發(fā)布時間：2018-05-24 22:12

  本文選題：Smac + 固相合成　； 參考：《華中科技大學》2009年博士論文

【摘要】： 實驗一擬Smac促凋亡多肽的合成及其生物活性的初步研究 目的探討擬Smac多肽的合成及其對膀胱癌細胞的促凋亡生物活性。方法應用固相多肽合成技術，合成具有細胞膜穿透性SmacN7融合多肽，經RP-HPLC純化、純度分析，用質譜儀定性鑒定；通過熒光顯微鏡觀察細胞凋亡形態(tài)、細胞增殖抑制率測定及流式細胞儀分析，研究其對低劑量絲裂霉素C誘導的膀胱癌T24細胞的凋亡促進作用。結果可穿透性融合多肽SmacN7產物峰純度達95％以上，分子量為3278.08，質譜鑒定結果與合成預期結果完全一致；50μg／L～500μg／L SmacN7作用12h～48h，腫瘤細胞出現典型的凋亡形態(tài)學改變；隨著SmacN7濃度的增加或作用時間的延長，細胞增殖抑制率出現明顯增加，藥物作用12h、24h、48h后增殖抑制率分別為(9.62±1.07)％～(61.48±1.15)％、(24.17±1.02)％～(72.86±1.68)％、(43.24±1.15)％～(84.91±1.74)％：腫瘤細胞凋亡率也明顯增加，分別為(6.12±1.16)％～(49.81±2.11)％、(13.47±1.15)％～(64.54±2.27)％、(28.91±1.08)％～(82.36±2.19)％。結論固相合成的擬Smac融合多肽SmacN7為高純度的目的肽，能夠穩(wěn)定地轉入細胞內且利用率高，并有明顯促進低劑量絲裂霉素C誘導的膀胱癌T24細胞凋亡的生物活性，為進一步研究膀胱腫瘤的生物治療積累了有價值的資料。 實驗二人工合成擬Smac融合多肽促低劑量絲裂霉素C誘導膀胱癌細胞凋亡的研究 目的探討人工合成擬Smac融合多肽(SmacN7)對低劑量絲裂霉素C(MMC)誘導的膀胱癌T24細胞凋亡的促進作用。方法運用固相多肽合成技術人工合成SmacN7細胞可穿透性融合多肽，0.05mg／ml MMC誘導的膀胱癌T24細胞與50μg／L～500μg／L的SmacN7融合多肽共同孵育4h～48h后，采用Annexin—V熒光染色檢測腫瘤細胞凋亡；流式細胞術和MTT比色檢測誘導后T24細胞凋亡率、增殖抑制率與SmacN7的時間和濃度效應關系。結果50μg／L～500μg／L SmacN7作用4h～48h，腫瘤細胞出現典型的凋亡形態(tài)學改變，隨著SmacN7濃度的增加或藥物作用時間的延長，腫瘤細胞凋亡率也增加，12h為5.67％～56.12％，24h為14.54％～65.24％，48h為31.48％～87.23％，同時細胞增殖抑制率出現明顯增加，藥物作用12h、24h、48h后增殖抑制率分別為9.58％～63.42％、28.94％～72.3％、44.7％～87.12％。結論SmacN7能夠有效地促進低劑量絲裂霉素C誘導的膀胱癌T24細胞凋亡，并具有時間和濃度依賴性，為膀胱腫瘤的生物治療提供了新思路。 實驗三人工合成擬Smac多肽增強膀胱癌細胞化療敏感性的實驗研究 目的：探討人工合成擬Smac細胞可穿透性促凋亡融合多肽SmacN7對膀胱癌化療藥物敏感性的促進作用。方法：人工合成細胞可穿透性促凋亡融合多肽SmacN7；噻唑藍(MTT)檢測SmacN7融合多肽對低劑量絲裂霉素C(MMC)誘導的膀胱癌T24細胞的相對存活率；Annexin V／PI雙標流式細胞術檢測T24細胞的凋亡；Western blot檢測SmacN7融合多肽與MMC聯(lián)用后T24細胞內XIAP、Caspase-3蛋白的表達；同時檢測Caspase-3活性及SmacN7融合多肽與MMC聯(lián)用對T24細胞的殺傷作用。結果：SmacN7融合多肽能穿透細胞并與內源性XIAP結合，增加低劑量MMC誘導的T24細胞凋亡并呈時間和濃度依賴性；能顯著降低細胞內XIAP的表達水平，增強Caspase-3的表達及活性；在24h和48h，SmacN7+MMC組與單用MMC組相比，T24細胞的存活率分別降低2.22倍和3.61倍。結論：人工合成擬Smac細胞可穿透性促凋亡融合多肽能夠促進化療藥物誘導的膀胱癌T24細胞凋亡，抑制細胞增殖，增強膀胱癌細胞對MMC的化療藥物敏感性。 實驗四擬Smac合成肽羧甲基殼聚糖磁性納米復合物的制備及性能研究 目的探討擬Smac合成肽羧甲基殼聚糖磁性納米復合物的制備及聯(lián)合恒定外磁場體外對膀胱癌細胞的凋亡促進作用。方法以羧甲基殼聚糖為骨架與具有超順磁性的Fe_3O_4納米粒合成納米載體粒子，將擬Smac合成肽SmacN7與其結合，制備擬Smac合成肽羧甲基殼聚糖磁性納米復合物，并通過透射電鏡、振動樣磁強計等考察其理化性質；Hoechst33258染色觀察外加磁場下納米復合物誘導膀胱癌T24細胞凋亡的形態(tài)，MTT比色分析法觀察其對腫瘤細胞的生長抑制作用。結果擬Smac合成肽羧甲基殼聚糖磁性納米粒子的粒徑約為46.2nm；磁化曲線提示具有超順磁性；載藥量和包封率分別為(31.8±3.6)％和(65.2±2.4)％并具有良好的藥物控釋性能；外加磁場下磁性納米粒子能使腫瘤細胞呈現明顯的凋亡形態(tài)改變并表現出顯著的生長抑制活性。結論擬Smac合成肽羧甲基殼聚糖磁性納米復合物具有粒徑小、較強的磁響應性、載藥量和包封率高和良好的藥物緩釋性能，聯(lián)合恒定外磁場具有明顯的促進腫瘤細胞凋亡的作用，為膀胱腫瘤的生物治療提供了新的切入點。 實驗五擬Smac合成肽磁性納米顆粒誘導膀胱癌細胞凋亡的體外實驗研究 目的研究擬Smac合成肽磁性納米顆粒(SmacN7-O-CMC-MNP_S)的制備及其協(xié)同恒定外磁場體外對人膀胱癌T24細胞的生長抑制作用并探討其作用機制。方法氧化還原法制備SmacN7-O-CMC-MNP_S并檢測其理化性質，，倒置顯微鏡和電鏡觀察細胞形態(tài)，TUNEL法檢測細胞凋亡，MTT、流式細胞術分別觀察SmacN7-O-CMC-MNP_S聯(lián)合化療藥物及恒定外磁場體外對人膀胱癌T24細胞的生長抑制作用，SABC法觀察Bcl-2／Bax蛋白的表達，Western blot檢測XIAP蛋白的表達。結果SmacN7-O-CMC-MNP_s磁性納米顆粒直徑46.2nm，呈球形，載藥量(31.8±3.6)％，磁響應性良好。含相同濃度SmacN7的單純SmacN7-O-CMC-MNP_S對膀胱腫瘤T24細胞的生長抑制率和凋亡率無明顯影響，但協(xié)同外磁場及在化療藥物的誘導下其對腫瘤細胞的生長抑制作用明顯增強；Bcl-2蛋白表達水平下調同時Bax蛋白的表達增強，外磁場組與化療藥物組差異有統(tǒng)計學意義(P＜0.05)，協(xié)同外磁場時Western blot檢測不同時間XIAP蛋白的表達顯著降低。結論SmacN7-O-CMC-MNP_S的制備不影響SmacN7的生物活性；恒定外磁場下其對腫瘤細胞的促凋亡作用明顯增強，為膀胱腫瘤的生物靶向治療奠定了實驗基礎。 實驗六擬Smac合成肽磁性納米顆粒在膀胱癌裸鼠移植瘤靶向治療的體內實驗和機制研究 目的研究擬Smac合成肽磁性納米顆粒對膀胱癌裸鼠移植瘤的體內靶向治療作用并探討其機制。方法40只荷瘤裸鼠隨機分成5組，每組8只，按不同組別(A、B、C、D、E組)給于不同治療，各組每天治療1次，連續(xù)1wk，磁場組在腫瘤部位施加0.8T外磁場30min。觀察裸鼠的進食、活動及生長情況、測瘤體積并計算抑瘤率；治療后32d處死動物，HE染色和電鏡觀察細胞結構，采用SABC法觀察Bcl-2／Bax蛋白的表達，采用RT—PCR法檢測各組腫瘤組織XIAPmRNA和Caspase-3mRNA的表達，Western blot法檢測各組腫瘤組織XIAP和Caspase-3蛋白表達。結果治療期間及治療后各組裸鼠的進食、活動及生長情況未見明顯異常，體重差異無統(tǒng)計學意義(p＞0.05)，SmacN7-O-CMC-MNP_S磁性納米顆粒加MMC聯(lián)合外加磁場組(E組)移植瘤體積增長最為緩慢且對膀胱癌移植瘤有明顯抑制作用，抑瘤率為58.4％，高于SmacN7-O-CMC-MNP_S磁性納米顆粒加MMC組(C組)(24.6％)和SmacN7-O-CMC-MNP_S磁性納米顆粒加外加磁場組(D組)(32.2％)；免疫組化SABC法檢測E組Bcl-2蛋白表達水平下調同時Bax蛋白的表達增強，與C、D組比較差異有統(tǒng)計學意義(p＜0.05)；RT—PCR結果示E組能顯著下調XIAPmRNA的表達，同時上調Caspase-3mRNA的表達；Western blot顯示E組能下調XIAP蛋白的表達同時上調Caspase-3蛋白的表達。結論在外加磁場和小劑量絲裂霉素C的共同作用下，SmacN7-O-CMC-MNP_S磁性納米顆粒在體內具有明顯的體內促進腫瘤凋亡、抑制腫瘤生長的作用，通過下調XIAP、上調Caspase-3在mRNA和蛋白方面的表達達到腫瘤靶向治療效果，為膀胱腫瘤的治療探索出一條新的途徑。
[Abstract]:Study on the synthesis and biological activity of Smac Pro apoptotic polypeptide
Objective to investigate the synthesis of pseudo Smac polypeptide and its biological activity for promoting apoptosis of bladder cancer cells. Methods the solid phase polypeptide synthesis technology was used to synthesize the SmacN7 fusion peptide with cell membrane penetration, and purified by RP-HPLC, the purity analysis was qualitatively identified by mass spectrometer, and the apoptosis morphology and inhibition rate of cell proliferation were measured by fluorescence microscope. And flow cytometry analysis, study its effect on the apoptosis of bladder cancer T24 cells induced by Low Dose Mitomycin C. Results the purity of the SmacN7 product peak of the penetrable fusion polypeptide is above 95%, the molecular weight is 3278.08. The results of the mass spectrometric identification are exactly the same as the expected results; 50 mu g / L to 500 mu g / L SmacN7 functions 12h to 48h, tumor The cells showed typical morphological changes of apoptosis. With the increase of SmacN7 concentration or the prolongation of action time, the inhibitory rate of cell proliferation increased obviously. The inhibition rate of proliferation was (9.62 + 1.07)% ~ (61.48 + 1.15)%, (24.17 + 1.02)% ~ (72.86 + 1.68)% and (43.24 + 1.15)% ~ (84.91 + 1.74)% (84.91 + 1.74)%) after drug action (24.17 + 1.02) and (43.24 + 1.15)% - (84.91 + 1.74)%: swelling The apoptosis rate of tumor cells also increased significantly (6.12 + 1.16)% ~ (49.81 + 2.11)%, (13.47 + 1.15)% ~ (64.54 + 2.27)% and (28.91 + 1.08)% ~ (82.36 + 2.19)%. Conclusion the pseudo Smac fusion polypeptide SmacN7 synthesized by solid phase was high purity of the target peptide, which could be steadily transferred into the cell with high utilization rate and obviously promoted low dose silk. The biological activity of C induced apoptosis in bladder cancer T24 cells has accumulated valuable information for further study of bladder tumor biotherapy.
Experiment two synthetic Smac fusion peptide promotes mitomycin C induced apoptosis in bladder cancer cells
Objective to investigate the effect of synthetic Smac fusion peptide (SmacN7) on the apoptosis of T24 cells induced by Low Dose Mitomycin C (MMC) in bladder cancer cells. Methods using solid-phase peptide synthesis technique to synthesize penetrable fusion peptide of SmacN7 cells, 0.05mg / ml MMC induced T24 cells of bladder cancer and 50 micron g / L to 500 mu After the peptides were incubated for 4H to 48h, Annexin V fluorescence staining was used to detect the apoptosis of tumor cells. Flow cytometry and MTT colorimetric assay were used to detect the apoptosis rate of T24 cells and the relationship between the proliferation inhibition rate and the time and concentration effect of SmacN7. The results were 50 mu g / L to 500 mu g / L SmacN7. With the increase of SmacN7 concentration or the prolongation of drug action time, the apoptosis rate of tumor cells also increased, 12h was 5.67% ~ 56.12%, 24h was 14.54% ~ 65.24%, 48h was 31.48% ~ 87.23%, and the inhibitory rate of cell proliferation increased obviously. The inhibition rate of proliferation of 12h, 24h and 48h was 9.58% to 63.42%, 28.94% to 72. after 48h, respectively. 3%, 44.7% to 87.12%. conclusion SmacN7 can effectively promote the apoptosis of bladder cancer T24 cells induced by Low Dose Mitomycin C, and it has time and concentration dependence, which provides a new idea for the biological treatment of bladder tumor.
Experiment three. Synthetic Smac peptide enhances the chemosensitivity of bladder cancer cells
Objective: To investigate the effect of synthetic Smac cells penetrating apoptosis fusion polypeptide SmacN7 on chemotherapeutic drug sensitivity of bladder cancer. Methods: synthetic cell penetrable apoptosis fusion peptide SmacN7, and thiazolium blue (MTT) detection of SmacN7 fusion peptide on T24 cells induced by Low Dose Mitomycin C (MMC) induced by MMC. Annexin V / PI double standard flow cytometry was used to detect the apoptosis of T24 cells, and Western blot was used to detect the expression of XIAP, Caspase-3 protein in T24 cells after the combination of SmacN7 fusion peptide and MMC. The cell was combined with endogenous XIAP to increase the time and concentration dependence of T24 cells induced by low dose MMC, which could significantly reduce the expression level of XIAP and enhance the expression and activity of Caspase-3. In 24h and 48h, the survival rate of T24 cells was reduced by 2.22 times and 3.61 times, respectively, compared with the single MMC group. Conclusion: the survival rate of the T24 cells was reduced by 2.22 times and 3.61 times respectively. The synthetic Smac cell penetrable apoptotic fusion peptide can promote the apoptosis of bladder cancer T24 cells induced by chemotherapeutic drugs, inhibit cell proliferation and enhance the chemosensitivity of bladder cancer cells to MMC.
Experiment four preparation and properties of Smac synthesized peptide carboxymethyl chitosan magnetic nanocomposite
Objective to study the preparation of quasi Smac synthetic peptide Carboxymethyl Chitosan Magnetic Nanocomposites and the effect of combined constant external magnetic field on the apoptosis of bladder cancer cells in vitro. Methods carboxymethyl chitosan was used as the skeleton and superparamagnetic Fe_3O_4 nanoparticles to synthesize nanoscale nanoparticles, and the quasi Smac peptide SmacN7 was combined to prepare quasi Smac. The physical and chemical properties of synthetic peptide carboxymethyl chitosan magnetic nanocomposites were investigated by transmission electron microscopy, vibration sample magnetometer, and so on. Hoechst33258 staining was used to observe the morphology of the apoptosis of bladder cancer T24 cells induced by nanocomposites under the applied magnetic field. The inhibitory effect of MTT on the growth of tumor cells was observed by MTT colorimetric analysis. Results the synthetic peptide carboxyl was synthesized by quasi Smac The particle size of the methyl chitosan magnetic nanoparticles was about 46.2nm, and the magnetization curve indicated that the magnetic nanoparticles were superparamagnetic, and the drug loading and encapsulation efficiency were (31.8 + 3.6)% and (65.2 + 2.4)%, respectively, and had good drug controlled release properties. Conclusion the quasi Smac synthetic peptide carboxymethyl chitosan magnetic nanocomposites have small particle size, strong magnetic responsiveness, high drug loading, high encapsulation efficiency and good drug release properties. The combined constant external magnetic field can obviously promote the apoptosis of tumor cells, which provides a new approach to the biological treatment of bladder tumor. Point.
Experimental five Smac synthetic peptide magnetic nanoparticles induce apoptosis of bladder cancer cells in vitro
Objective to study the preparation of magnetic nano particles (SmacN7-O-CMC-MNP_S) of quasi Smac synthetic peptide (SmacN7-O-CMC-MNP_S) and its synergistic effect on the growth of human bladder cancer T24 cells in vitro and explore its mechanism. Methods to prepare SmacN7-O-CMC-MNP_S and detect its physical and chemical properties by oxidation-reduction method and observe cell morphology by inverted microscope and electron microscope, TUNEL method Detection of apoptosis, MTT, flow cytometry to observe the inhibitory effect of SmacN7-O-CMC-MNP_S combined with chemotherapeutic drugs and constant external magnetic field on human bladder cancer T24 cells. SABC method was used to observe the expression of Bcl-2 / Bax protein and Western blot to detect the expression of XIAP protein. Results SmacN7-O-CMC-MNP_s magnetic nanoparticles were spherical in diameter. The drug loading was (31.8 + 3.6)% and the magnetic responsiveness was good. The simple SmacN7-O-CMC-MNP_S containing the same concentration of SmacN7 had no significant effect on the growth inhibition rate and apoptosis rate of bladder tumor T24 cells, but the inhibitory effect of the external magnetic field and the chemotherapeutic drugs on the growth of the tumor cells was obviously enhanced, and the expression level of Bcl-2 protein was downregulated at the same time B The expression of ax protein was enhanced, and the difference between the external magnetic field group and the chemotherapeutic drug group was statistically significant (P < 0.05). The expression of XIAP protein was significantly decreased when the Western blot was detected in the external magnetic field at different time. Conclusion the preparation of SmacN7-O-CMC-MNP_S did not affect the bioactivity of SmacN7, and the apoptosis promoting effect on the tumor cells was significantly increased under the constant external magnetic field. It provides an experimental basis for biological targeted therapy of bladder tumor.
In vivo experiment and mechanism study of the six Smac synthetic peptide magnetic nanoparticles in targeted therapy of bladder cancer xenograft in nude mice
Objective to study the effect of quasi Smac synthetic peptide magnetic nanoparticles on the target treatment of xenografts in nude mice of bladder cancer and explore its mechanism. Methods 40 tumor bearing nude mice were randomly divided into 5 groups, 8 rats in each group were treated in different groups (A, B, C, D, E) for different treatments. Each group was treated for 1 times a day, 1wk was continuous, magnetic field group applied 30min outside the magnetic field 30min at the tumor site. Observe the feeding, activity and growth of nude mice, measure the tumor volume and calculate the tumor suppressor rate. After 32D, the animals were killed, the cell structure was observed by HE staining and electron microscopy. The expression of Bcl-2 / Bax protein was observed by SABC method. The expression of XIAPmRNA and Caspase-3mRNA in each group of tumor tissues was detected by RT PCR method, and Western blot method was used to detect the tumor tissues of each group. XIAP and Caspase-3 protein expression. Results during and after treatment, the feeding, activity and growth of nude mice were not significantly abnormal, and there was no significant difference in weight difference (P > 0.05). SmacN7-O-CMC-MNP_S magnetic nanoparticles plus MMC combined magnetic field group (E group) increased the volume of transplanted tumor most slowly and was obvious to the tumor of bladder cancer. The inhibition rate was 58.4%, which was higher than that of SmacN7-O-CMC-MNP_S magnetic nanoparticles plus MMC group (group C) (24.6%) and SmacN7-O-CMC-MNP_S magnetic nanoparticles plus magnetic field group (D group) (32.2%). The expression of Bcl-2 protein in E group was down regulated and the expression of Bax protein was enhanced by immunohistochemical SABC method, and the difference was statistically significant compared with C, D group. Meaning (P < 0.05); RT - PCR results showed that group E could significantly down regulate the expression of XIAPmRNA and increase the expression of Caspase-3mRNA; Western blot showed that the E group could down regulate the expression of XIAP protein and up regulate the expression of Caspase-3 protein. In vivo, the body has a significant role in promoting tumor apoptosis and inhibiting tumor growth. By down regulation of XIAP, up regulation of the expression of Caspase-3 in mRNA and protein to achieve the therapeutic effect of tumor targeting, and to explore a new way for the treatment of bladder tumor.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R341

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