日本血吸虫细胞选择培养与用SV40大T抗原基因转染的研究

发布时间：2018-05-03 17:56

本文选题：日本血吸虫 + 童虫细胞　；参考：《中南大学》2012年博士论文

【摘要】：第一章3种选择培养基用于日本血吸虫细胞培养的研究 [目的]探索3种选择培养基对日本血吸虫(Schistosoma japonicum,S.j)细胞作选择培养,观察和鉴定各自对细胞培养的效果,探索通过选择性培养基培养获得单一种类血吸虫细胞的可能性。[方法]分别从感染家兔获取S.j-12d童虫和42d成虫,按常规方法制备细胞。采用生殖类细胞改良培养基对S.j-12d童虫和S.j-42d成虫细胞进行选择培养；同时采用上皮细胞培养基和1640-40综合培养基对S.j-12d童虫细胞进行培养。观察日本血吸虫细胞经这3种选择培养基培养后-般生长状况和形态特点；通过染色体核型分析、AKP染色、超微结构观察对培养细胞进行鉴定；利用BrdU掺入法或3H-TdR掺入法检测部分培养细胞的增殖能力,以及运用Dot-ELISA方法检测培养细胞的抗原性。[结果]日本血吸虫细胞经这3种选择性培养基培养后,均有不同程度的增殖,细胞分裂相明显,并且细胞活力状态良好。经生殖类细胞改良培养基培养的童虫和成虫来源细胞在早期分裂现象明显,呈葡萄串样生长繁殖,形成细胞团；第3周可见大量形态结构相近的细胞呈半贴壁生长状态。对培养细胞进行鉴定发现细胞有DNA合成,染色体具有单倍体和双倍体两种核型；AKP染色为强阳性,超微结构鉴定可见胞质中含有大量囊泡状小体,此为生殖类细胞的特征之一；培养4周细胞开始出现退化死亡。日本血吸虫童虫细胞经上皮细胞培养基培养后的鉴定结果显示：培养3d的细胞出现类上皮样细胞生长,培养1周呈半贴壁生长；细胞形态多呈长梭形,结构完整,核质分明,细胞核清晰可见,核仁明显；超微结构显示细胞膜完整,细胞器无扩张、水肿现象,核仁和核膜清晰；并且童虫细胞成分能被血吸虫细胞免疫血清所识别。根据细胞的形态特征和抗原性,初步认为经上皮细胞培养基培养的童虫细胞为上皮类细胞。用1640-40综合培养基培养的日本血吸虫细胞经鉴定后结果显示：培养细胞分裂相多样,培养早期(30d)细胞状态和活力较好,增殖相明显；超微结构观察显示培养30d细胞形态结构基本正常,进行细胞活力检测达到70%；细胞增殖试验结果显示细胞具有一定的增殖能力；染色体分析符合血吸虫二倍体核型；对细胞进行冻存复苏发现基本保持细胞原有的特性和活力。[结论]日本血吸虫混合细胞经生殖类细胞改良培养基和上皮细胞培养基培养后,能使混合细胞中类似生殖类细胞和上皮细胞进行选择性增殖,并具有该类特定细胞的部分标志性特征,可以达到初步分选细胞的目的,但连续培养时间不长。1640-40综合培养基能明显促进细胞增殖,改善细胞生长状态,延长细胞在体外的存活时间。第二章SV40大T抗原基因重组腺病毒转染日本血吸虫童虫与其细胞后的效果观察 [目的]构建含SV40大T抗原(SV40LT)基因的重组腺病毒表达载体,观察重组腺病毒转染日本血吸虫童虫及童虫细胞后SV40LT基因的表达情况,探讨重组腺病毒载体转染日本血吸虫童虫和童虫细胞的可能性,观察转染的外源SV40LT基因对童虫和童虫细胞生长状况以及细胞增殖的影响。[方法]将国外赠送的携带SV40LT基因的重组腺病毒质粒(AdHu5-SV40LT)采用热休克法重新转化Stbl2感受态菌,获得重组腺病毒质粒。对质粒用限制性内切酶酶切、PCR扩增法进行鉴定。鉴定正确后的质粒经Pac I酶切线性化后用脂质体共转染293A细胞,待出现细胞病变效应(cell pathological effect, CPE)后收集细胞,经反复冻融细胞离心收集细胞裂解上清,用氯化铯超速离心法对病毒上清进行浓缩和纯化,获得具有感染性的重组腺病毒(Ad-SV40LT)。采用50%细胞培养感染法(50%cell culture infectious dose,CCID50)测定腺病毒的滴度。日本血吸虫尾蚴常规感染家兔获得12d童虫,一部分用改良841培养基培养,另一部分按常规方法制备细胞,添加1640-40综合培养基培养。两者均培养3d后用重组腺病毒进行转染,继续培养6-10d分别收集虫体和细胞,通过PCR、RT-PCR、 Western blotting和免疫组织化学方法检测转染童虫及童虫细胞中SV40LT基因的转录和表达情况。[结果]转化Stbl2感受态菌后提取的AdHu5-SV40LT质粒绎Pac Ⅰ酶切和PCR鉴定为日的质粒。质粒成功转染293A细胞并可在293A细胞内进行有效的复制；从转染293A细胞裂解上清中提取病毒DNA进行PCR检测证实含有SV40LT目的基因；同时Western blotting结果显示在质粒转染的293A细胞中有SV40LT蛋白的表达；免疫组织化学染色也证实了同样的结果。腺病毒转染的293A细胞裂解上清经氯化铯超速离心浓缩后测得病毒滴度为2.6×109cfu/ml。常规制备的S.j-12d童虫细胞用1640-40综合培养基培养3d后可见细胞生长状态良好,部分细胞分裂相明显。用重组腺病毒转染的S.j-12d童虫及童虫细胞经PCR检测均扩增出了外源SV40LT基因558bp的目的产物,进一步用RT-PCR也证实了有目的基因mRNA的转录表达；Western blotting检测结果显示重组腺病毒转染的童虫及其细胞内有SV40LT蛋白的表达；免疫组织化学染色检测也证实在虫体被膜下、口腹吸盘以及童虫细胞内均有该蛋白的表达。病毒转染后童虫活力未受影响,而童虫细胞转染初期增殖较快,此后生长逐渐减慢,细胞出现崩解、死亡。[结论]用质粒AdHu5-SV40LT和脂质体共转染293A细胞后,成功获得了具有感染能力的携带外源SV40LT基因的腺病毒；重组腺病毒转染日本血吸虫童虫及童虫细胞后有目的基因SV40LT的转录和蛋白表达,但转染的外源SV40LT基因未能使童虫细胞获得无限增殖能力而达到细胞永生化。本实验结果表明腺病毒能转染日本血吸虫童虫及童虫细胞,并能将外源基因成功导入,为血吸虫的转基因研究提供实验依据。
[Abstract]:Chapter 1 3 selective media for cell culture of Schistosoma japonicum
[Objective] to explore the selection culture of 3 kinds of selective medium for Schistosoma japonicum (S.j) cells, observe and identify the effect of each cell culture, explore the possibility of obtaining single type of Schistosoma cells through selective culture medium. [Methods] to obtain S.j-12d and 42d adult from infected rabbits, respectively, according to the routine. Methods the cells were prepared. The S.j-12d and S.j-42d adult cells were selected by reproductive cell improvement medium. At the same time, the epithelial cell culture medium and 1640-40 comprehensive medium were used to culture the S.j-12d worm cells. The growth status and morphological characteristics of Schistosoma japonicum cells were observed after the culture of these 3 kinds of culture medium. The culture cells were identified by chromosome karyotype analysis, AKP staining and ultrastructural observation. The proliferation ability of some cultured cells was detected by BrdU incorporation or 3H-TdR incorporation, and the antigenicity of the cultured cells was detected by Dot-ELISA method. [results] the cells of Schistosoma japonicum were cultured by these 3 selective medium. The cell division phase was obvious, and the cell viability was good. The early division phenomenon was evident in the early division of the children and the adult cells cultured on the improved cell culture medium. The cell clusters were produced in series, and the cells with similar morphologic structures were half adhered to the wall in the third week. The cells were identified as DNA synthesis, and the chromosomes were haploid and diploid two karyotypes; AKP staining was strong positive. Ultrastructural identification showed that the cytoplasm contained a large number of vesicular bodies, which was one of the characteristics of reproductive cells, and the cells began to degenerate for 4 weeks. The cells of Schistosoma japonicum by epithelia cells The results of the culture of culture medium showed that the cells in the culture of 3D appeared like epithelioid cells, and the cells were half attached to the wall for 1 weeks. The cell morphology was long spindle shaped, the structure was complete, the nucleolus was clear, the nucleus was clearly visible and the nucleolus were clear. The ultrastructure showed that the cell membrane was intact, the organelle was not dilated, the edema, nucleolus and nuclear membrane were clear. And the cell components of the children can be identified by the sera of Schistosoma cells. According to the morphological characteristics and antigenicity of the cells, the primary culture of the epithelioma cells cultured in the epithelial cell culture is epithelial cells. The results of the culture of Schistosoma japonicum cells cultured in 1640-40 comprehensive medium show that the cultures of the cultured cells are diverse and cultured. The state and vitality of 30d cells were better and the proliferation phase was obvious. Ultrastructural observation showed that the morphology and structure of the cultured 30d cells were basically normal, the detection of cell viability reached 70%, and the cell proliferation test showed that the cells had certain proliferation ability, and the chromosome analysis accords with the diploid karyotype of Schistosoma japonicum, and the cells were frozen and recovered. [Conclusion] the hybrid cells of Schistosoma japonicum can selectively proliferate like reproductive and epithelial cells in the mixed cells after the cultivation of the improved cell culture medium and epithelial cell culture medium, and have some characteristic characteristics of this kind of cell, which can be achieved. The aim of cell was preliminarily divided, but the continuous culture time of.1640-40 comprehensive culture medium could obviously promote cell proliferation, improve cell growth state and prolong the survival time of cells in vitro.
Second chapter SV40 large T antigen gene recombinant adenovirus transfection of Schistosoma japonicum larvae and its cells
[Objective] to construct a recombinant adenovirus expression vector containing SV40 large T antigen (SV40LT) gene and observe the expression of SV40LT gene in the transfected adenovirus of Schistosoma japonicum and the child's cell, and to explore the viability of the recombinant adenovirus vector transfection to the children and the cells of the Schistosoma japonicum. The growth status of the cell and the effect of cell proliferation. [Methods] the recombinant adenovirus plasmid (AdHu5-SV40LT), a recombinant adenovirus carrying the SV40LT gene, was converted into recombinant adenovirus vector by heat shock method, and the recombinant adenovirus plasmid was obtained by heat shock method. The plasmid was identified by restriction endonuclease digestion and PCR amplification. The correct plasmid was identified. After the tangent of Pac I enzyme, the 293A cells were co transfected with liposomes, and the cells were collected after the appearance of the cytopathic effect (cell pathological effect, CPE). The cells were collected by the centrifugation and centrifugation of the frozen thawing cells. The virus supernatant was concentrated and purified by cesium chloride overspeed centrifugation. The infected recombinant adenovirus (Ad-SV40LT) was obtained. 50%cell culture infectious dose (CCID50) was used to determine the titer of adenovirus. The cercariae of Schistosoma japonicum were routinely infected with 12D, some of which were cultured with modified 841 medium. The other part was prepared by conventional methods and cultured in 1640-40 comprehensive medium. Both of them were cultured for 3D and used recombinant adenovirus. Transfection, continue to culture 6-10d to collect insect body and cell respectively. Through PCR, RT-PCR, Western blotting and immunohistochemical method, the transcription and expression of SV40LT gene in the transfected children and children's cells were detected. [results] the AdHu5-SV40LT plasmids extracted from the Stbl2 receptive bacteria were transformed and the Pac I enzyme cut and PCR were identified as the plasmid of the day. The plasmid was successfully transfected into 293A cells and could be effectively replicated in the 293A cells. The PCR detection of the virus DNA from the transfected 293A cell cleavage supernatant confirmed the SV40LT target gene, and the Western blotting results showed the expression of SV40LT protein in the plasmid transfected 293A cells, and the immunohistochemical staining was also confirmed. The same results. The adenovirus transfected 293A cell cleavage supernatant was concentrated after cesium chloride overspeed centrifugation to detect the virus titer of 2.6 x 109cfu/ml., and the cell growth condition was good and the cell division phase was obvious. The S.j-12d worm transfected with recombinant adenovirus and the recombinant adenovirus transfected by recombinant adenovirus were in good condition. The target products of the exogenous SV40LT gene 558bp were amplified by PCR detection, and the transcriptional expression of the target gene mRNA was confirmed by RT-PCR, and the results of Western blotting detection showed that the recombinant adenovirus transfected with the expression of SV40LT protein in the cells and their cells, and the immunohistochemical staining was also proved to be in the insect body. Under the membrane, the expression of this protein was found in the stoma sucker and the cells of the worm. The vitality of the worm was not affected by the transfection of the virus, and the cell proliferation was faster in the early stage of transfection, then the growth slowed down gradually, and the cells disintegrated and died. [Conclusion] the infection ability was successfully obtained after transfecting 293A cells with plasmid AdHu5-SV40LT and liposomes. The adenovirus carrying the exogenous SV40LT gene; the recombinant adenovirus transfected with the target gene SV40LT of Schistosoma japonicum and the child's worm cells had the transcription and protein expression, but the transfected exogenous SV40LT gene failed to achieve the immortalization of the cells with unlimited proliferation ability. The results showed that the adenovirus could transfect the Schistosoma japonicum. The larvae and worm cells can successfully import exogenous genes, and provide experimental evidence for transgenic research of Schistosoma japonicum.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392.1

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