated with 5 脳 10-3 mg / ml AIFP for 2448 and 72 h, the G2 / M phase of SKOV3 cells was prolonged (7.158.81 / 12.00, respectively) and that of the blank control group (2.32mg / ml) was 2.32 / 2.93% and 3.02, respectively, compared with that of the control group (2.32% 2.93%, P < 0.05), which was significantly different from that of the control group (7.158.81% 12.00) after treated with 5 脳 10 -3 mg / ml AIFP for 24 48 h and 72 h later, respectively. After treated with 5 脳 10 ~ (-6) mg / ml AIFP for 24 h and 72 h, the G _ 2 / M phase was prolonged (5.25% 6.90 and 8.14%, respectively, as compared with the blank control group, 2.32mg / ml AIFP). The results showed that the expression level of BcL-xl gene was down-regulated and the expression of Bax was up-regulated after SKOV3 cells were treated with 5 脳 10 ~ (-6) mg / ml AIFP, and the expression level of BcL-xl gene was down-regulated in SKOV3 cells after treatment with 5 脳 10 ~ (-6) mg 路ml / ml AIFP (5.25% 6.90%, respectively), and the expression of Bax was up-regulated after treatment with 5 脳 10 ~ (-6) mg / ml AIFP. Compared with the control group, the difference was statistically significant (P < 0.05). The results of Western Blot test showed that the expression level of BcL-xl and CDC25 cyclin B1 increased and the expression of Bax protein decreased. Compared with the blank control group, the stable section of the growth curve of Escherichia coli in the AIFP group was lower than that in the control group, indicating that the bacterial growth was inhibited. In the disk agarose diffusion test, the bacteriostasis ring appeared in the AIFP group. The diameter of bacteriostasis ring was smaller than that of ampicillin group, but significantly larger than that of blank control group, and the difference was statistically significant (P < 0.05). Conclusion 1. Primary ovarian cancer cells were successfully cultured and their purity could be tested in the next step. 2. 2 AIFP can inhibit the proliferation of primary ovarian cancer cells and SKOV3 cells. AIFP can promote apoptosis of ovarian cancer cells. 4 the effect of AIFP on ovarian cancer cell cycle is demonstrated by its ability to block ovarian cancer cell cycle in G 2 / M phase. 5AIFP could down-regulate the expression of Bax gene and protein and up-regulate the expression of Bax gene and protein. AIFP affects the growth curve of Escherichia coli and has a certain antibacterial effect on Escherichia coli.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

【参考文献】