本文選題：乳源抗菌--免疫調(diào)節(jié)融合肽　切入點(diǎn)：卵巢癌　出處：《安徽醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的探討乳源抗菌-免疫調(diào)節(jié)融合肽（antibacterial-immunomodulating fusionpeptide，AIFP）對卵巢癌細(xì)胞增殖、凋亡和周期的影響，探究其可能的分子機(jī)制，并初步研究其抗菌活性。 方法免疫熒光染色鑒定原代培養(yǎng)細(xì)胞的純度；MTT法檢測AIFP對卵巢癌細(xì)胞增殖的影響；流式細(xì)胞技術(shù)檢測AIFP對卵巢癌細(xì)胞凋亡和周期的影響；RT-PCR和Western Blot檢測AIFP對卵巢癌細(xì)胞Akt、CDC25C、cyclinB1、bax和Bcl-xl基因和相關(guān)蛋白表達(dá)水平影響；細(xì)菌生長曲線的繪制和紙片瓊脂糖擴(kuò)散法檢測AIFP的抗菌活性。 結(jié)果免疫熒光鑒定原代卵巢癌細(xì)胞細(xì)胞純度的鑒定結(jié)果顯示，原代卵巢癌細(xì)胞的純度可達(dá)84.61%，可以進(jìn)行下一步實(shí)驗(yàn)。MTT法顯示，AIFP作用于SKOV3細(xì)胞24、48和72h的ID50分為2.75×10-4mg/ml、3.96×10-5mg/ml和3.96×10-6mg/ml，與對陰性照組相比較，具有時(shí)間和劑量依賴性，流式細(xì)胞技術(shù)結(jié)果顯示：AIFP的濃度為5×10-3mg/ml，作用于原代卵巢癌細(xì)胞24、48和72h的凋亡率分別可達(dá)6.10%、19.31%和40.63%，與陰性對照組相比較差異用統(tǒng)計(jì)學(xué)意義（p＜0.05）。AIFP的濃度為×10-3mg/ml，作用于SKOV3細(xì)胞24、48和72h的凋亡率分別可為2.82%、7.82%和29.81%，與陰性對照組比較差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。用5×10-3mg/mlAIFP作用于SKOV3細(xì)胞24、48和72h后G2/M期延長（分別為7.15%、8.81%、12.00%）與空白對照組（2.32%、2.93%、3.02%）比較差異有統(tǒng)計(jì)學(xué)意義，用5×10-6mg/ml AIFP作用于SKOV3細(xì)胞24、48和72h后，G2/M期延長（分別為5.25%、6.90%、8.14%），與空白對照組（2.32%、2.93%、3.02%）比較差異有統(tǒng)計(jì)學(xué)意義。PCR實(shí)驗(yàn)顯示，AIFP作用于卵巢癌細(xì)胞后，Akt、CDC25C、cyclinB1、BcL-xl基因的表達(dá)水平下調(diào)， Bax的表達(dá)水平上調(diào)，與空白對照組比較差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。Western Blot實(shí)驗(yàn)結(jié)果顯示，Akt、CDC25C、cyclinB1和BcL-xl蛋白表達(dá)水平上升，Bax蛋白表達(dá)水平下降的，與空白對照組比較差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。AIFP組大腸桿菌生長曲線的穩(wěn)定區(qū)段位于對照組的下方，表明細(xì)菌生長受到抑制。紙片瓊脂糖擴(kuò)散實(shí)驗(yàn)中，AIFP組出現(xiàn)抑菌環(huán)，抑菌環(huán)的直徑小于氨芐青霉素組，但明顯大于空白對照組，且差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。 結(jié)論1、成功培養(yǎng)了原代卵巢癌細(xì)胞，經(jīng)鑒定其純度可以進(jìn)行下一步實(shí)驗(yàn)。 2、AIFP能夠抑制原代卵巢癌細(xì)胞和SKOV3細(xì)胞株的增殖。 3、AIFP能夠促進(jìn)卵巢癌細(xì)胞的凋亡。 4、AIFP對卵巢癌細(xì)胞周期的影響表現(xiàn)在其可以將卵巢癌細(xì)胞周期阻止在G2/M期 5、AIFP可以下調(diào)卵巢癌細(xì)胞Akt、CDC25C、cyclinB1、BcL-xl基因及蛋白的表達(dá)水平，，上調(diào)Bax基因及蛋白的表達(dá)水平。 6、AIFP影響大腸桿菌的生長曲線，對大腸桿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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.31

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