拟Smac新型促凋亡多肽分子对凋亡下游蛋白质组的影响及诱导凋亡的研究

发布时间：2018-05-24 22:12

本文选题：Smac + 固相合成　；参考：《华中科技大学》2009年博士论文

【摘要】： 实验一拟Smac促凋亡多肽的合成及其生物活性的初步研究目的探讨拟Smac多肽的合成及其对膀胱癌细胞的促凋亡生物活性。方法应用固相多肽合成技术，合成具有细胞膜穿透性SmacN7融合多肽，经RP-HPLC纯化、纯度分析，用质谱仪定性鉴定；通过荧光显微镜观察细胞凋亡形态、细胞增殖抑制率测定及流式细胞仪分析，研究其对低剂量丝裂霉素C诱导的膀胱癌T24细胞的凋亡促进作用。结果可穿透性融合多肽SmacN7产物峰纯度达95％以上，分子量为3278.08，质谱鉴定结果与合成预期结果完全一致；50μg／L～500μg／L SmacN7作用12h～48h，肿瘤细胞出现典型的凋亡形态学改变；随着SmacN7浓度的增加或作用时间的延长，细胞增殖抑制率出现明显增加，药物作用12h、24h、48h后增殖抑制率分别为(9.62±1.07)％～(61.48±1.15)％、(24.17±1.02)％～(72.86±1.68)％、(43.24±1.15)％～(84.91±1.74)％：肿瘤细胞凋亡率也明显增加，分别为(6.12±1.16)％～(49.81±2.11)％、(13.47±1.15)％～(64.54±2.27)％、(28.91±1.08)％～(82.36±2.19)％。结论固相合成的拟Smac融合多肽SmacN7为高纯度的目的肽，能够稳定地转入细胞内且利用率高，并有明显促进低剂量丝裂霉素C诱导的膀胱癌T24细胞凋亡的生物活性，为进一步研究膀胱肿瘤的生物治疗积累了有价值的资料。实验二人工合成拟Smac融合多肽促低剂量丝裂霉素C诱导膀胱癌细胞凋亡的研究目的探讨人工合成拟Smac融合多肽(SmacN7)对低剂量丝裂霉素C(MMC)诱导的膀胱癌T24细胞凋亡的促进作用。方法运用固相多肽合成技术人工合成SmacN7细胞可穿透性融合多肽，0.05mg／ml MMC诱导的膀胱癌T24细胞与50μg／L～500μg／L的SmacN7融合多肽共同孵育4h～48h后，采用Annexin—V荧光染色检测肿瘤细胞凋亡；流式细胞术和MTT比色检测诱导后T24细胞凋亡率、增殖抑制率与SmacN7的时间和浓度效应关系。结果50μg／L～500μg／L SmacN7作用4h～48h，肿瘤细胞出现典型的凋亡形态学改变，随着SmacN7浓度的增加或药物作用时间的延长，肿瘤细胞凋亡率也增加，12h为5.67％～56.12％，24h为14.54％～65.24％，48h为31.48％～87.23％，同时细胞增殖抑制率出现明显增加，药物作用12h、24h、48h后增殖抑制率分别为9.58％～63.42％、28.94％～72.3％、44.7％～87.12％。结论SmacN7能够有效地促进低剂量丝裂霉素C诱导的膀胱癌T24细胞凋亡，并具有时间和浓度依赖性，为膀胱肿瘤的生物治疗提供了新思路。实验三人工合成拟Smac多肽增强膀胱癌细胞化疗敏感性的实验研究目的：探讨人工合成拟Smac细胞可穿透性促凋亡融合多肽SmacN7对膀胱癌化疗药物敏感性的促进作用。方法：人工合成细胞可穿透性促凋亡融合多肽SmacN7；噻唑蓝(MTT)检测SmacN7融合多肽对低剂量丝裂霉素C(MMC)诱导的膀胱癌T24细胞的相对存活率；Annexin V／PI双标流式细胞术检测T24细胞的凋亡；Western blot检测SmacN7融合多肽与MMC联用后T24细胞内XIAP、Caspase-3蛋白的表达；同时检测Caspase-3活性及SmacN7融合多肽与MMC联用对T24细胞的杀伤作用。结果：SmacN7融合多肽能穿透细胞并与内源性XIAP结合，增加低剂量MMC诱导的T24细胞凋亡并呈时间和浓度依赖性；能显著降低细胞内XIAP的表达水平，增强Caspase-3的表达及活性；在24h和48h，SmacN7+MMC组与单用MMC组相比，T24细胞的存活率分别降低2.22倍和3.61倍。结论：人工合成拟Smac细胞可穿透性促凋亡融合多肽能够促进化疗药物诱导的膀胱癌T24细胞凋亡，抑制细胞增殖，增强膀胱癌细胞对MMC的化疗药物敏感性。实验四拟Smac合成肽羧甲基壳聚糖磁性纳米复合物的制备及性能研究目的探讨拟Smac合成肽羧甲基壳聚糖磁性纳米复合物的制备及联合恒定外磁场体外对膀胱癌细胞的凋亡促进作用。方法以羧甲基壳聚糖为骨架与具有超顺磁性的Fe_3O_4纳米粒合成纳米载体粒子，将拟Smac合成肽SmacN7与其结合，制备拟Smac合成肽羧甲基壳聚糖磁性纳米复合物，并通过透射电镜、振动样磁强计等考察其理化性质；Hoechst33258染色观察外加磁场下纳米复合物诱导膀胱癌T24细胞凋亡的形态，MTT比色分析法观察其对肿瘤细胞的生长抑制作用。结果拟Smac合成肽羧甲基壳聚糖磁性纳米粒子的粒径约为46.2nm；磁化曲线提示具有超顺磁性；载药量和包封率分别为(31.8±3.6)％和(65.2±2.4)％并具有良好的药物控释性能；外加磁场下磁性纳米粒子能使肿瘤细胞呈现明显的凋亡形态改变并表现出显著的生长抑制活性。结论拟Smac合成肽羧甲基壳聚糖磁性纳米复合物具有粒径小、较强的磁响应性、载药量和包封率高和良好的药物缓释性能，联合恒定外磁场具有明显的促进肿瘤细胞凋亡的作用，为膀胱肿瘤的生物治疗提供了新的切入点。实验五拟Smac合成肽磁性纳米颗粒诱导膀胱癌细胞凋亡的体外实验研究目的研究拟Smac合成肽磁性纳米颗粒(SmacN7-O-CMC-MNP_S)的制备及其协同恒定外磁场体外对人膀胱癌T24细胞的生长抑制作用并探讨其作用机制。方法氧化还原法制备SmacN7-O-CMC-MNP_S并检测其理化性质，，倒置显微镜和电镜观察细胞形态，TUNEL法检测细胞凋亡，MTT、流式细胞术分别观察SmacN7-O-CMC-MNP_S联合化疗药物及恒定外磁场体外对人膀胱癌T24细胞的生长抑制作用，SABC法观察Bcl-2／Bax蛋白的表达，Western blot检测XIAP蛋白的表达。结果SmacN7-O-CMC-MNP_s磁性纳米颗粒直径46.2nm，呈球形，载药量(31.8±3.6)％，磁响应性良好。含相同浓度SmacN7的单纯SmacN7-O-CMC-MNP_S对膀胱肿瘤T24细胞的生长抑制率和凋亡率无明显影响，但协同外磁场及在化疗药物的诱导下其对肿瘤细胞的生长抑制作用明显增强；Bcl-2蛋白表达水平下调同时Bax蛋白的表达增强，外磁场组与化疗药物组差异有统计学意义(P＜0.05)，协同外磁场时Western blot检测不同时间XIAP蛋白的表达显著降低。结论SmacN7-O-CMC-MNP_S的制备不影响SmacN7的生物活性；恒定外磁场下其对肿瘤细胞的促凋亡作用明显增强，为膀胱肿瘤的生物靶向治疗奠定了实验基础。实验六拟Smac合成肽磁性纳米颗粒在膀胱癌裸鼠移植瘤靶向治疗的体内实验和机制研究目的研究拟Smac合成肽磁性纳米颗粒对膀胱癌裸鼠移植瘤的体内靶向治疗作用并探讨其机制。方法40只荷瘤裸鼠随机分成5组，每组8只，按不同组别(A、B、C、D、E组)给于不同治疗，各组每天治疗1次，连续1wk，磁场组在肿瘤部位施加0.8T外磁场30min。观察裸鼠的进食、活动及生长情况、测瘤体积并计算抑瘤率；治疗后32d处死动物，HE染色和电镜观察细胞结构，采用SABC法观察Bcl-2／Bax蛋白的表达，采用RT—PCR法检测各组肿瘤组织XIAPmRNA和Caspase-3mRNA的表达，Western blot法检测各组肿瘤组织XIAP和Caspase-3蛋白表达。结果治疗期间及治疗后各组裸鼠的进食、活动及生长情况未见明显异常，体重差异无统计学意义(p＞0.05)，SmacN7-O-CMC-MNP_S磁性纳米颗粒加MMC联合外加磁场组(E组)移植瘤体积增长最为缓慢且对膀胱癌移植瘤有明显抑制作用，抑瘤率为58.4％，高于SmacN7-O-CMC-MNP_S磁性纳米颗粒加MMC组(C组)(24.6％)和SmacN7-O-CMC-MNP_S磁性纳米颗粒加外加磁场组(D组)(32.2％)；免疫组化SABC法检测E组Bcl-2蛋白表达水平下调同时Bax蛋白的表达增强，与C、D组比较差异有统计学意义(p＜0.05)；RT—PCR结果示E组能显著下调XIAPmRNA的表达，同时上调Caspase-3mRNA的表达；Western blot显示E组能下调XIAP蛋白的表达同时上调Caspase-3蛋白的表达。结论在外加磁场和小剂量丝裂霉素C的共同作用下，SmacN7-O-CMC-MNP_S磁性纳米颗粒在体内具有明显的体内促进肿瘤凋亡、抑制肿瘤生长的作用，通过下调XIAP、上调Caspase-3在mRNA和蛋白方面的表达达到肿瘤靶向治疗效果，为膀胱肿瘤的治疗探索出一条新的途径。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R341

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