槲皮素缓释化疗治疗脑胶质瘤的实验研究
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摘要
局部缓释化疗是颅内胶质瘤治疗的一种重要方式,廉价适用的新型注射微球载体的研发和应用,为胶质瘤化疗提供了更大的方便。槲皮素是目前已知的最强的化疗药物之一,它对多种肿瘤的抑制作用已被国内外的许多学者所证实,但对于胶质瘤细胞的治疗作用机理仍不十分清楚,目前,脑胶质瘤应用槲皮素注射微球的缓释化疗尚未见报导。本文采用槲皮素对体外培养的大鼠C6胶质瘤细胞和采用槲皮素注射微球对荷瘤动物模型的脑C6胶质瘤进行了诱导凋亡、增殖抑制作用的实验研究,目的在于探讨大鼠C6胶质瘤凋亡相关基因表达的变化,为槲皮素治疗胶质瘤提供科学依据。
本实验首次制备了槲皮素缓释剂并进行了红外吸收光谱定性分析及电镜观察,并且采用槲皮素对大鼠C6胶质瘤细胞增殖、四氮唑比色法、DNA电泳、电镜、流式细胞技术,免疫组织化学、末端脱氧核苷酸转移酶介导的dUTP切口末端标记(TUNEL法)等技术及槲皮素注射微球对C6胶质瘤模型治疗前后影像学变化进行了观察、免疫组化检测了C6胶质瘤相关基因的表达变化以及TUNEL法检测了凋亡。研究发现,槲皮素对体内外生长的肿瘤细胞均表现出明显的增殖抑制、诱导凋亡的作用,其对Bcl-2和PCNA表达有显著的抑制作用,对P53、Caspase-3表达有明显的激活作用,对C-myc在体内外的表达未发现明显影响。本实验研究结果表明槲皮素治疗胶质瘤的主要作用机理与上述基因变化导致的细胞凋亡有关。
The glioma is the most familiar malignant tumour of intracranial tumors,accouting for about 30-50%s of all encephalic tumours, because it grows in theattacked means without boundary between the normal brain and glioma, and thewhole tumour is impossibly cut off by the surgical operation, although surgicaloperation and radiotherapy could make long the life of parts of sufferers, the abilityof them is limited, most sufferers still die in the relapse or progress of their glioma,as one of the main therapeutical methods of the malignant tumor, chemotherapyplays an irreplaceable role by that of operation and radiotherapy. in the recent 30years, with the development of the new medicine and the furtherly foundational andclinical research of the tumors , the chemotherapy has been strengthened. theeffective rate of more than 10 kinds of malignant tumor has been over 50%currently.However,conflict between the tumor chemotherapy research and theclinical need are still obvious, as far as chemotherapy of intracranial tumors isconcerned, because of existence of the blood brain barrier, high-efficient andlow-poisonous chemotherapeutical medicines that can be used in clinic are too few,and the partial medicine density in tumor is too low, the increasing dose of drugbrings about the great side effect of the whole body.
In the sieving researchment on low-poisonous and high-efficient chemotherapymedicine, we have found that the flavonoid compound has the very stronganti-tumor function and is from the natural plant directly, and is being payed anextensive attention among the researchers, as a kind of flavonoid compound, the
research of quercetin to various kinds of tumors proved that quercetin is one of thestrongest anti-tumor medicines currently, and the toxicity of which is low, andwhich is the most important flavonoid in mankind's diet , the study on themechanism of anti-tumor about quercetin indicated that it might carry out the anti-tumor function by repressing the proliferation of tumor cell and inducing theapoptosis of tumor cell, and interferring the expression of related genes etc.But thereare few reports about the glioma treated by quercetin.The growth of the glioma shows its specific character: local growing in attackedmeans, and integrity of the blood brain barrier in the tumor edge. Moreover, therelapse of glioma within 2 cms from the primary tumour, without occurrence ofextracranial tumors. These characteristics are regarded as the obstacles of thetraditional chemotherapy of glioma, but strongly proved the basis for thedurative-releasing anti-tumor medicine without influence of the blood brain barrierand without the increase of the toxicity of the whole body, the partial medicinedensity was raised consumedly.the sustained -release chemotherapy is the effectivemethod of killing cell of tumour, which is to wrap medicine in a certain polymerloader, which is transplanted into the glioma, with the dissolvement of the polymerand released in a slow-continual way with high density in the part of tumour.Inrecent years, research and application of the dissolvable and injectable microspheresprovide the larger room of development in chemoterapy of glioma ,we adoptted thevaporizing method of the melting agent to prepare this kind of sustained–release themicrosphere .The research work of this experiment:1. In the preliminary experiment we make sure of the effective density ofquercetin used in vitro and in vito.2. My experiment in vitro, we used the MTT assay to examine the A value ofeach group and caculate the inhibited rate of cell, the flow cytometry was used to
check the cycle of cell and apoptotic ratio of cell C6 treated by 50 and 100μmol·L-1quercetin for 48 hs, the DNA electrophoresis and the observation of themicrostructure under electron microscope and the function of quercetin to induce theapoptosis of C6 cell was proved by using TUNEL method, the methods ofimmunohistochemistry was used to examine the expression of P53, Bcl-2, Caspase-3,C-myc and PCNA genes.3. In the third part of experiment, we prepare the sustained -releasechemomicrosphere by the vaporization of melting agent, and we still make use of theinfrared spectrum instrument to authenticate the chemotherapy medicine, by electronmicroscope and make an observation on the microsphere.4. In the experiment in vito, we prepare the rat brain tumor model of the gliomaC6 cell, and we still observed the state of rat, the picture of tumour growth throughMRI, by the immuno*histochemical method, we still examine the expression of theP53, Bcl-2, Caspase-3, C-myc and PCNA gene .we also use the TUNEL method toexamine the apoptosis, by the experiment in vitro and in vito, we aim at clarifyingthe mechanism tha. quercetin inhibits the proliferation of glioma C6 cell andproviding the proof for the clinical apply of quercetin.The result of this experiment:1. in the preliminary experiment, we discover the quercetin can inhibit theproliferation of rat glioma cell in vitro from 25 to 100μmol·L-1, with the increase ofdrug concentration and the extension of treatment time, the trend of the inhibitationis obviously enhansed, the untreated cell presents the shuttle form or triangles, closearranging, clear boundary, strong reflet light, the axon intertexture to form the net,smooth cell membrane without tuber, the circular cell nucleus in the center withoutvisible nucleolus, but with active propagate, after we treated the cells, the cellnumber along with the wall reduces with the medicine density increase, undermicroscope it is clear that the cell becomes circular with the grain material inside the
cell, a little part of cell membrane breaks, the boundary of most cell is not clear, withthe grain material in the cytoplasm and split tuber.DSMO group is similar ininhibitation of cell with the natri chlorid group, the cells still grow actively in the twogroups;The effective medical concentration from 25 to 100 mmol·L-1can treat thetumour under skin, with the medicine density increase and the extension of treatmenttime, the tumour turns small gradually, we observe that from the 50 mmol·L-1of thequercetin and above ,the tumour treated by quercetin is obviously inhibited, butDSMO make no differnce to the tumour2. In the experiment in vitro, the inhibited proliferation of C6 cell treated bydifferent density quercetin is dependent on time of treatment and medical dosage.3. The MTT method is used to examine the A value of the C6 cell treated byquercetin of different concentration, we find that the A value becomes gradually litlebut IR becomes large.4. By the flow cytometery, we find that, with the increase of concentration ofquercetin, apoptotic ratio of C6 cell increases, the cells stopping at G0/ G1 phaseincrease, but the cells stopping at S/ G2/ M phase reduce. the proportion of celltreated by 50μmol·L-1and 100μmol·L-1quercetin at G0/G1 is respectively 66.4±1.8%and 82.3±1.7%, compared with the 54.1±1.2% of control group, the difference ismarkedly showed;apoptotic rate of two group cells is respectively 13±2% and31±1.9%, compared with 3.5±1.6% of the control, the difference is markedlyshowed too.5. The immunohistochemical method that we used showed, Compared with thecontrol group, the low-expressions of Bcl-2 and PCNA, the high-expression ofCaspase-3 and P53, but the expression of C-myc is not obviously changed.6. Under the electron microscope, we saw that the cell of C6 treated byquercetin, becomes small and shrinkage with the separate chromatin and theconcentrated cytoplasm and crimpled cell membrane and nuclear membrane, and the
nuclear is splited into the part, finally the apoptotic body is formed in differentnumber7. TUNEL examination: Compared with the control group, the cells treated by100μmol·L-1 and 50μmol·L-1quercetin, in which the nuclear and cytoplasm of thecell are dyed into yellow, become increasing8. The DNA electronphoresis of cell treated by the 100μmol·L-1 quercetin for48hs shows the ladder of DNA after it is added into gelose gelatin9. The sustained-release microspheres of quercetin and PLGA are the multilayerones, the size of which is in uniformity, and the diameter of which is about 25 nm.10. By the stereostatic inject method, we establishes glioma C6 model, rate ofmaking tumour is 84%, compared with the method that researchers always used ofestablishing the animal model by the empty-handed means or abidingly embeddedstainless steel tuber in the past, the rate of forming tumour is high, without thetransferred tumour outside and the need of the pipe to planted11. The situation of growth of the rat with or without tumour, life time andpicture of MRI of the rat with tumour: most rats shows cachexia and lazy and weakappetite, etc.after the rats are injected with glioma C6 cell into their brain. But therats treated by the drug for three days or five days showes active and good appetite.MRI is done at the 7th day, the average diameter of the rat cerebraltumour is 4.3±0.6mm, the average diameter of the rat treated by the sustained -releasechemotherapeutical medicine for a week is 3.1±0.4 mm and 1.4±0.3 mm for threeweeks, compared with the control group tumor average diameter 5.4±0.8 mm at the14th day, we find the tumour turns small obviously, the average life time of the rat isof 19.3±2.8 days in control group but of 60.5±12.8 days in the treated rats12. compared with the control group, there is high expression of P53 andCaspase-3, low the expression of Bcl-2 and PCNA, the c-myc does not changeobviously, the increasing number of apoptotic cells is also proved by the TUNEL,
the experimental results are in accordance in vitro and in vito.Creative point of this experiment:1. The first study of the quercetin used for treatment of glioma is made on theinhibited proliferation though the P53, Bcl-2, Caspase-3, C-myc, PCNA genes2. The sustained -release chemotherapeutical microsphere of PLGA andquercetin is first prepared by the melting agent vaporization and first applied to treatthe C6 glioma through sustained -release chemotherapy
本实验首次制备了槲皮素缓释剂并进行了红外吸收光谱定性分析及电镜观察,并且采用槲皮素对大鼠C6胶质瘤细胞增殖、四氮唑比色法、DNA电泳、电镜、流式细胞技术,免疫组织化学、末端脱氧核苷酸转移酶介导的dUTP切口末端标记(TUNEL法)等技术及槲皮素注射微球对C6胶质瘤模型治疗前后影像学变化进行了观察、免疫组化检测了C6胶质瘤相关基因的表达变化以及TUNEL法检测了凋亡。研究发现,槲皮素对体内外生长的肿瘤细胞均表现出明显的增殖抑制、诱导凋亡的作用,其对Bcl-2和PCNA表达有显著的抑制作用,对P53、Caspase-3表达有明显的激活作用,对C-myc在体内外的表达未发现明显影响。本实验研究结果表明槲皮素治疗胶质瘤的主要作用机理与上述基因变化导致的细胞凋亡有关。
The glioma is the most familiar malignant tumour of intracranial tumors,accouting for about 30-50%s of all encephalic tumours, because it grows in theattacked means without boundary between the normal brain and glioma, and thewhole tumour is impossibly cut off by the surgical operation, although surgicaloperation and radiotherapy could make long the life of parts of sufferers, the abilityof them is limited, most sufferers still die in the relapse or progress of their glioma,as one of the main therapeutical methods of the malignant tumor, chemotherapyplays an irreplaceable role by that of operation and radiotherapy. in the recent 30years, with the development of the new medicine and the furtherly foundational andclinical research of the tumors , the chemotherapy has been strengthened. theeffective rate of more than 10 kinds of malignant tumor has been over 50%currently.However,conflict between the tumor chemotherapy research and theclinical need are still obvious, as far as chemotherapy of intracranial tumors isconcerned, because of existence of the blood brain barrier, high-efficient andlow-poisonous chemotherapeutical medicines that can be used in clinic are too few,and the partial medicine density in tumor is too low, the increasing dose of drugbrings about the great side effect of the whole body.
In the sieving researchment on low-poisonous and high-efficient chemotherapymedicine, we have found that the flavonoid compound has the very stronganti-tumor function and is from the natural plant directly, and is being payed anextensive attention among the researchers, as a kind of flavonoid compound, the
research of quercetin to various kinds of tumors proved that quercetin is one of thestrongest anti-tumor medicines currently, and the toxicity of which is low, andwhich is the most important flavonoid in mankind's diet , the study on themechanism of anti-tumor about quercetin indicated that it might carry out the anti-tumor function by repressing the proliferation of tumor cell and inducing theapoptosis of tumor cell, and interferring the expression of related genes etc.But thereare few reports about the glioma treated by quercetin.The growth of the glioma shows its specific character: local growing in attackedmeans, and integrity of the blood brain barrier in the tumor edge. Moreover, therelapse of glioma within 2 cms from the primary tumour, without occurrence ofextracranial tumors. These characteristics are regarded as the obstacles of thetraditional chemotherapy of glioma, but strongly proved the basis for thedurative-releasing anti-tumor medicine without influence of the blood brain barrierand without the increase of the toxicity of the whole body, the partial medicinedensity was raised consumedly.the sustained -release chemotherapy is the effectivemethod of killing cell of tumour, which is to wrap medicine in a certain polymerloader, which is transplanted into the glioma, with the dissolvement of the polymerand released in a slow-continual way with high density in the part of tumour.Inrecent years, research and application of the dissolvable and injectable microspheresprovide the larger room of development in chemoterapy of glioma ,we adoptted thevaporizing method of the melting agent to prepare this kind of sustained–release themicrosphere .The research work of this experiment:1. In the preliminary experiment we make sure of the effective density ofquercetin used in vitro and in vito.2. My experiment in vitro, we used the MTT assay to examine the A value ofeach group and caculate the inhibited rate of cell, the flow cytometry was used to
check the cycle of cell and apoptotic ratio of cell C6 treated by 50 and 100μmol·L-1quercetin for 48 hs, the DNA electrophoresis and the observation of themicrostructure under electron microscope and the function of quercetin to induce theapoptosis of C6 cell was proved by using TUNEL method, the methods ofimmunohistochemistry was used to examine the expression of P53, Bcl-2, Caspase-3,C-myc and PCNA genes.3. In the third part of experiment, we prepare the sustained -releasechemomicrosphere by the vaporization of melting agent, and we still make use of theinfrared spectrum instrument to authenticate the chemotherapy medicine, by electronmicroscope and make an observation on the microsphere.4. In the experiment in vito, we prepare the rat brain tumor model of the gliomaC6 cell, and we still observed the state of rat, the picture of tumour growth throughMRI, by the immuno*histochemical method, we still examine the expression of theP53, Bcl-2, Caspase-3, C-myc and PCNA gene .we also use the TUNEL method toexamine the apoptosis, by the experiment in vitro and in vito, we aim at clarifyingthe mechanism tha. quercetin inhibits the proliferation of glioma C6 cell andproviding the proof for the clinical apply of quercetin.The result of this experiment:1. in the preliminary experiment, we discover the quercetin can inhibit theproliferation of rat glioma cell in vitro from 25 to 100μmol·L-1, with the increase ofdrug concentration and the extension of treatment time, the trend of the inhibitationis obviously enhansed, the untreated cell presents the shuttle form or triangles, closearranging, clear boundary, strong reflet light, the axon intertexture to form the net,smooth cell membrane without tuber, the circular cell nucleus in the center withoutvisible nucleolus, but with active propagate, after we treated the cells, the cellnumber along with the wall reduces with the medicine density increase, undermicroscope it is clear that the cell becomes circular with the grain material inside the
cell, a little part of cell membrane breaks, the boundary of most cell is not clear, withthe grain material in the cytoplasm and split tuber.DSMO group is similar ininhibitation of cell with the natri chlorid group, the cells still grow actively in the twogroups;The effective medical concentration from 25 to 100 mmol·L-1can treat thetumour under skin, with the medicine density increase and the extension of treatmenttime, the tumour turns small gradually, we observe that from the 50 mmol·L-1of thequercetin and above ,the tumour treated by quercetin is obviously inhibited, butDSMO make no differnce to the tumour2. In the experiment in vitro, the inhibited proliferation of C6 cell treated bydifferent density quercetin is dependent on time of treatment and medical dosage.3. The MTT method is used to examine the A value of the C6 cell treated byquercetin of different concentration, we find that the A value becomes gradually litlebut IR becomes large.4. By the flow cytometery, we find that, with the increase of concentration ofquercetin, apoptotic ratio of C6 cell increases, the cells stopping at G0/ G1 phaseincrease, but the cells stopping at S/ G2/ M phase reduce. the proportion of celltreated by 50μmol·L-1and 100μmol·L-1quercetin at G0/G1 is respectively 66.4±1.8%and 82.3±1.7%, compared with the 54.1±1.2% of control group, the difference ismarkedly showed;apoptotic rate of two group cells is respectively 13±2% and31±1.9%, compared with 3.5±1.6% of the control, the difference is markedlyshowed too.5. The immunohistochemical method that we used showed, Compared with thecontrol group, the low-expressions of Bcl-2 and PCNA, the high-expression ofCaspase-3 and P53, but the expression of C-myc is not obviously changed.6. Under the electron microscope, we saw that the cell of C6 treated byquercetin, becomes small and shrinkage with the separate chromatin and theconcentrated cytoplasm and crimpled cell membrane and nuclear membrane, and the
nuclear is splited into the part, finally the apoptotic body is formed in differentnumber7. TUNEL examination: Compared with the control group, the cells treated by100μmol·L-1 and 50μmol·L-1quercetin, in which the nuclear and cytoplasm of thecell are dyed into yellow, become increasing8. The DNA electronphoresis of cell treated by the 100μmol·L-1 quercetin for48hs shows the ladder of DNA after it is added into gelose gelatin9. The sustained-release microspheres of quercetin and PLGA are the multilayerones, the size of which is in uniformity, and the diameter of which is about 25 nm.10. By the stereostatic inject method, we establishes glioma C6 model, rate ofmaking tumour is 84%, compared with the method that researchers always used ofestablishing the animal model by the empty-handed means or abidingly embeddedstainless steel tuber in the past, the rate of forming tumour is high, without thetransferred tumour outside and the need of the pipe to planted11. The situation of growth of the rat with or without tumour, life time andpicture of MRI of the rat with tumour: most rats shows cachexia and lazy and weakappetite, etc.after the rats are injected with glioma C6 cell into their brain. But therats treated by the drug for three days or five days showes active and good appetite.MRI is done at the 7th day, the average diameter of the rat cerebraltumour is 4.3±0.6mm, the average diameter of the rat treated by the sustained -releasechemotherapeutical medicine for a week is 3.1±0.4 mm and 1.4±0.3 mm for threeweeks, compared with the control group tumor average diameter 5.4±0.8 mm at the14th day, we find the tumour turns small obviously, the average life time of the rat isof 19.3±2.8 days in control group but of 60.5±12.8 days in the treated rats12. compared with the control group, there is high expression of P53 andCaspase-3, low the expression of Bcl-2 and PCNA, the c-myc does not changeobviously, the increasing number of apoptotic cells is also proved by the TUNEL,
the experimental results are in accordance in vitro and in vito.Creative point of this experiment:1. The first study of the quercetin used for treatment of glioma is made on theinhibited proliferation though the P53, Bcl-2, Caspase-3, C-myc, PCNA genes2. The sustained -release chemotherapeutical microsphere of PLGA andquercetin is first prepared by the melting agent vaporization and first applied to treatthe C6 glioma through sustained -release chemotherapy
引文
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