叶酸修饰壳聚糖小干扰RNA纳米粒靶向逆转卵巢癌多药耐药的研究
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摘要
第一部分叶酸-壳聚糖复合载体的制备及性质鉴定
目的合成叶酸修饰壳聚糖复合载体，并鉴定叶酸-壳聚糖复合载体的成功合成，测定叶酸偶联率。
方法1．用还原酰胺化法合成叶酸修饰壳聚糖复合载体；2．通过对合成样本的红外光谱测定，确认叶酸修饰壳聚糖复合载体；3．紫外扫描确定叶酸成功偶联于壳聚糖；4．通过对比FA浓度-吸光度标准曲线，测定叶酸偶联率。
结果1．获得了纯化的叶酸修饰壳聚糖复合载体，为淡黄色细致均匀的粉末，溶液为无味，淡黄色透明液体；2．红外吸收光谱显示叶酸修饰壳聚糖复合载体在1563cm~(-1)处出现新的特征性-CO-NH-振动波，说明-CO-NH-键成功在叶酸和壳聚糖间形成；3．紫外吸收光谱显示叶酸修饰壳聚糖复合载体在280nm处出现宽大吸收波，此为叶酸的特征性紫外吸收波长，证实叶酸成功与壳聚糖偶联；4．叶酸浓度在5.2-30.9μg／mL范围内与叶酸吸光度(A)呈良好的线性关系。回归方程为A=0.0127C+0.0016，R~2=0.9983。通过改变叶酸及壳聚糖不同的反应比，得到不同的叶酸偶联率，共合成四种不同偶联率的叶酸修饰壳聚糖复合载体，分别是3％、7％、11.2％和17％。
结论通过还原胺法成功合成叶酸修饰壳聚糖复合载体，在加入不同比例反应物后，共合成4种不同叶酸偶联率的叶酸修饰壳聚糖复合载体。提示可以利用与壳聚糖分子上的活性氨基的化合反应，成功修饰壳聚糖，改变其物理化学性能。
第二部分叶酸修饰壳聚糖siRNA纳米粒的制备及性质检测
目的合成叶酸修饰壳聚糖siRNA纳米粒，并测定其形态及直径大小。研究纳米粒对不同细胞的毒性及保护DNA免受降解的功能。
方法1．采用复凝聚法合成叶酸修饰壳聚糖PshRNA纳米粒(FA-CS-PshRNA)和壳聚糖pshRNA纳米粒(CS-PshRNA)；2．透射电镜观察纳米粒形态、大小，并测定不同叶酸偶联率下，纳米粒的平均直径；3．酶保护实验检测不同纳米粒对质粒DNA的成功包裹及保护功能；4．MTT法检测纳米粒对于不同妇科肿瘤细胞系(SKOV3、A2780、CAOV3、HeLa和MCF-7)产生的毒性；5．选择叶酸高表达的卵巢癌细胞系SKOV3，乳腺癌细胞系MCF-7和宫颈癌细胞系HeLa作为实验细胞，在体外培养过程中导入叶酸修饰壳聚糖siRNA纳米粒，荧光倒置显微镜下观察细胞形态及绿色荧光蛋白表达量，流式细胞仪检测不同细胞的转染效率。
结果1．本研究通过复凝聚法，成功合成了叶酸修饰壳聚糖PshRNA纳米粒和壳聚糖PshRNA纳米粒，纳米粒溶液为透明、无色、无味溶液；2．透射电镜显示纳米粒为近似球型，表面光滑、均质的纳米粒颗粒。壳聚糖PshRNA纳米粒粒径为138.4±0.7nm，叶酸修饰壳聚糖PshRNA纳米粒(叶酸偶联率：3％、7.5％、11.2％和17％)直径依次为289.6±0.7nm、78.1±0.3nm、186.6±0.6nm和212.2±0.5±nm；3．酶保护实验结果表明壳聚糖及叶酸修饰壳聚糖皆能有效结合和浓缩DNA形成相应纳米粒，壳聚糖及叶酸修饰壳聚糖皆能保护DNA不受DNAaseI的降解；4．细胞毒性实验显示叶酸修饰壳聚糖PshRNA纳米粒和壳聚糖PshRNA纳米粒对于不同细胞系亦有不同毒性。经壳聚糖PshRNA纳米粒处理后，MCF-7、A2780、CAOV3、SKOV3和HeLa的细胞活力分别是处理前的87.9±2.4％、91.4±1.0％、42.9±2.1％、102.0±4.0％和97.4±1.1％，而经叶酸修饰壳聚糖PshRNA纳米粒处理后，上述细胞的细胞活力分别为处理前的63.0±2.5％、90.6±1.3％、50.5±0.7％、106.5±1.8％和99.3±1.6％；5．对于MCF-7，SKOV3细胞系，叶酸修饰壳聚糖PshRNA纳米粒转染效率(16.8±1.2％和24.3±0.7％)明显高于壳聚糖PshRNA纳米粒(0.3±0.1％和0.7±0.1％)(P＜0.05)，壳聚糖PshRNA纳米粒和叶酸修饰壳聚糖PshRNA纳米粒对于HeLa细胞的转染效率分别为4.2±0.4％和4.8±0.9％，二者无统计学差异(P＞0.05)。
结论通过复凝聚法成功合成叶酸修饰壳聚糖PshRNA纳米粒和壳聚糖PshRNA纳米粒。不同叶酸偶联率影响纳米粒直径大小，在7.5％的叶酸偶联率下，纳米粒直径最小(78.1±0.3nm)。叶酸修饰壳聚糖PshRNA纳米粒和壳聚糖PshRNA纳米粒皆能稳定包裹质粒DNA，并能保护其免受DNAaseⅠ的酶解。在叶酸介导下，叶酸修饰壳聚糖PshRNA纳米粒的转染效率明显高于壳聚糖PshRNA纳米粒。提示通过叶酸修饰，明显提高了壳聚糖PshRNA纳米粒的转染效率。
第三部分卵巢癌SKOV3耐药细胞株建立及耐药性的研究
目的培养经泰素诱导SKOV3多药耐药细胞株，检测MDR1编码蛋白P-gp在SKOV3亲本株和耐药株中的表达差异，探讨MDR1基因表达与多药耐药的相关性。为研究靶向MDR1的叶酸修饰壳聚糖siRNA纳米粒逆转卵巢癌多药耐药提供细胞学基础。
方法1．以泰素为诱导剂，采用体外浓度梯度递增法建立卵巢癌SKOV3-ts多药耐药细胞株；2．MTT法检测SKOV3亲本株和耐药株中对紫杉醇和阿霉素的IC_(50)的改变；3．Western-blot和激光共聚焦法检测SKOV3亲本株和耐药株中P-gp蛋白的表达差异。
结果1．本章通过浓度梯度递增法成功建立了卵巢癌SKOV3多药耐药细胞株SKOV3-ts。该细胞株由体积相对较小的梭型变为体积膨胀的不规则形状，有些细胞呈星状和分支状结构，较SKOV3细胞大；2．应用半定量Western-blot检测出在SKOV3-ts及SKOV3细胞系中P-gp的表达量分别是1.15±0.02、0.08±0.01，差异有统计学意义(P＜0.05)。激光共聚焦法显示SKOV3细胞中只有微量P-gp表达，而SKOV3-ts细胞的细胞膜上显示较强P-gp的表达；3．SKOV3和SKOV3-ts对紫杉醇的IC_(50)分别是0.0048±0.0002和0.3957±0.0075，差异有统计学意义(P＜0.05)，对阿霉素的IC_(50)分别是0.0066±0.0006和0.2210±0.0046，差异有统计学意义(P＜0.05)。
结论通过浓度梯度递增法成功建立了卵巢癌SKOV3多药耐药细胞株SKOV3-ts，耐药细胞SKOV3-ts的P-gp表达量明显高于亲本细胞系SKOV3，对紫杉醇和阿霉素的IC_(50)较亲本细胞有明显升高。提示MDR1的过度表达与SKOV3细胞的多药耐药的产生有明显相关性，SKOV3-ts细胞耐药可对阿霉素产生交叉耐药，同时获得细胞膜P-gp高表达。故SKOV3-ts细胞可以作为卵巢癌耐药细胞模型，用作叶酸受体介导的纳米靶向修饰逆转卵巢癌多药耐药的研究。
第四部分叶酸修饰壳聚糖siRNA纳米粒靶向逆转SKOV3-ts细胞耐药的研究
目的检测叶酸修饰壳聚糖siRNA纳米粒与壳聚糖siRNA纳米粒对SKOV3-ts细胞系MDR1表达的影响，探讨经叶酸修饰后，纳米粒逆转SKOV3-ts细胞系耐药性的改变。
方法1．在SKOV3-ts细胞体外培养过程中导入叶酸修饰壳聚糖PshRNA纳米粒和壳聚糖PshRNA纳米粒，其中PshRNA为可表达靶向MDR1基因的siRNA的真核表达质粒，故而称为叶酸修饰壳聚糖siRNA纳米粒和壳聚糖siRNA纳米粒；2．RT-PCR法检测纳米粒转染SKOV3-ts细胞前后，MDR1mRNA表达改变；3．用Western-blot法检测纳米粒转染SKOV3-ts细胞前后，MDR1编码蛋白P-gp的表达改变；4．MTT法检测纳米粒转染SKOV3-ts细胞前后，对紫杉醇耐药IC_(50)的改变。
结果1．半定量RT-PCR检测出壳聚糖siRNA纳米粒与叶酸修饰壳聚糖siRNA纳米粒转染SKOV3-ts细胞后，MDR1mRNA表达量分别是0.75±0.01、0.27±0.01，差异有显著意义(P＜0.05)；2．半定量Western-blot法检测出壳聚糖siRNA纳米粒与叶酸修饰壳聚糖siRNA纳米粒转染SKOV3-ts细胞后，P-gp表达量分别是0.62±0.01、0.12±0.01，差异有显著意义(P＜0.05)；3．MTT法检测壳聚糖siRNA纳米粒与叶酸修饰壳聚糖siRNA纳米粒转染SKOV3-ts细胞后，针对PTX的IC_(50)分别为0.3830±0.0096和0.0353±0.0006，差异有统计学意义(P＜0.05)。
结论通过叶酸靶向修饰，叶酸修饰壳聚糖siRNA纳米粒能有效降低靶细胞SKOV3-ts的靶基因MDR1的mRNA和蛋白P-gp表达。逆转SKOV3-ts细胞针对PTX的IC_(50)，能有效治疗卵巢癌细胞基于MDR1诱导的多药耐药。
PartⅠPreparation and identification of folate-chitosancomplex vectors
Objective: To synthesize folate-chitosan complex vectors and identify the successfulcombination of folate-chitosan complex vectors, determine the folate coupling radio offolate.
Methods: 1.Folate-chitosan complex vectors were synthesized using reductive amidation.2.The combined samples were detected in light within the infrared spectrum using theFourier transform infrared (FTIR) spectrometer to determine the folate-chitosan complexvectors.3.Samples were scaned by the UV/Vis spectrophotometer.4.Folate coupling radioswere determined using folate concentration- absorbance standard curve.
Results: 1.The purified folate-chitosan complex vectors were obtained and the sampleswere stramineous, exquisite and adqulis fine powder.The solutions were tasteless,stramineous and transparent.2.The results of infrared absorption spectrum showed thereaction key -CO-NH- between the chitosan and folate corresponding to a new bigabsorption peak at 1563~(-1)cm in the folate-chitosan complex samples.3.The results detectedby the UV/Vis spectrophotometer showed a big ultraviolet absorption peak at 280nm in thefolate-chitosan complexes which was the signal of successful coupling of folate.4.Therewas a good linear relationship between the absorbance of folate and the amount of folate infolate-chitosan complexes from 5.15ug/ml to 30.9μg/ml.The folate tandard curveregression equation was A = 0.0127C + 0.0116, R~2=0.9983.There were four folate-chitosan complexes with different folate coupling radio (3%, 7.5%, 11.2% and 17%).
Conclusions: We successfully synthesized the folate-chitosan complex vectors usingreductive amidation.By adding different amount of folate and chitosan, we all synthesizedfour folate-chitosan complex vectors with different folate coupling radio at the samereaction condition.The results illustrated that the modification of chitosan can be obtainedby the combination reaction to the active amines of chitosan molecules, and so can changethe physical chemistry function of them.
PartⅡPeparation of folate modified chitosan siRNA nanoparticles andmeasurement of properties
Objective: To synthesize folate modified chitosan PshRNA nanoparticles (FA-CS-PshRNA)and chitosan PshRNA nanoparticles (CS-PshRNA) and detected their appearance anddiameters.To study the cytotoxity and transfect efficiency to different cell lines andprotective function for DNA of nanoparticles.
Methods: 1.Folate modified chitosan PshRNA nanoparticles (FA-CS-PshRNA) and chitosanPshRNA nanoparticles (CS-PshRNA) were synthesized using the complex coacervationmethod.2.The appearance and diameters of samples with different folate coupling radiowere detected using transmission electron microscope.3.The protective and packingfunction of different nanoparticles for plasmid DNA was identified by the enzymeprotection experiment.4.MTT method was used to detected the cytotoxity of nanoparticlesto different gynecologic tumor cell lines ((SKOV3、A2780、CAOV3、HeLa and MCF-7).5.The transfection efficiency nanoparticles was examined in different gynecologic cancer cells, such as cervical cancer cell lines HeLa, ovarian cancer cell lines SKOV3 and breastcancer cell lines MCF-7 which were all over-expressing folate receptor.The transfectionwas carried in vitro culture environment.The transfection efficiency of nanoparticles wasanalyzed with flow cytometry and cells were observed by invert fluorescence microscopeand taken photos.
Results: 1.Folate modified chitosan PshRNA nanoparticles (FA-CS-PshRNA) and chitosanPshRNA nanoparticles (CS-PshRNA) were successfully synthesized using the complexcoacervation method and the nanoparticle solutions were transparent, colorless andtasteless.2.The results of transmission electron microscope showed that the nanoparticleswere found to be near spherical appearance with homogeneous structure and smoothsurface.The particle diameter of chitosan PshRNA
nanoparticles was 138.4±0.7nm, and the diameter of 4 kinds of folate modified chitosanPshRNA nanoparticles (folate coupling Radio: 3%、7.5%、11.2%和17% )were 289.6±0.7nm、78.1±0.3nm、186.6±0.6nm and 212.2±0.5±nm respectively.3.The result of the enzymeprotection experiment declared that chitosan and folate modified chitosan can effectivelycombine and condense the DNA and can protect them from degradation of DNAaseⅠ.4.The cototoxity of folate modified chitosan PshRNA and chitosan PshRNA nanoparticles todifferent cells were different.After treatment with chitosan PshRNA nanoparticles, the cellvitality of different cells (MCF-7、A2780、CAOV3、SKOV3 and HeLa) were 87.9±2.4%、91.4±1.0%、42.9±2.1%、102.0±4.0 and 97.4±1.1% respectively compared with the cellsbefore treatment, after treatment with folate modified chitosan PshRNA nanoparticles, the cellvitality were 63.0±2.5%、90.6±1.3%、50.5±0.7%、106.5±1.8% and 99.3±1.6% respectivelycompared with the cells before treatment.5.For MCF-7, SKOV3 cells, the transfectionefficiency were 16.8±1.2% and 24.3±0.7% using folate modified chitosan PshRNAnanoparticles and were significantly increased compared to the transfection efficiency(0.3±0.1% and 0.7±0.1%) using chitosan PshRNA nanoparticles (P<0.05).But for HeLacell, there was no statistics disparity between chitosan PshRNA nanoparticles and folate modified chitosan PshRNA nanoparticles (P>0.05).
Conclusions: We successful synthesized the folate modified chitosan siRNA nanoparticelsand chitosan siRNA nanoparticles using the complex coacervation method.The differentfolate coupling radio can affect the diameters of nanoparticles and when the folate couplingradio was 7.5%, the diameter was smallest (78.1±0.3nm).The results illustrated thatchitosan and folate modified chitosan can all effectively combined and condensed the DNAand can protect them from degradation of DNAaseⅠ.The transfection efficiency of folatemodified chitosan siRNA nanoparticles were significantly increased compared to thetransfection efficiency of chitosan siRNA nanoparticles, this declared that frommodification of folate, the transfection efficiency of chitosan nanoparticles wassignificantly increased.
PartⅢEstablishment of the drug resistant SKOV3 ovarian cancer cell lineand study of drug resistance
Objective: To induce and culture the multidrug resistant SKOV3 ovarian cancer cell line(SKOV3-ts) using Paclitaxel, and measured the expression of MDR1 gene encodingprotein P-gp in parent cell line SKOV3 and drug resistant cell line SKOV3-ts.To explorethe correlation between the expression of multidrug resistant gene 1 (MDR1) and multidrugresistance (MDR).To supply the cytology foundation for the study of the reversal effectof folate modified chitosan siRNA nanoparticles on ovarian cancer MDR.
Methods: 1.The SKOV3-ts cell line was induced and culture by concentration gradientmethod in vitro and paclitaxel was used as inductor.2.IC_(50) of the cells to paclitaxel andadriamycin in parent cell line SKOV3 and drug resistant cell line SKOV3-ts were measuredby MTT assay.3.Western-blot and laser scanning confocal microscope (LSCM) were usedto detect the expression of P-gp in parent cell line SKOV3 and drug resistant cell lineSKOV3-ts.
Results: 1.The SKOV3-ts cell line was successfully established using concentrationgradient method.The cells were bigger than the parent cell line SKOV3.The appearance ofcell was changed from spindle shape to irregular shape and some cells were star like andbranch like patterns.2.The results of western-blot showed that expression level of P-gpwas significant increased in SKOV3-ts cells (1.15±0.02) compared with SKOV3 cells(0.08±0.01) (P<0.05).Photos of LSCM illustrated that there was trace quantity ofP-gp expression in SKOV3 cells.But in SKOV3-ts cell membranes, there was strong P-gpexpression.3.IC_(50) of SKOV3 and SKOV3-ts cells to paclitaxel were 0.0048±0.0002 and0.3957±0.0075 respectively, there was significant statistics meaning (P<0.05), and IC_(50)of SKOV3 and SKOV3-ts cells to adriamycin were 0.0066±0.0006 and 0.2210±0.0046respectively, there was significant statistics meaning (P<0.05).
Conclusions: We successfully established the multidrug resistant SKOV3 ovarian cancercell line SKOV3-ts by concentration gradient method in vitro.The expression of P-gp ofdrug resistant cells SKOV3-ts was significantly higher than parent cell line SKOV3, andthe IC_(50) of SKOV3-ts cells to paclitaxel and adriamycin were increased compared withSKOV3 cells.This illustrated that there was apparent correlation between over-expressionof MDR1 and MDR of SKOV3-ts cells.So SKOV3-ts cells obtained drug resistance andcan be used as cell model for the study of the reversal effect of folate modified chitosansiRNA nanoparticles on ovarian cancer MDR.
PartⅣTargeted reversal of multidrug resistance in SKOV3-ts cells by folatemodified chitosan siRNA nanoparticles
Objective: To detect the effect of chitosan PshRNA nanoparticles and folate modifiedchitosan PshRNA nanoparticles on the expression of MDR1 in SKOV3 cells.To explorethe changes of drug resistance in SKOV3-ts cells treated by nanoparticles aftermodification of folate.
Methods: 1.The folate modified chitosan PshRNA and chitosan PshRNA nanoparticleswere transfected into the SKOV3-ts cells in vitro culture process.(PshRNA was theeukaryonic expression plasmid whicj can express siRNA targeting MDR1 gene, so called chitosansiRNA and chitosan siRNA nanoparticles.2.The expression level of MDR1 mRNA inSKOV3-ts cells were detected by RT-PCR before and after transfection.3.The expressionlevel of P-gp in SKOV3-ts cells were detected by western-blot before and after transfection.4.The IC_(50) of SKOV3-ts cells to paclitaxel were detected by MTT assay before and aftertransfection.
Results: 1.The results of the half quantitative RT-PCR showed that the MDR1 expressionin SKOV3-ts cells transfected by chitosan siRNA and folate modified chitosan siRNAnanoparticles were 0.75±0.01, 0.27±0.01, and there was significant statistic meaning (P<0.05).2.The results of the half quantitative western-blot showed that the P-gpexpression in SKOV3-ts cells transfected by chitosan siRNA and folate modified chitosansiRNA nanoparticles were 0.62±0.01、0.12±0.01, and there was significant statisticmeaning (P<0.05).3.The results of MTT assay showed that the IC_(50) of SKOV3-ts cells to paclitaxel transfected by chitosan siRNA and folate modified chitosan siRNAnanoparticles were 0.3830±0.0096和0.0353±0.0006, and there was significant statisticmeaning (P<0.05).
Conclusions: Through folate targeted modification, folate modified chitosan siRNAnanoparticles can effectively decreased the expression of MDR1 mRNA and P-gp and canreverse the IC_(50) of SKOV3-ts cells to paclitaxel.Folate modified chitosan siRNAnanoparticles can reverse the MDR of ovarian cancer mediated by MDR1.
目的合成叶酸修饰壳聚糖复合载体，并鉴定叶酸-壳聚糖复合载体的成功合成，测定叶酸偶联率。
方法1．用还原酰胺化法合成叶酸修饰壳聚糖复合载体；2．通过对合成样本的红外光谱测定，确认叶酸修饰壳聚糖复合载体；3．紫外扫描确定叶酸成功偶联于壳聚糖；4．通过对比FA浓度-吸光度标准曲线，测定叶酸偶联率。
结果1．获得了纯化的叶酸修饰壳聚糖复合载体，为淡黄色细致均匀的粉末，溶液为无味，淡黄色透明液体；2．红外吸收光谱显示叶酸修饰壳聚糖复合载体在1563cm~(-1)处出现新的特征性-CO-NH-振动波，说明-CO-NH-键成功在叶酸和壳聚糖间形成；3．紫外吸收光谱显示叶酸修饰壳聚糖复合载体在280nm处出现宽大吸收波，此为叶酸的特征性紫外吸收波长，证实叶酸成功与壳聚糖偶联；4．叶酸浓度在5.2-30.9μg／mL范围内与叶酸吸光度(A)呈良好的线性关系。回归方程为A=0.0127C+0.0016，R~2=0.9983。通过改变叶酸及壳聚糖不同的反应比，得到不同的叶酸偶联率，共合成四种不同偶联率的叶酸修饰壳聚糖复合载体，分别是3％、7％、11.2％和17％。
结论通过还原胺法成功合成叶酸修饰壳聚糖复合载体，在加入不同比例反应物后，共合成4种不同叶酸偶联率的叶酸修饰壳聚糖复合载体。提示可以利用与壳聚糖分子上的活性氨基的化合反应，成功修饰壳聚糖，改变其物理化学性能。
第二部分叶酸修饰壳聚糖siRNA纳米粒的制备及性质检测
目的合成叶酸修饰壳聚糖siRNA纳米粒，并测定其形态及直径大小。研究纳米粒对不同细胞的毒性及保护DNA免受降解的功能。
方法1．采用复凝聚法合成叶酸修饰壳聚糖PshRNA纳米粒(FA-CS-PshRNA)和壳聚糖pshRNA纳米粒(CS-PshRNA)；2．透射电镜观察纳米粒形态、大小，并测定不同叶酸偶联率下，纳米粒的平均直径；3．酶保护实验检测不同纳米粒对质粒DNA的成功包裹及保护功能；4．MTT法检测纳米粒对于不同妇科肿瘤细胞系(SKOV3、A2780、CAOV3、HeLa和MCF-7)产生的毒性；5．选择叶酸高表达的卵巢癌细胞系SKOV3，乳腺癌细胞系MCF-7和宫颈癌细胞系HeLa作为实验细胞，在体外培养过程中导入叶酸修饰壳聚糖siRNA纳米粒，荧光倒置显微镜下观察细胞形态及绿色荧光蛋白表达量，流式细胞仪检测不同细胞的转染效率。
结果1．本研究通过复凝聚法，成功合成了叶酸修饰壳聚糖PshRNA纳米粒和壳聚糖PshRNA纳米粒，纳米粒溶液为透明、无色、无味溶液；2．透射电镜显示纳米粒为近似球型，表面光滑、均质的纳米粒颗粒。壳聚糖PshRNA纳米粒粒径为138.4±0.7nm，叶酸修饰壳聚糖PshRNA纳米粒(叶酸偶联率：3％、7.5％、11.2％和17％)直径依次为289.6±0.7nm、78.1±0.3nm、186.6±0.6nm和212.2±0.5±nm；3．酶保护实验结果表明壳聚糖及叶酸修饰壳聚糖皆能有效结合和浓缩DNA形成相应纳米粒，壳聚糖及叶酸修饰壳聚糖皆能保护DNA不受DNAaseI的降解；4．细胞毒性实验显示叶酸修饰壳聚糖PshRNA纳米粒和壳聚糖PshRNA纳米粒对于不同细胞系亦有不同毒性。经壳聚糖PshRNA纳米粒处理后，MCF-7、A2780、CAOV3、SKOV3和HeLa的细胞活力分别是处理前的87.9±2.4％、91.4±1.0％、42.9±2.1％、102.0±4.0％和97.4±1.1％，而经叶酸修饰壳聚糖PshRNA纳米粒处理后，上述细胞的细胞活力分别为处理前的63.0±2.5％、90.6±1.3％、50.5±0.7％、106.5±1.8％和99.3±1.6％；5．对于MCF-7，SKOV3细胞系，叶酸修饰壳聚糖PshRNA纳米粒转染效率(16.8±1.2％和24.3±0.7％)明显高于壳聚糖PshRNA纳米粒(0.3±0.1％和0.7±0.1％)(P＜0.05)，壳聚糖PshRNA纳米粒和叶酸修饰壳聚糖PshRNA纳米粒对于HeLa细胞的转染效率分别为4.2±0.4％和4.8±0.9％，二者无统计学差异(P＞0.05)。
结论通过复凝聚法成功合成叶酸修饰壳聚糖PshRNA纳米粒和壳聚糖PshRNA纳米粒。不同叶酸偶联率影响纳米粒直径大小，在7.5％的叶酸偶联率下，纳米粒直径最小(78.1±0.3nm)。叶酸修饰壳聚糖PshRNA纳米粒和壳聚糖PshRNA纳米粒皆能稳定包裹质粒DNA，并能保护其免受DNAaseⅠ的酶解。在叶酸介导下，叶酸修饰壳聚糖PshRNA纳米粒的转染效率明显高于壳聚糖PshRNA纳米粒。提示通过叶酸修饰，明显提高了壳聚糖PshRNA纳米粒的转染效率。
第三部分卵巢癌SKOV3耐药细胞株建立及耐药性的研究
目的培养经泰素诱导SKOV3多药耐药细胞株，检测MDR1编码蛋白P-gp在SKOV3亲本株和耐药株中的表达差异，探讨MDR1基因表达与多药耐药的相关性。为研究靶向MDR1的叶酸修饰壳聚糖siRNA纳米粒逆转卵巢癌多药耐药提供细胞学基础。
方法1．以泰素为诱导剂，采用体外浓度梯度递增法建立卵巢癌SKOV3-ts多药耐药细胞株；2．MTT法检测SKOV3亲本株和耐药株中对紫杉醇和阿霉素的IC_(50)的改变；3．Western-blot和激光共聚焦法检测SKOV3亲本株和耐药株中P-gp蛋白的表达差异。
结果1．本章通过浓度梯度递增法成功建立了卵巢癌SKOV3多药耐药细胞株SKOV3-ts。该细胞株由体积相对较小的梭型变为体积膨胀的不规则形状，有些细胞呈星状和分支状结构，较SKOV3细胞大；2．应用半定量Western-blot检测出在SKOV3-ts及SKOV3细胞系中P-gp的表达量分别是1.15±0.02、0.08±0.01，差异有统计学意义(P＜0.05)。激光共聚焦法显示SKOV3细胞中只有微量P-gp表达，而SKOV3-ts细胞的细胞膜上显示较强P-gp的表达；3．SKOV3和SKOV3-ts对紫杉醇的IC_(50)分别是0.0048±0.0002和0.3957±0.0075，差异有统计学意义(P＜0.05)，对阿霉素的IC_(50)分别是0.0066±0.0006和0.2210±0.0046，差异有统计学意义(P＜0.05)。
结论通过浓度梯度递增法成功建立了卵巢癌SKOV3多药耐药细胞株SKOV3-ts，耐药细胞SKOV3-ts的P-gp表达量明显高于亲本细胞系SKOV3，对紫杉醇和阿霉素的IC_(50)较亲本细胞有明显升高。提示MDR1的过度表达与SKOV3细胞的多药耐药的产生有明显相关性，SKOV3-ts细胞耐药可对阿霉素产生交叉耐药，同时获得细胞膜P-gp高表达。故SKOV3-ts细胞可以作为卵巢癌耐药细胞模型，用作叶酸受体介导的纳米靶向修饰逆转卵巢癌多药耐药的研究。
第四部分叶酸修饰壳聚糖siRNA纳米粒靶向逆转SKOV3-ts细胞耐药的研究
目的检测叶酸修饰壳聚糖siRNA纳米粒与壳聚糖siRNA纳米粒对SKOV3-ts细胞系MDR1表达的影响，探讨经叶酸修饰后，纳米粒逆转SKOV3-ts细胞系耐药性的改变。
方法1．在SKOV3-ts细胞体外培养过程中导入叶酸修饰壳聚糖PshRNA纳米粒和壳聚糖PshRNA纳米粒，其中PshRNA为可表达靶向MDR1基因的siRNA的真核表达质粒，故而称为叶酸修饰壳聚糖siRNA纳米粒和壳聚糖siRNA纳米粒；2．RT-PCR法检测纳米粒转染SKOV3-ts细胞前后，MDR1mRNA表达改变；3．用Western-blot法检测纳米粒转染SKOV3-ts细胞前后，MDR1编码蛋白P-gp的表达改变；4．MTT法检测纳米粒转染SKOV3-ts细胞前后，对紫杉醇耐药IC_(50)的改变。
结果1．半定量RT-PCR检测出壳聚糖siRNA纳米粒与叶酸修饰壳聚糖siRNA纳米粒转染SKOV3-ts细胞后，MDR1mRNA表达量分别是0.75±0.01、0.27±0.01，差异有显著意义(P＜0.05)；2．半定量Western-blot法检测出壳聚糖siRNA纳米粒与叶酸修饰壳聚糖siRNA纳米粒转染SKOV3-ts细胞后，P-gp表达量分别是0.62±0.01、0.12±0.01，差异有显著意义(P＜0.05)；3．MTT法检测壳聚糖siRNA纳米粒与叶酸修饰壳聚糖siRNA纳米粒转染SKOV3-ts细胞后，针对PTX的IC_(50)分别为0.3830±0.0096和0.0353±0.0006，差异有统计学意义(P＜0.05)。
结论通过叶酸靶向修饰，叶酸修饰壳聚糖siRNA纳米粒能有效降低靶细胞SKOV3-ts的靶基因MDR1的mRNA和蛋白P-gp表达。逆转SKOV3-ts细胞针对PTX的IC_(50)，能有效治疗卵巢癌细胞基于MDR1诱导的多药耐药。
PartⅠPreparation and identification of folate-chitosancomplex vectors
Objective: To synthesize folate-chitosan complex vectors and identify the successfulcombination of folate-chitosan complex vectors, determine the folate coupling radio offolate.
Methods: 1.Folate-chitosan complex vectors were synthesized using reductive amidation.2.The combined samples were detected in light within the infrared spectrum using theFourier transform infrared (FTIR) spectrometer to determine the folate-chitosan complexvectors.3.Samples were scaned by the UV/Vis spectrophotometer.4.Folate coupling radioswere determined using folate concentration- absorbance standard curve.
Results: 1.The purified folate-chitosan complex vectors were obtained and the sampleswere stramineous, exquisite and adqulis fine powder.The solutions were tasteless,stramineous and transparent.2.The results of infrared absorption spectrum showed thereaction key -CO-NH- between the chitosan and folate corresponding to a new bigabsorption peak at 1563~(-1)cm in the folate-chitosan complex samples.3.The results detectedby the UV/Vis spectrophotometer showed a big ultraviolet absorption peak at 280nm in thefolate-chitosan complexes which was the signal of successful coupling of folate.4.Therewas a good linear relationship between the absorbance of folate and the amount of folate infolate-chitosan complexes from 5.15ug/ml to 30.9μg/ml.The folate tandard curveregression equation was A = 0.0127C + 0.0116, R~2=0.9983.There were four folate-chitosan complexes with different folate coupling radio (3%, 7.5%, 11.2% and 17%).
Conclusions: We successfully synthesized the folate-chitosan complex vectors usingreductive amidation.By adding different amount of folate and chitosan, we all synthesizedfour folate-chitosan complex vectors with different folate coupling radio at the samereaction condition.The results illustrated that the modification of chitosan can be obtainedby the combination reaction to the active amines of chitosan molecules, and so can changethe physical chemistry function of them.
PartⅡPeparation of folate modified chitosan siRNA nanoparticles andmeasurement of properties
Objective: To synthesize folate modified chitosan PshRNA nanoparticles (FA-CS-PshRNA)and chitosan PshRNA nanoparticles (CS-PshRNA) and detected their appearance anddiameters.To study the cytotoxity and transfect efficiency to different cell lines andprotective function for DNA of nanoparticles.
Methods: 1.Folate modified chitosan PshRNA nanoparticles (FA-CS-PshRNA) and chitosanPshRNA nanoparticles (CS-PshRNA) were synthesized using the complex coacervationmethod.2.The appearance and diameters of samples with different folate coupling radiowere detected using transmission electron microscope.3.The protective and packingfunction of different nanoparticles for plasmid DNA was identified by the enzymeprotection experiment.4.MTT method was used to detected the cytotoxity of nanoparticlesto different gynecologic tumor cell lines ((SKOV3、A2780、CAOV3、HeLa and MCF-7).5.The transfection efficiency nanoparticles was examined in different gynecologic cancer cells, such as cervical cancer cell lines HeLa, ovarian cancer cell lines SKOV3 and breastcancer cell lines MCF-7 which were all over-expressing folate receptor.The transfectionwas carried in vitro culture environment.The transfection efficiency of nanoparticles wasanalyzed with flow cytometry and cells were observed by invert fluorescence microscopeand taken photos.
Results: 1.Folate modified chitosan PshRNA nanoparticles (FA-CS-PshRNA) and chitosanPshRNA nanoparticles (CS-PshRNA) were successfully synthesized using the complexcoacervation method and the nanoparticle solutions were transparent, colorless andtasteless.2.The results of transmission electron microscope showed that the nanoparticleswere found to be near spherical appearance with homogeneous structure and smoothsurface.The particle diameter of chitosan PshRNA
nanoparticles was 138.4±0.7nm, and the diameter of 4 kinds of folate modified chitosanPshRNA nanoparticles (folate coupling Radio: 3%、7.5%、11.2%和17% )were 289.6±0.7nm、78.1±0.3nm、186.6±0.6nm and 212.2±0.5±nm respectively.3.The result of the enzymeprotection experiment declared that chitosan and folate modified chitosan can effectivelycombine and condense the DNA and can protect them from degradation of DNAaseⅠ.4.The cototoxity of folate modified chitosan PshRNA and chitosan PshRNA nanoparticles todifferent cells were different.After treatment with chitosan PshRNA nanoparticles, the cellvitality of different cells (MCF-7、A2780、CAOV3、SKOV3 and HeLa) were 87.9±2.4%、91.4±1.0%、42.9±2.1%、102.0±4.0 and 97.4±1.1% respectively compared with the cellsbefore treatment, after treatment with folate modified chitosan PshRNA nanoparticles, the cellvitality were 63.0±2.5%、90.6±1.3%、50.5±0.7%、106.5±1.8% and 99.3±1.6% respectivelycompared with the cells before treatment.5.For MCF-7, SKOV3 cells, the transfectionefficiency were 16.8±1.2% and 24.3±0.7% using folate modified chitosan PshRNAnanoparticles and were significantly increased compared to the transfection efficiency(0.3±0.1% and 0.7±0.1%) using chitosan PshRNA nanoparticles (P<0.05).But for HeLacell, there was no statistics disparity between chitosan PshRNA nanoparticles and folate modified chitosan PshRNA nanoparticles (P>0.05).
Conclusions: We successful synthesized the folate modified chitosan siRNA nanoparticelsand chitosan siRNA nanoparticles using the complex coacervation method.The differentfolate coupling radio can affect the diameters of nanoparticles and when the folate couplingradio was 7.5%, the diameter was smallest (78.1±0.3nm).The results illustrated thatchitosan and folate modified chitosan can all effectively combined and condensed the DNAand can protect them from degradation of DNAaseⅠ.The transfection efficiency of folatemodified chitosan siRNA nanoparticles were significantly increased compared to thetransfection efficiency of chitosan siRNA nanoparticles, this declared that frommodification of folate, the transfection efficiency of chitosan nanoparticles wassignificantly increased.
PartⅢEstablishment of the drug resistant SKOV3 ovarian cancer cell lineand study of drug resistance
Objective: To induce and culture the multidrug resistant SKOV3 ovarian cancer cell line(SKOV3-ts) using Paclitaxel, and measured the expression of MDR1 gene encodingprotein P-gp in parent cell line SKOV3 and drug resistant cell line SKOV3-ts.To explorethe correlation between the expression of multidrug resistant gene 1 (MDR1) and multidrugresistance (MDR).To supply the cytology foundation for the study of the reversal effectof folate modified chitosan siRNA nanoparticles on ovarian cancer MDR.
Methods: 1.The SKOV3-ts cell line was induced and culture by concentration gradientmethod in vitro and paclitaxel was used as inductor.2.IC_(50) of the cells to paclitaxel andadriamycin in parent cell line SKOV3 and drug resistant cell line SKOV3-ts were measuredby MTT assay.3.Western-blot and laser scanning confocal microscope (LSCM) were usedto detect the expression of P-gp in parent cell line SKOV3 and drug resistant cell lineSKOV3-ts.
Results: 1.The SKOV3-ts cell line was successfully established using concentrationgradient method.The cells were bigger than the parent cell line SKOV3.The appearance ofcell was changed from spindle shape to irregular shape and some cells were star like andbranch like patterns.2.The results of western-blot showed that expression level of P-gpwas significant increased in SKOV3-ts cells (1.15±0.02) compared with SKOV3 cells(0.08±0.01) (P<0.05).Photos of LSCM illustrated that there was trace quantity ofP-gp expression in SKOV3 cells.But in SKOV3-ts cell membranes, there was strong P-gpexpression.3.IC_(50) of SKOV3 and SKOV3-ts cells to paclitaxel were 0.0048±0.0002 and0.3957±0.0075 respectively, there was significant statistics meaning (P<0.05), and IC_(50)of SKOV3 and SKOV3-ts cells to adriamycin were 0.0066±0.0006 and 0.2210±0.0046respectively, there was significant statistics meaning (P<0.05).
Conclusions: We successfully established the multidrug resistant SKOV3 ovarian cancercell line SKOV3-ts by concentration gradient method in vitro.The expression of P-gp ofdrug resistant cells SKOV3-ts was significantly higher than parent cell line SKOV3, andthe IC_(50) of SKOV3-ts cells to paclitaxel and adriamycin were increased compared withSKOV3 cells.This illustrated that there was apparent correlation between over-expressionof MDR1 and MDR of SKOV3-ts cells.So SKOV3-ts cells obtained drug resistance andcan be used as cell model for the study of the reversal effect of folate modified chitosansiRNA nanoparticles on ovarian cancer MDR.
PartⅣTargeted reversal of multidrug resistance in SKOV3-ts cells by folatemodified chitosan siRNA nanoparticles
Objective: To detect the effect of chitosan PshRNA nanoparticles and folate modifiedchitosan PshRNA nanoparticles on the expression of MDR1 in SKOV3 cells.To explorethe changes of drug resistance in SKOV3-ts cells treated by nanoparticles aftermodification of folate.
Methods: 1.The folate modified chitosan PshRNA and chitosan PshRNA nanoparticleswere transfected into the SKOV3-ts cells in vitro culture process.(PshRNA was theeukaryonic expression plasmid whicj can express siRNA targeting MDR1 gene, so called chitosansiRNA and chitosan siRNA nanoparticles.2.The expression level of MDR1 mRNA inSKOV3-ts cells were detected by RT-PCR before and after transfection.3.The expressionlevel of P-gp in SKOV3-ts cells were detected by western-blot before and after transfection.4.The IC_(50) of SKOV3-ts cells to paclitaxel were detected by MTT assay before and aftertransfection.
Results: 1.The results of the half quantitative RT-PCR showed that the MDR1 expressionin SKOV3-ts cells transfected by chitosan siRNA and folate modified chitosan siRNAnanoparticles were 0.75±0.01, 0.27±0.01, and there was significant statistic meaning (P<0.05).2.The results of the half quantitative western-blot showed that the P-gpexpression in SKOV3-ts cells transfected by chitosan siRNA and folate modified chitosansiRNA nanoparticles were 0.62±0.01、0.12±0.01, and there was significant statisticmeaning (P<0.05).3.The results of MTT assay showed that the IC_(50) of SKOV3-ts cells to paclitaxel transfected by chitosan siRNA and folate modified chitosan siRNAnanoparticles were 0.3830±0.0096和0.0353±0.0006, and there was significant statisticmeaning (P<0.05).
Conclusions: Through folate targeted modification, folate modified chitosan siRNAnanoparticles can effectively decreased the expression of MDR1 mRNA and P-gp and canreverse the IC_(50) of SKOV3-ts cells to paclitaxel.Folate modified chitosan siRNAnanoparticles can reverse the MDR of ovarian cancer mediated by MDR1.
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