昆虫抗菌肽对肿瘤细胞作用的效果观察
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摘要
肿瘤治疗中的放疗、化疗对人体造成的伤害一直是未解决的难题,而抗菌肽的出现给这一领域带来了一线光明。昆虫抗菌肽能杀伤肿瘤细胞而对人体的正常细胞无毒或毒副作用甚小。本研究通过提取天然的昆虫体内抗菌肽,利用一些实验方法观察其对肿瘤细胞的作用,探讨其作用机制、作用范围和作用效果。
首先,将所选的昆虫家蝇成虫、幼虫和蛹、淡色库蚊、美洲大蠊、德国小蠊以针刺的方法感染细菌,诱导其产生抗菌肽,然后经研磨、加热、离心等方法提取出昆虫血淋巴,再经阳离子交换柱层析等步骤提取这几种昆虫的抗菌肽;将所提的抗菌肽作用于大肠杆菌、金黄葡萄球菌、绿脓杆菌和白色念珠菌以观察其抑菌效果;再将提取的抗菌肽作用于肿瘤细胞人髓样白血病细胞(K562)、人淋巴瘤细胞株(daudi)、人食管癌细胞株(109)和人膀胱癌细胞株(T24),用流式细胞仪观测其抑制、杀伤肿瘤细胞的作用;然后用扫描电镜观察这些昆虫抗菌肽作用的肿瘤细胞膜表面的受伤状况;最后再用聚丙烯酰氨凝胶电泳方法分离提纯这6组昆虫抗菌肽,了解各组昆虫抗菌肽的种类、数量。
在抗肿瘤实验中,家蝇的幼虫和成虫产生的抗菌肽效果最好,它们对4种受试的肿瘤细胞杀伤力都在86%以上;用生理盐水代替磷酸缓冲液(PBS)提取抗菌肽可以使抗菌肽对正常人体细胞的损伤降到几乎为零;在抗菌实验中,家蝇幼虫经不同方法产生的抗菌肽对同种细菌的抑制效果就不同,同种细菌诱导产生的抗菌肽对不同细菌抑制效果也不同;扫描电镜观察证实了抗菌肽对肿瘤细胞的杀伤力是通过细胞膜穿孔的途径;聚丙烯酰氨凝胶电泳显示了这6组昆虫抗菌肽各自都由数种分子量不同的抗菌肽链构成,其中家蝇幼虫有7种抗菌肽链、家蝇成虫和淡色库蚊有5种抗菌肽链、家蝇蛹有4种而两种蟑螂各有3种抗菌肽链。
抗肿瘤细胞的实验表明,各种抗菌肽对肿瘤细胞的作用效果不一。有些可以很有效地杀死肿瘤细胞,如家蝇幼虫、成虫产生的抗菌肽;有些昆虫抗菌肽,如美洲大蠊,不仅不能杀死肿瘤细胞,而且还有保护和促进其生长的作用。因此,在利用抗菌肽抑制肿瘤细胞时要对昆虫抗菌肽进行筛选。在抗肿瘤细胞实验中我们还看到,以生理盐水代替磷酸缓冲液提取的抗菌肽效果更好。抗菌肽对正常人体细胞无害也是本实验得出的一个结论。抗菌实验表明,昆虫抗菌肽的诱导方式不同,产生的抗菌肽就不同,其抗菌谱也不同。扫描电镜结果表明,尽管可能还有其它的机制存在,但可以证明抗菌肽对肿瘤细胞的杀伤机制是通过膜的穿孔来进行的。聚丙烯酰氨凝胶电泳结果表明,每种昆虫都可产生多种抗菌肽,而改进诱导和提取方法,我们可能会得到更多的抗菌肽种类。
It is difficult for treatment of the cancer patients to reduce the hurt caused by medicine and rays.The appear of antibacterial peptide bring a much of hope.The peptides could kill germs and some tumour cells,and do not affect human body.
In this research,five groups of natural antibacterial peptides were extracted from the insects when the insects were injured by some bacteriums with a needle.we study the machine-process,the useful scoperum and the effect of the peptides which worked on the tumour cell through some exprements.
First,the insects such as Musca domestica、Blattella germanica、Periplaneta americana、Culex pipiens pallens were infected by bacteria with a needle,in order to be induced to product some antibacterial peptides.These peptides were extracted by a series of steps protocol including grinding,heating,centrifugal and CM-cillulose cation-exchange chromatography.Then the peptides worked on the turnout cells K562,daudi,109 and T24.We detected the effect of the peptides by Flow cytometer(FCM).Furthermore,Scanning electron microscopy(SEM)was be used to observe the injure of the tumour cell membrane.In the last exprement,the six groups of antibacterial peptides were isolated by Polyacrylamide Gel Electrophoreses(PAGE),to show their peptide kinds.
The anticancer experiments tell us that,Musca domestica lavae have the best result, because they kill the four species of cancer cell over 86%.If normal saline instead of PBS, the extracted peptides could cause little hurt to humanbody.The bacteriostatic experiments shows that,the same group of peptides which induced by diffent germ could made different results.The observing of Scanning Electron Microscope(SEM)confirmed that,the machine-process of the peptides killing the cancer cell was drilling some holes on the membrane of the cell.The PAGE shows that,all of groups of peptides consist of different molecular mass peptides.Such as Musca domestica lavae,consist of seven kinds peptides, Blattella germanica three、Periplaneta americana three、Culex pipiens pallens firve and so on.
We know from anticancer experiment that,the diffent peptides could make diffent results from the same tumour cell.some peptides could kill the cell beneficially,such as fly lavae and the adult;while some peptides such as Periplaneta Americana,not only kill the cancer cell,but also promote the cell.From the bacteriostatic experiments we see that,different peptide from the same insect could be made with different inducing methods and the different results could be made from the same bacteriostatic experiments using the same insecta antibacterial peptids.
Concludes as follows:We must select the peptide when we hope to make a frofit of it;It is better that the normal saline instead of PBS when piptides were extracted from insects;The peptides do not hurt human body;The machine-prOcess of the peptides killing tumour cell,is drilling some hole on the cell mabrane;The results of SDS-PAGE show that,every insect could have many kinds of peptides when we change the mothod to extract it.
首先,将所选的昆虫家蝇成虫、幼虫和蛹、淡色库蚊、美洲大蠊、德国小蠊以针刺的方法感染细菌,诱导其产生抗菌肽,然后经研磨、加热、离心等方法提取出昆虫血淋巴,再经阳离子交换柱层析等步骤提取这几种昆虫的抗菌肽;将所提的抗菌肽作用于大肠杆菌、金黄葡萄球菌、绿脓杆菌和白色念珠菌以观察其抑菌效果;再将提取的抗菌肽作用于肿瘤细胞人髓样白血病细胞(K562)、人淋巴瘤细胞株(daudi)、人食管癌细胞株(109)和人膀胱癌细胞株(T24),用流式细胞仪观测其抑制、杀伤肿瘤细胞的作用;然后用扫描电镜观察这些昆虫抗菌肽作用的肿瘤细胞膜表面的受伤状况;最后再用聚丙烯酰氨凝胶电泳方法分离提纯这6组昆虫抗菌肽,了解各组昆虫抗菌肽的种类、数量。
在抗肿瘤实验中,家蝇的幼虫和成虫产生的抗菌肽效果最好,它们对4种受试的肿瘤细胞杀伤力都在86%以上;用生理盐水代替磷酸缓冲液(PBS)提取抗菌肽可以使抗菌肽对正常人体细胞的损伤降到几乎为零;在抗菌实验中,家蝇幼虫经不同方法产生的抗菌肽对同种细菌的抑制效果就不同,同种细菌诱导产生的抗菌肽对不同细菌抑制效果也不同;扫描电镜观察证实了抗菌肽对肿瘤细胞的杀伤力是通过细胞膜穿孔的途径;聚丙烯酰氨凝胶电泳显示了这6组昆虫抗菌肽各自都由数种分子量不同的抗菌肽链构成,其中家蝇幼虫有7种抗菌肽链、家蝇成虫和淡色库蚊有5种抗菌肽链、家蝇蛹有4种而两种蟑螂各有3种抗菌肽链。
抗肿瘤细胞的实验表明,各种抗菌肽对肿瘤细胞的作用效果不一。有些可以很有效地杀死肿瘤细胞,如家蝇幼虫、成虫产生的抗菌肽;有些昆虫抗菌肽,如美洲大蠊,不仅不能杀死肿瘤细胞,而且还有保护和促进其生长的作用。因此,在利用抗菌肽抑制肿瘤细胞时要对昆虫抗菌肽进行筛选。在抗肿瘤细胞实验中我们还看到,以生理盐水代替磷酸缓冲液提取的抗菌肽效果更好。抗菌肽对正常人体细胞无害也是本实验得出的一个结论。抗菌实验表明,昆虫抗菌肽的诱导方式不同,产生的抗菌肽就不同,其抗菌谱也不同。扫描电镜结果表明,尽管可能还有其它的机制存在,但可以证明抗菌肽对肿瘤细胞的杀伤机制是通过膜的穿孔来进行的。聚丙烯酰氨凝胶电泳结果表明,每种昆虫都可产生多种抗菌肽,而改进诱导和提取方法,我们可能会得到更多的抗菌肽种类。
It is difficult for treatment of the cancer patients to reduce the hurt caused by medicine and rays.The appear of antibacterial peptide bring a much of hope.The peptides could kill germs and some tumour cells,and do not affect human body.
In this research,five groups of natural antibacterial peptides were extracted from the insects when the insects were injured by some bacteriums with a needle.we study the machine-process,the useful scoperum and the effect of the peptides which worked on the tumour cell through some exprements.
First,the insects such as Musca domestica、Blattella germanica、Periplaneta americana、Culex pipiens pallens were infected by bacteria with a needle,in order to be induced to product some antibacterial peptides.These peptides were extracted by a series of steps protocol including grinding,heating,centrifugal and CM-cillulose cation-exchange chromatography.Then the peptides worked on the turnout cells K562,daudi,109 and T24.We detected the effect of the peptides by Flow cytometer(FCM).Furthermore,Scanning electron microscopy(SEM)was be used to observe the injure of the tumour cell membrane.In the last exprement,the six groups of antibacterial peptides were isolated by Polyacrylamide Gel Electrophoreses(PAGE),to show their peptide kinds.
The anticancer experiments tell us that,Musca domestica lavae have the best result, because they kill the four species of cancer cell over 86%.If normal saline instead of PBS, the extracted peptides could cause little hurt to humanbody.The bacteriostatic experiments shows that,the same group of peptides which induced by diffent germ could made different results.The observing of Scanning Electron Microscope(SEM)confirmed that,the machine-process of the peptides killing the cancer cell was drilling some holes on the membrane of the cell.The PAGE shows that,all of groups of peptides consist of different molecular mass peptides.Such as Musca domestica lavae,consist of seven kinds peptides, Blattella germanica three、Periplaneta americana three、Culex pipiens pallens firve and so on.
We know from anticancer experiment that,the diffent peptides could make diffent results from the same tumour cell.some peptides could kill the cell beneficially,such as fly lavae and the adult;while some peptides such as Periplaneta Americana,not only kill the cancer cell,but also promote the cell.From the bacteriostatic experiments we see that,different peptide from the same insect could be made with different inducing methods and the different results could be made from the same bacteriostatic experiments using the same insecta antibacterial peptids.
Concludes as follows:We must select the peptide when we hope to make a frofit of it;It is better that the normal saline instead of PBS when piptides were extracted from insects;The peptides do not hurt human body;The machine-prOcess of the peptides killing tumour cell,is drilling some hole on the cell mabrane;The results of SDS-PAGE show that,every insect could have many kinds of peptides when we change the mothod to extract it.
引文
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